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package tfe
import (
"bytes"
"context"
"encoding/json"
"errors"
"fmt"
"io"
"math/rand"
"net/http"
"net/url"
"os"
"reflect"
"strconv"
"strings"
"time"
"github.com/google/go-querystring/query"
"github.com/hashicorp/go-cleanhttp"
retryablehttp "github.com/hashicorp/go-retryablehttp"
"github.com/svanharmelen/jsonapi"
"golang.org/x/time/rate"
)
const (
userAgent = "go-tfe"
headerRateLimit = "X-RateLimit-Limit"
headerRateReset = "X-RateLimit-Reset"
// DefaultAddress of Terraform Enterprise.
DefaultAddress = "https://app.terraform.io"
// DefaultBasePath on which the API is served.
DefaultBasePath = "/api/v2/"
)
var (
// ErrWorkspaceLocked is returned when trying to lock a
// locked workspace.
ErrWorkspaceLocked = errors.New("workspace already locked")
// ErrWorkspaceNotLocked is returned when trying to unlock
// a unlocked workspace.
ErrWorkspaceNotLocked = errors.New("workspace already unlocked")
// ErrUnauthorized is returned when a receiving a 401.
ErrUnauthorized = errors.New("unauthorized")
// ErrResourceNotFound is returned when a receiving a 404.
ErrResourceNotFound = errors.New("resource not found")
)
// RetryLogHook allows a function to run before each retry.
type RetryLogHook func(attemptNum int, resp *http.Response)
// Config provides configuration details to the API client.
type Config struct {
// The address of the Terraform Enterprise API.
Address string
// The base path on which the API is served.
BasePath string
// API token used to access the Terraform Enterprise API.
Token string
// Headers that will be added to every request.
Headers http.Header
// A custom HTTP client to use.
HTTPClient *http.Client
// RetryLogHook is invoked each time a request is retried.
RetryLogHook RetryLogHook
}
// DefaultConfig returns a default config structure.
func DefaultConfig() *Config {
config := &Config{
Address: os.Getenv("TFE_ADDRESS"),
BasePath: DefaultBasePath,
Token: os.Getenv("TFE_TOKEN"),
Headers: make(http.Header),
HTTPClient: cleanhttp.DefaultPooledClient(),
}
// Set the default address if none is given.
if config.Address == "" {
config.Address = DefaultAddress
}
// Set the default user agent.
config.Headers.Set("User-Agent", userAgent)
return config
}
// Client is the Terraform Enterprise API client. It provides the basic
// connectivity and configuration for accessing the TFE API.
type Client struct {
baseURL *url.URL
token string
headers http.Header
http *retryablehttp.Client
limiter *rate.Limiter
retryLogHook RetryLogHook
retryServerErrors bool
Applies Applies
ConfigurationVersions ConfigurationVersions
OAuthClients OAuthClients
OAuthTokens OAuthTokens
Organizations Organizations
OrganizationTokens OrganizationTokens
Plans Plans
Policies Policies
PolicyChecks PolicyChecks
PolicySets PolicySets
Runs Runs
SSHKeys SSHKeys
StateVersions StateVersions
Teams Teams
TeamAccess TeamAccesses
TeamMembers TeamMembers
TeamTokens TeamTokens
Users Users
Variables Variables
Workspaces Workspaces
}
// NewClient creates a new Terraform Enterprise API client.
func NewClient(cfg *Config) (*Client, error) {
config := DefaultConfig()
// Layer in the provided config for any non-blank values.
if cfg != nil {
if cfg.Address != "" {
config.Address = cfg.Address
}
if cfg.BasePath != "" {
config.BasePath = cfg.BasePath
}
if cfg.Token != "" {
config.Token = cfg.Token
}
for k, v := range cfg.Headers {
config.Headers[k] = v
}
if cfg.HTTPClient != nil {
config.HTTPClient = cfg.HTTPClient
}
if cfg.RetryLogHook != nil {
config.RetryLogHook = cfg.RetryLogHook
}
}
// Parse the address to make sure its a valid URL.
baseURL, err := url.Parse(config.Address)
if err != nil {
return nil, fmt.Errorf("invalid address: %v", err)
}
baseURL.Path = config.BasePath
if !strings.HasSuffix(baseURL.Path, "/") {
baseURL.Path += "/"
}
// This value must be provided by the user.
if config.Token == "" {
return nil, fmt.Errorf("missing API token")
}
// Create the client.
client := &Client{
baseURL: baseURL,
token: config.Token,
headers: config.Headers,
retryLogHook: config.RetryLogHook,
}
client.http = &retryablehttp.Client{
Backoff: client.retryHTTPBackoff,
CheckRetry: client.retryHTTPCheck,
ErrorHandler: retryablehttp.PassthroughErrorHandler,
HTTPClient: config.HTTPClient,
RetryWaitMin: 100 * time.Millisecond,
RetryWaitMax: 400 * time.Millisecond,
RetryMax: 30,
}
// Configure the rate limiter.
if err := client.configureLimiter(); err != nil {
return nil, err
}
// Create the services.
client.Applies = &applies{client: client}
client.ConfigurationVersions = &configurationVersions{client: client}
client.OAuthClients = &oAuthClients{client: client}
client.OAuthTokens = &oAuthTokens{client: client}
client.Organizations = &organizations{client: client}
client.OrganizationTokens = &organizationTokens{client: client}
client.Plans = &plans{client: client}
client.Policies = &policies{client: client}
client.PolicyChecks = &policyChecks{client: client}
client.PolicySets = &policySets{client: client}
client.Runs = &runs{client: client}
client.SSHKeys = &sshKeys{client: client}
client.StateVersions = &stateVersions{client: client}
client.Teams = &teams{client: client}
client.TeamAccess = &teamAccesses{client: client}
client.TeamMembers = &teamMembers{client: client}
client.TeamTokens = &teamTokens{client: client}
client.Users = &users{client: client}
client.Variables = &variables{client: client}
client.Workspaces = &workspaces{client: client}
return client, nil
}
// RetryServerErrors configures the retry HTTP check to also retry
// unexpected errors or requests that failed with a server error.
func (c *Client) RetryServerErrors(retry bool) {
c.retryServerErrors = retry
}
// retryHTTPCheck provides a callback for Client.CheckRetry which
// will retry both rate limit (429) and server (>= 500) errors.
func (c *Client) retryHTTPCheck(ctx context.Context, resp *http.Response, err error) (bool, error) {
if ctx.Err() != nil {
return false, ctx.Err()
}
if err != nil {
return c.retryServerErrors, err
}
if resp.StatusCode == 429 || (c.retryServerErrors && resp.StatusCode >= 500) {
return true, nil
}
return false, nil
}
// retryHTTPBackoff provides a generic callback for Client.Backoff which
// will pass through all calls based on the status code of the response.
func (c *Client) retryHTTPBackoff(min, max time.Duration, attemptNum int, resp *http.Response) time.Duration {
if c.retryLogHook != nil {
c.retryLogHook(attemptNum, resp)
}
// Use the rate limit backoff function when we are rate limited.
if resp.StatusCode == 429 {
return rateLimitBackoff(min, max, attemptNum, resp)
}
// Set custom duration's when we experience a service interruption.
min = 700 * time.Millisecond
max = 900 * time.Millisecond
return retryablehttp.LinearJitterBackoff(min, max, attemptNum, resp)
}
// rateLimitBackoff provides a callback for Client.Backoff which will use the
// X-RateLimit_Reset header to determine the time to wait. We add some jitter
// to prevent a thundering herd.
//
// min and max are mainly used for bounding the jitter that will be added to
// the reset time retrieved from the headers. But if the final wait time is
// less then min, min will be used instead.
func rateLimitBackoff(min, max time.Duration, attemptNum int, resp *http.Response) time.Duration {
// rnd is used to generate pseudo-random numbers.
rnd := rand.New(rand.NewSource(time.Now().UnixNano()))
// First create some jitter bounded by the min and max durations.
jitter := time.Duration(rnd.Float64() * float64(max-min))
if resp != nil {
if v := resp.Header.Get(headerRateReset); v != "" {
if reset, _ := strconv.ParseFloat(v, 64); reset > 0 {
// Only update min if the given time to wait is longer.
if wait := time.Duration(reset * 1e9); wait > min {
min = wait
}
}
}
}
return min + jitter
}
// configureLimiter configures the rate limiter.
func (c *Client) configureLimiter() error {
// Create a new request.
req, err := http.NewRequest("GET", c.baseURL.String(), nil)
if err != nil {
return err
}
// Attach the default headers.
for k, v := range c.headers {
req.Header[k] = v
}
req.Header.Set("Accept", "application/vnd.api+json")
// Make a single request to retrieve the rate limit headers.
resp, err := c.http.HTTPClient.Do(req)
if err != nil {
return err
}
resp.Body.Close()
// Set default values for when rate limiting is disabled.
limit := rate.Inf
burst := 0
if v := resp.Header.Get(headerRateLimit); v != "" {
if rateLimit, _ := strconv.ParseFloat(v, 64); rateLimit > 0 {
// Configure the limit and burst using a split of 2/3 for the limit and
// 1/3 for the burst. This enables clients to burst 1/3 of the allowed
// calls before the limiter kicks in. The remaining calls will then be
// spread out evenly using intervals of time.Second / limit which should
// prevent hitting the rate limit.
limit = rate.Limit(rateLimit * 0.66)
burst = int(rateLimit * 0.33)
}
}
// Create a new limiter using the calculated values.
c.limiter = rate.NewLimiter(limit, burst)
return nil
}
// newRequest creates an API request. A relative URL path can be provided in
// path, in which case it is resolved relative to the apiVersionPath of the
// Client. Relative URL paths should always be specified without a preceding
// slash.
// If v is supplied, the value will be JSONAPI encoded and included as the
// request body. If the method is GET, the value will be parsed and added as
// query parameters.
func (c *Client) newRequest(method, path string, v interface{}) (*retryablehttp.Request, error) {
u, err := c.baseURL.Parse(path)
if err != nil {
return nil, err
}
// Create a request specific headers map.
reqHeaders := make(http.Header)
reqHeaders.Set("Authorization", "Bearer "+c.token)
var body interface{}
switch method {
case "GET":
reqHeaders.Set("Accept", "application/vnd.api+json")
if v != nil {
q, err := query.Values(v)
if err != nil {
return nil, err
}
u.RawQuery = q.Encode()
}
case "DELETE", "PATCH", "POST":
reqHeaders.Set("Accept", "application/vnd.api+json")
reqHeaders.Set("Content-Type", "application/vnd.api+json")
if v != nil {
buf := bytes.NewBuffer(nil)
if err := jsonapi.MarshalPayloadWithoutIncluded(buf, v); err != nil {
return nil, err
}
body = buf
}
case "PUT":
reqHeaders.Set("Accept", "application/json")
reqHeaders.Set("Content-Type", "application/octet-stream")
body = v
}
req, err := retryablehttp.NewRequest(method, u.String(), body)
if err != nil {
return nil, err
}
// Set the default headers.
for k, v := range c.headers {
req.Header[k] = v
}
// Set the request specific headers.
for k, v := range reqHeaders {
req.Header[k] = v
}
return req, nil
}
// do sends an API request and returns the API response. The API response
// is JSONAPI decoded and the document's primary data is stored in the value
// pointed to by v, or returned as an error if an API error has occurred.
// If v implements the io.Writer interface, the raw response body will be
// written to v, without attempting to first decode it.
//
// The provided ctx must be non-nil. If it is canceled or times out, ctx.Err()
// will be returned.
func (c *Client) do(ctx context.Context, req *retryablehttp.Request, v interface{}) error {
// Wait will block until the limiter can obtain a new token
// or returns an error if the given context is canceled.
if err := c.limiter.Wait(ctx); err != nil {
return err
}
// Add the context to the request.
req = req.WithContext(ctx)
// Execute the request and check the response.
resp, err := c.http.Do(req)
if err != nil {
// If we got an error, and the context has been canceled,
// the context's error is probably more useful.
select {
case <-ctx.Done():
return ctx.Err()
default:
return err
}
}
defer resp.Body.Close()
// Basic response checking.
if err := checkResponseCode(resp); err != nil {
return err
}
// Return here if decoding the response isn't needed.
if v == nil {
return nil
}
// If v implements io.Writer, write the raw response body.
if w, ok := v.(io.Writer); ok {
_, err = io.Copy(w, resp.Body)
return err
}
// Get the value of v so we can test if it's a struct.
dst := reflect.Indirect(reflect.ValueOf(v))
// Return an error if v is not a struct or an io.Writer.
if dst.Kind() != reflect.Struct {
return fmt.Errorf("v must be a struct or an io.Writer")
}
// Try to get the Items and Pagination struct fields.
items := dst.FieldByName("Items")
pagination := dst.FieldByName("Pagination")
// Unmarshal a single value if v does not contain the
// Items and Pagination struct fields.
if !items.IsValid() || !pagination.IsValid() {
return jsonapi.UnmarshalPayload(resp.Body, v)
}
// Return an error if v.Items is not a slice.
if items.Type().Kind() != reflect.Slice {
return fmt.Errorf("v.Items must be a slice")
}
// Create a temporary buffer and copy all the read data into it.
body := bytes.NewBuffer(nil)
reader := io.TeeReader(resp.Body, body)
// Unmarshal as a list of values as v.Items is a slice.
raw, err := jsonapi.UnmarshalManyPayload(reader, items.Type().Elem())
if err != nil {
return err
}
// Make a new slice to hold the results.
sliceType := reflect.SliceOf(items.Type().Elem())
result := reflect.MakeSlice(sliceType, 0, len(raw))
// Add all of the results to the new slice.
for _, v := range raw {
result = reflect.Append(result, reflect.ValueOf(v))
}
// Pointer-swap the result.
items.Set(result)
// As we are getting a list of values, we need to decode
// the pagination details out of the response body.
p, err := parsePagination(body)
if err != nil {
return err
}
// Pointer-swap the decoded pagination details.
pagination.Set(reflect.ValueOf(p))
return nil
}
// ListOptions is used to specify pagination options when making API requests.
// Pagination allows breaking up large result sets into chunks, or "pages".
type ListOptions struct {
// The page number to request. The results vary based on the PageSize.
PageNumber int `url:"page[number],omitempty"`
// The number of elements returned in a single page.
PageSize int `url:"page[size],omitempty"`
}
// Pagination is used to return the pagination details of an API request.
type Pagination struct {
CurrentPage int `json:"current-page"`
PreviousPage int `json:"prev-page"`
NextPage int `json:"next-page"`
TotalPages int `json:"total-pages"`
TotalCount int `json:"total-count"`
}
func parsePagination(body io.Reader) (*Pagination, error) {
var raw struct {
Meta struct {
Pagination Pagination `json:"pagination"`
} `json:"meta"`
}
// JSON decode the raw response.
if err := json.NewDecoder(body).Decode(&raw); err != nil {
return &Pagination{}, err
}
return &raw.Meta.Pagination, nil
}
// checkResponseCode can be used to check the status code of an HTTP request.
func checkResponseCode(r *http.Response) error {
if r.StatusCode >= 200 && r.StatusCode <= 299 {
return nil
}
switch r.StatusCode {
case 401:
return ErrUnauthorized
case 404:
return ErrResourceNotFound
case 409:
switch {
case strings.HasSuffix(r.Request.URL.Path, "actions/lock"):
return ErrWorkspaceLocked
case strings.HasSuffix(r.Request.URL.Path, "actions/unlock"):
return ErrWorkspaceNotLocked
case strings.HasSuffix(r.Request.URL.Path, "actions/force-unlock"):
return ErrWorkspaceNotLocked
}
}
// Decode the error payload.
errPayload := &jsonapi.ErrorsPayload{}
err := json.NewDecoder(r.Body).Decode(errPayload)
if err != nil || len(errPayload.Errors) == 0 {
return fmt.Errorf(r.Status)
}
// Parse and format the errors.
var errs []string
for _, e := range errPayload.Errors {
if e.Detail == "" {
errs = append(errs, e.Title)
} else {
errs = append(errs, fmt.Sprintf("%s\n\n%s", e.Title, e.Detail))
}
}
return fmt.Errorf(strings.Join(errs, "\n"))
} | The code is a Go package that allows users to access the Terraform Enterprise API. It includes functions for making API requests, handling authentication, and parsing responses. The code also defines constants and error types. External libraries are used for handling HTTP requests and JSON parsing. | stacksmol |
Protein function is encoded within protein sequence and protein domains. However, how protein domains cooperate within a protein to modulate overall activity and how this impacts functional diversification at the molecular and organism levels remains largely unaddressed. Focusing on three domains of the central class Drosophila Hox transcription factor AbdominalA (AbdA), we used combinatorial domain mutations and most known AbdA developmental functions as biological readouts to investigate how protein domains collectively shape protein activity. The results uncover redundancy, interactivity, and multifunctionality of protein domains as salient features underlying overall AbdA protein activity, providing means to apprehend functional diversity and accounting for the robustness of Hox-controlled developmental programs. Importantly, the results highlight context-dependency in protein domain usage and interaction, allowing major modifications in domains to be tolerated without general functional loss. The non-pleoitropic effect of domain mutation suggests that protein modification may contribute more broadly to molecular changes underlying morphological diversification during evolution, so far thought to rely largely on modification in gene cis-regulatory sequences. How the diversity of animal body plans is established remains a central question in developmental and evolutionary biology [1], [2]. A key step towards understanding the molecular basis underlying diversity is to decipher mechanisms controlling proper genome expression, and how variations in these mechanisms have been at the origin of developmental and evolutionary diversity. While a large number of studies have focused on the impact of cis-regulatory sequences organization (reviewed in [3]), deciphering the intrinsic functional organization of trans-acting transcription factors remains largely unaddressed. Studies have identified functional domains ([4]–[9] and [7], [10], [11] for reviews), but how different protein domains jointly and collectively act for defining the overall activity has been poorly assessed. Yet, a recent study highlights that the synthetic shuffling of protein domains within proteins of the yeast-mating signaling pathway results in the diversification of the mating behavior, demonstrating the importance of protein domain interactions for functional diversification [12]. Hox genes, which encode homeodomain (HD) -containing transcription factors, provide a suitable paradigm to decipher how function is encoded within protein sequence, and how associated changes may constitute the origin of functional specification and diversification. Hox genes have arisen from duplication events of ancestral genes, followed by sequence divergence that promoted the emergence of up to 14 paralogous groups in vertebrates. Hox paralogue proteins display distinct regulatory functions, promoting axial morphological diversification in all bilaterian animals [13]–[17]. Previous work has established that sequence changes in the HD, the DNA binding domain, and a few additional protein domains, have played a major role in the diversification of Hox protein function [4]–[9], [18]–[21]. However, how protein domains functionally interact to shape overall protein activity remains elusive. We focused on three protein domains from the Drosophila central Hox paralogue protein Abdominal (AbdA, Figure 1). These domains are related by their demonstrated or potential involvement in the recruitment of the Extradenticle (Exd) cofactor, homologous to vertebrate PBX proteins, known to have key roles in establishing Hox functional specificity. The first domain, known as hexapeptide (HX) or PID (Pbx Interacting Domain), with a core YPWM sequence, is found in all Hox paralogue groups, with the exception of some posterior Hox proteins. Biochemical, structural and functional studies have shown that this motif mediates interaction with the Exd/PBX class of Hox cofactors (collectively referred as PBC). The second domain, termed UbdA (UA) is specifically found in the central Hox proteins AbdA and Ultrabithorax (Ubx). This paralogue-specific domain was recently shown to be required for Exd recruitment in the repression of the limb-promoting gene Distalless (Dll) [8], [22]. The third domain (TD), similar in sequence (TDWM) to the YPWM motif, is also paralogue-specific. The TD motif retains the W that provides strong contact with the PBC class proteins, and matches the sequence of the HX motif in some Hox proteins (eg., Hoxa1). Evidence for an Exd recruiting role of the TD domain in AbdA however remains to be demonstrated. To start unraveling how protein domains collectively shape Hox protein activity, the effect of single, combined double or triple domain mutations were analyzed using most known AbdA functions as biological readouts. The large functional window covered by the study allows identifying functional attributes of protein domains taken in isolation and collectively, and a quantitative analysis by hierarchical clustering highlights the functional organization of the Hox protein AbdA. Given the phylogeny of the studied protein domains, the work has also implication regarding the mechanisms underlying the evolution of AbdA protein function. AbdA variants bearing single or all possible combinations of protein domain mutations (Figure 1A) were ectopically expressed through the binary UAS-Gal4 expression system [23]. Protein levels following induced expression were quantified and experimental conditions ensuring levels close to that of endogenous AbdA were selected (see Materials and Methods). Impact of AbdA variants on target gene control, phenotypic traits and locomotion behavior (Figure 1B), covering AbdA functions of increasing complexity in different tissues, were evaluated in the anterior region where the endogenous AbdA protein is absent. Quantified results (see Text S1) are presented as loss (and in few cases as gain) of regulatory potential. Eleven functional assays were used to assess domain requirements for AbdA activity (Figure 1B). Four assays rely on the regulation of AbdA target genes, for which evidence of a direct regulation has been previously reported, including the regulation of Distalless (Dll) [8], [24], [25] and Antennapedia (Antp) [26] in the epidermis, and the regulation of wingless (wg) [27] and decapentaplegic (dpp) [28], [29] in the visceral mesoderm. Six assays rely on analysis of phenotypic traits. One of these phenotypic trait, oenocyte specification, results from the regulation of a single target gene [30]. Others, cerebral branch [31], somatic muscles [32], A2 epidermal morphology [33], [34], neuroblast [35], [36] and heart cell lineage specification [37] likely depend of the coordinated regulation of several target genes. Finally, we also used a behavioral trait, larval locomotion, thought to rely on integrated AbdA function in two distinct tissues, the somatic musculature and the nervous system [38]. In the somatic musculature, the abdominal specific pattern is characterized by the presence of muscle located ventrally and absent in thoracic segments, a feature that can be visualized by the expression of nautilus (nau) [32]. This distinction was previously shown to result, at least in part, from the activity of AbdA [32]. Accordingly, anterior ectopic expression of AbdA using the mesodermal driver (24B-Gal4) results in ectopic ventral expression of Nau in anterior segments (Figure 2). We found however that none of the AbdA protein domains under study, alone or in combination, was required to specify the abdominal specific features of the somatic musculature (Figure 2 and Figure S1). In the same conditions, a point mutation at position 50 of the homeodomain that impairs AbdA binding to DNA resulted in the loss of Nau inducing capacity (Figure 2 and Figure S1). The dispensability of the HX, TD and UA domains for specifying abdominal features of somatic muscle pattern is consistent with the fact that nau activation by AbdA is not dependent upon Exd activity [32], although results below argue that these domains assume other functions than Exd recruitment. In the embryonic central nervous system, a subset of 30 neuroblasts (NB' s) found in each hemisegment, including the NB5–6, generate a larger lineage in the thorax than in the abdomen. Recent studies demonstrated that posterior Hox genes, such as abdA, impose in the abdomen a smaller NB5–6 lineage by triggering an early cell cycle exit [39]. Misexpression of AbdA within NB5–6 in the thorax using ladybird (K) -Gal4 result in an early lineage truncation, mimicking the situation that normally occurs in the abdomen, ultimately leading to a smaller thoracic NB5–6 lineage size (Figure 3). Average number of NB5–6 cells in wild type thoracic and abdominal segments was previously estimated at 16 and 6 cells respectively: these values were considered as references for full (100%) or complete loss (0%) of repressive activities of AbdA variants on NB5–6 lineage. Intermediate repressive levels upon ectopic expression with ladybird (K) -Gal4 were deduced from the quantification of NB5–6 lineage cell numbers in thoracic segments T2/3 (see methods). Results obtained indicate that lineage truncation triggered by AbdA is similarly affected following UA, HX/UA, TD/UA and HX/TD/UA mutations (Figure 3), which can be best explained by a unique requirement of the UA domain for AbdA function. In the visceral mesoderm, AbdA is expressed in parasegment (PS) 8–12. The target genes wg and dpp are respectively activated (in PS8) and repressed (in PS8–12) by AbdA in the visceral mesoderm. Restricted (PS8) activation of wg by AbdA results from the action of the Dpp signal, locally produced by PS7 cells under the control of the Ubx protein [40]. Accordingly, anterior ectopic expression of AbdA only results in a mild activation of wg, as activation only occurs in cells experiencing partial repression of dpp [27]. Previous work has shown that the HX mutation results in a protein that activates dpp instead of repressing it, and consequently more efficiently activates wg [41]. AbdA variants were expressed with the 24B-Gal4 driver. Levels of regulatory activities were deduced following fluorescent in situ hybridization against dpp or wg in the visceral mesoderm of stage 14 embryos in PS1–PS7, ie anterior to endogenous AbdA expressing cells (PS8–12; Figure 4 and Figures S2 and S3). Arbitrary values have been assigned to regulatory activities of AbdA variants. For dpp (Figure 4A and Figure S2), no effect on dpp expression was scored by 0, normal repression of dpp expression in PS7 by 100 (partial repression was never observed) and ectopic activation (instead of repression) of dpp was scored by negative values (depending of the number of ectopic sites (see Text S1). For wg, in a manner similar to dpp, no effect was scored by 0, and positive and negative values were respectively assigned to normal (activation) or abnormal (repression) activities on wg expression (Figure 4B and Figure S3; see Text S1). Results obtained allow two conclusions. First, single domain mutations result in strong modification of AbdA activity. Second, domain mutations often result not only in a quantitative, but also in a qualitative (neomorphic) modification of activity, changing AbdA from an activator to a repressor, or reversely from a repressor to an activator. Oenocytes form under AbdA control in segments A1–A7. This occurs through AbdA-dependent activation of Rhomboid (Rho) in a chordotonal organ precursor cell called C1. Expression of Rho then enables the secretion of the EGF ligand Spitz that will instruct neighboring epidermal cells to differentiate into oenocytes [30]. In absence of AbdA, the EGF pathway is not locally activated and oenocytes are not specified [30]. Reversely, ectopic expression of AbdA induces oenocytes in thoracic segments. AbdA variants were ubiquitously expressed with the armadillo (arm) -Gal4 driver. Oenocyte inducing potential of AbdA variants, visualised with the seven-up (svp) -lacZ enhancer trap reporter construct, was deduced from the number of thoracic segments that contain ectopic oenocytes (see Text S1). This inductive potential is reduced following single mutations of the UA domain and combined mutation of the HX/TD or TD/UA domains, and is abolished following HX/UA and HX/TD/UA mutations (Figure 5 and Figure S4). These observations suggest an additive contribution of the HX, TD and UA protein domains for oenocyte induction by AbdA, consistent with protein domains acting independently of each other, and contributing uniquely through additive contribution to protein activity. The tracheal cerebral branch forms dorsally exclusively in the second thoracic segment T2, in response to repressive activities of Bithorax Hox proteins in T3-A8 segments [42]. This phenotypic trait can be followed by a breathless (btl) driven GFP reporter that extends posteriorly in the absence of Bithorax complex genes, and that is suppressed in T2 following Btl-driven expression of AbdA in the tracheal system (Figure 6A). Only full repression of cerebral branches was considered and repressive activities of AbdA variants thus correspond to either 0% (no repression) or 100% (full repression) (see Text S1). We found that the repression of the cerebral branch by AbdA is impaired following TD/UA and HX/TD/UA but not HX/UA or HX/TD mutations, revealing a functional redundancy between the TD and UA domains (Figure 6A, and Figure S5). In the embryonic heart, abdominal segments are made of six pairs of cells, instead of four in thoracic segments [37]. This difference was shown to result from AbdA (and Ubx) promoting the six cell lineage in the abdomen [37], and in the thorax following AbdA ubiquitous expression in the mesoderm driven by the 24B-Gal4 driver ([37], Figure 6B). The visualization of the lineage is facilitated by a Dorsocross (Doc) staining, that labels two cells in each hemisegment, allowing to unambiguously identify each hemisegment. Effects of AbdA variants in cardiac cells specification were visualized by double fluorescent immunostaining against AbdA and Dorsocross (Doc). The six cell lineage inductive capacity of AbdA was scored by counting the number of cardiac cells in the T2 and T3 segments (see Text S1). Results showed that the six cell lineage inductive ability of AbdA is lost following HX/UA and HX/TD/UA mutations (Figure 6B and Figure S6). These observations again highlight functional redundancy, but between the UA and HX domains, instead of TD and UA domain as observed in cerebral branch specification. Additional examples of functional redundancy, yet in more complex pattern of interactions between protein domains were found in the biological contexts described below. The limb-promoting gene Distalles (Dll) and Hox gene Antennapedia (Antp) are direct targets of AbdA [26], [43]. The ability of AbdA variants, following ubiquitous expression through the arm-Gal4 driver, to repress Dll (Figure 7A and Figure S7) and Antp (Figure 7B and Figure S8) was evaluated by examining the activity of a Hox responsive Dll enhancer (DME, [44]) and the expression of the Antp protein, respectively (see Text S1). Single domain mutations do not strongly affect repressive activities of AbdA on Dll and Antp, leading to a mean loss of 40%, with the exception of the TD mutation, which affects more (60%) the repressive activities on Antp. Combining domain mutations leads to stronger effects: in the case of Dll, simultaneous mutation of the HX and UA domains almost completely abolishes AbdA repressive activities, while in the case of Antp simultaneous mutation of the HX and UA domains or TD and UA domains results in a loss of 70% of AbdA repressive activity. More surprisingly, simultaneous mutation of the HX, TD and UA domains does not compromise further AbdA activity but instead restores a significant level of repressive activity, comparable to that of single domain mutated AbdA variants. This indicates that the three protein domains do not provide independent regulatory input, but likely act in interactive and mutually inhibitory ways. A similar yet more complex pattern of domain interactions was observed in the specification of A2 epidermal morphology. In this tissue, AbdA promotes the formation of a stereotyped trapezoidal arrangement of denticle belts (Figure 7C). The potential of AbdA variants to specify A2 epidermal morphology was assessed following arm-Gal4 driven expression by scoring the denticle belts morphology and organisation in transformed A1 and thoracic segments (Figure 7C and Figure S9). Epidermal specification was not impaired by HX and slightly reduced by UA or TD mutations. Simultaneous mutation in two domains suggests functional redundancy between HX and TD, UA and HX but not between UA and TD domains. As noticed previously for the regulation of Dll and Antp in the epidermis, mutating the three domains simultaneously restores the activity, generating a protein that displays an activity close to the wild type protein. In many animals including vertebrates, locomotion results from the coordinated action of regionally distinct sets of movements. Drosophila larvae crawl by means of three region specific movements [38]. The locomotion cycle starts by a contraction of the most posterior abdominal segments (A8/A9), followed by a wave of peristaltic movement in A1–A7, where each segment is transiently lifted up (D/V movement), pulled forward and lowered, starting from A7. When the wave reaches A1, the thoracic and head segments start moving by a telescopic type of movement (A/P movement), occurring through contraction of anterior segments [38]. It was established that AbdA is necessary and sufficient to specify the abdominal type of movement, namely abdominal peristalsis [38]. The potential of wild type and AbdA variants to promote abdominal peristalsis was evaluated following arm-Gal4 driven expression (Figure 7B), by scoring in the T3 thoracic segment D/V movements (see Text S1). Single domain mutations do not significantly alter promotion of abdominal peristalsis (Figure 7D and Figure S10). Again, two types of functional redundancy were observed: between the TD and UA domains, and to a lesser extent between the HX and UA domains. As in the case of Dll and Antp regulation and A2 epidermal morphology specification, triple domain mutation corrected the effects of double mutations, with a protein promoting abdominal peristalsis as efficiently as the wild type protein, providing an additional example of mutually suppressive activity of protein domains. Previous studies have established that Exd is required for Dll [25] and wg [45] regulation, oenocytes [30] and epidermal morphology specification [46], and neuroblast lineage commitment [37], while dispensable for Antp [46] and dpp [47] regulation. In the case of cerebral branch specification, no conclusion could be reached since loss of Exd results in the absence of cerebral branch formation in the T2 segment [48]: this positive input of Exd hinders the assessment of a possible contribution for AbdA mediated cerebral branch repression in abdominal segments. The potential implication of Exd in AbdA-mediated heart lineage commitment and larval locomotion is not known. Staining for Doc1 in embryos deprived for maternal and zygotic Exd showed that the abdominal hemi segments adopt the AbdA-dependent six cell lineage, showing the dispensability of Exd for this AbdA function (Figure 6C). The requirement of Exd for larval locomotion has been examined in homothorax (hth) mutant that impairs Exd nuclear transport and mimics exd maternal and zygotic loss [49]. The absence of peristaltic waves in this genetic context indicates a strict requirement of Exd for abdominal peristalsis (Figure S10). Taken together with the protein domain requirement results, the exd dependency indicates that the HX, UA and TD domains, known (HX and UA) or candidate (TD) Exd recruiting domains, are also required for Exd-independent function. This is supported by the HX/UA requirement for heart lineage specification, by the HX and UA requirement for proper regulation of the dpp target gene, the HX/TD requirement for Antp repression and the requirement of TD for dpp target regulation. Collectively, this highlights that the HX and UA (and likely TD) protein domains are multifunctional, serving in some biological context Exd interaction function, while in others, they are used differently, for a molecular activity that still remains to be defined. The complete set of quantitative data was analyzed using a hierarchical clustering method (Figure 8; see Materials and Methods). Clustering according to biological readouts does not reveal any clear grouping, regarding for instance developmental stage or tissue type, suggesting that the forces that govern domain usage and interaction between protein domains mostly reside in the regulated target gene. By contrast, clustering according to protein domains clearly reveals a hierarchical requirement of the domains for the various AbdA functions analyzed here. A bipartition of AbdA variants is observed, with the mutants for the HX, the TD and HX/TD domains on the one hand, and variants mutant for the UA domain, alone or in combination, on the other hand. Such bipartition suggests the existence of two functional modules that can be distinguished based on UA domain requirement. The first module, which relies mostly on the HX and TD domains, is used for a small subset of AbdA functions only. The second module relies on the activity of the HX, TD and UA domains, yet the requirements of the HX and TD domains are revealed only in UA deficient context. Thus, the driving force in this second functional module is the UA domain, as its mutation unmasks the requirement for the HX and TD domains, which is not revealed by their single or combined mutations. These results identify a prominent role of the UA domain in AbdA function. Studies towards deciphering the mode of action of Hox proteins have so far essentially concentrated on how individual protein domains contribute to protein function. These focused approaches allowed in depth analyses, unraveling the intimate molecular and sometimes structural details of how protein domains contribute to protein function, providing decisive insights into how Hox proteins reach specificity. This work provides a different complementary approach towards deciphering the mode of action of Hox proteins. First it aims at studying protein domains in combinations, using combined and not only single protein domain mutations, considering that the overall protein activity is likely not a sum of the activity of individual protein domains, and that novel properties may emerge from interactions between protein domains. Second, it uses extensive in vivo biological readout, (most of the known AbdA functions), instead of a single or a few functions. While impairing the in depth analyses of previous focused approaches, the large functional window covered by this study allows the identification of features underlying the intrinsic functional organization of the Hox protein AbdA. Although the approach taken relies on a gain of function strategy, special care was taken to select experimental conditions where proteins were expressed closed to physiological levels of expression. Biological readouts considered are functions that AbdA can sustain in ectopic places, suggesting that availability of AbdA protein partners is not a limitation of the experimental strategy chosen. Finally, the effects of expressing the AbdA variants (in all eleven biological readouts) were scored in regions anterior to the endogenous AbdA expression domain (ie in cells where the endogenous wild type gene product is not present), avoiding any further complexity that may result from competition with the endogenous AbdA protein. Below, we summarize how results obtained shed light on the mode of action of the Hox protein AbdA and discuss the evolutionary implications. This study identifies salient features underlying the intrinsic functional organization of the AbdA Hox transcription factor. Protein domains often display functional redundancy, with strong effects in most cases requiring simultaneous mutations of two or three domains. Redundancy was frequently observed between the HX and UA domains, or between the TD and UA domains, while redundancy between the HX and TD domains is less frequent (Figure 8). This indicates that redundancy does not necessarily rely on functional compensation through structurally related domains, since the HX and TD are closely related domains, while the UA domain is completely unrelated. Thus, functional redundancy rather reflects the potential to perform similar activities through distinct molecular strategies. This property likely confers robustness to Hox protein activity, accommodating mutations in protein domains without generally impacting on regulatory activities. Protein domains within AbdA also generally do not act as independent functional modules, but instead display a high degree of interactivity, as demonstrated by the non-additive effects of domain mutations in the majority of the biological readouts studied. In addition, protein domains are often multifunctional, in the sense that they serve different molecular functions. This is illustrated by the fact that the HX and UA domains, previously described to mediate Exd recruitment, are also required for Exd-independent processes. Thus domain interactivity and multifunctionality are hallmarks of AbdA regulatory activity. These properties provide means to apprehend the bases underlying Hox functional diversity with a restricted number of functional modules, and therefore may account for the variety of Hox-controlled biological functions. Protein domain usage and interaction between protein domains in AbdA strongly depends on the biological readout, suggesting that domain usage largely depends on the regulated target gene, and hence on the identity of the gene cis regulatory sequences. Recent reports support that DNA sequences impact on Hox protein activity: Hox binding site neighboring sequences are important for proper regulation of the reaper downstream target [50]; Sex combs reduced changes its conformation and activity depending on the cognate sequence [51]. Of note, a role for the target sequence in controlling the structure and activity of the glucocorticoid receptor has also been recently reported [52], indicating that this may generally apply for many DNA binding transcription factors. Our results also have implication on how modifications in protein sequences are translated into changes in protein function during evolution. The HX domain, common to all Hox proteins, is ancient and found in all bilaterians, and provides a generic mode of PBC interaction (Figure 9). The UA domain, specific to some central Hox proteins (AbdA and Ubx in Drosophila), was acquired later, at the time of protostome/deuterostome radiation. It provides a distinct yet to be characterised PBC interaction mode, specific to some Hox paralogues only, allowing fine-tuning of Hox protein activity [22]. TD is found only in insect AbdA and not in Ubx proteins, suggesting that it arose after the duplication that generated Ubx and AbdA in the common ancestor of insects (Figure 9). Remarkably, within AbdA arthropod proteins, the HX domain has significantly diverged in some lineages like anopheles, while the TD domain has been strictly conserved. Conceptually, two non-exclusive models could account for the evolution of protein function following the acquisition of a novel protein domain. In the first one, the acquisition provides a novel molecular and functional property, which adds to pre-existing ones. This is for example the case for the acquisition of the QA domain that confers repressive function to Ubx [6], and the acquisition/loss of HX or LRALLT domains by Futzitarazu (Ftz) from distinct insect species, which provides Ftz with the capacity to recruit either Exd or FtzF1 cofactors and switches its activity from a Hox to a segmentation protein [53]. In the second model, the acquisition of a novel protein domain interferes with the activity of pre existing domains, reorganizing the intrinsic functional organization of the protein. This view is supported by the predominant role of the UA domain and the widespread domain interactivity seen in this study. Evolutionary changes in animal morphology is thought to mostly rely on changes in cis-regulatory sequences [1]. This is conceptually supported by the modular organization of cis-regulatory sequences, allowing subtle and cell specific changes in gene expression not deleterious for the animal. Experimentally, it is largely supported by the correlation between expression of key developmental regulatory genes and morphological changes (for example see [54]), and by changes in cis-regulatory sequences that impact on morphological traits [55]–[59]. Changes in animal morphology could also result from changes in protein sequence and function, as shown for Hox proteins in the morphological diversification in arthropods [4], [6]. However, changes in protein function are not believed to broadly contribute to morphological diversification during animal evolution, based on the assumption that changes in protein sequences are expected to have pleiotropic effects, which as such, do not provide a mean to convey subtle and viable evolutionary changes. Our work grasps redundancy and selectivity in protein domain usage and as salient features of AbdA transcription factor intrinsic regulatory logic: even the HX domain, evolutionarily conserved in all Hox proteins, is essential for only one AbdA function, and often acts in a redundant way with the TD or the UA protein domains. Selective use of protein domains is also supported by findings of a few smaller scale studies of three other Drosophila Hox proteins: viable missense or small deletion mutations within the Scr protein coding sequences falls in different allelic series when examined for three distinct biological readouts [60]; deletion of C-terminal sequences of the Ubx protein, starting from an insect specific QA protein domain preferentially affects a subset of Ubx function [61]; dispensability of the HX was reported for the leg inducing capabilities of the Antp Hox protein, while required for other Antp functions [62]. This context dependent selective mode of protein domain usage, or differential pleiotropy, may be essential for the evolution of Hox protein functions, as it ensures developmental robustness of a Hox-controlled program while being permissive to evolutionary changes endowing novel functions to preexisting protein domains. In addition, our work also establishes that interactivity between protein domains is highly context dependent, suggesting that Hox protein function not only relies on selective mode of protein domain usage but also on selective mode of protein domain interactivity. Altogether, these observations challenge the view that changes in protein sequences necessarily have pleiotropic effects, giving more room for protein changes in the evolution of animal body plans. 24B-Gal4 and arm-Gal4 were used as embryonic mesodermal and ubiquitous drivers, respectively. Btl-Gal4 and lbe (K) -Gal4 for specific expression in the tracheal system and NB5–6 neuroblasts, respectively. The DME-lacZ and svp-lacZ lines are respectively from R. Mann (Columbia Univ., NY, USA) and S. Zaffran (IBDML, Marseille, France). exdXP11 and hthP2 alleles were used. Embryo collections, cuticle preparations, in situ hybridizations, and immunodetections were performed according to standard procedures. Digoxigenin RNA-labelled probes were generated according to the manufacturer' s protocol (Boehringer Mannheim, Gaithersburg, MD) from wg and dpp cDNAs cloned in Bluescript. Primary antibodies used are: anti-Antp (4C3, dilution 1/100, Developmental Studies Hybridoma Bank (DSHB) ); rabbit anti-AbdA (1/1000); guinea-pig anti-Doc2+3 (1/400) and rabbit anti-Dmef2 (1/2000) from L. Perrin (IBDML, Marseille, France); rabbit anti-Exd (1/1000) from R. Mann; rabbit anti-Nau (1/100) from BM Paterson (University of Texas Southwestern Medical Center, Dallas, TX); rabbit (1/500) or mouse (1/200) anti-GFP (1/500) from Molecular Probes; chicken anti-GFP (1/1000) from Aves labs; mouse anti-β-galactosidase (1/1000) from Promega; rabbit anti-β-galactosidase (1/1000) from MP Biomedical; anti-digoxigenin coupled to biotin (1/500) from Jackson. Secondary antibodies coupled to Alexa 488, Alexa 555 (Molecular Probes) or to biotin (Jackson) were used at a 1/500 dilution. AbdA variant were generated by PCR. Domain mutations were YPWM→AAAA; TDWM→AVAI; KEINE→KAAAA. The homeodomain point mutation alleviating DNA binding is a mutation of position 50 (Q→K; [47]). Constructs were cloned in pUAST or pUASTattB vectors for transgenic line establishment. Lines were crossed with the appropriate driver, and collected embryos were stained with anti-AbdA to select the conditions (line and temperature) that result in expression levels similar (+/−15%) to AbdA wild type levels in A2 (see [22] for a detailed description of the procedure). Procedures used for quantification of biological readouts using at least 10 embryos of each genotype are provided in Text S1. A matrix containing the values corresponding to the readout was built. The extreme values were given to the total loss of activity (value 0), and to the wild type activity (value 1 for 100% of activity). A hierarchical clustering algorithm (with Euclidian distance and average linking) was applied to the matrix using the MeV software suite [63]. The jacknife method was used for re-sampling the data and provides a statistical support for each tree node. Boxplots drawn using the R-Software. Boxplot depicts the value distribution obtained for each tested genotype. Black points correspond to individual counts. | Proteins perform essential regulatory functions, including control of gene transcription, a process central to development, evolution, and disease. While protein domains important for protein activity have been identified, how they act together to define the activity of a protein remains poorly explored. The predominant view influenced by prokaryotic transcription factors is that protein domains constitute independent functional modules, required for all aspects of protein activity. In this study, we used Hox proteins, evolutionarily conserved transcription factors playing key roles in the establishment of animal body plans, to examine how protein domains collectively shape protein activity. Results obtained using a broad range of biological readouts highlight a context-dependency in protein domain usage and interaction, revealing that protein domains are non-pleoitropic in nature. This suggests that protein modification may contribute more broadly to molecular changes underlying morphological diversity, so far thought to rely largely on modification of gene cis-regulatory sequences. | lay_plos |
Genome-scale metabolic models are available for an increasing number of organisms and can be used to define the region of feasible metabolic flux distributions. In this work we use as constraints a small set of experimental metabolic fluxes, which reduces the region of feasible metabolic states. Once the region of feasible flux distributions has been defined, a set of possible flux distributions is obtained by random sampling and the averages and standard deviations for each of the metabolic fluxes in the genome-scale model are calculated. These values allow estimation of the significance of change for each reaction rate between different conditions and comparison of it with the significance of change in gene transcription for the corresponding enzymes. The comparison of flux change and gene expression allows identification of enzymes showing a significant correlation between flux change and expression change (transcriptional regulation) as well as reactions whose flux change is likely to be driven only by changes in the metabolite concentrations (metabolic regulation). The changes due to growth on four different carbon sources and as a consequence of five gene deletions were analyzed for Saccharomyces cerevisiae. The enzymes with transcriptional regulation showed enrichment in certain transcription factors. This has not been previously reported. The information provided by the presented method could guide the discovery of new metabolic engineering strategies or the identification of drug targets for treatment of metabolic diseases. Systems Biology aims to use mathematical models to integrate different kinds of data in order to achieve a global understanding of cellular functions. The data to be integrated differ both in their nature and measurability. The availability of DNA microarrays allows for the comparative analysis or mRNA levels between different strains and conditions. These data provide genome-wide information, and changes in expression at different conditions are expressed in statistical terms such as p-values or Z-scores that quantify the level of significance in transcriptional changes. The availability of annotated genome-scale metabolic networks allowed mapping of the transcriptional changes in metabolic genes on to their corresponding metabolic pathways and defining significantly up or down regulated sub-networks [1]. Even though this allows for identification of transcriptional hot-spots in metabolism, this does still not provide information about whether there are any changes in metabolic fluxes in these pathways, as it has been shown that in general there is no clear correlation between gene expression and protein concentration [2] or metabolic flux [3], [4]. Metabolic fluxes are the result of a complex interplay between enzyme kinetics, metabolite concentrations, gene expression and translational regulation. Metabolic fluxes can be directly measured using 13C labeling experiments [5]. However, flux data obtained using this approach differ from gene expression data in two main features: 1) their determination is only possible for a relatively small subset of all the reactions in a genome-scale metabolic network and 2) they are indirect data in the sense that the fluxes are quantified obtained by fitting measured labeling patterns using a simple metabolic model. The complexity of the mRNA-flux dependence and the disparity in the nature of both kinds of data make their integration an important challenge. In this paper we propose a method to integrate gene expression data with flux data by transforming a limited amount of quantitative flux data into a genome-scale set of statistical scores similar to the one obtained from DNA microarrays. In order to do that, a set of experimental exchange fluxes are fixed for each of the studied conditions or for each of the strains investigated, and a sampling algorithm is then used to obtain a set of flux distributions satisfying the experimental values. This approach allows for obtaining means and standard deviations for each flux in the genome-scale network. From the mean and standard deviation it is possible to derive statistical scores for the significance of flux change between conditions [6], [7]. Random sampling in the region of feasible flux distributions has been previously used to study the statistical distribution of flux values and determine a flux backbone of reactions carrying high fluxes [8] as well as to define modules of reactions whose fluxes are positively correlated [9], [10]. Also mitochondria related diseases have been analyzed using random sampling [11]. All the works published so far used the Hit and Run algorithm to perform the sampling [7]. By dividing the average difference among two conditions (e. g. carbon sources or mutant strains) by its standard deviation, it is possible to obtain Z scores for each metabolic flux. These scores can be transformed into p-values that measure the significance of change of each flux (see methods). By comparing these p-values with the p-values derived from gene-expression arrays, the enzymes in the network can be classified as: 1) enzymes that have a significantly correlated change both in flux and expression level (reactions showing transcriptional regulation), 2) enzymes that show a significant change in expression but not in flux (we will refer to them as showing post-transcriptional regulation) and 3) enzymes that show significant changes in flux but not a change in expression (metabolic regulation). Hereby we provide a framework that allows for global classification of reaction fluxes into those that are transcriptionally regulated, post-transcriptionally regulated and metabolically regulated (see Fig. 1). This will have substantial impact on the field of metabolic engineering where changes in gene-expression are often used as the key means to alter metabolic fluxes. In the paper we show the use of the presented framework for the analysis of the yeast Saccharomyces cerevisiae grown at different growth conditions and for the analysis of different deletion mutants. The combined use of random sampling of genome-scale metabolic networks and expression data allows for global mapping of reactions that are either transcriptionally or metabolically regulated. This information can be used to guide the engineering of microbial strains or as a diagnosis tool for studying metabolic diseases in humans. In particular we should highlight that reactions in which there is no relation between gene transcription level and metabolic flux are not suitable targets for flux increase via gene over-expression. Through analysis of different data sets the method revealed that many changes in gene expression are not correlated with a corresponding change in metabolic fluxes. The use of gene-expression data alone can therefore be misleading. However, our method allowed for identification of many specific reactions that are indeed transcriptionally regulated, and we further identified that the expression of these enzymes is regulated a few key transcription factors. This fact suggests that the regulation of metabolism has evolved to contain a few flux-regulating transcription factors that could be the target for genetic manipulations in order to redirect fluxes. Here we propose a sampling method that finds extreme solutions among the feasible flux distributions of the metabolic network. These solutions correspond to the corners in the region of allowed flux distributions, and in mathematical terms they are elements of the convex basis of the region of feasible solutions (which is a convex set). The COBRA Toolbox [12] includes a random sampling option that uses the Hit and Run algorithm [13] to obtain points uniformly distributed in the region of allowed solutions. The difference between the two sampling methods is illustrated in Fig. 2. In order to assess the accuracy of our sampling method to estimate the average fluxes and their standard deviations, we compared a set of internal fluxes measured with 13C labeling [14] with predictions using 500 sampling points obtained using the sampling method in the convex basis and 500 sampling points obtained using the sampling algorithm implemented in the COBRA Toolbox. The results are summarized in Table 1 where our method is labeled Convex Basis (CB), because it samples elements of the convex basis of the region of allowed solutions (see above), and the method from the COBRA Toolbox is labeled Hit and Run (HR). The Z values in the table are the number of standard deviations that the real value is deviating from the calculated mean. The means obtained by the two sampling methods are very similar for most of the reactions; however the standard deviations found using the HR algorithm are significantly smaller. With the HR method the real values for the fluxes in many cases deviate several standard deviations from the mean, A high value of Z indicates that the real value has a very low chance of being obtained using the considered sampling method (or in other words: the real value does not belong to the family of solutions that is generated by the sampling method). The number of samples with the HR algorithm was increased up to 5000 to check possible effects of the sample size on the standard deviation. Only small increases were observed for the standard deviations of the studied fluxes. Using the CB algorithm we obtain higher standard deviations and the real flux is for most reactions less than one standard deviation away from the mean flux. We can therefore conclude that the CB sampling method gives more realistic standard deviations for the fluxes. This is important if we want to compare the significance of flux changes between conditions. An underestimated standard deviation would make some flux changes appear as being significant even though they may not be in reality, and our method therefore gives a more conservative list of significantly changed reaction fluxes than the HR algorithm. To evaluate our method we used data for the yeast S. cerevisiae. Data from growth on four different carbon sources (glucose, maltose, ethanol and acetate) in chemostat cultures and five deletion mutants (grr1Δ, hxk2Δ, mig1Δ, mig1Δmig2Δ and gdh1Δ) grown in batch cultures were used. The exchange fluxes and gene expression data for the mentioned conditions have been published earlier [15]–[17]. Our method obtains probability scores for each enzyme in the metabolic network (see methods) and this allowed us to classify the enzymes as transcriptionally regulated (correlation between flux and gene expression), post-transcriptionally regulated (changes in gene expression don' t cause changes in flux) and metabolically regulated (changes in flux are not caused by changes in gene-expression). The cut-off chosen for this classification was a probability score above 0. 95. Tables 2 and 3 show the 10 top scoring enzymes in each group (or fewer when less than 10 enzymes had a score exceeding 0. 95). The method is illustrated in Fig. 3. The method to identify the significance of flux changes relies on a set of measured external fluxes, and in some cases strains that don' t show significant changes in external fluxes have changes in internal fluxes [18]. These changes cannot be identified with our method, and our estimations of the significance of flux changes can therefore be seen as conservative estimates. The lists of transcriptionally and metabolically regulated reactions are therefore more reliable than the list of post-transcriptionally regulated reactions (in which some fluxes may be changed in reality but their change pass undetected). The reactions showing transcriptional regulation form a set of putative targets where enzyme over-expression or down regulation will influence the flux through these reactions. The reactions showing metabolic regulation points to parts of the metabolism where the pools of metabolites are possibly increasing or decreasing in connection with transcriptional changes and hereby counteracting possible changes in enzyme concentration as a result of transcriptional changes. This knowledge can be used to identify whether one should target changes in enzyme concentration (vmax changes), e. g. through over-expression, or changes in enzyme affinity (Km changes), e. g. through expression of heterologous enzymes, in order to alter the fluxes. The steady state condition and the irreversibility of some reactions impose limitations on the flux distributions attainable by the cell [18]. The set of feasible solutions can be further constrained by fixing some fluxes to their experimental values. In general, the fluxes most accessible to experimental determination are those corresponding to uptake or secretion rates. After fixing a subset of fluxes, genome scale models still have a large number of degrees of freedom. In this study we used the genome scale model iFF708 for S. cerevisiae [27]. Random sampling has previously been performed [7] by enclosing the region of allowed solutions in a parallelepiped with the same dimensions as solution space (the null space of the stoichiometric matrix) and generating random points inside this parallelepiped. The points that lie inside the region of possible solutions are then selected. The COBRA Toolbox [12] uses a Hit and Run algorithm to generate random points in this way. In this work instead of sampling inside the region of allowed solutions we sampled at its corners. In order to obtain corners in the space of allowed solutions we used the simplex method with a random set of objective functions to be maximized. The maximization of each of these objective functions will give a corner in the space of solutions. The constraints imposed upon each optimization are: (1) (2) (3) The values of the measured fluxes (vexp) are different between conditions. This fact changes the shape of the region of feasible solutions between different conditions. S is the stoichiometric matrix of the network. In order to reduce the effects of internal loops we first identified all the reactions that can get involved in loops using the FVA (Flux Variability Analysis) option in the COBRA Toolbox. The reactions that can be involved in loops are unbounded and show the default maximal or minimal value set in the COBRA Toolbox (1000 or −1000). If these bounds were kept, the means and standard deviations for these reactions would be unrealistic [6] and cannot be used for further analysis. In order to reduce the effect of loops, the default maximal and minimal fluxes for the reactions involved in loops, were set to a smaller value in order to reduce the loop effect. In order to select an appropriate value the bounds were increased from 0 in steps of 0. 1 until the minimal value that allows obtaining flux distributions consistent with the experimental fluxes is found. These values went from 1 to 15 mmol h−1g-DW−1 depending on each condition. Also no weights (eq. 4) were assigned to the reactions involved in loops in order to avoid objective functions that maximize the activity of loops. Random objective functions were generated by selecting random pairs of reactions and assigning them random weights (the reactions involved in loops were excluded from these choices). The weights (wi) assigned to each reaction were generated by dividing a random number between 0 and 1 by the maximal flux for this reaction obtained using FVA. This normalization was made to account for the different size orders of the different reactions. The objective functions take the form: (4) One solution is obtained for each of the objective functions generated. Our objective is to obtain means and standard deviations for each flux in each of the compared conditions and use them to get a Z-score quantifying the significance of change in each flux between the considered conditions. This score is equal to the difference between the means in each of the conditions divided by the standard deviation of this difference (note that the variance of the difference is the sum of the two variances and the standard deviation its square root). (5) The difference between averages in the numerator follows a normal distribution (according to the central limit theorem) with a standard deviation equal to the standard deviation of the flux (the denominator in eq. (5) ) divided by the square root of the number of samples. Therefore, Z itself follows a normal distribution with a standard deviation equal to the inverse of the square root of the number of samples. The Z score measures the significance of change in terms of standard deviations. If the error in the Z score is lower than 0. 15, no information would be lost in terms of classifying a reaction as significantly changed or not. The order of size of a genome-scale model is about 1000 reactions. A reasonable accuracy for the Z-scores would be to expect errors higher than 0. 15 on the Z score only for 1 reaction in the whole model. This means a p-value of 0. 001. If we want to keep the error on the Z score under 0. 15 with a probability of 0. 999 we need 500 samples, and this was therefore selected as the sampling number. The Z-scores can be transformed into probabilities of change by using the cumulative Gaussian distribution. Once we have Z-scores for the significance of flux changes and Z-scores for the significance of gene-expression changes we can obtain probabilities of having correlated expression and flux changes for each enzyme. An increase in enzyme expression can result in an increase of flux (transcriptional regulation). In order to evaluate the probability for a reaction of being transcriptionally regulated we multiply the probability of its enzyme level changing by the probability of its flux changing in the same direction (obtained using the cumulative normal distribution). (6) (7) If there is a decrease in expression and a decrease in flux, both Z-scores are negative and we will use the absolute values of the Zs in eq. (6). If there is an increase in expression and a negative flux becomes more negative, we will use the absolute value of the Z-score for the flux change. If the direction of the flux changes between conditions, this change must be driven by the metabolic concentrations and no by transcriptional regulation, therefore a Ptri of zero is assigned by default. In the same way as in eq. (6) we can define probabilities for the expression level changing and for the flux not changing (post transcriptional regulation). (8) (9) Now we use the error function because we want to evaluate the probability of change in any direction. The absolute value of Z is used in all the cases. The probability of a change in flux but not in transcription (metabolic regulation) can be obtained for each reaction as follows: (10) Each of these three probabilities can be associated to each enzyme in the metabolic network. Table 4 summarizes the criteria to assign each type of regulation. | The sequencing of full genomes and the development of high-throughput analysis technologies have made available both genome-scale metabolic networks and simultaneous transcription data for all the genes of an organism. Genome-scale metabolic models, with the assumption of steady state for the internal metabolites, allow the definition of a region of feasible metabolic flux distributions. This space of solutions can be further constrained using experimental flux measurements (normally production or uptake rates of external compounds). Here a random sampling method was used to obtain average values and standard deviations for all the reaction rates in a genome-scale model. These values were used to quantify the significance of changes in metabolic fluxes between different conditions. The significance in flux changes can be compared to the changes in gene transcription of the corresponding enzymes. Our method allowed for identification of specific reactions that are transcriptionally regulated, and we further identified that these reactions can be ascribed to a few key transcription factors. This suggests that the regulation of metabolism has evolved to contain a few flux-regulating transcription factors that could be the target for genetic manipulations in order to redirect fluxes. | lay_plos |
At least 25 inherited disorders in humans result from microsatellite repeat expansion. Dramatic variation in repeat instability occurs at different disease loci and between different tissues; however, cis-elements and trans-factors regulating the instability process remain undefined. Genomic fragments from the human spinocerebellar ataxia type 7 (SCA7) locus, containing a highly unstable CAG tract, were previously introduced into mice to localize cis-acting “instability elements, ” and revealed that genomic context is required for repeat instability. The critical instability-inducing region contained binding sites for CTCF—a regulatory factor implicated in genomic imprinting, chromatin remodeling, and DNA conformation change. To evaluate the role of CTCF in repeat instability, we derived transgenic mice carrying SCA7 genomic fragments with CTCF binding-site mutations. We found that CTCF binding-site mutation promotes triplet repeat instability both in the germ line and in somatic tissues, and that CpG methylation of CTCF binding sites can further destabilize triplet repeat expansions. As CTCF binding sites are associated with a number of highly unstable repeat loci, our findings suggest a novel basis for demarcation and regulation of mutational hot spots and implicate CTCF in the modulation of genetic repeat instability. Trinucleotide repeat expansion is the cause of at least 25 inherited neurological disorders, including Huntington' s disease (HD), fragile X mental retardation, and myotonic dystrophy (DM1) [1]. One intriguing aspect of trinucleotide repeat disorders is ‘anticipation’ – a phenomenon whereby increased disease severity and decreased age-of-onset are observed as the mutation is transmitted through a pedigree [2]. In spinocerebellar ataxia type 7 (SCA7), for example, disease onset in children, who inherit the expanded repeat, averages 20 years earlier than in the affected parent [3]. The basis of the profound anticipation in SCA7 stems from a significant tendency to undergo large repeat expansions upon parent-to-child transmission [4]. Other similarly-sized, disease-linked CAG/CTG repeat tracts do not exhibit strong anticipation, and are much more stable upon intergenerational transmission, as occurs at the spinobulbar muscular atrophy (SBMA) disease locus [5]. Drastic differences in the stability of CAG/CTG repeats, depending upon the locus at which they reside, strongly support the existence of cis-acting DNA elements that modulate repeat instability at certain loci. Furthermore, dramatic variation in CAG tract instability in tissues from an individual patient, together with disparities in the timing, pattern, and tissue-selectivity of somatic instability between CAG/CTG disorders, indicates a role for epigenetic modification in DNA instability [1], [6]–9. While the existence of cis-elements regulating disease-associated instability is widely accepted, the identities of cis-elements that define the mutability of any repeat are still unknown. Proposed cis-elements that regulate repeat instability include: the sequence of the repeat tract, the length and purity of the repeat tract, flanking DNA sequences, surrounding epigenetic environment, replication origin determinants, trans-factor binding sites, and transcriptional activity [10]–[12]. Such cis-elements may enhance or protect against CAG tract instability. To identify cis-elements responsible for CAG expansion at the SCA7 locus, we previously introduced SCA7 CAG-92 repeat expansions into mice, either on 13. 5 kb ataxin-7 genomic fragments or on ataxin-7 cDNAs. Comparison of CAG repeat length change revealed that ataxin-7 genomic context drives repeat instability with an obvious bias toward expansion, while SCA7 CAG repeats introduced on ataxin-7 cDNAs were stable [13]. To localize the cis-acting elements responsible for this instability tendency, we derived lines of transgenic mice based upon the original 13. 5 kb ataxin-7 genomic fragment, deleting a large region (∼8. 3 kb) of human sequence beyond the 3′ end of the CAG tract (α-SCA7-92R construct). As deletion of the 3′ region in the α-SCA7-92R transgenic mice significantly stabilized the CAG-92 tract [13], we hypothesized that cis-elements within this 3′ region modify repeat instability at the SCA7 locus. To identify cis-acting instability elements at the SCA7 locus and the trans-acting proteins that regulate them, we evaluated the critical genomic region 3′ to the CAG repeat for sequences that might regulate genetic instability. In the case of SCA7 and a number of other highly unstable CAG/CTG repeat loci, including HD, DM1, SCA2, and dentatorubral-pallidoluysian atrophy, binding sites for a protein known as CTCF (i. e. the “CCCTC binding factor”) have been found [14]. CTCF is an evolutionarily conserved zinc-finger DNA binding protein with activity in chromatin insulation, transcriptional regulation, and genomic imprinting [15], [16]. As CTCF affects higher order chromatin structure [17], [18], we wondered if CTCF binding at the SCA7 locus might regulate CAG repeat instability. To test this hypothesis, we derived SCA7 genomic fragment transgenic mice with CTCF binding site mutations, and found that impaired CTCF binding yielded increases in both intergenerational and somatic instability at the SCA7 locus. Detection of increased somatic instability in association with hypermethylation of the CTCF binding site indicated a role for epigenetic regulation of SCA7 CAG repeat stability. Our results identify CTCF as an important modifier of repeat instability in SCA7, and suggest that CTCF binding may influence repeat instability at other tandem repeat expansion disease loci. At the SCA7 locus, there are two CTCF binding sites that flank the CAG repeat tract; the CTCF-I binding site is located 3′ to the CAG repeat (Figure S1), within the critical region deleted from the SCA7 genomic fragment in the α-SCA7-92R mice (Figure 1A). As CTCF binding sites are associated with highly unstable repeat loci [14], and CTCF binding can alter chromatin structure and DNA conformation [17], [18], we hypothesized that CTCF binding might be involved in SCA7 repeat instability. To test this hypothesis, we decided to compare SCA7 CAG repeat instability in mice carrying either the wild-type CTCF binding site or a mutant CTCF binding site that would be incapable of binding CTCF. To define the CTCF binding sites, we performed electrophoretic mobility shift assays to confirm that CTCF protein specifically binds to the putative CTCF-I binding site, and we found that both the CTCF DNA binding domain fragment and full-length CTCF protein bind to the SCA7 repeat locus 3′ region (Figure 1B). When we mapped the CTCF-I contact regions at the SCA7 repeat locus by methylation interference and DNA footprinting, we defined a region that is protected from DNase I treatment upon CTCF binding and subject to altered CTCF binding upon methylation treatment (Figure 1C). We then introduced point mutations at 11 nucleotides within this 3′ CTCF-I binding site, including eight contact nucleotides contained within the footprinted region (Figure 1C; Figure 1A, bottom). After confirming that CTCF binding was abrogated by these point mutations in electrophoretic mobility shift assays (Figure 1B), we derived a RL-SCA7 94R 13. 5 kb genomic fragment construct, that was identical to our original RL-SCA7 92R genomic fragment construct [13], except for: i) the presence of a mutant CTCF-I binding site, and ii) a minor repeat size increase to 94 CAG repeats. The RL-SCA7 94R CTCF-I-mutant construct was microinjected, and two independent lines of RL-SCA7 94R CTCF-I mutant transgenic mice were generated (hereafter referred to as the SCA7-CTCF-I-mut line mice – to distinguish them from the original RL-SCA7-92R transgenic mice with an intact CTCF-I binding site, hereafter referred to as the SCA7-CTCF-I-wt line mice). To assess in vivo occupancy of the CTCF-I binding site in SCA7-CTCF-I-wt and SCA7-CTCF-I-mut mice, we performed chromatin immunoprecipitation (ChIP) assays. To distinguish between the two CTCF binding sites, separated by a distance of 562 bp, we used two primer sets, including one extending 3′ to the CAG repeat. Quantitative PCR amplification with a primer set (‘A’) within ∼800 bp of the CTCF-I and CTCF-II sites yielded comparable CTCF occupancy in SCA7-CTCF-I-wt and -mut mice. As most sheared DNA fragments isolated by ChIP exceed 1 kb, intact CTCF-II sites and the primer set ‘A’ amplicon will be present in sheared DNA fragments isolated by ChIP from SCA7-CTCF-I-wt and -mut mice, accounting for comparable CTCF occupancy with primer set A. However, a significant reduction in CTCF occupancy at the CTCF-I site was observed in the SCA7-CTCF-I-mut mice for primer set B, which is closer to the CTCF-I binding site (at a distance of ∼700 bp) than the CTCF-II binding site (at a distance of ∼1,200 bp, thereby exceeding the size of most sheared DNA fragments isolated by ChIP) (Figure 1D; p = 0. 02, one-way ANOVA). Thus, ChIP analysis indicated that in vivo CTCF-I occupancy is significantly diminished in the cerebellum of SCA7-CTCF-I-mut mice. We assessed intergenerational repeat length instability in 3 month-old SCA7-CTCF-I-wt and SCA7-CTCF-I-mut mice by PCR amplification of the CAG repeat from tail DNAs, and found that mutation of the CTCF-I site destabilized the CAG repeat during intergenerational transmission (p = 0. 002, Mann-Whitney two-tailed test) (Figure 2A). Increased intergenerational instability in the SCA7-CTCF-I-mut mice was reflected by a broader range of repeat length change, as mean expansion and deletion sizes were greater for SCA7-CTCF-I-mut mice in comparison to SCA7-CTCF-I-wt mice (+4. 4 CAG' s/−4. 7 CAG' s vs. +2. 6 CAG' s/−2. 0 CAG' s). Analysis of repeat length instability between the two SCA7-CTCF-I-mut lines revealed similar intergenerational repeat instability (p = 0. 93, chi-square), and there was no difference in expansion bias between the two lines (p = 0. 25, chi-square). Thus, the SCA7-CTCF-I-mut mice did not show integration site effects, suggesting that increased instability in the two lineages results from altered CTCF binding. We then assessed germ line repeat instability by small-pool PCR of individual alleles in sperm DNAs from mice at age 2 months and 16 months (Figure 2B–C). As the mice aged, the CAG repeat in SCA7-CTCF-I-mut mice became increasingly unstable (p = 0. 009, Mann-Whitney two-tailed test), as mean expansion and deletion sizes were significantly greater for 16 month-old SCA7-CTCF-I-mut mice in comparison to SCA7-CTCF-I-wt mice (+24. 3 CAG' s/−15. 5 CAG' s (mut) vs. +9. 2 CAG' s/−1. 0 CAG (wt) ). Increasing CAG repeat instability with aging in SCA7-CTCF-I-mut mice suggests a role for CTCF in DNA instability during spermatogenesis, or for the male germ line-restricted CTCF-like paralogue (CTCFL), also known as brother of the regulator of imprinted sites, or ‘BORIS’ [19]. A potential role for CTCFL/BORIS in male germ line instability in the SCA7-CTCF-I-mut mice is plausible, as mutation of the SCA7-CTCF-I site also prevented binding of CTCFL/BORIS in electrophoretic mobility shift assays (Figure S2). Another intriguing feature of repeat instability is variation in repeat size within and between the tissues of an individual organism. This tissue-specific instability, or “somatic mosaicism”, occurs in human patients with repeat diseases, and in mouse models of repeat instability and disease [1], [8], [11]. While shown to be age-dependent, the mechanistic basis of inter-tissue variation, which even occurs in postmitotic neurons [20], is unknown. To determine if somatic CAG mosaicism at the SCA7 locus involves CTCF binding, we surveyed repeat instability in various tissues from SCA7-CTCF-I-wt and SCA7-CTCF-I-mut mice. At two months of age, the SCA7 CAG repeat was remarkably stable in all analyzed tissues (Figure 3A). However, by ∼10 months of age, SCA7-CTCF-I-wt and SCA7-CTCF-I-mut mice displayed large CAG repeat expansions in the cortex and liver (Figure 3B). The liver also exhibited a bimodal distribution of repeat size (i. e. two populations of cells with distinct tract lengths) (Figure 3B). The most pronounced somatic instability differences existed in the kidney, with large expansions for SCA7-CTCF-I-mut mice, but stable repeats in the SCA7-CTCF-I-wt mice (Figure 3B). This pattern of increased kidney and liver repeat instability was present in both SCA7-CTCF-I-mut transgenic lines (Figure 3B; Figure S3). Indeed, comparable somatic instability was also detected in both SCA7-CTCF-I-mut transgenic lines at five months of age (Figure S4). When we closely examined repeat instability in the cortex by small-pool PCR, we observed significantly different repeat sizes (p = 8. 6×10−5, Mann-Whitney), with a range of 39 to 152 CAG repeats in SCA7-CTCF-I-wt mice and 26 to 245 CAG repeats in SCA7-CTCF-I-mut mice (Figure 3C; Table 1). The increased somatic instability occurred in both SCA7-CTCF-I-mut transgenic lines, as an expansion bias was apparent in both lineages upon small-pool PCR analysis (Figure 3D; Table 1). These findings suggest that CTCF binding stabilizes the SCA7 CAG repeat in certain tissues. Thus, as noted for the germ line and documented for two independent lines of SCA7-CTCF-I-mut transgenic mice, SCA7 somatic CAG instability is dependent upon age and the presence of intact CTCF binding sites. CTCF binding can be regulated by CpG methylation, as methylation at CTCF recognition sites abrogates binding [16]. This finding was confirmed for un-methylated and methylated versions of the SCA7 CTCF-I recognition site (Figure 4A; Figure S5). Highly variable levels of instability have been documented in the kidneys of transgenic repeat instability mouse models [21], [22], although the reasons for pronounced instability in this tissue are unknown. Interestingly, one mouse with a wild-type CTCF-I binding site (SCA7-CTCF-I-wt) displayed marked CAG repeat instability in its kidney DNA (Figure 4B), paralleling the considerable instability observed in the SCA7-CTCF-I-mut mice (Figure 3B). Bisulfite sequencing of kidney DNA from this SCA7-CTCF-I-wt mouse revealed high levels of CpG methylation at the wild-type CTCF-I binding site, including the central CTCF contact site (Figure S6); whereas methylation was not observed in kidney DNAs from 14 other SCA7-CTCF-I-wt mice that displayed only modest levels of CAG instability (Figure 4C). The high levels of CAG instability and the CpG methylation in this mouse were restricted to the kidney, as the cerebellum and tail DNAs of the same mouse, which showed limited CAG instability (Figure 4B), were completely unmethylated (Figure 4C). This finding suggests a direct link between methylation status of the CTCF binding site and CAG repeat instability. Of all the tissues analyzed from SCA7-CTCF-I-wt mice, liver exhibits the greatest amount of somatic mosaisicm, with the largest repeat expansions (Figure 3B). We hypothesized that the high levels of CAG repeat instability in the liver of SCA7-CTCF-I-wt mice might result from methylation of the CTCF-I binding site. To address this question, we performed bisulfite sequencing analysis of liver DNAs from SCA7-CTCF-I-wt mice, and documented moderately high levels of methylation at the CTCF-I binding site (Figure 4D; Figure S7). These results indicate a correlation between CpG methylation and CAG repeat instability. Thus, in SCA7 transgenic mice, decreased CTCF binding, either by CpG methylation or mutagenesis of the CTCF-I binding site, enhanced CAG repeat instability. We have identified a CTCF binding site as the first cis-element regulating CAG tract instability at a disease locus. Furthermore, binding of the trans-factor CTCF to this cis-element influences CAG instability, and this interaction is epigenetically regulated. At the SCA7 locus and four other CAG/CTG repeat loci known to display pronounced anticipation, functional CTCF binding sites occur immediately adjacent to the repeats, and CTCF binding can affect DNA structure and chromatin packaging at such loci, and elsewhere [14], [23]–[26]. Although an interplay between GC-content, CpG islands, epigenetic modification, chromatin structure, repeat length, and unusual DNA conformation has long been postulated to underlie trinucleotide repeat instability [11], [27]–[29], the mechanistic basis of this process is ill-defined. CTCF insulator and genomic imprinting functions are subject to epigenetic regulation, as methylation status is a key determinant of CTCF action at certain “differentially methylated domains” and methylation changes at CTCF binding sites are linked to oncogenic transformation [16], [18]. At the SCA7 locus, methylation status of the CTCF-I binding site may be similarly important for its ability to tamp down repeat instability, as hypermethylation of the CTCF-I site was associated with a dramatic enhancement of somatic instability in the SCA7 genomic fragment transgenic mouse model. Thus, inability to bind CTCF at sites adjacent to CAG tracts, because of binding site mutation or CpG methylation in the case of the SCA7-CTCF-I site, can promote further expansion of disease-length CAG repeat alleles (Figure 5). In both human patients and transgenic mice with expanded repeat tracts, the repeat displays high levels of instability. The flanking sequence has been thought to contain elements that may protect or enhance repeat instability. Our results show that CTCF binding is a stabilizing force at the SCA7 repeat locus, suppressing expansion of the CAG repeat in the germ line and soma. Interestingly, deletion of ∼8. 3 kb of 3′ genomic sequence in our previous SCA7 transgenic mouse, including the CTCF-I site, stabilized the repeat [13]. The CAG-92 stabilization, arising from the ∼8. 3 kb 3′ genomic fragment deletion, suggests the existence of positive cis-regulators that were “driving” CAG instability. One such element could be a replication initiation site that was mapped within the genomic region 3′ to the CTCF-I binding site at the SCA7 locus [30]. Hence, the 8. 3 kb 3′ deletion could grossly alter the chromatin organization of the adjacent repeat, and would likely ablate replication origin activity, stabilizing the CAG repeat tract. However, this ∼8. 3 kb genomic region likely also contained negative cis-regulators of CAG repeat instability, whose dampening effects would not be apparent due to the coincident loss of instability drivers. Our results indicate that CTCF binding negatively regulates expanded CAG repeat instability at the SCA7 locus. CTCF regulation of repeat instability potential is consistent with its many roles in modulating DNA structure. CTCF can mediate long-range chromatin interactions and can co-localize physically distant genomic regions into discrete sub-nuclear domains [17], [18]. CTCF insulates heterochromatin and silenced genes from transcriptionally active genes, as CTCF binding sites occur at transition zones between X-inactivation regions and genes that escape from X-inactivation [24]. CTCF has been implicated in genomic imprinting, although recent studies indicate that such transcription insulator events may involve the coordinated action of CTCF with cohesin [31]–[33]. CTCF binding at the DM1 locus sequesters repeat-driven heterochromatin formation to the immediate repeat region, while repeat expansion-induced loss of CTCF binding may permit spreading of heterochromatin to adjacent genes, accounting for the mental retardation phenotype in congenital DM1 [23]. As DNA structural conformation and transcription activity are two highly intertwined processes that appear fundamental to the instability of expanded tandem repeats [10], [11], CTCF appears a likely candidate for modulation of trinucleotide repeat instability. At the SCA7 locus, a pronounced tendency for repeat expansion has been associated with transmission through the male germ line [3], [4], [34]. Although we have hypothesized that CTCF is principally responsible for modulating SCA7 CAG repeat instability both in the germ line and in the soma, we considered a possible role for the related CTCF-like factor BORIS. BORIS and CTCF share identical 11 zinc-finger domains for DNA binding [19]; hence, both CTCF and BORIS can bind to the CTCF binding sites at the SCA7 locus. Upon mutation or methylation of the CTCF binding site 3′ to the SCA7 CAG repeat, neither CTCF nor BORIS can bind (Figure 1C; Figure 4A; Figure S8). As BORIS can bind to the H19 differentially methylated domain even when it is methylated [35], our results suggest that the methylation dependence of BORIS binding is locus specific. BORIS and CTCF expression patterns overlap very little, if at all, and in the male germ line, BORIS appears restricted to primary spermatocytes, while CTCF occurs almost exclusively in post-meiotic cells, such as round spermatids [19]. Interestingly, neither BORIS nor CTCF could be detected by immunostaining proliferating spermatogonia. In human HD patients and transgenic mouse models of CTG/CAG instability, large repeat expansions have been documented in spermatogonia, but not in post-meiotic spermatids or spermatozoa [36]–[39]. Thus, absence or low levels of BORIS or CTCF in spermatogonia — the cells in which the largest and most frequent repeat expansions occur — may contribute to the paternal parent-of-origin expansion bias common to most CAG/CTG repeat diseases. In spermatocytes, BORIS may stabilize expanded CAG repeats, just as CTCF binding appears to promote repeat stability in somatic tissues. Thus, in the SCA7-CTCF-I-mut mice, abrogated binding of BORIS may contribute to increased repeat instability and expansion bias in the male germ line. Our findings suggest that CTCF is a trans-acting factor that specifically interacts in a methylation-dependent manner with the adjacent cis-environment to prevent hyper-expansion of disease length CAG repeats. In a Drosophila model of polyglutamine repeat disease, expression of the mutant gene product modulated repeat instability by altering transcription and repair pathways [10]. Similarly, uninterrupted repeat sequences, and in particular, runs of CG-rich trinucleotide repeats, can affect replication machinery, DNA repair pathways, and nucleosome positioning, though in cis, by altering the structure and conformation of the DNA regions within which they reside [40], [41]. Association of adjacent CTCF binding sites with repeat loci is a common feature of unstable microsatellite repeats [14]. We propose that acquisition of CTCF binding sites at mutational hot spots represents an evolutionary strategy for insulating noxious DNA sequences [42], and our findings indicate that CTCF binding site utilization at a mutational hot spot is subject to epigenetic regulation. We thus envision a predominant role for CTCF in modulating genetic instability at DNA regions containing variably-sized repeats, unstable sequence motifs, or other repetitive sequence elements. To derive the SCA7-CTCF-I-mut transgenic construct, we synthesized a PCR primer with randomly mutated nucleotides introduced at the CTCF-I contact sites for recombineering into the RL-SCA7-92R (SCA7-CTCF-I-wt) construct [13], and then confirmed loss of CTCF binding by the mutated fragment by electrophoretic mobility shift assay (protocol provided below). Using a standard recombineering approach [43], we PCR-generated a SCA7-CTCF-I targeting cassette containing a Chloramphenicol resistance gene and Cla I restriction site flanked by SCA7-CTCF-I region sequences with the following primer set: hSCA7-wt-CAM-F, 5′-tcccccctgcccccctcctgtatcgatgtttaagggcaccaataactgc-3′ & hSCA7-mut-CAM-R, 5′-catctctgcccctcgatttttatcgatatcgataatgatgagcacttttcgaccg-3′. After recombineering the SCA7-CTCF-I-mut targeting cassette into the SCA7-CTCF genomic fragment carried on a plasmid, selection, and PCR screening, we deleted the Chloramphenicol gene by Cla I digestion and ligation. We verified the sequence of the SCA7-CTCF-I-mut construct prior to linearization with Sal I – Spe I digestion, gel purification, and microinjection into C57BL/6J×C3H/HeJ oocytes. Transgene-positive founders were backcrossed onto the C57BL/6J background for more than 12 generations to yield incipient congenic mice before repeat instability analysis commenced. All experiments and animal care were performed in accordance with the University of Washington IACUC guidelines. We amplified a 161 bp DNA fragment (SCA7-CTCF-I) from the SCA7 locus with primers (5′-ctccccccttcaccccctcgagac-3′ & 5′-gtgacgcacactcacgcacgcacgg-3′) labeled at their 5′ ends by γ-32P-ATP. We gel-purified the 5′ end-labeled fragment, and used it for electrophoretic mobility shift assays, with in vitro translated proteins, as previously described [14]. We synthesized the CTCF-11 zinc finger (ZF) DNA binding domain, full length CTCF and full length CTCFL/BORIS proteins using the pCITE-11ZF, pCITE-7. 1, and pCITE-BORIS expression constructs [14], [19], [44], with the TnT reticulocyte lysate coupled in vitro transcription-translation system (Promega). For “super-shifts”, we used an anti-CTCF antibody (Upstate Biotechnology) or anti-BORIS antibody [19], [44]. We methylated the end-labeled SCA7-CTCF-I fragment with Sss I methyl-transferase (New England Biolabs) in the presence of 0. 8 mM S-adenosylmethionine. We confirmed the methylation status by restriction enzyme digestion with Nru I, and used unmethylated fragment as a control [14]. We PCR-amplified the SCA7-CTCF-I fragment and labeled it at the 5′ end on either the coding or anti-sense strand, incubated the purified probes with CTCF and then partially digested them with DNase I, or partially methylated them at guanine residues with dimethyl sulfate, and then incubated them with CTCF. Details of these protocols, as well as our methods for isolation and analysis of free probe DNA fragments on sequencing gels, have been described [14]. Bisulfite treatment of tissue DNAs was done as previously described [45], and PCR primers spanning the SCA7-CTCF-I region were designed so that they excluded CpG dinucleotides within the binding region. PCR products were then cloned into a Topo TA vector and sequenced. Sequencing of positive control samples, treated with Sss I to methylate all cytosines in CpG dyads, were included in every run, and revealed lack of C to T conversion at all CpG dyads in all control samples analyzed. We prepared tissues, cross-linked proteins to DNA, and processed tissue samples essentially as we have done previously [46]. However, we doubled the length of the sonication step, and, prior to immunoprecipitation, we fractionated supernatant DNAs on agarose gels to gauge the extent of shearing. After confirming that the bulk of sheared DNAs migrated in the 500–1,000 bp range, we performed immunoprecipitation with an anti-CTCF antibody (Upstate Biotechnology), as described [14]. DNAs were isolated and then subjected to real-time qPCR analysis with different SCA7 genomic region primer and probe sets (available upon request) on an ABI-7700 sequence detection system. For each CTCF ChIP sample, we normalized SCA7 locus occupancy to a control region of the Myc locus lacking CTCF binding sites [14]. All primer and probe sequence sets are available upon request. We PCR-amplified the SCA7 CAG repeat from genomic DNA samples in the presence of 0. 1µCi of α-32P-ATP, and resolved the radiolabeled PCR products on 1. 8% agarose gels [13]. For small-pool PCR, dilution of genomic DNA' s, yielding 1–5 genome equivalents, was performed prior to amplification and sizing [4]. In all experiments, at least three mice/genotype, or three samples/time point, were analyzed. All primer sequences are available upon request. | The human genome contains many repetitive sequences. In 1991, we discovered that excessive lengthening of a three-nucleotide (trinucleotide) repeat sequence could cause a human genetic disease. We now know that this unique type of genetic mutation, known as a "repeat expansion," occurs in at least 25 different diseases, including inherited neurological disorders such as the fragile X syndrome of mental retardation, myotonic muscular dystrophy, and Huntington' s disease. An interesting feature of repeat expansion mutations is that they are genetically unstable, meaning that the repeat expansion changes in length when transmitted from parent to offspring. Thus, expanded repeats violate one major tenet of genetics-i. e., that any given sequence has a low likelihood for mutation. For expanded repeats, the likelihood of further mutation approaches 100%. Understanding why expanded repeats are so mutable has been a challenging problem for genetics research. In this study, we implicate the CTCF protein in the repeat expansion process by showing that mutation of a CTCF binding site, next to an expanded repeat sequence, increases genetic instability in mice. CTCF is an important regulatory factor that controls the expression of genes. As binding sites for CTCF are associated with many repeat sequences, CTCF may play a role in regulating genetic instability in various repeat diseases-not just the one we studied. | lay_plos |
Background Interoperable communications is not an end in itself. Rather, it is a necessary means for achieving an important goal—the ability to respond effectively to and mitigate incidents that require the coordinated actions of first responders, such as multi-vehicle accidents, natural disasters, or terrorist attacks. Public safety officials have pointed out that needed interoperable communications capabilities are based on whether communications are needed for (1) “mutual-aid responses” or routine day- to-day coordination between two local agencies; (2) extended task force operations involving members of different agencies coming together to work on a common problem, such as the 2002 sniper attacks in the Washington, D.C. metropolitan area; or (3) a major event that requires response from a variety of local, state, and federal agencies, such as major wildfires, hurricanes, or the terrorist attacks of September 11, 2001. A California State official with long experience in public safety communications breaks the major event category into three separate types of events: (1) planned events, such as the Olympics, for which plans can be made in advance; (2) recurring events, such as major wildfires and other weather events, that can be expected every year and for which contingency plans can be prepared based on past experience; and (3) unplanned events, such as the September 11th attacks, that can rapidly overwhelm the ability of local forces to handle the problem. Interoperable communications are but one component, although a key one, of an effective incident command planning and operations structure. As shown in figure 1, determining the most appropriate means of achieving interoperable communications must flow from an comprehensive incident command and operations plan that includes developing an operational definition of who is in charge for different types of events and what types of information would need to be communicated (voice, data, or both) to whom under what circumstances. Other steps include: defining the range of interoperable communications capabilities needed for specific types of events; assessing the current capabilities to meet these communications needs; identifying the gap between current capabilities and defined requirements; assessing alternative means of achieving defined interoperable developing a comprehensive plan—including, for example, mutual aid agreements, technology and equipment specifications, and training—for closing the gap between current capabilities and identified requirements. Interoperable communications requirements are not static, but change over time with changing circumstances (e.g., new threats) and technology (e.g., new equipment), and additional available broadcast spectrum. Consequently, both a short- and long-term “feedback loop” that incorporates regular assessments of current capabilities and needed changes is important. In addition, the first responder community is extensive and extremely diverse in size and the types of equipment in their communications systems. According to SAFECOM officials, there are over 2.5 million public safety first responders within more than 50,000 public safety organizations in the United States. Local and state agencies own over 90 percent of the existing public safety communications infrastructure. This intricate public safety communications infrastructure incorporates a wide variety of technologies, equipment types, and spectrum bands. In addition to the difficulty that this complex environment poses for federal, state, and local coordination, 85 percent of fire personnel, and nearly as many emergency management technicians, are volunteers with elected leadership. Many of these agencies are small and do not have technical expertise; only the largest of the agencies have engineers and technicians. In the past, a stovepiped, single jurisdiction, or agency-specific communication systems development approach prevailed—resulting in none or less than desired interoperable communications systems. Public safety agencies have historically planned and acquired communications systems for their own jurisdictions without concern for interoperability. This meant that each state and local agency developed communications systems to meet their own requirements, without regard to interoperability requirements to talk to adjacent jurisdictions. For over 15 years, the federal government has been concerned with public safety spectrum issues, including communications interoperability issues. A variety of federal departments and agencies have been involved in efforts to define the problem and to identify potential solutions, such as the Department of Homeland Security (DHS), the Department of Justice (DOJ), the Federal Communications Commission (FCC), and the National Telecommunications and Information Agency (NTIA) within the Department of Commerce (DOC), among others. Today, a combination of federal agencies, programs, and associations are involved in coordinating emergency communications. DHS has several agencies and programs involved with addressing first responder interoperable communication barriers, including the SAFECOM program, the Federal Emergency Management Agency (FEMA), and the Office for Domestic Preparedness (ODP). As one of its 24 E-Gov initiatives, the Office of Management and Budget (OMB) in 2001 created SAFECOM to unify the federal government’s efforts to help coordinate the work at the federal, state, local, and tribal levels to establish reliable public safety communications and achieve national wireless communications interoperability. The SAFECOM program was brought into DHS in early 2003. In June 2003, SAFECOM partnered with the National Institute of Standards and Technology (NIST) and the National Institute of Justice (NIJ) to hold a summit that brought together over 60 entities involved with communications interoperability policy setting or programs. Several technical factors specifically limit interoperability of public safety wireless communications systems. First, public safety agencies have been assigned frequencies in new bands over time as available frequencies become congested and as new technology made other frequencies available for use. As a result, public safety agencies now operate over multiple frequency bands—operating on these different bands required different radios because technology was not available to include all bands in one radio. Thus, the new bands provided additional capabilities but fragmented the public safety radio frequency spectrum, making communications among different jurisdictions difficult. Another technical factor inhibiting interoperability is the different technologies or different applications of the same technology by manufacturers of public safety radio equipment. One manufacturer may design equipment with proprietary technology that will not work with equipment produced by another manufacturer. Nature and Scope of Interoperable Communication Problems Nationwide Are Unknown The current status of wireless interoperable communications across the nation—including the current interoperable communications capabilities of first responders and the scope and severity of the problems that may exist—has not been determined. Although various reports have documented the lack of interoperability of public safety first responders wireless communications in specific locations, complete and current data do not exist documenting the scope and severity of the problem at the local, state, interstate, or federal levels across the nation. Accumulating this data may be difficult, however, because several problems inhibit efforts to identify and define current interoperable communications capabilities and future requirements. First, current capabilities must be measured against a set of requirements for interoperable communications, and these requirements vary according to the characteristics of specific incidents at specific locations. Who needs to talk to whom, when they need to talk, and what set of communications capabilities should be built or acquired to satisfy these requirements depends upon whether interoperable communications are needed for day- to-day mutual aid, task force operations that occur when members of different agencies come together to work on a common problem such as the National Capitol Region sniper investigation, or major events such as a terrorist attack. Requirements for interoperable communications also may change with the expanding definition of first responders—from the traditional police, fire, and emergency medical providers to include such professions as health care providers and other professions—and the evolution of new technology. Establishing a national baseline for public safety wireless communications interoperability will be difficult because the definition of who to include as a first responder is evolving, and interoperability problems and solutions are situation specific and change over time to reflect new technologies and operational requirements. In a joint SAFECOM/AGILE program planning meeting in December 2003, participants agreed that a national baseline is necessary to know what the nation’s interoperability status really is, to set goals, and to measure progress. However, at the meeting, participants said they did not know how they were going to define interoperability, how they could measure interoperability, or how to select their sample of representative jurisdictions; this was all to be determined at a later date. SAFECOM has embarked on an effort to establish a national baseline of interoperable communications capabilities by July 2005, but SAFECOM is still working out the details of the study that would be used to develop the baseline. At the time of our review, SAFECOM officials acknowledged that establishing a baseline will be difficult and said they are working out the details of their baseline study but still expect to complete it by July 2005. DHS also has other work under way that may provide a tool for such self- assessments by public safety officials. An ODP official in the Border and Transportation Security Directorate of DHS said ODP is supporting the development of a communications and interoperability needs assessment for 118 jurisdictions that make up the Kansas City region. The official said the assessment will provide an inventory of communications equipment and identify how the equipment is used. He also said the results of this prototype effort will be placed on a CD-Rom and distributed to states and localities to provide a tool to conduct their own self assessments. SAFECOM officials said they will review ODP’s assessment tool as part of a coordinated effort and use this tool if it meets the interoperability requirements of first responders. Second, technical standards for interoperable communications are still under development. Beginning in 1989, a partnership between industry and the public safety user community developed what is known as Project 25 (P- 25) standards. According to the Public Safety Wireless Network (PSWN) program office, Project 25 standards remain the only user- defined set of standards in the United States for public safety communications. DHS purchased radios that incorporate the P-25 standards for each of the nation’s 28 urban search and rescue teams. PSWN believes P-25 is an important step toward achieving interoperability, but the standards do not mandate interoperability among all manufacturers’ systems. Standards development continues today as new technologies emerge that meet changing user needs and new policy requirements. Third, new public safety mission requirements for video, imaging, and high-speed data transfers, new and highly complex digital communications systems, and the use of commercial wireless systems are potential sources of new interoperability problems. Availability of new spectrum can also encourage the development of new technologies and require further development of technical standards. For example, the FCC recently designated a new band of spectrum, the 4.9 Gigahertz (GHz) band, for use and support of public safety. The FCC provided this additional spectrum to public safety users to support new broadband applications such as high- speed digital technologies and wireless local area networks for incident scene management. The FCC requested in particular comments on the implementation of technical standards for fixed and mobile operations on the band. NPSTC has established a task force that includes work on interoperability standards for the 4.9 GHz band. Federal Leadership and Intergovernmental Cooperation Is Needed The federal government, states, and local governments have important roles to play in assessing interoperability needs, identifying gaps in meeting those needs, and developing comprehensive plans for closing those gaps. The federal government can provide the leadership, long-term commitment, and focus to help state and local governments meet these goals. For example, currently national requirements for interoperable communications are incomplete and no national architecture exists, there is no standard database to coordinate frequencies, and no common nomenclature or terminology exists for interoperability channels. States alone cannot develop the requirements or a national architecture, compile the nationwide frequency database, or develop a common nationwide nomenclature. Moreover, the federal government alone can allocate communications spectrum for public safety use. Need to Establish National Requirements and a National Architecture One key barrier to the development of a national interoperability strategy has been the lack of a statement of national mission requirements for public safety—what set of communications capabilities should be built or acquired—and a strategy to get there. A key initiative in the SAFECOM program plan for the year 2005 is to complete a comprehensive Public Safety Statement of Requirements. The Statement is to provide functional requirements that define how, when, and where public safety practitioners communicate. On April 26, 2004, DHS announced the release of the first comprehensive Statement of Requirements defining future communication requirements and outlining future technology needed to meet these requirements. According to DHS, the Statement provides a shared vision and an architectural framework for future interoperable public safety communications. DHS describes the Statement of Requirements as a living document that will define future communications services as they change or become new requirements for public safety agencies in carrying out their missions. SAFECOM officials said additional versions of the Statement will incorporate whatever is needed to meet future needs but did not provide specific details. A national architecture has not yet been prepared to guide the creation of interoperable communications. An explicit, commonly understood, and agreed-to blueprint, or enterprise architecture, is required to effectively and efficiently guide modernization efforts. For a decade, GAO has promoted the use of enterprise architectures, recognizing them as a crucial means to a challenging goal—agency operational structures that are optimally defined in both business and technological environments. SAFECOM officials said development of a national architecture will take time because SAFECOM must first assist state and local governments to establish their communications architectures. They said SAFECOM will then collect the state and local architectures and fit them into a national architecture that links federal communications into the state and local infrastructure. Standard Databases and Common Nomenclature Not Yet Established Technology solutions by themselves are not sufficient to fully address communication interoperability problems in a given local government, state, or multi-state region. State and local officials consider a standard database of interoperable communications frequencies to be essential to frequency planning and coordination for interoperability frequencies and for general public safety purposes. Police and fire departments often have different concepts and doctrines on how to operate an incident command post and use interoperable communications. Similarly, first responders, such as police and fire departments, may use different terminology to describe the same thing. Differences in terminology and operating procedures can lead to communications problems even where the participating public safety agencies share common communications equipment and spectrum. State and local officials have drawn specific attention to problems caused by the lack of common terminology in naming the same interoperability frequency. The Public Safety National Communications Council (NCC), appointed by the Federal Communications Commission (FCC) was to make recommendations for public safety use of the 700 MHz communications spectrum. The NCC recommended that the FCC mandate (1) Regional Planning Committee use of a standard database to coordinate frequencies during license applications and (2) specific names be designated for each interoperability channel on all pubic safety bands. The NCC said that both were essential to achieve interoperability because public safety officials needed to know what interoperability channels were available and what they were called. In January 2001, the FCC rejected both recommendations. It said that the first recommendation was premature because the database had not been fully developed and tested. The FCC directed the NCC to revisit the issue of mandating the database once the database was developed and had begun operation. The FCC rejected the common nomenclature recommendation because it said that it would have to change the rules each time the public safety community wished to revise a channel label. In its final report of July 25, 2003, the NCC renewed both recommendations. It noted that the FCC had received a demonstration of a newly developed and purportedly operational database, the Computer Assisted Pre-Coordination Resource and Database System (CAPRAD), and that its recommendations were consistent with previous FCC actions, such as the FCC’s designating medical communications channels for the specifc purpose of uniform useage. Converting SAFECOM’s Functions To A Long-Term Program In 2001, the Office of Management and Budget (OMB) established SAFECOM to unify the federal government’s efforts to help coordinate work at the federal, state, local, and tribal levels in order to provide reliable public safety communications and achieve national wireless communications interoperability. However, SAFECOM was established as an OMB E-Gov initiative with a goal of improving interoperable communications within 18-24 months—a timeline too short for addressing the complex, long-term nature of the interoperability problem. In addition, the roles and responsibilities of various federal agencies within and outside DHS involved in communications interoperability have not been fully defined, and SAFECOM’s authority to oversee and coordinate federal and state efforts has been limited in part because it has been dependent upon other federal agencies for cooperation and funding and has operated without signed memorandums of understanding negotiated with various agencies. DHS, where SAFECOM now resides, announced in May 2004 that it had created an Office for Interoperability and Compatibility within the Science and Technology Directorate, to coordinate the federal response to the problems of wireless and other functional interoperability and compatibility. The new office is responsible for coordinating DHS efforts to address interoperability and compatibility of first responder equipment, to include both communications equipment and equipment such as personal protective equipment used by police and fire from multiple jurisdictions. The plan as approved by the Secretary of DHS states that by November 2004 the new office will be fully established and that action plans and a strategy will be prepared for each portfolio (type or class of equipment). The plan presents a budget estimate for creation of the office through November 2004 but does not include costs to implement each portfolio’s strategy. The plans for the new office do not clarify the roles of various federal agencies or specify what oversight authority the new office will have over federal agency communications programs. As of June 2004, the exact structure and funding for the office, including SAFECOM’s role within the office, were still being developed. Multiple Federal Agencies Have Roles And Responsibilities For Interoperability DHS has not defined how it will convert the current short-term program and funding structures to a permanent program office structure. When it does, DHS must carefully define the SAFECOM mission and roles in relation to other agencies within DHS and in other federal agencies that have missions that may be related to the OMB-assigned mission for SAFECOM. SAFECOM must coordinate with multiple federal agencies, including ODP within DHS, AGILE and the Office for Community Oriented Policing Services (COPS) in DOJ, the Department of Defense, the FCC, the National Telecommunications and Information Administration within the Department of Commerce, and other agencies. For example, AGILE is the DOJ program to assist state and local law enforcement agencies to effectively and efficiently communicate with one another across agency and jurisdictional boundaries. The Homeland Security Act assigns the DHS Office for Domestic Preparedness (ODP) primary responsibility within the executive branch for preparing the United States for acts of terrorism, including coordinating or, as appropriate, consolidating communications and systems of communications relating to homeland security at all levels of government. An ODP official said the Homeland Security Act granted authority to ODP to serve as the primary agency for preparedness against acts of terrorism, to specifically include communications issues. He said ODP is working with states and local jurisdictions to institutionalize a strategic planning process that assesses and funds their requirements. ODP also plans to develop tools to link these assessments to detailed interoperable communications plans. SAFECOM officials also will face a complex issue when they address public safety spectrum management and coordination. The National Telecommunications and Information Administration (NTIA) within the Department of Commerce is responsible for federal government spectrum use and the FCC is responsible for state, local, and other nonfederal spectrum use. The National Governors’ Guide to Emergency Management noted that extensive coordination will be required between the FCC and the NTIA to provide adequate spectrum and to enhance shared local, state, and federal communications. In September 2002, GAO reported that FCC and NTIA’s efforts to manage their respective areas of responsibility were not guided by a national spectrum strategy and had not implemented long- standing congressional directives to conduct joint, national spectrum planning. The FCC and the NTIA generally agreed with our recommendation that they develop a strategy for establishing a clearly defined national spectrum plan and submit a report to the appropriate congressional committees. In a separate report, we also discussed several barriers to reforming spectrum management in the United States. On June 24, 2004, the Department of Commerce released two reports entitled Spectrum Policy for the 21st Century, the second of which contained recommendations for assessing and managing public safety spectrum. SAFECOM’s Authority To Coordinate Federal And State Efforts Is Limited SAFECOM has limited authority to coordinate federal efforts to assess and improve interoperable communications. Although SAFECOM has developed guidance for use in federal first responder grants, SAFECOM does not have authority to require federal agencies to coordinate their grant award information. SAFECOM is currently engaged in an effort with DOJ to create a “collaborative clearinghouse” that could facilitate federal oversight of interoperable communications funding to jurisdictions and allow states access to this information for planning purposes. The database is intended to decrease duplication of funding and evaluation efforts, de-conflict the application process, maximize efficiency of limited federal funding, and serve as a data collection tool for lessons learned that would be accessible to state and locals. However, SAFECOM officials said that the challenge to implementing the coordinated project is getting federal agency collaboration and compliance. As of February 2004, the database contained award information from the 2003 COPS and FEMA interoperability communications equipment grants, but no others within or outside DHS. SAFECOM’s oversight authority and responsibilities are dependant upon its overall mission. OMB officials told us that they are currently in the process of refocusing the mission of the SAFECOM program into three specific parts: (1) coordination of federal activities through several initiatives, including participation in the Federal Interagency Coordination Council and establishment of a process for federal agencies to report and coordinate with SAFECOM on federal activities and investments in interoperability; (2) developing standards; and (3) developing a national architecture for addressing communications interoperability problems. They said identification of all current and planned federal agency communications programs affecting federal, state, and local wireless interoperability is difficult. According to these officials, OMB is developing a strategy to best utilize the SAFECOM program and examining options to enforce the new coordination and reporting process. SAFECOM officials said they are working to formalize the new reporting and coordination process by developing written agreements with other federal agencies and by obtaining concurrence of major state and local associations to the SAFECOM governance structure. SAFECOM officials noted that this newly refocused SAFECOM role does not include providing technical assistance or conducting operational testing of equipment. They said that their authority to conduct such activities will come from DHS enabling directives. SAFECOM officials also said that they have no enforcement authority to require other agencies to use the SAFECOM grant guidance in their funding decisions or to require agencies to provide grant program information to them for use in their database. State and Local Governments Can Play a Central Role States, with broad input from local governments, can serve as focal points for statewide planning to improve interoperable communications. The FCC has recognized the important role of states. In its rules and procedures, the FCC concluded that because states play a central role in managing emergency communications and are usually in control at large scale-events and disasters, states should administer the interoperability channels within the 700 MHz band of communications spectrum. States can play a key role in improving interoperable communications by establishing a management structure that includes local participation and input to analyze and identify interoperability gaps between “what is” and “what should be,” developing comprehensive local, state, and regional plans to address such gaps, and funding these plans. The states we visited or contacted—California, Florida, Georgia, Missouri, Washington and a five state Midwest consortium—were in various stages of formulating these management structures. However, states are not required to establish a statewide management structure or to develop interoperability plans, and there is no clear guidance on what should be included in such plans. In addition, no requirement exists that interoperability of federal communications systems be coordinated with state and local government communications systems. The use of a standard database on communications frequencies by public safety agencies within the state and common terminology for these frequencies in preparation and implementation of these statewide interoperable plans are essential but are also not required. Without planning, coordination, and applicable standards—in other words, without a commonly understood and accepted blueprint or national architecture—the communications systems developed between and among locations and levels of government may not be interoperable. States are key players in responding to normal all-hazards emergencies and to terrorist threats. Homeland Security Presidential Directive 8 notes that awards to states are the primary mechanism for delivery of federal preparedness assistance for these missions. State and local officials also believe that states, with broad local and regional participation, have a key role to play in coordinating interoperable communications supporting these missions. The Public Safety Wireless Network (PSWN), in its report on the role of the state in providing interoperable communications, agreed. According to the PSWN report, state leadership in public safety communications is key to outreach efforts that emphasize development of common approaches to regional and statewide interoperability. The report said that state officials have a vested interest in establishing and protecting statewide wireless infrastructures because public safety communications often must cross more than one local jurisdictional boundary. However, states are not required to establish a statewide capability to (1) integrate statewide and regional interoperability planning and (2) prepare statewide interoperability plans that maximize use of spectrum to meet interoperability requirements of day-to-day operations, joint task force operations, and operations in major events. Federal, state, and local officials are not required to coordinate federal, state, and local interoperability spectrum resources that, if successfully addressed, have significant potential to improve public safety wireless communications interoperability. As a result, states may not prepare comprehensive and integrated statewide plans that address the specific interoperability issues present in each state across first responder disciplines and levels of government. Several state and local agencies that we talked with emphasized that they are taking steps to address the need for statewide communications planning. State officials also told us that statewide interoperability is not enough because incidents first responders face could cross state boundaries. Thus, some states are also taking actions to address interstate interoperability problems. For example, Illinois, Indiana, Kentucky, Michigan, and Ohio officials said that their states have combined efforts to form the Midwest Public Safety Communications Consortium to promote interstate interoperability. According to these officials, they also have taken actions to form an interstate committee to develop interoperability plans and solicit support from key players, such as local public safety agencies. Statewide Interoperable Communications Committees Offer Potential for Coordinated Statewide Planning FCC recognized a strong state interest in planning and administering interoperability channels for public safety wireless communications when it adopted various technical and operational rules and polices for the 700 MHz band. In these rules and policies, FCC concluded that administration of the 2.6 MHz of interoperability channels in that band (approximately 10 percent) should occur at the state-level in a State Interoperability Executive Committee (SIEC). FCC said that states play a central role in managing emergency communications and that state-level organizations are usually in control at large-scale events and disasters or multi-agency incidents. FCC also found that states are usually in the best position to coordinate with federal government emergency agencies. FCC said that SIEC administrative activities could include holding licenses, resolving licensing issues, and developing a statewide interoperability plan for the 700 MHz band. Other SIEC responsibilities could include the creation and oversight of incident response protocols and the creation of chains of command for incident response and reporting. Available data indicate that 12 to 15 states did not create SIECs but have relied on Regional Planning Committees or similar planning bodies. Content and Scope of Statewide Interoperability Plans Not Established A comprehensive statewide interoperable plan can provide the guiding framework for achieving defined goals for interoperability within a state and for regions within and across states (such as Kansas City, Mo and Kansas City, Kans.). NCC recommended that all SIECs prepare an interoperability plan that is filed with FCC and updated when substantive changes are made or at least every three years. NCC also recommended to FCC that SIECs, for Homeland Security reasons, should administer all interoperability channels in a state, not merely those in the 700 MHz band. According to NCC, each state should have a central point identified for information on a state’s interoperability capability. None of the four states we visited had finished preparation and funding of their state interoperability plans. Washington and Florida were preparing statewide interoperability plans at the time we visited. Georgia officials said they have a state interoperability plan but that it is not funded. However, one other state we contacted, Missouri, has extended SIEC responsibility for interoperability channels beyond the 700 MHz band. The Missouri SIEC has also designated standard operational and technical guidelines as conditions for the use of these bands. SIEC requires applicants to sign a MOU agreeing to these conditions in order to use these channels in the state of Missouri. The Missouri SIEC Chairman said the state developed its operational and technical guidelines because FCC had not established its own guidelines for these interoperability channels in the VHF and UHF bands. The chairman said Missouri borders on eight other states and expressed concern that these states will develop different guidelines that are incompatible with the Missouri guidelines. He said FCC was notified of Missouri’s actions but has not taken action to date. In another example, California intends to prepare a statewide interoperability plan. California’s SIEC is re-examining California’s previous stove piped programs of communications interoperability (separate systems for law enforcement, fire, etc.) in light of the need to maintain tactical channels within disciplines while promoting cross-discipline interoperability. Coordination of Federal and State Interoperable Frequencies in Statewide Plans FCC designated frequency coordinators told FCC that planning for interoperability channels should include federal spectrum designated for interoperability with state and local governments. We found several examples in our field work that support inclusion of federal agencies in future state and local planning for interoperable communications. For example, a Washington State official told us that regional systems within the state do not have links to federal communications systems and assets. In another example, according to an emergency preparedness official in Seattle, a study of radio interoperable communications in a medical center also found that federal agencies such as FBI are not integrated into hospital or health communications systems, and other federal agencies have no radio infrastructure to support and participate in a health emergency such as a bio-terrorism event. He told us that he has no idea what the federal communications plan is in the event of a disaster; he said he does not know how to talk to federal health officials responding to an incident or what the federal government needs when they arrive. The federal government is developing a system that could improve interoperable communications on a limited basis between state and federal government agencies. The Integrated Wireless Network (IWN) is a radio system that is intended to replace the existing radio systems for the DOJ, Treasury, and DHS. IWN is an exclusive federal law enforcement communications system that is intended to interact and interface with state and local systems as needed but will not replace these systems. According to DOJ officials, IWN is intended to improve federal to state/ local interoperability but will not address interoperability of state and local systems. However, federal interoperability with state and local wireless communications systems is hindered because NTIA and FCC control different frequencies in the VHF and UHF bands. To enhance interoperability, NTIA has identified 40 federal government frequencies that can be used by state and local public safety agencies for joint law enforcement and incident response purposes. FCC, however, designated different frequencies for interoperability in the VHF band and in the UHF band from spectrum it controls for use by state and local public safety agencies. Federal Grant Structure Does Not Support Statewide Planning Total one-time replacement of the nation’s communications systems is very unlikely, due to the costs involved. A 1998 study cited the replacement value of the existing public safety communication infrastructure nationwide at $18.3 billion. DHS officials said this estimate is much higher when infrastructure and training costs are taken into account. Furthermore, DHS recently estimated that reaching an accelerated goal of communications interoperability will require a major investment of several billion dollars within the next 5 to 10 years. As a result of these extraordinary costs, federal funding is but one of several resources state and local agencies must use in order to address these costs. Furthermore, given the high costs, the development of an interoperable communications plan is vital to useful, non-duplicative spending. However, the federal funding assistance programs to state and local governments do not fully support regional planning for communications interoperability. Federal grants that support interoperability have inconsistent requirements to tie funding to interoperable communications plans. In addition, uncoordinated federal and state level grant reviews limit the government’s ability to ensure that federal funds are used to effectively support improved regional and statewide communications systems. Local, state and federal officials agree that regional communications plans should be developed to guide decisions on how to use federal funds for interoperable communications; however, the current funding requirements do not support this planning process. Although recent grant requirements have encouraged jurisdictions to take a regional approach to planning, current federal first responder grants are inconsistent in their requirements to tie funding to interoperable communications plans. States and locals are not required to provide an interoperable communications plan as a prerequisite to receiving some federal grant funds. As a result, there is no assurance that federal funds are being used to support a well- developed strategy for improving interoperability. For example, the fiscal year 2004 Homeland Security Grant (HSG) and Urban Areas Security Initiative (UASI) grants require states or selected jurisdictions to conduct a needs assessment and submit a Homeland Security Strategy to ODP. However, the required strategies are high-level and broad in nature. They do not require that project narratives or a detailed communications plan be submitted by grantees prior to receiving grant funds. In another example, fiscal year 2003 funding provided by COPS and FEMA for the Interoperable Communications Equipment Grants did not require that a communications plan be completed prior to receiving grant funds. However, grantees were required to provide documentation that they were actively engaged in a planning process and a multi-jurisdictional and multidisciplinary project narrative was required. In addition to variations in requirements to create communications interoperability plans, federal grants also lack consistency in defining what “regional” body should conduct planning. Grant Submissions and Performance Period Time Frames Also Present Challenges to Short- and Long-Term Planning State and local officials also said that the short grant application deadlines for recent first responder grants limited their ability to develop cohesive communications plans or perform a coordinated review of local requests. Federal officials acknowledged that the limited submission timeframes presents barriers to first responders for developing plans prior to receiving funds. For example, several federal grant programs—the Homeland Security Grant, UASI grant, COPs and FEMA communication equipment grants, Assistance to Firefighters Grant—allow states only 30 or 60 days from the date of grant announcement to submit a grant proposal. These time frames are sometimes driven by appropriations language or by the timing of the appropriations enactment. Furthermore, many grants have been awarded to state and locals for communications interoperability that have 1- or 2-year performance periods, and according to state and local officials, do not support long-term solutions. For example, Assistance to Fire Fighters Grants, COPS/ FEMA’s Interoperable Communications Equipment Grants, and National Urban Search and Rescue grants all have 1-year performance periods. UASI, HSG program, and Local Law Enforcement Block Grants have 2-year performance periods. No Coordinated Federal or State Grant Review Exists to Ensure Funds are Used to Improve Regional or Statewide Communications Interoperability The federal and state governments lack a coordinated grant review process to ensure that funds allocated to local governments are used for communication projects that complement each other and add to overall statewide and national interoperability. Federal and state officials said that each agency reviews its own set of applications and projects, without coordination with other agencies. As a result, grants could be given to bordering jurisdictions that propose conflicting interoperability solutions. In fiscal year 2003, federal officials from COPS and FEMA attempted to eliminate awarding funds to conflicting communication systems within bordering jurisdictions by coordinating their review of interoperable communications equipment grant proposals. However, COPS and FEMA are only two of several federal sources of funds for communications interoperability. In an attempt to address this challenge, in 2003 SAFECOM coordinated with other agencies to create the document Recommended Federal Grant Guidance, Public Safety Communications and Interoperability Grants, which lays out standard grant requirements for planning, building, and training for interoperable communications systems. The guidance is designed to advise federal agencies on who is eligible for the first responder interoperable communications grants, the purposes for which grant funds can be used, and eligibility specifications for applicants. The guidance recommends standard minimum requirements, such as requirements to “…define the objectives of what the applicant is ultimately trying to accomplish and how the proposed project would fit into an overall effort to increase interoperability, as well as identify potential partnerships for agreements.” Additionally, the guidance recommends, but does not require, that applicants establish a governance group consisting of local, tribal, state, and federal entities from relevant public safety disciplines and purchase interoperable equipment that is compliant with phase one of Project-25 standards. The House Committee on Appropriations report for the DHS FY 2004 appropriation states that the Committee is aware of numerous federal programs addressing communications interoperability through planning, building, upgrading, and maintaining public safety communication systems, among other purposes. The Committee directed that all DHS grant programs issuing grants for the above purposes incorporate the SAFECOM guidance and coordinate with the SAFECOM program when awarding funding. To better coordinate the government’s efforts, the Committee also encouraged all other federal programs issuing grants for the above purposes to use the guidelines outlined by SAFECOM in their grant programs. However, SAFECOM officials said that they have no enforcement authority to require other agencies to use this guidance in their funding decisions or to require agencies to provide grant program information to them for use in their database. Conclusions A fundamental barrier to successfully addressing interoperable communications problems for public safety has been the lack of effective, collaborative, interdisciplinary, and intergovernmental planning. Jurisdictional boundaries and unique public safety agency missions have often fostered barriers that hinder cooperation and collaboration. No one first responder agency, jurisdiction, or level of government can “fix” the nation’s interoperability problems, which vary across the nation and often cross first responder agency and jurisdictional boundaries. Changes in spectrum available to federal, state and local public safety agencies— primarily a federal responsibility conducted through the FCC and NTIA— changes in technology, and the evolving missions and responsibilities of public safety agencies in an age of terrorism all highlight the ever-changing environment in which interoperable communications needs and solutions must be addressed. Interdisciplinary, intergovernmental, and multi- jurisdictional partnership and collaboration are essential for effectively addressing interoperability shortcomings. Recommendations We are making recommendations to DHS and OMB to improve the assessment and coordination of interoperable communications efforts. We recommend that the Secretary of DHS: in coordination with the FCC and National Telecommunications and Information Administration, continue to develop a nationwide database of public safety frequency channels and a standard nationwide nomenclature for these channels, with clear target dates for completing both efforts; establish requirements for interoperable communications and assist states in assessing interoperability in their states against those requirements; through DHS grant guidance encourage states to establish a single, statewide body to assess interoperability and develop a comprehensive statewide interoperability plan for federal, state, and local communications systems in all frequency bands; and at the appropriate time, require through DHS grant guidance that federal grant funding for communications equipment shall be approved only upon certification by the statewide body responsible for interoperable communications that grant applications for equipment purchases conform with statewide interoperability plans. We also recommend that the Director of OMB, in conjunction with DHS, review the interoperability mission and functions now assigned to SAFECOM and establish those functions as a long-term program with adequate authority and funding. In commenting on a draft of this report, the Department of Homeland Security discusses actions the department is taking that are generally consistent with the intent of our recommendations but do not directly address specific steps detailed in our recommendations with respect to establishment of statewide bodies responsible for interoperable communications within the state, the development of comprehensive statewide interoperability plans and tying federal funds for communications equipment directly to those statewide interoperable plans. OMB did not provide written comments on the draft report. This concludes my prepared statement, Mr. Chairman, and I would be pleased to answer any questions you or other members of the Subcommittee my have at this time. This is a work of the U.S. government and is not subject to copyright protection in the United States. It may be reproduced and distributed in its entirety without further permission from GAO. However, because this work may contain copyrighted images or other material, permission from the copyright holder may be necessary if you wish to reproduce this material separately. | Lives of first responders and those whom they are trying to assist can be lost when first responders cannot communicate effectively as needed. This report addresses issues of determining the status of interoperable wireless communications across the nation, and the potential roles that federal state, local governments can play in improving these communications. In a November 6, 2003, testimony, GAO said that no one group or level of government could "fix" the nation's interoperable communications problems. Success would require effective, collaborative, interdisciplinary and intergovernmental planning. The present extent and scope nationwide of public safety wireless communication systems' ability to talk among themselves as necessary and authorized has not been determined. Data on current conditions compared to needs are necessary to develop plans for improvement and measure progress over time. However, the nationwide data needed to do this are not currently available. The Department of Homeland Security (DHS) intends to obtain this information by the year 2005 by means of a nationwide survey. However, at the time of our review, DHS had not yet developed its detailed plans for conducting this survey and reporting its results. The federal government can take a leadership role in support of efforts to improve interoperability by developing national requirements and a national architecture, developing nationwide databases, and providing technical and financial support for state and local efforts to improve interoperability. In 2001, the Office of Management and Budget (OMB) established the federal government's Wireless Public Safety Interoperable Communications Program, SAFECOM, to unify efforts to achieve national wireless communications interoperability. However, SAFECOM's authority and ability to oversee and coordinate federal and state efforts has been limited by its dependence upon other agencies for funding and their willingness to cooperate. OMB is currently examining alternative methods to implement SAFECOM's mission. In addition, DHS, where SAFECOM now resides, has recently announced it is establishing an Office for Interoperability and Compatibility to coordinate the federal response to the problems of interoperability in several functions, including wireless communications. The exact structure and funding for this office, which will include SAFECOM, are still being developed. State and local governments can play a large role in developing and implementing plans to improve public safety agencies' interoperable communications. State and local governments own most of the physical infrastructure of public safety communications systems, and states play a central role in managing emergency communications. The Federal Communications Commission recognized the central role of states in concluding that states should manage the public safety interoperability channels in the 700 MHz communications spectrum. States, with broad input from local governments, are a logical choice to serve as a foundation for interoperability planning because incidents of any level of severity originate at the local level with states as the primary source of support. However, states are not required to develop interoperability plans, and there is no clear guidance on what should be included in such plans. | gov_report |
RELATED APPLICATIONS [0001] This application claims the benefit of Japanese Application No. JP2002-361900, filed Dec. 13, 2002, which is incorporated by reference herein in its entirety. TECHNICAL FIELD OF THE INVENTION [0002] The present invention relates to a film forming agent for coating plants and a method for supplying ingredients to plants using such film forming agent. BACKGROUND [0003] For plants such as vegetables and fruits, the proper taste and flavor of the plant is only obtained when a sufficient amount of the nutrients appropriate for the respective plant have been supplied in its growth process. [0004] Currently, the appropriate nutrients are obtained from the soil where plants are cultivated. However, the soil has a tendency to become barren because of continuous cultivation and deterioration of land environment, etc., and it is becoming more difficult to supply sufficient amounts of such appropriate nutrients in recent years. For example, these days the soil often lacks proper mineral ingredients and therefore the taste of vegetables and fruits often lacks flavor and sweetness. In addition, nutrients supplied from compost don't always supplement the soils adequately. [0005] Thus, to supply mineral nutrients to plants, one conventional method is to pump up deep-sea water, dilute it with water, and spray it over the surface of plant leaves, thereby directly supplying mineral nutrients to the leaf surfaces and allowing them absorb mineral ingredients. However, the minerals supplied by deep-sea water diluted and sprayed onto leaves using this method is dissolved by rainwater and washed away, and therefore spraying of the deep-sea water is necessary every time it rains, which is troublesome and costly. [0006] Another method is to directly spray mineral nutrients over the soil and let plants absorb them from their roots. However, the supply of mineral nutrients to the roots has only a limited effect and has the same problem that they are easily washed away with rainwater. [0007] In addition, agricultural chemicals are sprayed to exterminate harmful insects, but such agricultural chemicals stick to plants and residual agricultural chemicals remain even after water washing plants, which is harmful not only to consumers but also agricultural producers and workers, etc. The use of agricultural chemicals by agricultural producers and workers are harmful to health particularly in the case of growing plants in greenhouse because it involves work in a closed room. Furthermore, the use of agricultural chemicals is harmful to health of not only agricultural producers and workers but also people in general when agricultural chemicals are applied to plants for public facilities such as street trees and park trees, etc. [0008] Thus there is a long felt need for a method of supplying mineral and other nutrients to plants in a manner that is not easily washed away by rain water. In addition, it would be beneficial if such method also limited or prevented insect damage to plants thus decreasing or even elimination the need for harmful agricultural chemicals such as pesticides. SUMMARY OF THE INVENTION [0009] The present invention meets the above long felt needs. As discussed above, to supply mineral ingredients to plants such as vegetables and fruits, the conventional methods include diluting deep-sea water containing sufficient mineral ingredients with water and then spraying it over leaf surfaces of plants or cultivating plants with soil containing added mineral ingredients, but the added mineral ingredients are washed away with rainwater and it is therefore necessary to spray deep-sea water after every time it rains, which is troublesome, takes a lot of man-hours and increases costs in aspects of labor efficiency and workability. The present invention solves this by supplying minerals to plant such that the minerals are not easily washed away. [0010] Moreover, agricultural chemicals are sprayed to exterminate harmful insects, but agricultural chemicals stick to plants and residual agricultural chemicals remain even after water washing plants, which is harmful not only to consumers but also to agricultural producers and workers, etc. The use of agricultural chemicals by agricultural producers and workers are harmful to health particularly in the case of growing plants in greenhouse because it involves work in a closed room. Furthermore, the use of agricultural chemicals is harmful to health of not only agricultural producers and workers but also people in general when agricultural chemicals are applied to plants for public facilities such as street trees and park trees, etc. Aspects of the present invention address these problems by providing an alternative means of protecting plants from insects that lowers exposure of humans to harmful agricultural chemicals. [0011] Thus it is an object of the present invention to provide a film forming agent for coating plants and a method for supplying ingredients to plants capable of supplying necessary ingredients to plants easily and reliably. [0012] It is another object of the present invention to provide a film forming agent for coating plants and a method for supplying ingredients to plants to make sure that damages to plants by harmful insects are prevented. [0013] It is a further object of the present invention to provide a film forming agent for coating plants and a method for supplying ingredients to plants which is absolutely harmless to the human body, capable of not only supplying necessary ingredients to plants easily and reliably but also making sure that damages to plants by harmful insects are prevented. [0014] In order to solve the above-described problems, the present invention provides a film forming agent for coating plants and a method for supplying nutrients to plants have the following characteristic configuration. One aspect of the present invention is a film forming agent for coating plants comprising cellulose which includes at least one of mineral ingredient and saccharide ingredient. In one embodiment, the mineral ingredient includes citric acid. [0015] In another embodiment, the film forming agent comprises a basic ingredient made of 2.5 to 3.5 weight percentage of hydroxypropylmethyl cellulose with a hydroxypropyl base of 4 to 12% by weight of the hydroxypropylmethyl cellulose and 96.5 to 97.5 weight percentage of water; a mineral content of 2.5 parts of the ingredient; and polysaccharide (sugar) content 5.0 parts of the solution 100 parts. In yet another embodiment, the mineral ingredient is composed of 40 parts natural salt, 60 parts natural magnesium chloride, and 3.0 part trisodium citrate. In still another embodiment, the polysaccharide is any one of sugar, fructose, maltose or glucose. In another embodiment, the film forming agent for coating plants comprises 2.5 to 3.5 parts hydroxypropylmethyl cellulose, 96.5 to 97.5 parts water; and 2 parts trisodium citrate. [0016] Another aspect of the present invention includes a method for supplying ingredients to plants by spraying any one of the above-described agents forming film coating plants over plants and thereby forming a cellulose film on the surface of said plants. DETAILED DESCRIPTION OF THE INVENTION [0017] The configurations and operations of embodiments of the film forming agent for coating plants and the method for supplying ingredients to plants according to the present invention will be explained in detail below. [0018] The film forming agent for coating plants and the method for supplying ingredients to plants form a thin cachet film (hereinafter referred to as “cell coat”) including ingredients necessary for a plant on the surface of plants by means of spraying, etc., and thereby directly supply the above-described necessary ingredients from the surface of the plant to the plant through the cell coat. The cell coat is made of a material used for medicine that is harmless to the human body, which is hardly washed away even if gotten wet with rain and therefore requires no repeated spraying every time it rains and provides excellent workability. [0019] In one embodiment, the film forming agent for coating plants (cellulose film forming agent) including the above-described cell coat according to the present invention is basically composed of hydroxypropylmethyl cellulose, sodium chloride, magnesium chloride and sodium citrate as principal ingredients, and may be mixed with approximately 70 kinds of other trace minerals and polysaccharides. [0020] A more specific composition of the film forming agent for coating plants according to the present invention is as in the following example: [0021] (1) A basic ingredient made of 2.5 to 3.5 weight percentage of hydroxypropylmethyl cellulose whose hydroxypropyl base is 4 to 12% by weight of the hydroxypropylmethyl cellulose and 96.5 to 97.5 weight percentage of water; [0022] mineral ingredient representing 2.5 percent by weight of the above-described basic ingredient; and [0023] polysaccharide (sugar) representing 5.0 percent by weight of the above-described basic ingredient [0024] is a preferred example of the film forming agent for coating plants. [0025] In one embodiment, the composition of the above-described mineral ingredient is: [0026] 40 parts natural salt; [0027] 60 parts natural magnesium chloride; and [0028] 3.0 parts trisodium citrate [0029] as a preferred example. [0030] In certain embodiments, the polysaccharide may be selected from the following group sugar, fructose, maltose or glucose, etc. [0031] Here, natural magnesium chloride is preferred as the mineral ingredient and the trisodium citrate acts as a catalyst to promote absorption at the surface of the plant. [0032] Another example of the present invention is as follows: [0033] (2) The film forming agent for coating plants is composed of: [0034] hydroxypropylmethyl cellulose: 2.5 to 3.5 parts [0035] water: 96.5 to 97.5 parts [0036] trisodium citrate: 2.0 parts [0037] Numerous suitable methods for forming the cell coat with the compositions of the present invention. By way of example, it is possible to use a spraying method utilizing a mechanical power sprayer, power sprayer, fixed power sprayer or knapsack power sprayer. The configuration of the film forming agent for coating plants used for spraying with each machine is as follows: [0038] 1. Mechanical power sprayer (SS): [0039] Film forming agent for coating plants in above example (1) or (2): 20 kg [0040] Water 500 l sprayed over 10 acres [0041] 2. Power sprayer: [0042] Film forming agent for coating plants in above example (1) or (2): 12 kg [0043] Water 300 l sprayed over 10 acres [0044] 3. Fixed power sprayer: [0045] Film forming agent for coating plants in above example (1) or (2): 8 kg [0046] Water 200 l sprayed over 10 acres [0047] 4. Knapsack power sprayer: [0048] Film forming agent for coating plants in above example (1) or (2): 4 kg [0049] Water 100 l sprayed over 10 acres [0050] Spraying and forming the cellulose film forming agent with the above-described configurations according to the present invention over the surface of a plant allows the surfaces of leaves and fruits of the plant to be wrapped with the cellulose film forming agent including a mineral ingredient and saccharide and allows the mineral ingredient and saccharide, etc., to be absorbed from the surfaces of leaves for a long stretch of time. This cellulose film forming agent forms film a on the surfaces of leaves and fruits in close contact with cell membranes, and can thereby protect the plant. [0051] This cellulose film forming agent has excellent weather resistance and durability, and is hardly washed away with rainwater, tasteless, odor-free, colorless, and harmless and provides excellent workability. The film typically has a thickness of 5 to 20 microns, which is made variable depending on the plant in such a way that it is thicker for those with permeability and thinner for those with less permeability. Mineral ingredients and saccharides are easily dissolved into water and therefore are easily washed away from leaves, but the use of them together with the cellulose film forming agent of the present invention allows for durable application of the mineral ingredients and saccharaides and can show significant effects on the plants even with a small amount thereof. [0052] Furthermore, since the cellulose film forming agent of the present invention forms a film on the surface of the plant, the plant is more resistant to viral infections and less susceptible to damage by harmful insects. [0053] Furthermore, the present invention has no residual agricultural chemicals and allows production of plants free of or with a reduced amount of agricultural chemicals and moreover this cellulose film is totally harmless even if it enters the human body through the mouth and is never washed away with rainwater. The present invention makes it possible for the plant to absorb minerals and saccharides, etc., through the cellulose film forming agent and thereby obtain plants which are strong, resistant to diseases and with a pleasant taste, and improve sugar content easily. [0054] It has been experimentally confirmed that forming a film on plants by spraying the cellulose film forming agent including various ingredients according to the present invention provides various effects as will be described below. [0055] It has been experimentally confirmed that including citric acid in minerals improves the absorbency of minerals by a plant and activates the plant, refreshes the leaf colors, increases chlorophyll and deepens the colors. [0056] Being coated with the cellulose film prevents evaporation of water content, last long and up taste sweet of vegetables and fruits. It has been experimentally confirmed that the presence of the cellulose prevents minerals and saccharides from being washed away with rainwater. [0057] Though bagging cultivation is used for many fruit trees, the coating with cellulose eliminates the need for it and further allows the plant to catch sunlight directly and thereby have a improve color and gross. It has been experimentally confirmed that light can be shielded by coating the plant with colored cellulose and wash it away with water and vinegar. [0058] In addition to the above-described effects, the following effects against harmful insects, insects and disease-causing germs can also be obtained: [0059] It has been experimentally confirmed that a hydroxypropyl base ranging from 4 to 12% by weight of the hydroxypropylmethyl cellulose can serve as a repellent against aphids, whiteflies, citrus red mites, red spiders, T.kanzawai (tea), etc. [0060] It has been experimentally confirmed that including mineral ingredients in the cellulose film has the bacteriostatic effect on disease-causing germs. [0061] It has been experimentally confirmed that including mineral ingredients in the cellulose film has the effect of repelling harmful insects. [0062] It has been experimentally confirmed that the cellulose film stops up pores of insects, causes them to have difficulty in breathing and prevents them from contacting leaves, etc., dropping off the leaves in approximately 3 to 7 minutes. This can avoid using agricultural chemicals for plants in public facilities such as street trees and park trees, making them completely harmless. [0063] It has been experimentally confirmed that being coated with the cellulose film prevents insects from inserting their stylets into or pulling them out of plant tissue and thereby prevents insect damage. [0064] It has been experimentally confirmed that being coated with the cellulose film prevents action of insects from hatching and becoming larvae and also prevents them from developing from larvae to imagoes. [0065] It has been experimentally confirmed that being coated with the cellulose film prevents infection by means of spores which are sources of infection and stay above the film. The coating reduces humidity, increases resistance to diseases, thus preventing diseases from spreading. [0066] Coating with the cellulose film and minerals causes disease-causing germs to be disinfected with minerals and lose their functions. Coating over an extended time period has a great effect. [0067] The effects of the present invention in an economical aspect include its advantage in labor efficiency and workability as it reduces the number of times spraying is required from 5 to 15 times in the conventional method for exterminating harmful insects to 2 to 5 times, 1/3 of the conventional one. [0068] Furthermore, compared to conventional agricultural chemicals, the present invention could reduce the total cost of materials used by 10 to 30%. [0069] The effects of the present invention in an environmental aspect include its harmlessness to workers as opposed to the use of agricultural chemicals which is harmful to health of agricultural producers and workers. Moreover, the health of consumers is also protected because there are no residual agricultural chemicals in harvested farm products and fruits. [0070] The effects of the present invention in a social aspect include its ability to make completely harmless plants for public facilities such as street trees and park trees, etc., to which agricultural chemicals have been conventionally applied, thus improving the environment, protecting nature and having no adverse effects on atopic people or people allergic to chemical substances. [0071] The configurations and operations of the preferred embodiments of the present invention have been described in detail so far. However, these embodiments are only illustrative examples of the present invention and do not limit the present invention. It is easily understandable to those skilled in the art that the present invention can be modified in various manners according to particular applications without departing from the spirit of the present invention or essential characteristics thereof. [0072] As described above, the film forming agent for coating plants and the method for supplying ingredients to plants of the present invention make it possible not only to supply necessary ingredients to plants easily and reliably but also to harmlessly and reliably prevent damage to plants caused by harmful insects. That is, it allows plants such as vegetables and fruits to easily absorb ingredients such as minerals and saccharides through the surfaces of leaves by a means that is not easily washed away by rainwater and easily supplies those ingredients, which promotes quality improvement and growth of plants. Furthermore, the present invention can eliminate damage to health of agricultural producers and workers caused by agricultural chemicals as measures against harmful insects and disease-causing germs. | The present invention provides compositions and methods of use and application of such compositions that not only ensure that necessary nutrients are supplied to plants but also harmlessly and reliably prevent damage to plants caused by harmful insects. The compositions include film forming agents for coating plants that include cellulose which contains at least one of a mineral ingredient and a saccharide ingredient, wherein the mineral ingredient preferably includes citric acid. This film forming agent for coating plants is sprayed over plants, and a cellulose film is thereby formed on the surfaces of the plant, supplying the nutrients to the plant and protecting the plant from insect damage. | big_patent |
[Scene: The Bay Mirror. Phoebe walks in and goes over to her assistant.]
Phoebe: Good morning. Any phone calls?
Assistant: Take your pick. We've got adulterers, cross-dressers, thirty-four year old virgins, and, oh, your nephew.
Phoebe: Oh, Chris called?
Assistant: No, Wyatt. Um, actually, I think Piper did the dialling, but. You have another nephew?
Phoebe: Um, no, uh, but-but, you know, maybe some day I will. You know what I mean? Okay.
(Phoebe goes into her office and Chris there. She gets a fright.)
Chris: Phoebe, I need your help.
Phoebe: I've been calling for you all week. Didn't you hear me?
Chris: For the first couple of days, yeah. Then I put you on mute.
Phoebe: You can put me on mute?
Chris: I had to, I was busy. Now, I need your help.
Phoebe: Oh, yeah, and I need yours too, because you come here, you drop this bombshell on me, and you expect me to keep this secret? And I don't even know why I'm keeping the secret.
Chris: Nobody can find out Piper and Leo are my parents. It can mess with the whole future.
Phoebe: Yeah, well, if you didn't want anybody to know, I don't know why you told me.
Chris: I told you because you busted me, and I'm glad you did. I have been so focused on protecting Wyatt, I've completely forgotten about me. This month is my conception date.
Phoebe: Your conception date?
Chris: That's where I've been. Oracles, fortune tellers, soothsayers they all say the same thing. If mum and dad don't screw, this month I'm screwed.
Phoebe: Okay, I'm just trying to get used to you being my nephew. I never hit on you, did I?
Chris: What? No.
Phoebe: Oh, thank god.
Chris: Can we focus here, please? Mum and dad need to have s*x. Now who's gonna tell them? You or me?
Phoebe: No, nobody's gonna tell them because we're not gonna reopen those wounds.
Chris: Okay. So how do we get them back together?
Phoebe: W-we? There is no we here. We don't. You're the one that split them up. And why did you split them up?
Chris: Leo had to become an Elder to make room for me as your Whitelighter. It was the only way I could protect Wyatt from turning evil.
Phoebe: You're unbelievable. I mean, the most kids who are the cause of their parents divorce actually feel guilty. And you're sitting here like it's part of your master plan.
Chris: I'm sensing some real issues here.
Phoebe: Oh, you're damn right there are issues. You can't just pop in from the future and play with people's lives because your big brother picked on you.
Chris: He picked on the world, Phoebe.
Phoebe: I'm not finished. Your parents were happy until you split them up. And now you want my help because you didn't think this all the way through?
Chris: Feel better?
Phoebe: Yes.
Chris: Will you help me?
Phoebe: No. Oh, I don't know.
Chris: If I'm not conceived in the next couple of weeks, I'll disappear forever. (Chris picks up a letter off Phoebe's desk.) You are willing to help complete strangers. How about family? (Phoebe snatches the letter off Chris and receives a premonition.) What is it? What'd you see?
Phoebe: A woman being attacked.
Chris: Where?
[Scene: Arabia. Cave. Phoebe and Chris are there.]
Phoebe: Looks like a dig site.
Chris: A desert in the middle east. Are you sure your scrying wasn't off?
Phoebe: Maybe Jenny is an archaeologist.
Chris: Yeah, why would an archaeologist in the middle east send a letter to an advice columnist in San Francisco?
Phoebe: She said she was with a controlling man.
Chris: Okay, you're missing my point. What happens if this is a trap? (Chris sees some bones on the ground.) What is that? (He bends down to have a look. A sword flies past above his head. They turn around to see two Arabians standing near the cave entrance. They shout words at Phoebe and Chris and move forward. Phoebe throws a potion at them and they are vanquished.) You think anyone heard them?
(A ball of light hits Chris in the shoulder and knocks him to the ground. A guy on a flying carpet flies in. He is holding a bottle. Chris uses telekinesis to knock down some trestles in front of the man. He stops suddenly and drops the bottle.)
Man: No!
(Phoebe throws a potion which doesn't harm the man but he flies away. Phoebe rushes to Chris's side.)
Phoebe: Are you okay?
Chris: I'll be fine. Was that a flying carpet?
Phoebe: What is that?
(Phoebe goes over to the bottle and picks it up. She wipes off the dirt and pink smoke escapes out of the bottle. Jinny, the Genie appears.)
Jinny: Thank you for responding to my letter.
Phoebe: Wait, are you Jinny?
Jinny: At your service, master.
Opening Credits
[Scene: Conservatory. Phoebe, Leo, Chris and Jinny are there. Leo is healing Chris's shoulder.]
Jinny: I could heal him, master. Your warrior needs his strength. My last master will be coming back for me.
Phoebe: I think he's got it under control.
Jinny: Good idea, save your wishes.
Leo: Did you get a good look at the demon?
Phoebe: I did, and when we're done here I'll go up to the Book of Shadows and check it out. I also called Paige to see if she can keep an eye on Jinny for me.
Chris: Thanks.
Jinny: There is no need to guard me. Even if I was not bound to serve you I would do it anyway for sparing me from Bosk.
Phoebe: Bosk?
Jinny: My last master. He's cruel, even for a demon. And I would know. My bottle has been passed around from demon to demon for centuries.
Leo: That's terrible.
Jinny: You can not begin to know. That is why I got a message to Phoebe. I knew if she had my bottle she would wish me free.
Phoebe: No wishes. I know all about Genies. You're tricksters.
Leo: Listen, I gotta get back up there. You think you can handle this without Piper?
Chris: Where is Piper?
Leo: On a date.
Chris: On a date in the middle of the day?
Phoebe: Yeah, Greg works nights.
Chris: (thinks) Greg, Greg. Greg, the fireman? You mean the one she's insanely sexually attracted to? Doesn't that bother you?
Leo: No. If it's makes her happy, that's all that matters.
Chris: Oh, come on! What about all this forbidden lovers, you and me against the world stuff? That just doesn't go away.
Leo: You know, Chris, it's a little late for male bonding. Especially since I'm petitioning the other Elders to send you back to your time.
Chris: What?
Phoebe: Wait, are you serious?
Jinny: You look tense, master. Neck rub?
Leo: Even though Chris's intensions are good, his methods have put us all at risk. So, he's going back.
Chris: You mean abandoning me again.
Leo: Look, you did your job, you warned us about an evil that was after Wyatt. I think we can handle it from here.
(Leo orbs out.)
Chris: I've gotta stop him.
Phoebe: Don't worry, I'll talk to Leo.
Chris: No, no, not Leo. Greg, the fireman. He's about to sleep with my mum.
(Chris orbs out.)
Phoebe: I really wish you wouldn't do that.
(Jinny puts her hands together and blinks. Chris orbs back in.)
Chris: What just happened?
Jinny: Your wish is my command, master.
(They hear the front door close.)
Paige: Alright, where's the Genie? (Phoebe and Jinny walk into the living room. Paige walks in.) Oh my god, you landed one.
Phoebe: She's a Genie, not a trout.
Jinny: You still have two wishes, master. I suggest you save one for Bosk.
Phoebe: I told you, no wishes. We're gonna do this our way.
Jinny: But you can not handle him. Nobody can. He has a flying carpet and an army of forty thieves.
Phoebe: Thirty-eight. I vanquished two.
Paige: Let me guess. He wanted a crew and a nice ride. Original for a demon's wish, yeah?
Jinny: Yes. And if Bosk gets me back, he will force me to grant his third wish.
Paige: What's his third wish?
(Large diamond earrings appear on Paige's earlobes.)
Phoebe: Did you do that?
Jinny: No, but they are lovely. Who conjured them for you?
Paige: My boyfriend, Richard. He's been showering me with gifts all week.
Phoebe: I thought you were gonna talk to him about binding his powers?
Paige: I have but every time I bring it up I just get another present. Luxury problem I know, but still.
Phoebe: Yeah, not good. Back to the demon. Uh, what was his third wish?
Jinny: Zanbar.
Phoebe: Zanbar?
Paige: What's Zanbar?
Jinny: The lost city. Before being swallowed up by the desert, it was the seat of power for an evil empire.
(Chris walks in.)
Chris: Phoebe? Will you do something, please? I can't orb.
(A large diamond bracelet appears on Paige's arm.)
Paige: Damn him.
Phoebe: You know, Paige, if he won't listen to you, maybe he'll listen to his family.
Paige: Most of them are dead. Remember, the feud.
Jinny: Please, we do not have time for this. If Bosk captures me, Zanbar will rise again from the dust.
Paige: It's just a city.
Jinny: A city of magic. Bosk has been using his thieves to search for his former site. If he finds it and wishes it back, there will be no stopping him. That is why you must wish me free, master. If I am not a Genie, it will solve your problems and mine. I beg you.
Chris: Hey, a little help here, please?
(Paige's clothes change into a black evening dress.)
Phoebe: Okay, I'm losing my mind. Uh, Paige, go to Richard, deal with it so you can help us.
Paige: Okay.
(Paige leaves.)
Phoebe: You, I will help you get your parents back together but it has to be on my terms. Agreed?
Chris: Agreed.
Phoebe: Go get Piper, we could use her help. Uh, I wish that he could orb. (Jinny puts her hands together and blinks. Chris orbs out.) And we need to find a vanquishing potion for that demon.
Jinny: Oh, yes, master.
Phoebe: Phoebe.
Jinny: Yes, master Phoebe.
[Scene: Greg's apartment. Piper and Greg are sitting on the couch, making out. Chris orbs in outside. He knocks on the door.]
Chris: Piper! Piper!
Piper: Forget it.
Chris: I know you're in there. Please open the door now.
Piper: Just a sec. (Piper gets up and goes over to the door. Chris continues to knock. She opens the door.) Go away.
Chris: We have an emergency.
(Greg walks over to the door.)
Greg: Is there a problem here?
Chris: Yeah, many problems, many levels. Piper has to come home now.
Greg: Excuse me? Who are you again?
Chris: I'm a friend of her husband's.
Piper: Ex-husband, and he's not really a great friend. Um, it's okay, I got it.
Greg: Well, I'm here if you need me.
(They kiss and Greg walks away. Piper goes out into the hallway with Chris and closes the door behind her.)
Piper: What is this big emergency? Can't it wait and hour or two?
Chris: No, it can't. There's a demon on the loose, a Genie running a muck, and it took me two wishes to get here.
Piper: You can't make wishes with Genies.
Chris: See, we need you. Come on, let's orb.
(He grabs her hand.)
Piper: No, no, no. Listen. I am not gonna leave him high and dry again without an explanation. So your demon can wait five minutes.
(She goes back inside.)
[SCENE_BREAK]
[Scene: Arabia. Cave. Bosk is there.]
Bosk: Open sesame.
(A door opens in the cave wall. He walks through, going into another cave where there are thieves and treasure.)
Thieve: What happened?
Bosk: The Genie was stolen, thanks to your warriors.
Thieve: They were my two best swordsmen.
Bosk: Yeah, well swords don't work real well against potions. What the hell are witches doing all the way out here?
Thieve: We have defences against their type. (The thieve picks up a pendant necklace.) The eye of Aghbar. It protects against witches magic.
Bosk: I need that Genie. All this work for nothing if we don't get her back.
Thieve: I'll gather my warriors, all my warriors.
Bosk: No. No, you and your men, you keep digging. I've got to find out where Zanbar's buried before I wish for its return. I can't risk another demon beating me to the throne.
Thieve: As you wish.
[Scene: Richard's House. Paige is there walking through the rooms.]
Paige: Richard? Richard.
(Richard suddenly appears behind Paige.)
Richard: You like your earrings?
(Paige gets a fright.)
Paige: Oh, you're materialising now.
Richard: Yeah. Pretty handy, huh? So, uh, you like your earrings?
Paige: Yeah, there's a bit of a problem.
Richard: They're too small? No, they're too big. I can shrink them. Let's see, um...
Paige: No, that's not the problem. I just, you've given me enough.
Richard: I'm just trying to make you happy. I want you to know that I care.
Paige: What would make me really happy is if you just stopped with all the potions and all the magic, just for a while.
Richard: Didn't we have this conversation?
Paige: Yeah, but apparently only one of us was listening.
Richard: No, I was listening. I mean, that's why I'm doing this, to prove that I can handle it. I'm not turning into a dark beast, right?
Paige: But that's not the point.
Richard: Most women would thank a guy for that, but you're treating me like a common criminal.
Paige: Well, I guess I'm not most women.
Richard: I gotta go. Hope you like the earrings.
(He walks off.)
[Scene: Manor. Attic. Phoebe and Jinny are there. Jinny is looking through the Book of Shadows.]
Jinny: He was my master once. And him too. And her.
Phoebe: Boy, you sure got around. How did so many demons get a hold of you, anyway?
Jinny: Some bought, some stole. I changed town so many times I lost track.
Phoebe: I'm sorry I can't set you free. But wishing is just too risky right now.
(Piper and Chris orb in.)
Piper: Okay, let's go. Greg's not gonna wait forever.
Chris: Well, then you should dump him.
Piper: What is that supposed to mean?
Phoebe: He's just being over protective.
Piper: I take it you're the Genie.
Jinny: Jinny.
Phoebe: Jinny the Genie.
Piper: Of course. Who's the demon?
Chris: Uh. (Chris goes over to the Book of Shadows and looks at the page on Bosk.) He's a low level demon with minimal powers. There's a vanquishing potion.
Phoebe: Yeah, that's what I'm working on.
Piper: Good. Then you're almost done with me too. Okay, so what you're planning is summon him to us?
Phoebe: Yeah, that's what I was thinking.
Chris: What's the rush?
Piper: Well, not that it's any of your business but Greg's shift starts in a few hours and I won't see him for three days. So I'm gonna go call him and I'd put the Genie back in the bottle just to be safe. No offence, but we've been burned before.
(Piper leaves the room.)
Phoebe: Do you mind?
Jinny: Yes, master.
(Jinny is sucked into the bottle.)
Phoebe: I feel so bad.
Chris: As well you should. If we don't do something soon, I can end up half fireman instead of half Whitelighter.
Phoebe: Oh, for goodness sake.
Chris: Look, I'm running out of time here. So what do you say we get to use that Genie to make mum and dad... you know.
Phoebe: That's vile. And against the rules. I would think you wouldn't want to be conceived that way.
Chris: Well, beats not being conceived at all.
Phoebe: Look, I told you I would help you on my terms, okay? So back off.
Chris: What are your terms?
Phoebe: Hmm, not really sure yet. But I am done with this potion. As soon as Piper gets back we are ready to go.
(Bosk comes crashing through the window on his flying carpet, knocking Chris to the floor. Phoebe throws the potion at Bosk but the pendent around his neck blocks it.)
Bosk: Not this time, witch.
Phoebe: Jinny, I wish you free!
(Pink smoke escapes out of the bottle and Jinny appears wearing black clothes.)
Jinny: Well, it's about time. Who's the master now?
(Jinny throws a fireball at Bosk and vanquishes him. Piper walks in. Jinny reaches for the bottle.)
Piper: Chris.
(Chris holds out his hand and the bottle flies into it. Piper tries to blow Jinny up but Jinny ducks. Jinny jumps on the flying carpet and it flies out the window. Chris gets up.)
Chris: Where's Phoebe?
Phoebe's Voice: Here. In here! (Chris looks in the bottle and sees Phoebe dressed in a blue Genie costume.) Hello, master.
[SCENE_BREAK]
[Scene: Manor. Attic. Piper and Chris are there. Piper is looking in the bottle.]
Piper: Will you come out of there, please?
Phoebe: I can't. I don't know how. Try commanding me.
Piper: Uh, okay. Get the hell outta there.
Phoebe: No, not you. My master.
(Piper walks away and Chris looks in the bottle.)
Chris: You mean me?
Phoebe: Well, yeah, you did pick up the bottle, didn't you?
Chris: Alright, get out of the bottle. I command you.
(Blue smoke rises out of the bottle and Phoebe appears. Piper laughs.)
Piper: You look ridiculous.
Phoebe: I feel ridiculous.
Piper: How am I supposed to get back to Greg now with this?
Phoebe: Is that all you care about? Would you look at me. I am trapped in pantaloons right now. Where is the mirror? (She walks over to the mirror.) Oh, and why do I always get stuck with the wig?
Piper: Trust me, you don't. Leo!
Chris: Have you ever noticed that Leo is the first person you call in your time of need?
(Leo orbs in.)
Leo: Uh-oh.
Phoebe: Yeah, right, uh-oh.
Piper: I still can't believe you made a wish with a Genie. You know better than that.
Phoebe: I thought she was an innocent. How was I to know that I was gonna unleash a demon.
(Leo picks up the bottle.)
Leo: It says so right here.
Phoebe: Oh, right, right there in Arabic.
Piper: There's a warning label on the Genie bottle?
Leo: Yeah. An ancient sorcerer condemned a demon into the bottle for not marrying him. It says whoever tried to free her they have to switch places with her. Missed a big one here, bud.
Chris: You wanna pin this on me?
Phoebe: Leo, it's not his fault, it's my fault.
Piper: How come your empathy thing didn't give her away?
Phoebe: She tricked me and obviously the book too.
Chris: Let's just figure out a way to fix this, okay?
Leo: Well, the only way to fix it is to get the demon to wish Phoebe free, reverse the magic.
Phoebe: That's what we need to do then.
Piper: I'll call Paige.
Phoebe: Yes. I do believe the element of surprise is very important here.
Chris: You sure? Because we could always take our time with this plan, you know, keep Piper around just a little bit longer.
Phoebe: Yes, master.
Chris: Good, I'm glad you agree.
Phoebe: Actually, I don't agree, but I-I can't... How am I supposed to take charge and take commands all at the same time?
Leo: Well, you won't have to. Chris is coming back up there with me. The Elders have agreed to send him back to his time.
Chris: What?
Phoebe: You don't know what you're doing here, Leo.
Leo: You don't belong here. And as your Whitelighter he's doing more harm than good.
Chris: You're so full of it. This isn't about me being a bad Whitelighter, it's about you feeling like I've let you down somehow. So whatever issues you might have with me, I wish you would just get over it already. (Phoebe puts her hands together and blinks.) Leo?
(Leo laughs.)
Leo: Of course I forgive you, man. You don't have to yell. All you gotta do is ask.
Chris: I did?
Leo: Yeah. And listen, with all that whole going back to the future thing, you know, don't even worry about it because I'll talk to the other Elders and we're gonna work it all out, okay? It's no big deal, okay? Come on, give me a hug.
(Leo hugs Chris.)
[Scene: Richard's house. Paige is there talking on the phone.]
Paige: What do you mean she didn't read the warning label?
Piper's Voice: I'll explain later. The bottom line is we need you home now.
Paige: Well, I can't. I'm kind of in the middle of saving Richard right now. (She looks over at Richard's relatives, dead and alive, sitting in the living room.) I'll be right with you. (to Piper) I took Phoebe's advice, I got his family here.
Piper: I thought most of his relatives are dead.
Paige: Uh-huh, they are.
Piper: You're holding a magical intervention with ghosts?
Paige: Well, I thought about and I realised that one of Richard's problems is that he's got no family here. He's got no support system. So the burden of helping him has kind of fallen on me.
Piper: Okay, fine. Hey, maybe since you have all those ghosts there, you can get one to help us out when you're done.
Paige: Help us do what?
(Richard walks in.)
Richard: What's going on here?
Paige: Uh, I'll call you back.
(Paige hangs up.)
Richard: What are they doing here?
Paige: Your family is here because they care about you. They've seen what happens to you when you use magic and I don't want it to happen again.
Richard: You summoned them?
Steve: We're here because we want to be here. We need you to listen to us.
Richard: This is crazy. This is an intervention, right? I'm getting outta here.
(Richard starts to leave.)
Paige: Richard, wait.
Richard: After everything I've done, this is how you thank me? By embarrassing me in front of my family?
(The spirits disappear.)
Paige: Look, if you keep doing this, something bad is going to happen, something terrible. I can feel it.
Steve: She's right, Richard. Our family, they died because of the magic, I can't let yourself end up like that.
Paige: I can make you a power stripping potion. It'll turn you back into yourself. Just let me help you.
(Richard disappears.)
Steve: What do we do now?
[Scene: Arabia. Cave. Jinny vanquishes one of the thieves.]
Jinny: Anyone else have a problem taking orders from an ex-Genie?
Thieve #1: We are at your service, my queen.
Jinny: Hmm, queen. I like the sound of that. Every queen deserves an empire. Have you found the location of the lost city yet?
Thieve #1: We believe we've discovered the site.
Jinny: Very good. Now all I need is that bottle. Gather your best fighters. We're going on a witch hunt.
[SCENE_BREAK]
[Scene: Manor. Attic. Leo is there writing a letter. Chris walks in.]
Chris: Hey, Piper says they're ready for those crystals. Genie Phoebe's getting on her last nerves down there. What are you doing?
Leo: Writing you an apology. I just, I can't seem to get it right.
Chris: Leo, come on, man, you don't need to do that.
Leo: No, I know I don't need to but I want to. It feels good to forgive.
Chris: Yeah, why don't you just hold onto that feeling, okay? We've gotta go help the sisters.
Leo: Alright, well, in a minute. This is just as important. "Dear Chris..."
Chris: Alright, alright, enough already. You said you're sorry, let's just not go overboard.
Leo: Okay, but after everything I've put you through, I feel like I owe it to you.
Chris: Honestly, a letter's not gonna mean a hell of a lot to me. I got plenty of them growing up.
Leo: I'm sorry.
Chris: Uh, from my father. He wasn't around much.
Leo: That's awful. You wanna talk about it?
Chris: No. What I want for you is to grab the crystals and go downstairs to where Piper is. Do you remember her?
Leo: Sure. But right now I'm a little more concerned about you. You seem a little stressed.
Chris: Yeah, you're damn right I'm stressed. I'm concerned about you two. You two need to get back together already. Any chance that's gonna happen?
Leo: I don't think so. But thanks for caring, man. It means a lot.
Chris: Wait. You still love her, I know you do. How could you just throw that away?
Leo: It's a little personal don't you think?
Chris: More than you know. Look, are you telling me that there is no chance that you and Piper are gonna hook up in let's say, I don't know, the next couple of weeks?
Leo: Actually, yeah, that's what I'm saying. We've both moved on, and nothing short of a miracle can make that happen.
[Cut to the conservatory. Piper and Phoebe are there. Piper is writing a spell. Phoebe looks over her shoulder.]
Phoebe: Uh-uh-uh, the wording's not quite right there. Jinny is an upper-level demon.
Piper: Hey, I don't need a bossy Genie on my back. I'm giving up a lot to be here. I'll write the vanquishing spell the way I want.
Phoebe: You should invoke the name of...
Piper: Do I need to call Chris to shut you up?
Phoebe: You wouldn't.
Piper: Keep pushing me. (Paige orbs in.) Back so soon?
Paige: Yeah, the intervention was a complete train wreck. Richard wouldn't listen to anyone.
Phoebe: Oh, Paige, I'm so sorry. (Paige laughs.) You're laughing at me? I'm trying to be sympathetic and you're laughing at me.
Paige: I'm sorry. Maybe I needed a laugh after what I just went through.
Phoebe: Yeah, it's okay. Is there anything I can do?
Paige: Yeah, but don't you need to go help Major Nelson?
(Piper and Paige laugh. Phoebe isn't impressed.)
Piper: What? It's funny, this is kinda funny.
Phoebe: Let's just finish the spell, okay?
Piper: Alright, hey, I want this done just as badly as you do. Did you find us a ghost?
Paige: Yeah, I got us Richard's dad. He's hanging out in limbo waiting for my call. Why do we need a ghost?
Piper: Well, once we capture Jinny, he can possess her and force her to wish Phoebe free.
(Leo and Chris walk in, chuckling. Leo is holding a box.)
Leo: Here's those crystals you wanted.
(He puts them on the table.)
Paige: You guys sure are chummy.
Leo: Yeah, I had a change of heart. Decided to let bygones be bygones.
Piper: Really?
Phoebe: Guys, there's something I have to tell you.
Chris: Ah, after we talk in the kitchen.
Phoebe: But...
Chris: Phoebe. (He holds up the bottle. Phoebe disappears into it.) Now that was cool. If you'll excuse me, I'm gonna have a little one on one with the help.
(Chris leaves the room.)
Piper: What's he hiding now?
(The doorbell rings.)
Paige: I'll get it.
(Paige leaves the room.)
Leo: You know, Chris is a hell of a guy. You too should give him a chance once in a while.
[Cut to the foyer. Paige opens the door. Richard stands there.]
Richard: Hey.
Paige: Richard. What are you doing here?
Richard: I, uh, came to apologise.
(A bunch of roses appears in his arms.)
[Cut to the kitchen. Phoebe and Chris are there.]
Phoebe: You wanna make them do what?
Chris: We finally got dad in a good mood and mum, she's still sexed up from the fireman. This is the perfect time to hit them with the whammy.
Phoebe: No, we are not going to make Piper and Leo sleep together, okay? We're gonna do this my way, mister.
Chris: Master.
Phoebe: Oh, you know what? Listen to me.
Chris: I'm sorry, Phoebe, but I'm running out of time here. A guy's gotta survive. I wish for Piper and Leo to sleep together tonight.
Phoebe: I'm not...
(Phoebe puts her hands together and blinks. They hear a loud thud come from the other room.)
Chris: What was that? (Phoebe and Chris rush into the conservatory. They see Piper and Leo fast asleep on the floor.) Oh, no. They're sleeping. You tricked me.
Phoebe: No, you made me wish for them to sleep together. And they're sleeping together.
Chris: This is a mess. I've only got one more wish to sort this thing out, so if you don't mind...
(He holds up the bottle.)
Phoebe: Oh, no, actually I do mind because Jinny could be here at any moment.
Chris: You know what? I'll summon you when she does. I command you back into the bottle.
Phoebe: When I get out this...
(She disappears into the bottle.)
[Cut to the foyer.]
Richard: You're gonna break up with me over some flowers?
Paige: Look, I'm sorry. It's me or magic. You just have to choose one.
Richard: You can't give me that kind of choice.
Paige: I just did.
Richard: You wanna talk about dependencies, why are you always running off to be with your sisters?
(They hear a crash in the conservatory.)
Chris: Ow!
Paige: Wait here.
[Cut to the conservatory. Chris is thrown across the room by one of the thieves. He drops the bottle. The thieve pulls out his sword. Paige runs in. Chris stands up.]
Chris: Mind the bottle, Phoebe's inside.
(The thieve charges for Chris and they fight. Paige heads for the bottle and another thieve attacks her. She orbs out and orbs back in behind him.)
Paige: Sword! (His sword orbs into Paige's hand and she stabs him, vanquishing him. Jinny shimmers in and heads for the bottle. Richard walks in and sends her flying across the room. Paige stabs the other thieve with the sword and vanquishes him.) Crystals! (The crystals in the box orbs out.) Circle! (The crystals orb back in making a circle around Jinny.) Got her.
(Jinny tries to walk but the crystals zap her.)
Chris: Where's the bottle?
(They turn around to see Richard holding the bottle.)
Paige: Richard.
(Richard disappears with the bottle.)
[SCENE_BREAK]
[Scene: Manor. Conservatory. Paige and Jinny are there.]
Jinny: You think these crystals can hold me?
(She reaches out and the crystals zap her.)
Paige: Nope, not for long. That's why we're putting you back in your bottle.
Jinny: When I form my empire, the first thing I'm gonna do is rid the world of witches.
Paige: Oh, yeah? Well, when you're back in your bottle, the first thing I'm gonna do is put you in the microwave. Ha ha. How do you like that?
[Cut to the living room. Piper, Leo and Chris are there. Piper and Leo are asleep next to each other on the couch. Chris covers them with a blanket. Paige walks in.]
Paige: Think she can scare me. They're still asleep? Have you tried smelling salts?
Chris: It won't work, trust me.
Paige: What's wrong with them?
Chris: It's a long story.
Paige: I don't know, why don't you give me the cliff notes version. Come on, Chris. You and Leo and Phoebe have all been acting weird since before the demon attacked. What is going on?
Chris: Alright, I made a little wish.
Paige: You did what?
Chris: Two little wishes.
Paige: Oh, great. It's not bad enough I have to worry about Richard, now I have to worry about you too. What did you wish for?
Chris: For Leo to forgive me, which by the way was an accident.
Paige: And?
Chris: For Piper and Leo to sleep together.
Paige: You! Oh my god, you are sick! What is wrong with you? You're disgusting!
Chris: No...
Paige: You are some creepy registered s*x offender from the future.
Chris: No, no, no...
Paige: Oh my god, you are so gross.
Chris: I'm Piper and Leo's son.
Paige: What?
Chris: They're my parents. I came back to save my family.
Paige: You're serious.
Chris: Yeah. Only now I've gotta save myself. Because if my mum doesn't get pregnant in the next month, there is no me.
Paige: This is all so wrong. And this has been such a long day.
Chris: Look, I'm gonna orb over to Richard's, okay, and grab the bottle.
Paige: No, you can't. He's, uh, he's crazy right now, he might hurt you, okay? I need to strip him of his powers. It's a whole thing.
Chris: Well, how's that gonna help?
Paige: Well, he's been corrupted by magic and if I don't strip him of his powers, I might not be able to save him. Who else knows about this?
Chris: About me? Just Phoebe.
Paige: Alright, you watch Jinny, I'm gonna go make this potion, okay?
Chris: Okay.
[Scene: Richard's house. Richard is there. The bottle is sitting on a table. Phoebe is inside the bottle running from side to side, trying to tip the bottle over.]
Phoebe: Come on, come on. (The bottle tips over and Phoebe escapes out of the bottle.) Oh, thank god. I thought a demon got me. Why didn't you let me out?
Richard: I'm not ready for you yet.
Phoebe: Hey, we're in the black magic vault. Um, is there a phone around because I'd really love to call Paige and just check in.
Richard: No, you're not. I know I've got a book of wishes around here somewhere.
(Richard looks through some drawers.)
Phoebe: Wishes?
Richard: Yeah. Gotta get the wording right.
Phoebe: Yeah, maybe you should just wish for sleep because I'm really good at that wish.
Richard: Look, I just want Paige to accept me the way I am with magic. It's the only way we'll work.
Phoebe: I don't think magic is the answer to your problems, I think it's the cause of your problems.
Richard: Got you brainwashed too. I'm gonna have to cast a spell on the entire family. Uh-huh. Here it is. (Paige orbs in.) Paige, I told you. Don't orb in and surprise me.
Phoebe: He's not himself right now.
Paige: How come you didn't tell me Chris was my nephew?
Phoebe: Yeah, maybe we could talk about that later because your boyfriend's about to whoo-whoo!
Richard: Look, I'm fine, alright? I just need to do some reading. I'll call you when I'm ready.
Paige: This is for your own good.
(Paige throws a potion at Richard and he uses telekinesis to send the potion and Paige flying.)
Phoebe: Paige!
[Scene: Manor. Conservatory. Jinny is there with her eyes closed and her hands held out in front of herself. The doorbell rings.]
[Cut to the foyer. Chris answers the door. Greg is standing there.]
Chris: Aren't you supposed to be at work or something?
Greg: I'm on a break. I came to surprise Piper.
Chris: Oh. Little booty call, huh?
Greg: Is she here?
Chris: No, sorry, she's sleeping.
(Chris starts to close the door but Greg stops him.)
Greg: I don't believe you.
Chris: Okay, see for yourself. (Greg walks in and looks in the living room. He sees Piper and Leo asleep on the couch. Chris waves his hand and Piper falls closer to Leo, and Leo's hand falls on her shoulder.) Do you want me to tell her you stopped by?
Greg: Uh, no, that won't be necessary.
(Greg leaves.)
[Cut to the conservatory. The flying carpet creeps under the door and flies over to Jinny. The carpet hits the crystal shield and gets zapped. Jinny steps out of the crystal circle.]
[Cut to the foyer. Chris closes the front door.]
Chris: Sorry, mum, it's for the best.
(Chris walks into the living room. Jinny is there holding a fireball.)
Jinny: Take me to the bottle.
[Cut to Richard's house. Richard helps Paige up.]
Richard: Are you okay? I'm sorry, I didn't mean to.
Paige: It's okay. It's fine.
Richard: I'll make it up to you.
Phoebe: No, no, no, not that way.
Paige: No more wishes.
Richard: No, no, it's okay. It's not for me. Phoebe, I wish you free. (A blue tornado of wind surrounds Phoebe, rises up and then surrounds Richard. It disappears.) Uh, what is this?
(Chris orbs in with Jinny.)
Phoebe: What are you doing here?
(Jinny throws a fireball at them and they duck. Chris attacks Jinny and she grabs his arm and throws him across the room. Jinny picks up the bottle.)
Jinny: Genie, I wish the Charmed Ones dead.
(Richard puts his hands together and nods his head. The girls fall to the floor, dead.)
Chris: No!
(Chris runs to their side.)
Jinny: Now, into the bottle. (Richard is sucked into the bottle.) My condolences.
(Jinny disappears.)
[SCENE_BREAK]
[Scene: Richard's house. Chris is kneeling beside Phoebe and Paige.]
Chris: No. You can't be dead yet. It's not your time. I know it's not your time. (Phoebe and Paige's spirits float out of their body.) I'm sorry, I'm so sorry, this is all my fault. She was gonna kill mum and dad.
[Cut to the manor. Living room. Piper and Leo are still asleep on the couch. Piper's spirit floats out of her body.]
Piper: Oh, no. Leo, wake up. (Leo stirs and a glowing light from his hand heals Piper.) But how...
(Piper's spirit floats back into her body.)
[Cut to Richard's house.]
Phoebe: Hey, Chris, we're not moving on.
Paige: Why aren't we moving on?
Chris: Who cares? You're still here.
Paige: Got any unfinished business?
Phoebe: No. You?
Paige: Yeah, now that you mention it, it would have been nice to find out I had another nephew before I died.
Phoebe: I was gonna tell you but I just didn't get a chance.
Chris: Guys we can fix this. We can reverse the magic. All we need to do is get the bottle.
Phoebe: That's true, we're ghosts, we can possess Jinny. Where is Jinny?
Paige: Well, she was planning to conjure the lost city.
Phoebe: Don't worry, we're gonna take care of this. Should we go check on Piper first?
Chris: No, no, no. Piper's fine. I mean, she has to be, right? If she was dead, I would have vanished.
Paige: How do we reach Jinny?
Chris: You're ghosts, you can haunt anybody you want. You should concentrate and it should wisp you right to her. (Phoebe and Paige close their eyes and they vanish.) Wait for me!
[Cut to Arabia. Jinny and thieves walk over to a two-headed animal skeleton laying in the sand.]
Jinny: You sure this is the site?
Thieve: One of the hounds of the Zanbar. They guarded the city for the Sultan.
Jinny: I could use a few of those myself. (to the bottle) You ready in there? I wish to resurrect the lost city of Zanbar. (A huge Arabian city rises out of the desert sands.) Finders, keepers.
Paige: I wouldn't unpack just now if I were you.
(They turn around to see ghost Phoebe, ghost Paige and Chris standing there.)
Jinny: Why haven't you moved on? You're dead.
Phoebe: So are you.
(Phoebe's spirit jumps into Jinny's body. One of the thieves runs towards Chris and Chris throws him across the room. The other two thieves pull out their swords and runs towards them.)
Paige: Watch out.
Chris: Phoebe, a little help here.
(Phoebe/Jinny throws a fireball at both the thieves, vanquishing them.)
Phoebe/Jinny: I think I've got control of the body.
(Chris grabs a sword and stabs the last thief, vanquishing him.)
Paige: Okay, all clear.
(Phoebe/Jinny picks up the bottle.)
Phoebe/Jinny: Richard, I wish you free.
(Phoebe's spirit exits Jinny's body. Jinny is sucked into the bottle and Richard appears in her place.)
Paige: Hi, honey.
Jinny: Let me out of here!
Richard: I wish the Charmed Ones alive again.
Jinny: Yes, master.
(Phoebe and Paige's spirit vanishes.)
Chris: You okay?
Richard: Take this, get it out of my site.
Chris: Sure. Could you get rid of that thing first?
Richard: Yeah. No problem.
[Scene: Richard's house. Living room. Richard is there holding a potion vial. Paige walks in.]
Paige: Hey. What's going on?
Richard: Just thinking.
Paige: Oh, yeah? What about?
Richard: Everything. I mean, sometimes it feels like my life's just one big disaster after the next, you know? Like right from the start, being born into this stupid feud, all that family hatred. I mean, it's amazing I've gone this far without ending up like one of them.
Paige: Well, Richard, you have. That's what's important, right? You are here for a reason, for a purpose, you just have to figure out what it is.
Richard: How can you have so much faith, Paige. I mean, with everything out there, all the evil.
Paige: Well, that's because I don't see all the evil, I see all the good too. Especially in you.
Richard: Sometimes I'm not sure.
Paige: I am.
Richard: Well, I hope you're right because I'm giving up a lot... to see it too. My whole life I've had powers and magic and even if I didn't use it, it was just apart of me. And now, uh, it's kind of scary to think I'm gonna be losing it, and losing you too.
Paige: Well, you have to take care of yourself first, right? And as long as I'm around and bringing magic into your life, you won't be able to.
Richard: I know.
Paige: Have you taken the power stripping potion yet?
(Richard tips the vial upside down to show it's empty. Tears fall from Paige's eyes.)
[Scene: Manor. Living room. Phoebe and Chris are there. The grandfather clock chimes.]
Chris: They're not waking up. How come they're not waking up?
Phoebe: Would you relax?
Chris: Relax? I'm sorry, did you say relax? Because I'm about to disappear, vanish forever, cease to exist.
Phoebe: Oh my god, you're so dramatic. (Piper and Leo wake up.) Hey, are you okay?
(Piper quickly pulls away from Leo.)
Piper: I think so. Wh-wh-what are you doing?
Leo: Uh, I don't know, I swear. (Leo stands up.) What's going on?
Phoebe: Arabic sleeping potion. Very strong, not good.
Piper: You mean Jinny did this?
Phoebe: Who else?
Piper: Well, we've gotta stop her.
Phoebe: We already did while you were sleeping. She's back in the bottle.
(Chris picks up the bottle.)
Chris: Which we were sort of hoping you could take care of for us.
(He gives Leo the bottle.)
Leo: So that's it? It's all over?
Chris: Pretty much. I mean, you still forgive me, right?
Leo: Of course.
Chris: Good.
Phoebe: I still wanna know why we all didn't die. What? I'm curious.
Chris: Oh, sure, you don't mind them knowing that they almost died but not that... Never mind.
Piper: What are you talking about?
Phoebe: Jinny made a wish for the Charmed Ones to die and we almost did, but then we turned into ghosts and...
Piper: Huh. So that wasn't a dream I had. I was floating over my body looking down at me and then, uh, you healed me.
Leo: I did?
Piper: Yeah. I called to you when you were sleeping and somehow you must have heard me and you wouldn't let me go.
Chris: Well, then that must be the reason why Phoebe and Paige's spirits didn't move on. See the wish was for all the Charmed Ones to die, so saving you must have saved them.
Piper: That was really sweet of you.
Leo: Any time. So you wanna go with me to get Wyatt?
Piper: Sure.
(Leo orbs out with Piper.)
Chris: Wait, wh... What about me?
Phoebe: I wouldn't give up. There may be hope for you yet. | Phoebe frees a genie, Jinny ( Saba Homayoon ), from a bottle, only to find that Jinny is a demon that has tricked her into becoming a genie herself. Anxious to get Leo and Piper back together so he can be conceived, Chris uses Phoebe to make his wish come true, but she takes his instructions too literally. When Chris reveals to Paige that he made the wish to make Piper and Leo sleep together, she calls him a pervert, and he reveals to her that Piper and Leo are his parents and that Piper needs to get pregnant within a few weeks or he will perish. Feeling that he is losing Paige, Richard opportunistically covets the genie's magic and steals the bottle so he can wish Paige back into his life. Paige and Richard reconciled to permanent separation, but Richard must drink a power-stripping potion for his own good. | summ_screen_fd |
Identification of functional genetic variation associated with increased susceptibility to complex diseases can elucidate genes and underlying biochemical mechanisms linked to disease onset and progression. For genes linked to genetic diseases, most identified causal mutations alter an encoded protein sequence. Technological advances for measuring RNA abundance suggest that a significant number of undiscovered causal mutations may alter the regulation of gene transcription. However, it remains a challenge to separate causal genetic variations from linked neutral variations. Here we present an in silico driven approach to identify possible genetic variation in regulatory sequences. The approach combines phylogenetic footprinting and transcription factor binding site prediction to identify variation in candidate cis-regulatory elements. The bioinformatics approach has been tested on a set of SNPs that are reported to have a regulatory function, as well as background SNPs. In the absence of additional information about an analyzed gene, the poor specificity of binding site prediction is prohibitive to its application. However, when additional data is available that can give guidance on which transcription factor is involved in the regulation of the gene, the in silico binding site prediction improves the selection of candidate regulatory polymorphisms for further analyses. The bioinformatics software generated for the analysis has been implemented as a Web-based application system entitled RAVEN (regulatory analysis of variation in enhancers). The RAVEN system is available at http: //www. cisreg. ca for all researchers interested in the detection and characterization of regulatory sequence variation. Medical genetics research has produced a continuous stream of successes in which functional defects in genes have been linked to disease phenotypes. Such advances can lead to improved diagnostics, therapies, and therapeutics. While most discovered genetic defects create missense or nonsense substitutions in protein-coding sequences, there remain disease-associated genes for which there is no difference in protein-coding information between individuals of different phenotypes. Examples of the latter include CD36 type II deficiency [1], phenotypic variances in plasma Dopamine-beta-hydroxylase [2], and serum angiotensin I converting enzyme levels [3]. Since there is no change in the encoded proteins, some of these genes may be aberrantly expressed as a result of genetic changes in key regulatory sequences affecting the binding of transcription factors and thereby the rate of transcription. There are few confirmed examples of regulatory genetic variants with a pronounced impact on disease. A common TA-repeat polymorphism in the cis-regulatory TATA box of the UGT1A1 gene causes Gilbert syndrome in homozygous humans [4]. The variation has pharmacokinetic consequences: the reduced expression of the protein leads to altered glucuronidation of several drugs. Another example is a noncoding sequence variant of the α-globin gene cluster, which has been shown to generate a new transcriptional promoter by creating a novel functional binding site for GATA-1 [5]. The mutation was detected in Melanesian patients with a variant type of the inherited blood disorder α-thalassemia, in which patients have reduced expression of α-globin genes and anemia. Other examples of regulatory genetic variations include two mutations in HNF-1 binding sites in the promoter of the alpha fetoprotein, causing hereditary persistence of the protein which is otherwise only expressed in the fetus [6]. Usually, however, the effect of genetic variation on gene transcription is less pronounced, or merely associated with increased disease risk [7–11]. Several groups have compiled lists of regulatory sequence variations associated with allele-specific expression patterns [12–14]. Large-scale genomics studies have demonstrated that allele-specific differences in gene expression are common (reviewed in [15]), and that a portion of the differences can be attributed to genetic variation in noncoding regulatory regions [16–18]. The high frequency of observed allelic expression differences in mice [19] has been supported by similar rates observed with human samples [20,21]. Genome-wide mappings of expression levels in model organisms have revealed significantly higher fractions of polymorphisms close to genes for which altered mRNA expression levels between individuals have been linked to the locus of the same gene (self-linkage) compared to genes with no self-linkage [22,23]. While numerous studies have identified genes with allelic differences in regulation, it is still a challenge to separate causal genetic variations from linked neutral variations. Computational methods to distinguish functional variations from neutral variations could prove useful to direct limited laboratory resources to sites most likely to exhibit a phenotypic effect. The identification of potential transcription factor binding sites (TFBSs) in DNA is fundamental to regulatory analysis. A traditional bioinformatics approach to predict TFBSs is through the application of binding site profile models known as position-specific weight matrices (PWMs) [24]. Such matrix models assign a score to each candidate binding sequence. In many cases, the models accurately predict in vitro binding properties of the corresponding transcription factors [25]. However, the low specificity of these models precludes their use for the detection of in vivo biologically relevant sites in the absence of additional information [26]. One type of additional information that is readily available is the evolutionary conservation of functional noncoding genomic sequence. This is the underlying principle behind phylogenetic footprinting, which can be used to eliminate regions less likely to contain cis-acting regulatory sites and thereby increase the specificity of predictions generated with position-specific weight matrices [27–29]. Restricting the search for genetic variation to predicted TFBSs in conserved regions can be expected to increase the likelihood of identifying functional variants. Such predictions are interesting if the putative TFBS is consistent with the biology of the associated disease. Here we present an in silico driven approach to selecting genetic variation likely to influence gene regulation. We combine phylogenetic footprinting with detection of effects of genetic variation on putative TFBSs to identify variation with the potential to alter gene regulation. We test our in silico approach on genes with documented regulatory genetic variation and compare the results to a large set of background SNPs, to evaluate the enrichment of regulatory SNPs by our selection method. We introduce RAVEN (regulatory analysis of variation in enhancers), a Web interface to our application, that enables the scientific community to apply our approach to their genes of interest. Stormo and colleagues have shown that PWM representations of TFBSs gives scores that are proportional to the binding energy between the DNA sequence and the protein [30] (see Figure S1A and S1B for a demonstration of two examples where this holds true). This implies that matrix models of TFBSs can be used to estimate the effect on the transcription factor binding affinity of genetic variation in a regulatory region. Our in silico prediction tool identifies polymorphisms for which the assigned score to a TFBS model differs between the two allelic sequences. To define the expected ranges of allelic differences in TFBS scores for various types of mutations, we generated panels of simulated binding sites based on the distribution of bases at each position of the TFBS frequency matrices in the JASPAR database [31], representing wild-type TFBS sequences. We then inserted mutations into the generated sequences to produce a collection of synthetic regulatory sequence variants. Various types of mutations, including 1–5 bp substitutions, 1 bp insertions, and 1 bp deletions were introduced into the generated sequences; see Methods for a detailed description of the mutation classes. For every generated sequence, we computed a TFBS score delta by comparing the TFBS score for the wild-type allele (the sequence generated from the frequency matrix of the TFBS) with the one obtained for the mutated allele. For an explanation of the scoring system and its scale, see [24]. The box plot in Figure 1 shows that the majority of the simple 1 bp substitutions gives score deltas below four, and mutation of additional bases gives higher score deltas. Insertion and deletion polymorphisms behave similarly to each other, with approximately the same median value of the score deltas as for the 1 bp substitutions, but with a higher value of the third quartile and more outliers. To assess the applicability of the approach to real sequences, we tested the PWM models of TFBSs on instances of genetic variation shown experimentally to affect the regulation of their corresponding genes. We compiled a list of 104 examples of experimentally verified regulatory 1 bp substitution polymorphisms. For comparison, we also compiled a list of 4,000 background 1 bp substitutions from dbSNP with a minor allele frequency exceeding 0. 05. We tested all SNPs for overlap with the TFBS models in the JASPAR database, and recorded the score deltas that fulfilled the criteria described in Methods. We then calculated the fractions of SNPs that were retained for various TFBS score delta thresholds. Figure 2 shows the fractions of retained regulatory and background SNPs for the score delta thresholds of 1 unit increments between one and nine. From Figure 2, it is apparent that the difference in the distributions of score deltas between the regulatory SNPs (rSNPs) and the background data is very small (median score deltas for the SNPs that overlap with a TFBS according to the search criteria are 4. 8 and 4. 9 for the regulatory and background SNPs respectively). When we restricted the search to TFBS models with a minimum specificity of ten bits (72 out of the 123 models in JASPAR fulfilled this more selective criterion), the fraction of retained SNPs decreased to approximately 75% for low TFBS score deltas. However, there was still no significant enrichment of rSNPs (unpublished data). These results indicate that the application of PWM models of TFBSs alone does not enrich for rSNPs. We tested the application of phylogenetic footprinting to assess the method' s capacity to enrich for bona fide rSNPs. The underlying principle behind phylogenetic footprinting is that functional noncoding elements are more likely to be evolutionarily conserved than nonfunctional surrounding sequence. Restricting the search for regulatory genetic variation to conserved regions is thus likely to increase the enrichment of functional sites. We therefore tested how often the upstream fraction of our experimentally verified regulatory polymorphisms was located within conserved genomic regions. Conservation of the rSNP positions was quantified using the phastCons scores [32] available at the UCSC genome browser from alignments between the May 2004 release of the human genome and chimp, mouse, rat, dog, chicken, fugu, and zebrafish. We performed similar testing for 26,044 background SNPs from dbSNP that are located within 10 kb upstream of human genes with known mouse orthologs. Figure 3 shows that the SNPs with documented effect on gene regulation are more frequently located within evolutionarily conserved sequences relative to background SNPs. For example, when using a phastCons score threshold of 0. 4 to define conserved regions, approximately 28% of the rSNPs were retained compared to only 9% of the background SNPs. Significant differences in the frequency of SNPs that fall within conserved regions for the rSNP dataset and the background set were observed for all phastCons score thresholds above 0. 1 (Figure 3). Genomic regions close to the transcription start sites (TSS) of genes are generally more conserved between species than genomic regions far away from genes. Since research on regulatory genetic variation traditionally has been focused on the proximal promoter regions, and since the background SNPs were relatively evenly distributed in the region from 10 kb upstream of the TSS to the TSS, there was an overrepresentation of SNPs within the proximal promoter in the set of rSNPs compared with the background SNPs. To evaluate if this bias affected the enrichment of rSNPs in conserved regions relative the background, we compared the distributions of phastCons scores in the two datasets for SNPs located at different distances from the TSS. We binned all SNPs located from 10 kb to 2 kb upstream of the TSS, all SNPs located from 2 kb to 500 bases upstream of the TSS, all SNPs located from 500 bases upstream of the TSS to the TSS, and all SNPs from 100 bases upstream of the TSS to the TSS. The four bins contained 8,20,76, and 23 rSNPs, respectively. Due to the relatively low number of available rSNPs, separation of SNPs into smaller intervals was unpractical. Figure 4 shows that for SNPs located from 10 kb to 2 kb upstream, as well as 2 kb to 500 bases upstream of the TSS of the respective genes, there was no significant difference between the phastCons scores for the regulatory and background SNPs. However, for SNPs in the interval from 500 bp upstream to the TSS as well as for the full dataset, the phastCons score values were significantly higher for the regulatory than for the background SNPs (p-values 0. 001 and 0. 0002, respectively). Also in the interval closest to the TSS the phastCons score, values were higher for the regulatory than for the background SNPs, but the difference was not statistically significant. In the intervals closest to the TSS, the bias in location closer to the TSS for rSNPs is small or eliminated (in the interval from −500 bp to the TSS the median distances to the TSS were 168 bp and 237 bp for the regulatory and background SNPs respectively, and in the interval from 100 bp upstream to the TSS the median distance for both datasets was 51 bases). This suggests that the higher fraction of rSNPs in conserved regions relative to the background is not simply an effect of the rSNPs being located closer to the TSS than the background SNPs. Although the TFBS analysis alone did not provide enrichment of rSNPs relative to the background, we tested if the intersection of TFBS analysis and phylogenetic footprinting could increase the enrichment given by phylogenetic footprinting alone. We counted the number of regulatory and background SNPs that both affected predicted TFBSs and were located in conserved regions (predicted rSNP), for phastCons score thresholds 0. 1 to 0. 9 and for TFBS score delta thresholds between one and nine. We also counted the number of predicted rSNPs based on phylogenetic footprinting alone. Figure 5 shows the sensitivity (fraction of predicted rSNPs) versus 1—specificity (fraction of nonpredicted background SNPs) for the different thresholds, where the curves correspond to different TFBS score delta thresholds. When the relative TFBS score threshold for the best matching allele was set to 80% (the left panel), there was virtually no difference in performance between phylogenetic footprinting alone and when TFBS score delta thresholds of less than five was applied, and for larger score delta thresholds the sensitivity was very low. Also when the relative TFBS score threshold for the best matching allele is increased to 90% (the right panel), the application of TFBS analysis provides no enrichment compared with phylogenetic footprinting alone. Variations in particularly high scoring TFBS fail to disrupt sites, thus rSNP predictions for higher scoring TFBS candidates (Figure 5, right panel) are less predictive than those for predicted sites of lower initial scores (Figure 5, left panel). Sometimes additional knowledge is available for the gene in which regulatory polymorphisms are searched, for example a researcher may know a set of transcription factors likely to be involved in the regulation of the gene in the context of the studied disease. When this type of information is available, it is possible to reduce the number of false positive TFBS predictions, since predictions of SNPs affecting the “wrong” transcription factor can be eliminated. We analyzed a dataset of genes in which known TFBSs have been disrupted either by naturally occurring SNPs or by mutagenesis experiments. In this set, the transcription factors involved in the regulation of the corresponding genes had been determined experimentally. We searched a region from −3,000 bp to +500 bp relative to the TSS of every gene in the dataset for potential rSNPs affecting a binding site for the transcription factor associated with the gene. Table 1 shows that 12 of the 20 rSNPs with “prior knowledge” overlapped and affected a predicted binding site for the verified transcription factor, and in eight of these cases the rSNPs were located in genomic regions with a phastCons score above 0. 4. Consistent with previous observations about the unusual predictive properties of an Sp1 binding profile [27], in two cases the Sp1 profile predicted decreased binding affinity toward a mutation in contrast to the published observation of increased affinity. The analyzed regions (3,000 bp upstream to 500 bp downstream of the TSS of the analyzed genes) contained between four and 43 SNPs in addition to the analyzed rSNP. When the regions were analyzed with all PWMs in JASPAR with information content higher than 10 bits, between two and 32 (mean value 13) SNPs were predicted to affect a TFBS, and between zero and 11 of these were also located in conserved regions (mean value three). When the regions were analyzed with the PWM corresponding to the verified TFBS, the number of SNPs affecting predicted TFBS (in addition to the rSNP) was between zero and eight, with mean value one. Between zero and two of these were also located in conserved regions (mean value 0. 3). This analysis indicates that when information regarding candidate transcription factors involved in the regulation of a gene is available, specificity is increased. We also compared the score deltas obtained from analyzing the regulatory polymorphisms with known affected TFBS to the score deltas obtained when analyzing the larger data set used in Figure 2. When analyzing the regulatory polymorphisms with prior knowledge with all PWMs from the JASPAR database, the results were similar to those obtained for the larger set and the background. However, when the regulatory polymorphisms were analyzed with the PWMs for the respective verified TFBSs, the score delta was higher (Figure 6). In the analyses using all PWMs, we selected the mean score delta over all matches between the analyzed PWMs and the SNP, whereas in the analysis with the PWM of the verified transcription factor we used the PWM match giving the largest score delta for that particular PWM. This may have caused the lack of overlap between the interquartile ranges of score deltas in the “all” and “prior knowledge” analyses in Figure 6. To facilitate efficient analyses of the type presented in this paper, computational methods and newly implemented algorithms were developed as an integrated framework for rSNP analysis. The framework includes all the components for the location and extraction of data from genome and SNP databases, pattern detection, phylogenetic footprinting, and SNP effect estimation. To facilitate user access, we developed a flexible Web application that enables researchers to easily detect potential regulatory gene variation in their gene of interest (http: //www. cisreg. ca). The organizational schema of the Web application is shown in Figure 7. The progression through analysis in RAVEN is event-driven, i. e., designed for a number of different application scenarios using different subcomponents of the system, and in different order. The components are: We have presented a computational method and a flexible, user-oriented Internet based tool for prediction of genetic variation with a potential to affect the regulation of the corresponding genes. In the absence of information about which transcription factor regulates a gene, our sequenced-based in silico analysis does not provide enough selectivity between polymorphisms with a documented impact on transcriptional regulation and background SNPs. However, if such prior information is available, we have demonstrated that our Web-based tool can aid experimental researchers in prioritizing which SNPs are most likely to affect the biological function of genomic regulatory elements. Synthetic SNP set. Panels of simulated binding sites for transcription factors were generated from the distributions of bases at each position of all the TFBS frequency matrices available in the JASPAR database (123 matrices) [31]. A random base was inserted before and after the generated motif so that the sequences were n+2 bp long, where n is the length of the TFBS model. The insertion of extra bases before and after the motif enables the TFBS to shift one base in the mutated sequence if that would give a better match between the TFBS model and the sequence. Mutations of different types were introduced into the synthesized sequences: 1 bp substitutions, 2 bp substitutions at adjacent positions, two randomly placed 1 bp substitutions, 3 bp substitutions both in adjacent and at random positions, four randomly placed base pair substitutions, five randomly placed substitutions, one randomly placed 1 bp insertion, and one randomly placed 1 bp deletion. In case of insertion mutations, the random base after the site was removed so that the alleles are of same length and also of the same length as the other sequences of the corresponding site, i. e., n+2 bp. In case of deletion mutation, an extra random base was introduced in the end of the deleted allele so that both alleles were of length n+2 bp. Ten synthetic TFBS sequences were generated from each TFBS model (123 models), and ten mutants of each type were created for each generated wild-type sequence. This makes a total of 123 × 10 × 9 × 10 sequences = 98,400 sequences. Regulatory SNP set. We searched the literature for variations with a documented regulatory role. All single base variations will be referred to as SNPs, although the frequency of the variations may be rare or unknown. The criteria for selecting SNPs to this set was that they should show allele-specific binding to nuclear extracts or transcription factors in vitro using electrophoretic mobility shift assays, and that the SNPs should show evidence of allele-specific transcription rates in reporter gene assays. We also used SNPs from previous collections of regulatory polymorphisms in human: 22 SNPs were taken from a collection created by Rockman and Wray [12], 17 were taken from the collection created by Buckland et al. [14], and 39 SNPs were extracted from the ORegAnno data base [13]. We did not require that the transcription factor affected by a particular SNP should be defined. rSNPs that were selected for in vitro studies based on the fact that they were located in evolutionarily conserved regions would have resulted in a bias in the phylogenetic footprinting analysis, and we have therefore not included such examples in our dataset. We limited the set to SNPs located in the regions from 10 kb upstream to the transcription start sites of human genes with available human–mouse orthologs (ortholog mapping was performed as in [45]). In total, 104 one-base substitution polymorphisms were collected. PubMed accession numbers and other details about the rSNP data set are shown in Dataset S1. Regulatory SNPs with known affected transcription factor binding site. To generate a collection of regulatory polymorphisms for which the involved transcription factor has been experimentally determined, we mined the literature for examples of human TFBSs containing regulatory variation, either naturally occurring or generated in mutagenesis experiments. We applied the same inclusion criteria as for the rSNP set above, but only polymorphisms affecting binding sites for transcription factors for which there are PWMs available in JASPAR were included. The sequences for the regulatory variations with known affected TFBSs are provided in Dataset S2. Background SNP sets. For background SNPs we downloaded from dbSNP those SNPs located in the upstream regions of human genes with a known mouse ortholog. We included SNPs in the regions stretching from −10 kb to the TSS (ortholog mappings and definitions of TSSs were determined as in [45]), unless a region is truncated by upstream genes in which case a smaller region was used. Only validated SNPs with minor allele frequency greater than 0. 05 (in all populations) were included in the set. The dbSNP125 version of the database was used, in which SNP positions are calculated on the NCBI35 version of the human genome assembly. The total background SNP dataset used for the phylogenetic footprinting analysis contains 26,044 SNPs. For the analysis of overlap between SNPs and TFBSs, we used a subset of the total background dataset (4,000 SNPs), in which SNPs were selected randomly from the total background set. The background SNP datasets are available in Dataset S3 (26,044 SNPs) and Dataset S4 (4,000 SNPs). Defining the background SNPs as nonfunctional is a necessary simplification. As the background SNPs are taken from dbSNP without any functional analysis prior to inclusion, it is likely that the background dataset contains SNPs that have a regulatory function and hence the false positive predictions on the background set might include some true positive predictions. However, given the absence of a dataset with experimentally verified neutral SNPs in proximal promoter regions and the reasonable expectation that any true positives would be rare, the background SNP set is the only suitable dataset to which the rSNPs can be compared. Saturation mutagenesis data used in Figure S1. Data was obtained from a saturation mutagenesis experiment of the heat shock element of the human HSP70. 1 gene promoter [46]. The authors systematically mutated every base in the motif to all other base variants. A total of 55 sequences were tested covering 18 bases (i. e., 54 mutations plus wild-type). For every sequence, the relative free energy of the DNA–protein interaction (relative to the wild-type sequence) was measured by gel shift densitometry. From this analysis we compiled a list of sequence variants with corresponding relative free energies. We also used data from a saturation mutagenesis experiment in the Mnt repressor of the Salmonella phage P22 [30]. The authors mutated nine positions in the Mnt repressor and measured the relative free energy for every mutant sequence (relative to the wild-type sequence). From their analysis we again compiled a list of sequence variants with corresponding relative free binding energies. In the study of the Mnt repressor, the authors also presented a frequency matrix for the Mnt repressor that we used for scoring the sequences. The sequences used in the analysis of saturation mutagenesis experiments are shown in Dataset S5. Analysis of synthetic mutated TFBS sequences. The mutated and wild-type versions of the synthetic TFBS sequences were scored with the PWM used to generate a particular wild-type sequence, using perl scripts based on the TFBS framework [47]. The best possible match between the TFBS model (the model that was used to generate the wild-type sequence) and the DNA was collected for each sequence. A score delta was then calculated as the absolute value of the difference between the score delta for the wild-type sequence and the mutated sequence. To collect the score delta for a wild-type and mutated sequence pair we required that the relative score for the best matching allele should be at least 80% of the maximum score for the respective TFBS [24]. TFBS analysis of documented regulatory SNPs and the background SNPs. PWMs corresponding to all TFBSs in the JASPAR data base were used to score both alleles of the SNPs in the background and rSNP datasets. Scoring was performed as above, but with two modifications. (1) We required a predicted binding site to be located at the same position in the sequence for both alleles of a SNP. This is the same methodology as is used in the Web interface to our application. (2) If several matches were obtained between a SNP and a particular TFBS model (fulfilling the requirement that the highest-scoring allele should give a score of at least 80% of the of the maximum score for the respective TFBS), we collected the hit that gave the largest score delta between the sequence variants since we wanted to calculate the fraction of retained SNPs for different TFBS score deltas. Sometimes a SNP overlapped potential binding sites for more than one TFBS model. To calculate the fraction of retained SNPs for various TFBS score delta thresholds we collected, for each SNP, the maximum score delta over all TFBS models (rather than the score delta for the TFBS model that gave the largest absolute score for the best matching allele). We measured the fraction of retained SNPs in respective datasets for the absolute TFBS score delta thresholds in one-unit increments from one to nine. Phylogenetic footprinting analysis of regulatory SNPs and the background SNPs. We used the phastCons scores from multiple alignments of eight vertebrate genomes available in the UCSC genome browser (the May 2004 release) to perform phylogenetic footprinting. SNPs were assigned the mean phastCons score in a window of 21 bases (ten bases upstream and downstream of the SNP). Several other window sizes were tested, from one to 41 bp, but the 21 bp window gave the best enrichment of rSNPs. Analysis of regulatory SNPs with known affected TFBS. The examples of regulatory variation from the literature for which the affected TFBS are known were analyzed using the Web interface to our application (RAVEN, see description below). The following parameters were used for the analysis: the phastCons threshold for sequence conservation between species was 0. 4. The threshold for a match between the top-scoring allele of a variation and a TFBS model was 80%. The absolute score difference between the two alleles of a variation was required to be greater than 1. 5 [24]. The TFBS analysis of the different SNP datasets shown in Figure 6 were performed as above, but with the exception that the average score delta from all PWMs that fulfilled the search criteria for a particular SNP was collected instead of collecting only the PWM that gave the largest score delta. Correlation analysis of relative free energy of mutated binding sites versus TFBS score delta for Figure S1. The mutated and wild-type sequences in the saturation mutagenesis experiments were scored with the PWMs corresponding to position frequency matrices for the respective transcription factors [24]. A score delta was computed for every sequence variant by subtracting the absolute score for the wild-type sequence from the score for the mutated sequence. For each sequence the optimal match between the TFBS model and the analyzed sequence was used, with the requirement that the motif should overlap the mutated base. The score deltas for all the sequence variants were then correlated to the experimental relative free binding energies. The position frequency matrix for the heat shock element was taken from Transfac [42]. The matrix has Transfac accession number M00146 and is defined by: For the analysis of mutated versions of the Salmonella phage P22 Mnt repressor, we used a published position frequency matrix [30]. The matrix is defined by: The frequency matrices were transformed into weight matrixes using the TFBS bioinformatics programming framework [47]. Functions from the same framework were also used for searching sequences for putative binding sites. Analyses of the correlation of TFBS score deltas and relative free binding energy for the sequence variants were performed in R [48]. The application is implemented in object-oriented Perl (Conway, 1999) with extensive use of Bioperl [49] and TFBS [47] bioinformatics programming frameworks. The system consists of several database components, an application engine layer, and a Web interface layer. It is deployed on a dual-CPU Linux/i686 server. Data. The dbSNP [34] sequence data was downloaded from NCBI ftp site (ftp: //ftp. ncbi. nlm. nih. gov/). Genomic sequence data with the positions of repeat sequences, mappings of cDNAs and dbSNP SNPs to the genome, as well as phastCons scores, were obtained from the UCSC Genome Informatics repository (http: //genome. ucsc. edu/) [50]. HGVbase (http: //www. hgvbase. org/) [35] data was obtained directly from the master database at Karolinska Institutet. | DNA sequence variations (polymorphisms) that affect the expression levels of genes play important roles in the pathogenesis of many complex diseases. Compared with genetic variations that alter the amino acid sequences of encoded proteins, which are relatively easy to identify, sequence variants that affect the regulation of genes are difficult to pinpoint among the large amount of nonfunctional polymorphisms located in the vicinity of genes. Computational methods to distinguish functional from neutral variations could therefore prove useful to direct limited laboratory resources to sites most likely to exhibit a phenotypic effect. In this paper we present a Web-based tool for the identification of genetic variation in potential transcription factor binding sites. This tool can be used by any scientist interested in the characterization of regulatory polymorphisms. Using experimentally verified regulatory polymorphisms and background data collected from the literature, we evaluate the method' s capacity to identify regulatory genetic variation, and we discuss the limitations of its application. | lay_plos |
Regulation of rod gene expression has emerged as a potential therapeutic strategy to treat retinal degenerative diseases like retinitis pigmentosa (RP). We previously reported on a small molecule modulator of the rod transcription factor Nr2e3, Photoregulin1 (PR1), that regulates the expression of photoreceptor-specific genes. Although PR1 slows the progression of retinal degeneration in models of RP in vitro, in vivo analyses were not possible with PR1. We now report a structurally unrelated compound, Photoregulin3 (PR3) that also inhibits rod photoreceptor gene expression, potentially though Nr2e3 modulation. To determine the effectiveness of PR3 as a potential therapy for RP, we treated RhoP23H mice with PR3 and assessed retinal structure and function. PR3-treated RhoP23H mice showed significant structural and functional photoreceptor rescue compared with vehicle-treated littermate control mice. These results provide further support that pharmacological modulation of rod gene expression provides a potential strategy for the treatment of RP. Retinitis pigmentosa (RP) is an inherited retinal degenerative disease with a prevalence of 1 to 3,000-5,000 births (Hartong et al., 2006; Parmeggiani, 2011; Boughman et al., 1980). More than 3,000 mutations in about 60 genes have been identified to be associated with RP (Hartong et al., 2006; Daiger et al., 2013). Most of these mutations are in genes essential for rod photoreceptor development and function (Hartong et al., 2006). There is currently no approved medical therapy that slows or prevents rod degeneration in these individuals. One emerging approach to treating retinal degeneration is through targeting the factors that regulate rod gene expression. Studies of retinal development have identified several transcription factors that regulate photoreceptor gene expression. For example, loss of function mutations in the rod-specific transcription factors Nrl or Nr2e3 cause rods to acquire a more cone-like identity (Mears et al., 2001; Montana et al., 2013; Haider et al., 2006; Haider et al., 2000; Cheng et al., 2011; Cheng et al., 2004; Corbo and Cepko, 2005; Peng et al., 2005; Chen et al., 2005; Zhu et al., 2017; Yu et al., 2017). Knockout/down strategies have shown that Nrl and Nr2e3 are necessary even in mature rods to maintain their normal levels of gene expression (Montana et al., 2013; Zhu et al., 2017; Yu et al., 2017). Moreover, the reductions in rod gene expression from deletion of Nrl or Nr2e3, with either conditional deletion or CRISPR-Cas9 deletion, were sufficient to promote the survival of photoreceptors in multiple models of recessive and dominant RP (Montana et al., 2013; Zhu et al., 2017; Yu et al., 2017). We have recently reported that this regulatory pathway can be modulated using small molecule modulators of rod gene expression that we have named Photoregulins (Nakamura et al., 2016). Treatment of developing or mature retina with Photoregulin1 (PR1) reduces rod gene expression and increases the expression of some cone genes. In addition, treatment of two mouse models of RP (mice with the RhoP23H and the Pde6bRd1 mutations) with PR1 slows rod degeneration in vitro (Nakamura et al., 2016). However, in vivo analyses of PR1 were limited by the compound' s potency, solubility, and stability in vivo. In this study, we have identified a structurally unrelated compound, Photoregulin3 (PR3) that also significantly represses rod gene expression, but is more amenable for in vivo studies. With PR3 treatment, we show anatomical and functional preservation of the retina in RhoP23H mice, providing in vivo proof-of-concept of this novel therapeutic strategy for the treatment of RP. In order to identify compounds that may target Nr2e3, we searched PubChem (https: //pubchem. ncbi. nlm. nih. gov/) for top-scoring hit compounds previously identified to interact with Nr2e3 in transfected CHO-S cells in a luciferase-based assay (PubChem Assay IDs: 602229,624378,624394, and 651849). As a secondary screen for the initial hits we used primary retinal cell cultures and assayed Rhodopsin expression because it is a well-defined target of Nr2e3 signaling and is expressed at high levels exclusively in rod photoreceptors (Cheng et al., 2004; Peng et al., 2005; Haider et al., 2009). We dissociated retina from postnatal day 5 (P5) mice and cultured them in media containing the small molecules. After treatment for 2 days, we assessed Rhodopsin expression with an immunofluorescence-based assay (Nakamura et al., 2016). One compound, PR3 (Figure 1A), showed robust reduction in Rhodopsin compared to DMSO and PR1 treatment (Figure 1B). We confirmed this finding with qPCR analysis using intact retinal explant cultures from P4 mice treated with a 0. 3 μM dose of PR1 or PR3. Similar to the immunofluorescence assay, treatment with PR3 resulted in reduced Rhodopsin but not Otx2, a rod transcription factor upstream of Nr2e3, compared to DMSO and PR1 (Figure 1C). Mutations in Nr2e3 result in an increased number of S Opsin+ photoreceptors as well as a reduction in rod gene expression (Haider et al., 2006; Haider et al., 2000; Cheng et al., 2011; Peng et al., 2005; Chen et al., 2005; Cheng et al., 2006). To determine if PR3 treatment also affects cone gene expression, we explanted intact retinas from P11 wild type mice in media containing DMSO or PR3 for 3 days. We used intact retinas for this experiment to assess changes in dorsal and ventral retina independently. After fixation and whole mount immunostaining, we counted S Opsin+ cells in the dorsal and ventral retina. Similar to Nr2e3 mutations, treatment with PR3 resulted in an increase in the number of S Opsin+ cells in the ventral, but not dorsal retina (Figure 1D–E). PR3 was initially identified as a chemical modulator of Nr2e3 in a luciferase-based assay that identified ligands by disruption of the Nr2e3-NCoR dimer complex and had a calculated IC50 of 0. 07 μM in this assay (PubChem Assay IDs: 602229,624378,624394, and 651849). To confirm a direct Nr2e3-PR3 interaction, we used isothermal titration calorimetry (ITC). Consistent with our other assays, ITC qualitatively showed a direct interaction between PR3 and Nr2e3 (Figure 1F; estimated Kd of 67 μM using a one site model). The initial screen that identified PR3 demonstrated its effects on Nr2e3 in a co-repression assay with NCoR; however, the effects we observed on rod gene expression suggested that PR3 also inhibits the co-activator function of Nr2e3. To explore this possibility, we assessed the effects of PR3 on the ability of Nr2e3 to cooperate with Nrl and Crx, two other transcription factors known to form a co-activation complex for rod gene expression. We first transfected HEK cells with Nr2e3, Nrl, and Crx and measured activation of the Rhodopsin promoter in the presence of DMSO or PR3. PR3 strongly reduced Rhodopsin promoter activation (Figure 1—figure supplement 1A). We next used co-immunoprecipitation to assess whether PR3 affects the interaction between Nr2e3 and Nrl in HEK 293 cells. We found that PR3 caused an increase in the binding of Nr2e3 to Nrl (Figure 1—figure supplement 1B–C). Several reports have shown that ligand binding to nuclear receptors stabilizes interactions with co-activators by stabilizing the structure of the ligand-binding domain and co-activator binding sites (Onishi et al., 2010; Jia et al., 2009; Forrest and Swaroop, 2012; Fu et al., 2014). It is not clear why stabilizing the complex would reduce its activity; however, it may be that stabilizing interactions with Nr2e3 prevents Nrl and Crx from interacting with consensus sites on the DNA, or alternatively prevents the recruitment of other components of the transcriptional machinery. Nevertheless, our results indicate that PR3 affects the formation or stabilization of the complex among these critical rod gene regulators. Although PR3 was identified in an assay for Nr2e3, it is also possible that it interacts with other nuclear receptors. Two related nuclear receptors that are expressed in photoreceptors are Errb and Rorb. Deletion of these genes in mice has effects on rod gene expression (Onishi et al., 2010; Jia et al., 2009; Forrest and Swaroop, 2012; Fu et al., 2014). However, small molecule modulators of ERRb induce rapid rod death (Onishi et al., 2010), and so it is unlikely that PR3 acts through this pathway. Loss of function Rorb mutations reduce rod gene expression and lead to a phenotype more like what we observe with PR3 treatment. To determine whether PR3 might also act through antagonism of RORb we transfected HEK 293 cells with RORb and Crx and measured activation of the S Opsin promoter, a known target of RORb. In transfected HEK 293 cells, RORb and Crx synergistically activate the S Opsin promoter (Srinivas et al., 2006; Liu et al., 2017). Treatment with PR3 did have a small effect on RORb-Crx driven activation of the S Opsin promoter (Figure 1—figure supplement 1D); however, this effect was much smaller than what we found for PR3 on activation of the Rhodopsin promoter by Nr2e3, Nrl, and Crx (Figure 1—figure supplement 1A). To further evaluate a role for PR3 as an RORb antagonist, we analyzed downstream RORb target gene expression in developing retina. One target that is specific to RORb, but not regulated by Nr2e3 is Prdm1 (Liu et al., 2017; Wang et al., 2014; Mills et al., 2017). Prdm1 expression was unchanged in P0 retinal explants treated with PR3 (Figure 1—figure supplement 1E), supporting our hypothesis that the effects of PR3 are primarily mediated through modulation of Nr2e3. To further evaluate whether PR3 has other targets in the retina, we tested PR3 in Rd7 retinas, which harbor a spontaneous mutation in Nr2e3 (Akhmedov et al., 2000; Haider et al., 2001; Chen et al., 2006). We explanted adult (P23-35) retinas from wild type and Rd7 mice in media containing DMSO or PR3 and measured Rhodopsin expression by qPCR. Consistent with the hypothesis that the effects of PR3 on rod gene expression are mediated through Nr2e3, PR3 caused a significant reduction in Rhodopsin expression in wild type retinas but not in retinas from Rd7 mice (Figure 1G). While this experiment confirms Nr2e3 specificity of PR3, it remains unclear why Rd7 retinas exhibit only moderate reductions in rod gene expression (Corbo and Cepko, 2005; Peng et al., 2005; Chen et al., 2005; Haider et al., 2001), while PR3 treatment or CRISPR-Cas9 deletion of Nr2e3 (Zhu et al., 2017) result in substantial decreases. This suggests that there is some developmental compensation that is not present when the gene is deleted or inhibited in postmitotic rods; alternatively, PR3 may be acting in a dominant-negative manner, and inhibiting the ability of the Nr2e3-Nrl-Crx complex to function properly. Nr2e3 signaling is important for rod photoreceptor cell fate, development and maturation, and maintenance of expression. To determine the effect of PR3 treatment on gene expression in postmitotic retinal cells, we systemically treated (intraperitoneal injection; IP) wild type mice with PR3 or vehicle at P12. At P13,24 hr after the injection, we collected the retinas for global transcriptome analysis by RNA sequencing. As hypothesized, we observed a decrease in most rod photoreceptor-specific transcripts (Figure 2A–B). Similar to conditional knockout of Nrl in adult mice and knockdown of Nrl by CRISPR/Cas9 in postmitotic photoreceptors, we did not observe global increases in cone gene expression (Montana et al., 2013; Yu et al., 2017). Nevertheless, we did observe an increase in the number of S Opsin+ cells in the ventral retina with PR3 treatment in vivo (Figure 2C–D). Genes expressed in both rod and cone photoreceptors, eg. Crx and Otx2, showed no difference in expression between control and PR3 treatment. We next used electron microscopy (EM) to perform an ultrastructural analysis of photoreceptor morphology after PR3 treatment. We carried out IP injections of vehicle or PR3 in wild type mice for three consecutive days starting at P11. At P14,24 hr after the third injection, we euthanized the mice and processed their retinas for EM. Photoreceptor outer segments begin to form during the second and third postnatal week; mutations in Nr2e3 lead to an impairment in rod outer segment formation, and we predicted that PR3 treatment would affect their development in a similar way (Haider et al., 2006). As predicted, PR3 treatment prevented outer segment development; outer segments of PR3-treated photoreceptors were strikingly truncated compared to controls (Figure 2E). We did not observe any indication of photoreceptor apoptosis induced by PR3 treatment upon examination of outer nuclear layer (ONL) nuclei (Figure 2E), further indicating that the effects on rod development were not due to an increase in cell death. We have recently shown that reductions in rod gene expression caused by treatment with PR1 were sufficient to slow the degeneration of RhoP23H photoreceptors in vitro (Nakamura et al., 2016). The RhoP23H mutation causes misfolding of Rhodopsin in rod photoreceptors, which leads to activation of the unfolded protein response and eventually results in rod and cone death (Nguyen et al., 2014). In RhoP23H mice, most rod photoreceptors undergo apoptosis by the end of the third postnatal week (Sakami et al., 2011). To determine whether we could prevent photoreceptor degeneration in this model, we treated RhoP23H mice with PR3 or vehicle from P12-P14 until P21, during the period of rod photoreceptor death (Figure 3A). At P21, we assessed visual function with ERGs and euthanized the mice for histological and qPCR analyses. At P21, control RhoP23H mice had only 2–3 rows of photoreceptors remaining in their ONL (Figure 3B–C). Rods were sparse and there were few remaining cones (S Opsin+ and Cone Arrestin+ photoreceptors). Control RhoP23H mice had minimal scotopic and photopic b-wave amplitudes by ERG analysis (Figure 4A, B, D and E). By contrast, retinas from PR3-treated RhoP23H mice had several rows of rod and cone photoreceptors in the ONL (Figure 3B–C). The surviving cones in the PR3-treated retinas were more elongated and healthier than in the DMSO control retinas. We confirmed our histological results with qPCR on retinas from control and PR3 RhoP23H mice and found that treated mice had more expression of Recoverin and Rhodopsin, indicating greater photoreceptor cell survival (Figure 3D). ERG analysis of PR3-treated RhoP23H mice showed significantly elevated scotopic and photopic b-wave amplitudes at most stimulation intensities compared to littermate controls (Figure 4A, C, D and F). Together, these data support the conclusion that PR3 treatment prevented structural and functional degeneration of photoreceptors in this model of RP. In this study we successfully prevented photoreceptor degeneration in the RhoP23H mouse, the first report of successful treatment of this RP model with a small molecule in vivo. Our strategy was to reduce the expression of photoreceptor genes by targeting the rod-specific nuclear receptor Nr2e3 with a small molecule modulator. Treatment with PR3 decreased rod gene expression, and was sufficient to functionally and structurally preserve photoreceptors in the RhoP23H mouse. Previous studies have shown that genetic manipulation of the rod photoreceptor differentiation pathway may be useful for the treatment of multiple RP models. Conditional deletion of Nrl in adult mouse rods prevents degeneration in the Rho−/− model of recessive RP (Montana et al., 2013). More recently, knockdown of Nrl or Nr2e3 by AAV-CRISPR/Cas9 gave long-term histological and functional preservation of photoreceptors in numerous RP models (Zhu et al., 2017; Yu et al., 2017). Our report now shows that a small molecule targeting this same regulatory pathway is also effective at slowing rod degeneration in a particularly aggressive RP model and provides a novel target for medical therapy of retinal degeneration. C57Bl/6 (Jackson Stock No: 000664), RhoP23H (Sakami et al., 2011) (Jackson Stock No: 017628), and Nr2e3Rd7 (Jackson Stock No: 004643) mice were used at the indicated ages. All mice were housed by the Department of Comparative Medicine at the University of Washington and protocols were approved by the University of Washington Institutional Animal Care and Use Committee. The research was carried out in accordance with the ARVO statement for the Use of Animals in Ophthalmic and Vision Research. For all experiments, a sample size of at least four mice per condition was chosen to ensure adequate power to detect a pre-specified effect size. From each litter, half of the animals were randomly assigned to the control group and the other half to the experimental group, and no animals were excluded. Photoregulin3 was identified by searching previous small molecule screens with PubChem for Nr2e3 interacting molecules. It was initially obtained from ChemDiv and then synthesized and purified in large quantities in the lab after initial screening. For in vivo experiments, mice were injected intraperitoneally with PR3 dissolved in DMSO at 10 mg/kg. Retinas were dissected from postnatal day 5 (P5) mice and dissociated by treatment with 0. 5% Trypsin diluted in calcium- and magnesium-free HBSS for 10 min at 37°C. Trypsin was inactivated by adding an equal volume of FBS and cells were pelleted by centrifugation at 4°C and resuspended in media (Neurobasal-A containing 1% FBS, 1% N2,1% B27,1% Pen/Strep, and 0. 5% L-Glutamine). For the immunofluorescence assay, cells were plated into 96-well black walled, clear bottom tissue culture plates at a density of 1 retina/5 wells. Small molecules were diluted in media and were added the day following dissociation. After two days of treatment, cells were fixed with 4% PFA for 20 min at room temperature, blocked with blocking solution (10% Normal Horse Serum and 0. 5% Triton X-100 diluted in 1X PBS) for 1 hr at room temperature, and incubated overnight at 4°C with primary antibodies generated against Rhodopsin (1: 250; Rho4D2, Gift from Dr. Robert Molday, UBC) diluted in blocking solution. The following day, wells were washed with 1X PBS and then incubated with species appropriate, fluorescently labeled secondary antibodies diluted in blocking solution for 1 hr at room temperature. Wells were washed three times, counterstained with ToPro3, and the entire plate was imaged using a GE Typhoon FLA 9400 imager. Optical density measurements were obtained from the plate scans using ImageJ software and Rhodopsin expression was normalized to ToPro3 nuclear stain. RNA from retinas was isolated using TRIzol (Invitrogen) and cDNA was synthesized using the iScript cDNA synthesis kit (Bio-Rad). SSO Fast (Bio-Rad) was used for quantitative real-time PCR. For analysis, values were normalized to Gapdh (ΔCT) and ΔΔCT between DMSO and compound-treated samples was expressed as percent of DMSO treated controls (100*2^ΔΔCt). t-tests were performed on ΔCT values. The following primer sequences were used: Gapdh (F: GGCATTGCTCTCAATGACAA, R: CTTGCTCAGTGTCCTTGCTG), Rhodopsin (F: CCCTTCTCCAACGTCACAGG, R: TGAGGAAGTTGATGGGGAAGC), Opn1sw (F: CAGCATCCGCTTCAACTCCAA, R: GCAGATGAGGGAAAGAGGAATGA), Recoverin (F: ACGACGTAGACGGCAATGG, R: CCGCTTTTCTGGGGTGTTTT), Otx2 (F: CCGCCTTACGCAGTCAATG, R: GAGGGATGCAGCAAGTCCATA), and Prdm1 (F: TTCTCTTGGAAAAACGTGTGGG, R: GGAGCCGGAGCTAGACTTG). Intact retinas without RPE from mice at the indicated ages and genotypes were explanted on 0. 4 μm pore tissue culture inserts in media (Neurobasal-A containing 1% FBS, 1% N2,1% B27,1% Pen/Strep, and 0. 5% L-Glutamine) containing DMSO or 0. 3–1. 0 μM PR3. Full media changes were performed every other day. Explants or eye cups were fixed with 4% PFA for 20 min at room temperature, blocked with blocking solution (10% normal horse serum and 0. 5% Triton X-100 diluted in 1X PBS) for 1 hr at room temperature, and incubated overnight at 4°C with primary antibodies generated against S Opsin (1: 400, SCBT, sc-14363). The following day, the retinas were washed with 1X PBS, and then incubated with a species appropriate, fluorescently-labeled secondary antibody diluted in blocking solution overnight, followed by washing with 1X PBS and DAPI staining. The retinas were transferred to slides and coverslipped with Fluoromount-G (SouthernBiotech). An Olympus FluoView FV1000 was used for confocal microscopy. Cells were counted from single plane confocal images taken at fixed settings. Eyecups were fixed in 4% PFA in 1X PBS for 20 min at room temperature and then cryoprotected in 30% sucrose in 1X PBS overnight at 4°C. Samples were embedded in OCT (Sakura Finetek), frozen on dry ice, and then sectioned at 16–18 μm on a cryostat (Leica). Slides were blocked with a solution containing 10% normal horse serum and 0. 5% Triton X-100 in 1X PBS for 1 hr at room temperature and then stained overnight at 4°C with primary antibodies (Rho4D2 at 1: 250 from Dr. Robert Molday, S Opsin at 1: 400 from SCBT: sc-14363, Cone Arrestin at 1: 1000 from Millipore: AB15282, Otx2 at 1: 200 from R and D Systems: BAF1979) diluted in blocking solution. Slides were washed three times with 1X PBS the following day and then incubated in fluorescently labeled secondary antibodies diluted in blocking solution for 2 hr at room temperature, stained with DAPI, washed, and coverslipped using Fluoromount-G (SouthernBiotech). An Olympus FluoView FV1000 was used for confocal microscopy. Cells were counted from single plane confocal images taken at fixed settings. Counts in the central retina were taken adjacent to the optic nerve head (50 μm from the nerve head on the ventral side) and counts in the peripheral retina were taken 50 μm from the peripheral edge on the ventral side. NR2E3 protein (aa 90–410) was expressed as an His8-MBP-TEV fusion protein from the expression vector pVP16 (DNASU Plasmid ID: HsCD00084154). E. coli BL21 (DE3) cells were grown to an OD600 of 1, and then induced with 0. 2 mM IPTG at 16°C overnight. Cells were harvested, resuspended in extract buffer (20 mM Tris pH 8,200 mM NaCl, 10% glycerol, 5 mM 2-mercaptoethanol, and saturated PMSF diluted 1: 1,000), and then lysed by sonication on ice. Lysates were centrifuged at 4°C and the supernatant was loaded onto an equilibrated column containing 5 mL of Ni-NTA agarose (Qiagen). The column was washed with 20 mM Tris pH 8,1M NaCl, 5 mM 2-mercaptoethanol, and 40 mM imidazole, and then the protein was eluted with 20 mM Tris pH 8,200 mM NaCl, 5 mM 2-mercaptoethanol, and 100 mM imidazole. The fusion protein was incubated with TEV overnight at 4°C and then the His8-MBP tags were separated from NR2E3 by ion exchange chromatography. For isothermal titration calorimetry, 100 μM PR3 was injected into 20 μM NR2E3 in 10 mM Sodium Phosphate buffer pH 8 with 50 mM NaCl and 0. 5% DMSO in a MicroCal ITC-200 (Malvern) and the data was analyzed with Origin 7. 0 software. RNA from retinas was isolated using TRIzol (Invitrogen) and total RNA integrity was checked using an Agilent 4200 TapeStation and quantified with a Trinean DropSense96 spectrophotometer. RNA-seq libraries were prepared from total RNA using the TruSeq RNA Sample Prep kit (Illumina) and a Sciclone NGSx Workstation (PerkinElmer). Library size distributions were validated using an Agilent 4200 TapeStation. Additional Library quality control, blending of pooled indexed libraries, and cluster optimization were performed using Life Technologies’ Invitrogen Qubit Fluorometer. RNA-seq libraries were pooled (4-plex) and clustered onto a flow cell lane. Sequencing was performed using an Illumina HiSeq 2500 in rapid mode employing a paired-end, 50 base read length (PE50) sequencing strategy. Mice were euthanized by CO2, and then perfused with 0. 9% saline followed by 4% glutaraldehyde in 0. 1 M sodium cacodylate buffer. Eye cups were fixed in 4% glutaraldehyde in 0. 1 M sodium cacodylate buffer, washed with 0. 1 M sodium cacodylate buffer, and then post-fixed in 2% osmium tetroxide. After fixation, eye cups were washed with water, dehydrated through a graded series of ethanol, incubated in propylene oxide and then epon araidite, polymerized overnight at 60°C, and then sectioned at a thickness of 70 nm. Images were obtained using a JEOL JEM-1230 electron microscope. Mice were dark adapted overnight (12–18 hr). All subsequent steps were carried out under dim red light. Mice were placed in an anesthesia chamber and anesthetized with 1. 5–3% isoflurane gas. Mice were transferred from the anesthesia chamber to a heated platform maintained at 37 ˚C and positioned in a nose cone to maintain a constant flow of Isoflurane. Drops of 1% tropicamide and 2. 5% phenylephrine Hydrochloride were applied to each eye. A reference needle electrode was placed subdermally on the top of the head and a ground needle electrode was placed subdermally in the tail. Drops of 1. 5% methyl cellulose were applied to each eye and contact lens electrodes were placed over each eye. Dim red light was turned off and the platform was positioned inside of an LKC Technologies UTAS BigShot ganzfeld and a series of flashes of increasing intensity were delivered scotopically. A series of photopic flashes were performed immediately following the series of scotopic flashes. HEK293 cells were transfected with 1 μg of the luciferase reporter BR-225Luc (Dr. Shiming Chen) or S Opsin 600 pGL3 (Dr. Douglas Forrest), 1 ng of the control pRL-CMV (Promega) and 100 ng of hNRL-pCMVSport6 (Open Biosystems), hCRX-pCMVSport6 (Open Biosystems), hNR2E3-pcDNA3. 1/HisC (Dr. Shiming Chen), or hRORB-pLenti6. 2/V5-DEST (DNASU) in 24-well plates using Lipofectamine 3000 reagent (Thermo Fisher Scientific). Transfection reagents were removed the following day and replaced with media containing DMSO or 1 μM PR3 for 2 days. Cells were lysed and firefly and renilla luciferase activity was measured with the Dual-Luciferase Reporter Assay System (Promega) using a 1420 Multilabel Victor3V plate reader. HEK293 cells were transfected in 6-well tissue culture plates with lipofectamine 3000 (Thermo Fisher Scientific) and 800 ng of each hNRL-pCMVSport6 (Open Biosystems) and hNR2E3-pcDNA3. 1/HisC (Dr. Shiming Chen) in Opti-MEM media. Transfection reagents were removed after 24 hr and replaced with media containing DMSO or 1 μM PR3 for 2 days. Cells were lysed with Co-IP lysis buffer (25 mM Tris-HCl pH 7. 5,150 mM NaCl, 1 mM EDTA, 1% Triton X-100,5% glycerol and 1X protease inhibitor cocktail). Sheep anti-mouse IgG magnetic Dynabeads were incubated with anti-Nr2e3 antibody (5 μg/precipitation) diluted in Co-IP buffer for 2 hr at 4°C. Equal volume of lysate were then added to the antibody-coated beads and incubated overnight at 4°C. The following day, beads were washed four times with Co-IP buffer and then incubated at 85°C for 15 min in 1X sample buffer diluted in Co-IP buffer. Samples were loaded and run in a 4–20% SDS gel (Bio-Rad). Protein was transferred to a PVDF membrane (Thermo Fisher Scientific), blocked (5% BSA and 0. 1% Tween 20 in 1X PBS) for at least 1 hr at room temperature and stained with primary antibodies anti-Nrl (Chemicon) or Anti-Nr2e3 (R and D systems) diluted in blocking solution overnight at 4°C. Membranes were washed with 0. 1% Tween 20 in 1X PBS and then incubated with Clean-Blot IP Detection Reagent (Thermo Fisher Scientific) diluted in blocking solution for 1 hr at room temperature. Signals were visualized on X-ray film with SuperSignal West Dura Extended Duration Substrate (Thermo Fisher Scientific) and quantified using ImageJ software. | There are several diseases that cause people to lose their eyesight and become blind. One of these diseases, called retinitis pigmentosa, kills cells at the back of the eye known as rod cells. At first, it affects vision in low light and peripheral vision, but later it affects vision during the daytime as well. There are no effective treatments for patients with retinitis pigmentosa. Yet previous genetic studies have shown that disrupting the activity of genes in rod cells can slow the progression of the disease and preserve vision in mice. As for all genes, proteins called transcription factors regulate the activity of rod cell genes. Nakamura et al. now report the discovery of a small drug-like molecule, that they name Photoregulin3, which alters the activity of a transcription factor that regulates rod genes. In follow-up experiments, mice with a mutation that replicates many of the features of retinitis pigmentosa were given Photoregulin3 to see if it could slow the progression of the disease. Indeed, Photoregulin3 could stop many of the rod cells from degenerating in the treated mice. At the end of the experiment, the mice treated with this small molecule had about twice as many rods as the control mice. The treated mice also responded better to flashes of light. Nakamura et al. hope that the findings will one day benefit patients with retinitis pigmentosa. But first more research needs to be done before testing Photoregulin3 in humans. For example, the drug-like molecule needs to be made more potent, and if possible adapted to work when given orally, meaning patients could take it as a pill. | lay_elife |
Neurocysticercosis is a disease caused by the oral ingestion of eggs from the human parasitic worm Taenia solium. Although drugs are available they are controversial because of the side effects and poor efficiency. An expressed sequence tag (EST) library is a method used to describe the gene expression profile and sequence of mRNA from a specific organism and stage. Such information can be used in order to find new targets for the development of drugs and to get a better understanding of the parasite biology. Here an EST library consisting of 5760 sequences from the pig cysticerca stage has been constructed. In the library 1650 unique sequences were found and of these, 845 sequences (52%) were novel to T. solium and not identified within other EST libraries. Furthermore, 918 sequences (55%) were of unknown function. Amongst the 25 most frequently expressed sequences 6 had no relevant similarity to other sequences found in the Genbank NR DNA database. A prediction of putative signal peptides was also performed and 4 among the 25 were found to be predicted with a signal peptide. Proposed vaccine and diagnostic targets T24, Tsol18/HP6 and Tso31d could also be identified among the 25 most frequently expressed. An EST library has been produced from pig cysticerca and analyzed. More than half of the different ESTs sequenced contained a sequence with no suggested function and 845 novel EST sequences have been identified. The library increases the knowledge about what genes are expressed and to what level. It can also be used to study different areas of research such as drug and diagnostic development together with parasite fitness via e. g. immune modulation. Taenia solium is a parasitic tapeworm infecting approximately 50 million people worldwide [1]. Humans are the definitive host whereas pigs are the intermediate. Cysticercosis is caused when humans accidentally act as the intermediate host and ingest the eggs from tapeworms. The cysts are developed into oncospheres which penetrate the epithelial cells, then migrate to different parts of the body. After migration the oncospheres are established in the tissues as cysts. The cysts can vary in number and size. Cysts which end up in the central nervous system most commonly give symptoms and are the cause of neurocysticercosis (NCC) [2]. The clinical picture of NCC can range from asymptomatic, mild headache and seizures to death [3]. Annually NCC is responsible for at least 50 000 deaths [4]. In parts of Latin America more than 80% of the population with seizures has been diagnosed with NCC [5], [6], [7], [8]. Also a correlation between epilepsy and NCC can be seen and in endemic countries NCC accounts for 30–50% of all late-onset epilepsy [9]. It has also been demonstrated that oncospheres or the cysts have a rich source of antigens which can be capable of stimulating a protective immune response [10]. Several projects are underway to identify an affordable and effective vaccine, but currently the treatment of choice is drugs [11], [12], [13]. Different lines of treatment are available, such as albendazole and praziquantel. Those treatments are controversial as they only are partially effective and side effects have been reported [14]. Another drawback is the availability diagnostic methods in endemic areas. They include the use of X-ray computed tomography (CT), a method not affordable to the majority of the patients with NCC. Other methods include detecting specific antibodies or antigens, which seldom are used as routine within local diagnostic settings [15]. The above problems have been recognized and projects have been initiated to use molecular tools in order to give aid to drug discoveries, vaccine candidates and diagnostic antigens. Recently an EST library from the pig cysticerca stage consisting of 812 unique ESTs was published [16] and a consortium has been established in order to sequence the T. solium genome [1]. At the National Center for Biotechnology Information (NCBI), there are different EST libraries from the genus Taenia published. The published ESTs from T. solium are distributed between the adult stage (45%) and the larval stage (55%). EST analysis is a very cost effective method to discover novel genes and to see expression of these and known genes together with identifying different protein groups (e. g. proteins with signal peptides). Once an EST library has been established it also can be used to identify genes behind different amino acid sequences which have derived from different peptides of interest [17], [18]. In order to extend our knowledge about these issues and to have a library for common use which contains sequence fragments from proposed expressed genes, an EST library from the cyst stage in the intermediate pig host has been made. The library consists of 4674 high quality sequences. Analyses of these sequences demonstrate that over 50% of the identified unique ESTs cannot be described, 25% previously identified within other worm species and 20% of the genes are conserved throughout the eukaryotic kingdom. Among the 25 most highly expressed genes identified, 3 are suggested in literature to be associated to the oncosphere surface and therefore potential targets for vaccine development. Also identified within this group is a novel proposed surface protein with homology to T. ovis 45W antigen, designated Tsol15. A cDNA library, Uni-ZAP XR (Stratagene), constructed from polyA+ selected mRNA from 1. 75g cysticerci isolated from muscle of a naturally infected Peruvian pig was used (kindly given by Kathy Hancock, CDC [19]). The library was transformed into the ampicillin resistant vector phagemid pBluescript SK (+/−) and host bacteria (XLOLR) using mass excision protocol as described by the manufacturer (ZAP Express cDNA Synthesis Kit and ZAP Express cDNAGigapack III Gold Cloning Kit, Instruction manual, Strategene). As insertion of the cDNA disrupts the expression of the LacZ gene, X-gal [20 mg/ml] was streaked on 14 cm Ø Petri dishes containing agar and ampicillin. To induce the expression of LacZ, IPTG [100 mg/ml] was also added prior to streaking on host bacteria. Clones without insertion and thus a functional LacZ gene are able to digest X-gal, resulting in a blue staining. Positive, uncolored colonies were picked and dropped into 96 well microtiter plates. Wells contained 5µl TempliPhi Denature Buffer (GE HealthCare). The toothpicks were lifted into new 96 well microtiter plate wells containing LB media, DMSO and ampicillin, incubated ∼20h, at 37°C and then frozen for storage. Microtiter plates with denature buffer and bacteria were heated 95°C for 3min, and after an addition of 5µl TempliPhi Premix (GE Healthcare) incubated at 30°C overnight. A rolling circle PCR program with reverse M13 primer was run prior to ethanol precipitation. Samples were dried and 5µl loading buffer was added prior to sequencing. Preparation and PCR were conducted accordingly to DYEnamic ET Dye Terminator Cycle Sequencing Kit for MegaBace DNA Analysis System (GE Healthcare). The reagent amounts and concentrations were modified and optimized at the Center for Genomics and Bioinformatics, Karolinska Institutet, Stockholm, Sweden. The sequencing process was done by a MegaBace 1000 sequencing system (Amersham Biosciences). Computational analyses were done using Phred for base calling and Phrap to assemble sequences. Cross match was used to do queries within the ESTs, cut out vector sequences and assemble contigs [20], [21], [22]. To compare the generated ESTs against database on both a protein and a gene level BLAST search was used (NCBI). To disregard irrelevant or low BLAST scores a cut off E-value of <10−5 was used. The translation of the DNA sequences into 3 different frames was performed using Virtual Ribosome - version 1. 1 [23]. Translation was done in 2 groups; first, all sequences were translated and second, only sequences with an initiation codon were translated. SignalP 3. 0 Server was used in order to predict signal sequences within all open reading frames (ORF) where the start codon ATG could be detected [24]. The D-score in the implemented Neural Network together with Hidden Markov models was chosen. The D-score is an average of S-mean and Y-max, where S-mean is a calculation of the length of the predicted signal peptide and Y-max gives a cleavage site prediction. The result of the D-score is given as: yes or no to the question of whether signal peptide is present. Besides the straightforward prediction of the presence of signal peptide, it also shows a probability of signal peptide. Of all the unique sequences the most probable putative protein of each contig and singlet were put into one of seven different hierarchic taxonomy categories. According to the BLASTX results, the organisms producing the putative proteins were set to the taxonomy group last shared with T. solium and BLASTX scores with E-value <10−5 were set as unknowns. The software Blast2GO was used as described earlier [25]. Sequences in fasta format were loaded into the program and the default settings were used to assign GO terms. From a BLAST search, the annotation of the sequences were performed and pie charts were made using 2nd level GO terms based on Biological processes, Molecular functions and Cellular components. InterProscan was also used within the Blast2GO software and result merged together with the GO terms, as described [25]. Analysis of metabolic pathways were also performed by Blast2GO and KOBAS [26], [27] using the KEGG data base [25]. Summarized in Table 1, a library consisting of 5760 ESTs from pig cysticercus was generated. Readable sequences were generated with 5551 ESTs (96. 4%). After conducting base call and disregarding short sequences and vector sequences, 4674 high quality ESTs were saved (GenBank accession numbers, GT889435–GT894161). Using Phred and Phrap [20], [21], [22] 1650 unique sequences could be identified (Table 1; Figure S1). The contigs were comprised of 3462 sequences, which corresponded to 438 unique contigs, mean of 7. 9 sequences per contig. Contigs mean length were 558 nucleotides (variation between 142–1886 nucleotides). The remaining 1212 sequences were identified as single unique sequences (Table 1). The percentage of ESTs identified with a poly-A tail was 18% and the majority of these were found within the contigs, 40%. The expression pattern of the 25 most abundant ESTs with a BLASTX cut off E-value of 10−5, revealed these to make up 34. 5% of all high quality sequences (Table 2; Table S1). The four most common are a set of conserved genes found within the eukaryotic kingdom, representing the antioxidant enzyme PHGPx, the structural proteins Tubulin and Actin and a lysomal enzyme, ATPase (Table 2). These enzymes and structural proteins accounted for 19% of all the ESTs sequenced. The other 21 putative proteins in the top 25 list are other conserved proteins specific to cestodes, unknown proteins and mitochondrial proteins. Among the highly expressed cestode proteins were the vaccine and diagnostic candidates Tsol18/HP6 [28] and T24 [19]. Also identified was a novel proposed surface protein with homology to T. ovis 45W antigen, ToW5/7 (Figure 1). The molecular weight of this protein is predicted to be 15 kDa and was therefore given the name Tsol15 (GenBank accession number, GU338867). Of the 1650 unique EST sequences, 754 different EST sequences could be identified as putative genes found in other organisms in the eukaryotic kingdom. A classification according to taxonomical closeness to T. solium using NCBI Entrez Taxonomy database was conducted (Figure 2) [29]. Unknown or novel sequences accounted for 54% of 1650 unique sequences. The second largest group was Platyhelminthes, which accounted for 25% of the ESTs. Within this group, 5% of the ESTs were unique to the Taeniidae and 3% unique to Taenia species. Conserved within the eukaryotic kingdom was 20% of all the different EST. Also found was a small group, less than 1%, shared with Nematoda. Secretory proteins have been suggested to play a major role in the pathogenesis of parasites [30], [31]. The secretory proteins may also act in immunomodulation processes [32]. We therefore searched our ESTs for putative signal peptides according to the criteria given by SignalP 3. 0 [24]. The ESTs were translated into 3 different reading frames and the longest ORF from each EST was chosen as the relevant putative protein and used in the SignalP 3. 0 prediction software. We found 154 ESTs sequences with predicted signal peptides, representing 9. 3% of the unique sequences. Of these, 119 (78%) were derived from ESTs with no significant BLASTX hit (E-value <10−5) to the NR database in Genbank. Among the 35 predicted signal peptides with a significant hit in the BLASTX database were 4 ESTs identified within the 25 most abundant groups of ESTs (Table 2, Table S2). These 4 ESTs were found to have similarity to an ATPase, Tso31d [33], HP6/Tsol18 [11], and an antigen earlier described in T. ovis (Figure 1, Table 2, Table S2). Notable, also among the predicted secreted proteins are at least 2 different proteinase inhibitors, a serine proteinase inhibitor, detected as a single EST (TSCC. R39. esd, Table S2) and a proteinase inhibitor belonging to the Kunitz family. The ESTs with homology to the Kunitz family consisted of three ESTs (TS. Contig318_rframe2_ORF, Table S2). Putative roles of these proteins are immunomodulation of the host [32] or to act and interfere with host physiological processes at the initial stages of infection [34]. All of the 1650 unique ESTs were analyzed for their similarities to proteins within the Genbank NR database. The automated software Blast2GO was used, which also annotated the different sequences when possible to a gene ontology (GO) group described by the Gene Ontology Consortium [35]. The annotations to GO groups were also performed with InterProScan. Results were merged together within the Blast2GO and further analyzed [25]. 754 ESTs scored a significant hit (E-value <10−5) and 634 of these were able to obtain a GO term (Figure 3, Table S3). Presented in Figure 3 are percentages of 2nd level GO terms as graphs. The 3 different graphs are presented as Cellular components (Figure 3a), Molecular functions (Figure 3b) and Biological processes (Figure 3c). In the Cellular components, 3 different major categories could be found; cell (46%), organelle (30%) and macromolecular complex (18%, Figure 3a). In the Molecular function category the main categories were ESTs involved in binding activities, 50%, and proteins involved in catalytic activities, 33% (Figure 3b). In the third category, Biological process, 26% belong to cellular processes and 20% to metabolic processes. Other ESTs were divided into smaller categories and no major category could be identified (Figure 3c). The ESTs with an assigned GO group were mapped into different metabolic pathways by 2 different web-based programs, KOBAS (KEGG Orthology-Based Annotation System) [26], [27] and Blast2GO [25]. The ESTs mapped in to 60 different metabolic pathways using Blast2GO and in most pathways several ESTs could be found (Figure S2). Similar results were also obtained from KOBAS web based program even if presented in a broader spectrum with no strict rule to metabolic pathways (Table S4). NCC is responsible for over 50 000 deaths annually. In order to increase our understanding about the disease causing stage we have sequenced over 5000 ESTs from this stage. Another EST library containing 817 sequences from the same stage can be found in the literature [16]. However, in order to get an overview of the expression profile more ESTs are needed. This library will add data to the understanding of T. solium and it is to our knowledge the biggest EST library from T. solium cysticerca analyzed to date. A BLAST search against published ESTs at NCBI from Taenia species was performed (Table S5). The result demonstrates that 845 (52% of the whole library) unique ESTs are added to the NCBI archive. The library can be divided into 2 groups, ESTs represented as a single sequence and as contigs which contain several ESTs (Table 1). Most of the unique ESTs are found within the single group, 62%, meanwhile the more expressed contigs have less unique ESTs, 28%. Previously described ESTs from different parasitic nematodes and trematodes suggest, in accordance with our study, that a large number of the ESTs belong to a group of unknown sequences with no similarity to other sequences found within the Genbank NR database [16], [36], [37]. The large size of this group is most likely due to the relatively few genome projects performed on trematodes to date (Figure 1). The only 2 whole genome sequences of trematodes that has been annotated and recently released to the public are the Schistosoma mansoni and S. japonicum genomes [38], [39]. This also is visualized in the GO hits demonstrated within this study; 57% of the ESTs are found to have similarity to genes in S. mansoni and S. japonicum. The GO predictions present data which differ from earlier described analyses by Almeida, C. R. et al [16]. In Figure 3c, a decrease in ESTs responsible for cellular processes is seen, from 61% to 26%. Meanwhile, the class for metabolic processes is found to be similar, 15% versus 20%. In the binding category (Figure 3b) an increase from 41% to 50% is seen and the catalytic activity increases from 29% to 33% [16]. The changes seen are most likely due to the size of the different data sets used, 96 annotated sequences [16] compared to 634 (this study). Among the ESTs found to be most highly expressed were structural proteins (e. g. tubulin and actin) together with different enzymes (e. g. PHGPx isoform 1 and ATPase, Table 2). Genes proposed to be expressed at the membrane of the human oncospheres and involved in the establishment of the human larval stage also is highly expressed. Examples of such genes are Tso31d [33], T24 [19] and HP6/Tsol18 [11], a candidate vaccine protein [40], [41]. Several of the highly expressed ESTs have unknown functions and further characterization are needed. Other highly expressed sequences have homology to genes found in other species and can therefore be proposed to a function or to become a target molecule for example, vaccine development. An example can be found in Table 2 where a cestode specific EST, predicted to contain a signal peptide, is the ninth most common expressed protein in the cysticercal stage. The whole sequence of this mRNA could be obtained from contig 492 (GenBank accession number, GU338867, Figure S1) and analysis of the same revealed an open reading frame (ORF) corresponding to a protein with a predicted molecular weight of 15. 3 kDa. Results from a BLAST search revealed a similarity to a family of 45W antigens from Taenia ovis, E-value <10−6 (Figure 2). The 45W family of antigens are known to be expressed at the surface of oncospheres [42]. The new protein was named Tsol15. Other Tsol-proteins have been suggested to be valuable target molecules for diagnostic and vaccine purposes and Tsol15 could also join in as a proposed diagnostic antigen. This EST library could also give crucial information with different word searches. The searches for molecules that have effects on immunomodulation of host defense are presented here. Several molecules have been described to play a role in host cellular immune responses. Examples of such molecules are different protein inhibitors, cystatin, prostaglandin, nonintegrin, TGF-beta and macrophage migration inhibitory factor [32], [43], [44]. Of these a general serine protein inhibitor and a Kunitz type 8 of serine protein inhibitors were identified within the group of predicted secreted molecules (Table S2). Both inhibitors have been identified in the cestode Echinococcus granulosus and suggested to be involved in the interface between parasite and host [34], [45]. Also cystatin and TGF-beta signal transducer were identified (Table S1). However, more work is needed in order to be able to draw any conclusions. Another example of the use is to identify drug targets. Patients suffering from schistosomiasis or cysticercosis will be treated (if at all) with Praziquantel, a drug that disrupts calcium homeostasis within the parasite. This drug is freely distributed among several (but not all) countries via the ‘Schistosomiasis Control Initiative’ (SCI; http: //www. schisto. org). The ubiquitous use of Praziquantel will most certainly lead to the development of resistance among various species of flatworm. Because Praziquantel, in many cases, is the only available drug, the development of resistance is a very serious problem. In order to identify new drug targets metabolic pathways could be studied and enzymes unique to the cestodes be found. Presented in Figure S2 are single ESTs which have been annotated and mapped in to 60 different metabolic pathways. The code of single ESTs are given and from there the nucleotide sequence be obtained. In Figure S2 proposed drug targets can be identified, e. g. 6-phosphofructokinase (TSBR. R48. esd), glutathione synthetase (TSBV. R85. esd) and thioredoxin glutathione reductase (TS. seq. Contig445) [46], [47]. Another way of identifying possible drug targets would be to use the strategy by Crowther G. J., et al [48]. The authors describe an in silico method to identify prioritized drug targets in neglected disease pathogens. The work also highlights the fact that data are lacking for less studied pathogens, e. g. helminths and how to overcome this by mapping data from homologues genes in well studied organisms [48]. Using a similar approach several ESTs from this library could be identified as having homology to S. mansoni drug targets. Examples are ATPase subunit alpha (TSAO. R53. esd), tubulin alpha (TSAF. R50. esd), guanine nucleotide-binding protein (TS. seq. Contig375), beta-tubulin (TSBB. R23. esd), 6-phosphofructokinase (TSBR. R48. esd) and proteasome subunit beta 1 (TSAB. R74. esd). Cysticercosis is a major health problem in many developing countries where this disease is endemic affecting both humans and animals. The persistence of this zoonosis is intimately associated to cultural patterns and mainly to the extreme poverty of the human populations. This disease is a neglected problem, probably partly due to it is being mainly restricted to low income countries. Although several predictions are made and EST libraries can be used to identify genes which might be important to novel drug discoveries or used in new diagnostic tools, the intriguing results are the vast majority of ESTs that have an unknown function today. A solution to increase the understanding of this group and possibly get data which will favor the patients with neurocysticercosis would be to increase the resources in the field of neglected parasitic worms. | A method used to describe expressed genes at a specific stage in an organism is an EST library. In this method mRNA from a specific organism is isolated, transcribed into cDNA and sequenced. The sequence will derive from the 5′-end of the cDNA. The library will not have sequences from all genes, especially if they are expressed in low amounts or not at all in the studied stage. Also the library will mostly not contain full length sequences from genes, but expression patterns can be established. If EST libraries are made from different stages of the same organisms these libraries can be compared and differently expressed genes can be identified. Described here is an analysis of an EST library from the pig cysticerca which is thought to be similar to the stage giving the human neglected disease neurocysticercosis. Novel genes together with putative drug targets are examples of data presented. | lay_plos |
The 2014/2015 West African Ebola Virus Disease (EVD) outbreak attracted global attention. Numerous opinions claimed that the global response was impaired, in part because, the EVD research was neglected, although quantitative or qualitative studies did not exist. Our objective was to analyse how the EVD research landscape evolved by exploring the existing research network and its communities before and during the outbreak in West Africa. Social network analysis (SNA) was used to analyse collaborations between institutions named by co-authors as affiliations in publications on EVD. Bibliometric data of publications on EVD between 1976 and 2015 was collected from Thomson Reuters’ Web of Science Core Collection (WoS). Freely available software was used for network analysis at a global-level and for 10-year periods. The networks are presented as undirected-weighted graphs. Rankings by degree and betweenness were calculated to identify central and powerful network positions; modularity function was used to identify research communities. Overall 4,587 publications were identified, of which 2,528 were original research articles. Those yielded 1,644 authors’ affiliated institutions and 9,907 connections for co-authorship network construction. The majority of institutions were from the USA, Canada and Europe. Collaborations with research partners on the African continent did exist, but less frequently. Around six highly connected organisations in the network were identified with powerful and broker positions. Network characteristics varied widely among the 10-year periods and evolved from 30 to 1,489 institutions and 60 to 9,176 connections respectively. Most influential actors are from public or governmental institutions whereas private sector actors, in particular the pharmaceutical industry, are largely absent. Research output on EVD has increased over time and surged during the 2014/2015 outbreak. The overall EVD research network is organised around a few key actors, signalling a concentration of expertise but leaving room for increased cooperation with other institutions especially from affected countries. Finding innovative ways to maintain support for these pivotal actors while steering the global EVD research network towards an agenda driven by agreed, prioritized needs and finding ways to better integrate currently peripheral and newer expertise may accelerate the translation of research into the development of necessary live saving products for EVD ahead of the next outbreak. The 2014/2015 West African Ebola Virus Disease (EVD) outbreak with more than 28,000 cases and 11,000 deaths, was a public health emergency of international concern [1,2]. Although EVD was discovered in the former Zaire (now: Democratic Republic of Congo) more than 40 years ago, the absence of treatment generated global alarm and raised questions on the state of EVD research. Studies analysing EVD transmission and clinical trials testing EVD treatments or vaccines have been difficult due to the small number of infected cases in previous outbreaks [3,4]. Moreover, the pharmaceutical industry has been criticized for neglecting EVD research because it is not profitable enough as EVD occurred rarely and mostly in impoverished African communities [3,5–7]. EVD outbreaks have attracted general public attention since the mid-90s, benefitting science funding, leading to increased publications, but EVD research funding is mostly spent outside of affected African countries and research capacity building there was neglected [8]. The World Health Organization (WHO) called for greater transparency and better sharing of results from clinical trials as being a necessary contribution to facilitate research and development (R&D) for the benefit of science and patients [9] and published a research priority agenda [10]. The necessity for increased transparency also applies to any existing EVD research and expertise to improve the value and efficiency of research efforts. In order to enhance the understanding of on-going EVD research activities and its communities, social network analysis (SNA) of bibliometric data of EVD related scientific publications can be used. Since co-authorships are the most visible and accessible indicator for collaborations, co-authorship-based SNA studies can be used to measure the presence of research collaborations and their evolution over time [11–13]. SNA metrics can reveal network patterns and identify its most central and influential actors [14–16]. The volume of publications, in combination with results from a co-authorship network analysis, can serve as a proxy indicator for R&D. Besides mapping the research landscape [17], especially co-authorship network analysis can provide insight into the degree of research governance and be relevant for strategic research planning [18,19]. Moreover, information from collaboration networks can be used to identify potential collaborations in order to improve research communication and therefore maybe also influence research outcomes [12,20]. The aim of this study is to identify EVD research activities and to analyse the structure of the evolving EVD research community network over time to map existing research collaborations and influential actors based on centrality network metrics. Bibliometrics of 2,528 articles resulting for our WoS search were exported as tab-delimited data and imported into MS Excel as one bibliometric data set (Fig 2). In the raw data set each entry referred to one publication. We included data on title, authors, address of authors’ affiliated institution, publication year, source, language, document type, cited references, funding agency, publisher and subject category in further analysis. Other columns were deleted from the data set. Information on addresses of author’s affiliated institution, e. g. institution name, sub-departments and institution address including city and country, were split into separate columns. Data processing and further cleaning was performed using the software AppleScript [22] and OpenRefine [23]. Name disambiguation, e. g. Centers for Disease Control and Prevention was abbreviated as CDC, Ctr Dis Contr and Centers Dis Cont, orders within names, e. g. Univ Washington and Washington Univ or name spellings, e. g., Univ Georgia, UNIV GEORGIA were identified and harmonised using OpenRefine algorithms or manually. Missing data, e. g. missing country information of an affiliated institution, were substituted by manual web search. If an institution name appeared with addresses in different locations in the data set, e. g. WHO with location Switzerland and location Copenhagen e. g. due to different regional offices, different locations were considered for construction of the network to account for institutions international representations. Institutions duplicates originating from publications with multiple co-authors affiliated with the same institutions were eliminated to ensure a single weighting of institutions. The free online application Table2net was used to extract network information from the refined data set to construct a Gephi readable file [24]. Network nodes (i. e. actors) are institutions named as authors’ affiliations in original research publications. Network edges are titles of joint publications from authors’ affiliated institutions. The free software Gephi was used to calculate network metrics and visualise the networks [25]. Network analysis provides various tools and metrics in order to assess different notions of importance of individual nodes and node groups. As the simplest metric of centrality we calculated each node' s degree, as the sum of direct links to other nodes. Nodes with more direct connections are considered more central. The average node degree captures the number of actors that each actor is connected with on average. The average weighted node degree also takes the weight of a connection between a pair of nodes into account [26,27]. Betweenness centrality measures the frequency with which a particular node lays on the shortest paths between all other node pairs. Therefore, nodes with a high betweenness are considered to have a broker position as they connect many other nodes and thus have a large influence on the transfer of items through the network, under the assumption that item transfer follows the shortest paths [26,28]. We used a betweenness calculation algorithm for weighted graphs as developed by Opsahl [29]. Besides positional properties of the nodes within the network, metrics are capturing topological aspects of the network as a whole. This information can provide an insight on the evolution at network level. Density measures were calculated to assess the connectivity of the network. The density of a network is defined as the total number of existing edges divided by the total number of possible connections. If edges exist between all nodes (density = 1) a network is considered completely dense [26,28]. Since density captures the probable feasible number of connections in a network, it is an indicator for possible community building [30] or innovation flow within a network [15]. Communities within the network were detected using Gephi’s modularity algorithm. Modularity measures the degree of separation of a network into modules or clusters (communities). While a modularity value of 1 indicates that the actors separate perfectly into self-contained clusters, a value of -. 5 suggest the opposite, a homogeneously connected network [27,31]. Networks with a high modularity score employ dense connections between nodes within the modules but sparse connections between nodes from different modules. For visual presentation of network metric calculations we used Gephi' s Force Atlas II algorithm in log-linear mode optimized towards hub dissuasion [32]. Systematic search in WoS for publications containing “Ebola*” yielded a total of 4,587 publications between 1976 and 2015, including original articles (2,531), editorial material (659), news items (437), reviews (415), letters (325), meeting abstracts (157), corrections (36), notes (14), reprints (7), biographical items (4) and book reviews (2). Amongst the 2,531 original articles were 75 article proceedings and five article book chapters. Three of those publications appeared with anonymous authors and were therefore deleted for social network analysis (Figs 1 & 2). The first EVD research article was published in 1977, shortly after the first noted EVD outbreak in 1976. Only few EVD publications were visible until the early nineties, whereas from 1994 onwards the number of yearly EVD publications increased continuously (Fig 3). Since 1994 a higher frequency of EVD outbreaks were recorded and more EVD cases were being detected in almost every year. Several localised EVD outbreaks in Africa have occurred with up to several hundred cases. The initial EVD outbreak in 1976, with a relatively high number of reported cases (>600), was followed by only a small number of publications on EVD research. No EVD outbreaks were reported between 1979 and 1994 and hardly any publications were published on the topic. The number of publications increased gradually and continuously after the second outbreak in 1994, although compared to the 1976 outbreak only about one-tenth of cases were reported (Fig 4). A substantial increase in EVD research publications occurred during the 2014/2015 West African outbreak. An almost 10-fold increase from 2013 (171), 2014 (772) to 2015 (1,621) was visible for almost all document types, but it was most pronounced for editorials (5,220,343), letters (1,75,213), news items (4,190,118) and meeting abstracts (9,5, 66) respectively. An increase in reprints, notes, biographical items and book reviews was not detected. Bibliometrics of 2,528 original research articles were used for social network analysis. Based on their co-authors’ affiliated institutions a global network including institutions from 101 different countries with 704 connections was constructed (Figs 5 & 6). Research institutions in the United States (US) are among the most highly connected institutions in EVD research (degree (d) = 80). They are mostly connected to institutions in Canada (d = 40) with an edge weight (ew) of 130 and Europe, especially Germany (d = 53, ew = 110), the United Kingdom (UK) (d = 60, ew = 90) and France (d = 57, ew = 51), but also to Japan (d = 32, ew = 99). Connections between US institutions and institutions in EVD affected African countries are less frequent (e. g. Guinea-USA ew = 14, Sierra Leone-USA ew = 32, Liberia-USA ew = 30). However, institutions in Sierra Leone and Guinea (both d = 32) and other African countries, especially Nigeria, Uganda and Ghana, are embedded in the global research network with connections to UK, Germany, France and Switzerland. The overall density of the global country-level EVD research network measures 0. 15, with an average degree of 14. 65 and an average weighted degree of 61. 01. Amongst all collaborations on country-level, nine research communities were identified using modularity-based community detection and visualised by different colours (Fig 6). The largest community (red) is centred around the US with strong collaborations to Canada, Germany and the UK, representing 59. 41% of the co-authorships collaborations (weighted edges). Another large community is a (mostly francophone) European–African community (blue) representing 31. 68% of all co-authorships connections. Among all published original research articles between 1976 and 2015 a total of 1,644 co-author' s affiliated institutions were named, which yielded 9,907 co-authorship connections in the overall research network (Fig 7). The main actors according to degree are the US government (CDC USA, d = 353; NIH, d = 315; USAMRIID, d = 283) and WHO (d = 256). Other prominent actors are from the US and European countries. Most central institutions are publicly funded (e. g. CDC USA, USAMRIID), government research institutions (e. g. BNI, ISERM), (mostly public) universities (e. g. Uni London, Univ Marburg) or international institutions (e. g. WHO) or non-governmental institutions (NGOs) (e. g. MSF). Modularity analysis reveals 166 communities within the network (Fig 7), whereas the largest community (blue) represents 17. 33% of the total network nodes and the second largest (green) represents 14. 44% of the network nodes. Numerous smaller and less connected communities exist in the periphery, with some being entirely disconnected from the main network. The temporal development of the research network is visualised over four 10-year time periods (Figs 8,9, 10 & 11). In the first decade 1976–1985, (Fig 8) the network consists of only a few actors, with one large central cluster surrounded by four smaller clusters. The German Bernhard-Nocht Institute (BNI) has the highest centrality degree (d = 11), closely followed by the Institut Pasteur, PHLS Center for Microbiology and Research (Salisbury, UK) and USAMRIID. The CDC USA is a central institution (d = 7) of a smaller cluster, publishing with African partners (Kenyan Ministry of Health) others. Smaller research groups in Kenya (Kemri Wellcome Trust, Institute of Primate Research, Kenya Trypanosomiasis Research Institute), UK and US published together, but had no connections with others. In the second decade 1986–1995, (Fig 9) two larger, but separate, research communities evolved. One francophone French-Swiss-African community with a homogenous structure in which the Institut Pasteur published mainly with the University of Basel, Institut de recherche pour le développement (IRD), Ecole national veterinaire Lyon and the Hospital Bichat Claude Bernard Paris. The other community consists mostly of American and German institutions, with three main actors (USAMRIID, CDC USA and the University of Marburg), where the USAMRIID and CDC USA connect this community. During this period the WHO had its first appearance as a disconnected actor. All institutions in the network of the second decade are public entities. With the occurrence of new EVD outbreaks in 1994/1995 the EVD research network grew in the third decade 1996–2005, (Fig 10) into a star-like structure with surrounding chains. During this decade the CDC USA evolved as the most central actor (d = 87). The University of Marburg (d = 54), USAMRIID (d = 52), WHO (d = 46) and NIH (d = 36) remain central but less prominent actors. The network of the fourth decade 2006–2015, (Fig 11) is skewed by publications in 2014/2015. During this time only few public research institutions and university actors dominate the research collaborations but numerous new actors appeared. Prominent cooperation exist between CDC USA and WHO and CDC, NIH and USAMRIID. While the transnational WHO was well embedded in the network over these last two decades, all main network actors are public institutions, mostly from the US and European countries. While the global EVD research network remains relatively consistent in the first two decades, the third and in particular the forth decade shows substantial overall increase in the number of institutions and the links between them (Table 1). Simultaneously the average node degree and weighted node degree increased over time, which indicates a growing number of collaborations and research activity per institution. The decreasing density of the network over all decades indicates a decreasing number of realised edges between nodes relative to the total number of possible edges. The increasing average node degree implies a growing number of research connections per institution. The number of communities increased in line with number of nodes. The high modularity values show that the solutions of the community detection algorithm reflect the substructures of the graph well, i. e. the increase in communities is unlikely to represent a sheer increase in volume, but rather seems to capture an evolution of the field of EVD into several smaller communities. A degree distribution analysis of the EVD research network in the fourth decade shows a skewed node-degree distribution (Fig 12). While almost 100 nodes appear with a degree of zero (d = 0), indicating no collaboration at all, only few institutions have a very high degree above 160 (mean 12. 24; median 5). Most institutions had a degree of less than five (d≤5) as they were named as affiliations by authors of few publications by authors that published with only few co-authors. The few very well connected institutions, such as NIH and CDC USA, are the key actors in this period. In fact the CDC USA has maintained a very central position in the network over all time periods. The private NGO Médecins Sans Frontières (MSF) has only recently emerged within the network and is centrally embedded with a high degree (d = 157). Since the first reported EVD outbreak in 1976 until today the total number of publications on EVD in WoS has exceeded more than 4500 publications, of which 2528 were original research articles. Like in scientometric analyses we used joint publishing as a proxy indicator of scientific collaboration [17] and thus knowledge exchange for our SNA of the co-authorship network [11,13,30]. Indeed for the EVD overall network we identified research contributions from 1,644 research institutions in 101 countries; most actors are indeed coming from the US [17]. Since 1994 EVD research publications have increased continuously, steadily and independently of the major West African outbreak. This growth in publications is mirrored by a growth in the number of institutions (from 30 to 1,489) and edges (from 60 to 9,176) and therefore on-going network growth accompanied by a decreasing network density. The overall network is an extensive aggregation of 166 different communities with a clearly dominant anglophone and francophone community. This same dominance is seen when analysing the most central actors by degree and betweenness centrality both confirming the dominance of 10 institutions in powerful, control or broker positions in the network [11,33,34]. The pattern of a growing EVD network in size but with a reducing density is characterised by some outliers (106 institutions not connected), frequently less connected contributions from developing countries and the private sector, but with a strong and stable core of dominant or ‘central’ institutions. These characteristics of the network are supported by many of the analyses we performed. For example the relatively and increasingly poorly connected nature of the network (network density), the heavily skewed node degree distribution with the median node degree remaining rather constant, the relatively compact nature of the network (path lengths) and the strong centralisation showing a dominance of a few very strongly connected actors and many poorly connected actors. Although we acknowledge that our analysis is weakened by the absence of a comparator network (a common challenge in emerging research fields), we also believe that our analysis bring some added value. For example SNA metrics for the overall network shows a density of 0. 007 and calculating network density for each decade individually showed progressively decreasing density from 0. 138 in the first decade to 0. 008 in the last decade. While this is largely influenced by both the size (the more actors a network includes, the more difficult it is for all actors to be connected) and also the correspondingly rapid growth in the network (connections take time to build), we still believe that these figures should raise questions about whether the network–and therefore research outputs–could benefit from greater connectivity and linkages and in doing so greater optimise knowledge transfer and the spread of innovation [15]. The node degree distribution (for the last decade from 2006–2015) further confirms both the observed increase in the average node degree is attributable to only a few central actors whereas the overall network was not well connected in this period. Thus, the network growth during the 2014/2015 epidemic diluted connectivity, at a time when collaboration was arguably most needed. These observations are built on when we look further at the node degree distribution for 2006–2015. This confirms that while most actors only had few connections during this time, some actors are extremely connected. This distribution form has been described as “power law” or “scale free distribution” and is typically observed amongst poorly connected networks [35,36]. This ‘concentrated core’ is corroborated by the high number of the average weighted node degree (17. 89), in contrast to the average node degree (12. 05), which is also an indicator that some actors in the EVD network are connected more strongly to each other than others due to repeated publishing [27]. It shows that these actors have on-going collaborations, share research results intensely by jointly publishing—but focus sharing amongst their co-authors. This latter finding is something confirmed by our SNA results, which show strong centralisation amongst six institutions (CDC USA, NIH, USAMRIID, WHO, the University of Marburg and the University of Harvard), suggesting that knowledge is mostly exchanged within the network between and/or through these actors. Centrality is a measure of power in SNA [37], this is especially the case for our central actors whose knowledge broker status is confirmed with regard to EVD research due to their high degree and betweenness centralities. Additionally, observation of the path lengths reveal further insight into the efficiency of information exchange, with the shorter the average path length of a network diameter, the more efficient is information exchanged within the network structure [26,35]. We found that the average paths lengths (3. 02) of the overall network is lower than the average node degree (12. 05), indicating both that some institutions have a lot of direct neighbours and that on average nodes can reach other nodes by crossing only two other nodes. The network diameter (8. 0) suggests that sub-graphs within the network do not span more than across a chain of eight nodes. Taking both aspects into account this implies that the overall structure of the network is characterized by isolated and weakly connected components, i. e. localized small networks that have only few relations amongst each other. Although our study cannot, unfortunately, reveal anything about the ‘type’ of research conducted, observations on the type of research institution maybe serve as a proxy for this insight. Two notable observations here were both the relative underrepresentation and disconnectedness in the overall network of both research institutions from affected countries and the private sector. Among the unconnected nodes appear some private industry actors (e. g., Novartis Vaccines, Biohelix Corp, Baxter Bioscience and Oravax Inc.), in addition to African universities such as the University of Benin and the University of Mbarara. While there may be many good reasons that explain the disconnectedness, for example proprietary restrictions to collaboration (in the case of industry), new entrants to the field or for resource-related barriers to International collaboration. This observation remains significant for a number of reasons, presumably both of these actor types posses’ unique and distinct knowledge and capabilities that could diversify and strengthen the expertise within the network if better and more broadly integrated, this is likely even more the case during a public health emergency of international concern. Also, this ability to identify disconnected but valuable nodes, demonstrates a great added value of tools such as SNA. Finally the recent entry into the network of non-traditional research actors such as MSF should be welcomed, especially as endemic country capacity is being developed and integrated into international networks, due to their unique position as being close to patients in the field yet able to advocate–distant funders–on the need for a well-supported, needs-driven research agenda [5,38]. We believe the structure, nature and evolution of the international EVD research network described in this paper presents some learnings for policy. Looking positively, the network itself has maintained a similar structure–a relatively compact network with a few consistent actors at its core–over the four decades studied, implying it is a stable constellation. This institutional memory provides a solid foundation for knowledge maintenance over time, indeed without central actors networks might be disrupted and knowledge exchange hampered [30]. The growth in the network over time through the entry of new actors, particularly since 2014/2015, is positive as it likely indicates the arrival of new ideas and approaches. However although collaboration has increased over time, our analysis found that the network remains relatively poorly connected. Hence there may be an additional role for the ‘central actors’ to expand their role beyond a hub for dissemination and exchange into a facilitator for integrating the newer actors and expertise into the network. Additional opportunities presented by the network analysis include: a reflection on the, perhaps, over-reliance or vulnerability to the network of all of the ‘central actors’ being public government or university institutions. The importance of predictable, sustainable, funding flows to their continued role as network ‘brokers’ feels more exposed in these current financially and politically turbulent times. While the dominance of these institutions is not surprising, we assume that they have the infrastructure, capability and public-financing, it may represent a weakness in two respects: firstly, with respect to its insufficiently diverse expertise mix, particularly with respect to the translation of this research into the development of tangible, context-relevant tools and capacity building in affected countries [8,39]; secondly, with respect to the risk of over-centralising expertise, resulting in the stifling or suppression of innovation and growth and development of new ideas. Finally, in small research areas for diseases predominantly impacting the lives of those in low-income countries such as EVD, the inherent market failures indicate that this reliance of public-financing will likely continue [Wölfel in: 3,5–7]. Given this, we believe, that a valuable insight from our study is to observe ways in which the network efficiency could be enhanced to extract greater patient-impact from the public financing inputs. For example: focused efforts on integrating new collaborators into the network, provision of tools to enhance the productivity and improved transparency and sharing of research data [9,40] the identification of expertise gaps and targeted filling of these gaps and lastly, but perhaps most importantly, National alignment, focus and financing coordination (strategic research planning) around the globally agreed prioritised research agenda [41]. Although many of these calls have already been made by many actors, particularly since the 2014/2015 EVD outbreak we believe this study represents an important empirical tool to support these calls and inform National and global policy development as the global community works to avert the next EVD outbreak. The use of bibliometric data has intrinsic limitations and restrictions related to any analysis of secondary data and where data ceases to provide information, in particular in relation to content or results of published research. Two major limitations to our study were identified and previously highlighted. The first being the absence of other publications with which to contextualise and compare our results. This absence of relativity in our conclusions limits the comparative value of our findings although the absolute data remain valid. Although SNA is increasingly being used as a tool to analyses research areas it remains a relatively new field so we are optimistic that this is a time-limited constraint. Secondly, we acknowledge that our study would be greatly enriched by an ability to analyse the data by ‘type’ of research not only type of publication i. e. basic, applied, clinical, implementation research, translation, health systems etc. However, at present, this is not a search field within WoS, so we were unable to attain the source data. Should key, public, medical, search engines enable this in the future, SNA such as ours would be an even more powerful tool to provide insight into research focus and productivity. This analysis we believe would have great value–supplementing existing financing and development pipeline analyses [42,43]—in providing a more granular understanding of product development gaps and the persistent absence of tools for the prevention, diagnosis and treatment of EVD [6,44]. Our analysis of decreasing network density over time could have been further triangulated with the use of an additional metric such as the percentage of the giant component or the clustering coefficient. Other limitations include reporting delays and the possibility that some publications were not included in the WoS database, however sample testing of other databases, including PubMed. gov, did not reveal other publications on EVD. Although the impact of missing publications was likely small future studies could aggregate studies from diverse databases and in particular try to assess contribution of private industries R&D. Despite manual and automated attempts to resolve challenges with institution name cleaning and disambiguation it cannot be excluded that some actors and/or relationships were not captured or were captured incorrectly. Although unlikely, errors of the software used cannot be completely excluded and different algorithms might lead to different presentations of results. Therefore network visualisations should be critically assessed in context to minimise misinterpretations. We further note that GeoLayout visualisation can be misleading since it locates the African continent in the map centre and visualised edges may overlap nodes. For this reason a country distribution was processed additionally with Force Atlas 2. The use of only free available software and easy accessible bibliometric data from WoS both facilitate the easy reproducibility of our study. We conducted the first systematic landscaping of published EVD global research bibliometrics using SNA tools for analysis and visualisation. Since 1976 Ebola outbreak EVD research, numbers of authors and affiliated institutions and links between them are constantly increasing, mostly independent from outbreaks and in-particular in the past two decades. The overall EVD research network is organised around a few co-authoring key actors, mostly publicly financed. Low network density indicates room for increased cooperation between institutions, in-particular links to less connected and more peripheral institutions could foster knowledge exchange and innovation. Key network actors, such as the CDC USA, maintained network coherence over time–and probably kept EVD research on-going. Limited scientific collaboration of research organisations from LMIC and the private industry, and how they utilise their expertise and knowledge, is neglected. However, the absence of effective treatments for EVD questions the existing EVD research network efficacy and efficiency and suggests the need for both direction and structure to optimize the network to focus on research relevant for treatments. Since most institutions in the global network are publicly funded, guidance to direct and re-orientate research might be facilitated by funders (through calls targeting knowledge and translation gaps) and be offered by supranational policy setting entities such as WHO and its Global Observatory on Health Research and Development. Further in-depth quantitative and qualitative analysis, e. g. text mining of publications abstracts, analysis of EVD research study methods and separate R&D product pipeline analysis, is recommended to ensure empirically based strategic research guidance and relevant to EVD product development. In any case, SNA of co-authorship networks is an innovative tool to evaluate research collaborations between individuals, organizations and countries, contributes to the understanding of the evolution of research networks and should be used for strategic research planning and a regular monitoring. | Ebola Virus Disease (EVD) research publications were used to analyse and visualise collaborations between institutions jointly publishing research results, using freely available social network analysis tools. Constructed co-authorship networks between author affiliated institutions showed EVD research publications increased and networks evolved over time. The global network is organised around a few co-authoring, mostly publicly financed key actors, highly connected with powerful and broker positions. The results present an extensive narrative how modern empirical scientific methods for data processing and translation can supplement evidence-based arguments for public discussion on the status and focus of global EVD research. Based on the network characteristics or concentration of expertise, we recommend a globally agreed and prioritized EVD research agenda may facilitate the translation of this research into new EVD tools. Also, to analyse research networks regularly to enable public discussion on the direction in which research could be organized and optimised. We would like to encourage others to utilize our methods with open access tools to enhance new methods to the field of NTD R&D. | lay_plos |
Hepatitis C virus (HCV) entry, translation, replication, and assembly occur with defined kinetics in distinct subcellular compartments. It is unclear how HCV spatially and temporally regulates these events within the host cell to coordinate its infection. We have developed a single molecule RNA detection assay that facilitates the simultaneous visualization of HCV (+) and (−) RNA strands at the single cell level using high-resolution confocal microscopy. We detect (+) strand RNAs as early as 2 hours post-infection and (−) strand RNAs as early as 4 hours post-infection. Single cell levels of (+) and (−) RNA vary considerably with an average (+): (−) RNA ratio of 10 and a range from 1–35. We next developed microscopic assays to identify HCV (+) and (−) RNAs associated with actively translating ribosomes, replication, virion assembly and intracellular virions. (+) RNAs display a defined temporal kinetics, with the majority of (+) RNAs associated with actively translating ribosomes at early times of infection, followed by a shift to replication and then virion assembly. (−) RNAs have a strong colocalization with NS5A, but not NS3, at early time points that correlate with replication compartment formation. At later times, only ~30% of the replication complexes appear to be active at a given time, as defined by (−) strand colocalization with either (+) RNA, NS3, or NS5A. While both (+) and (−) RNAs colocalize with the viral proteins NS3 and NS5A, only the plus strand preferentially colocalizes with the viral envelope E2 protein. These results suggest a defined spatiotemporal regulation of HCV infection with highly varied replication efficiencies at the single cell level. This approach can be applicable to all plus strand RNA viruses and enables unprecedented sensitivity for studying early events in the viral life cycle. Hepatitis C virus (HCV) belongs to the Flaviviridae family of enveloped, positive-stranded RNA viruses. Following productive entry into hepatocytes, the 9. 6 kb HCV genome is translated to produce a single large polyprotein [1], which is cleaved by viral and host proteases to yield ten distinct protein products [2]. These proteins include the structural proteins (core, E1 and E2) and the non-structural proteins (p7, NS2, NS3, NS4A, NS4B, NS5A, and NS5B). The five “replicase” proteins NS3 to NS5B are essential and sufficient for HCV RNA replication [3,4]. Similar to all other positive strand RNA viruses, HCV induces rearrangements of intracellular membranes to create a favorable microenvironment for RNA replication to occur [5–8]. Replication complex formation appears to require the viral NS4B and NS5A proteins [5,9]. NS5B, the viral RNA-dependent RNA polymerase is the key enzyme of the replicase complex [10,11]. Using the (+) strand genome as a template, NS5B first synthesizes a complementary (−) strand, resulting in a double-stranded (ds) RNA intermediate, and then proceeds to transcribing progeny (+) strands. Newly synthesized (+) strand RNAs are then thought to be shuttled out of replication compartments to serve as templates for further translation by cellular ribosomes or become encapsidated into assembling virions on the surface of lipid droplets (LDs) [12]. Although these processes are likely linked, a single viral (+) strand RNA can only be involved in either translation, replication or packaging at a given time, and the switch from one process to another has to be regulated [13]. For HCV, the switch from translation to replication is unclear. The cellular protein Ewing sarcoma breakpoint region 1 (EWSR1) binds to the viral RNA cis acting replication element (CRE), and has been proposed to regulate the switch from translation to replication by modulating the kissing interaction between the CRE and a RNA stem-loop structure in the HCV 3’ UTR [14]. Similarly, for polioviruses, the switch from translation to replication is regulated by the action of viral proteases on a cellular protein binding to the 5’cloverleaf viral RNA structure [15]. The switch from replication to assembly is not well understood, however it has been suggested that the phosphorylation state of NS5A might regulate the process [16]. It is also possible that HCV (+) RNA fate is spatially regulated by the distinct subcellular localizations of translation, replication and assembly. It is not clear how HCV spatially and temporally regulate its lifecycle within the host cell. Mathematical models have been developed to study HCV RNA dynamics during primary infection of chimpanzees [17], and humans (liver transplantation [18,19], response to IFN [20] and ribavirin treatment [21]), in addition to HCV replicons in cell culture [22–25]. Recently, data on the dynamics of infectious HCV RNA in cell culture became available [26]. While these past studies were focused on whole cell populations, organs and organisms, data on viral RNA kinetics in single cells are lacking. Advances in single RNA detection methods using ISH followed by amplification [27] enable the analysis of HCV RNA at the single cell level. Previous studies have utilized this approach to detect (+) strand HCV RNAs in hepatoma cell lines [28] as well as in infected human liver biopsies [29]. To better understand the spatiotemporal organization of HCV infection, we have developed RNA detection methods that allow for simultaneous visualization of HCV (+) and (−) RNA strands at the single cell level. We have combined this approach with 4-color high-resolution confocal microscopy to study the localization of (+) and (−) HCV RNAs with viral and host proteins of interest. Using this single cell RNA detection approach, we can interrogate the cell-to-cell variability of HCV infection kinetics. Moreover, we can determine whether (+) strand fates are regulated temporally. The approach developed in this study can be generically applicable to all RNA viruses and enables unprecedented sensitivity for studying early events in the viral life cycle, which are unappreciated for viruses of relatively low replicative capacity, including some viruses of the Flaviviridae, due to limits of detection. We used the QuantiGene ViewRNA ISH detection system to specifically detect HCV (+) and (−) strand RNAs in single cells. Briefly, non-overlapping oligonucleotide probe sets specific to either the (+) or (−) strand of HCV NS3/4A region were hybridized to target RNAs and sequential hybridization steps provided up to 8,000-fold signal amplification. To validate the specificity of labeling, wild type or polymerase defective (GND) subgenomic JFH1 HCV replicon RNAs were electroporated into Huh-7. 5 cells, which were then fixed and processed for HCV RNA detection (Fig. 1A) and quantification (Fig. 1B). (+) strand RNA, comprising a mix of input and newly synthesized RNA, was abundant at 6 hours post-electroporation (hpe) and increased up to 20 fold by 96 hpe for the wild type, but not the polymerase defective replicon containing cells. (−) strand RNA was also detected at 6 hpe and increased at a similar rate as the (+) strand by 96 hpe for wild type HCV. There was no (−) strand detected at any of the time points for the GND replicon, thus confirming that polymerase function is required for (−) strand RNA detection. As expected, there was no signal for either (+) or (−) HCV RNAs detected in mock infected cells. To further confirm the specificity of our (−) strand RNA labeling in the context of HCV infection, we used the HCV direct-acting antivirals Sofosbuvir and Daclatasvir. Sofosbuvir is a nucleotide analogue inhibitor of the NS5B polymerase [30] already approved for anti-HCV therapy in combination with ribavirin or other direct acting antivirals such as Daclatasvir, Ledispavir, or Simeprevir [31]. Daclatasvir is a potent inhibitor of the NS5A protein [32] that is already approved for anti-HCV therapy in Europe and Japan [33]. As expected, there was very little (−) strand accumulation at 6 and 48 hours post-infection (hpi) in cells treated with either Sofosbuvir or Daclatasvir at time of infection. In contrast, (−) strand was detected at 6 hpi and increased 10-fold by 48 hpi in DMSO treated cells (S1 Fig.). We did not observe an effect of either inhibitor on (+) strand levels at 6 hpi, however by 48 hpi there was a drastic decrease in (+) strand numbers due to the replication defect imposed by the antiviral drugs. The kinetics of accumulation and ratios of HCV (+) and (−) strand RNAs are unknown at the single cell level. To this end, we quantified HCV (+) and (−) strand RNAs at the single cell level over a time course of infection. As expected, we observed (+) and (−) strand HCV RNA numbers increased over time (Fig. 2A). Quantitation of (+) and (−) strand RNAs showed considerable cell-to-cell variability (>10-fold) with respect to the number of RNA puncta per cell plane (Fig. 2B). (+) strand puncta could be detected as early as 2 hpi in ~80% of cells, which likely reflected input genomic RNA. (−) strand puncta were reliably detected in cells at 6 hpi, suggesting that viral RNA replication is established by 6 hpi. Consistent with this interpretation, we observe modest increases in (+) RNA at 6 and 12 hpi. Much larger increases in (+) and (−) RNA accumulation occurred between 12 and 24 hpi, suggesting that this time point correlates with more robust HCV replication. By 48 hpi, an average of 331±52 (+) RNAs and 49±12 (−) RNAs were observed per cell plane. In order to correlate this value with HCV RNAs per cell, we performed Z-stack analysis of HCV infected cells at 48 hpi and found that the average per cell number was 415±39 (+) RNAs and 94±11 (−) RNAs per cells (S1 Movie). Thus, the fluorescence per cell plane captures the majority of the whole cell RNAs. This high percentage reflects the high fluorescence of the RNA ISH signal, in addition to the relative flatness of Huh-7. 5 cells. We observed a (+) strand to (−) strand ratio of about 10: 1 throughout most of the time course, which is in agreement with the ratio determined in infected hepatocytes in humans [34] as well as previous reports using subgenomic replicon systems [3,35]. Notably, this ratio was about 6: 1 early in infection (6 hpi), which correlates with the ratio of (+): (−) strands inside replication complexes [35]. Although the average (+): (−) RNA ratio of ~10 was relatively constant, there was significant cell-to-cell variability in ratio, ranging from 1–35. We next developed a series of microscopy assays to visualize the association of HCV RNA with markers of specific stages of the viral life cycle. HCV translation was defined as the colocalization of (+) RNAs with actively translating ribosomes (Fig. 3). Active RNA replication was defined as colocalization of HCV (−) RNA with (+) RNA or replicase components NS3 and NS5A (Fig. 4, Fig. 5). HCV assembly was defined as colocalization of (+) RNA with core and intracellular virions were defined as colocalization of (+) RNA with virion E2 (Fig. 6). A caveat to the interpretation of these assays is that colocalization of HCV RNA with these markers does not define a physical interaction. (+) RNA viruses cannot be simultaneously translated (with ribosomes moving 5’ to 3’) and replicated (with the replicase moving 3’ to 5’) [13]. These processes need to be coordinately regulated, either by spatially separating (+) strand RNAs destined for translation and replication, or by regulating the initiation of these processes. In the case of HCV, the mode of regulation is not yet known, nor is it known whether translation occurs in the proximity of replication compartments. To address these questions, we applied a ribosome-bound nascent chain puromycylation assay [36] that detects actively translating ribosomes in our single molecule HCV RNA detection assay. Briefly, puromycin, a Tyr-tRNA mimetic, enters the ribosome A site and terminates translation. Puromycylated nascent chains are stalled on ribosomes by cycloheximide, a chain elongation inhibitor, and detected via fixed cell immunofluorescence using an anti-puromycin monoclonal antibody. In the absence of puromycin, we detect no fluorescent signal, while puromycin labeling is readily detected in the presence of puromycin (Fig. 3A), primarily in the cytoplasm but also some in the nucleus, consistent with a previous report [36]. To confirm the specificity of puromycin labeling, cells were pre-treated with anisomycin, a competitive inhibitor of translation, prior to puromycylation. As expected, anisomycin drastically decreased puromycin labeling (Fig. 3A). We next performed the puromycylation assay in combination with RNA ISH over a time course of HCV infection (Fig. 3B, C). (+) RNA translation, as defined by (+) RNA localization with puromycylated ribosomes, occurred as early as 2 hpi, with a peak in the percent of (+) RNAs undergoing translation (70%) at 6 hpi. Between 12 and 24 hpi, a steady state level of translated (+) RNAs was achieved at ~35%. While the (−) RNA is unlikely to be associated with actively translating ribosomes, we do observe that a high percentage of HCV (−) RNAs also colocalize in the proximity of puromycylated ribosomes. This indicates that sites of viral RNA replication and translation may be in close proximity. Interestingly, we observed an unusual puromycylation staining pattern in ~20% of the cells at 6 hpi, in which enlarged puromycylation puncta were detected (Fig. 3B). It is possible that these enlarged puncta reflect ER rearrangements (Fig. 3B). In support of this interpretation, we observed a similar localization pattern of the ER marker calnexin at this time point of infection (Fig. 3D). HCV RNA synthesis involves a dsRNA intermediate, which localizes inside the viral induced replication complexes [9,37]. We next quantified (+) and (−) RNA colocalization over a time course of infection as a marker of HCV RNA replication (Fig. 4A, B). We observed that (−) strand puncta colocalizing with (+) strand puncta became detectable as early as 4 hpi; however, it was generally a low frequency event from 4–12 hpi. The frequency of HCV (+) and (−) RNA colocalization increased significantly between 12 and 24 hpi, with ~25–35% of (−) strand puncta colocalized with (+) strand puncta. This percentage remained relatively constant from 24–72 hpi, suggesting that only ~1/3 of (−) strands are actively engaged in RNA replication at a given time. Unfortunately, attempts to correlate the (+) and (−) RNA colocalization with dsRNA localization using the commonly used J2 dsRNA antibody were unsuccessful, due to incompatible fixation and permeabilization conditions. We next examined the localization of HCV RNAs with viral protein components of the replication complex. HCV NS5A is a multifunctional protein involved in both the replication and assembly stages of HCV life cycle [4,38–40]. NS5A appears to have at least two functions in HCV RNA replication. It is part of the replicase complex that binds viral RNA [3,4] and it also promotes the formation of double membrane vesicles, which are thought to be the sites of viral RNA replication [9]. The function of NS5A in replication complex formation is regulated in part by the interaction of NS5A with its host cofactor, phosphatidylinositol-4-kinase III-α (PI4KA) [41–43]. Additionally, NS5A partially colocalizes with core protein on surface of lipid droplets and is required for virion assembly [12,38]. We performed a time course of (+) and (−) strand RNA colocalization with the NS5A protein during HCV infection (Fig. 5A). Since NS5A protein levels early in infection are undetectable by standard immunofluorescence analysis we used a tyramide signal amplification system (TSA) to detect NS5A at 6 and 12 hpi. We observed limited colocalization of NS5A with either (+) or (−) RNA at 6 hpi; however, there was a dramatic, specific increase in NS5A colocalization with (−) RNA at 12 hpi (~90% of (−) RNA colocalized with NS5A, Fig. 5B). At later time points, ~30% of NS5A colocalized with (−) RNA, which would suggest, only ~30% of (−) strand RNAs undergo replication at later times of infection, which is consistent with the observed colocalization of (−) with (+) RNA. The localization of NS5A with (+) RNA increased over time, consistent with its role in virion assembly (Fig. 5B). NS3 is also part of the replicase complex and its helicase activity is required for HCV replication and possibly assembly [7,44,45]. In contrast to NS5A, NS3 did not preferentially colocalize with (−) strands at early time points (Fig. 5C, D). At later time points, we observed that NS3 had similar levels of RNA colocalization as was observed for NS5A (~30%). The distinct frequencies of (−) RNA localization with NS3 and NS5A are consistent with the interpretation that NS5A, but not NS3, is involved in the initial formation of replication compartments, while both NS3 and NS5A are involved in replicase function. The current model for HCV assembly is that ER-derived HCV replication complexes are in close proximity to the sites of virion assembly, intracellular lipid droplets (LDs). The viral core (capsid) protein accumulates on or near the surface of LDs and mutants that attenuate virion assembly lead to a hyper-accumulation of core at the LD [6,46,47]. Viral RNA containing capsids are then enveloped at the ER and egress cells via the secretory pathway in association with components of the VLDL machinery [48–50]. We first evaluated the compatibility of the RNA ISH protocol with detection of virion associated HCV RNA. Buoyant density purified HCV virions were processed for RNA detection followed by immunostaining for core protein. As shown in S2 Fig., ~60% (+) RNA puncta (red) colocalize with core puncta (green). Thus, single molecule virion RNAs can be detected by the RNA ISH protocol. We then quantified (+) and (−) strand RNA colocalization with HCV core at 24,48 and 72 hpi (Fig. 6A, B). As expected, we observed (+) strand colocalization with core that increases with time. ~15% of (+) RNA is colocalized with core at 24 hpi, which increases to ~35% at 72 hpi. We did observe some (−) strand colocalization with core; however it did not increase over the time course, suggesting that it was unrelated to virion assembly. An intensity line profile (Fig. 6C) showed that the (−) strand (magenta) signal, although “colocalized” does not identically overlap with LD-associated core, but is instead juxtaposed to the LD-associated core. In contrast, the (+) strand (red peak) overlaps the core (green) peak suggesting full colocalization. Thus, residual localization of (−) RNA with core likely reflects the close proximity of sites of replication and assembly. To quantify intracellular assembled virions we made use of an antibody (CBH-5) that recognizes E2 on fully assembled virions [51]. This E2 localization pattern is very different from the pattern of E2 localization using E2 antibodies that recognize ER localized E2 (S3 Fig.). Furthermore, CBH-5 staining in an established HCV assembly mutant (NS2-G10P) [52] is at background levels similar to an uninfected control (S4 Fig.). At 48 and 72 hpi, approximately 10% of (+) strand puncta colocalize with E2 (Fig. 6D, E). We observe that virtually all of the E2 puncta (green) colocalize with (+) strand RNAs (red), while minimal colocalization of E2 was observed with (−) strand RNAs (magenta). We next plotted the kinetics of the HCV life cycle based on quantitation of the localization of HCV RNAs with markers of translation ( (+) strands with puromycylated ribosomes), replication compartment formation ( (−) RNA localized with NS5A), active replication (localization of (−) RNA with (+) RNA and NS3), and assembly and intracellular virions ( (+) RNA colocalization with core and virion E2, respectively). The standard quantitation of HCV RNA by quantitative RT-PCR (Fig. 7A) and infectious HCV (Fig. 7B) show a typical increase of both over time. HCV RNA increased at 12–24 hpi and plateaued between 48 and 72 hpi, while intracellular virions peaked between 24 and 48 hpi and plateaued by 72 hpi. We first quantified the total (+) RNAs devoted to translation, replication, assembly, or virions as determined in our previous assays (Fig. 7C). We observed similar kinetics of (+) RNA accumulation as compared to the qRT-PCR assay. HCV (+) RNAs were initially associated with translation, while (+) RNAs associated with replication increased from 12 to 48 hpi and then slightly decreased. HCV RNAs associated with virion assembly increased between 24 and 48 hpi and plateaued. We then quantified the relative amounts of (+) RNA devoted to each process (Fig. 7D). (+) strand RNAs display a defined temporal kinetics, with the majority of (+) RNAs associated with actively translating ribosomes at early times of infection, followed by a peak of replication between 12 and 24 hpi, followed by virion assembly and detection of assembled virions in the cell cytoplasm. Interestingly, after the peaks of (+) RNA association with translation and then replication were displaced, both populations achieved a steady state level of ~25% of total (+) RNAs each, which slowly decayed over time as the levels of virion-associated (+) RNAs increased. In comparing the relative kinetics of each process in the viral life cycle, the slowest kinetic delay is from HCV assembly to accumulation of intracellular virions (Fig. 7D). Quantitation of HCV (−) RNA colocalization with markers of replication revealed distinct phases of HCV replication (Fig. 7E). Transient active replication could be observed at early time points (4 hpi), however, robust active replication did not occur until between 12 and 24 hpi. This was preceded by a high level of colocalization of NS5A with (−) RNA. These data are consistent with a model wherein low levels of HCV RNA replication precede replication compartment (or membranous web) formation, replication compartments are then formed at 12 hpi, which is then followed by robust HCV RNA replication. The percent of HCV (−) RNAs engaged in active RNA replication then remains relatively constant throughout the time course of infection at ~35%. Recent improvements in multiplexed single molecule RNA FISH enable sensitive single cell detection of viral RNAs in infected cells. This approach has been used to detect RNA from a number of viruses, including the (+) strand RNA of HCV in vitro and in vivo [29,53]. In this study, we developed assays for the specific co-detection of HCV (+) and (−) RNA in the same infected cell, in combination with high-resolution microscopy to quantify the kinetics of HCV RNAs at the single cell level. We verified the specificity of these assays. (+) RNA detection was specific to HCV infected- or transfected-cells. RNA puncta increased in abundance during infection, which was precluded by HCV inhibitors. HCV (−) RNA detection had similar properties, in addition to being specific for cells transfected with wild type, but not polymerase defective, HCV RNAs (Fig. 1 and S1 Fig.). We confirmed that this approach has single molecule sensitivity and that the fixation/detection method is compatible with detection of HCV RNAs in infectious virions (S2 Fig.). We then used this assay to perform a kinetic analysis of HCV infection at the single cell level (Fig. 2). HCV (+) RNA was readily detected at 2 hpi, while the (−) RNA was detected at 6 hpi. Both (+) and (−) RNA accumulation increased over a 48 hour time course, as would be expected. There was considerable cell-to-cell variability in both (+) and (−) RNA accumulation with both showing an ~10-fold range in values at a given time point. This variability was also reflected in (+) / (−) RNA ratio, which ranged from less than one to greater than 35 depending on the infected cell. This large variability in (+) / (−) ratio suggests that distinct host factors that differentially regulate (+) and (−) RNA synthesis are limiting in individual Huh-7. 5 cells. Despite this cell-to-cell variability, the mean (+) / (−) RNA ratio of ~10 was relatively constant and is consistent with previous estimates using strand-specific northern blot analysis of HCV replicon cells [3,35] as well as qRT-PCR analysis of HCV-infected whole cell lysates [26]. We next developed microscopic assays to identify HCV RNAs associated with actively translating ribosomes, replication complexes, nucleocapsid assembly, and intra-cellular virions. The colocalization of HCV (+) RNAs with puromycylated ribosomes indicated actively translating HCV RNAs. At 6 hpi, the majority of (+) RNAs were associated with translating ribosomes, and this association subsequently decreased to a steady state of ~30% of total HCV RNAs over the time course of infection (Fig. 3). Interestingly, there was also a high level of colocalization of puromycylated ribosomes with HCV (−) RNAs. While it is possible that HCV (−) RNAs are associated with ribosomes, a more likely interpretation is that sites of HCV translation and replication are in close proximity. (+) RNAs cannot be simultaneously translated and replicated due to steric hindrance between ribosomes traveling 5’ to 3’ and the HCV replicase moving 3’ to 5’ [13]. Models for the differential regulation of HCV translation and replication include spatial separation (e. g. the exclusion of ribosomes from replication compartments) and/or the differential regulation of these processes via protein-RNA and RNA-RNA interactions. The viral polymerase NS5B can only initiate RNA synthesis from the same RNA that it has been translated from (a cis requirement) [54]. Additionally, viral translation is dependent on active RNA replication [55], suggesting that the translation complexes have to be in very close proximity to the replication complexes. In this case, the decision whether to translate or replicate the viral RNA may be modulated by regulation of HCV RNA-RNA or RNA-protein interactions. For example, the HCV RNA kissing loop interaction between the HCV cis-acting replication element and the 3’UTR by EWSR1 may promote a switch between HCV translation and replication [14]. Our data suggest that replication and translation are spatially linked. Development of puromycylation assays compatible with immuno-EM may answer the question of whether active translation occurs within, or adjacent to, HCV replication compartments. Detection of the (−) RNA, the replication intermediate, is indicative of a viral replication complex. We detect (−) RNA as early as 4 hpi in infected cells, some of which is involved in active replication, as defined by (+) RNA colocalization (Fig. 4). By 12 hpi the majority of (−) RNA is associated with NS5A, but not NS3 or (+) RNA. NS5A has been implicated in the formation of HCV replication compartments [9], which may explain this selective HCV (−) RNA/NS5A colocalization at 12 hpi. We also observe high levels of colocalization of (−) RNA at the 6 hpi time point with large globular fluorescent labeling of ER markers, including ribosomes and calnexin, in a subset of cells (Fig. 3B, D). Given the kinetic coincidence between (−) RNA appearance, NS5A association, and altered ER morphology, it is possible that this represents large scale alterations of the ER associated with replication complex formation. Alternatively, it may be a stress response to infection, such as induction of the ER stress response. The appearance of (−) RNA and low levels of transient dsRNA replication intermediates at 4 hpi appears to precede HCV replication compartment/membranous web formation, as defined by extensive (−) RNA/NS5A colocalization at 12 hpi, and robust HCV RNA replication (12–24 hpi). The proposed kinetics of membranous web formation is consistent with previous EM data that described the initial appearance of intracellular double membrane vesicles at 16 hpi and further accumulation until 24 hpi [9]. The transient HCV replication prior to replication compartment formation suggests a strategy wherein HCV synthesizes limited amounts of (+) and (−) RNAs early during infection to decrease its reliance in the integrity of the initially infecting HCV (+) RNA. Any damage or modifications to the initial (+) RNA would result in an abortive infection, this it makes sense that the virus would synthesize a small pool of viral RNAs early to maximize the probability of a productive infection. It is unclear whether these early replication intermediates (4 hpi) are shielded by a small amount of membrane remodeling that precedes the robust replication compartment formation at 12 hpi. If these replication intermediates are not membrane-protected, they may be vulnerable to cytosolic RNA sensors, such RIG-I-like RNA sensors and dsRNA activated protein kinase R (PKR). It remains to be determined whether these RNAs are recognized by innate immune sensors or alternatively, whether HCV has evolved a mechanism to protect these RNAs from detection. Future studies will examine the localization of innate immune RNA sensors with HCV (+), (−), and dsRNAs. Higher proportions of active replication complexes, as defined by colocalization of (−) RNA with either (+) RNA or NS3 are detected later in infection, between 12 and 24 hpi. This time point corresponds to increases in (+) and (−) RNA fluorescent puncta (Fig. 4), in addition to HCV RNA levels by quantitative real time RT-PCR (Fig. 7A). The proportion of HCV replication complexes that are defined as active (% of (−) RNAs that colocalize with NS3 or (+) RNAs) is consistently ~30% after 24 hpi. This is consistent with predictions from cryo-EM analysis of HCV replication complexes, which indicate that some HCV replication compartments are not compatible with active replication complexes [9]. The specific viral (−) strand detection is of practical interest, since despite extensive efforts; there are few good markers of HCV replication complexes for microscopy analysis. Only a small fraction of the NS5B polymerase (<5%) resides inside replication complexes [35] and other replicase proteins (NS3 and NS5A) have multiple localizations beyond the replication complex. Similarly, there are no cellular proteins have been described that are consistently localized solely to replication complexes. We and many other labs have used a dsRNA antibody to detect putative viral RNA replication complexes [37,56]. Although, this antibody recognizes antigen within replication compartments and is specific to infected cells [9], it is possible that the antibody may also detect structured viral RNAs. We attempted to assess the degree of colocalization of the dsRNA antibody with (−) RNA; however, the fixation and permeabilization steps of the two detection methods were not compatible. HCV (+) RNAs associated with core both in the proximity of lipid droplets, which are putative sites of nucleocapsid assembly, and in discrete puncta, which may represent assembled virions. The percent HCV (+) RNA that colocalized with core increased during the time course from ~15% at 24 hpi to ~35% at 72 hpi (Fig. 6). Similarly the colocalization with (+) RNA and an E2 antibody that preferentially recognizes E2 incorporated into virions increased over the time course from virtually none at 24 hpi to ~12% of (+) RNAs at 72 hpi. This kinetics mirrors the production of infectious HCV (Fig. 7B). We observed a juxtaposition of ~15% of HCV (−) RNAs with core, consistent with the spatial linkage between sites of RNA replication and assembly. No HCV (−) RNA colocalized with virion E2. The development of assays to define (+) RNAs associated with translation, replication, nucleocapsid assembly, and intra-cellular virions allowed us to quantify the total number of (+) RNAs, in addition to the proportion of (+) RNAs, associated with each process (Fig. 7). We observed an initial association of (+) RNAs with translation, followed rapidly by replication. The number of (+) RNAs associated with translation or replication increased with similar kinetics and abundance until 48 hpi, after which they diminished slightly. Association of (+) RNAs with nucleocapsid assembly was detected at 24 hpi and peaked at 48 hpi, after which it plateaued as the most abundant class of HCV (+) RNAs in infected cells. Putative association of (+) RNAs with intracellular virions was detected at 48 hpi and increased slightly at 72 hpi. Analysis of the proportion of (+) RNAs associated with each process in the viral life cycle revealed a tightly coordinated regulation. An initial peak of HCV translation at 6 hpi was rapidly displaced by HCV replication at 12–24 hpi, after which both maintained a steady state level of ~25% each of total HCV (+) RNAs. The proportion of (+) RNA associated with nucleocapsid assembly began to displace the replication peak at 24 hpi, after which it gradually increased throughout the time course. Finally, ~10% of HCV (+) RNA associated with intracellular virions was detected at 48 hpi and beyond. The slowest kinetic delay that we observed is from HCV assembly to accumulation of intracellular virions (Fig. 7D). This is consistent with the observation that core in HCV JC1-infected cells (similar to the virus used in this study) has reduced localization at LDs as compared with the less efficiently assembled HCV JFH1 virus [57], suggesting that virion nucleocapsid assembly is not a rate-limiting event in core trafficking, but that HCV envelopment may be rate-limiting. Future studies will investigate viral and cellular factors that regulate the temporal kinetics of (+) RNA fate, and thus the coordinated integration of the viral life cycle. Huh-7. 5 cells [58] were grown in Dulbecco’s modified high glucose media (DMEM; Invitrogen) supplemented with 10% fetal bovine serum (FBS; Atlanta Biologicals), nonessential amino acids (NEAA, 0. 1mM; Gibco), 1% penicillin-streptomycin (Gibco), and maintained in 5% CO2 at 37°C. For viral stock propagation, Huh-7. 5 cells were electroporated with HCV genotype 2a infectious clone pJFHxJ6-CNS2C3 RNA [59], which is similar to the HCV JC1 virus [60], and viral supernatants were collected for up to 5 passages of the electroporated cells. Viral titers were determined by limiting dilution and immunohistochemical staining using an antibody directed to NS5A (9E10) [61]. Drugs were dissolved in DMSO and used at indicated concentrations. Sofosbuvir (PSI-7977) (Medchem Express), 10 μM; Daclatasvir (SelleckChem) 1nM. The QuantiGene ViewRNA ISH Cell Assay kit (QVC0001) and probes were purchased from Affymetrix, CA. The (+) strand probe set (VF1–10121) is designed to anneal to bases 3733–4870 of the HCV JFH1 genome, while the (−) strand probe set (VF6–11102) anneals to the negative strand with corresponding bases 4904–5911 on the positive strand. For RNA ISH, Huh-7. 5 cells seeded onto 12 mm round coverslips at a confluency of 60–70% were infected with HCV at a multiplicity of infection (MOI) of 1. 5. At indicated times post-infection, coverslips were fixed in 4% paraformaldehyde for 30 minutes and permeabilized with 70% ethanol for 1 hour at 4°C. Subsequently, coverslips were co-incubated with the (+) and (−) HCV specific probes (1: 100 in Probe Set Diluent QF) for 3h at 40°C. Washing and signal amplification were performed as per manufacturer’s instructions. Following the last wash, coverslips were blocked with 20% goat serum in PBS followed by overnight incubation with primary antibodies as follows: 1: 25000 anti-NS5A 9E10 (a kind gift of Charles Rice, Rockefeller University), 1: 200 anti-NS3 (Virogen), 1: 1000 anti-calnexin (Enzo Life Sciences), 1: 100 anti-core (Virostat), 1: 100 anti-E2 CBH-5 (a kind gift from Steven Foung, Stanford University), 1: 2000 anti-E2 1C1 (a kind gift from Arash Grakoui, Emory University). Following primary antibody incubation, coverslips were washed two times with PBS and incubated with AlexaFluor 488 conjugated secondary antibody (1: 1000) for 1 hour before mounting on Prolong Gold with DAPI reagent (Invitrogen). The tyramide signal amplification kit (Molecular Probes; T20912) was used as per manufacturer’s instructions. Following QuantiGene ViewRNA ISH labeling, cells in coverslips were incubated in peroxidase quenching buffer for 1 hour to quench endogenous peroxidase activity. Following blocking with 1% provided blocking reagent, coverslips were incubated with primary antibodies (1: 25000 anti-NS5A or 1: 200 anti-NS3) for 1 hour. Coverslips were washed two times with PBS and then incubated with a 1: 150 dilution of the HRP conjugate for 1 hour. Following two washes in PBS, coverslips were labeled with the tyramide working solution for 5 minutes before mounting on Prolong Gold reagent (Invitrogen). Huh-7. 5 cells seeded onto poly-lysine treated coverslips were incubated in media supplemented with 91 μM puromycin (Invitrogen) and 208 μM emetine (Sigma) for 5 minutes at 37°C. Cells were then incubated for 2 minutes on ice with permeabilization buffer (50 mM Tris-HCl, pH 7. 5,5 mM MgCl2,25 mM KCl, 355 μM cycloheximide, EDTA-free protease inhibitors, 10 U/ml RNaseOut and 0. 015% digitonin. After this extraction step, cells were washed once with PBS, fixed in 4% paraformaldehyde and processed for QuantiGene ViewRNA ISH. Following RNA labeling and blocking, cells were incubated with the PMY-2A4 monoclonal antibody developed by Jonathan Yewdell, National Institutes of Health, and obtained from the Developmental Studies Hybridoma Bank, University of Iowa. Coverslips were imaged for (+) strand HCV RNAs by using the PMT detector set for the 566–608 nm wavelength range, (−) strand by using the HyD detector set for the 650–714 nm wavelength range, AlexaFluor 488 was visualized by using the HyD detector set for the 495–535 nm wavelength range, and DAPI stained nuclei were visualized by using the PMT detector set for the 424–465 nm wavelength range. Immunofluorescence images were taken using a Leica SP5 II AOBS Tandem Scanner Spectral confocal microscope with a 100X 1. 46 oil objective. Post-acquisition, images were processed using Fiji (Image J) software. Specifically, each red, green, magenta and blue stack was separated into individual channels. Each channel was processed by using the Filters/Unsharp Mask tool and then merged into a single image. Colocalization was determined by using the JACoP plugin post image threshold. The Manders’ coefficients were converted to % colocalization. Each graph data point represents the average of 25 images (single cells) and depending on the time point the average total number of (−) strand puncta analyzed ranges from ~25 to ~2500 while the total number of (+) strand puncta analyzed ranges from ~25 to ~10000. At indicated times post-infection, cells were washed twice with PBS and lysed in RLT lysis buffer (Qiagen). RNA was isolated using the RNAeasy kit (Qiagen). HCV RNA levels were analyzed using the Platinum qRT-PCR Thermoscript One-Step System (Applied Biosystems) and a custom designed primer probe set: Forward: 5’-ACTTCATTAGCGGCATCCAATAC, Reverse: 5’-CGGCACTGAATGCCATCAT, Probe: 5’-6FAM-CAGGATTGTCAACACTGCCAGGGAACC-Iowa Black. RNA was reverse transcribed for 30 min at 50°C followed by an inactivation step (95°C for 6 min). The cDNA was then amplified for 50 cycles of 95°C for 15 seconds, 60°C for 1 min. HCV RNA levels were normalized to 18S ribosomal RNA. All assays were performed on an ABI 7300 system and analyzed with SDS 1. 3 software (Applied Biosystems). T-test was performed to compare data sets. P-values less than 0. 05 were considered significant. | The stages of the viral life cycle are spatially and temporally regulated to coordinate the infectious process in a way that maximizes successful replication and spread. In this study, we used RNA in situ hybridization (ISH) to simultaneously detect HCV (+) and (−) RNAs and analyze the kinetics of HCV infection at the single cell level as well as visualize HCV RNAs associated with actively translating ribosomes, markers of viral replication compartment formation, active RNA replication, nucleocapsid assembly, and intracellular virions. We observed a spatial linkage between sites of viral translation and replication, in addition to replication and assembly. HCV (+) RNAs follow a tight temporal regulation. They are initially associated with translating ribosomes, followed by a peak of replication that achieves a steady state level. The remaining HCV (+) RNAs are then devoted to virion assembly. Analysis of HCV (−) RNAs revealed that low levels of transient RNA replication occur early after infection prior to the formation of devoted replication compartments and robust replication. This suggests that HCV synthesizes additional (+) and (−) strands early in infection, likely to decrease its reliance on maintaining the integrity of the initially infecting (+) RNA. | lay_plos |
Introduction Congress has many techniques for obtaining documents from the executive branch, including simple requests, committee investigations, subpoenas, and holding executive officials in contempt. One procedure, used only in the House of Representatives, is the resolution of inquiry, which "is a simple resolution making a direct request or demand of the President or the head of an executive department to furnish the House of Representatives with specific factual information in the possession of the executive branch." It has been the practice to use the verbs "request" in asking for information from the President and "direct" when addressing department heads. Resolutions of inquiry are often much more effective in obtaining information from the executive branch than one would expect from viewing committee and floor action. Administrations have often released a substantial amount of information, leading the committee of jurisdiction to conclude that the dispute is moot and it is therefore appropriate to report the resolution adversely and table it on the floor. As examples in this report demonstrate, the sponsor of a resolution will often support an adverse report and tabling action because the Administration has substantially complied with the resolution. There is no counterpart in current Senate parliamentary practice for resolutions of inquiry, although there are precedents dating to the end of the 19 th century and an effort in 1926. Nothing prevents the Senate from passing such resolutions, but apparently the Senate is satisfied with the leverage it has through other legislative means, including the nomination process and Senate "holds." Unlike the House, the Senate has no special practices for expediting consideration through committee discharge or non-debatable motions, and resolutions are not generally privileged for immediate consideration. Origins of Practice From its very first years, Congress has requested information from the executive branch to further legislative inquiries. Initially, these requests did not depend on a House rule. They were made pursuant to the implied authority of Congress to investigate the executive branch. For example, in 1790 the House investigated the receipts and expenditures of public moneys during Robert Morris's term as Superintendent of Finance during the Continental Congress. Congress sought documents from the executive branch in 1790 to judge the size of an annuity to be given Baron Frederick von Steuben. As part of its 1792 investigation into the military losses suffered by the troops of Major General Arthur St. Clair, the House received a substantial number of documents from the War Department. These early investigations differed in scope and procedure from the House resolution of inquiry, which depends not on Congress operating as the "Grand Inquest" but by a special rule that grants privileged status to a lawmaker's motion to obtain documents from the executive branch. Early House rules contained no procedure for requesting information from the President or cabinet officials. Throughout its first two decades, however, the House made repeated requests to the President and departmental heads for information, sometimes to be returned to Congress, and sometimes to the states. For example, on January 5, 1797, the House took up this resolution (involving the concurrence of both chambers): Resolved, by the Senate and House of Representatives of the United States of America in Congress assembled, That the President of the United States be requested to give information to the several States who were, by the Commissioners appointed to settle accounts between the United States and the individual States, found indebted to the United States of the several sums in which they were so found indebted.... This type of resolution differed from the resolution of inquiry, because it lacked privileged status under House rules. Similarly, in 1811, the House considered a resolution requesting the President to lay before the House "a list of the whole number of persons impressed, seized, and otherwise unlawfully taken from on board vessels sailing under the United States' flag on the high seas or rivers, in ports and harbors." In 1820, the House clarified its rules for requesting information from the executive branch. There was concern that the House had not been acting with sufficient consideration before making such requests. In offering an amendment to House rules on December 12, 1820, Representative Charles Rich noted that "six clerks had been constantly employed, from the close of the last session to the present time, in collecting the materials to enable one of the departments to answer a call at the last session." Rich offered this change to the rules: A proposition, requesting information from the President of the United States, or directing it to be furnished by the Secretary of either of the Executive Departments, or the Postmaster General, shall lie upon the table one day for consideration, unless otherwise ordered, with the unanimous consent of the House. On the following day, the House agreed to Rich's proposition. Two years later, the House made another change to its rules governing resolutions of inquiry, requiring not merely a day's delay but also committee consideration: "And shall be taken up for consideration on the next day, in the order in which they were presented, immediately after reports are called for from select committees, and, when adopted, the Clerk shall cause the same to be delivered." The House rule now read: A proposition, requesting information from the President of the United States, or directing it to be furnished by the head of either of the Executive Departments, or by the Postmaster General, shall lie on the table one day for consideration, unless otherwise ordered by the unanimous consent of the House; and all such propositions shall be taken up for consideration in the order they were presented, immediately after reports are called for from select committees; and, when adopted, the Clerk shall cause the same to be delivered. This language survived until 1879, when the House Rules Committee reported language to eliminate the need for lawmakers to seek unanimous consent from the chamber in order to seek executive documents. Speaker Samuel J. Randall explained that it was "very seldom that it is in order for a member to offer a resolution calling for information; that is the difficulty. Any one member at any time may prevent a call for information." Granting this advantage, Representative Roger Q. Mills objected to the procedure for committee referral: "What is the necessity for having a resolution calling for information from one of the Executive Departments referred to a committee? What is the use of my offering a resolution of that kind and having it referred to a committee and there buried?" Representative James A. Garfield explained that the purpose of committee referral was to avoid the "constant danger of gentlemen upon this floor duplicating calls for information. Some one may want some information and offer a resolution calling for it and it passes by unanimous consent, and the same thing may have been asked already by somebody else and nobody has paid any attention to the fact that the same thing has already been called for." Garfield thought it better that legislative requests for information "be referred to the committees, in order that they may not be duplicated so as to put the Departments to the necessity of employing a large number of clerks for a useless purpose." The House Committee on Rules recommended language that gave committees of jurisdiction full discretion over resolutions referred to them: "Under this call resolutions for information from the Executive Departments of the Government may be offered for reference to the appropriate committees, such committees to have the right to report at any time." The language "under this call" referred to a procedure that required resolutions calling for executive information to be offered only during the morning hour of every Monday. Representative Mills objected to this procedure, pointing out that a resolution calling for information might be "of a partisan character," because a member of the minority wanted information in the possession of an executive officer of the majority party in the House. Did anyone believe, he asked, "that such a resolution would get out of any committee against the vote of a majority of its members, when the design of the resolution was, perhaps, to expose the malfeasance of some officer belonging to the party of the majority?" Representative William H. Calkins found Garfield's argument about duplication unpersuasive. If a lawmaker asked for information that an executive department had already made available to another lawmaker, "it would be a full answer to the resolution for such Department to reply that the information had already been given, and the Department would not be required to go over it again." As to Mills's argument that a committee could use its majority party power to block any action on a resolution, Speaker Randall noted that members of the majority party could block floor action on the resolution, because "a single member of that majority could object to it." Mills conceded that point, but said "there would be a record." Representative John H. Baker thought that too much power had been centered in the committees of jurisdiction. Upon receiving a resolution requesting information, it should be "imperative for the committee to report either for or against the resolution, so as to allow the question to come before the House for its determination." Speaker Randall considered that "a very good suggestion" that did not occur to the Rules Committee. Representative Harry White sharpened Baker's proposal by requiring the committee to report "within one week." Baker's amendment, as modified, was agreed to, resulting in this language: "And such committees shall report thereon within one week thereafter." Committee and Floor Procedures Under House Rule XIII, clause 7, a Member may address a resolution of inquiry "to the head of an executive department." The resolution is privileged and may be considered at any time after it is properly reported or discharged from committee. If the resolution is not reported to the House within 14 legislative days after its introduction, a motion to discharge the committee from its further consideration is privileged. Should the committee or committees of referral report (or be discharged under a time limit placed by the Speaker) within the 14 day period, only a designee of the committee can move to proceed to the consideration of the resolution on the floor. Typically, the House debates a resolution of inquiry for not more than one hour before voting on it. When a committee reports a resolution, the time for consideration is generally given to the committee chairman, who may decide to grant half the time to the ranking member of the committee or subcommittee. The deadline for a committee to report was extended from one week to 14 legislative days in 1983. In calculating the days available for committee consideration, the first day and the last day are not counted. A resolution of inquiry is usually referred to the committee that has jurisdiction over the subject matter, but on a number of occasions two or more committees have been involved in responding to a resolution of inquiry. After a resolution of inquiry is introduced and referred to committee, the committee sends the resolution to the Administration for action, requesting a timely response to allow the committee to act within the deadline for a committee report. While waiting for information from the executive branch, the committee may decide to act on the resolution in the form in which it was referred or consider amendments to it. The committee then votes to report the resolution favorably or adversely. It may also decide not to report at all, forcing the Member who introduced the resolution to make a motion to discharge the committee. In most cases the committee reports, either positively or negatively. If the committee concludes that the Administration's response is in substantial compliance with the resolution, it may offer a motion on the House floor to table the resolution on the ground that the congressional interest has been satisfied. When a resolution of inquiry is reported from committee, the chairman of the committee calls up the resolution and becomes floor manager, either to pass the resolution or table it. If the committee decides not to report, the sponsor of the resolution can call up the resolution as privileged business. The privileged status of the resolution applies only to requests for facts within the Administration's control and not for opinions or investigations. In 1905, a Member of the House asked unanimous consent for a resolution that requested the Secretary of the Interior "to furnish to Congress a report on the progress of the investigation of the black sands of the Pacific slope... and for his opinion as to whether or not this investigation should be continued." Another Member pointed out that the Geological Survey, in a letter to the Senate, had already reported on the result of the investigation. Because of a possible duplication of printing, objection was heard to the resolution of inquiry. The sponsor of the resolution asked: "Is not this a privileged resolution?" Speaker Joseph G. Cannon replied, "The Chair thinks the first part of the resolution privileged. The latter part is not privileged; and that destroys the privilege of the whole resolution." Resolutions of inquiry are directed "to the head of an executive department." There have been parliamentary challenges to resolutions that are directed to executive officials who are not considered the head of an executive department. In 1891, a Member offered a resolution of inquiry to the Regents of the Smithsonian Institution for information regarding expenditures of the National Zoological Park. The following dialogue occurred between Representative Benjamin A. Enloe and Speaker Charles F. Crisp: The SPEAKER. The rule applies only to resolutions of inquiry addressed to the heads of Executive Departments. Mr. ENLOE. On that point, Mr. Speaker, I desire to say that the reason why the resolution was framed as it is and also the reason why I consider this as presenting a question of privilege is because it is addressed to the Regents of the Smithsonian Institution, who are made trustees for the disbursement of this fund and for the organization of this park and are not under the control of any Department of the Government. The SPEAKER. "Head of Executive Departments" is the language of the rule. Mr. ENLOE. I understand; but the Regents of the Smithsonian are not under the jurisdiction of any Department of the Government. A MEMBER. And consequently do not come under the rule. Mr. ENLOE. They are virtually the head of a Department, and I should think they come within the meaning of the rule. The SPEAKER. They are not heads of any Executive Department. Mr. ENLOW. Well, Mr. Speaker, I believe it is privileged; but, instead of arguing that proposition, I will ask unanimous consent that the resolution be now considered by the House. Objection was heard. Following Enlow's resolution, a Member announced that he had a resolution reported back from the Committee on Commerce, asking for certain information from the Interstate Commerce Commission. Again Speaker Crisp ruled: "The Chair thinks it is not privileged. The Interstate Commerce Commission is not the head of a department." There are cases when the Chair rules against a resolution of inquiry because it is not directed to an executive department, but the Member prevails through a unanimous consent motion. In 1904, a member called up a resolution of inquiry to obtain information from the Civil Service Commission. After the Chair ruled that the resolution was not a privileged matter because it did not call upon "the head of Department, but upon the Civil Service Commission," the Member asked unanimous consent for its immediate consideration. There was no objection, "and the resolution was accordingly considered and adopted." Although the President is not "the head of an executive department," resolutions of inquiry are directed to the President without the parliamentary challenge that the President is not technically a departmental head. Administrative Discretion Some House resolutions of inquiry give the Administration discretion in providing factual information to Congress, particularly when they are directed to the President. In 1811, a resolution requested from the President, "as far as practicable," a list of Americans impressed by other countries, "with such other information on this subject as he in his judgment may think proper to communicate." In the same year, a resolution requested from the President information relative to the situation in the Indiana Territory, "which may not be improper to be communicated." Early in 1812, a resolution requested the President to furnish the House with copies of instructions given to the U.S. Minister at London, regarding the impressment of American seamen into the naval service of Great Britain, "excepting so much as it may be improper to disclose, on account of any pending negotiation." In 1876, the House passed a resolution requesting President Ulysses S. Grant to inform the House "if, in his opinion, it is not incompatible with the public interest," whether since March 4, 1869 (the date his term began) any executive offices, acts, or duties had been performed at a distance from "the seat of Government established by law, and for how long a period at any one time, and in what part of the United States; also, whether any public necessity existed for such performance, and, if so, of what character, and how far the performance of such executive offices, acts, or duties, at such distance from the seat of Government established by law was in compliance with the act of Congress of the 16 th day of July, 1790." This resolution was not taken up as a resolution of inquiry. Instead, the rules were suspended by the necessary two-thirds majority and the resolution adopted. President Grant could have withheld information on the ground stated in the resolution, that disclosure was not compatible with the public interest. He chose to set forth constitutional reasons for declining the information. First, he said he could find nothing in the Constitution to justify congressional interest as to where the President discharged official acts and duties. What the House could require in terms of information from the executive branch was limited "to what is necessary for the proper discharge of its powers of legislation or of impeachment," neither of which, he said, applied. Asking where executive acts are performed and at what distance from the seat of Government "does not necessarily belong to the province of legislation. It does not profess to be asked for that object." Second, if the House sought the information to assist in the impeachment process, "... it is asked in derogation of an inherent natural right, recognized in this country by a constitutional guaranty which protects every citizen, the President as well as the humblest in the land, from being made a witness against himself." This position was not well taken. Other Presidents have made it clear that if the House sought information as part of impeachment proceedings, the information would be supplied. In denying the House the papers it requested on the Jay Treaty, President George Washington stated that the only ground on which the House might have legitimately requested the documents was impeachment, "which the resolution has not expressed." President James Polk recognized that the power of impeachment gives the House "... the right to investigate the conduct of all public officers under the Government. This is cheerfully admitted. In such a case the safety of the Republic would be the supreme law, and the power of the House in the pursuit of this object would penetrate into the most secret recesses of the Executive Department. It could command the attendance of any and every agent of the Government, and compel them to produce all papers, public or private, official or unofficial, and to testify on oath to all facts within their knowledge." Third, Grant pointed out that previous Presidents found it necessary to discharge official business outside the nation's capital, and that "during such absences I did not neglect or forego the obligations of the duties of my office." To his letter to the House he appended a study on the number of days other Presidents had conducted official business outside the nation's capital. Fourth, with regard to the statute of July 16, 1790, Grant said that no act of Congress could limit his constitutional duty to discharge governmental functions outside the nation's capital, and that the 1790 statute made no attempt to do so. He noted that on March 30, 1791, shortly after passage of the statute cited in the resolution, President Washington issued a proclamation "having reference to the subject of this very act from Georgetown, a place remote from Philadelphia, which then was the seat of Government.... " In 1952, the House debated a resolution of inquiry to "direct" the Secretary of State to transmit to the House, "at the earliest practicable date, full and complete information with respect to any agreements, commitments, or understandings which may have been entered into" by President Harry Truman and Prime Minister Winston Churchill in the course of their conversations during January 1952, "and which might require the shipment of additional members of the Armed Forces of the United States beyond the continental limits of the United States or involve United States forces in armed conflict on foreign soil." The resolution came to the floor accompanied by an adverse report from the Committee on Foreign Affairs. During debate on the resolution, which passed 189 to 143, those who supported the resolution regarded it as non-binding. For example, Representative John Martin Vorys advised his colleagues that "we cannot by this resolution make the Executive answer. We cannot make the President, we cannot make the Secretary of State, say anything. That has been passed on time and again under the precedents of this House. We can put a question up to them. All we can do, if we pass this resolution, is to say to the Secretary of State and the Department of State: "Please try again. That answer you sent down was not very good." Representative James P. Richards, who voted against the resolution, said, regarding this resolution, "it is within the province of the President to refuse to divulge information that he considers would be dangerous or incompatible with the interests of our Nation." Discretion over the release of information to Congress has also been given expressly to department heads. In 1971 the House considered a resolution directing the Secretary of State to furnish certain information respecting U.S. operations in Laos, but the language of the resolution included the phrase "to the extent not incompatible with the public interest." The House tabled this resolution, 261 to 118. In 1979, in the midst of an energy crisis, a resolution of inquiry ( H.Res. 291 ) requested certain facts from the President, "to the extent possible," regarding shortages of crude oil and refined petroleum products, refinery capacity utilization, and related matters. It was adopted 340 to 4. Committee Review A committee has a number of choices after a resolution of inquiry is referred to it. It may vote on the resolution up or down or amend it. It can report favorably or adversely, but an "adverse report" is often accompanied by a substantial amount of information prepared by the executive branch. The quality and quantity of this information can bring the Administration into compliance with the resolution, making further congressional action unnecessary. Usually a committee issues a report on a resolution of inquiry; if it does not, it can be discharged. Committee Amendments Resolutions of inquiry may be amended at the committee level before action on the House floor. In 1980, the House acted on H.Res. 745, a resolution directing President Jimmy Carter to furnish information on the role of Billy Carter, the President's brother, as an agent of the government of Libya. The House Judiciary Committee, after considering and adopting a number of amendments, reported the resolution favorably by a vote of 27 to 0. The amendments included two that had been adopted by the Foreign Affairs Committee. A third committee, the Permanent Select Committee on Intelligence, reported on the resolution with regard to classified material that touched on the relationship between Libya and Billy Carter. It concluded that the Administration was in substantial compliance with H.Res. 745. During floor action, the chairman of the House Judiciary Committee, Representative Peter Rodino, asked unanimous consent that the committee amendments be considered en bloc. There was no objection to his request and the committee amendments were agreed to. He then noted that out of the previous 33 resolutions of inquiry, dating back to 1932, motions to table carried 25 times, largely because there had been substantial compliance to the committee on jurisdiction. It was Rodino's judgment that the Administration had substantially complied with H.Res. 745 and that the issue was therefore "moot" and he would make a motion to table the resolution. Representative Robert McClory, a member of the Judiciary Committee, disagreed with Rodino's position and his proposal to table the resolution. In McClory's view, "there has been something less than substantial compliance with the terms of the resolution," and that one omission from the materials assembled by the Administration was President Carter's "conversation on June 17, 1980, with the Attorney General concerning the Billy Carter investigation." Rodino's motion to table the resolution was rejected on a vote of 124 to 260, after which the House voted to agree to the resolution. In defeating the tabling motion, 116 Democrats joined 144 Republicans. Adverse Reports The fact that a committee reports a resolution of inquiry adversely does not mean the committee opposes the resolution or that the Administration has declined to supply information. The documents delivered by the executive branch may bring it in substantial compliance with the resolution, thus making it unnecessary for the committee to report the resolution favorably for floor action. An example typifying this executive-legislative exchange comes from 1979, when 81 Members supported H.Res. 291, a resolution that directed President Carter to provide the House with information on the energy crisis: shortages of crude oil and refined petroleum products, methods used in allocating oil supplies, possible actions within the private industry to withhold or reduce oil supplies, and any reduction in the supply of crude oil from any foreign country. Within a week, 21 additional Members joined as sponsors of the resolution. The House Committee on Interstate and Foreign Commerce reported the resolution unfavorably and recommended that it not pass. However, the committee had been seeking the information in a number of hearings, and had asked the Department of Energy to provide the information requested in the resolution. The committee stated that much of the information could be found in departmental publications, and that some of the information had been obtained in the course of committee investigations. Yet it also faulted the Administration: "it cannot be said that all information necessary to a full understanding of the supply problem is collected by the DOE, nor that the information which is collected is timely. To the contrary, the Committee has found the DOE lacking vital information on such matters as secondary stocks and actual sales of products." The information supplied by the department was "rarely timely, as a result of long lag times in sending out forms and retrieving them," and the department was "heavily reliant on unverified industry data despite the clear directives from the Congress in a variety of statutes, such as the Energy Supply and Environmental Coordination Act of 1974, and the Department of Energy Organization Act." The committee offered several reasons for reporting the resolution adversely: (1) the department had provided "all of the requested documents which were available at the time resolution was considered, and has promised to provide the Committee additional information when it becomes available;" (2) much of the information was of a confidential or proprietary nature, which was appropriate to share with the committee of jurisdiction but less appropriate to share with the entire Congress; (3) the cost of reproducing the documents was substantial and unnecessary; (4) whatever information was available to the department had been shared with the committee and Congress; and (5) the data requested would probably not "quell public skepticism relating to the Nation's gasoline problems" The committee then added a sixth reason: The Committee wishes to make clear that it is extremely interested in reliable information concerning the nature of our petroleum supply problems. The information currently available is far from adequate, and the Committee in reporting this resolution adversely does not suggest that the Congress and the public have been fully informed concerning these matters. Nor does the Committee wish to indicate that the Congress does not have a right to such information. To the contrary, the Congress clearly has such a right. Rather, the use of a resolution of inquiry is not the appropriate mechanism for obtaining this readily available data: it simply will not result in any new data. When the resolution came to the floor on June 14, Representative John Dingell pointed to a desk covered with information provided by the Energy Department, including "the tables, data, and other documents. The total is a stack of papers nearly a foot high." Yet he also conceded that all of the committee members "believe that the Department's gathering system is inadequate and that data concerning the energy supplies, demands, and prices is not timely provided." Representative Dingell said he was not critical of those who filed the resolution of inquiry: "I do believe that continued inquiry by the Congress is highly desirable. I believe that the information must be made plain." Instead of the mass of material sitting on the desk, several Members wanted a summary of what the documents contained. Representative Dingell said the department had prepared a summary but it was not yet available from the printer. After several Members objected to voting on the resolution without a summary, Representative Dingell agreed to withdraw his initial motion for the immediate consideration of the resolution. Debate continued the next day, with a number of Members expressing dissatisfaction with the quality of departmental data. Minority Leader John J. Rhodes, who had introduced the resolution, said that "as far as the technicalities of the situation are concerned, those questions were answered, but they were answered in such a way as to be almost incomprehensible, and certainly not to inform with the House or the American people as to the reasons for the existence of these shortages." A move to table the resolution of inquiry lost on a vote of 4 to 338. As the debate moved along, with Members of both parties expressing support for the resolution, Representative Dingell said "I understand the temper of the House very clearly. I want to have my colleagues know that we have had the resolution on inquiry fully and fairly and properly complied with by the DOE, and it will be further fully, fairly, and properly complied with according to the letter of the rules of the House if this resolution is adopted." He wanted his colleagues to know "I have no objection to the vote which will take place, and I want them to know that the vote will, I regret to advise them, procure no new information other than that which was available at the committee table and which was made available to my Republican colleagues yesterday in response to the resolution." He pledged to "persist in my efforts to procure the information which I and my colleagues desire to have on this particular matter, and that the motion to table made earlier by me was simply to save the time of the House and to see to it that the information requested by the sponsors of the resolution of inquiry was presented to the House in a proper and appropriate fashion." The resolution of inquiry passed on a vote of 340 to 4. Another example comes from 1986, after Representative Leon Panetta introduced H.Res. 395 to receive documents regarding the Administration's use of $27 million in appropriated funds for humanitarian assistance for the Nicaraguan democratic resistance. A subcommittee of the House Foreign Affairs Committee held a hearing on the resolution and made a tentative recommendation that the resolution be reported favorably to the full committee. The subcommittee reviewed documents provided by the Administration, and agreed to recommend that the full committee report adversely if the subcommittee received information covering six categories. This second effort by the Administration convinced both the subcommittee and Representative Panetta that the executive branch was in essential compliance with the resolution, but the subcommittee and Representative Panetta also agreed that the documents demonstrated that the Administration "has not complied with the law requiring it to set up appropriate monitoring procedures with respect to the so-called humanitarian assistance for the Contras authorized by the Congress." Representative Panetta, having met with representatives from the Central Intelligence Agency to review classified documents, wrote to the chairman of the full committee that the Administration had complied with his resolution of inquiry. Competing Investigations A committee may decide to report a resolution of inquiry adversely because it competes with other investigations that are regarded as more appropriate. In 1980, for example, H.Res. 571 directed the Attorney General to furnish the House with "all evidence compiled by the Department of Justice and the Federal Bureau of Investigation against Members of Congress in connection with the Abscam investigation," which was a Justice Department undercover operation that led to charges of criminal conduct against certain Members of Congress. The resolution also asked for "the total amount of Federal moneys expended in connection with the Abscam probe." The House Judiciary Committee reported the resolution adversely. Committee opposition to the resolution was unanimous. The Justice Department "vigorously oppose[d]" the resolution. The objections raised by the department, with which the committee agreed, centered on the concern that disclosure of evidence to the House would jeopardize the ability of the department to successfully conduct grand jury investigations and to prosecute any indictments, and that the release of unsifted and unevaluated evidence "would injure the reputations of innocent people who may be involved in no ethical or legal impropriety." Other considerations were present. The House Standards of Official Conduct Committee, conducting its own inquiry into Abscam, unanimously opposed the resolution of inquiry. The committee had begun the process of negotiating with the Justice Department to obtain access to evidence needed for investigation by the House. Moreover, two subcommittees of the House Judiciary Committee were planning hearings into the proper standards for the Justice Department to conduct undercover operations, particularly against Members of Congress. During House debate, Representative John J. Cavanaugh expressed concern that Abscam "raises serious questions of the separation of powers and the ability of one branch of our Government—the executive—to employ investigative methods that are capable of subverting and intimidating and compromising the independence, the constitutional independence, of another and separate branch of our Government." In this case, Congress chose not to interrupt or interfere with Justice Department prosecutions because it might appear to be self-serving. Representative William J. Hughes stated: "I can think of nothing that would be more damaging to the Congress than to be perceived as having obstructed an active criminal investigation." One Member was concerned that forcing the Justice Department to release evidence might help some Members who faced criminal prosecution and look as though lawmakers had greater protection than the average citizen. By a vote of 404 to 4, the House decided to table the resolution of inquiry. In other situations, Congress may choose to investigate a scandal even if jeopardizes successful prosecutions. In terms of public policy, it may be more important to investigate a matter promptly rather than wait for the Justice Department or an Independent Counsel to investigate, prosecute, and pursue appeals. Such was the case with Iran-Contra, where both Houses of Congress concluded that the value of timely legislative investigation outweighed the needs of prosecutors. Lawrence Walsh, the independent counsel for Iran-Contra, recognized that if Congress "decides to grant immunity, there is no way that it can be avoided. They have the last word and that is a proper distribution of power.... The legislative branch has the power to decide whether it is more important perhaps even to destroy a prosecution than to hold back testimony they need." Discharging a Committee If a committee receives a resolution of inquiry and fails to report it within the requisite number of days, a motion to discharge the committee is privileged. That procedure was used in 1971 after Representative James M. Collins introduced H.Res. 539 directing the Secretary of Health, Education, and Welfare (HEW) to furnish certain documents. The resolution directed the release, "to the extent not incompatible with the public interest," of any documents containing a list of the public school systems, from August 1, 1971 to June 30, 1972, that would be receiving federal funds and would be engaging in busing schoolchildren to achieve racial balance. Also requested were any documents regarding HEW rules and regulations with respect to the use of any federal funds administered by the department for busing to achieve racial balance. The resolution was referred to the Committee on Education and Labor. When the committee failed to report the resolution during the deadline, which was seven days in 1971, Representative Collins moved to discharge the committee. His motion was agreed to, 252 to 129. Representative Thomas P. (Tip) O'Neill, Jr.,who at that time was the House Majority Whip, voted against the discharge motion but admitted that he was uncertain about the meaning of the resolution: "What does the resolution do? Is there anything wrong? Is it a serious resolution? Is it something we should have had up today? Is it of that import?" He said that when Members came to the floor they were told: "Well, if you are for busing, you vote 'nay.' If you are against busing, you vote 'yea.'" He now realized that the guidance given to Members was "inaccurate." The vote was not for or against busing, but for or against receiving information from HEW. With this new understanding, Representative O'Neill announced that he had no objection to the resolution and that "I will, and I hope all other Members will vote for the resolution." Representative O'Neill asked the chairman of the Education and Labor Committee, Representative Carl Perkins, why the committee had not acted on the resolution. Perkins explained: "To be perfectly truthful and frank... I forgot about it... [I]t was of the nature that the sponsor of the resolution could have picked up the telephone and gotten the information from HEW." Representative Edith Green emphasized that the resolution "is simply a request for information," not "a bill to legislate," and asked the HEW Secretary "in a perfectly orderly fashion to supply within 60 days the amount of money that is now being spent and in which districts for busing and the guidelines, rules and regulations which HEW has drawn up to enforce this busing to achieve some magical racial balance." With the purpose of the resolution clarified, the House passed it 351 to 36. Military Operations in Vietnam The House has frequently used resolutions of inquiry to obtain information on matters of defense and military policy. A particularly heavy use of resolutions of inquiry came during the Vietnam War. In 1971, the House voted on two resolutions to give Members access to the "Pentagon Papers," the Defense Department study entitled "United States-Vietnam Relationships, 1945-1967." One of the cosponsors of the resolution, Representative Bella Abzug, stated that the procedures adopted by the House Armed Services Committee, which had a single copy of the study, did not provide Members adequate access to the 47-volume study: "they cannot take notes, cannot have staff people review and comment, cannot report on what they have read. Under such limitations, a Congressman must have an elephantine memory to retain the facts that would enable him to exercise his constitutional duty." H.Res. 489 directed the President "to furnish the House of Representatives within fifteen days after the adoption of this resolution with the full and complete text" of the Pentagon Papers. The House Armed Services Committee reported the resolution adversely, 25 to 2, and it was tabled on the floor, 272 to 113. H.Res. 490, containing the identical language, was also reported adversely and tabled. Also in 1971, the House considered three resolutions of inquiry to obtain information about U.S. covert operations in Laos. H.Res. 492 directed the Secretary of State, "to the extent not incompatible with the public interest," to provide the House with any documents containing policy instructions or guidelines given to the U.S. Ambassador in Laos regarding covert CIA operations in Laos, Thai and other foreign armed forces operations in Laos, U.S. bombing operations other than those along the Ho Chi Minh Trail, U.S. armed forces operations in Laos, and U.S. Agency for International Development operations in Laos that assisted, directly or indirectly, military or CIA operations in Laos. The resolution was accompanied by an adverse report from the House Foreign Affairs Committee. Representative Benjamin Rosenthal, a cosponsor of the resolution, explained its purpose: This administration has steadfastly refused to report to the people and to the Congress the nature of the CIA covertly declared war in Laos where the CIA agents are advising the Meo tribesmen. The administration has steadfastly refused to admit that we are hiring Thai mercenaries and ferrying them to Laos in American aircraft to conduct a war in defense of the Laotian Government—a war which this administration has not declared... Yet it is widely reported in the papers—the New York Times and the Washington Post and other newspapers, Life magazine and the Christian Science Monitor—that all of these events are taking place. We in Congress are forced to depend on what we are advised of in the public newspapers as to our involvement in Laos. The resolution was tabled, 261 to 118. Another resolution of inquiry, directing the Secretary of State—"to the extent not incompatible with the public interest"—to furnish the House with additional information regarding U.S. policy involving Laos, was also tabled. House resolutions of inquiry are typically reported from committee after a committee meeting and a roll-call vote, but usually without holding hearings. However, in 1972 the House Armed Services Committee held hearings on H.Res. 918, a resolution of inquiry introduced by Representative Abzug to obtain information on U.S. bombing in Vietnam. Most of the resolution requested specific facts on U.S. military personnel in South Vietnam, the number of sorties flown during specific periods, the tonnage of bombs and shells fired or dropped during specific periods, and other statistics. In testifying on the resolution at the hearings, Representative Abzug stated that the level of bombing constituted "the most dramatic proof yet that the Nixon administration is entirely committed to a full-scale and long-term U.S. air war in Indochina instead of negotiating a full withdrawal in return for the release of our captured pilots." At these hearings, Dennis J. Doolin, Deputy Assistant Secretary of Defense for East Asia and Pacific Affairs, provided information on some of the elements in H.Res. 918. The resolution was reported adversely, 32 to 4. During floor debate, the chairman of the House Armed Services Committee, Representative F. Edward Hébert, explained that the information sought in the resolution was in committee files, "available to any Member of the House for his examination, subject, of course, to the rules established by the committee which preclude the release or public use of such information without the consent of the committee." After describing the material available in the committee's sessions, both open and closed, he said that the resolution "is directed to giving the Congress the information which is here printed for them to see. Every question is answered." Later in the debate, Representative William J. Randall, a member of the Armed Services Committee, noted that the when the committee went into executive session, "[a]ll afternoon the answers to the questions propounded by the Member from New York [Representative Abzug] were spread upon the record. We were given the very latest facts and figures on all of the things asked for in the resolution." House floor debate on the Abzug resolution, occupying 87 pages in the Congressional Record, includes the transcript from the open hearings before the Armed Services Committee and a number of articles on military operations in Vietnam. Some of the Members who voted to table the resolution objected only to one part: the part asking the Administration to give the target date for full independence for Saigon. Otherwise, said Representative Paul Findley, "the resolution seems to deal entirely with facts of past actions that should be available to Congress." The House voted 270 to 113 to table the resolution. Although the resolution was not agreed to, it forced the delivery of information from the Administration to the Armed Services Committee, and from there to individual Members. A similar pattern emerged in 1973, when the House acted on H.Res. 379, which directed the Secretary of Defense to furnish the House information on military operations in Cambodia and Laos: the number of sorties flown by the U.S. during certain periods, the tonnage of bombs and shells fired or dropped during certain periods, the number and nomenclature of U.S. aircraft lost over Cambodia and Laos, and other statistics. The House Armed Services Committee held a hearing to review the 19 specific questions addressed in the resolution. Chairman Hébert asked the Defense Department "to be as responsive as possible to each of the questions, and to the maximum extent possible provide this information in open session." If necessary, the committee would go into closed session to "receive such additional classified information as may be necessary to permit the Department to be fully responsive to this privileged resolution." In open session, Deputy Assistant Secretary Doolin provided answers to each of the questions, with two exceptions. He told the committee that he would not be able to provide the answer for Question 10 for another 24 hours, at which time the committee received the information and placed it in the hearing record. He also noted that Question 18, regarding the legal authority for U.S. military activity in Cambodia and Laos since January 27, 1973, would be addressed by DOD General Counsel J. Fred Buzhardt, who proceeded to provide a legal analysis. As noted in the following exchange with Representative Charles Wilson, all of the information given by Doolin and Buzhardt was in open session: Mr. CHARLES WILSON. There was no difficulty in presenting this to us in open session, was there? Mr. DOOLIN. No, sir. I have tried to be as forthcoming as possible. Mr. CHARLES WILSON. This information could have been furnished by a resolution asked for by any Members of the Congress, I assume? Mr. DOOLIN. Yes, sir. Toward the end of the hearing, Chairman Hébert noted that the resolution "asks for certain information to be brought to the attention of the Congress. That information is now before the attention of the Congress. Therefore, making, in effect, the resolution a moot question." The sponsor of the resolution, Representative Robert L. Leggett, agreed that "we answered all of the questions I think really very well." When Chairman Hébert said "the resolution becomes moot," Leggett responded: "I concur in that." The committee then voted 36 to zero to report the resolution adversely. The answers to the 19 questions were placed in the Congressional Record, at which point the resolution was tabled. Forcing Other Legislative Actions Some resolutions of inquiry have caused Congress to take other legislative actions to address the lack of information received from the Administration. The two examples included here relate to the calling of supplemental hearings and the adoption of substitute legislation. Supplemental Hearings A resolution of inquiry, after being partially satisfied by answers from the Administration, can trigger supplemental information obtained through congressional hearings. This was the result of H.Res. 552, introduced by Representative Benjamin Rosenthal on June 18, 1975, to seek information about the Administration's proposed sale of Hawk and Redeye missiles to Jordan. On the following day, the House Committee on International Relations forwarded the resolution to President Gerald R. Ford, requesting a prompt reply. The White House responded on June 25, providing responses to the 20 questions put by the resolution. However, committee chairman Thomas E. Morgan questioned whether the resolution was a bona fide "privileged resolution of inquiry" under House rules. On June 26, the committee voted to table the resolution on the ground that it was not restricted to factual answers, but instead required "investigation" on the part of the President to answer several of the questions. Representative Rosenthal, having announced his intention to call up H.Res. 552 for House action because the committee had not reported on his resolution, agreed to withhold that motion in exchange for committee hearings. Representative Morgan advised Representative Rosenthal that the committee "should get the facts regarding the proposed sale, and I will be glad to cooperate with him in making that happen." The hearings were important because Congress was in the process of deciding whether to block the sale by passing a resolution of disapproval under Section 36(b) of the Foreign Military Sales Act. On July 9, Representative Rosenthal said that information about the proposed sale "was leaked to the press, not formally announced," and that "[n]o attempt was made to inform the Congress about the sale in the past two months, and there would have been none were it not for the questions posed in House Resolution 552, the resolution of inquiry." When the Administration acknowledged the sale, it indicated that formal notice would be reported to Congress in late July or early August. Representative Rosenthal pointed out that "Congress probably will be in recess at that time and unable to act on this very important arms sale and policy decision." Formal notice of the sale reached Congress on July 10. Under Section 36(b), Congress had 20 calendar days to pass a concurrent resolution of disapproval. Legislative action on the disapproval resolution therefore had to be completed by July 30. On July 14, Representative Jonathan Bingham and 10 other Members introduced H.Con.Res. 337 to disapprove the sale. On July 16 and 17, a subcommittee of the House International Relations Committee held two days of hearings on the proposed sale. Administration officials defended the sale on the first day; eight Members of Congress raised their objections the following day. With the disapproval resolution moving toward a vote, President Ford withdrew the proposed sale on July 28 and entered into negotiations with Congress. The Administration announced a compromise on September 16, limiting the missiles to "defensive and non-mobile antiaircraft weapons." Triggering Legislation In 1991, just prior to U.S. military operations against Iraq, Representative Barbara Boxer and six Democratic colleagues introduced H.Res. 19 to call for certain information regarding casualty estimates, biological and chemical weapons, financial assistance from other countries (burdensharing), and other information. Members of both parties recognized that the House was entitled to budgetary and other information from the executive branch, but decided on a different approach. After the war began, Representatives Charles Schumer and Leon Panetta introduced H.R. 586 on January 18, for the purpose of requiring regular reports from the Administration on U.S. expenditures for military operations and the financial contributions from other countries. Action on a bill would avoid the 14-day deadline imposed by a resolution of inquiry. On February 21, the House moved to suspend the rules to pass H.R. 586. During debate on the bill, several Members discussed that the General Accounting Office had not been given access to any of the costs incurred in connection with the war. Representative Schumer said that until the resolution of inquiry and his bill were introduced, "we just were not getting those answers when we asked questions." Lawmakers received information on what allies had pledged but not "about how much they had actually paid." Representative Boxer announced that she would support H.R. 586 and the tabling of her resolution. In a letter dated February 20, Brent Scowcroft, National Security Adviser to President George H.W. Bush, provided specific information in response to H.Res. 19. After H.R. 586 passed 393 to 1, the House engaged in a brief debate on H.Res. 19 before tabling it by a vote of 390 to 0. In discussing the resolution of inquiry, Representative Dante Fascell said that it "has proven to be a catalyst for the executive branch to be more forthcoming with the Congress in providing necessary and appropriate information in order to satisfy the oversight responsibilities of the Congress." Mexico Rescue Package Another use of a resolution of inquiry occurred in 1995, after the Clinton Administration offered a multibillion dollar rescue package for the Mexican peso. As initially introduced by Representative Marcy Kaptur, the resolution ( H.Res. 80 ) did not contain discretion for the Administration. It requested the President, within 14 days after the adoption of the resolution, "to submit information to the House of Representatives concerning actions taken through the exchange stabilization fund to strengthen the Mexican peso and stabilize the economy of Mexico." The House Banking and Financial Services Committee voted 37 to 5 to report the resolution favorably, but with a substitute directing the President to submit the documents "if not inconsistent with the public interest." The committee explained that its requests for documents "should not be construed to include drafts of documents provided in final form, nor any notes of any individual." On March 1, the House adopted the committee substitute and agreed to the resolution, 407 to 21. Although the resolution established a deadline of 14 days, White House Counsel Abner J. Mikva sent a letter to Speaker Newt Gingrich that the Administration would not be able to provide the documentary material until May 15, or two months after the date set in the resolution. By April 6, the Treasury Department had supplied Congress with 3,200 pages of unclassified documents and 475 pages of classified documents, with additional materials promised. The White House said it was in "substantial compliance" with the resolution. Iraq's Declaration on WMD On February 12, 2003, Representative Dennis Kucinich introduced a resolution of inquiry to give the House access to the 12,000-page Iraqi declaration on its weapons of mass destruction. The declaration had been provided to the UN Security Council on December 7, 2002. In his floor statement on H.Res. 68, Representative Kucinich said that if the Administration was intent on going to war against Iraq, "I believe it is incumbent upon them to make the document which was portrayed as evidence of an Iraqi threat available for all to evaluate." He asked that "the primary documents be transmitted in their complete and unedited form." The Administration gave a copy of the declaration to the House on March 7, after which the House International Relations Committee voted to report H.Res. 68 adversely. Representative Doug Bereuter, who chaired the committee markup, said that the Administration's release of the document rendered the resolution moot: "I would say, in short, Mr. Kucinich has won his point." When the declaration reached the House on March 7, the Speaker directed the Permanent Select Committee on Intelligence to retain custody because of its facilities for handling classified documents. The declaration was made available for review by Members and to House staff with appropriate security clearances who have executed a nondisclosure oath or affirmation. Resolutions in the 108th-111th Congresses While no resolutions of inquiry were introduced in the 110 th Congress (2007-2008), the number introduced in the 108 th and 109 th Congresses (2003-2006) represented a substantial increase over prior years. Between the 102 nd and 107 th Congresses (1991-2002), an average of one resolution of inquiry was introduced in each Congress. In the 108 th Congress, however, 14 such resolutions were introduced, and 39 resolutions of inquiry were introduced in the 109 th Congress. The 53 resolutions of inquiry introduced in the 108 th and 109 th Congresses exceed the total number of such resolutions introduced over the previous 25 years combined. In fact, in no Congress since the 76 th Congress (1939-1940) were more resolutions of inquiry introduced than the number introduced during the 109 th Congress. As of the date of this report, eight resolutions of inquiry have been introduced in the 111 th Congress (2009-2010). Conclusion House resolutions of inquiry have historically been an effective means of obtaining factual material from the executive branch. In the past, even when committees report the resolutions adversely or succeed in tabling them on the House floor, a substantial amount of information has usually released to Congress. In fact, arguments that the Administration has complied with a resolution are frequently the reason for reporting a resolution adversely and tabling it. On occasion, a resolution of inquiry is reported adversely because it competes with other investigations (either in Congress or in the executive branch) that are considered the more appropriate avenue for inquiry. In some situations, resolutions of inquiry have been instrumental in triggering other congressional methods of obtaining information, such as through supplemental hearings or alternative legislation. Recent Congresses have shown an increase in the use of these privileged resolutions. Members turn to resolutions of inquiry for different reasons. A Member may introduce such a resolution if he or she has been unable to do so through other channels (e.g., committee investigations and hearings). The committee of jurisdiction might have advised the lawmaker that it had no intention of investigating the matter. Also, a resolution of inquiry is often a useful way for a Member to bring attention to an issue, receive basic information from the Administration, and perhaps trigger more extensive legislative investigations. | The resolution of inquiry is a simple House resolution that seeks factual information from the executive branch. Such resolutions are given privileged status under House rules and may be considered at any time after being properly reported or discharged from committee. Such resolutions apply only to requests for facts-not opinions-within an Administration's control. This report explains the history, procedure, specific uses of resolutions of inquiry, and notes recent increases in their usage. The examples in this report demonstrate that, historically, even when a resolution of inquiry is reported adversely from a committee and tabled on the floor, it has frequently led to the release of a substantial amount of information from the Administration. Data from more recent Congresses suggest a potential change in the use and efficacy of these privileged resolutions. For other CRS reports regarding legislative techniques for obtaining information from the executive branch, see CRS Report 95-464, Investigative Oversight: An Introduction to the Law, Practice and Procedure of Congressional Inquiry, by [author name scrubbed], and CRS Report RL30240, Congressional Oversight Manual, by [author name scrubbed] et al. This report will be updated as events warrant. | gov_report |
At least three young Minnesota women are now believed to have traveled to Syria to give aid to the ISIS terror group responsible for the brutal beheadings of American journalists, MailOnline has learned. The trio left some three weeks ago, Omar Jamal, a leader of the Somali community in the state capital, St. Paul, tells MailOnline. They said they intended to become nurses tending to fighters injured in ISIS' violent surge in Syria and Iraq. The news comes as 19-year-old suburban Denver woman Shannon Conley who federal authorities say intended to wage jihad has pleaded guilty to trying to help the Islamic State militant group in Syria. American girl: Colorado teen Shannon Conley pleaded guilty to terror charges in Denver federal court today after her April arrest for trying to flee the country and join ISIS. Searching: Conley (pictured on the right, with a friend) tried out several different religions in college before selecting Islam. Abroad, police fear two young girls who fled Austria are inspiring other teenagers to join Islamic State ranks after they successfully fled the country saying they were going to Syria. Samra Kesinovic was aged just 16 and her friend Sabina Selimovic 15 when the two vanished this year from their homes in the Austrian capital Vienna. The case of one of the Minnesota girls, a 19-year-old, has already been widely reported, after the girl's family called the FBI, but Jamal said he believes at least two more girls have gone to the Middle East hotspot. 'Their identity is not known because their families have not contacted the authorities,' said Jamal. 'They have gone to Syria but as there are no official reports of them we do not know who they are.' The shocking new revelation comes as MailOnline can reveal that the FBI has subpoenaed the 19-year-old girl's family to appear before a Grand Jury later this month, as authorities attempt to discover who bought her ticket, gave her money and provided her with a false passport. There is no suggestion that the family is under criminal investigation, said one law enforcement source with knowledge of the subpoena. 'They are looking for evidence on exactly what the family knows,' said the source. Included in the subpoena is a demand for cell phone records as the girl called her brother from Turkey and later, after she crossed the border, from Syria. Teen jihadis: Left is Samra Kesinovic, 16, who is thought to have fled to Syria to join the Islamic State from Austria. On the right is 15-year-old Sabina Selimovic who went with her. Austrian teens: Samra Kesinovic and Sabina Selimovic in a photo they posted online. Police now fear the two are inspiring other girls to flee to Syria to take up holy war. However, Jamal said the girl's family is worried about what may happen at the Grand Jury hearing and feels 'betrayed' by the FBI's action as it believed it had done the right thing by contacting authorities in the first place while the families of the two unidentified girls do not have to appear. FBI's Minnesota spokesman Kyle Loven told MailOnline he 'is not in a position to comment' on the subpoena because the case of the missing girl is an ongoing investigation. 'I cannot confirm and will not deny its existence,' he said. The teen, who turns 20 next month, was about to start nursing classes at St. Paul College but decided to leave to give aid to wounded fighters. Jamal said he is sure she will not be placed into front line battle but it is possible she would be married off to one of the ISIS fighters. She is the middle of five children brought up by a single Somali mother, living off St. Paul's Selby Avenue. She had recently started taking classes at al-Farook Youth and Family Center in the Minneapolis suburb of Bloomington, said Bob Fletcher, a former sheriff in St. Paul, who has dedicated himself to helping the Twin Cities' large Somali community since being defeated in an election in 2010. Al-Farook has been at the center of claims that Amir Meshal, an alleged recruiter for ISIS, had been talking to youngsters on its site. The mosque kicked the New Jersey-born man off campus, but now the Minneapolis-St. Paul Fox TV station has raised questions of whether Meshal is actually an FBI mole rather than an ISIS recruiter. Possible recruiting ground: Al-Farook has been at the center of claims that Amir Meshal, an alleged recruiter for ISIS, had been talking to youngsters on its site. 'What we don't know is whether al-Farook is part of the problem or part of the solution right now,' said Jamal. 'And who is Amir Meschal? Where did he come from? Did he recruit these girls?' Meshal sued the FBI in federal court in 2009 claiming he was tortured and held for four months in three different countries after being apprehended crossing the Somali border into Kenya. The young woman who is known to have gone to Syria traveled on a 'borrowed' passport. However the passport holder did not report it was missing until the girl had already entered Syria, the Minneapolis Star-Tribune reported. In the family's only comments, a male relative asked in the Star-Tribune: 'How many more kids have to die before we do something about it? 'Those animals who are taking our children are what we’re concerned about. We love this country more than anything else. I love America. We don’t want to see anything bad happen here. This is the most dangerous thing happening to this country.' ISIS has rocketed to infamy over the summer due to its savage rampage through Iraq and Syria where it has murdered thousands of civilians and soldiers. American journalists Jim Foley and Steven Sotloff were both beheaded in videos that the group distributed to the west. Despite the violent imagery, Fletcher, the retired sheriff, said recruitment to ISIS is appealing to young Muslims because they get the feeling they are in at the start of something new, an Islamic state, hat they believe can offer them a future they fail to see in America. 'Many Somalis are struggling as their transition to the American life has been difficult. They have been here a shorter time than most immigrants and their arrival coincided with a downturn in the economy so many have not been able to find work. 'Many also came here without fathers, who had either been killed in the war or did not make the trip, so on occasion the extremist religious leader becomes their father-figure.' He said that going to be a nurse in an ISIS stronghold like Raqqa in northern Syria is also attractive because it is portrayed as relatively stable away from the frontline battle and a place where volunteers can start to build the infrastructure of a new city | Three girls left Minnesota three weeks ago to join ISIS insurgents in Syria. Their identities have not yet been revealed. The FBI has subpoenaed a 19-year-old girl's family to appear before a Grand Jury later this month. She was about to start nursing classes at St. Paul College but decided to leave to give aid to wounded fighters. The girl is one of five children to a single mother originally from Somalia. She had recently started taking classes at al-Farook Youth and Family Center, an alleged recruiting ground for ISIS. | cnn_dailymail |
Vision is the dominant sensory modality in many organisms for foraging, predator avoidance, and social behaviors including mate selection. Vertebrate visual perception is initiated when light strikes rod and cone photoreceptors within the neural retina of the eye. Sensitivity to individual colors, i. e., peak spectral sensitivities (λmax) of visual pigments, are a function of the type of chromophore and the amino acid sequence of the associated opsin protein in the photoreceptors. Large differences in peak spectral sensitivities can result from minor differences in amino acid sequence of cone opsins. To determine how minor sequence differences could result in large spectral shifts we selected a spectrally-diverse group of 14 teleost Rh2 cone opsins for which sequences and λmax are experimentally known. Classical molecular dynamics simulations were carried out after embedding chromophore-associated homology structures within explicit bilayers and water. These simulations revealed structural features of visual pigments, particularly within the chromophore, that contributed to diverged spectral sensitivities. Statistical tests performed on all the observed structural parameters associated with the chromophore revealed that a two-term, first-order regression model was sufficient to accurately predict λmax over a range of 452–528 nm. The approach was accurate, efficient and simple in that site-by-site molecular modifications or complex quantum mechanics models were not required to predict λmax. These studies identify structural features associated with the chromophore that may explain diverged spectral sensitivities, and provide a platform for future, functionally predictive opsin modeling. Many living organisms rely upon vision as the dominant sensory modality for foraging, predator avoidance, and social behaviors including mate selection. Vertebrate visual perception is initiated when light strikes rod and cone photoreceptors within the neural retina of the eye. Within photoreceptors, the visual pigments absorb light and interact with downstream intracellular signaling pathways. Visual pigments consist of seven-transmembrane, G-protein-coupled receptor proteins (GPCR) called opsins, together with a chromophore covalently bound through a Schiff base attachment at a lysine residue. The spectral sensitivities of visual pigments are a function of the type of chromophore (11-cis retinal or 11-cis-3,4-didehydro retinal) and the amino acid sequence of the associated opsin protein [1–4]. Color vision is possible when different cone opsins with distinct peak spectral sensitivities are expressed in separate cone photoreceptor populations, providing differential input to downstream retinal neurons. Cone opsins are under strong natural selection [5–8], and minor changes in their amino acid sequences can result in large changes in spectral sensitivities of their corresponding pigments [4]. For example, the human green cone opsin is 96% identical at the amino acid level to the human red cone opsin, but their corresponding pigments show peak spectral sensitivities that are 28 nm different [9]. Vertebrate cone opsins are grouped into four families: SWS1, SWS2, RH2, and LWS, which typically produce pigments sensitive to very short wavelengths (UV-violet, 360–450 nm), short wavelengths (blue, 450–495 nm), medium wavelengths (green, 495–560 nm), and long wavelengths (yellow-red, 560–700 nm), respectively [10]. However, there is a large amount of spectral variation within each cone opsin family. For example, some SWS1 pigments are maximally sensitive to blue wavelengths (e. g. human blue cone opsin), and some LWS pigments are maximally sensitive to green (e. g. human green cone opsin) [11]. Fueling this variability within both primate and fish genomes is the presence of numerous tandemly-replicated cone opsin genes [12]. The phylogenies in Fig 1 show several examples of tandem replications in the rh2 opsin genes of selected teleosts. The zebrafish, Danio rerio, has four rh2 genes, which arose by multiple duplication events after the divergence of otomorpha (like D. rerio) from euteleosteomorpha (like medaka, Oryzias latipes). Tandem duplications also occurred in the common ancestor of O. latipes, Poecilia reticulata and Metriaclima zebra, and again in M. zebra (rh2Aα and rh2Aβ). Experimentally measured peak spectral sensitivities (λmax) of these opsins reconstituted with chromophore are also indicated on the phylogenies, along with λmax for inferred ancestral sequences for the ancestors of the extant Rh2 opsins (Fig 1) [13]. Mutation studies have shown that position 122 in teleost Rh2 opsins predicts green-shifted λmax (> 495 nm) when occupied by a Glu (E), and blue-shifted λmax (< 495 nm) when occupied by a Gln (Q); this substitution alone has been demonstrated to account for ~15 nm of spectral shift [13] (see SI S1 Fig for the E122Q substitution). There are two equally parsimonious explanations for the evolutionary timing of substitutions at position 122 that resulted in the λmax of ancestral and extant opsins (Fig 1A and 1B). However, given that the opsin genes are under strong selection, the most parsimonious explanation may not reflect the true evolution of these proteins. Replicated opsin genes therefore provided the raw genetic material for tremendous diversity in spectral sensitivities through mutation and neofunctionalization. This diversity contributes to organismal colonization of, and persistence within, novel environments. The high rate of mutation and unequal recombination within cone opsin genes in humans can also have deleterious consequences, including numerous types of color blindness and some cone degenerative diseases that drastically reduce visual function [14–16]. The ability of a pigment to absorb light at a specific λmax is determined by the conformation adopted by the chromophore; this conformation depends on the shape and composition of the binding pocket and the counter-ions that stabilize the Schiff base in the dark or ground state [17–19]. Laborious residue-by-residue substitution approaches, followed by reconstitution of opsin with chromophore and subsequent measurement of absorbance, have identified a small number of key residues that contribute to spectral shift within minor subsets of each cone opsin family. For example, the “five sites” rule states that the identities of five specific residues within the binding pocket of some mammalian LWS opsins can predict peak spectra [20], and the E122Q substitution described above predicts green vs. blue λmax in teleost Rh2 visual pigments [13]. However, the “five sites” rule does not extend beyond LWS opsins [21], and is not predictive beyond selected mammals [22]. In teleost Rh2 pigments, E122Q predicts green vs. blue, but further spectral differences cannot be explained by specific contributions of identified amino acid replacements [13]. Despite the functional significance of the evolution of color vision, there is currently no simple strategy for predicting λmax of a chromophore-bound cone pigment [4]. We report here an alternative, efficient and more accurate approach to predicting spectral peak sensitivities of cone opsins, using a spectrally-diverse group of 14 teleost Rh2 opsins for which sequences and λmax are known (Fig 1). Through the generation of homology structures and atomistic molecular dynamics (MD) simulations [23], we identified two parameters of chromophore conformation and fluctuation that together accurately predict peak spectral sensitivities. Furthermore, these studies identify structural features associated with the chromophore that explain diverged spectral sensitivities, and provide a platform for future, functionally predictive opsin modeling. To develop a model that predicts peak spectral sensitivities from amino acid sequence, we selected a spectrally diverse set of Rh2-type teleost cone opsins. The Rh2 opsin proteins were chosen because they are most closely related phylogenetically to Rh1 opsin proteins. RH1 opsins are present in vertebrate rods, and form the rhodopsin visual pigments [10], and the mammalian RH1 opsins are the only vertebrate opsins for which experimental protein structures are available [24,25]. Moreover, amino acid sequences and corresponding spectral sensitivities of pigments reconstituted with 11-cis retinal are known for many teleost Rh2 opsins and show a wide range of λmax (452–528 nm) (Fig 1; Table 1). We obtained sequence information for four Rh2 opsins from Danio rerio (zebrafish) [26], three zebrafish ancestral Rh2 opsins inferred by likelihood-based Bayesian statistics [13], two Rh2 opsins from Oryzias latipes (medaka) [27], two Rh2 opsins from Poecilia reticulata (guppy) [21], and three Rh2 opsins from Metriaclima zebra (cichlid) [28] (Table 1; SI S1 Fig). With this sequence information we built three-dimensional homology structures using the bovine rhodopsin (RH1 opsin + 11-cis retinal chromophore) structure as a template (PDB ID: 1U19) [24]. These structures were embedded in explicit membrane bilayers and water with the chromophore bound covalently to the lysine residue in the binding pocket (Fig 2), and were then subjected to 100 ns classical MD [23] simulations using the protocol described in the methods section (SI S1 Movie). We analyzed the MD simulations for all 14 pigments and identified structural features associated with the chromophore and attached lysine that could potentially be used to explain spectral sensitivity differences. For each pigment we examined a total of 19 angles (15 torsion angles and four geometric angles) formed by the heavy atoms of the lysine attached to 11-cis retinal (LYS+RET) (Fig 3A and 3C; SI S2 Fig). Several angles discriminated blue- (λmax < 495 nm) from green-sensitive (λmax > 495 nm) pigments, Torsions 1,4, 14,15, and Angle 3 (see Torsion 15 in Fig 3C; see Torsion 1–14 and Angle 1–4 in SI S2 Fig). For the majority of the 19 angles examined, we obtained a single peak in the distribution (Fig 3C and S2 Fig). One notable exception was Torsion 1 (C5 –C6 –C7 –C8), which returned a single peak with a negative value of −63° ± 53° for zebrafish, medaka, and guppy Rh2 pigments (SI S2 Fig), but returned two peaks for all cichlid Rh2 pigments (SI S2 Fig; SI S3 Fig). This two-peak distribution was previously documented for the rhodopsin (RH1) visual pigment, using combined quantum mechanics/molecular mechanics (QM/MM) simulations [29]. To understand the dynamics of the chromophore within the opsin binding pocket we visualized the chromophore conformations seen in blue- vs green-sensitive pigments (Fig 3B). A compact cluster of conformations was observed for blue-sensitive pigments, in contrast to a more broadly distributed cluster in green-sensitive pigments. We calculated root mean square fluctuations (RMSF) of all the heavy atoms of the chromophore and the attached lysine residue (LYS+RET) (Fig 3D) for each pigment as follows: RMSF (V) =1T∑t=1T (vt−v¯), where T is total number of molecular dynamics trajectory frames (V). The atoms within the blue-sensitive pigments clearly show lower RMSF values than the green-sensitive pigments (Fig 3D). The results describing the conformations and dynamics of the chromophore suggested that these parameters may be used to distinguish green- from blue-sensitive Rh2 pigments and potentially to accurately predict λmax. We used a standard model selection procedure to determine the simplest linear regression model that fit the data. Possible model parameters were the median values for five angles that appeared to predict blue- vs green-sensitive λmax (Torsions 1,4, 14,15, and Angle 3; Fig 3C; SI S2 Fig), the area under the curve (AUC) of the RMSF values of heavy atoms of the β-ionone ring only (RMSF (ring) ), and the AUC of RMSF values of all the heavy atoms of LYS+RET (RMSF (LYS+RET) ). The best linear regression model for the 14 teleost Rh2 pigments contained two terms, the median value of Torsion 15 (C7 –C6 –C5 –C18) and the AUC of RMSF (LYS+RET). This predictive model is: λmax (predicted) = 475. 628 + (-8. 720*Torsion 15) + (34. 925*RMSF (LYS+RET) ). Larger values for Torsion 15 are therefore predicted to blue-shift λmax, and larger values for RMSF (LYS+RET) are predicted to green-shift λmax. Fig 4A shows the empirically determined λmax values [13,21,26–28] vs the predicted values for each Rh2 pigment analyzed. Spectral peaks predicted by our model correlate very well with the experimental values (R2 = 0. 94). We next used a leave-one-out approach to further test the statistical model. Each Rh2 pigment was removed from the regression analysis to obtain the coefficients for a model using Torsion 15 and RMSF (LYS+RET) as parameters, and then the λmax of the removed pigment was predicted based upon the new linear model. This test showed that there were no individual Rh2 opsins disproportionately influencing our results, and that the correlation of the individual predictions based upon only 13 pigments was also high (R2 = 0. 91) (Fig 4A). These results demonstrate that our approach can be used to predict spectral peak sensitivities for a wide range of Rh2 pigments with high accuracy. We next wished to further test the approach itself, rather than the specific regression model described above. Therefore, we generated a linear model based upon only a subset of the Rh2 pigments: 11 pigments of medaka, guppy, zebrafish, and zebrafish (cyprinid) ancestors. This resulted in a linear model utilizing Torsions 4 and 14 (1190. 6208+ (-4. 9097*Torsion 4) + (2. 9445*Torsion 14), that predicted λmax of these 11 Rh2 pigments with high accuracy (R2 = 0. 94) (Fig 4B). A leave-one-out approach to test this model also showed good predictive ability (R2 = 0. 89) (Fig 4B). This model was then used to predict spectral peaks for the three cichlid Rh2 pigments (which were not used to generate this particular model) using Torsion 4 and Torsion 14 data obtained from their MD simulations. The predictive accuracy was again rather high (Fig 4B; cyan symbols); particularly surprising given that the λmax of two of these cichlid Rh2 pigments (519 nm; Rh2-Aβ; 528 nm, Rh2-Aα) reside outside of the wavelength range of the 11 Rh2 pigments used to generate this model (467–516 nm). Indeed, the predicted value for Rh2-Aβ was 519 nm, matching its experimental value. These results demonstrate the utility and generality of the overall approach: statistical models derived from MD simulations of predicted visual pigment structures have the power to predict their λmax. We have developed a new approach for the prediction of cone pigment peak spectral sensitivity with a high degree of accuracy over a large range of λmax (452–528 nm). The approach required only template pigment structure and opsin protein sequence data as input. MD simulations were performed on the protein structures, and parameters describing the conformation of the opsin were then used in a statistical model. This in silico process revealed structural features of visual pigments, particularly within the chromophore and attached lysine residue that predict λmax. The approach is accurate, efficient and simple in that site-by-site molecular modifications or complex quantum mechanics models were not required. Instead, a two-term, first-order regression model was sufficient to achieve high correlations with empirical data. Although cone pigment homology models have been built using a rhodopsin template [30], to our knowledge this is the first report of a molecular modeling approach that predicts peak spectral sensitivities of vertebrate cone pigments. Previous strategies to predict visual pigment λmax include site-by-site amino acid substitutions followed by measurement of pigment spectra to identify potential contributions of specific amino acid residues to spectral shift. These approaches are informative but not able to provide accurate predictions over a wide range of spectra or opsins. For example, the “five sites” rule established for some LWS opsins, states that the amino acid changes H197Y, Y277F, T285A, and A308S shift λmax of these pigments toward lower wavelengths, with each change contributing in an additive manner [20,31,32]. However, this rule largely fails to predict relative λmax beyond selected mammals, even in other LWS opsins [21,22]. Similarly, the amino acid change E122Q in teleost Rh2 opsins predicts a change from green- to blue-sensitive λmax [13], but provides no further insights into specific λmax (Fig 1), and it is not known how broadly this rule applies to other vertebrate opsin classes. The approach described here does not require site-by-site manipulations and does not rely on identifying key residues. Rather, it considers each opsin sequence in its entirety, and provides highly accurate predictions of both green vs blue and specific λmax values across a wide range of Rh2 pigments. One of the key elements of our predictive model is the AUC of RMSF (LYS+RET); larger values correspond to greater fluctuation of heavy atoms of LYS+RET, and green-shifting of the λmax. We believe these findings provide new insight into the mechanisms of how E122Q contributes to spectral shift. The chromophore’s β-ionone ring resides in close proximity to the amino acid residue at position 122 [33]. The negative charge on E122 may encourage the hydrophobic β-ionone ring to explore other space in the binding pocket and increase its fluctuation, resulting in a green-shifted λmax, whereas Q122 discourages such fluctuations. If true, this increased motion of chromophore in green-shifted pigments in the dark state may raise the energy of this (ground) state, thereby decreasing the energy difference between the ground and excited states. We speculate that this decreased energy difference may in part underlie the higher λmax [4]. Another strategy previously described for the prediction of λmax focused on the shift in spectral sensitivity that takes place due to chromophore association with an opsin protein; this has been referred to as “opsin shift” [4,34]. This approach has only been applied to RH1 opsins (rhodopsins). The chromophore 11-cis retinal, in a Schiff base-bound state, absorbs at 360 nm, but shifts to 440 nm when the Schiff base is protonated, as in the environment of an opsin binding pocket [29,35]. Motto et al. [36] further explored mechanisms of spectral shift in bovine rhodopsins with specific mutations affecting amino acids lining the binding pocket, using combined quantum mechanical and molecular mechanical (QM/MM) methods, and MD simulations. They suggested that rotation along the chromophore’s single bond C6 –C7 (Torsion 1 of the present study) blue-shifts the rhodopsin by providing a reduced degree of conjugation. The MD simulations of the present study also identified Torsion 1 as appearing predictive of blue- vs green-sensitive λmax (SI S2 Fig), but this parameter did not emerge as a key element of our predictive model. We identified two distribution peaks identified for several torsions, including Torsion 1 (C5—C6—C7—C8) within cichlid Rh2 pigments, but not the other teleost Rh2 pigments. Several previous studies have estimated the torsional angle of C5 –C6 –C7 –C8 in bovine rhodopsin (RH1). Spooner et al. [37,38] estimated this value to be −28° ± 7° based on solid state NMR data derived from 13C labeled 11-Z-retinal substrate. Sugihara et al. [38] carried out geometry optimization and constrained MD simulation residues within 4. 5 Å of the chromophore (27 amino acids) using the self-consistent-charge density-functional-based-tight-binding (SCC-DFTB) method to identify the preferred conformation of the chromophore in the active site. They obtained a value of −35°. Rajamani et al. [29] performed combined QM/MM simulations using the chromophore in the rhodopsin-membrane-water configuration and tracked the instantaneous value of C5 –C6 –C7 –C8 torsion (Torsion 1). Interestingly, they also obtained two distribution peaks; a large fraction (86%) of the structures had a negative torsional angle of −68° ± 55° and a smaller fraction had +68° ± 25° with statistical weighted average of −49°. In our studies Torsion 1 was predictive of blue- vs green-sensitive spectra (SI S2 Fig), even when the two peaks for cichlid pigments were included. However, this angle was not identified as a key element of our statistical model for predicting λmax. The success of the present study in predicting λmax for Rh2 opsins points to elements of interest–Torsion 15 of the chromophore, and chromophore fluctuation (RMSF) –for future examination in determining mechanisms of color tuning in vertebrate visual pigments. While our correlational analyses provide predictive power, quantum mechanical studies are needed to mechanistically explain differences in λmax. Accuracy of the present approach also suggests numerous potential applications of this and similar approaches for biology, biophysics, and bio-engineering. The development of atomistic MD models for the other vertebrate visual pigment classes could potentially be used to predict λmax of any rod or cone pigment for which opsin sequence information is available. It is important to note, however, that in the current approach we restricted the analysis to Rh2 pigments, a class of pigments with high sequence similarity to each other and to the RH1 pigment template, and representing less than the entire range of vertebrate visual pigment λmax. A next logical step will be to build structural homology models for more divergent classes of vertebrate visual pigments. If successful, such models would lead to a more accurate understanding of mechanisms underlying spectral shift, and the range of evolutionary trajectories that lead to these shifts. Models could also be used to understand destabilizing effects of mutations associated with disease, and to design novel vertebrate opsins with specific spectral sensitivities for optogenetic applications as alternatives to channelrhodopsin [4]. The amino acid sequences of Rh2 cone opsins from zebrafish (Danio rerio) [13,26], medaka (Oryzias latipes) [27], guppy (Poecilia reticulate) [21] and cichlid (Metriaclima zebra) [28] were downloaded from UniProt (http: //www. uniprot. org/) (14 total sequences; see Table 1 for accession numbers). A template structure search was first carried out using MODELLER v9. 15 [39]. The bovine rhodopsin (RH1) structure (PDB ID: 1U19) [24] was chosen as a template for all of the teleost Rh2 opsins studied here because it satisfied the following criteria: i) sequence identity >60% (Table 1); ii) >95% sequence coverage with the target Rh2 sequence; iii) presence of 11-cis retinal bound to the binding pocket and occupied palmitoylation sites; iv) high X-ray crystal resolution (2. 2 Å); and v) no mutations in the crystal structure protein. MODELLER v9. 15 was then used to perform the sequence alignments and generate three-dimensional structures of Rh2 cone opsins. For each opsin sequence we generated five homology structures. Stereochemical checks were performed using the SWISS-MODEL structure assessment tool (https: //swissmodel. expasy. org/) on all five structures and the best was chosen based on minimal stereochemical deviation and high QMEAN score. The final selected structure of each Rh2 opsin was first uploaded on the web server, Prediction of Proteins in Membranes (http: //opm. phar. umich. edu/server. php). Membrane boundaries provided by this server along with the protein model were then uploaded onto the CHARMM-GUI server (http: //charmm-gui. org/) for further processing. To obtain the 11-cis retinal chromophore within the binding pocket of each structure, we changed the three letter amino acid code of the lysine residue that binds covalently with the chromophore and forms the Schiff base, from LYS to LYR in the PDB file. This modification allowed CHARMM-GUI to recognize and build the Schiff base and use appropriate forcefield parameters available on the server. We chose to include the palmitate moiety only in zebrafish Rh2 opsins because, among all Rh2 opsin sequences used in this study, only zebrafish Rh2 sequences shared a conserved palmitoylation site (C323) (SI S1 Fig) with the template bovine rhodopsin sequence. The C323 residue in all seven zebrafish opsin structures was linked to a palmitate molecule using the “add palmitoylation sites” option in CHARMM-GUI. Protonation states of amino acid residues were assigned at the physiological pH of 7. 4. The protein was embedded in an unsaturated homogeneous bilayer consisting 1-steroyl-2-docosahexaenoyl-sn-glycero-3-phosphocholine lipids to provide a realistic representation of the phospholipids found in the cone outer segment. The replacement method [40] was used to pack the opsin model with lipid bilayer. Lipid layer thickness was chosen to be 1. 6 (~70 lipids in top leaflet and ~70 lipids in bottom leaflet). Each system was placed in a rectangular solvent box, and a 10 Å TIP3P water layer (15 Å in the case of cichlid Rh2 opsins to prevent boundary effects) was added to solvate intra-and extra-cellular space. Charge neutrality of the system was achieved by adding Na+ and Cl− ions at a concentration of 0. 15 mol/L to the water layers. CHARMM-GUI (incorrectly) assumed the retinal was in 11-trans conformation and thus after preparing the system we replaced the coordinates of the retinal with 11-cis conformation obtained from template bovine rhodopsin structure. The CHARMM36 forcefield [41] parameters were used for the protein and lipids. Each system was first minimized using steepest descent for 5,000 steps. To allow equilibration of the water each system was then simulated for a total of 550 ps with the positions of all heavy atoms in the protein, phosphorus atoms in the lipid head group and all dihedral angles in the lipid carbon chains harmonically restrained. Each restrained simulation was divided in six steps where the restraints were gradually relaxed for each step. During the restrained simulations, the temperature of the system was set to 300 K and the pressure was maintained at 1 atm using the Berendsen algorithm. Production NPT simulations for each system were then carried out for 100 ns using Parrinello-Rahman barostat [42] with semi-isotropic pressure coupling and Nosѐ-Hoover thermostat [43] for maintaining the temperature. For all simulations, the LINCS algorithm was used to constrain all bonds involving hydrogen atoms to their ideal lengths. Particle mesh Ewald [44] was used for electrostatics with a real-space cutoff of 1. 2 nm. Van der Waals interactions were cut off at 1. 2 nm with the Force-switch method for smoothing interactions. Each trajectory was 100 ns long with time step of 2 fs and updated neighbor lists every 20 steps. Trajectory snapshots were saved every 10 ps. All systems were prepared using the CHARMM-GUI (http: //www. charmm-gui. org) web server. Molecular dynamics (MD) simulations were carried out using GROMACS v5. 1. 2 [45]. Analysis of the internal degrees of freedom i. e. torsion and bond angles was carried out using plumed-driver tool from PLUMED v2. 2. [46]. Molecular visualization of MD simulations was done in VMD [47]. The best linear regression model to predict spectral peak for the samples of teleost Rh2 opsin proteins was determined using seven parameters obtained from the MD simulations: the medians for Torsion 1, Torsion 4, Torsion 14, Torsion 15, and Angle 3 (Fig 2C; SI S2 Fig), and the areas under the curves (AUC) for root mean square fluctuation RMSF (ring) and RMSF (LYS+RET). A best subsets procedure was used in which regression models with number of covariates from one to seven were fitted using the regsubsets option in the “leaps” R library (https: //cran. r-project. org/web/packages/leaps/leaps. pdf), and the seven best models for each number of covariates was retained. Each of these 49 regression models was ranked based upon their Bayesian Information Criterion (BIC) value [48]. The best fitting statistical model based upon the BIC was examined for influential data points using a leave-one-out test, as follows. For each of the molecular Rh2 structures under consideration, one protein was removed from the regression analysis for the best fitting statistical model, and then the peak spectral sensitivity of the removed protein was predicted based upon the new linear model. | Vertebrate color vision is possible when cone visual pigments with distinct peak spectral sensitivities (λmax) are expressed in separate cone populations and provide differential input to downstream neurons. The λmax is a function of the type of chromophore (such as 11-cis retinal) and the amino acid sequence of the associated opsin protein. In this study we utilize a molecular modeling approach to predict with high accuracy the λmax of cone visual pigments, using only the amino acid sequences of the corresponding opsin proteins as input. Such a functionally predictive, genome to phenome, opsin modeling has been elusive for decades, and now carries high potential for future applications in evolutionary biology, biophysics, bio-engineering, and vision science. | lay_plos |
As a fresh batch of leaked files gave new urgency to worldwide controversy surrounding the publication of US diplomatic communiqués, there was continued mystery over the motivation, intentions and whereabouts of the man at the heart of the whistle-blowing website Wikileaks. Despite accusations that Julian Assange is on the run, The Independent has learnt that Scotland Yard has been in contact with his legal team for more than a month but is waiting for further instruction before arresting him. Police forces around the globe have been asked to arrest the enigmatic Wikileaks founder, who is wanted in Sweden to answer a series of sexual allegations against him. But the 39-year-old Australian supplied the Metropolitan Police with contact details upon arriving in the UK in October. Police sources confirmed that they have a telephone number for Mr Assange and are fully aware of where he is staying. Britain's Serious Organised Crime Agency (Soca) has received the so-called "red notice" – an international arrest warrant – but has so far refused to authorise the arrest of Mr Assange, who is thought to be in South-east England. Until it does, police forces cannot act. The delay is said to be a technical one, with sources suggesting Soca needed clarifications about the European Arrest Warrant issued by Swedish prosecutors for Mr Assange, a fast-track system for arresting suspects within the EU. His name was added to Interpol's worldwide wanted list on 20 November, but only publicly revealed on Tuesday night. It has focused even greater attention on the man who has been lying low since releasing 250,000 mostly classified cables from US embassies across the globe. Outrage at the data breach intensified yesterday, with the Russian Prime Minister Vladimir Putin and his Turkish counterpart Recep Tayyip Erdogan among those to complain. Friends of Mr Assange said he has been working through the night to protect his website from repeated attempts by hackers to bring it down. Last night, it emerged that Amazon had agreed to stop hosting Wikileaks after pressure from US senators. Wikileaks had temporarily switched to Amazon's servers during the cyber attacks. Mr Assange's associates refused to reveal his current whereabouts because death threats have been made against him. His lawyer, Mark Stephens, insisted last night Mr Assange was not hiding. He has conducted brief media interviews from his current location. He is wanted in Sweden on suspicion of rape, sexual molestation and unlawful coercion, but no formal charges have been filed. Mr Assange has always denied the accusations, saying he had consensual sex with two women during a trip to Sweden in August. His mother, Christine Assange, spoke of her distress at the Interpol alert issued against her son, adding that she was concerned for his wellbeing. "He's my son and I love him and obviously I don't want him hunted down and jailed," she told the Australian Broadcasting Corporation from her Queensland home. "A lot of stuff that is written about me and Julian is untrue." The enemies he has accumulated abroad rounded on him yesterday. One former adviser to the Canadian Prime Minister, Stephen Harper, even suggested Mr Assange should be assassinated. Hillary Clinton, the US Secretary of State, has said the site acted illegally and added there would be "aggressive steps" taken against those who released the information. Mr Assange's legal team has lodged an appeal with Sweden's highest court against the order to have him returned to Stockholm. Mr Stephens launched a counterattack against Swedish prosecutors yesterday, saying they had failed to meet basic legal obligations such as informing him about the allegations he faces. "Given that Sweden is a civilised country, I am reluctantly forced to conclude that this is a persecution and not a prosecution," Mr Stephens said. "There is no suggestion that he is in any way a fugitive. The police and the security services here know exactly where he is located." Mr Assange has hinted in the past he may head to Switzerland. He has also been offered safe haven by the government of Ecuador. However, now the Interpol notice has been issued, it will be much more difficult for him to move around the globe unnoticed. PARIS - For Julian Assange, the WikiLeaks founder and guiding spirit, it must have been sweet. Secretary of State Hillary Rodham Clinton was trying to smooth over the embarrassment, while the White House huffed that a criminal investigation was underway. The French government was decrying "the height of irresponsibility," and Pakistan was reckoning with a rebuke from its traditional patron, Saudi Arabia. Meanwhile, the pasty-faced Assange, a self-appointed knight-errant who has been doing battle with official secrecy since 2006, was in the shadows somewhere - few knew where - undoubtedly savoring the ruckus caused by WikiLeaks' exposure of confidential State Department cables. In an uncomfortable irony, however, he had to do the savoring in a secret location, because Interpol, the international anti-crime organization, has issued a high-priority "red notice" asking member countries to arrest him. President Obama's spokesman, Robert Gibbs, has said the U.S. government might want to prosecute Assange, a 39-year-old Australian, for violating U.S. secrecy laws and perhaps even for espionage. But that is not the reason for the notice - at least not yet. Swedish authorities on Nov. 18 asked Interpol to help them take Assange into custody for questioning on accusations of rape and sexual harassment. The charges were brought by two Swedish women in August after what both described as consensual sexual encounters in Sweden that escalated into something unwanted and illegal. Assange, who has long expressed fear of reprisal from Washington and other governments, denied anything but consensual sex and suggested that the two women were part of a plot to smear his name and undermine his campaign to get government secrets into the open. Before the women came forward, Assange had sought a Swedish residence visa, hoping to benefit from the country's strong protection of press freedoms. But since then, he has been traveling constantly and staying below the radar, popping up in London, appearing on a videoconference in Amman, Jordan, and answering questions from Time magazine via Skype, reluctant to show himself in flesh and blood. In the Time Q&A, he said Clinton should resign because of her cable suggesting that U.S. diplomats surreptitiously gather personal data on their counterparts, something Assange said would violate the Vienna Convention on diplomatic activity. Gibbs dismissed that idea as "ridiculous" and said the problem is rather that Assange violated the diplomatic convention. An underground existence is nothing new for Assange, his associates have pointed out. Driven by his anti-secrecy crusade and convinced that governments are out to get him, he has long avoided fixed residences, borrowing travel funds and sleeping on other people's couches between marathon sessions at the computer. WikiLeaks announced via Twitter, meanwhile, that it has been the target of repeated hacker attacks since the publication of more than 250,000 leaked State Department communications, a claim in line with Assange's frequent assertions that U.S. intelligence agencies are intruding on his work. While Assange stayed out of sight, his Stockholm lawyer, Bjorn Hurtig, filed an appeal Wednesday against the Swedish government's arrest order. At the same time, his London attorney, Mark Stevens, said the Stockholm prosecutor's tactics show that she is out to get Assange for more than legal reasons. "Since Sweden is a civilized country, I have to come to the conclusion that this is persecution and not prosecution," Stevens said in an e-mail to the Associated Press. One news report suggested that Assange is considering asking for asylum in Switzerland. But that country is known for keeping secrets, so that seemed an unlikely solution. Moreover, WikiLeaks has announced that its next big project is to reveal the confidential documents of a major bank, believed to be Bank of America, which could make the land of numbered accounts an awkward haven for Assange. A surprising possibility seemed to arise Monday, when a deputy foreign minister of Ecuador, Kintto Lucas, said Assange would be welcome in that South American country. "We are going to invite him to come to Ecuador so he can freely present the information he possesses and all the documentation, not just over the Internet but in a variety of public forms," Lucas announced. By Wednesday, however, that idea had fallen apart. President Rafael Correa said neither he nor his foreign minister had approved the invitation to Assange and gave a strong impression that they never would. [email protected] The U.S. government, apparently aided by freelance computer hackers, chased WikiLeaks from an American commercial computer network and temporarily stopped the leak of embarrassing diplomatic documents. But within hours, the website was back online, publishing from a fortified bunker in Sweden. FILE - In this Oct. 23, 2010 file photo, WikiLeaks founder Julian Assange speaks during a news conference in London. Interpol on Tuesday, Nov. 30, 2010 placed Assange on its most-wanted list after Sweden... (Associated Press) A picture of Wikileaks founder Julian Assange is shown in this photo of the cover of the Wednesday, Dec. 1, 2010 edition of the New York Post, photographed in New York, Wednesday, Dec. 1, 2010. Police... (Associated Press) The virtual chase Wednesday was mirrored by a real-life pursuit as European authorities hunted for the site's fugitive founder, Julian Assange, who is wanted in Sweden on rape charges. Undeterred, Assange continued releasing confidential government documents. Some showed how the Obama administration and Congress helped persuade Spain not to pursue charges against members of George W. Bush's administration for allowing torture of terrorism suspects. Amazon.com Inc. prevented WikiLeaks from using the U.S. company's computers to distribute State Department communications and other documents, WikiLeaks said Wednesday. The WikiLeaks site was inaccessible for several hours before it returned to servers owned by its previous Swedish host, Bahnhof, which are housed in a protective Cold-War era bunker. Tech blogs have compared it to a lair from a James Bond movie. "We have been under attack and we have had to move to different servers," WikiLeaks spokesperson Kristinn Hrafnsson said. "But we have ways and means to bypass any closure in our services." Amazon's move to evict WikiLeaks from its servers came after congressional staff called the company to inquire about its relationship with WikiLeaks, Sen. Joe Lieberman, the Connecticut independent, said Wednesday. "The company's decision to cut off WikiLeaks now is the right decision and should set the standard for other companies WikiLeaks is using to distribute its illegally seized material," Lieberman said in a statement. He added that he would have further questions for Amazon about its dealings with WikiLeaks. The White House said it was taking new steps to protect government secrets after WikiLeaks released thousands of sensitive U.S. diplomatic cables. Officials said national security adviser Tom Donilon has appointed a senior aide to identify and develop changes needed in light of the document dump. Australian Prime Minister Julia Gillard called WikiLeaks' publication of classified documents "an illegal thing to do" on Thursday. But Gillard did not indicate Australia was about to take legal action against Australian-born Assange. The country's attorney general reiterated that authorities are investigating whether Assange has broken any Australian laws, but have not yet reached a conclusion. The White House on Wednesday spurned a call from Assange for Secretary of State Hillary Rodham Clinton to step down if she had any role in directing U.S. diplomats' spying on other foreign leaders. "Mr. Assange's suggestion is ridiculous and absurd, and why anyone would find his opinion here relevant is baffling," said spokesman Tommy Vietor, adding Clinton was doing an "extraordinary" job. The White House said U.S. diplomats do not engage in spying. Clinton was in Astana, Kazakhstan, enduring repeated comments about the WikiLeaks disclosures as she met with foreign officials at a conference of international leaders. Among those she met with was Italian Prime Minister Silvio Berlusconi, who had been described in newly released U.S. diplomatic cables as "feckless" and a party animal. "We have no better friend, we have no one who supports the American policies as consistently as Prime Minister Berlusconi has, starting in the Clinton administration, through the Bush administration and now the Obama administration," she said during a summit of the Organization for Security and Cooperation in Europe. The WikiLeaks matter was discussed in virtually all of Clinton's private one-on-one meetings with European leaders and foreign ministers during the summit meeting Wednesday. Assange remained a fugitive Wednesday, shadowed by the Europe-wide arrest warrant. A German security official, speaking on condition of anonymity because no authorization was given to discuss the legal steps, confirmed that a warrant for Assange had been issued in that country. Hrafnsson noted the "interesting timing" of the warrant but said Assange had gone into hiding for another reason: "If you think about the threats we've heard about our people, and against Julian, it's very natural and understandable that his location is kept secret." Assange's London-based lawyer, Mark Stephens, complained his client had yet to receive formal notice of the allegations he faces _ something Stephens described as a legal requirement under European law. The lawyer added that Assange had repeatedly offered to answer questions about the investigation, to no avail. The exact nature of the allegations facing Assange isn't completely clear. Stephens has in the past described them as part of "a post-facto dispute over consensual, but unprotected sex." Even Swedish prosecutors have disagreed about whether to label the most serious charge as rape. Formal charges have not been filed, but a detention order issued Nov. 18 at the request of Marianne Ny, Sweden's Director of Public Prosecution, remains in force pending an appeal by Assange. The case is now before Sweden's Supreme Court. ___ Associated Press writers Malin Rising in Stockholm; Matthew Lee and Lolita C. Baldor in Washington; Peter Svensson in New York; Robert Burns in Astana, Kazakhstan; Raphael G. Satter in London; and Kirsten Grieshaber in Berlin contributed to this report. | With Interpol seeking to bring him into custody on rape charges and people including Sarah Palin and Bill O'Reilly calling for his head, it may be a while before Julian Assange turns up at a press conference-but the Independent claims to know where he is. It reports that the WikiLeaks founder provided Britain's Metropolitan Police with his contact details when he arrived in the country in October; police sources say that they do indeed have a telephone number for Assange, and know where he is staying-likely in southeast England, notes the paper. Authorities there have received the international arrest warrant for Assange, but sources say they aren't authorizing Assange's arrest yet due to some technical questions. He may be planning to seek asylum in Switzerland, despite that country's fondness for secrecy, the Washington Post reports. WikiLeaks itself has also been forced to find a new home. After being evicted from servers it was renting from Amazon, the site shifted back to its old home in Sweden, in servers housed in a Cold War-era bunker. "We have been under attack and we have had to move to different servers," a WikiLeaks spokesperson tells AP. "But we have ways and means to bypass any closure in our services." | multi_news |
The possibility that a new black independent film movement — or even a genuinely crossover cinema — would emerge in the wake of “Precious: Based on the Novel ‘Push’ by Sapphire,” now seems as remote as it did before this art-house phenomenon made its way, assisted by Oprah Winfrey and Tyler Perry, from the Sundance Film Festival to the Oscars. But while Ms. Winfrey and Mr. Perry, as executive producers, brought attention to this story of an abused black teenage girl, “Precious” now looks more like a one-off than a harbinger of change, much like another of last year’s nominees, “The Princess and the Frog,” the first Disney movie with a black princess. (The latest Disney princess, Rapunzel in “Tangled,” is as blond as Sleeping Beauty back in 1959.) What happened? Is 2010 an exception to a general rule of growing diversity? Or has Hollywood, a supposed bastion of liberalism so eager in 2008 to help Mr. Obama make it to the White House, slid back into its old, timid ways? Can it be that the president’s status as the most visible and powerful African-American man in the world has inaugurated a new era of racial confusion — or perhaps a crisis in representation? Mr. Obama’s complex, seemingly contradictory identity as both a man (black, white, mixed) and a politician (right, left, center) have inspired puzzlement among his supporters who want him to be one thing and detractors who fear that he might be something else. In their modest way American movies helped pave the way for the Obama presidency by popularizing and normalizing positive images of black masculinity. Actors like Mr. Poitier and Harry Belafonte made the leap, allowing black men to move beyond porters and pimps to play detectives, judges, the guy next door, the God upstairs and the decider in the Oval Office. At the same time, while the variety of roles increased, the commercially circumscribed representational conservatism of American cinema — with its genre prerogatives and appetite for uplift, its insistence on archetypes and stereotypes, villains and heroes — meant that these images tended to fit rather than break or bend the mold. Certainly this isn’t a cinema that jibes with what, in his 1995 memoir “Dreams From My Father,” Mr. Obama called “the fluid state of identity.” Photo The recognition of that fluidity, and the exploitation of it for creative and commercial ends, has, from the swing era through hip-hop, been much more the province of America’s popular music than its movies. Partly because movies remain a top-down, capital-intensive art form, they have been more cautious and apt to cater to rather than to subvert the perceived prejudices of the audience. In Hollywood race has often been a social problem to be earnestly addressed (and then set aside), or a marketing challenge. In the 1960s the studios congratulated themselves for making sober, correct-thinking dramas that often starred Mr. Poitier in films like “In the Heat of the Night” and “Guess Who’s Coming to Dinner,” both released in 1967 and which together reaped 17 Oscar nominations. A few years later, when a new generation of actors and filmmakers emerged from the rubble of the old studio system, Mr. Poitier was no longer alone as African-Americans began to appear on screen and behind the camera to an unprecedented extent. Faces and voices that had once been found only in “race movies” or art-house films by the likes of Shirley Clarke (“The Cool World”) filtered into the mainstream. There were blaxploitation hits like “Shaft,” as well as crossover dramas (“Sounder”) and popular comedies, including the trilogy “Uptown Saturday Night,” “Let’s Do It Again” and “A Piece of the Action,” directed by Mr. Poitier and starring him and Bill Cosby. The independent world saw the emergence of off-Hollywood directors like Charles Burnett, Haile Gerima, Billy Woodberry and Julie Dash. Race in American cinema has rarely been a matter of simple step-by-step progress. It has more often proceeded in fits and starts, with backlashes coming on the heels of breakthroughs, and periods of intense argument followed by uncomfortable silence. To that end, the 1980s were, with a few exceptions — or maybe just the exception of Eddie Murphy in “48 Hours,” Trading Places” and “Beverly Hills Cop” — marked as much by racial retrenchment as by the consolidation of the blockbuster mentality. More hopefully, the end of that decade ushered in a new generation of do-it-yourself black filmmakers, most famously and outspokenly Spike Lee, who tried to beat the system and then joined it. Newsletter Sign Up Continue reading the main story Please verify you're not a robot by clicking the box. Invalid email address. Please re-enter. You must select a newsletter to subscribe to. Sign Up You agree to receive occasional updates and special offers for The New York Times's products and services. Thank you for subscribing. An error has occurred. Please try again later. View all New York Times newsletters. Mr. Murphy, Mr. Lee and the African-American stars who ascended in the 1990s and the decade that followed — notably Will Smith, Morgan Freeman, Jamie Foxx and of course Mr. Washington — often had to shoulder the burden of representing their race even as they pursued their individual ambitions. For the most part, these stars rode to the top of the box office in stories that did not engage or address race, while the films that did take the subject on more directly — like “Ali” and “Dreamgirls” — often did so at a historical safe distance. It was almost as if, with the ascendancy of individual black movie stars, Hollywood no longer felt the need to tell stories about black people as a group. This retreat from race by the big studios partly explains the emergence of a newly separate black cinema with its own stars (Morris Chestnut, Vivica A. Fox), auteurs (Ice Cube, Tyler Perry) and genres (including tales of buppie courtship like “Two Can Play That Game” and of neighborhood striving like the “Barbershop” franchise). Emerging from outside the mainstream and indie world, the prolific Mr. Perry has become one of the most successful directors and producers of any color. Last year he directed a much-maligned adaptation of Ntozake Shange’s “For Colored Girls Who Have Considered Suicide When the Rainbow Is Enuf.” Some complained that Mr. Perry had bowdlerized that’s famous feminist work, but he had made it his own, complete with melodramatic flourishes and divas like Janet Jackson. Advertisement Continue reading the main story Mr. Lee has been among Mr. Perry’s critics. “We’ve got a black president, and we’re going back,” Mr. Lee said in 2009. “The image is troubling, and it harkens back to Amos ’n’ Andy.” The philosopher Cornel West has been more charitable (“Brother Tyler can mature”) and last year he put a larger frame around the issue of race and the movies in America, noting that with “all the richness in black life right now,” that “the only thing Hollywood gives us is black pathology. Look at the Oscars. Even ‘Precious,’ with my dear sister Mo’Nique, what is it? Rape, violation, the marginalized. Or else you get white missionary attitudes toward black folk. ‘The Blind Side?’ Oh my God! In 2010? I respect Sandra Bullock’s work, but that is not art.” “The Blind Side” might not have been art, but along with three other best picture nominees — “Precious,” “District 9” and “Avatar” — it did take on questions of black and white relations. At the center of “The Hurt Locker,” which won best picture, was the volatile friendship between two soldiers, a hot-headed white bomb-disposal specialist played by Jeremy Renner and his cautious black sergeant, played by Anthony Mackie. Race in that movie was not a theme or a problem to be solved, but rather a subtle, complex fact of life. These nominees suggested a range of approaches to a volatile subject, from allegory to melodrama to various kinds of realism. Did filmmakers somehow exhaust the subject? Or has the cultural ground shifted and, with the economic crisis, made other kinds of stories feel more urgent? While it might be a stretch to yoke together the differently privileged milieus of “The King’s Speech,” “The Kids Are All Right” and “The Social Network,” it is hard to escape the impression that class made something of a comeback in 2010. “The Fighter” relates the story of working-class, boxing brothers from a former Massachusetts mill town. Set in the Ozarks, “Winter’s Bone” involves the violent, clannish world of crystal meth producers whose grandfathers likely ran moonshine. “The Town,” which brought Mr. Renner his second nomination in two years, depicts a similar milieu, but with Boston bank robbers. So is class the new race? It may be tempting to think so, given the state of the economy and the political discourse, but the two have never really stood apart in American life. And the racial complexity of American life seems, at least at the moment, to have stymied the collective imagination of the movie industry. Perhaps this next year will bring more change we can believe in. For now, though, looking down the roster of the most popular movies released in 2010, only one seems capable of acknowledging, and making a story of the perfectly ordinary reality of a black man with a blue-collar job. That was Tony Scott’s “Unstoppable,” starring none other than Denzel Washington. The rest of Hollywood, it seems, will always be chasing him. Whoopi Goldberg Misfires in Attack on Article About Black Oscar Winners Email This Whoopi Goldberg used her bully pulpit on 'The View' on Monday to blast The New York Times and two of its marquee writers for what she believed was a "When you win an Academy Award, that's part of what you've done, your legacy. I will always be Academy Award winner Whoopi Goldberg," she said during her lengthy rant, referring to her best supporting actress win for 'Ghost.' "This is not hidden information. And to these two critics, the head critics of The New York Times. It's hard to not take it personally." Whoopi Goldberg used her bully pulpit on 'The View' on Monday to blast The New York Times and two of its marquee writers for what she believed was a disrespectful article about race and the Academy Awards. "I'm embarrassed to tell you it hurt me terribly," the actress said in a segment focusing on a piece written by Manohla Dargis and A. O. Scott that listed several black Oscar winners dating back to 1940, but didn't include Goldberg."When you win an Academy Award, that's part of what you've done, your legacy. I will always be Academy Award winner Whoopi Goldberg," she said during her lengthy rant, referring to her best supporting actress win for 'Ghost.' "This is not hidden information. And to these two critics, the head critics of The New York Times. It's hard to not take it personally." "Real change seemed to have come to movies or at least the Academy, which had given statuettes to a total of seven black actors in the previous 73 years." "The error lies with those who are reading the story incorrectly. The point of the piece was not to name every black actor or actress who has been awarded an Oscar, it was to draw a comparison between the number who won prior to 2002 (the year Halle Berry and Denzel Washington won) and those who have won since. And the story states very clearly that in 73 years, prior to 2002, only seven black actors/actresses won Oscars." The article, titled 'Hollywood's Whiteout,' apparently riled Goldberg up because it seemed to list all of the black Americans who have won an Oscar. A closer look, however, proved otherwise. The authors write that nine years ago, when Denzel Washington and Halle Berry won their historic Oscars:The article then notes that after Washington and Berry came a string of Oscar wins for Jamie Foxx, Forest Whitaker, Morgan Freeman, Jennifer Hudson and Mo'Nique.It was presumably this passage, along with mentions of Sidney Poitier and Hattie McDaniel, that caused Goldberg to believe she had been forgotten. "People in Somalia know. People in China know," she said on 'The View.' "I know it's hard to believe, but I'm a worldwide person who's known."But if a correction is what she desires, Goldberg will be waiting a very, very long time.reached out to the Times, and a source at the newspaper clarified that Goldberg won her Oscar for 1990's 'Ghost,' more than a decade before the time period that the authors were focusing on. In the article, Dargis and Scott were discussing race and the cinematic representation of people of color since 2002, when Berry and Washington were triumphant.Later on Monday, the Times released a statement that tweaks Goldberg without naming her:Also not mentioned in the article were Louis Gossett, Jr. (1982) and Cuba Gooding, Jr. (1996).This year's Oscars have raised ire because there are no persons of color nominated in the acting categories.Goldberg has not responded to requests to clarify her comments on 'The View,' which included some pretty pointed attacks on the journalists and the newspaper."I don't know what to say about what you've done. It's just, well, I can say, is that you're sloppy in your work and you're supposed to be better than this," she said. "This is The New York Times, not some bozo newspaper from Hoochi-Coochie land. This is The New York Times. I just, you know, it hurts."► Read the New York Times article here. As many have pointed out, there are zero black acting nominees for the Oscars this year. The New York Times did an article on the subject entitled “Hollywood’s Whiteout”, in which they discussed the very small amount of winners over the years. They listed every one most…except for Whoopi Goldberg. And don’t think she didn’t notice. On today’s View, Goldberg lashed out at the Times for its “sloppy journalism” going so far as to bring her Oscar to the set to prove that, yes, it does exist. After Barbara Walters mentioned the apparent snub and asked how she felt, Goldberg said it “hurt [her] terribly,” while admitting that she was “embarrassed” for feeling that way. However, it was clear just how stung she was. “It’s hard to not take it personally. It’s very hard not to take it personally. You know, there’s a lot of stuff that people say and do but this is sloppy journalism because this is not a hidden thing. Everybody kind of knows. People in Somalia know, people in China know, because I, I know it’s hard to believe, because I am a world-wide person whose known. Because there wasn’t anyone like me and it was 70 years between Hattie McDaniel, the first black woman to win, and me. So this omission, I don’t know what to say, there’s nothing I can say except you’re sloppy in your work and you’re supposed to be better than this. This is the New York Times. It’s not some bozo paper from Hoochie-Coochie Land.” It would be easy to look quickly and condemn Goldberg as just another actress diva, however, it should be noted that she is only one of thirteen black actors to win the award, something that she clearly counts as one of the biggest moments of her life. You’d think there’d be some room to mention number four in an article of more than 2,000 words. Still, there’s no reason to be angry at the Times. And there’s no reason to be angry at Goldberg. Who we should be angry at is Hollywood. Thirteen? Only thirteen? Seriously? (h/t TMZ) UPDATE: This post originally stated that the Times article listed every winner except Goldberg. This isn’t true as they also didn’t mention Louis Gossett, Jr.. I should have been clearer that the original article never claimed to list every black winner, just those in recent years. However, I still understand why Goldberg was hurt. Like I said, though, the real anger shouldn’t be directed at the Times or the actress. The real problem is that there are so few black winners that any one of them has legitimate reason to feel left out when not mentioned. Check out the clip from The View below: | Whoopi Goldberg has lashed out at the New York Times for an article on how no black actors are being considered for Academy Awards this year. While Goldberg can sympathize with the article's main thrust, it's the fact that the authors-movie critics AO Scott and Manohla Dargis-seem to list every black Academy Award winner except her, Mediaite reports. Goldberg took her grievances-and her actual Oscar-to the View. "It's hard to not take it personally. It's very hard not to take it personally. You know, there's a lot of stuff that people say and do but this is sloppy journalism because this is not a hidden thing. Everybody kind of knows. People in Somalia know, people in China know, because I, I know it's hard to believe, because I am a world-wide person who's known," Goldberg said. Yet PopEater did some digging, and even contacted the Times, which noted that the list was not supposed to be comprehensive, and focused on race in cinema since 2002-12 years after Goldberg won her honors for 1990's Ghost. Fellow black '90s winners Louis Gossett Jr and Cuba Gooding Jr were also excluded, yet have remained silent on the issue. | multi_news |
Glioblastoma (GB) is a highly invasive and lethal brain tumor due to its universal recurrence. Although it has been suggested that the electroneutral Na+-K+-Cl− cotransporter 1 (NKCC1) can play a role in glioma cell migration, the precise mechanism by which this ion transporter contributes to GB aggressiveness remains poorly understood. Here, we focused on the role of NKCC1 in the invasion of human primary glioma cells in vitro and in vivo. NKCC1 expression levels were significantly higher in GB and anaplastic astrocytoma tissues than in grade II glioma and normal cortex. Pharmacological inhibition and shRNA-mediated knockdown of NKCC1 expression led to decreased cell migration and invasion in vitro and in vivo. Surprisingly, knockdown of NKCC1 in glioma cells resulted in the formation of significantly larger focal adhesions and cell traction forces that were approximately 40% lower than control cells. Epidermal growth factor (EGF), which promotes migration of glioma cells, increased the phosphorylation of NKCC1 through a PI3K-dependant mechanism. This finding is potentially related to WNK kinases. Taken together, our findings suggest that NKCC1 modulates migration of glioma cells by two distinct mechanisms: (1) through the regulation of focal adhesion dynamics and cell contractility and (2) through regulation of cell volume through ion transport. Due to the ubiquitous expression of NKCC1 in mammalian tissues, its regulation by WNK kinases may serve as new therapeutic targets for GB aggressiveness and can be exploited by other highly invasive neoplasms. Glioblastoma (GB) is the most common malignant primary brain tumor. GBs are aggressive and display key features of invasion and infiltration of healthy brain tissue [1]. Due to its invasive nature, GB is not curable through surgical resection [2], [3]. The surgical and medical treatment for patients with this disease has evolved in the last 20 years, however the prognosis remains dismal due to tumor recurrence [4]. Thus, understanding the mechanisms that GB cells utilize during migration and invasion into normal brain tissue is paramount in the development of novel, effective therapies. Volume regulation, cytoskeletal rearrangements, and adhesion dynamics are major determinants of cell migration and are essential processes in invasion [5], [6]. Migration of mammalian cells is accompanied by volume changes. For instance, neutrophils [7] and dendritic cells [8] undergo cell volume increases when exposed to signals leading to migratory responses. Indeed, it has been hypothesized that inhibition of cell volume regulation impairs cell migration [9], [10]. NKCC1, a transporter that belongs to the SLC12A family of cation-chloride cotransporters, is a fundamental transporter utilized in the regulation of intracellular volume and in the accumulation of intracellular Cl− [11], [12]. NKCC1 mediates the movement of Na+, K+, and Cl− ions across the plasma membrane using the energy stored in the Na+ gradient, generated by the Na+/K+ ATPase. Recent work supports the notion that intracellular volume regulation by NKCC1 [13], [14], as well as aquaporin 4 (AQP4) [15], may indeed promote glioma cell invasion. However, whether cell volume regulation is the only or primary mechanism mediating NKCC1 effects is unclear. It is equally unclear if NKCC1 is differentially regulated in invasive cells. In addition to cell volume regulation, ion transporters can participate in anchoring the cytoskeleton to the plasma membrane by binding to ezrin-radixin-moesin (ERM) proteins [16], [17]. ERM proteins associate directly with actin and integral membrane proteins, which connect the cytoskeleton to the plasma membrane [18]. Anion exchangers (AE) 1,2, and 3, Na+/H+ exchanger 1 (NHE1), and a Na+/Ca++ exchanger are all able to act as cytoskeletal anchors by interacting with ERM proteins [19]. It has been shown that ERM proteins bind to clusters of positive amino acids in the juxtamembranous domain of NHE1, CD44, CD43, and ICAM-2 [16], [20] and that these interactions regulate cell migration and contractility, as well as focal adhesion turnover [21], [22]. The interaction between ion transporters, as integral membrane proteins, and the cytoskeleton mediates the transduction of contractile forces generated from within the cell to the extracellular matrix and promotes migration. However, the mechanistic action of NKCC1 on cell contractility and focal adhesion dynamics in the context of GB cell migration and invasion are entirely unknown. Activation of NKCC1 transport activity requires phosphorylation of key threonine residues in the NKCC1 N-terminal domain [23]. Phosphorylation of NKCC1 is mediated by at least three members of a novel family of unusual kinases that lack a key lysine in their catalytic domain, the WNK kinases (With No K-lysine) [24], [25]. These kinases have been implicated in the pathogenesis of hypertension and epilepsy [26], [27]. Of these, WNK3 is the most abundantly expressed in the brain [28]. Interestingly, WNK1 is a substrate for Akt-mediated phosphorylation [29]. Hence, it is possible that Akt may regulate NKCC1 activity through the regulation of the WNK kinases. Intracellular signaling pathways, such as phosphoinositide 3-kinase (PI3K) -Akt, are frequently altered in GBs [30]. Akt is able to regulate various cellular functions through phosphorylation of a conserved substrate sequence, and altered regulation of this pathway can lead to aberrant cell behavior, such as increased proliferation and migration [31]–[33]. Importantly, intracellular signaling pathways of promigratory factors such as epidermal growth factor (EGF) [34]–[36], and integrin signaling pathways converge on Akt, modulating cell processes such as cell cycle, apoptosis, and migration [37]. PI3K, the activator of Akt, is thought to be critical in mediating both chemotactic and random cell migration [38]. Therefore, the regulation of NKCC1 by the interaction between Akt signaling and WNK kinases may be important in determining the invasive properties of GB cells. To further our understanding of the role of NKCC1 in GB cell migration and invasion, we investigated (1) whether the expression of NKCC1 in human tumors correlates with tumor grade, (2) whether NKCC1 affects cell contractility and migration, (3) whether NKCC1 can have an effect on the interaction between the cells and the cells' adhesion substratum, and (4) whether a signaling mechanism involved in the regulation of NKCC1 by promigratory factors exists in GB cells. We found that NKCC1 expression indeed correlates with in vivo glioma aggressiveness and that the transporter activity modulates migration speed and invasiveness of cells derived from various human GBs. Furthermore, we show that NKCC1 expression affects GB cell traction forces, possibly by regulating focal adhesion dynamics. Moreover, the regulation of NKCC and KCC transport by WNK3 may determine the invasive behavior of GB cells. Additionally, we show evidence of NKCC1 phosphorylation regulation by Akt through WNK3 phosphorylation upon stimulation with a promigratory factor, EGF. This suggests an important link between the activation of WNK3 by Akt as well as changes in the activity of ion transport systems in glioma cells. Taken together, these findings strongly suggest that ion transport regulation might be integrated into the control of glioma cell invasiveness in a complex fashion that extends beyond regulation of cell volume and involves the interplay between cell adhesion and growth factor signaling. The understanding of these complex interactions may assist in the design of novel therapeutic strategies. Prior data implicating NKCC1 in GB invasiveness were based on established, model GB cell lines, rather than primary cells or tissues. We therefore first evaluated and characterized whether primary cells isolated from human GB indeed supported the role of NKCC1 in invasiveness as suggested previously [13]. We assayed invasiveness using the transwell invasion assay in the presence or absence of the NKCC1 inhibitor bumetanide [39]. Inhibition of NKCC1 transport in various primary human glioma cells exposed to 25 and 50 µM of bumetanide led to a dose-dependent decrease in the number of invasive cells (Figure 1A and Figure S1A). Significant inhibition of invasion was seen in GB cells tested at a concentration of 50 µM (Figure S1B), a concentration at which bumetanide does not exhibit considerable non-specific effects on other cation-chloride transporters [40], [41]. To further examine whether the effect of bumetanide on cell invasion is due to inhibition of NKCC1, we performed stable knockdown of NKCC1 using lentiviral particles carrying NKCC1 shRNA. Knockdown of NKCC1 in GB cells (NS561, NS567, NS501, and NS318) was successfully established in 4 GB cell lines and the efficiency of knockdown was assessed by immunoblot of whole cell lysates of these GB cells (Figure S1C). We confirmed that, as previously shown by Haas and colleagues [13], knockdown of NKCC1 significantly reduced the invasiveness of all these cells (Figures 1B, S1D). Taken together, these data suggest that NKCC1 may indeed play a role in invasiveness of primary GB cells, supporting prior results [13] obtained in non-primary cell cultures. Since NKCC and KCC transporters work in a concerted inverse manner to regulate intracellular volume and intracellular chloride concentration ([Cl−]i) [42], we tested whether inhibition of KCC transport, an important Cl− extrusion mechanism, might mimic NKCC1 overexpression and lead to increased invasion. To test this hypothesis we performed transwell invasion experiments in the presence of DIOA (R (+) -Butylindazone), a potent K+-Cl− transport inhibitor that has no effect on NKCC transport activity. Consistent with this hypothesis, inhibition of KCC transport with DIOA resulted in increased cell invasion. This effect was statistically significant in two of the four cell lines tested (NS221 and NS318) (Figure 1C). DIOA is a non-specific inhibitor of KCC co-transporters, which may be the cause of a heterogeneous effect on GB cells observed. To avoid this confounding result we induced the genetic knockdown of KCC4 (Figure S1E), a KCC family member implicated in cervical and ovarian cancer invasiveness [43]. KCC4 had similar expression levels in the cell lines used for the experiments (Figure 2D). Knockdown of KCC4 in NS318 showed a significant increase in the number of invading cells (Figure 1D). These data suggest that KCC transport inhibition could lead to an increase in [Cl−]i promoting invasive behavior of GB cells. It is thought that GB tumor stem cells may be the core component of the invasive cell population [44]. Therefore, in addition to invasiveness of primary GB cells in vitro, we explored the role of NKCC1 in the invasion of primary brain tumor stem cells (BTSC) in vivo. Tumor area and area of invasion in the corpus callosum were then quantified to evaluate differences in tumor size and invasive ability of BTSCs carrying the control shRNA as well as BTSCs carrying NKCC1 shRNA. We found that tumors generated after the implantation of BTSCs with control shRNA were significantly smaller than tumors generated with the NKCC1 shRNA harboring BTSC line (Figure 1E). Consistent with previous results by Haas and colleagues using commercial GB cell lines [13], the invaded area in the corpus callosum of mice that were implanted with BTSCs carrying the control shRNA was significantly larger than that of mice implanted with BTSCs carrying NKCC1 shRNA (Figure 1F). NKCC1 knockdown did not affect the proliferative potential of the BTSCs injected in vivo (Figure S2). These results suggest that NKCC1 may be an important determinant of primary and GB stem cell invasiveness, in congruence with prior suggestions based on commercial GB cell lines [13]. To evaluate the potential clinical importance of NKCC1 in glioma invasion in vivo, we characterized NKCC1 expression in a large array of glioma tissue samples using a tissue microarray (TMA) containing several tumors of different grades ranging from World Health Organization (WHO) Grade II to WHO Grade IV (Table S1). The results revealed that NKCC1 protein expression was significantly higher in GB and anaplastic astrocytoma (AA) tissue samples compared with expression in Grade II astrocytomas and normal brain (Figure 2A and 2B). Epithelial tissues included in the TMA were used as positive controls (intestinal mucosa and tissue from the distal collecting duct in the kidney) (Figure 2B). As a corollary to this analysis and a complement to the results in Figure 1, we characterized the expression levels of NKCC1 protein in multiple primary human GB cells and found that all cell lines tested showed substantial expression of NKCC1 (Figure 2C). The data obtained from this set of samples showed that NKCC1 protein expression indeed correlates with glioma grade, in that tissues from GB and AA expressed higher NKCC1 protein levels than low-grade astrocytomas and normal brain. This correlation between NKCC1 expression with glioma grade suggests that NKCC1 may contribute to the increased invasiveness of high-grade tumors. Our results so far strongly suggest that NKCC1 may indeed be an important determinant of GB cell invasion. While all prior analyses attempted to link the role of NKCC1 in cell migration to its role as a cell volume regulator [11], [13], [14], [45], we examined whether NKCC1 plays an essential role in the regulation of polarization of cell morphology and migration of GB cells. We were particularly interested in whether NKCC1 might affect cell migration and how this migratory behavior may depend on the mechanical cues mimicking the extracellular matrix components. In this study we employed nanoscale grooves to analyze the migratory behavior of glioma cells. Our substrate mimics ECM features, such as myelinated fiber tracts, upon which brain cancer cells have been shown to migrate [46], [47]. This model offers the advantage of allowing biased cell migration along the nano-ridges of the textured surface that can be quantified in terms of cell speed and migration (Figure S3A–C). We found a significant reduction in the cell migration speed of human primary GB cells stably transduced with NKCC1 shRNA (Figure 3A–B). Similarly, a significant decrease in migration speed was observed when GB cells were treated with bumetanide (Figure S3D). Migration directionality was quantified by measuring the ratio of cell movements parallel to the ridges on the pattern versus those that were perpendicular to the pattern. This metric tended to correlate with the speed of migration, showing significant decreases in directionality for cells expressing NKCC1 shRNA (Figure 3C–D). Overall, GB cells stably transduced with NKCC1 shRNA displayed a lower speed of migration and showed more random migration as demonstrated by the decrease in directionality. The cell migration data indicated that NKCC1 can directly or indirectly affect cell motility, but the mechanism of how an ion transporter can be involved in this process is not immediately apparent. It is therefore of interest to note that at least some ion transporters have been reported to associate with the ERM complex to anchor actin to the plasma membrane, affecting cell migration [16], [19]. The ERM complex proteins bind to clusters of positive amino acids such as lysine (K) and arginine (R) in proteins that are known to bind ERM proteins and to serve as anchors for the actin cytoskeleton such as CD44, CD43, and ICAM-2 [20]. Also, NHE1, a Na+-H+ exchanger, acts as an anchor for the cytoskeleton in migrating cells, through the interaction with ERM proteins [16]. Based on these data, we studied the sequence of the juxtamembrane carboxy-terminus domain of human NKCC1 and found clusters of positively charged amino acids identical to those found in other ERM binding proteins. These clusters of positive amino acids are conserved in the human, mouse, and rat NKCC1 sequences (Figure 4A). These amino acids may be important in the interaction between ERM proteins and NKCC1 and may be similar to other ERM-integral membrane protein binding [20]. To assess the possibility that NKCC1 may affect GB cell migration through a mechanism other than cell volume regulation, we compared the size of focal adhesions formed by NKCC1 knockdown cells and cells transduced with the control shRNA. Focal adhesions were stained with an antibody against vinculin and paxillin, cytoskeletal proteins that are part of focal adhesions that also regulate mechanical coupling of the cytoskeleton to the extracellular matrix (ECM). We observed small, thin, and elongated focal adhesions primarily in the extending processes in control virus shRNA cells, whereas in NKCC1 shRNA cells, focal adhesions were much larger (Figure 4B–C and Figure S4), indicative of focal adhesion maturation [22], [48]. The area of focal adhesions was significantly larger in NKCC1 shRNA cells when compared to control virus cells (Figure 4D). The increased focal adhesion area was also seen when we used paclitaxel (a drug that stabilizes microtubules dynamics and disrupts focal adhesion formation) as a positive control for this experiment [49]. These results suggest that NKCC1 expression not only regulates cell volume but may also be important in modulating focal adhesion dynamics and maturation. Cells exert traction forces on their environment during migration and invasion in response to different mechanical and chemical cues in the extracellular matrix. These forces are applied through points of cell adhesion via focal adhesion-mediated integrin-ECM connections [21], [22]. To evaluate whether the increase in size of focal adhesions after NKCC1 depletion had a functional effect on the generation of contractile forces by GB cells, we quantified cell traction forces exerted by adherent living GB cells (control virus versus NKCC1 shRNA). We found that NKCC1-deficient cells exerted significantly lower cell traction forces than control virus cells (Figure 5A–B). Compared to control virus cells, NKCC1 shRNA cells exhibited approximately a 40% decrease (NS501,44% decrease; NS561,37% decrease; p<0. 002, nested ANOVA) in net contractile moments, which is a scalar measure of cell contractile strength (Figure 5C–D, Figure S5). No within-group differences existed between both tested cell lines. To further support the interaction of NKCC1 and ERM proteins, we immunoprecipated endogenously expressed NKCC1 and probed the immunoprecipated lysate with an antibody against Ezrin. We found that endogenous Ezrin associates with immunoprecipitated NKCC1. These results strongly suggest that in primary human GB cells, Ezrin is an NKCC1 binding partner. As expected, actin, a binding partner of Ezrin, also co-immunoprecipitated with NKCC1 (Figure 6A). We also performed the reverse experiment where Ezrin was immunoprecipitated and then probed for NKCC1 on the immunblot. In multiple primary human GB cell lines, we found that after performing immunoprecipitation of Ezrin, NKCC1 was also pulled down (Figure 6B). As expected, actin was also co-immunoprecipitated. To assess whether the association of NKCC1 and Ezrin is important for the generation of cell traction forces, we mutated the two clusters of basic amino acids found in the juxtamembranous domain of NKCC1 (putative Ezrin-binding sites) and measured the functional consequences in GB cells. We found that the net contractile movements of cells expressing the Ezrin-binding null NKCC1 were significantly lower than cells expressing wild type-NKCC1 (Figure 6C). Furthermore, cells expressing the Ezrin-binding null-NKCC1 had a lower projected cell area than cells expressing wild-type NKCC1 (Figure 6D). Thus, the absence of NKCC1 expression may lead to the formation of more mature focal adhesions. In turn, these mature focal adhersions may further enhance the adhesion of cells to the substratum, which decreaces the migration speed of NKCC1 shRNA cells, as observed above. Mature focal adhesions do not participate in the generation of contractile forces in migrating cells; rather, they participate in anchoring cells to the substrate [21]. On the other hand, nascent adhesions apply forces to the substratum to drive cell movement [21]. Hence, our results suggest that NKCC1 affects cell-ECM interactions by stimulating higher traction force generation and lower substratum adhesion, thus enhancing cell motility in a synergistic fashion. The pronounced effect of NKCC1 expression on focal adhesion formation and cell migration suggests the importance of its partial intracellular localization. We initially approached this issue by performing immunocytochemistry experiments on multiple GB cells. We found that all GB cells had a polarized subcellular expression of NKCC1. In cells that appeared to have a more stationary phenotype with multiple projections, the expression of NKCC1 was primarily correlated with these projections (Figure 7A and Figure S6). Specifically, NKCC1 was localized either to the apparent leading edge of a moving cell or to its rear, frequently in a mutually exclusive pattern (Figure 7B and Figure S6). Expression of NKCC1-EGFP fusion protein in GB cells supported that NKCC1-EGFP expression was mainly localized to the plasma membrane of the extending processes confirming the results obtained by immunofluorescence (Figure 7B, Figure S7, and Video S1 and Video S2). In cells spreading on nano-structured substrata, NKCC1-EGFP localization oscillated between the two transiently existing edges, before a prominent single edge was formed. Furthermore, when using immunocytochemistry, we examined the sub-cellular localization of WNK3, a serine/threonine kinase that regulates the transport activity of NKCC1 through phosphorylation [26], [27], [50]. We observed partial co-localization of WNK3 immunoreactivity with NKCC1 immunoreactivity in the edges of extending processes (Figure 7A and Figure S6). These findings suggest that the cellular localization of NKCC1 is spatially heterogeneous during GB cell migration. Although the localization patterns were diverse in different cell states, the overall pattern that emerged from this analysis was that NKCC1 is associated with extending processes of the cell. This finding correlates with the suggestion that NKCC1 is important in the formation of new focal adhesions and controlling existing focal adhesions and active cytoskeletal components. The aforementioned data suggest that NKCC1 transport activity is important for glioma cell migration and invasion, at least in part through direct regulation of the cytoskeletal and ECM-cell adhesion dependent processes. NKCC1 transport activity is known to be regulated through phosphorylation and de-phosphorylation events mediated by members of the novel serine/threonine kinase family WNKs [26]. NKCC transport is activated by stimulation with EGF in corneal epithelial cells [51]. It is well established that EGF promotes astrocytic [34] and glioma cell migration [36], [52], [53]. Thus, we examined the effect of EGF on the phosphorylation of NKCC1 as an indication of NKCC1-activation using an NKCC1 phospho-specific antibody [23]. After stimulating glioma cells with EGF, NKCC1 phosphorylation increased in a time-dependent and dose-dependent manner in NS318 and NS567 cells (Figure 8A). To gain insight into the regulation of phosphorylation of NKCC1 in an unbiased cellular system, we stimulated HEK-293 cells with EGF in the presence or absence of wortmannin (WM), a PI3K inhibitor. After exposure of HEK-293 cells to EGF, NKCC1 phosphorylation increased significantly. However, in the presence of WM, EGF-induced NKCC1 phosphorylation was blocked (Figure 8B). These findings together demonstrate that the EGF-induced increase in phosphorylation of NKCC1 requires activation of the PI3K-Akt pathway. The activity of cation-chloride cotransporters is regulated in a coordinated manner by the novel family of serine-threonine kinases WNK [50]. WNK3 promotes phosphorylation and activation of NKCC1 transporters while promoting phosphorylation and inactivation of KCC transporters [50]. It has also been shown that WNK1, another member of the WNK family, is phosphorylated and activated by Akt (protein kinase B) [29], [54]. Therefore, we decided to test if Akt phosphorylates WNK3 after stimulation with EGF. We immunoprecipitated total WNK3 from HEK-293 cells exposed to serum-free media, and we stimulated with EGF, or EGF in the presence of the PI3K inhibitor WM. Samples were immunoblotted with an antibody that recognizes phosphorylated Akt substrates (αPAS antibody) (Figure 8C). Data obtained from this experiment showed a basal phosphorylation level of WNK3, which increases with exposure to EGF. This increase in phosphorylation is repressed by inhibition of PI3K with WM, suggesting that Akt phosphorylates WNK3. The same samples were immunoblotted against phosphorylated Akt, and as expected, we found that WM also inhibited the phosphorylation of Akt (Figure 8D). In silico analysis of the protein sequence of WNK3 revealed that two putative Akt phosphorylation motifs are present and are conserved in available WNK3 protein sequences of the human and rat, as previously found in WNK1 (Figure 8E) [54]. These findings indicate that NKCC1 may be activated by factors that stimulate migration of astrocytic or glioma cells, such as EGF via kinases of the WNK family, a family of kinases that have been shown to regulate the transport activity of multiple members of the SLC12A family of transporters. The nearly universal recurrence of GB after surgical resection is largely due to invasion of glioma cells into healthy brain tissue and presents a major impediment in the improvement of GB patient survival. In this study, we tested the hypothesis that NKCC1 expression and transport activity are crucial elements of cell migration and invasion in primary human GB cell lines. Here we provide evidence for the participation of NKCC1 in the migration and invasion of primary human glioma cells, highlighting its possible role in anchoring the actin cytoskeleton to the plasma membrane. Additionally, through this anchoring, NKCC1 mediates transduction of the cellular contractile forces to focal adhesions that interact with the extracellular matrix. These in vitro findings were confirmed functionally in an in vivo model using primary human BTSCs, where their invasion was decreased significantly after NKCC1 knockdown. Given these findings, NKCC1 inhibition could potentially be used in the clinic to improve glioblastoma treatment, given that Bumetanide (a commonly used diuretic, FDA-approved) decreases the invasive potential of glioma cells in vivo [13]. This could potentially improve surgical resection of the tumor mass, as tumor cells lacking NKCC1 activity would form less invasive tumors. Our work shows that NKCC1 protein expression in multiple glioma samples is higher in high-grade gliomas such as GB and anaplastic astrocytomas. Inhibition of NKCC1 transport pharmacologically, as well as genetic inhibition of NKCC1 expression, decreases invasion of multiple primary human GB cell lines. These results are in accordance with previous findings using commercial human glioma cell lines [13]. Our data further indicate that migration speed of GB cell lines in a 2-D nanopatterned substrate is decreased by pharmacological inhibition and by shRNA-based silencing of NKCC1 expression. Interestingly, pharmacological and genetic inhibition of the K+-Cl− cotransporters leads to a more invasive behavior of GB cells in vitro. Moreover, our results suggest that NKCC1 may affect the morphology of focal adhesions, perhaps due to a putative ERM binding motif in the cytoplasmic domain of NKCC1. We also found that NKCC1 is located at the extending processes of GB cells and that NKCC1 polarization may precede migration towards the direction of this pole. Furthermore, exposure of GB cells to EGF, a factor that promotes migration and invasion of normal and tumor cells [34]–[36], [55], [56], induces phosphorylation (activation) of NKCC1 through PI3K-Akt-WNK3 pathway. The concerted action of local anchoring of the actin microfilaments to the plasma membrane and volume regulation may be important for the polarization of cells during migration. Coupling the actin cytoskeleton to the plasma membrane is essential for the regulation of cell morphology and migration [6]. Our immunocytochemistry and live cell imaging experiments using a GFP-NKCC1 fusion protein demonstrate that NKCC1 is localized to the extending processes of migrating GB cells. During the migratory process, cells acquire a polarized morphology where actin, integrin receptors, and ion transporters among other proteins, become asymmetrically distributed in the cell. Some examples of ion transporters that show a polarized localization to the leading edge of the cell include NHE1 and AE2 [9], [16], [17], which also have K+ channels that are polarized to the rear end of the cell [57]. Ezrin-radixin-moesin (ERM) proteins bind actin filaments and anchor them to integral plasma membrane proteins. Some of these integral membrane proteins include NHE1, CD44, and intercellular cell adhesion molecule 2, which have ERM binding motifs (ICAM-2) [16], [18]. The ERM binding motif consists of clusters of positive amino acids, such as lysine and arginine residues in juxtamembranous intracytoplasmic domains of these proteins. By analyzing the peptide sequence of NKCC1, we found clusters of lysine and arginine residues in the N-terminal cytoplasmic domain that are conserved across mammalian species, which may bind ERM proteins. Indeed, we found that NKCC1 is able to bind to Ezrin and actin with our co-immunoprecipitation assay. The generation of advancing membrane protrusions is necessary for migrating cells to achieve cell translocation. ERM protein binding to NHE1 is necessary during migration to promote extension of advancing processes to anchor the cytoskeleton [16], [17]. Our results show that NKCC1 is polarized to the extending processes of migrating glioma cells; therefore, it is likely that NKCC1 is necessary during migration to anchor the cytoskeleton, aiding in the extension of lamellipodia, and mediate local volume changes at the same time [14]. NKCC1 expression may be an important determinant of the response of migrating cells to external physical cues. The extracellular matrix surrounding cells presents topographical features ranging from nanometers (nm) to microns (µm) and affects cell behavior. For example, collagen fibrils form with diameters from 20–200 nm and influence cell polarity and migration through “contact guidance” [58]–[65]. Recent studies have employed more intricate substrates presenting nanoscale features (e. g., grooves, ridges, bumps, and pillars) to more closely model the cellular microenvironment [66]–[68]. In this study, we have employed nanoscale features mimicking the ECM found in the brain. These features include myelinated fiber tracts, upon which brain cancer cells have been shown to migrate [47]. It is known that cell migration is governed by many molecular processes, including attachment to the cell substrate. Our nanopattern provides a quasi-3-D platform that can examine these interactions with ECM by examining speed, direction, and morphologies of migrating cells. While cells move in 2-D, they respond to topographical cues from the substrate presented in 3-D [46], [69]. By examining cell migration after genetic changes to NKCC1 expression, we sought changes that might inhibit the overall motility of glioma cells. Our observed changes in migratory behavior upon simulated ECM suggests that these cells will be less migratory and invasive, eventually leading to improved medical outcomes. These changes in migratory behavior were further supported by our experiments employing more classical techniques (e. g., transwell). It has been shown that formation of actin stress fibers precedes the formation of nascent focal adhesions in the lamellipodium of fibroblasts [70]. However, stress fiber formation depends on the anchorage of actin bundles to the plasma membrane, which has been shown in the interaction of NHE1 with ERM proteins [16]. Nascent adhesions are present at the front of the cell and exert traction forces that lead to cell repositioning [21]. As focal adhesions increase in size, they mature and the traction forces that they exert decrease considerably [21]. In migration studies of cells that do not express focal adhesion kinase (FAK), a major regulator of focal adhesion turnover, it was shown that these cells possess larger focal adhesions and display lower cell spreading [71], [72]. In fact, these observations were also seen in GB cells when NKCC1 is knocked down; NKCC1 knockdown cells display larger focal adhesions and smaller projected cell area than control shRNA cells. These changes in focal adhesion size were accompanied by a decrease in the generation of contractile forces by GB cells. These findings suggest that the localized distribution of NKCC1 to the extending processes plays a role in the modulation of focal adhesion turnover and generation of nascent focal adhesion to maintain cell contractility and traction for efficient migration. Cell volume changes are expected in migrating cells since alterations in shape during extension and retraction occur throughout migration. Multiple ion transport mechanisms are responsible for regulating and maintaining cellular volume in response to changes in extracellular osmolarity and during cell migration [5]; in addition, ion gradients and local volume changes have been described in migrating cells [73], [74]. These mechanisms include ion transporters such as NKCC1, NHE1, KCC transporters, and also ion channels. For instance, neutrophils undergo an increase in intracellular volume in response to the chemotactic factor N-formylmethionyl-leucyl-phenylalanine; this increase in cell volume and increased migration is blunted by NHE1 transport inhibitors and by exposure to hyperosmolar solutions, suggesting that NHE1-mediated volume increase is necessary for neutrophil migration [7], [75]. Na+-K+-Cl− transport inhibition has also shown to decrease Madin-Darby canine kidney cell migration [74]. Our results are in accordance with the findings discussed above where inhibition of NKCC transport decreases migration. We also show that NKCC1 knockdown decreases GB cell migration, confirming the effects of pharmacologic inhibitors. Pharmacological inhibitors and shRNA-based approaches may have off-target effects, but the fact that the effect of both on GB cell migration is the same confirms that the results seen by manipulating NKCC1 expression/transport are consistent. EGFR activation promotes migration of normal neuroblasts, astrocytes, and glioma cells [34], [53], [76]–[78]. EGF signaling affects migration through diverse mechanisms, such as actin polimerization [79], [80], focal adhesion kinase regulation [81]–[83], and matrix metalloproteinase expression [84]. EGF mediates its effects on cell migration and proliferation through activation of its receptor-tyrosine kinase and the various downstream signaling pathways, which include the PI3K-Akt pathway. NKCC1 phosphorylation by WNK3 after activation of the PI3K-Akt pathway supports the hypothesis that NKCC1 activity is necessary for GB cell migration. Furthermore, WNK3 activation after EGF stimulation suggests that phosphorylation and activation of NKCC1 and phosphorylation and inhibition of KCC transporters may result in GB cell migration. Therefore it seems this balance between these opposing transport activities is important in the determination of GB cell invasion. The PI3K-Akt signaling pathway, among many other cell functions, is central in the control of cell motility and polarization. PI3K is activated by receptor tyrosine kinases and Ras [85]. It modulates these functions by bringing diverse proteins that are able to bind phosphatidyl-inositol triphosphate (PIP3) close to the membrane. A notable example of the proteins that are recruited to the membrane is Akt, which is activated after binding to PIP3 and phosphorylated by 3′-phosphoinositide-dependent kinase 1 (PDK1); AKT is recruited in a polarized manner to the leading edge of the migrating cell membrane [86]. It is well known that PI3K activation mediates cytoskeletal rearrangements and cell polarization through the action of the guanine nucleotide exchange factors (GEFs) [87], [88]. Moreover, PI3K modulates actin polymerization and membrane insertion at the leading edge of a cell by regulating the activity of Arf [89]. Its activation also promotes cell polarization through Rac regulation [90]. It is still necessary to assess if the cytoskeletal rearrangements and cell polarization mediated by PI3K are important in the generation of the partial distribution of NKCC1 to the extending processes of GB cells. WNK3 regulates ion transport through phosphorylation: it phosphorylates and activates NKCC1 and phosphorylates and inhibits transport of the KCC transporters in a reciprocal manner [27], [50]. Furthermore, Haas et al. have shown that following a hyperosmotically induced decrease in cell volume, WNK3 may regulate NKCC1. Also, the reduced expression of WNK3 by shRNA diminished the ability of glioma cells to migrate in vitro [91]. Our results show that inhibition of NKCC and KCC transport result in opposite effects in GB cell migration. Similar to WNK1, our immunoprecipitation experiments show that EGF induces phosphorylation of WNK3 through Akt [29], [54]. When phosphorylated, WNK3 then phosphorylates NKCC1 and possibly KCC transporters. This mechanism is similar to the mechanism proposed for increased excitability of neurons, where WNK3 signaling is impaired, resulting in GABA-mediated excitation of neurons and seizure activity [27]. Therefore, it is conceivable that WNK3 activation results in activation of NKCC1 and inhibition of KCC transport, causing increased migration of GB cells. In this study, we show that NKCC1 transport expression and activity are necessary for GB cells to migrate and invade. The mechanism affecting cell contractility that we report in this article may be independent from regulation of volume changes and may be due to regulation of focal adhesion formation and turnover. We also show that EGF regulates NKCC1 phosphorylation through an Akt-WNK3 pathway, linking the PI3K-Akt pathway to the WNK3 kinase. This suggests that WNK3 may have a role in determining GB cell migratory properties. Furthermore, given that NKCC1 is ubiquitously expressed, it is possible that it plays a very similar role in physiological migration such as inflammatory cell diapedesis or neural precursor migration during development, as well as in the process of metastasis of other highly aggressive cancers. Patient samples of glioma tissues were obtained at the Johns Hopkins Hospital under the approval of the Institutional Review Board (IRB). All human brain tumor cell lines were derived from intraoperative tissue samples from patients treated surgically for newly diagnosed glioblastoma multiforme without prior treatment as listed in Table S2. Differentiation potential of cell lines for an in vivo experiment was evaluated by immunohistochemistry against GFAP, TuJ1, and NG2 (Figure S9). Detailed culture methodology has been previously described [92], [93]. VSV-G pseudotyped virus was produced by co-transfecting 293T cells with a shRNA transducing vector and two packaging vectors: psPAX2 and pMD2. G. The shRNA sequence used was 5′-TAG TGC TCT CTA CAT GGC ATG GTT AGA AGC TCT ATC TAA GGA CCT ACC ACC AAT CCT C-3′. Seventy-two hours after transduction, cells were cultured in the presence of puromycin for selection of cells expressing the shRNA. Knockdown was assessed by quantitative PCR (Figure S8) and immunoblot (inset in Figure 1F and Figure S1C). The sequence of human NKCC1 (SLC12A2, accession number NM001046) was amplified by PCR using gene-specific primers. The sequence of the primers employed is as follows: sense, 5′- GCG TGC TGC CGG AGA CGT CC-3′; antisense, 5′- AGT CAC CAT TCG CCA TTG TGA TGT T-3′. The resulting PCR product was cloned into pCR-XL-TOPO (Invitrogen). The cloned sequence was verified in its entirety to confirm the absence of mutations. The EGFP fusion protein was made by cloning the NKCC1 open reading frame into pcDNA3-EGFP using standard cloning procedures. All other procedures are listed in Supplemental Experimental Procedures (Text S1). Total RNA was extracted from primary glioma cell lines using the RNAeasy kit (Qiagen) and reverse transcribed using the SuperScript III First-Strand Synthesis System for RT-PCR (Invitrogen). The target cDNAs were analyzed using SYBR Green PCR master mix (Applied Biosystems) in a 7300 Real-Time PCR system (Applied Biosystems). For relative quantification, the results obtained were compared to the levels of target mRNA expression present in the control cell line and normalized for GAPDH expression. Primers are listed in Supplemental Experimental Procedures (Text S1). NKCC, WNK3, Akt, Ezrin, and actin were detected using rabbit and mouse primary antibodies. Detection was done with the appropriate horseradish-peroxidase conjugated secondary antibodies and using the enhanced chemiluminescence reagent (GE Healthcare Life Sciences). Antibodies are listed in Supplemental Experimental Procedures (Text S1). Cell lysates (150 µg of protein) were incubated with anti-NKCC antibody (T4 antibody, 1 µg; DSHB) and anti-Ezrin (cell signaling cat: 3145,1∶100) overnight at 4 °C on a shaking platform. Indirect immunoprecipitation was done with protein G magnetic beads (Millipore). Proteins were then eluted and denatured in LDS protein loading buffer (Invitrogen). Fifty thousand cells were plated in the top chamber of a matrigel-coated membrane (24-well insert; pore size, 8 mm; BD Biosciences). Cells were plated in medium containing 0. 5% of serum, whereas medium with 2% serum was used as a chemo-attractant in the lower chamber. After 48 h cells that invaded were stained and counted for comparison. Migration of glioma cells was quantified using a novel directional migration assay using nano-ridges/grooves constructed of transparent poly (urethane acrylate) (PUA), and fabricated using UV-assisted capillary lithography (see Figure S3A–C) [94]. Nanopattern surfaces were coated with laminin (3 µg/cm2). Cell migration was quantified using timelapse microscopy (Video S3). Long-term observation was done on a motorized inverted microscope (Olympus IX81) equipped with a Cascade 512B II CCD camera and temperature and gas controlling environmental chamber. Phase-contrast and epi-fluorescent cell images were automatically recorded under 10× objective (NA = 0. 30) using the Slidebook 4. 1 (Intelligent Imaging Innovations, Denver, CO) for 15 h at 10–20-min intervals. A custom-made MATLAB script was used to identify cell boundaries from phase-contrasted images and to measure cell centroid positions. Average individual cell speed was calculated from individual cell trajectories and durations of the image acquisition. Mean squared displacements at various time intervals were calculated using a previously published method [95]. The spindle shape factor was defined as the ratio of the length of maximum cell width (maximal axis) to the minimum value of the cell width in the direction perpendicular to maximum axis, regardless of the orientation with respect to nanogrooves. For each condition, over 60 cells were quantified in total. For quantitative analysis of cell orientation, cells were fixed and stained for F-actin with phalloidin. The orientation angle of polarized cell was determined by measuring the acute angle between the major axis of the cell and the direction of grooves. More than 100 cells for each group were used to construct the polarization angle distributions with range −90° and 90°. A summary of all the migration assays used is presented in Table S3. The contractile stress arising at the interface between an adherent cell and its substratum was measured with traction microscopy [96]. For each cell analyzed, the traction field was computed using Fourier transform traction cytometry as described previously. The computed traction field was used to obtain the net contractile moment, which is a scalar measure of the cell' s contractile strength (Figure S5) [97]. Cells were fixed in 4% paraformaldehyde in phosphate-buffered saline (pH 7. 4) for 1 h and blocked with 10% normal donkey serum in PBS for 1 h. Subsequently, fixed cells were incubated with primary antibody at 4 °C overnight. The preparation was then incubated with Alexa Fluor-conjugated secondary antibodies (Invitrogen) and mounted using Aquamount (VWR). All antibodies and their dilutions are listed in Supplemental Experimental Procedures (Text S1). All animal protocols were approved by the Johns Hopkins Animal Care and Use Committee. In vivo invasion and tumorigenesis of cells expressing NKCC1 shRNA were assessed in 4- to 6-wk-old male mice (nude/athymic mice, NCI) using our brain tumor model as previously described [98]. Mice were sacrificed 8 wk after injection. Brains were fixed using transcardiac perfusion, postfixed overnight at 4 °C in 4% formalin, embedded in OCT compound (Tissue-Tek), and frozen, sectioned, and stained with an antibody against human nestin (1∶500, MAB5326 Millipore). Stained cryosections were used to calculate tumor size and invasiveness by computer-based morphometrics using Image J. Please refer to Text S1 for detailed description of the intracranial injection of GB BTSCs. Primary human GB cells expressing the control shRNA and NKCC1 shRNA were treated with 10 µM 5-ethynyl-20-deoxyuridine (EdU). Cells were harvested for detection of EdU incorporation using Click-iT EdU Flow Cytometry Assay Kits (Invitrogen, Cat. No. C35002) following the manufacturer' s instructions. The percentage of cells that incorporated EdU was measured using flow cytometric detection of EdU. Data were analyzed using Kaluza software (Beckman Coulter). A tissue microarray was designed and built according to previously established methods [99]. Cores were taken from each tumor mass or control tissue (see Table S1). The tissue that was included in the cores of the microarray was representative of the tissue blocks from where the cores were obtained. Analysis and correction for cell number was done using the FRIDA software (free web-based tissue microarray analysis software). Unless otherwise noted, data are presented as mean ± standard error of the mean. A t test was used to compare two groups; one-way analysis of variance (ANOVA) was used in multiple group comparisons with Bonferroni' s post hoc test. Mann-Whitney rank-sum test was used to evaluate the statistical significance in quantification of spindle shape factor where indicated. In order to satisfy the distributional assumptions associated with the ANOVA, cell traction force data were first converted to log scale prior to analyses. For the comparisons between treatments, we used a nested ANOVA. All analyses were performed in Sigma Plot 9. 0 (Systat Software Inc., San Jose, CA) SAS Version 9. 2 (SAS Institute, Cary, NC), and a two-sided p value less than 0. 05 was considered significant. | Treatment of many cancers has been hampered by the invasive ability of tumor cells. A notable example is brain cancer, which is incurable due to its invasiveness and resulting high tumor recurrence after surgical resection. Here, we analyze further the function of NKCC1, an ion transporter that is known to regulate cell volume and intracellular chloride concentration, and to play an important role in brain tumor cell invasion. Our findings suggest that in addition to its conventional function as an ion transporter, NKCC1 may also interact with the cytoskeleton and affect brain tumor cell migration by acting as an anchor that transduces contractile forces from the plasma membrane to the extracellular matrix en route to cell migration. Moreover, we show that regulation of NKCC1 by a family of unconventional enzymes, the WNK kinases, is an important factor that affects the activity of NKCC1 and may determine the invasive ability of brain tumor cells. We postulate that NKCC1 has multiple functions in brain tumor cell migration and that together with its regulatory enzymes may be therapeutic targets in the treatment of brain tumors or other types of cancer, given the wide expression of these proteins throughout the body. | lay_plos |
DNA polymerase ν (pol ν), encoded by the POLN gene, is an A-family DNA polymerase in vertebrates and some other animal lineages. Here we report an in-depth analysis of pol ν–defective mice and human cells. POLN is very weakly expressed in most tissues, with the highest relative expression in testis. We constructed multiple mouse models for Poln disruption and detected no anatomic abnormalities, alterations in lifespan, or changed causes of mortality. Mice with inactive Poln are fertile and have normal testis morphology. However, pol ν–disrupted mice have a modestly reduced crossover frequency at a meiotic recombination hot spot harboring insertion/deletion polymorphisms. These polymorphisms are suggested to generate a looped-out primer and a hairpin structure during recombination, substrates on which pol ν can operate. Pol ν-defective mice had no alteration in DNA end-joining during immunoglobulin class-switching, in contrast to animals defective in the related DNA polymerase θ (pol θ). We examined the response to DNA crosslinking agents, as purified pol ν has some ability to bypass major groove peptide adducts and residues of DNA crosslink repair. Inactivation of Poln in mouse embryonic fibroblasts did not alter cellular sensitivity to mitomycin C, cisplatin, or aldehydes. Depletion of POLN from human cells with shRNA or siRNA did not change cellular sensitivity to mitomycin C or alter the frequency of mitomycin C-induced radial chromosomes. Our results suggest a function of pol ν in meiotic homologous recombination in processing specific substrates. The restricted and more recent evolutionary appearance of pol ν (in comparison to pol θ) supports such a specialized role. In mammalian cells, a diverse group of DNA polymerases carry out genomic DNA replication and genome maintenance. These include the core enzymes for semi-conservative DNA replication (pols α, δ, ε and telomerase), base excision repair (pol β), mitochondrial DNA replication and repair (pol γ and Primpol), non-homologous end-joining and immunological diversity (pols λ, μ, θ and terminal-deoxynucleotidyl transferase), and DNA damage tolerance by translesion synthesis (η, ι, κ, ζ, and Rev1). Some of these enzymes have roles in more than one pathway of DNA processing [1,2]. The consequences of genetic disruption of an individual DNA polymerase vary widely, ranging from embryonic lethality to no discernable phenotype. Pol ν, encoded by the POLN gene in mammalian cells, is not assigned in the preceding list, and it is the only identified polymerase for which an analysis of knockout animals remains to be described. The catalytic domain of pol ν belongs to the A-family of DNA polymerases, and is related to the pol domain of pol θ (encoded by mammalian POLQ / Drosophila Mus308) [3–6]. Pol θ participates in a pathway of DNA double-strand break repair by alternative end joining [7,8], and consequently defects in Mus308 or POLQ confer hypersensitivity to various DNA damaging agents [8–10]. The function of pol ν is currently uncertain, but several roles have been suggested. For example, it was reported that siRNA-mediated knockdown of POLN sensitizes human cells to DNA crosslinking agents [11,12]. Mammalian genomes encode three Mus308 homologs: POLN, POLQ, and DNA helicase HELQ [3,13,14]. Pol θ possesses both a helicase-like and a DNA polymerase domain while pol ν has only a DNA polymerase domain and HELQ has only a helicase domain. Our phylogenetic analysis of the distribution of these three genes is shown in Fig 1. POLQ genes are found throughout most of the eukaryotic lineage, in both animals and in plants, and are inferred to be present in the last eukaryotic common ancestor, 1500 million years ago. Even the unicellular green algae Ostreococcus (the smallest free-living eukaryote) contains POLQ. HELQ is found in most eukaryotic branches, but not in plants. HELQ orthologs are also present in both euryarchaea and crenarchaea [15]. In contrast, POLN genes are restricted to a much more limited range of genomes, in some metazoan groups. POLN appeared during genomic evolution much more recently than POLQ. The POLN gene arose, either by gene duplication or horizontal transfer, in the last common ancestor of the metazoa, around 600 million years ago. POLN genes are distinguished by characteristic sequence insertions in the polymerase domain (S1 Fig), with the location of insert 3 distinct from that of POLQ [5]. Human pol ν (900 amino acid residues) has a strong strand-displacement activity and low fidelity, with exceptionally efficient insertion of T opposite template G in the steady state [4,16,17]. Purified pol ν can bypass the major groove DNA lesion 5S-thymine glycol (Tg) by inserting A, and bypasses major groove DNA-peptide and some DNA-DNA crosslinks [4,18], but does not bypass other DNA modifications including an abasic (AP) site, a cisplatin-induced intrastrand d[GpG] crosslink, a cyclobutane pyrimidine dimer, a 6–4 photoproduct, or minor groove DNA-peptide or DNA-DNA crosslinks [4,18]. A unique cavity in the polymerase domain allows pol ν to generate and accommodate a looped-out primer strand [6]. However, it is currently unknown how these unique biochemical properties are implemented in vivo. To help illuminate pol ν function, we examined POLN gene expression patterns in mice, and phenotypes of mice and cells where POLN was disrupted. The results suggest a specific function in germ cells, but do not support a role for pol ν in tolerance of DNA crosslinks. To explore possible biological functions of pol ν, we established two different Poln knockout mouse models. In one model, the second exon was deleted in mice of C57BL/6J; 129Sv background (Fig 2 and S2 Fig). This exon encodes the ATG initiation codon and the first 46 amino acids, including an N-terminal protein domain that is highly conserved in vertebrates [21]. We avoided targeting the first exon, because POLN shares this exon with HAUS3, an essential gene encoded within the first intron of POLN [21]. In another model, a zinc finger nuclease (ZFN) -mediated 4 or 13 bp frameshift was introduced in the DNA polymerase domain of pol ν (FVB/NCrl background) (Fig 3A and S3 Fig). ZFN-mediated Poln mutant mice are missing critical parts of the DNA polymerase domain including the highly conserved motifs 5 and 6 (Fig 3B). A universally conserved Asp residue in motif 5 of A-family DNA polymerases (D804 in pol ν) that coordinates bivalent metal ions for interaction with an incoming nucleotide, is lost [22]. In all of the engineered mice studied here, a long noncoding RNA gene that overlaps with Poln exons 3–6 remains intact. Both homozygous Poln-deficient mouse models were viable and fertile. Comparing them to their wild-type littermates, no reproductive or developmental differences were observed. Heterozygous Poln Ex2 mutant mouse crosses yielded pups with normal litter sizes and within expected proportions of genotypes and genders (Table 1). Homozygous Poln knockout mouse crosses for the ΔEx2, del4 and del13 constructs all yielded normal litter sizes and with normal sized pups at birth. A phenotype analysis, which included a complete gross necropsy, hematology and serum chemistry, full histopathology on all tissues, and survey radiographs was completed with adult ZFN-mediated Poln knockout mice. A comparison between wild-type mice and Poln-deficient mice did not reveal any obvious differences in size, body weight, organ development (Table 2), or skeletal appearance. Additionally, no significant differences between genotypes were observed in hematology or serum chemistry analysis (Table 2). Poln+/+, Poln+/ΔEx2, and PolnΔEx2/ΔEx2 mice were monitored for over 2 years to assess survival, general health, and tumor incidence. Loss of functional Poln did not affect overall survival compared to wild-type and heterozygous littermate controls (Fig 4). There were no significant differences in tumor prevalence, multiplicity or distribution of tumor types (Tables 3 and 4). Non-neoplastic lesions were of types and incidence commonly found in aging laboratory mice, and there appeared to be no pattern of susceptibility related to genotype (Table 5) or sex. All mice had a variety of age-related lesions that were considered incidental and not included in non-neoplastic diagnoses. These included atrophies of testicles, ovaries, uterus, and thymus, mild multifocal lymphoid infiltrates in various organs, and mild glomerulopathy. Similarly, no obvious alterations in lifespan, general health or cause of death were noted in Poln del4 and del13 mice. We previously reported that the POLN transcript is most prominent in adult testis from the human, mouse, and zebrafish [3,21]. To quantify absolute transcript levels, we compared expression of Poln, Polq, and the replicative polymerase Pold1 in different mouse tissues. Poln expression was highest in testis by an order of magnitude, much higher than Polq and Pold1 in this organ. However, Poln transcript was almost undetectable in other tissues, in contrast to Polq and Pold1 (Fig 5A). To investigate when Poln starts expressing during testis development, we isolated testes from young male mice of various ages. Poln expression gradually increased and was higher than Polq and Pold1 after 16-days, when the population of spermatocytes in the pachytene stage increases [23]. In contrast, the amounts of mRNA of Polq and Pold1 were not markedly increased during testis development (Fig 5B). Fractions enriched in spermatids and pachytene spermatocytes were prepared by centrifugal elutriation, and Poln expression was higher in the pachytene-enriched fraction (Fig 5C). These data suggested a possible meiosis-related function of pol ν in testis. Testis morphology was normal in Poln knockout mice (Fig 6A), and testes size measurements, histology and breeding experiments described above did not suggest a marked perturbation of spermato- and spermiogenesis. To test for an overt role of pol ν in meiotic recombination, chromosome spreads were stained with SYCP3 (a component of the synaptonemal complex) and MLH1 (a crossover marker) to assess progression through meiotic prophase I (Fig 6B). MLH1 foci numbers per nucleus were not statistically different between pol ν proficient and Polndel4/del4 mice (Fig 6C). PolnΔEx2/ΔEx2 spermatocytes had one less MLH1 focus per nucleus than controls (p = 0. 03) (Fig 6D). The results suggest that while pol ν is not essential for crossing over or meiosis, it may be required for efficient crossing over at specific loci, at least in some genetic backgrounds (see legend to S4 Fig) We examined the potential role of pol ν in meiotic homologous recombination-associated DNA synthesis. Crossovers, arising by recombination at one meiotic hotspot A3 [24,25], were isolated from sperm of PolnΔEx2/ΔEx2 C57BL/6J x DBA/2J and Polndel4/del4 FVB/NCrl x DBA/2J F1 hybrid mice, and control animals. The crossover frequency at A3 in FVB/NCrl x DBA/2J is half that of C57BL/6J x DBA/2J (Fig 6E and 6F). The A3 locus in FVB/NCrl lacks a high affinity PRDM9 binding site (S4 Fig) and, therefore it likely does not receive meiotic DSBs to initiate recombination [26]. As such, the FVB/NCrl x DBA/2J F1 hybrid receives meiotic DSBs on only the DBA/2J allele, rather than at both the C57BL/6J and DBA/2J alleles as in the C57BL/6J x DBA/2J F1 hybrid. Consistent with this model, the A3 locus shows reciprocal crossover asymmetry, an altered distribution of crossovers depending upon their orientation, which is a hallmark of meiotic hotspots with biased DSB formation on only one of the two parental alleles (S4 Fig). A total of 155,157,185,180 crossovers from Polndel4/del4, Poln+/del4, PolnΔEx2/ΔEx2, and Poln+/ΔEx2 males were isolated and mapped. Consistent with reduced MLH1 foci, the Poisson corrected crossover frequency in male PolnΔEx2/ΔEx2 mice was 80% of that in heterozygous littermates (p = 0. 04) (Fig 6F). The crossover frequency in Polndel4/del4 mutants was not significantly reduced compared to controls (p = 0. 09, Fig 6E), but combining both datasets showed a statistically significant (p = 0. 01) reduction in Poln homozygous mutants to about 80% of the control frequency (S5 Fig). We further examined the so-called ‘crossover refractory zone’ (CRZ) at the A3 hotspot in Poln mutant mice. Crossover formation in this 258 bp region of the A3 hotspot is strongly inhibited, even though DSBs are formed there abundantly [24,27]. The region contains secondary structure-forming sequence that may inhibit heteroduplex formation [24]. The combined Poln mutant data (S5 Fig) showed a statistically significant (p = 0. 04) 50% reduction in crossover frequency at this region (10 of 340 crossovers in knockouts, 21 out of 337 crossovers in controls). Whole genome transcriptome analysis by RNAseq was performed to determine whether Poln disruption influences gene expression in testis from PolnΔEx2/ΔEx2 mice. Using rRNA-depleted total RNA, we obtained an average of 65 million reads per sample (range 53 to 73 million) with an average mapping rate of 90% to the reference mouse genome. Poln disruption did not perturb overall transcription (Fig 7A). Only a few genes had significantly altered expression, at a false discovery rate (FDR) ≤ 0. 05. Expression of only four of these genes was increased more than 2-fold (S1 Table), comprising two that encode proteins (Lgi2 and 9330182L06Rik), and two specifying noncoding RNAs (BB283400 and Speer4cos). Expression of only two genes was reduced more than 2-fold, Egfem1 and Poln itself. Because these genes have no close physical linkage and their functions are unknown, we have not pursued them further at present. As expected, expression of the targeted exon 2 of Poln was not detected in PolnΔEx2/ΔEx2 mice. All other Poln exons showed greatly reduced or absent expression, likely related to the disruption of normal splicing. Haus3 expression was not influenced by deletion of the targeted Poln exon (Fig 7B). We asked whether pol ν influences levels of ionizing radiation-induced DNA damage in testis. This could be the case if pol ν is directly involved in DSB repair (by analogy with the related pol θ) or if pol ν is necessary for translesion DNA synthesis of radiation-induced lesions such as thymine glycol (Tg). Pol ν is proficient in bypass of Tg, which blocks the progression of replicative DNA polymerases [4]. A dose dependent increase in γ-H2AX foci (a surrogate for DNA damage including DSB) was observed in wild-type, PolnΔEx2/ΔEx2, and Polq-/- mice (Fig 8A). The number of γ-H2AX foci in the absence of irradiation was higher in Polq-/- mice than in wild-type or PolnΔEx2/ΔEx2 mice. This is consistent with the higher basal level of γ-H2AX foci in pol θ-suppressed human cells [28]. Following irradiation of mice with 1 or 2 Gy, the average γ-H2AX foci numbers in round spermatids of testis sections was measured [29]. After 5 hr, the number of foci was reduced to a similar level in all mice (Fig 8B), irrespective of Poln and Polq status. As a control experiment, blood was taken from killed mice after irradiation and micronuclei were measured in reticulocytes. Frequencies of micronuclei were increased in Polq-/- mice after IR, as previously reported [10,30,31]. Poln status did not influence the level of micronuclei in reticulocytes (Fig 8C). We also examined PolnΔEx2/ΔEx2 Polq-/- double knockout mice and found that Poln deletion did not further increase the frequency of micronuclei above that in Polq-/- reticulocytes (Fig 8C). Because pol θ can participate in end-joining of DNA breaks produced during immunoglobulin class-switch recombination (CSR) [8], we tested whether pol ν is also involved in CSR. Naïve B cells isolated from the spleens of wild-type and PolnΔEx2/ΔEx2 mice were stimulated for IgM to IgG class switching, and then the fraction of IgG1-positive B cells was measured by flow cytometry. Parallel B-cell cultures were incubated with NU7026, an inhibitor of DNA-PKcs that increases the proportion of CSR with >1 bp insertion at the junction [8,32]. We found that stimulated B cells from Poln-proficient and deficient mice had similar overall frequencies of IgG1 (6. 1%). Inhibition of DNA-PKcs increased the frequency of CSR in both genotypes by 2-fold (11. 7% in wild-type, 12. 5% in PolnΔEx2/ΔEx2) (S2 Table), as observed previously with wild-type and Polq-/- mice [8]. The Sμ-Sγ1 junction was then sequenced from 100 clones of each group of IgG1-positive B cells. Insertions of >1 bp at Sμ-Sγ1 junctions were pol θ-dependent [8], but pol ν-independent (Fig 9A and 9B). We also performed qPCR analysis of transcript expression in splenic B cells for Polq, Poln, Helq, Haus3, and Pold1. Interestingly, among those genes only Polq expression was increased after B-cell activation by lipopolysaccharide and interleukin 4 treatment (Fig 9C). Cells with inactivation of pol θ or its ortholog HELQ show increased sensitivity to DNA damaging agents, such as bleomycin (pol θ) [8] and mitomycin C or cisplatin (HELQ) [33–35]. To determine whether inactivation of pol ν also sensitizes cells to DNA damage, we examined the relative sensitivity of PolnΔEx2/ΔEx2 MEFs to mitomycin C (MMC), cisplatin, bleomycin, hydroxyurea, olaparib, formaldehyde and acetaldehyde. Aldehyde sensitivity was tested because of the observation that purified pol ν can bypass some DNA-peptide crosslinks [18]. However, we detected no hypersensitivity of immortalized MEFs to any of the tested DNA damaging agents (Fig 10). We also examined primary Polndel4/del4 MEFs and found no hypersensitivity to MMC, olaparib, etoposide or 5-fluorouracil (S6 Fig). Rev3L null MEFs were used as positive controls in the same experiment, and they showed sensitivity to MMC as reported [36]. We generated PolnΔEx2/ΔEx2 Polq−/− MEFs by crossing PolnΔEx2/ΔEx2 and Polq−/− mice, isolating double mutant MEFs from the progeny and immortalizing with SV40 Tag. Polq−/− MEFs were more sensitive to bleomycin, as reported [8]. PolnΔEx2/ΔEx2 MEFs were not more sensitive to bleomycin and Poln ablation did not further increase the bleomycin sensitivity of Polq−/− MEFs (S7A Fig). The cellular sensitivity to MMC was not increased in Poln or Polq single knockout MEFs or in the double homozygous mutants (S7B Fig). These results contrast with previous reports that siRNA directed against POLN causes an increased sensitivity to MMC and cisplatin [11,12]. Therefore, we performed siRNA-mediated knockdown in human cells using the previously reported targeting sequence [11]. In that study, the efficiency of siRNA-mediated knockdown was assessed using an antibody raised against pol ν. We have developed ~80 Pol ν monoclonal antibodies and two polyclonal antibodies against pol ν -77 [37], and identified several of them that can immunoblot and immunoprecipitate overexpressed pol ν in human cells (e. g. Mab#40 in S8 Fig). However, we have not been able to detect endogenous pol ν with these or other antibodies, and so we turned to a different approach to monitor the efficiency of siRNA-mediated POLN knockdown. Pol ν was expressed in a doxycycline-inducible manner in 293T-Rex cells. We confirmed complete knockdown of tagged protein expression, showing that the POLN-specific siRNA construct very effectively inactivates POLN mRNA (Fig 11A, top panel). We also performed quantitative PCR assays for POLN and HAUS3 as described [21] and detected significant reduction of doxycycline-induced POLN transcript (P = 0. 01) as well as endogenous POLN transcript (P = 0. 03) by the POLN-specific siRNA (S9 Fig). Note that only incomplete transcripts of POLN have been detected in 293T cells [21]. HAUS3 expression was not affected by this siRNA (S9 Fig). Nevertheless, neither pol ν-depletion (siN) nor pol ν-overexpression (293T-REx (POLN) + Dox, siC) had any influence on mitomycin C sensitivity in human cells (Fig 11B and S8 Fig). It was also proposed that pol ν depletion caused an increased formation of radial chromosomes in human cells treated with MMC [11]. We therefore analyzed metaphase spreads of 293T-REx depleted of pol ν, and 293FT cells depleted of pol ν, FANCA, or FANCD2 (Fig 11A). Suppression of FANCA and FANCD2 resulted in an increased frequency of radial chromosomal formation after treatment with mitomycin C as expected for these Fanconi anemia gene products. However, pol ν depletion did not increase the frequency of radial chromosome formation (Fig 11C and 11D). The investigations presented here answer many long-standing questions about pol ν function and expression. An analysis of mice with genetic disruptions of Poln has not been reported previously. Because Poln is expressed only in some metazoan lineages, many widely-used model organisms are not relevant for biological analysis of pol ν. We found that Poln is not essential for mouse embryonic development or for viability. Three independently established and targeted mouse models that disrupt Poln were fully viable, in C57BL/6J; 129Sv and FVB/NCrl backgrounds. The Poln deficient mice have a normal lifespan and lack abnormalities for many investigated anatomic features. Consistent with this, the International Mouse Phenotyping Consortium (mousephenotype. org) has recently listed mice with a homozygous knockout of exon 6. No abnormalities in adults or embryos were detected in a battery of anatomical, physiological and behavioral tests. No expression of Poln was detected in embryos, also consistent with our reported observations [21]. Our observations point to a function of pol ν in the testis. We discovered that Poln is uniquely upregulated during mouse testicular development and that it is enriched in spermatocytes. This may be a conserved feature in organisms where POLN occurs. For example, we found that POLN expression was essentially undetectable in somatic adult cells or embryonic cells from the zebrafish, but was present in adult zebrafish testis [21]. Further, the phylogenetic analysis described here shows that POLN genes exist only in metazoan animals that use sexual reproduction and deploy sperm. This is consistent with a specialized function for pol ν in discrete spermatocyte-forming organs. The evolutionary distribution of POLN is a contrast to the wide distribution of POLQ genes in animals and plants, including many unicellular species and some that reproduce asexually. The DNA polymerase activity of pol ν appears to be critical for its function, as all organisms encoding the enzyme retain the six conserved DNA polymerase motifs with the residues necessary for catalytic activity. Expression of recombinant protein from the human, mouse and zebrafish POLN cDNAs all produce active DNA polymerase [21]. Importantly, it is uncertain whether the pol ν protein is expressed significantly in mammalian somatic cells or tissues. Most POLN transcripts in somatic human cells are inactive, alternatively spliced variants [3]. The protein is not detectable with the antibodies currently available, although mass spectrometry experiments detect a few peptides representing pol ν in various mouse, human, and rat tissues including brain, heart, kidney, liver, lung, pancreas, spleen, and testis [38]. The low level expression of mostly inactive transcripts is consistent with the lack of phenotypes in somatically derived cells, described here. Pol ν may be important at specific recombination hot spots where DNA forms unique DNA secondary structures which provide challenges for other DNA polymerases. We detected a modest but significant reduction in meiotic recombination frequencies at the A3 hot spot in Poln deficient mice. The frequency was lower in the crossover refractory zone of A3 in Poln deficient mice. This zone harbors insertion/deletion polymorphisms and possibly forms a hairpin structure inhibiting replicative DNA polymerases [24]. The strand displacement activity of pol ν [4] may be effective in helping to synthesize the secondary structure-forming sequence. In fact, the crystal structure of pol ν reveals a cavity in the polymerase domain, which could accommodate a looped-out primer strand [6]. Such a looped-out primer could be formed during annealing of insertion/deletion polymorphisms during meiotic recombination. Further, we previously reported proteins associated with pol ν when it was overexpressed in human cells. Mass spectrometry analysis showed associations of pol ν with homologous recombination factors including BRCA1, FANCJ, BRCA2, and PALB2 [21]. This is consistent with the participation of pol ν in a specialized homologous recombination reaction. Our results show that mammalian cells have normal responses to DNA crosslinking agent exposure following pol ν elimination or depletion. This contrasts with the reported increased sensitivity to the DNA crosslinking agents MMC and cisplatin in human cells after an siRNA-mediated knockdown of POLN [11,12], Our conclusions are based on several independent knockouts of Poln function in the mouse, and on controlled siRNA and shRNA-depletion of POLN in human cells. These cells showed normal survival responses to crosslinks, and no evidence of increased frequency of MMC-induced radial chromosomes. We did not detect cellular crosslink-sensitivity after suppression of POLN. In studying pol ν, it is also crucially important to insure, as we have done here, that knockdown of POLN does not interfere with the expression of the essential HAUS3 gene encoded in intron 1 of vertebrate POLN. Further, we emphasize that antibodies cannot be used currently to monitor endogenous knockdown of POLN. Finally, we have described that there is little evidence for expression of pol ν in somatic cells, which would explain the lack of phenotypes in non-germline cells when the gene is disrupted. Current evidence points towards a specific germ cell function. Although pol ν shares homology with the DNA polymerase domain of Drosophila Mus308, which is involved in resistance to DNA crosslinking agents [9], it appears that a different Mus308 homolog is more involved in crosslink sensitivity in mammalian cells. HELQ, which shows similarity to the Mus308 helicase domain, is involved in DNA crosslink tolerance [34,33,35]. Our results are more in line with the lack of sensitivity to DNA crosslinking agents that was reported for POLN−/− chicken DT40 cells [39,40]. The ability of pol ν to perform limited bypass of a major groove DNA-peptide adduct (N6-A-peptide) and a DNA-DNA (N6-A-N6-A) crosslink residue [18] may be more broadly related to properties of pol ν that allow it to bypass blocks such as the secondary DNA structures that exist within meiotic recombination hotspots. In many organisms, it is a common observation that knocking out a single DNA repair protein generates little or no DNA damage or growth-related phenotype. Double mutations, or disruption of backup pathways, are frequently necessary to reveal significant phenotypes. For example, no phenotype of pol ι disruption has been detected as a single mutant. In contrast, pol η /pol ι double-deficient mice show an altered spectrum of UV radiation-induced tumors [41,42]. We constructed pol ν /pol θ double-defective mice, and as described here, this double mutation did not exacerbate cellular sensitivity to DNA damaging agents beyond what we observed with pol θ defective animals. Further screening of double mutants seems a possible approach to unveil the biological function of pol ν in vertebrates. The present work provides a major resource and foundation that paves the way for further exploration of the function of this unique human enzyme. Research mice were handled according to the policies of the MD Anderson Cancer Center Institutional Animal Care and Use Committee, under approved protocol number 00001119-RN00. The strategy for cre-loxP based knockout and targeting vector construction was designed and performed by genOway (Lyon, France). The genomic region of interest containing the murine Poln locus was isolated by PCR from genOway' s 129Sv BAC library. PCR fragments were subcloned into the pCR4-TOPO vector (Invitrogen). The following primers were used. For the short homology arm: 5’-CATAACAGGTCAGAGTCACAAAACAGATATGC-3’and 5’- TATCTCACAGACATCAAAACCTACACATGCC-3’; for proximal long homology arm: 5’-CCAGGTAATTTAGATGTGTGAACCAGATGC-3’ and 5’- ACAAACTTTCCAGAACAAGGACAATGACC-3’; for distal long homology arm: 5’-GGGACAGAATAGAAACAAAATGACAAAATAGACC-3’ and 5’-GCTTATGCAAGTAGAGATTCAAAGTTGATGTAAGG-3’. The resulting sequenced clones (containing intron 1 to intron 2) were used to construct the targeting vector. A region including exon 2 was flanked in the adjacent introns by a Neo cassette (LoxP site—FRT site—PGK promoter—Neo cDNA—FRT site) and by a distal loxP site (Fig 2A). This allowed generation of constitutive and conditional knockout lines which had deleted the 145 bp Poln exon 2 containing the translational initiation codon, 760 bp of the upstream intron and 63 bp of the downstream intron. In the knockout, the deleted region is replaced by a 50 bp fragment containing the distal loxP site, simultaneously disrupting an endogenous AflII restriction enzyme site. Linearized targeting vector was transfected into 129Sv ES cells (5 x 106 ES cells in presence of 40 μg of linearized plasmid, 260 V, 500 μF). Positive selection was started 48 hr after electroporation, by addition of 200 μg/mL of G418. 2547 resistant clones were isolated and amplified in 96-well plates. Duplicates of 96-well plates were made. The set of plates containing ES cell clones propagated on gelatin were genotyped by both PCR and Southern blot analysis. For PCR analysis, one primer pair was designed to amplify sequences spanning the 5’ homology region. This primer pair designed to specifically amplify the targeted locus was: sense, 5’ GAAAAGCCTCGAAGATATGGGCACC-3', anti-sense (Neo cassette) 5' - GCCTCCCCTACCCGGTAGAATTAGATC-3'. Targeting was confirmed by Southern blot analysis using internal and external probes on both 3’ and 5’ ends. Two clones were identified as correctly targeted at the Poln locus. Clones were microinjected into C57BL/6J blastocysts, and gave rise to male mosaics with a significant ES cell contribution (as determined by an agouti / black coat color). Mice were bred to C57BL/6J mice expressing Flp recombinase to remove the Neo cassette (B6; 129Sv-Polntm1. 1Rwd designated as Polnlox allele) and to female C57BL/6J mice expressing Cre recombinase to generate a germline deletion of Poln (B6; 129Sv-Polntm1. 2Rwd designated as PolnΔEx2 allele). The following genotyping primers were used. Polnlox: forward, 5' - GAACCAGATGCTTGTTTGTTCTTTTCACC-3' ; reverse, 5' - GTGTACTGAAATACTCCTCAGTTCTAAAAACGACC-3' ; wild-type 152-bp product, floxed 266-bp product. PolnΔEx2: forward, 5' - CAGGTAATTTAGATGTGTGAACCAGATGCTTG-3' ; reverse, 5' - GGCAGTACAATAACAGAAACACTTCTCTTATGACC-3' ; wild-type 1402-bp product, deleted 484-bp product (S2 Fig). Animals were validated by Southern blot analysis of AflII-digested DNA using a 5’ probe (Fig 2) of 364-bp probe for the Southern blot was generated by PCR on genomic DNA using the primer set: 5’-TGGGCAGTTAACTTAGTGGCAACTCTACT-3’ and 5’- GTGTACCTGATCTGTTCCATGTCTTCATATAATC-3’. Sixteen ZFN pairs targeting Poln were designed and assembled by PCR subcloning into the pZFN plasmid by Sigma. All pairs were tested for efficiency of generating double strand breaks using the Surveyor Mutation assay in cultured mouse Neuro2A cells. The selected ZFN pair, targeting exon 21, was used for subsequent microinjections into FVB/NCrl embryos. The ZFN target site (cleavage site in lowercase) is: 5’-GGCCTCTCCCGAGGAtctgtGCACAGGACCAGCAG-3’. Validation used forward primer: 5’-CCCTGGGAATACTTGGGACT and reverse primer: 5’-ACTACCAGGCAGGACAGGTG. Embryos were obtained from superovulated Charles River FVB/NCrl female mice. ZFN pairs were microinjected and transferred into pseudopregnant foster females. Thirty-six pups were born. The pups were sampled for genotyping at approximately 3 weeks of age. Four founders (11% efficiency) have been identified. Three of the four founders resulted in early stop codons. Founder #22 has a 4 base pair deletion (FVB/NCrl. Cg-Polnem1Rwd designated as Polndel4 allele). Founder #26 has a 13 base pair deletion (FVB/NCrl. Cg-Polnem2Rwd designated as Polndel13 allele) and #27 is chimeric with a 4 and 13 base pair deletion. An ApaL1 restriction site is present in the ZFN targeted site and the sequence is lost in the 4-bp and 13-bp-deleted allele. This is used to validate the genotyping result (S3 Fig). Thirty mice (15 males and 15 females) of each genotype (PolnΔEx2/ΔEx2, Poln+/ΔEx2, and Poln+/+) were monitored over time for tumor incidence and survival. Mice were housed in an SPF modified barrier facility under standard light cycle and temperature conditions. Sentinal animals were free of multiple murine infectious and parasitic agents including Helicobacter and mouse parvoviruses. Mice were fed irradiated standard rodent diet (Harlan Laboratories Irradiated Teklad 22/5 Rodent Diet #8940) ad libitum and provided with reverse osmosis purified water via an automated system. Mice were monitored from approximately 3 weeks of age until they reached a moribund state. Mice were monitored daily for overall health status. Additionally, body condition score [43] and body weight were assessed weekly and recorded. Mice were removed from the study if any of the following criteria were met: Loss of greater than 20% body weight in one week, body condition score of 1 or 2 [43], respiratory or ambulatory difficulties, or non-healing dermatitis. Mice removed from study were killed by carbon dioxide inhalation followed by cervical dislocation before complete necropsies were performed. Body weight and weights of the following organs were obtained: spleen, kidneys, liver, adrenals, testes, heart, thymus, and brain. All tissues were examined grossly, fixed for 24–48 hr in neutral buffered formalin, and stored in 70% ethanol. For all mice, H&E-stained slides of the following tissues were prepared by standard techniques and examined by a board-certified veterinary pathologist: kidneys, liver, heart, lungs, thymus, spleen, testes or ovaries, brain, all gross lesions. For all suspected lesions of histiocytic sarcoma or lymphoma, immunohistochemistry was performed to identify T cells (CD3), B-cell (CD45R), and macrophages (F4/80 or MAC). Additional immunohistochemistry was performed as required for specific diagnosis. Physiological serum parameters were measured using the VetScan VS2 Analyzer with the Comprehensive Diagnostic Profile reagent rotor (Abaxis). The primary mouse embryonic fibroblasts (MEFs) were derived from e13. 5 embryos with genotypes Poln+/+, Poln+/ΔEx2 and PolnΔEx2/ΔEx2 (C57BL/6J mouse, in which exon 2 encoding the first methionine was deleted) and Poln+/+, Poln+/del4 and Polndel4/del4 (FVB/NCrl mouse, in which a Zinc Finger Nuclease-mediated 4 bp frameshift was introduced in the DNA polymerase domain of Poln). Primary MEFs were cultured in medium containing high glucose, glutamax-DMEM (Invitrogen), 15% Hyclone FBS (Thermo Scientific), non-essential amino acids, sodium pyruvate, MEM vitamin solution, penicillin/streptomycin (Invitrogen) and maintained in air-tight containers filled with a gas mixture containing 93% N2,5% CO2 and 2% O2 (Praxair) at 37°C. MEFs were immortalized with SV40 Tag as reported [36]. Immortalized MEFs were cultured in medium containing high glucose, glutamax-DMEM (Invitrogen), 10% FBS (Atlanta Biologics) and penicillin/streptomycin and maintained in a humidified 5% CO2 incubator at 37°C. We prepared a cDNA library was prepared from 129Sv mouse testis. The full-length open reading frame (ORF) was amplified with primers (5’-caccaaaatggaaaattatgaggcatgtg and 5' -caggagatcctgggctcagccgactacaaagacgatgacgacaagtaggaattcatatat), and cloned into pENTR/D-TOPO vectors and then recombined into pDEST17 vectors (Invitrogen). Total RNA was extracted using TRIzol (ThermoFisher Scientific), and RNA integrity assessed using the Agilent 2100 bioanalyzer (Agilent Technologies, Inc.). Total RNA (1 μg) was used as template to synthesize cDNA with the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems). qPCR was then performed on the ABI 7900HT Fast Real-Time PCR System (Applied Biosystems). Custom assays for mouse Poln, Polq, and Pold1 were designed using FileBuilder 3. 1 software (Applied Biosystems) and ordered from Applied Biosystems. TaqMan primer and probe sets for each gene are shown in Table 6. The absolute quantity (AQ) of transcripts for Poln and Polq was determined using the generated standard curves and Applied Biosystems’ Sequence Detection Software version 2. 2. 2 (ABI). Standard curves for each gene were determined using the plasmids pDEST17 carrying cDNA coding full length of Poln, and Polq clone (MGC: 189905, IMAGE: 9088092). For relative quantification, TaqMan primers and the probe set for Gapdh were purchased from Applied Biosystems. Triplicate qPCR reactions each containing cDNA representing 40 ng of reverse-transcribed total RNA were then assayed for transcript quantity with Gapdh serving as endogenous controls to normalize input RNA levels. Cell suspensions were prepared from 25 adult mice (~100 days old) by previously published methods [44,45]. Seminiferous tubules were isolated by incubating the decapsulated testes with collagenase (0. 5 mg/mL) and DNase I (200 μg/mL) in enriched DMEM/F12 (Invitrogen), to which 0. 1 mM non-essential amino acids (Invitrogen), 1 mM sodium pyruvate (Invitrogen), and 5 mM sodium lactate (Sigma) were added. This decapsulated testis tissue was shaken for 15 min at 35°C in a water bath, until it was mostly dispersed into tubules. The dispersed tubules were allowed to settle and, after removal of the supernatant, were resuspended in 32 mL of DMEM/F12 solution. In each of four 50 mL tubes, 8 mL of this suspension was layered onto 40 mL of 5% Percoll solution (Sigma), and the tubules were allowed to settle until most of the larger tubules and clumps were at the bottom. The supernatants were removed and the settled tubules were washed with DMEM/F12 solution and then further digested with trypsin (1 mg/mL) and DNase I (200 μg/mL) in enriched DMEM/F12 for 20 min at 35°C with shaking. Fetal bovine serum was added to 10%, and the cells were dispersed by pipetting. Total cell suspensions were separated by centrifugal elutriation (JE-6B rotor, Beckman) to obtain fractions enriched in spermatids (flow rate: 15. 6 mL/min, rotor speed: 2250 rpm) and pachytene primary spermatocytes (37 mL/min, 2250 rpm). The purified pachytene fraction was obtained by plating the Percoll-enriched fraction on DSA (Sigma) coated dishes, to which Sertoli cells bind strongly, and the pachytene spermatocytes were recovered in the unbound cell fraction. The Sertoli cells recovered from the elutriator were purified by plating them on the DSA-coated dishes, removing the unbound and loosely bound cells, and directly extracting the bound Sertoli cells with RLT lysis buffer (QIAGEN). The purity of each fraction was initially determined by cell smears stained with periodic acid Schiff-hematoxylin. Testes were removed from killed mice and fixed in neutral-buffered formalin for 24 hr before being processed for histologic sectioning (4 μm) and stained with hematoxylin and eosin (H&E). The samples were analyzed using a BX41 Olympus microscope with 10X objective. In order to detect recombinant molecules at the A3 locus in sperm, F1 hybrid animals carrying heterozygous A3 alleles were generated. The PolnΔEx2 allele in the C57BL/6J background and the Polndel4 allele in the FVB/NCrl background were backcrossed into DBA2/J strain. The C57BL/6J or FVB/NCrl strains share an ~1. 8% polymorphism density at A3 with the DBA2/J strain allowing detection of recombinant molecules from sperm DNA. F1 hybrid males were generated from matings of parents carrying each of the Poln mutant alleles in one of the background strains. Spermatocyte spreads were prepared according to previously published protocol [46]. Briefly, slides rinsed with PBS were blocked for 30 min at room temperature in Gelatin Block Solution (GBS: 0. 2% v/v fish gelatin (Sigma G-7765), 0. 2% v/v IgG-free BSA (Jackson ImmunoResearch 001-000-162), 0. 05% w/v Tween-20). Next, covered slides were incubated overnight at 4°C with 100 μl of primary antibody diluted in GBS. Slides were then washed with GBS on a rotating platform shaker (one quick rinse followed by 5,10 and 15 min rinses). Next, covered slides were incubated for 45 min at 37°C with 100 μl of secondary antibody diluted (1: 200) in GBS. Slides were washed with GBS as above, followed by two 5 min washes with 0. 4% PhotoFlo 200 (Kodak 1464510). Slides were then dried in the dark and mounted with Prolong® Gold antifade with DAPI. Antibodies were used in two combinations in this study (dilution): Rabbit anti-SCP3 (1: 500; Santa Cruz Biotechnology sc-33195) and Mouse anti-MLH1 (1: 20; BD Pharmingen 551092) or Mouse anti-SCP3 (1: 200; Santa Cruz Biotechnology sc-74569); and Rabbit anti-SCP1, (1: 200; Abcam ab15090); secondary antibodies (all 1: 200) were: Goat anti-Rabbit 594 (Life Technologies A11037) and Goat anti- Mouse 488 (Life Technologies A11029) or Goat anti-Mouse 594 (Life Technologies A11029) and Goat anti-Rabbit 488 (Life Technologies A11034). Images were acquired on a Zeiss Axio Imager M2 with a Plan-Apochromat 100x/1. 4 oil immersion objective. DNA samples were isolated from sperm from epididymides of adult (2–5 m. o.) F1 hybrid animals, as previously described [24,25]. Two animals were analyzed for each allele, per genotype. Briefly, a standard phenol/ chloroform/ isoamyl alcohol DNA isolation protocol was followed by ethanol precipitation. An aliquot of each DNA sample was used to quantify DNA concentration by UV absorbance and by comparison to a dilution series by agarose gel electrophoresis, using high quality sperm DNA of defined concentration. The number of amplifiable DNA molecules/pg was determined by performing 12–24 PCR reactions per sample seeded with 12 pg per reaction (equivalent to 2 amplifiable molecules/well). The crossover assay for A3 was as described previously [24,25]. Briefly, recombinant molecules were identified after two rounds of nested PCR by 0. 8% agarose gel electrophoresis and positive reactions (putative crossovers) were then PCR-amplified, together with positive and negative controls. Products in a 96-well format were dot-blotted onto nylon hybridization membrane (Roche, 11417240001) and genotyped by Southern blotting with allele-specific oligonucleotide (ASO) probes. Somatic DNA from spleen or liver for each assayed animal was used as a negative control at total DNA inputs equivalent to or higher than sperm DNA. No crossovers were detected in somatic controls (frequency < 1. 05 x 10−5). A detailed version of the Southern blotting protocol for the A3 hotspot has been described [24,25]. Briefly, ASO probes were radiolabeled using T4 polynucleotide kinase and hybridized with nylon membranes containing PCR products from the crossover assay. After hybridization, blots were exposed for 4–5 hr on phosphorimager screens and scanned using the Typhoon FLA 9500 phosphorimager (GE Healthcare). Scans were then scored and positive signals used to generate crossover breakpoint maps. Each ASO is an 18-bp oligonucleotide designed to specifically hybridize with one of the two parental genotypes. At the A3 locus, most of the FVB/NCrl sequence is identical to the C57BL/6J strain. Wild-type and PolnΔEx2/ΔEx2 mice were generated from Poln heterozygous knockout crosses, and Polq-/- mice were generated from Polq heterozygous knockout crosses. 8–12 week-old mice received whole-body irradiation with 1 Gy or 2 Gy at 2 Gy/min, 160 kV peak energy (Rad Source 2000 irradiator, Suwanee, GA). For DSB induction, three wild-type mice per dose were analyzed at 30 min post-irradiation. For the DSB repair kinetics three mice per time-point were analyzed at 0. 5,5, 24, and 48 hr after irradiation with 2 Gy. In each experiment three mock-irradiated mice served as controls. After animals were sacrificed, testes were immediately removed and placed in fixative. Formalin-fixed tissues were embedded in paraffin and sectioned at an average thickness of 4 μm. Tissue sections were incubated with primary antibody against γ-H2AX (Bethyl) followed by Alexa Fluor-488-conjugated secondary antibody (Invitrogen). Finally, sections were mounted in VECTASHIELD mounting medium with 4′, 6-diamidino-2-phenylindole (DAPI) (Vector Laboratories). For quantitative analysis, radiation-induced foci were counted by eye using a Leica DMI 6000 microscope equipped with a 63X oil objective. Tissue sections were incubated with primary antibody against γ-H2AX. To evaluate potential differences in DSB repair kinetics, the two-way ANOVA test was performed for each dose and repair-time. The criterion for statistical significance was p ≤ 0. 05. For RNA-seq analysis, four biological replicates were prepared for Poln+/+ and PolnΔEx2/ΔEx2 mice. RNA was purified from testis of 68-day old male mice with RNeasy kit (QIAGEN) with on-column DNase treatment. The libraries were prepared using the Illumina TruSeq stranded total RNA kit according to the manufacturer’s protocol, except that the PCR amplification cycle was reduced to 10. The libraries were sequenced on HiSeq 2000 (Illumina), generating 53–73 million pairs of 75 bp reads per sample. Each pair of reads represents a cDNA fragment from the library. The reads were mapped to mouse genome (mm10) by TopHat (Version 2. 0. 7) [47]. By reads, the overall mapping rate is 90–96%. 83–93% fragments have both ends mapped to mouse genome. The number of fragments in each known gene from RefSeq database 48 (downloaded from UCSC Genome Browser on March 6,2013) was enumerated using htseq-count from HTSeq package (version 0. 5. 3p9) [48]. Genes with less than 10 fragments in all the samples were removed before differential expression analysis. The differential expression between conditions was statistically accessed by R/Bioconductor package edgeR (version 3. 0. 8) [49]. Genes with false discovery rate ≤0. 05 and fold change ≥2 were called significant. Gene clustering and heatmap were done by Cluster 3. 0 and TreeView. GO analysis was performed using Ingenuity Pathway Analysis (IPA) software. The transcriptional profiling plot (Fig 7A) was made using R software. Approximately 100 μL of blood obtained from individual mice by cardiac puncture were collected into tubes containing 350 μL of heparin solution and fixed in ultra-cold methanol according to the protocol in the Mouse MicroFlowBasic Kit (Litron Laboratories). The fixed samples were stored at −80°C until the flow cytometry analysis was performed. Methanol-fixed blood samples were washed and labeled with anti-CD71-FITC, anti-CD61-PE and PI for high speed flow cytometry using CellQuest software, v5. 2 (Becton Dickinson, San Jose, CA). For each sample, 2 × 104 CD71-positive reticulocytes were analyzed for the presence of micronucleated reticulocytes. Flow cytometers were calibrated by staining Plasmodium berghei-infected rodent blood (malaria biostandards) in parallel with test samples on each day of analysis. Statistical analysis was performed using the Student' s t-test or one-way ANOVA followed by Tukey' s test. B cells were isolated from mouse spleens, purified by negative selection with anti-CD43 depletion (Miltenyi) and stimulated with IL-4 and Lipopolysaccharide and IL-4 (Sigma) for 72 hr. Where indicated, cultures were incubated with DNA-PKcs inhibitor 20 μM NU7026 (Tocris) dissolved in DMSO, or mock-treated. Three mice were analyzed in triplicate and cell count numbers and viability were similar for all groups. The culture, flow-cytometric analysis for CSR analysis and junction analysis has been described [32,50]. Sμ-Sγ1 CSR junctions were amplified by PCR using the following conditions for 25 cycles at 95°C (30 s), 55°C (30 s), 68°C (180 s) using the primers (FWD 5′-AATGGATACCTCAGTGGTTTTTAATGGTGGGTTTA-3′; REV 5′ CAATTAGCTCCTGCTCTTCTGTGG-3′) and Pfu Turbo (Stratagene). To the PCR reaction, 5 U of Taq polymerase (Promega) was added and incubated at 72°C for 10 min. The resulting product was TOPO TA cloned and transformed into Top10 E. coli cells (Life Technologies) and plasmids were purified and sent for sequencing using M13 FWD and REV primers in addition to the amplification primers for sequencing. 100 clones for each group were analyzed for mutations, deletions, insertions, and sequence overlaps at the junction and both 30 nt upstream and downstream of the junction. T-REx 293 cells (Invitrogen) were transfected with the pcDNA5/FRT/TO TOPO TA construct. Full-length pol ν with six His residues at the N-terminus and a FLAG tag at the C-terminus [4,21] was inserted into the plasmid vector. Stable clones were selected with hygromycin and blasticidin. Expression of the Flag-tagged pol ν was induced with 0. 1 μg/mL doxycycline (SIGMA) for 24 hr. Basal repression and doxycycline-induced expression of pol ν were confirmed by immunoblotting. The POLN-specific RNAs (designated ‘siN’, 5’- AAGCACCCAAUUCAGAUUACU) (Dharmacon), the FANCA-specific RNAs (designated ‘siA’, 5’-AAGGGUCAAGAGGGAAAAAUA-3’) (Invitrogen), the FANCD2-specific Stealth RNAs (designated ‘siD2’, 5’-CCAUGUCUGCUAAAGAGCGUUCAUU-3’) (Invitrogen) and ON-TARGETplus Non-Targeting siRNAs as a negative control designated ‘siC’ (Thermo Scientific) were used. The siRNAs were introduced into 293FT or 293T-REx POLN cells. 24 hr prior to transfection, cells were plated in a 6-well plate at 2. 0 x 105 cells/well. For each well, 5 pmol of siRNAs was diluted into 250 μl of Opti-MEM (Invitrogen). In a separate tube, 5 μl of Lipofectamine RNAiMAX reagent (Invitrogen) was diluted into 250 μl of Opti-MEM and incubated at room temperature for 10 min. The Lipofectamine RNAiMAX dilution was added into the diluted siRNA duplex and incubated at room temperature for 20 min. Before the transfection, medium was replaced with fresh 2. 5 mL of DMEM supplemented with 10% fetal bovine serum for each well. The Lipofectamine RNAiMAX-siRNA complex was added dropwise to the cells and incubated at 37°C. After 24 hr the cells were washed, trypsinized, and plated with fresh DMEM medium supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin (Invitrogen). To measure the levels of proteins, whole cell crude extracts were prepared 48 hr after the RNA transfection and analyzed by immunoblotting with anti-pol ν (PA434) [3], anti-FANCA (Bethyl, A301-980A, 1: 10,000 dilution), anti-FANCD2 (GeneTex Inc., EPR2302,1: 2000 dilution), and anti-α-tubulin (Sigma, T5168,1: 8000 dilution) antibodies. The following sequence was cloned into pSIF-H1-copGFP vector to target POLN: 5’-GATCCTCTTTGGCGAGTTAGAGCTGTACTTCCTGTCAGATGCAGCTTTAACTTGCCAAAGAGGATTTTTT-3’, underlined sequence indicates the target sequence (System Biosciences). Lentiviral particles were generated by transfection of three plasmids (the expression plasmid, e. g., pSIF-H1-copGFP-hPOLN. 1; plus pFIV-34N and pVSV-G) into 293FT cells using FuGene 6. Culture media from transfected cells was collected 48 hr after transfection to isolate the viral particles, passed through 0. 45 μm filters, used immediately or stored at −80°C in single-use aliquots. Lentiviral transduction was completed as follows: Briefly, 6. 0 × 104 cells were seeded into 6-well plate and incubated for 24–30 hr at 5% CO2 at 37°C. Cells were transduced for 18 hr with shRNA-expression lentiviral stocks at 32°C and cultured for 72 hr at 37°C as described [51]. Stable cell lines were selected by detecting GFP expression (a co-expressed marker gene). To measure the levels of proteins, immunoblotting was performed with anti-pol ν (Mab#40) and anti-PCNA (Santa Cruz, sc-56,1: 1,000 dilution) antibodies. For the ATPlite assay (PerkinElmer), 1,250 cells were plated per well into white 96 well plates and incubated twenty-four hr prior to inducing DNA damage. The cells were incubated with DNA damage inducing agents for the indicated time. After incubation, the cells were immediately lysed and assayed for ATPlite luminescence as described in the manufacturer' s instructions. For the clonogenic assay, 1. 0 x 105 cells were plated in 60 mm culture plates and incubated for 24 hr prior to DNA damage induction. Groups of plates were exposed to indicated doses of mitomycin C for 1 hr. After making a dilution series for each group, cells were returned to the incubator until colonies could be detected in the samples (7 to 14 days), and then were fixed, stained, and scored for survival. Cells were treated with 40 ng/mL of mitomycin C for 48 hr. At forty-four hr after mitomycin C treatment, cells were treated with 0. 03μg/mL colcemid solution (Sigma) for 4 hr. The cells were then trypsinized and exposed to 0. 075M KCl for 15 min at 37°C, and were fixed in 3: 1 methanol: glacial acetic acid. The cells were spread on glass slides, Giemsa stained and metaphases were analyzed using a BX41 Olympus microscope, with 60X or 100X oil objectives. Photographs were taken with the 60X oil objective on a Spot Idea 5 color digital camera. 100 metaphases per sample were analyzed to identify cell population with radial chromosome. | The work described here fills a current gap in the study of the 16 known DNA polymerases in vertebrate genomes. Until now, experiments with genetically disrupted mice have been reported for all but pol ν, encoded by the POLN gene. To intensively analyze the role of mammalian pol ν we generated multiple Poln-deficient murine models. We discovered that Poln is uniquely upregulated during testicular development and that it is enriched in spermatocytes. This, and phylogenetic analysis indicate a testis-specific function. We observed a modest reduction in meiotic recombination at a recombination hotspot in Poln-deficient mice. Pol ν has been suggested to function in DNA crosslink repair. However, we found no increased DNA crosslink sensitivity in Poln-deficient mice or POLN-depleted human cells. This is a major difference from some previous findings, and we support our conclusion by multiple experimental approaches, and by the very low or absent expression of functional pol ν in mammalian somatic cells. The present work represents the first description and comprehensive analysis of mice deficient in pol ν, and the first thorough phenotypic analysis in human cells. | lay_plos |
Background PTSD can develop following exposure to combat, natural disasters, terrorist incidents, serious accidents, or violent personal assaults like rape. People who experience stressful events often relive the experience through nightmares and flashbacks, have difficulty sleeping, and feel detached or estranged. These symptoms may occur within the first 4 days after exposure to the stressful event or be delayed for months or years. Symptoms that appear within the first 4 days after exposure to a stressful event are generally diagnosed as acute stress reaction or combat stress. Symptoms that persist longer than 4 days are diagnosed as acute stress disorder. If the symptoms continue for more than 30 days and significantly disrupt an individual’s daily activities, PTSD is diagnosed. PTSD may occur with other mental health conditions, such as depression and substance abuse. Clinicians offer a range of treatments to individuals diagnosed with PTSD, including individual and group therapy and medication to manage symptoms. These treatments are usually delivered in an outpatient setting, but they can include inpatient services if, for example, individuals are at risk of causing harm to themselves. DOD’s Post-Deployment Process and Screening for PTSD DOD’s screening for PTSD occurs during its post-deployment process. During this process, DOD evaluates servicemembers’ current physical and mental health and identifies any psychosocial issues commonly associated with deployments, special medications taken during the deployment, and possible deployment-related occupational/environmental exposures. The post-deployment process also includes completion by the servicemember of the post-deployment screening questionnaire, the DD 2796. DOD uses the DD 2796 to assess health status, including identifying servicemembers who may be at risk for developing PTSD following deployment. In addition to questions about demographics and general health, including questions about general mental health, the DD 2796 includes four questions used to screen servicemembers for PTSD. The four questions are: Have you ever had any experience that was so frightening, horrible, or upsetting that, in the past month, you have had any nightmares about it or thought about it when you did not want to? tried hard not to think about it or went out of your way to avoid situations that remind you of it? were constantly on guard, watchful, or easily startled? felt numb or detached from others, activities, or your surroundings? The completed DD 2796 is reviewed by a DOD health care provider who conducts a face-to-face interview to discuss any deployment-related health concerns with the servicemember. Health care providers that review the DD 2796 may include physicians, physician assistants, nurse practitioners, or independent duty medical technicians—enlisted personnel who receive advanced training to provide treatment and administer medications. DOD provides guidance for health care providers using the DD 2796 and screening servicemembers’ physical and mental health. The guidance gives background information to health care providers on the purpose of the various screening questions on the DD 2796 and highlights the importance of a health care provider’s clinical judgment when interviewing and discussing responses to the DD 2796. Health care providers may make a referral for a further mental health or combat/operational stress reaction evaluation by indicating on the DD 2796 that this evaluation is needed. When a DOD health care provider refers an OEF/OIF servicemember for a further mental health or combat/operational stress reaction evaluation, the provider checks the appropriate evaluation box on the DD 2796 and gives the servicemember information about PTSD. The provider does not generally arrange for a mental health evaluation appointment for the servicemember with a referral. See figure 1 for the portion of the DD 2796 that is used to indicate that a referral for a further mental health or combat/operational stress reaction evaluation is needed. DOD and VA Health Care Systems DOD’s health care system, TRICARE, delivers health care services to over 9 million individuals. Health care services, which include mental health services, are provided by DOD personnel in military treatment facilities or through civilian health care providers, who may be either network providers or nonnetwork providers. A military treatment facility is a military hospital or clinic on or near a military base. Network providers have a contractual agreement with TRICARE to provide health care services and are part of the TRICARE network. Nonnetwork providers may accept TRICARE allowable charges for delivering health care services or expect the beneficiary to pay the difference between the provider’s fee and TRICARE’s allowable charge for services. VA’s health care system includes medical facilities, community-based outpatient clinics, and Vet Centers. VA medical facilities offer services which range from primary care to complex specialty care, such as cardiac or spinal cord injury. VA’s community-based outpatient clinics are an extension of VA’s medical facilities and mainly provide primary care services. Vet Centers offer readjustment and family counseling, employment services, bereavement counseling, and a range of social services to assist veterans in readjusting from wartime military service to civilian life. Vet Centers are also community points of access for many returning veterans, providing them with information and referrals to VA medical facilities. DOD’s Quality Assurance Program In January 2004, DOD implemented the Deployment Health Quality Assurance Program. As part of the program, each military service branch must implement its own quality assurance program and report quarterly to DOD on the status and findings of the program. The program requires military installation site visits by DOD and military service branch officials to review individual medical records to determine, in part, whether the DD 2796 was completed. The program also requires a monthly report from the Army Medical Surveillance Activity (AMSA), which maintains a database of all servicemembers’ completed DD 2796s. DOD uses the information from the military service branches, site visits, and AMSA to develop an annual report on its Deployment Health Quality Assurance Program. For Veterans, DOD Offers a Benefit for a Specific Period of Time and VA Offers Various Health Care Services DOD offers an extended health care benefit to some OEF/OIF veterans for a specific period of time, and VA offers health care services that include specialized PTSD services. For some OEF/OIF veterans, DOD offers three health care benefit options through the Transitional Assistance Management Program (TAMP) under TRICARE, DOD’s health care system. The three benefit options are offered for 180 days following discharge or release from active duty. In addition, OEF/OIF veterans may purchase health care benefits through DOD’s Continued Health Care Benefit Program (CHCBP) for 18 months. VA also offers health care services to OEF/OIF veterans following their discharge or release from active duty. VA’s health benefits include health care services, including specialized PTSD services, which are delivered by clinicians who have concentrated their clinical work in the area of PTSD treatment and who work as a team to coordinate veterans’ treatment. DOD Offers Mental Health Benefits to OEF/OIF Veterans for 180 Days or More Through TAMP, DOD provides health care benefits that allow some OEF/OIF veterans to obtain health care services, which include mental health services, for 180 days following discharge or release from active duty. This includes services for those who may be at risk for developing PTSD. These OEF/OIF veterans can choose one of three TRICARE health care benefit options through TAMP. While the three options have no premiums, two of the options have deductibles and copayments and allow access to a larger number of providers. The options are TRICARE Prime—a managed care option that allows OEF/OIF veterans to obtain, without a referral, mental health services directly from a mental health provider in the TRICARE network of providers with no cost for services. TRICARE Extra—a preferred provider option that allows OEF/OIF veterans to obtain, without a referral, mental health services directly from a mental health provider in the TRICARE network of providers. Beneficiaries pay a deductible and a share of the cost of services. TRICARE Standard—a fee-for-service option that allows OEF/OIF veterans to obtain, without a referral, mental health services directly from any mental health provider, including those outside the TRICARE network of providers. Beneficiaries pay a deductible and a larger share of the costs of services than under the TRICARE Extra option. See Table 1 for a description of the beneficiary costs associated with each TRICARE option. In addition, OEF/OIF veterans may purchase DOD health care benefits through CHCBP for 18 months. CHCBP began on October 1, 1994, and like TAMP, the program provides health care benefits, including mental health services, for veterans making the transition to civilian life. Although benefits under this plan are similar to those offered under TRICARE Standard, the program is administered by a TRICARE health care contractor and is not part of TRICARE. OEF/OIF veterans must purchase the extended benefit within 60 days after their 180-day TAMP benefit ends. CHCBP premiums in 2006 were $311 for individual coverage and $665 for family coverage per month. Reserve and National Guard OEF/OIF veterans who commit to future service can extend their health care benefits after their CHCBP or TAMP benefits expire by purchasing an additional benefit through the TRICARE Reserve Select (TRS) program. As of January 1, 2006, premiums under TRS are $81 for individual coverage and $253 for family coverage per month. DOD also offers a service, Military OneSource, that provides information and counseling resources to OEF/OIF veterans for 180 days after discharge from the military. Military OneSource is a 24-hour, 7-days a week information and referral service provided by DOD at no cost to veterans. Military OneSource provides OEF/OIF veterans up to six free counseling sessions for each topic with a community-based counselor and also provides referrals to mental health services through TRICARE. VA Offers Health Services, Including Specialized PTSD Services, to OEF/OIF Veterans VA also offers health care services to OEF/OIF veterans, and these services include mental health services that can be used for evaluation and treatment of PTSD. VA offers all of its health care services to OEF/OIF veterans through its health care system at no cost for 2 years following these veterans’ discharge or release from active duty.21, 22 VA’s mental health services, which are offered on an outpatient or inpatient basis, include individual and group counseling, education, and drug therapy. For those veterans with PTSD whose condition cannot be managed in a primary care or general mental health setting, VA has specialized PTSD services at some of its medical facilities. These services are delivered by clinicians who have concentrated their clinical work in the area of PTSD treatment. The clinicians work as a team to coordinate veterans’ treatment and offer expertise in a variety of disciplines, such as psychiatry, psychology, social work, counseling, and nursing. Like VA’s general mental health services, VA’s specialized PTSD services are available on both an outpatient and inpatient basis. Table 2 lists the various outpatient and inpatient specialized PTSD treatment programs available in VA. See 38 U.S.C. § 1710(e)(1)(D), 1712A(a)(2)(B) (2000), and VHA Directive 2004-017, Establishing Combat Veteran Eligibility. OEF/OIF veterans can receive VA health care services, including mental health services, without being subject to copayments or other cost for 2 years after discharge or release from active duty. After the 2-year benefit ends, some OEF/OIF veterans without a service- connected disability or with higher incomes may be subject to a copayment to obtain VA health care services. VA assigns veterans who apply for hospital and medical services to one of eight priority groups. Priority is generally determined by a veteran’s degree of service-connected or other disability or on financial need. VA gives veterans in Priority Group 1 (50 percent or higher service-connected disabled) the highest preference for services and gives lowest preference to those in Priority Group 8 (no disability and with income exceeding VA guidelines). In addition to the 2-year mental health benefit, VA’s 207 Vet Centers offer counseling services to all OEF/OIF veterans with combat experience, with no time limitation or cost to the veteran for the benefit. Vet Centers are also authorized to provide counseling services to veterans’ family members to the extent this is necessary for the veteran’s post-war readjustment to civilian life. VA Vet Center counselors may refer a veteran to VA mental health services when appropriate. Based on DOD Data, About 5 Percent of OEF/OIF Servicemembers May Have Been at Risk for Developing PTSD and Over 20 Percent Received Referrals Using data provided by DOD from the DD 2796s, we found that about 5 percent of the OEF/OIF servicemembers in our review may have been at risk for developing PTSD, and over 20 percent received referrals for further mental health or combat/operational stress reaction evaluations. About 5 percent of the 178,664 OEF/OIF servicemembers in our review responded positively to three or four of the four PTSD screening questions on the DD 2796. According to the clinical practice guideline jointly developed by VA and DOD, individuals who respond positively to three or four of the four PTSD screening questions may be at risk for developing PTSD. Of those OEF/OIF servicemembers who may have been at risk for PTSD, 22 percent were referred for further mental health or combat/operational stress reaction evaluations. About 5 Percent of OEF/OIF Servicemembers May Have Been at Risk for Developing PTSD Of the 178,664 OEF/OIF servicemembers who were deployed in support of OEF/OIF from October 1, 2001, through September 30, 2004, and were in our review, 9,145—or about 5 percent—may have been at risk for developing PTSD. These OEF/OIF servicemembers responded positively to three or four of the four PTSD screening questions on the DD 2796. Compared with OEF/OIF servicemembers in other service branches of the military, more OEF/OIF servicemembers from the Army and Marines provided positive answers to three or four of the PTSD screening questions—about 6 percent for the Army and about 4 percent for the Marines (see fig. 2). The positive response rates for the Army and Marines are consistent with research that shows that these servicemembers face a higher risk of developing PTSD because of the intensity of the conflict they experienced in Afghanistan and Iraq. We also found that OEF/OIF servicemembers who were members of the National Guard and Reserves were not more likely to be at risk for developing PTSD than other OEF/OIF servicemembers. Concerns have been raised that OEF/OIF servicemembers from the National Guard and Reserve are at particular risk for developing PTSD because they might be less prepared for the intensity of the OEF/OIF conflicts. However, the percentage of OEF/OIF servicemembers in the National Guard and Reserves who answered positively to three or four PTSD screening questions was 5.2 percent, compared to 4.9 percent for other OEF/OIF servicemembers. Twenty-two Percent Who May Have Been at Risk for Developing PTSD Received Referrals Of the 9,145 OEF/OIF servicemembers who may have been at risk for developing PTSD, we found that 2,029 or 22 percent received a referral— that is, had a DD 2796 indicating that they needed a further mental health or combat/operational stress reaction evaluation. The Army and Air Force servicemembers had the highest rates of referral—23.0 percent and 22.6 percent, respectively (see fig. 3). Although the Marines had the second largest percentage of servicemembers who provided three or four positive responses to the PTSD screening questions (3.8 percent), the Marines had the lowest referral rate (15.3 percent) among the military service branches. DOD Cannot Provide Reasonable Assurance That OEF/OIF Servicemembers Who Need Mental Health Referrals Receive Them During the post-deployment process, DOD relies on the clinical judgment of its health care providers to determine which servicemembers should receive referrals for further mental health or combat/operational stress reaction evaluations. Following a servicemember’s completion of the DD 2796, DOD requires its health care providers to interview all servicemembers. For these interviews, DOD’s guidance for health care providers using the DD 2796 advises the providers to “pay particular attention to” servicemembers who provide positive responses to three or four of the four PTSD screening questions on their DD 2796s. According to DOD officials, not all of the servicemembers with three or four positive responses to the PTSD screening questions need referrals for further evaluations. Instead, DOD instructs health care providers to interview the servicemembers, review their medical records for past medical history and, based on this information, determine which servicemembers need referrals. DOD expects its health care providers to exercise their clinical judgment in determining which servicemembers need referrals. DOD’s guidance suggests that its health care providers consider, when exercising their clinical judgment, factors such as servicemembers’ behavior, reasons for positive responses to any of the four PTSD screening questions on the DD 2796, and answers to other questions on the DD 2796. However, DOD has not identified whether these factors or other factors are used by its health care providers in making referral decisions. As a result, DOD cannot provide reasonable assurance that all OEF/OIF servicemembers who need referrals for further mental health or combat/operational stress reaction evaluations receive such referrals. DOD has a quality assurance program that, in part, monitors the completion of the DD 2796, but the program is not designed to evaluate health care providers’ decisions to issue referrals for mental health and combat/operational stress reaction evaluations. As part of its review, the Deployment Health Quality Assurance Program requires DOD’s military service branches to collect information from medical records on, among other things, the percentage of DD 2796s completed in each military service branch and whether referrals were made. However, the quality assurance program does not require the military service branches to link responses on the four PTSD screening questions to the likelihood of receiving a referral. Therefore, the program could not provide information on why some OEF/OIF servicemembers with three or more positive responses to the PTSD screening questions received referrals while others did not. DOD is conducting a study that is intended to evaluate the outcomes and quality of care provided by DOD’s health care system. This study is part of DOD’s National Quality Management Program. The study is intended to track those who responded positively to three or four PTSD screening questions on the DD 2796 and used the form as well to indicate they had other mental health issues, such as feeling depressed. One of the objectives of the study is to determine the percentage of those who were referred for further mental health or combat/operational stress reaction evaluations, based on their responses on the DD 2796. Conclusions Many OEF/OIF servicemembers have engaged in the type of intense and prolonged combat that research has shown to be highly correlated with the risk for developing PTSD. During DOD’s post-deployment process, DOD relies on its health care providers to assess the likelihood of OEF/OIF servicemembers being at risk for developing PTSD. As part of this effort, providers use their clinical judgment to identify those servicemembers whose mental health needs further evaluation. Because DOD entrusts its health care providers with screening OEF/OIF servicemembers to assess their risk for developing PTSD, the department should have confidence that these providers are issuing referrals to all servicemembers who need them. Variation among DOD’s military service branches in the frequency with which their providers issued referrals to OEF/OIF servicemembers with identical results from the screening questionnaire suggests the need for more information about the decision to issue referrals. Knowing the factors upon which DOD health care providers based their clinical judgments in issuing referrals could help explain variation in the referral rates and allow DOD to provide reasonable assurance that such judgments are being exercised appropriately. However, DOD has not identified the factors its health care providers used in determining why some servicemembers received referrals while other servicemembers with the same number of positive responses to the four PTSD screening questions did not. Recommendation for Executive Action We recommend that the Secretary of Defense direct the Assistant Secretary of Defense for Health Affairs to identify the factors that DOD health care providers use in issuing referrals for further mental health or combat/operational stress reaction evaluations to explain provider variation in issuing referrals. Agency Comments and Our Evaluation In commenting on a draft of this report, DOD concurred with our conclusions and recommendation. DOD’s comments are reprinted in appendix II. DOD noted that it plans a systematic evaluation of referral patterns for the post-deployment health assessment through the National Quality Management Program and that an ongoing validation study of the post-deployment health assessment and the post-deployment health reassessment is projected for completion in October 2006. Despite its planned implementation of our recommendation to identify the factors that its health care providers use to make referrals, DOD disagreed with our finding that it has not provided reasonable assurance that OEF/OIF servicemembers receive referrals for further mental health evaluations when needed. To support its position, DOD identified several factors in its comments that it stated may explain why some OEF/OIF servicemembers with the same number of positive responses to the four PTSD screening questions are referred while others are not. For example, DOD health care providers may employ watchful waiting instead of a referral for a further evaluation for servicemembers with three or four positive responses to the PTSD screening questions. Additionally, DOD stated in its technical comments that providers may use the referral category of “other” rather than place a mental health label on a referral by checking the further evaluation categories of mental health or combat/operational stress reaction. DOD also stated in its technical comments that health care providers may not place equal value on the four PTSD screening questions and may only refer servicemembers who indicate positive responses to certain questions. Although DOD identified several factors that may explain why some servicemembers are referred while others are not, DOD did not provide data on the extent to which these factors affect health care providers’ clinical judgments on whether to refer OEF/OIF servicemembers with three or four positive responses to the four PTSD screening questions. Until DOD has better information on how its health care providers use these factors when applying their clinical judgment, DOD cannot reasonably assure that servicemembers who need referrals receive them. DOD’s plans to develop this information should lead to reasonable assurance that servicemembers who need referrals receive them. DOD also described in its written comments its philosophy of clinical intervention for combat and operational stress reactions that could lead to PTSD. Central to its approach is the belief that attempting to diagnose normal reactions to combat and assigning too much significance to symptoms when not warranted may do more harm to a servicemember than good. While we agree that PTSD is a complex disorder that requires DOD health care providers to make difficult clinical decisions, issues relating to diagnosis and treatment are not germane to the referral issues we reviewed and were beyond the scope of our work. Instead, our work focused on the referral of servicemembers who may be at risk for PTSD because they answered three or four of the four PTSD screening questions positively, not whether they should be diagnosed and treated. Further, DOD implied that our position is that servicemembers must have a referral to access mental health care, but there are other avenues of care for servicemembers where a referral is not needed. We do not assume that servicemembers must have a referral in order to access these health care services. Rather, in this report we identify the health care services available to OEF/OIF servicemembers who have been discharged or released from active duty and focus on how decisions are made by DOD providers regarding referrals for servicemembers who may be at risk for PTSD. DOD also provided technical comments, which we incorporated as appropriate. VA provided comments on a draft of this report by e-mail. VA concurred with the facts in the draft report that related to VA. We are sending copies of this report to the Secretary of Veterans Affairs; the Secretary of Defense; the Secretaries of the Army, the Air Force, and the Navy; the Commandant of the Marine Corps; and appropriate congressional committees. We will also provide copies to others upon request. In addition, the report is available at no charge on the GAO Web site at http://www.gao.gov. If you or your staff members have any questions regarding this report, please contact me at (202) 512-7101 or [email protected]. Contact points for our Offices of Congressional Relations and Public Affairs may be found on the last page of this report. GAO staff members who made major contributions to this report are listed in appendix III. Appendix I: Scope and Methodology To describe the mental health benefits available to veterans who served in military conflicts in Afghanistan and Iraq—Operation Enduring Freedom (OEF) and Operation Iraqi Freedom (OIF), we reviewed the Department of Defense (DOD) health care benefits and Department of Veterans Affairs (VA) mental health services available for these veterans. We reviewed the policies, procedures, and guidance issued by DOD’s TRICARE and VA’s health care systems and interviewed DOD and VA officials about the benefits and services available for post-traumatic stress disorder (PTSD). We defined an OEF/OIF veteran as a servicemember who was deployed in support of OEF or OIF from October 1, 2001, through September 30, 2004, and had since been discharged or released from active duty status. We classified National Guard and Reserve members as veterans if they had been released from active duty status after their deployment in support of OEF/OIF. We interviewed officials in DOD’s Office of Health Affairs about health care benefits, including length of coverage, offered to OEF/OIF veterans who are members of the National Guard and Reserves and have left active duty status. We attended an Air Force Reserve and National Guard training seminar in Atlanta, Georgia, for mental health providers, social workers, and clergy to obtain information on PTSD mental health services offered to National Guard and Reserve members returning from deployment. To obtain information on DOD’s Military OneSource, we interviewed DOD officials and the manager of the Military OneSource contract about the services available and the procedures for referring OEF/OIF veterans for mental health services. We interviewed representatives from the Army, Air Force, Marines, and Navy about their use of Military OneSource. We interviewed VA headquarters officials, including mental health experts, to obtain information about VA’s specialized PTSD services. We reviewed applicable statutes and policies and interviewed officials to identify the services offered by VA’s Vet Centers for OEF/OIF veterans. In addition, to inform our understanding of the issues related to DOD’s post-deployment process, we interviewed veterans’ service organization representatives from The American Legion, Disabled American Veterans, and Vietnam Veterans of America. To determine the number of OEF/OIF servicemembers who may be at risk for developing PTSD and the number of these servicemembers who were referred for further mental health evaluations, we analyzed computerized DOD data. We worked with officials at DOD’s Defense Manpower Data Center to identify the population of OEF/OIF servicemembers from the Contingency Tracking System deployment and activation data files. We then worked with officials from DOD’s Army Medical Surveillance Activity (AMSA) to identify which OEF/OIF servicemembers had responded positively to one, two, three, or four of the four PTSD screening questions on the DD 2796 questionnaire. AMSA maintains a database of all servicemembers’ completed DD 2796s. The DD 2796 is a questionnaire that DOD uses to identify servicemembers who may be at risk for developing PTSD after their deployment and contains the four PTSD screening questions that may identify these servicemembers. The four questions are: Have you ever had any experience that was so frightening, horrible, or upsetting that, in the past month, you have had any nightmares about it or thought about it when you did not want to? tried hard not to think about it or went out of your way to avoid situations that remind you of it? were constantly on guard, watchful, or easily startled? felt numb or detached from others, activities, or your surroundings? Because a servicemember may have been deployed more than once, some servicemembers’ records at AMSA included more than one completed DD 2796. We obtained information from the DD 2796 that was completed following the servicemembers’ most recent deployment in support of OEF/OIF. We removed from our review servicemembers who either did not have a DD 2796 on file at AMSA or completed a DD 2796 prior to DOD adding the four PTSD screening questions to the questionnaire in April 2003. In all, we reviewed DD 2796’s completed by 178,664 OEF/OIF servicemembers. To determine the criteria we would use to identify OEF/OIF servicemembers who may have been at risk for developing PTSD, we reviewed the clinical practice guideline for PTSD developed jointly by VA and DOD, which states that three or more positive responses to the four questions indicate a risk for developing PTSD. Further, we reviewed a retrospective study that found that those individuals who provided three or four positive responses to the four PTSD screening questions were highly likely to have been previously given a diagnosis of PTSD prior to the screening. To determine the number of OEF/OIF servicemembers who may be at risk for developing PTSD and were referred for further mental health evaluations, we asked AMSA to identify OEF/OIF servicemembers whose DD 2796 forms indicated that they were referred for further mental health or combat/operational stress reaction evaluations by a DOD health care provider. To examine whether DOD has reasonable assurance that OEF/OIF veterans who needed further mental health evaluations received referrals, we reviewed DOD’s policies and guidance, as well as policies and guidance for each of the military service branches (Army, Navy, Air Force, and Marines). Based on electronic testing of logical elements and our previous work on the completeness and accuracy of AMSA’s centralized database, we concluded that the data were sufficiently reliable for the purposes of this report. NDAA also directed us to determine the number of OEF/OIF veterans who, because of their referrals, accessed DOD or VA health care services to obtain a further mental health or combat/operational stress reaction evaluation. However, as discussed with the committees of jurisdiction, we could not use data from OEF/OIF veterans’ DD 2796 forms to determine if veterans accessed DOD or VA health care services because of their mental health referrals. DOD officials explained that the referral checked on the DD 2796 cannot be linked to a subsequent health care visit using DOD computerized data. Therefore, we could not determine how many OEF/OIF veterans accessed DOD or VA health care services for further mental health evaluations because of their referrals. We conducted our work from December 2004 through April 2006 in accordance with generally accepted government auditing standards. Appendix II: Comments from the Department of Defense Appendix III: GAO Contact and Staff Acknowledgments GAO Contact Acknowledgments In addition to the contact named above, key contributors to this report were Marcia A. Mann, Assistant Director; Mary Ann Curran, Martha A. Fisher, Krister Friday, Lori Fritz, and Martha Kelly. | Many servicemembers supporting Operation Enduring Freedom (OEF) and Operation Iraqi Freedom (OIF) have engaged in intense and prolonged combat, which research has shown to be strongly associated with the risk of developing post-traumatic stress disorder (PTSD). GAO, in response to the Ronald W. Reagan National Defense Authorization Act for Fiscal Year 2005, (1) describes DOD's extended health care benefit and VA's health care services for OEF/OIF veterans; (2) analyzes DOD data to determine the number of OEF/OIF servicemembers who may be at risk for PTSD and the number referred for further mental health evaluations; and (3) examines whether DOD can provide reasonable assurance that OEF/OIF servicemembers who need further mental health evaluations receive referrals. DOD offers an extended health care benefit to some OEF/OIF veterans for a specified time period, and VA offers health care services that include specialized PTSD services. DOD's benefit provides health care services, including mental health services, to some OEF/OIF veterans for 180 days following discharge or release from active duty. Additionally, some veterans may purchase extended benefits for up to 18 months. VA also offers health care services to OEF/OIF veterans following their discharge or release from active duty. VA offers health benefits for OEF/OIF veterans at no cost for 2 years following discharge or release from active duty. After their 2-year benefit expires, some OEF/OIF veterans may continue to receive care under VA's eligibility rules. Using data provided by DOD, GAO found that 9,145 or 5 percent of the 178,664 OEF/OIF servicemembers in its review may have been at risk for developing PTSD. DOD uses a questionnaire to identify those who may be at risk for developing PTSD after deployment. DOD providers interview servicemembers after they complete the questionnaire. A joint VA/DOD guideline states that servicemembers who respond positively to three or four of the questions may be at risk for PTSD. Further, we reviewed a retrospective study that found that those individuals who provided three or four positive responses to the four PTSD screening questions were highly likely to have been previously given a diagnosis of PTSD prior to the screening. Of the 5 percent who may have been at risk, GAO found that DOD providers referred 22 percent or 2,029 for further mental health evaluations. DOD cannot provide reasonable assurance that OEF/OIF servicemembers who need referrals receive them. According to DOD officials, not all of the servicemembers with three or four positive responses to the PTSD screening questions will need referrals for further mental health evaluations. DOD relies on providers' clinical judgment to decide who needs a referral. GAO found that DOD health care providers varied in the frequency with which they issued referrals to OEF/OIF servicemembers with three or more positive responses; the Army referred 23 percent, the Marines about 15 percent, the Navy 18 percent, and the Air Force about 23 percent. However, DOD did not identify the factors its providers used in determining which OEF/OIF servicemembers needed referrals. Knowing the factors upon which DOD health care providers based their clinical judgments in issuing referrals could help explain variation in the referral rates and allow DOD to provide reasonable assurance that such judgments are being exercised appropriately. | gov_report |
Active sensing organisms, such as bats, dolphins, and weakly electric fish, generate a 3-D space for active sensation by emitting self-generated energy into the environment. For a weakly electric fish, we demonstrate that the electrosensory space for prey detection has an unusual, omnidirectional shape. We compare this sensory volume with the animal' s motor volume—the volume swept out by the body over selected time intervals and over the time it takes to come to a stop from typical hunting velocities. We find that the motor volume has a similar omnidirectional shape, which can be attributed to the fish' s backward-swimming capabilities and body dynamics. We assessed the electrosensory space for prey detection by analyzing simulated changes in spiking activity of primary electrosensory afferents during empirically measured and synthetic prey capture trials. The animal' s motor volume was reconstructed from video recordings of body motion during prey capture behavior. Our results suggest that in weakly electric fish, there is a close connection between the shape of the sensory and motor volumes. We consider three general spatial relationships between 3-D sensory and motor volumes in active and passive-sensing animals, and we examine hypotheses about these relationships in the context of the volumes we quantify for weakly electric fish. We propose that the ratio of the sensory volume to the motor volume provides insight into behavioral control strategies across all animals. The motor volume is the swept volume of positions a body occupies for a given trajectory or set of trajectories. It is a function of the way the body moves, as well as the geometric extent of the body. To define it more clearly, we first present a definition of the time-limited reachable set. We then use this definition to informally define the motor volume and the stopping motor volume (the swept volume of the body over trajectories that bring the body to a halt), with the precise definitions presented in Text S1. For simplicity of description, we treat an organism as a rigid 3-D body. We define the configuration space as the six-dimensional space representing the rigid-body degrees of freedom (typically the (x, y, z) position of the center of mass, and θ, ϕ, and Ω, the yaw, pitch, and roll angles, respectively). The state space X consists of the six configuration components and their time rates of change (vx, vy, vz, vθ, vϕ, vΩ), resulting in a total of twelve dimensions. The dynamics of the system are given by where x is the instantaneous state and u is the instantaneous control input from the space U℘ of all feasible instantaneous control inputs. The time-limited reachable set R (x0, T) is a construct from nonlinear control system theory [14–16] referring to all points in the state space X that can be reached by a system of the form given by Equation 1 from an initial state x0 given any feasible control history u of duration T. A feasible control history is a control input to the system as a function of time, such as muscle activations for a musculoskeletal system or steering wheel angles for a car. The reachable set is defined as: This is the set of states reachable in time exactly T. In our subsequent definitions, we will use the union of all reachable sets from t = 0 to t = T, denoted as: To illustrate the concept of the time-limited reachable set, consider the simple one-dimensional case of a locomotive moving along a train track (Figure 4A). The state space X is simply the locomotive' s configuration space (the x position of its center of mass), as well as the time rate of change of this one configuration component (vx). The initial condition x0= (0 m, 5 m/s). The control inputs are limited to the set U℘ = [–1 m/s2, +1 m/s2]. Figure 4A shows two dashed curves representing state space (x, vx) trajectories under constant acceleration (+1 m/s2) and deceleration (−1 m/s2), starting from an initial velocity of 5 m/s and running for 5 s; all time-limited reachable sets of 5 s or less from this initial condition must be within these two curves. For example, for t = 3 s, the time-limited reachable set R [ (0 m, 5 m/s), ≤ 3 s] is the union of sectors “1” through “3. ” Figure 4B shows time-limited reachable sets R [ (0 m, 0 m/s), ≤ 5 s] —identical conditions as for Figure 4A but with zero initial velocity. For organisms with internal degrees of freedom (multi–rigid-body systems with joints or flexible bodies), the same concepts apply but now the state space needs to include the additional degrees of freedom, and their time rates of change. The motor volume (Text S1, Equation S1) is similar to the reachable set but with several important differences. First, rather than a set of points in state space, the motor volume is the volume defined by the set of (x, y, z) coordinates of all positions occupied by the body over the time interval of interest. Second, these points are in a coordinate frame that is aligned to the body at the onset of the behavior for which the motor volume is being constructed. For the locomotive example, the thick green line between x = −2 and x = 38 in Figure 4A represents the motor volume (more correctly, the motor “line” in this case) MV (5 m/s, ≤ 5 s). As defined in Text S1, the motor volume is derived from the time-limited reachable set, where this set is computed from Equations 1 and 3 [17,18]. However, in our case, we do not have access to either the equation of motion (Equation 1) for the fish or to the set of feasible control histories; thus, we will estimate the motor volume empirically by examining motion capture data collected from over 100 prey capture sequences (see Methods). In this paper, we will consider the motor volume of the fish over all initial velocities that are typical within our prey capture sequence dataset. Prey capture sequences were selected to begin about 0. 5 s before the onset of behavioral response and end at prey engulfment [11], and thus contain both pre- and post-detection behavior. The stopping motor volume (Text S1, Equation S2) designates the set of all (x, y, z) coordinates of all positions occupied by the body from a given initial velocity to the location at which the body can be halted in the shortest amount of time. For the locomotive example, MVstop (5 m/s) is equal to MV (5 m/s, ≤ 5 s) with control inputs limited to the set U℘ = [–1 m/s2], and is shown by the red line in Figure 4A. If the train had started at rest, then MVstop would collapse to a single point as illustrated by the red dot in Figure 4B. In this paper, MVstop will be ascertained through analysis of motion capture data (see Methods); if a mechanical model exists, MVstop can be estimated by computing optimal stopping trajectories from each initial velocity. The sensory volume (SV) for a given object is the volume defined by the set of (x, y, z) coordinates at which that object can be reliably detected, in body-fixed or sensory system–fixed coordinates. The SV depends on a number of factors, such as target size, orientation, velocity, properties of the sensory system, properties of the detection algorithm, and so on. In this paper, we will estimate SV (prey) for the active electrosensory detection of small water fleas (D. magna). With these definitions in hand, we can explore possible functional relationships between sensory and motor volumes. Restricting our consideration to the stopping motor volume, consider Figure 5, which shows three possible relationships between sensory and stopping volume geometries. In Figure 5A (“collision mode”), the sensory volume is smaller than the stopping volume. We hypothesize that this situation should rarely occur in nature when the SV is for objects that the animal needs to avoid colliding with. Anywhere that MVstop is not covered by the SV represents a region in which unintended collisions could occur. Figure 5B (“reactive mode”) illustrates a situation in which the SV and MVstop are fully overlapping. We hypothesize that this condition represents a functional lower bound on the size and shape of the SV, when the SV is computed for obstacles that the animal should avoid. Also, if the animal' s prey capture strategy demands that it come to full stop to consume or “handle” the prey, then the reactive mode can be considered a lower bound on the size and shape of the SV for prey capture. Finally, Figure 5C (“deliberative mode”) illustrates the case in which the SV is much larger than, and fully encloses MVstop. This case is typical for visually guided predators in terrestrial environments hunting in full daylight, where visual range can far exceed stopping distance. In the context of foraging behavior, the deliberative mode would generally lead to higher foraging efficiency. However, for active-sensing systems, the sensing range could be limited by factors such as energetic costs, clutter, and quartic attenuation, among others, leading to a situation closer to the reactive mode (Figure 5B) than the deliberative mode (Figure 5C). If these constraining factors are particularly severe, then we predict that the SV should approximate MVstop, as in the reactive mode. Finally, we hypothesize that if the SV and MVstop approximately match, then the animal may make behavioral adjustments to either the sensory volume (e. g., by changing the probe intensity) or to the stopping volume (e. g., by changing their “cruising” velocity) to maintain a matched relationship between the SV and the MVstop, particularly where the absence of such adjustments would bring the animal into collision mode. Here we examine SV–MV relationships in the context of prey capture behavior of the black ghost knifefish. From earlier behavioral studies, we know that the fish comes to a near halt before engulfing prey [11]. Thus, we predict that SV (prey) should fully enclose MVstop over the velocities that characterize pre-detection swimming. Our results will show that the relationship between SV (prey) and MVstop for the black ghost is best described by the reactive mode shown in Figure 5B. For this mode, we predict that the animal might use behavioral adjustments to maintain a match between SV and MVstop. We test this hypothesis by reanalyzing results of our prior work with prey capture behavior in water of different electrical conductivities, which influences the distance at which prey can be detected [11]. The evidence suggests that as the SV (prey) shrinks in size due to increased water conductivity, the fish decreases its mean prey search velocity, which causes a corresponding decrease in the size of MVstop. Using synthetic prey capture trajectories, the sensory volume for active electrosensory prey detection in A. albifrons was estimated by computing a detection isosurface surrounding the fish, such that every point on the surface generates a threshold level of activation after summing and filtering the electrosensory afferent signals (see Methods). Every point contained within this bounding isosurface would yield a suprathreshold signal. The estimated SV was found to be omnidirectional (Figure 3), extending in all directions from the body surface. On average, the sensory surface was 34 ± 5 mm (N = 7,056 detection points, all numbers quoted as mean ± standard deviation unless otherwise noted) from the fish' s body surface. As described in more detail below, this is also consistent with the empirically determined Daphnia detection distance of 28 ± 8 mm (N = 38) reported in an earlier behavioral study [19] once the sensorimotor delay time is factored in. In addition to using synthetically generated linear prey capture trajectories, we also tested detection performance using empirically measured fish and prey trajectories from a previous study, which have more complex spatiotemporal profiles [11]. The computationally estimated detection distance was 33 ± 7 mm (N = 38 trials × 10 repeats, or 380, see Methods), compared with the measured detection distance of 28 ± 8 mm (N = 38 trials). However, the latter empirical value is actually the “reactive distance” at which a motor response is first observed and does not include the sensorimotor delay between detection and movement. The estimated sensorimotor delay for behavioral reactions in the knifefish is 115 ms (see Methods). Incorporating the sensorimotor delay, we obtain an estimate for the “neural” detection distance in the empirical data of 35 ± 9 mm (N = 38), which is in good agreement with the simulation result of 33 ± 7 mm for these trajectories. We examined the motor volume as defined above for the entire body, MVbody as well as the motor volume for the mouth alone, MVmouth, at fourteen discrete times, ranging from 117 to 700 ms. Motor volumes at three of these time points are illustrated in Figure 6. The motor volume that maximally overlaps the SV (t = 432 ms) is shown in interactive 3-D Figure S4 along with the SV. Because of the unusually high maneuverability of the fish, including its backward-swimming capability, the shape of the body motor volume is omnidirectional and approximately cylindrical on short time scales, extending both in front of the head and behind the tail of the fish. The size and shape MVmouth, while also omnidirectional, is more compact in the rostrocaudal direction. Figure 7 shows the relationship between the SV and the stopping motor volume. The SV fully encloses MVstop except for a rostral protrusion. In the present study, we find that the time-limited motor volumes for A. albifrons are omnidirectional and extend equally in front and behind the fish (Figure 6; interactive 3-D version Figures S2, S3, and S5). This confirms the previously reported propensity of weakly electric knifefish to spend a significant fraction of time swimming backward at velocities comparable to their forward-swimming velocities [13]. The motor volume is found to be cylindrical, indicating that the fish has considerable lateral and dorsoventral mobility [11,12]. The time-limited motor volume introduced in this study provides a quantitative measure of the fish' s motor capabilities as a function of time interval. The body motor volume was closer in shape to the sensory volume and exhibited greater overlap than the mouth motor volume (Figure 6). Functionally, the mouth motor volume is more closely associated with prey capture behavior, whereas the body volume may be more relevant to obstacle avoidance and general navigation in complex environments. The sensory volume seems better matched to the body motor volume, which suggests that general navigational capabilities, habitat complexity, and obstacle avoidance should all be considered when examining relationships between sensory and motor volumes in the context of prey capture behavior. We find that the active sensory volume for prey detection is also omnidirectional and approximately cylindrical (Figure 3). The omnidirectional shape of the SV is similar to the shape of the isopotential surfaces of the self-generated electric field surrounding the fish (Figure 1A), but exhibits less of a bulge in the caudal tail region due to anisotropies in the sensor density (Figure 1B), field intensity (Figure 1A), reduced sensory surface area due to the tapering body morphology (Figure 1B), and the sensitivity of the primary electrosensory afferents to prey-induced perturbations [20,21]. The relative importance of these factors in determining the precise shape of the sensory volume was not examined. It will be particularly interesting in future studies to explore the extent to which the higher electroreceptor density in the head region of the fish influences prey-detection distance versus spatial resolution of prey position. The simulations of prey detection in this study combined quantitative models of each of these factors in order to arrive at an estimated sensory volume for electrosensory-mediated prey detection. As shown in Figure 2, there was good agreement between the computationally estimated sensory volume and the empirical distribution of prey detections found in an earlier study [11]. The empirical study had relatively low N (38 prey capture events for the water conductivity that yielded maximum detection range), and the detection points were biased toward the dorsal surface of the fish, because prey were introduced into the tank from above. The computational approach used here allowed us to obtain a more complete and less biased estimate of the fish' s sensory detection volume. Our prediction that the fish avoids collision mode is supported by Figure 7 (interactive 3-D version Figure S6), which shows nearly full enclosure of the stopping volume by the sensory volume. The stopping volume was taken from just before detection (which always occurred during forward movement) to the point of zero forward velocity as the fish reverses to capture the prey. Thus, unlike the time-limited motor volume, which sampled all initial velocities including negative velocities, the stopping volume is more strongly biased in the forward direction. However, the extent of the sensing range is restricted to nearly the lower limit of the reactive mode (Figure 5B) where the SV and MV are matched. We expect this is due to constraints that include the metabolic cost for emitting energy into the environment and the interference caused by interaction between the emitted signal and nearby clutter. Energetic costs scale steeply with sensing range, approximately as a quartic power of the range due to geometric spreading effects on the outbound and return paths of active probe signal [1,3]. For quartic scaling, doubling the active-sensing range would require a 16-fold increase in emitted energy. To appreciate the effect of this scaling, consider that an active 15-g A. albifrons requires approximately 300 J/day [22]. If we assume that ≈1% is used for the field, as estimated for another weakly electric fish [23], then 3 J/day is needed for the field. To double the detection distance for D. magna from the measured ≈30 mm to ≈60 mm would therefore require 48 J/day for the field; to double this again to ≈120 mm (still less than one body length for a 15-g fish) would then require 768 J/day, more than double the entire energy budget of the fish. Thus, the high energetic costs associated with extending the active-sensing range is likely to place strong selective pressure on the shape and extent of the active sensory volume. In comparison, the high acuity passive visual system of a typical raptor allows it to spot prey from over a kilometer away, or about 10,000 body lengths. Although it is more difficult to provide a quantitative metric for the interference effects from clutter, the great reduction in emission power observed in dolphins and bats when surrounded by clutter or when nearing a target (see Introduction) suggests that this may also be an important factor in limiting the desirable range of an emitted signal. Returning to the automobile scenario, driving at night in a dense fog provides a practical example of where backscatter is a limiting constraint. Increasing headlight intensity under these conditions (e. g., switching from low beams to high beams) can actually degrade detection performance because the “noise” from backscattered light makes it more difficult to detect the “signal” that is reflected back from a target of interest. Given that the black ghost is in reactive mode, we predict that the fish may make behavioral adjustments to either the sensory volume or to the stopping volume to maintain a matched relationship between the SV and the MVstop, particularly where the absence of such adjustments would bring the animal into collision mode. We are able to qualitatively evaluate this hypothesis by examining how search (predetection) swimming velocity varies with water conductivity. We have shown that water conductivity changes the range at which prey are detected [11]. In the previous study [11], we found that the mean detection distance decreased from 28 mm at a water conductivity of 35 μS/cm to 12 mm at 600 μS/cm. Over this conductivity range, the fish' s predetection swimming velocity decreased 30% from 99 mm/s to 71 mm/s. At the shorter detection distances associated with higher conductivity water (600 μS/cm), we have previously estimated that multiple sensory modalities, including passive electrosense and lateral line mechanosense, are playing a role [24]. Thus, quantitative evaluation of the matching hypothesis would require modeling the SV for these other sensory modalities, which is outside the scope of this study. The size of the estimated sensory volume, and to a lesser degree the shape of the volume, are influenced by the properties of the neural detection algorithm. A more sensitive algorithm will result in a larger detection range. The detection algorithm used here is not intended to model the fish' s actual detection performance in detail. Doing so would require a more extensive analysis of additional factors, such as sensory reafference associated with tail bending, environmental background noise, contributions of other sensory modalities, neuroanatomical constraints on sensory convergence, etc. Rather, the detection volume reconstructed here is intended as an estimate of “best-case” detection performance based solely on changes in active electrosensory afferent spike activity. Echolocating bats emit ultrasonic energy into the environment to detect prey [25]. While the precise size and shape of the bat' s sensory volume will vary with many factors (species, call intensity, duration, etc.), the sensory volume for echolocation is generally a cone that extends in front of the head of the bat with an angular range of approximately ±30° in azimuth and elevation [26]. The angular coverage may extend as much as ±75° relative to the body axis when head and pinnae movements are included [25,27,28]. For the detection of flying insects by pipistrelle bats, the sensory volume extends at least 100–200 cm in front of the animal [25,29] based on the reactive distance to prey. The detailed shape of the bat' s motor volume has not been reported. The stopping distance can be estimated by combining information on the initial velocity of the bat, maximal deceleration, and sensorimotor time delay. Taking a representative bat cruising velocity of 5 m/s [25], a maximal deceleration of 15 m/s2 (estimated from a sample trajectory in [25]), and an estimated sensorimotor delay of 100–200 ms [30] yields an estimated stopping distance in the range of 130–180 cm. Although there is a great deal of uncertainty in these estimates, the stopping distance of the bat seems comparable to the sensory range for prey detection. This suggests that bats, like electric fish, have an active sensory volume for prey detection that may be comparable to their stopping volume. Quantitative comparisons of sensory and motor volumes for a single bat species would help clarify these relationships. Odontocetes (toothed whales, dolphins, and porpoises) also use ultrasonic energy for prey detection. Dolphins can detect prey-sized objects at distances on the order of 100 m [6,31]. The 100-m sensing range of dolphins is certain to be significantly beyond their stopping volume, although there is little published data with which to make quantitative comparisons. This suggests that energetic and clutter-related constraints on active sensing may not be as significant for dolphins as they are for bats and electric fish. Both the active electrosensory volume for prey detection and the time-limited motor volume of A. albifrons are omnidirectional and approximately cylindrical. This is in striking contrast to most other animals, which tend to exhibit a strong forward bias in both sensory and motor volumes. This forward bias is observed for passive-sensing systems such as visually guided fish [32–37]. Figure 8 (interactive 3-D version Figure S7) compares the omnidirectional prey sensory volume in the black ghost knifefish (A. albifrons) with a more typical forward-biased passive sensory volume. The latter is illustrated by the volume for visually mediated prey detection in the stone moroko (Pseudorasbora parva), a fish of comparable size that also feeds on Daphnia [36]. The angular coverage of the visual volume is approximately 100° in azimuth and 60° in elevation, with a range that varies from about 60–120 mm, depending on environmental conditions [36]; the 120-mm range is shown in Figure 8. The active electrosensory volume of A. albifrons and the passive visual volume of P. parva for prey detection are similar in size (approximately 1,000 cm3 each) but quite different in shape. We propose that the ratio of the sensory volume to stopping volume (SV: MVstop) is a useful metric for both active and passive sensory systems when considering whether sensorimotor control systems are in collision, reactive, or deliberative mode. Collision mode occurs when the ratio is below unity. Reactive mode occurs when the ratio approximates unity, as appears to be the case for knifefish and bats. In this mode, sensorimotor control algorithms are likely to be reactive, with relatively fast, direct coupling between sensation and action. Movement options are largely conditioned by mechanical considerations such as inertia and minimal turning radius. For example, some sensor-based motion planning algorithms in robotics are based on estimating the stopping volume for the nearest obstacle; as the robot becomes more massive, the range of any active-sensing system for obstacle detection must be extended accordingly [38]. Deliberative mode occurs when the ratio is large, as for dolphin echolocation and for many passive visual and auditory systems. In this mode, an animal can acquire sensory data from targets that are far outside its stopping volume. This allows the animal a greater range of movement options, because there is adequate time for complex motion planning before reaching the target [39]. For example, in the context of prey capture behavior, a dolphin with a high SV: MVstop ratio is able to engage in long-range tracking of distant prey, whereas a weakly electric fish with a ratio near unity must use a reactive strategy for chance encounters with nearby prey. Quantitative analyses of sensory and motor volumes for both active and passive-sensing systems can highlight important functional relationships between sensing, movement, and behavioral control in animals. In a previously published study [11], adult weakly electric fish (A. albifrons) were videotaped in a light-tight enclosure under infrared illumination. Individual water fleas (D. magna, 2–3 mm in length) were introduced near the water surface and drifted downward; prey capture behavior was recorded using a pair of video cameras oriented along orthogonal axes. Relative to the fish' s velocity (∼100 mm/s) the prey were relatively stationary (prey velocity < 20 mm/s). Prey capture events (from shortly before detection to capture) were subsequently digitized, and 3-D motion trajectories of the fish surface and prey were obtained using a model-based tracking system with a spatial resolution of 0. 5 mm and a temporal resolution of 1/60 s [40]. The time of prey detection was defined by the onset of an abrupt longitudinal deceleration as the fish reversed swimming direction to capture the prey [11]. These reversals are characteristic of most prey capture encounters. This is related to the fact that the fish tends to swim forward with its head pitched downward, such that the dorsum forms the leading edge as the fish moves through the water. Initial prey encounters thus tend to be uniformly distributed along the entire length of the body, so a reversal of swimming direction is typically required to intercept the prey. The volume of space supporting prey detection by the active electric sense was estimated computationally using measurements and empirically constrained models of the prey, electric field, fish body and sensor distribution, electrosensory images, afferent firing dynamics, and behavior. Model parameter values and their sources are summarized in Table 1. Electric field. The electric field vector Efish (mV/cm) at a 3-D point in space x was computed using an empirically tested model of the electric field [41]. This model sums the individual contributions to the field from each of a series of charged poles used to model the electric organ of the fish: where x is a point in space (cm), is the location of pole i of n total poles, q is a normalization constant (mV cm) that scales the overall magnitude of the field, σmes is the conductivity of the water that the field measurements were performed in (which establishes the q value), and σmod is the conductivity of the water for the simulated field. The quantity q is analogous to electric charge in an electrostatic model and is distributed such that the first m poles have a “charge” of q/m and the remaining poles have a charge of –q/ (n – m), resulting in a total net charge of zero. For our simulations, n = 267, m = 266 (all but one pole at the tail was positive), q = 10, and the pole locations xp ran from the nose to the tail of the fish along the central axis of the fish body with equidistant spacing. These values resulted in field values within 10% of measurements of the electric field vector Efish of A. albifrons obtained by other researchers (B. Rasnow, C. Assad, P. Stoddard, unpublished data) in water of conductivity σmes = 210 μS/cm using a multiaxis electrode array [20,42] (Figure 1A). The term scales the field strength to the water conductivity used in simulation σmod = 35 μS/cm. This scaling is based on empirical measurements of the knifefish field at different water conductivities [43], which suggest the electric organ can be idealized as constant current source. We selected 35 μS/cm because our earlier study [11] found that the detection range was highest for trials at this conductivity, and this conductivity is most similar to rivers of the fish' s native habitat. Body model with electroreceptor distribution. We used a prior survey [44] of the density of probability type (P-type) tuberous electroreceptor organs (hereafter electroreceptors) on the surface of A. albifrons. These are the dominant electroreceptor type for A. albifrons [45,46]. Each electroreceptor is connected uniquely to a primary afferent, which generates action potentials with a probability that varies with stimulus intensity. The receptor locations were mapped onto a high-resolution polygonal model of the fish derived from a 3-D scan of a body cast [19,24] in accordance with the measured sensor densities [44] (Figure 1B). This resulted in a total of 13,857 mapped electroreceptors, in close agreement with neuroanatomically derived counts from A. albifrons [44]. Prey model. Based on prior measurements of live prey (D. magna), it was modeled as a 1. 5-mm-radius conductive sphere with an electrical conductance of σobj = 300 μS/cm [19,24]. Idealizing the prey as a sphere allows us to use an analytical model for the stimulus caused by the prey, described below. Electrosensory image formation. The voltage perturbation Δϕ (mV) at an electroreceptor on the fish surface, arising from a small spherical target object, was computed using an empirically tested model [47]: where Efish (mV/cm) is the electric field vector at the prey, r (cm) is the vector from the center of the spherical object to the electroreceptor on the fish surface, a is the radius of the sphere (cm), σobj is the conductivity of the sphere, and σw is the conductivity of the water (μS/cm). Simulations were run with water conductivity σw set to 35 μS/cm (see Methods, Electric field). Primary afferent spiking activity. Computed voltage perturbations at each sensory receptor on the fish body were transformed into primary afferent spiking activity using a previously published adaptive threshold model of P-type (probability coding) primary electrosensory afferent response dynamics [48]. This model gives rise to negative correlations in the interspike interval (ISI) sequence, which lead to long-term spike train regularization. This correlation structure has been shown to increase information transfer and improve detection performance for weak signals [48–50]. The electric organ discharge (EOD) frequency was taken as 1 kHz, which is typical of A. albifrons [51]. P-type afferents fire at most one spike per EOD cycle. Thus, afferent activity was modeled as a binary spike train with a sampling period equal to the EOD period (1 ms). On each EOD cycle (n), the following update rules are evaluated in order: Equation 6 implements low-pass filtering of the voltage perturbation Δϕ[n] with gain β and time constant τm. The state variable u[n] can be conceptualized as a membrane potential; it is initialized to 0, corresponding to the steady-state value with no stimulus present (Δϕ = 0). Equation 7 adds random noise to u[n] to create a noisy membrane potential v[n]; the noise w[n] is modeled as zero-mean Gaussian noise with variance σ2. The actual noise distribution is likely to be more complex, but the Gaussian approximation adequately captures available empirical data. Equation 8 describes the behavior of an adaptive spike threshold θ[n] that decays toward a baseline threshold θ0 with a time constant τθ. Equation 9 represents the process of spike generation, where s[n] is the binary spike output (s = 1, spike; s = 0, no spike); H is the Heaviside function, defined as H (x) = 0 for x < 0 and H (x) = 1 for x ≥ 0. A spike is generated whenever the noisy membrane potential v[n] exceeds the threshold θ[n]. Equation 10 implements a relative refractory period by elevating the threshold θ[n] by an amount b immediately following a spike. (The threshold level subsequently decays toward its steady state value according to Equation 8.) Parameter values common among all afferents in the model were β = 2. 0, b = 0. 09, σ = 0. 04, θ0 = −1, and τm = 2 [48]. The values of the time constant τθ were generated independently for each afferent according to τθ = 21 – 18 ln (z), where z is a uniformly-distributed random number between 0 and 1. This distribution for τθ results in a distribution of baseline firing probabilities in the model that is matched to the empirically observed distribution [52] (Figure 9A). The initial value for the threshold level θ[n] was drawn randomly from a Gaussian distribution with mean (0. 064) and standard deviation (0. 045) matched to the steady-state distribution of threshold values obtained from the model with no stimulus present (Δϕ = 0). As detailed in Brandman and Nelson [48], these parameter choices result in a distribution of baseline firing rates, gains, and amplitude modulation–frequency tuning properties similar to empirically measured values [21,53], as shown in Figure 9. At the time of prey detection, the peak change in transdermal voltage is on the order of 1 μV, and the peak change in afferent firing rate is on the order of 1 spike/s [12,54]. Simulated prey encounter trajectories. During the search phase of prey capture behavior observed in earlier studies, A. albifrons typically swims forward with a mean longitudinal velocity of ∼100 mm/s, with its head pitched downward at an angle of ∼30° relative to horizontal [11]. The slowly moving prey were relatively stationary, with typical velocities less than ∼20 mm/s [11]. In the current simulation study, we modeled this relative motion between the fish and prey by moving the prey target along horizontal rays at 100 mm/s toward a stationary model fish pitched downward at 30° (Figure 10A). Two sets of such prey trajectories were simulated, one set consisting of trajectories from head-to-tail, the other set from tail-to-head, since the fish swims forward and backward. A static model of the fish (140 mm long) was centered within a rectangular box at a pitch of 30°. The box size was chosen such that any prey trajectory originating on a box face would begin well outside the detection range of the fish. The shortest distance between a point on any face of the box and any point on the fish was 60 mm. This distance was set by examining preliminary simulations and empirical measurements, which showed typical detection distances of 20–40 mm. The resulting dimension of the box was 245 mm (length), 129 mm (width), and 193 mm (height). For the head-to-tail trajectories, horizontal prey positions from the front plane to the back plane were generated with starting and ending points centered on a grid with spacing of 5 mm (a total of 1,014 trajectories). Variation in starting and ending points was achieved by adding random values ranging from negative to positive one-half the grid spacing. Intervening points on the trajectory were on a straight line, at a time interval of 1/60th of a second. The same number of tail-to-head trajectories were generated in the same manner. For each simulated trajectory, we use a detection algorithm to estimate the point at which the prey should have become detectable based on changes in afferent spike activity. The detection points that emerge from this analysis are then used to estimate the size and shape of the electrosensory prey detection volume. The approach used here is not intended to model the fish' s actual detection performance in detail. Doing so would require a more extensive analysis of additional factors, such as sensory reafference associated with tail bending, environmental background noise, contributions of other sensory modalities, neuroanatomical constraints on sensory convergence, etc. Rather, the detection volume reconstructed here is intended as an estimate of “best-case” detection performance, based solely on changes in electrosensory afferent spike activity. The voltage perturbation corresponding to each prey position was computed from Equation 5 across the full complement of 13,857 sensory receptors. The resulting history of voltage perturbations at each receptor was interpolated to produce values at each millisecond and used as input for Equation 6. Because of the stochastic nature of the afferent model, ten spike trains were generated for each afferent voltage history for each trial. Output spike trains for these synthetic trials were individually filtered using a boxcar filter with a window size τ = 200 ms, which in previous studies has corresponded to the best weak signal detection capability [52]. At the end of each millisecond, we extract and sum the scalar activity level of each of the 13,857 afferents. The motivation for this approach is that we assume that any neural detection algorithm will require pooling of information from multiple afferents; our goal here is not to specify exactly how this pooling process takes place, but rather to evaluate a best-case scenario. Using the “no-stimulus” condition, a detection threshold was established at a level that yielded one false detection per ten repeats. This detection strategy is a population-level extension of the algorithm described by Goense and Ratnam [54] for detection of weak signals in an individual spike train with ISI correlations. The prey detection distance was defined by the position of the prey at the time of threshold crossing for the “stimulus” condition. For each prey trajectory, the median and standard deviation of the detection distances were computed over ten repeats. The number of detections over all trajectories formed a bimodal distribution, with one peak near one, corresponding to “false alarms” for trajectories outside of the detection range, and a second peak near ten, corresponding to “true hits” for detections well within sensory range. There was a minimum in the bimodal distribution around seven, and we retained all trajectories with eight or more detections (out of ten) for further analysis. The mean and standard deviation of the detection distance for the resulting cloud of detection points was computed. To create the SV, this point cloud was triangulated into a smooth 3-D surface representation using a commercial software package (RapidForm, INUS Technology, Seoul, South Korea). Measured prey encounter trajectories. The synthetic prey capture distance results were validated by following the same methodology outlined above using measured prey encounter trajectories from an earlier study [11]. The earlier study found best detection performance for trials in low conductivity water most similar to the water of the fish' s native habitat. Therefore, we examined only this subset (35 μS/cm, N = 38). In our prior study, the measured detection locations were estimated by examining the first change in behavior near to the prey [11] and therefore include sensorimotor delay time. Thus, to compare these to the computed neural detection points, we retrieved the distance between the prey and the fish at the detection time minus the sensorimotor delay time (115 ms [55]). The estimation of MV from motion capture data does not assume the fish is stationary at the initial time step; rather, MV is estimated over all the initial velocities typical of our prey capture behavioral segments starting from just before detection to capture, which includes forward as well as backward velocities, in addition to heave (up in the body frame), and angular velocities such as roll and pitch. It is defined in the coordinate frame at the fish' s initial position (see Text S1 for details). We consider both the, where is the initial velocity in the coordinate frame fixed to the fish' s initial position, for the entire body surface, MVbody, as well as the 3-D motor volume for the mouth alone, MVmouth. The MV was computed from the 3-D fish surface trajectory data obtained from 116 reconstructed prey-capture trials from an earlier study [11]. Because the motor volume for the mouth is a subset of this space, we will simply refer to the full volume as the MV, unless the distinction between mouth and body volumes is relevant. was estimated from the trajectory data at fourteen different times T, ranging from 117 to 700 ms. For a given T, the nodes on the surface of the polygonal fish model at time tinit + T were transformed back into the body-centered coordinate system of the fish at time tinit. This was repeated over all possible starting times tinit for each trajectory (every 1/60th of a second up to the length of the trial minus T), thus uniformly sampling all observed velocities. The result of this procedure was a dense point cloud, representing where points on the fish' s surface can reach over time T. The points on the surface of this cloud delineate an empirical estimation of (Equation S1) in the absence of the equation of motion (Equation 1) and feasible control histories, as discussed in the Introduction. The surface of the motor volume at each of the 14 intervals was defined by binning of the point cloud around the fish into voxels (5 × 5 × 5 mm), smoothing the data with a 3-D Gaussian convolution kernel (standard deviation, 5 mm), and setting a threshold to include all voxels with point counts up to the 95th percentile. Each resulting point cloud was triangulated to form closed polyhedra for further analysis using commercial software (RapidForm, INUS Technology, Seoul, South Korea). The motor volume for the mouth was constructed following the same procedure as for the body, but using only a single node at the rostrum of the polygonal body model. Because only a single node was used, the resulting point cloud was less dense than the body point cloud. To accommodate the lower density, we maintained all voxels with point counts up to the 90th percentile (rather than the 95th percentile used for the body) when constructing MVmouth. The stopping volume was constructed similarly, but for comparison of the stopping volume to the SV, we restrict our selection of trials to those of the same conductivity as used for estimating the SV, a total of 38 trials at 35 μS/cm. Unlike the MV, MVstop is not a function of a fixed time T but rather depends on the set of initial velocities from which we monitor movement until zero velocity is reached (see Text S1). Thus, we examine the volume swept by the body from the behavioral reaction (detection) time minus the sensorimotor delay time (115 ms, [55]) to the time at which the longitudinal velocity of the body is zero. We take the union of these 38 volumes to derive the stopping volume over all 35 μS/cm trials. Computations were performed on a 54 CPU (2 GHz G5,1 GB RAM) cluster of Xserves (Apple Computer, Cupertino, California, USA) running OS X. An open-source distributed computing engine (Grid Engine, Sun Microsystems, Santa Clara, California, USA) was used to manage the computation across the nodes. Simulation and analysis programs were written in MATLAB (The Mathworks, Nantick, Massachusetts, USA) and compiled to portable executables for execution on the cluster. Interactive 3D visualizations of the sensory volume for prey (water fleas, D. magna) (SV), time-limited motor volume (MV), and stopping motor volume (MVstop) for A. albifrons, the black ghost knifefish. These Virtual Reality Markup Language (VRML) models can be viewed using downloadable web browser plugins and external viewers available for many platforms. As of the date of publication, one of the following is recommended, in order of preference: Cortona VRML plugin (Windows and Mac OS X; available at: http: //www. parallelgraphics. com/products/cortona). Octaga VRML player (Windows, Mac OS X. and Linux; available at: http: //www. octaga. com/). Xj3D viewer (Windows, Mac OS X. and Linux; available at: http: //www. web3d. org/x3d/xj3d/). | Most animals, including humans, have sensory and motor capabilities that are biased in the forward direction. The black ghost knifefish, a nocturnal, weakly electric fish from the Amazon, is an interesting exception to this general rule. We demonstrate that these fish have sensing and motor capabilities that are omnidirectional. By combining video analysis of prey capture trajectories with computational modeling of the fish' s electrosensory capabilities, we were able to quantify and compare the 3-D volumes for sensation and movement. We found that the volume in which prey are detected is similar in size to the volume needed by the fish to stop. We suggest that this coupling may arise from constraints that the animal faces when using self-generated energy to probe its environment. This is similar to the way in which the angular coverage and range of an automobile' s headlights are designed to match certain motion characteristics of the vehicle, such as its typical cruising speed, turning angle, and stopping distance. We suggest that the degree of overlap between sensory and movement volumes can provide insight into the types of control strategies that are best suited for guiding behavior. | lay_plos |
By Ceri JacksonBBC News When the young Aristotle Onassis would sail in Cardiff's Tiger Bay, he always headed for one place. Away from the high commerce and architectural splendour of the dockland's banking halls and buildings - a stately procession of Georgian, Victorian and Edwardian-style magnificence befitting the world's coal metropolis - he would walk a short distance towards the legendary Bay. The young Onassis, destined to amass the largest privately owned shipping fleet and beguile the 20th Century as one of its richest and most celebrated men, would turn into George Street. There close to the bustling back-to-back terraces - a harmonious, cacophonous, cheek-by-jowl melting pot of 50-plus nationalities, faiths, costumes, food, music and customs - a Spanish immigrant who had settled in Tiger Bay ran a delicatessen. Onassis, then a humble teenage seaman, would walk under the rows of chorizo hanging from the rafters and make his way to a dining room upstairs. There he would join five or six fellow Greeks some of whom lived in the area, among them Alexanderos Callinicos, a local ship chandler. He was a friend of the shop's owner Josefina Hormaechea's and he would regularly ask her to prepare a meal - typically soup and roast lamb with potatoes - so the men could meet and discuss business. Whenever, years later, Josefina was reminded of her encounters with Onassis and people would ask her what he was like, her daughter Gloria Del Gaudio remembers she would exclaim: "He was a skinny, ugly little kid." To Josefina the man whose company the most glamorous and influential in the world would clamour for, was just another itinerant seamen washed ashore into Tiger Bay from the Seven Seas on an unrelenting, rhythmic human tide. "Cardiff was like Saudi Arabia at the turn of the 20th Century," says Neil Sinclair, author, historian and the only genuine Cardiff accent to feature in J Lee Thompson's 1959 movie Tiger Bay, which starred John Mills and his daughter Hayley in her first starring role at the age of 12. "But instead of oil it was the prized anthracite coal of the south Wales Valleys which was exported to the rest of the world. "It had been transformed from an insignificant village in the middle of marshland to a city at the centre of everything. Coal, iron and steel industry from the south Wales valleys had ignited Britain's' industrial revolution." The 'black gold' of the Rhondda created untold wealth. Cardiff docks made'millionaires by the minute', its financial quarter second only to the Square Mile of London. The thronging trading hall of the magnificent Coal Exchange, now largely derelict, set the price of coal worldwide and it was where the signing of the first million pound cheque took place. And along with the financial prosperity came a societal richness. The high demand for labour led to the creation of one of Britain's first and arguably most distinctive and successful multi-cultural communities. "There was no place quite like it on earth," says Neil. "No matter where you were from, colour, religion, ethnicity, it didn't matter. Everyone thrived as one big community. "It was like walking into an Aladdin's Cave. It had the flavour of a Kasbah. At one time 57 different nationalities lived and worked in respectful harmony." The acclaimed Welsh poet, writer and broadcaster Gwyn Thomas said of Tiger Bay during a visit in the 1950s: "Whenever any two children of different races play together, humanity grows an inch or two; another ancient fear, another mouldering prejudice is told to mind its manners and behave." It is after all what we are famous for, so had the Welsh succeeded in teaching the world to sing in harmony? "Tiger Bay was in the simplest words possible a symbol of racial, ethnic, religious and ecumenical harmony," says Neil. "My mother used to say 'the League of Nations could learn a thing or two from Tiger Bay'." What a legacy for Cardiff; one to be cherished for generations to come you might think. But get off the train at Cardiff Central today and ask for directions to Tiger Bay and you are likely to be met with a scratching of heads. Just the other side of the railway track, almost every last vestige of the area has been wiped from the face of the earth; the fact it once existed let alone its staggering history largely unknown by many of those living in the city today. But then that, some bitterly protest, was precisely the plan. Cardiff, one of Europe's youngest capitals, is an emerging city on the UK map. A popular sporting, weekend and holiday destination it is prized for its vibrant nightlife, culture, shopping and gateway to outstanding rural and coastal wildernesses. But as the ghosts of the past would attest, the 'Diff' as it is has become affectionately known is no 'new kid on the block'. The city's re-discovered swagger was woven into its DNA generations ago. Neil Sinclair grew up in Tiger Bay. He still lives in the area in a high-rise council tower block of flats, built on the bulldozed rubble of his childhood home. What happened to it was, he says, "one of the most torrid pages of meanness and spitefulness to be found in the annals of Welsh history". That history begins with John Crichton-Stuart, the 2nd Marquess of Bute. A wealthy Scottish aristocrat, landowner and industrialist he realised vast wealth lay in the south Wales coalfields and set about exploiting it. In 1839 the first in a series of docks was built - Bute West Dock, Bute East Dock, Roath Basin, Roath Dock followed and finally in 1907 the Queen Alexandra Dock. Fine buildings sprung up and squares of decorative four-storey town houses were built around tranquil parks to accommodate magnates, merchants and sea captains. Loudoun Square was chief among them and as the wealthy later began their migration to more verdant suburbs emerging in Cardiff, such as Cathedral Road, it become the beating heart of Tiger Bay. Surrounding it were the terraces of Butetown which ran off the main artery of Bute Street and had been built as a model village in the early 19th Century for workers. Christina Street, Maria Street, Angelina Street, Sophia Street - addresses which remain today - were named after children in Crichton-Stuart's family. By the later 1800s Butetown had taken on its unofficial name as the legendary Tiger Bay, the source of tales once told by sailors around the world. "Local folklore has it that there was a woman who used to walk around Loudoun Square with two tigers but then seamen were known for their tall tales," says Neil. "Portuguese sailors are believed to have come up with the name. The tides in the area are notoriously difficult. After successfully docking they would say that sailing into Cardiff was like sailing through a bay of tigers. And so it was - Tiger Bay stuck." Another theory is that its reputation as a wild hotbed of hedonism, rough house boozers, crime, prostitution and illegal gambling earned it sole use of a once generic term long used by sailors for raucous ports everywhere. Some of the nicknames given to the area's 97 pubs - House of Blazes, Bucket of Blood, Snakepit - infamous for brawling sailors and prostitutes could add some weight to that. It was a tough, hard life. No doubt about that. But many who grew up there would say that theory was just part of the conspiracy. Other pubs were equally as well known for fantastic jazz and superb French cuisine, a magnet to out-of-town Bohemian types. "This was a sea port and sea ports have had this sort of reputation for millennia," Neil says. "If you have a Dickensian view of dockland life anywhere in the world you would cast aspersions on Tiger Bay. But that was purely an outsider's view. "This was a major industrial port and thousands of workers descended upon it daily. Come 4 o'clock when the whistle blew they came out in what seemed like their millions. "They got on their bikes but they didn't go home and slap the money on the table for their mams, they headed for the pubs and the place was jumping. "And that's where your prostitutes come in. When the men were paid it was like moths to flames. But that's how the city of Cardiff gained its wealth, off the backs of those people. "Yes, if you wanted trouble you could find trouble but that stigma obscures the fact that the majority of the men who lived there were hard working members of the community, not pimps running whores. Far from it. "Everyone looked out for one another and nobody ever locked their door. If this was such a dreadful place then surely we'd have all been murdered in our beds? "It was stigmatised. Good old fashioned racism. This was commonly stated on the streets of Tiger Bay - 'if you're black stand back, if you brown stick around, and if you're white, you're alright'. "The Victorian Imperial mentality was very much 'oh, what do black people do after dark?' They became the boogie man. Upper class families used to say to their kids who wouldn't go to bed 'I'll take you to Loudoun Square and leave you there!'." By the beginning of the 20th Century the district had a burgeoning population of inter-racial married couples and their families. Among them Shirley Bassey, rugby legends Billy Boston MBE and Colin Dixon, heavyweight boxer Joe Erskine and swimmer and water polo Olympian Paolo Radmilovic, his father a Tiger Bay pub landlord from Croatia and his mother, the Cardiff-born daughter of Irish immigrants. He won four gold medals across three successive Olympic Games, a team GB record only broken by Sir Steve Redgrave. Many of the local women who had married foreign men were commonly denounced as prostitutes who had shamed their chapel-going families by marrying 'heathens'. "Part of the reason why multiculturalism worked was down to our Welsh mams and Welsh-speaking grandmothers," insists Neil. "The sea men who were the heads of the family were more away at sea than they were at home. The white women in particular created a strong sisterhood, a Celtic matriarchy that kept us in line. "Many of them came from the valleys and when it was discovered that they'd married an Arab or a Malay or an African, they were ostracised. "They couldn't go back home. So Tiger Bay became their home and they formed a strong alliance. We were one big family. "Cardiff was a place people would head for. My grandfather docked in Bristol from Barbados. As a seaman of colour he was confronted with racism. But the word had got out 'if you want to feel at home, get to Tiger Bay'." Others did not share the enthusiasm. Around the time of the Great War as the docks was at its zenith, the self-proclaimed moral arbiters of the day made no secret of their loathing, and stories spread about "Cardiff girls sold into slavery every night". Newspapers reported stories about policemen patrolling the streets armed with sabres and of grave concerns of a "great increase in alien floating population… earning high wages able to buy property for fancy prices and clear out British residents". In truth seamen and dockers were poorly paid and their families led a hard life. And it was about to get even harder. Cardiff had enjoyed the boom, now it was time for the bust. Oil was growing in importance as a maritime fuel and by 1932, in the depths of the depression, coal exports had plummeted. Despite intense activity during the Second World War, exports continued to fall, ceasing altogether in 1964. There was mass unemployment and parts of Tiger Bay's housing, which had no indoor toilets or bathrooms, had fallen further into dilapidation. There was also a rise in tuberculosis and social deprivation. In the early 1960s under the orders of the city's fathers, the bulldozers began moving in. Tiger Bay, along with all of the area's 97 pubs, bar one, the Packet, was razed. Residents were moved to other areas of the city with the promise they could return to newly-built council homes. But after a period of a couple of years, many had settled and did not come back. The community had broken up. "They said it was a slum and all the lies you can imagine," Neil says. "From an insider's perspective we were targeted, something to be got rid of. "Rather than renovate housing in stages as they'd done in neighbouring Grangetown for instance, they wanted us gone and one way to do that was to knock it all down. "All they succeeded in doing was destroying the architectural legacy of the Marquess of Bute for Cardiff and for Welsh history. "Replace it with the first council estate to be dropped on to an inter-generational, multicultural, multiethnic, multiecumenical community. It was a tragedy. "As a child you think the whole world is like your immediate environment. It was only as I got older that I realised the rest of the world didn't live as we did, they didn't have respect and tolerance for one another. "If I'd known then that it was only ever going to be a moment in time, I'd have screamed blue murder. But you thought it was going to be this way for the rest of your life." Neil says when they witnessed the old trees being ripped from the park of Loudoun Square, they knew the game was up, a community which had grown over 150 years was gone. "Loudoun Square was the jewel in the crown," he says. "I've been in London wandering round and turn a corner and my heart drops because I see a square reminiscent of it and I think 'our Cardiff could've been so fabulous'. "All those houses were solidly built. Some of the terraces had granite foundations. The wrecking balls were hitting the walls eight to nine times before they would give. "If they were here today, they would be the houses tourists would flock to see. Instead we have a lacklustre council estate, an architectural monstrosity." In a BBC Wales interview in 1982 the then Lord Mayor of Cardiff Philip Dunleavy described the building of the tower blocks as "a disaster" and one which Cardiff would not repeat. "They're anti-social," he said. "I don't know if you've been on to the top floors but it's like living in the country, one's very remote except for the corridor on which one lives. "People tend to get lonely and the neighbourliness they experienced when they lived in terraced houses has disappeared." By the early 1980s the once thriving docks was a neglected wasteland of dereliction and mudflats. Its residents say they suffered from social exclusion, above average unemployment and social services which used their home as a "dumping ground" for problem cases. The docklands had given the city its wealth but had then been disinherited. In 1987 redevelopment plans were announced. Its aim clearly stated: To put Cardiff on the international map as a superlative maritime city which will stand comparison with any such city in the world, thereby enhancing the image and economic well-being of Cardiff and Wales as a whole. The irony was not lost on some. After all that description sounded painfully familiar. In 1999 the construction of a controversial barrage was complete, built to impound the rivers Taff and the Ely to create a massive fresh-water lake. The area - now home to the Senedd, Dr Who studios, a sports village, restaurants, bars and the Wales Millennium Centre - was re-branded as Cardiff Bay. Meanwhile, a stone's throw away in Butetown, the grievances persist. "The stigma's still there," says Neil. "That's the Cardiff mentality. "Living in the towers is like living in a prison; cameras on every floor. That's not just my point of view. Many people will say the same thing. "People of my age and older are walking around with a pain in the heart. "I might go to sleep every night in the new Loudoun Square but when my head hits the pillow I'm back in the old Loudoun Square." Neil wrote his book 'Endangered Tiger' in a bid to restore a sense of pride for Tiger Bay. In it he cites a poem called Sweet Tiger Bay attributed only to 'M' which was published in the Western Mail on Saturday, August 23rd 1902. Its final lines encapsulate the love and lament felt by so many, living and dead, for the magic and mayhem of the place: I live in troubles, but they pass like bubbles, When Fancy conjures up the days that were: Put Death before me and an Angel o'er me, To bear me upward-this to him I'd say: "Young friend, your attitude has won my gratitude; But please, another night in Tiger Bay!" | On Monday #towerlives kicks off, a week-long BBC festival of storytelling and music, on air and on the ground, in and around the council estate tower blocks of Butetown in Cardiff. Its aim is to give a platform to voices from a community often talked about but rarely heard. In the first of a series of stories from the tower blocks the BBC news website talks to one resident, who witnessed the extraordinary events that led to their construction, a history of fortunes - both financial and social - made and lost. | xlsum_en |
FIELD OF THE INVENTION [0001] The present invention relates to intravascular neutron capture therapy. More particularly, the present invention provides methods and apparatus for making and using an implantable stent comprising a material capable of reducing restenosis, thereby improving long-term patency of the implanted stent. BACKGROUND OF THE INVENTION [0002] Over the past 20 years, the number of percutaneous coronary revascularization procedures has increased to more than one million per year. About 50% of these procedures include stent implantation. A stent is often designed as a thin metal wire mesh, which keeps a fabric in a desired shape, for instance forming a very thin tube providing an open channel for a fluid. FIG. 1 illustrates an example of such a stent, which is commercially available from JOMED AB, Helsingborg, Sweden. A polyfluorotetraethylene (“PFTE”) graft material integrated into the stent is used to seal off a perforated or ruptured artery wall. [0003] One drawback associated with previously known stents is the restenosis effect, i.e., the epithelial cells of the vessel walls adjacent to the ends of the stent and surrounding the stent may experience excess growth of cells, thereby clogging the vessel. [0004] One way to decrease the risk of restenosis is to irradiate the vessel in the vicinity of the stent. The cell proliferation rate is thereby decreased, and the vessel remains patent. Several ways to apply local radiation doses have been investigated, including temporarily placing balloons filled with radioactive solution inside the stent area or placing radioactive wires. Previously known radioactive stents used in clinical trials are activated by reactor or accelerator irradiation. Irradiation by small X-ray tubes inserted into the coronary arteries through a guide catheter has also been suggested, and there is still considerable discussion about the best radiation delivery system. [0005] U.S. Pat. No. 5,728,042 to Schwager describes an appliance comprising a core wire on which is mounted a coil of radioactive material. A first proximal radiopaque coil configuration and a second distal radiopaque coil configuration maintain and locate the radioactive radiation coil on the core wire, thereby ensuring positioning thereof on the core and ensuring accurate visualization via X-ray fluoroscopy. [0006] U.S. Pat. No. 5,782,742 to Crocker et al. discloses a balloon catheter with an inflatable balloon having thereon a radiation carrier such as a radiation delivery metal foil, such as gold. The foil is irradiated, and the balloon is thereafter positioned at a treatment site in a vessel and expanded to bring the metal foil layer into close proximity with the vessel wall. In another embodiment, the radiation carrier is in the form of a dopant in the balloon material. A PE or PET multi-layer or single layer balloon can be extruded with sodium phosphate (monobasic, dibasic or tribasic) as a filler. The phosphate filled balloon can be placed in a neutron beam to produce sodium phosphate P-32. Other suggested radiation delivery sources are Y-90, Au-198, Ir-192 and Mo-99. [0007] German publication DE 197 54 870 A1 to Alt discloses a stent with an expandable perforated tube. The tube has a cover containing a biocompatible carrier containing a radioactive material, which is P-32 or Au-198. The radioactive material has an activity level of about one micro-Curie. [0008] U.S. Pat. No. 5,730,698 to Fischell et al. discloses an expandable temporary stent system, including an over-the-wire balloon angioplasty catheter. The balloon angioplasty catheter has a proximal section that remains outside the body. A stent assembly is slidably mounted on the balloon angioplasty catheter in a coaxial manner and has a proximal section and a distal section, where a temporary stent is located at the distal section. The system further comprises a radiation shield over the stent assembly. The patent also discloses a method for treatment of arterial stenosis by means of the stent system. [0009] Several problems are common to all devices for this type of intravascular brachytherapy. Dimensions are small, and misplacement of the radiation source by as little as a few millimeters can give rise to a very inaccurate dose distribution. Normally, radiation is delivered in conjunction with balloon catheterization, before one knows whether or not radiation therapy is necessary. Furthermore, working with radioactive sources in a catheterization laboratory is problematic, as a new catheterization has to be performed, thereby adding risk to the patient and costs to the treatment. [0010] In view of the foregoing, it would be desirable to provide apparatus and methods for neutron capture therapy that provide temporal separation during a stenting procedure between balloon dilatation following stenting and delivery of radiation. [0011] It further would be desirable to provide methods and apparatus that ensure neutron capture therapy is only provided to patients where radiation exposure is expected to provide therapeutic benefit. [0012] It still further would be desirable to provide methods and apparatus for neutron capture therapy that allow therapy to be repeated as needed. SUMMARY OF THE INVENTION [0013] In view of the foregoing, it is an object of the present invention to provide methods and apparatus for neutron capture therapy that provide temporal separation during a stenting procedure between balloon dilation following stenting and delivery of radiation. [0014] It is another object to provide methods and apparatus that ensure neutron capture therapy is only provided to patients where radiation exposure is expected to provide therapeutic benefit. [0015] It is yet another object to provide methods and apparatus for neutron capture therapy that allow therapy to be repeated as needed. [0016] These and other objects are accomplished by providing a stent having a stable target nuclide with a large capture cross-section for thermal neutrons. This nuclide is preferably incorporated as an alloy in the stent. When there is a clinical need for neutron capture therapy, the stent is irradiated with thermal neutrons, thereby giving rise to ionization radiation around the stent device. Concentration of target nuclide and thermal neutron flux determines dose rate around the stent. Since radiation is applied by an external source, it can be delivered at any time after placement of the stent and easily may be repeated. [0017] Methods for making the stent according to the present invention are also provided. BRIEF DESCRIPTION OF THE DRAWINGS [0018] The above and other objects and advantages of the present invention will be apparent upon consideration of the following detailed description, taken in conjunction with the accompanying drawings, in which: [0019] FIG. 1 is an isometric view of a prior art stent; [0020] FIG. 2 is a graph illustrating calculation of the KERMA dose rate around a point source created by irradiation of 1 mg Gd-157 with 10 8 thermal neutrons per second per cm 2 ; [0021] FIG. 3 is a graph illustrating build-up zones from different qualities of gamma and X-ray radiation; and [0022] FIG. 4 is an isometric view of a stent in accordance with the present invention. DETAILED DESCRIPTION OF THE INVENTION [0023] The present invention provides a stent comprising a stable nuclide element that may be externally activated by thermal neutrons, thereby providing localized neutron capture therapy in the vicinity of the vessel around the stent. Since radiation is applied by an external source, therapy may be delivered at any time after placement of the stent and easily may be repeated. Furthermore, unlike other known radiation techniques, the present invention ensures that neutron capture therapy is only provided to patients where radiation exposure is expected to provide therapeutic benefit. [0024] In accordance with the principles of the present invention, a stent is constructed including a material having a high neutron capture cross-section, for example, greater than 10 3 barns, and that provides a high quality of radiation emission. As will of course be apparent, the irradiation dose provided by the stent after irradiation by an external source also depends upon the amount of stable nuclide element that is incorporated into the stent. Preferred stable nuclides suitable for use in a stent constructed in accordance with the present invention are listed below with their corresponding thermal neutron capture cross-sections. Atomic element Cross-section (barn) 157 Gd 254000 155 Gd 60900 149 Sm 40140 113 Cd 20600 151 Eu 5900 [0025] Bulk materials with which these nuclide elements may be combined to form a stent or other interventional device for neutron capture therapy are provided below, again with corresponding thermal neutron capture cross-sections. Preferably, the bulk materials have significantly lower neutron cross-sections compared to the elements employed for neutron capture, generally less than 10 2 barns. Atomic element Cross-section (barn) 198 Au 98 59 Co 20 48 Ti 7.8 109 Ag 4.5 107 Ag 3.0 56 Fe 2.6 While these bulk materials may produce a small amount of ionization radiation when subjected to thermal neutron radiation, the contribution of this ionization radiation to a composite absorbed dose is negligible. [0026] In a preferred embodiment, a stent for neutron capture therapy comprises gadolinium as the stable nuclide. Gadolinium is a trivalent metallic element and is a member of the rare earth group. Its atomic number is 64, and it has a relative atomic mass of 157.25. Gadolinium has the largest known thermal neutron capture cross-section (254000 barn) of any element. The most frequent stable Gadolinium nuclide is denoted as Gd-157. Gd-157 makes up 15.65% of all Gadolinium, and it primarily radiates energy in the form of high-energy gamma radiation. [0027] A previously known stent, for example, such as depicted in FIG. 1, typically may weigh on the order of 40 mg. The amount of stable nuclide incorporated into such a stent in accordance with the principles of the present invention may be chosen based upon a variety of design considerations. For the purposes of illustration, assume an enriched target of N atoms of Gd-157 and a neutron flux of n thermal neutrons/cm2/s. The number of neutron captures per second, Ac, may be computed as: Ac=n·N·2.54·10 −9. 1 mg of Gd-157 (N=3.8·10 18 atoms), which radiates neutrons at a rate of approximately n=10 8 neutrons/cm 2 /s, provides an Ac of about 9.7·10 7 captures per second. This is equivalent to a radioactive source with a strength of 9.7·10 7 Bq. [0028] From the gamma spectrum of Gd-157, the Γ-constant may be determined as 1.28 Gy/h/m 2, and the dose rate distribution may be determined for a point source containing the 1 mg of Gd-157. FIG. 2 presents this dose rate distribution in a thermal neutron field of 10 8 n/cm 2 /s. The dose obtained from this calculation reflects a KERMA value rather than an actual absorbed dose. Gamma energies emitted are fairly high, on the order of several MeV. These energies may have build-up zones of several millimeters, as seen in FIG. 3. The build-up zones level out the dose close to the source and compensate for the square-law dependence at the closest distances from the stent. This is an advantage of the present invention when compared to other radiation techniques that use high-energy beta or low-energy gamma sources having negligible build-up zones. [0029] According to these illustrative calculations, a therapeutic radiation dose may be delivered within a few seconds (<10 seconds in a distributed source). The dose contribution of the neutrons themselves, distributed as a general background, yield a dose far below biologically dangerous levels. [0030] In this example, the dose rate is expected to be about 1 Gy/second in the area closest to the source, as seen in FIG. 2. The required dose may then be delivered in 10-30 seconds, or somewhat longer (in a couple of minutes) if the source is extended to offer a larger area, such as stent 20 of FIG. 4. Stent 20 is fabricated from a material incorporating stable nuclide element S. This example indicates that therapeutic dose rates may be delivered within clinically acceptable parameters. Corresponding calculations according to the above example also may be performed for the other listed atomic elements. It will be apparent to those of skill in the art that the amount of stable element S may be tailored to specific patient populations. [0031] Referring still to FIG. 4, stent 20 may comprise metal wire mesh 22 that is fabricated from an alloy or mix incorporating from a few tens to a few hundreds of micrograms of the desired stable nuclide S. In another embodiment, wire mesh 22 may comprise hollow wires in which stable element S is located. Wire mesh 22 is preferably coated with a biocompatible material B to prevent direct contact between body tissue and the wire mesh metal containing stable nuclide S. Also, stent 20 optionally may include fabric 24, thereby providing a continuous tubular profile to stent 20. [0032] The % composition, as well as the nuclide or nuclides comprising stable element S, may be varied within stent 20 to obtain a differentiation of radiation along stent 20. In some applications, creating a larger radiation dose at the ends of the stent, where restenosis may be more pronounced, is expected to be advantageous. [0033] A method of using stent 20 is now described. Stent 20 is deployed at a treatment site within a patient's vasculature using well known percutaneous or subcutaneous techniques. When neutron capture therapy is deemed therapeutically beneficial, the patient is subjected to external radiation near the treatment site at clinically-acceptable levels that minimize damage to biological tissue. Due to its high neutron capture cross-section, stable nuclide element S preferentially absorbs and emits the radiation to tissue at the treatment site surrounding stent 20, thereby providing localized radiation therapy in a concentrated dose. The emitted radiation acts on surrounding tissue to provide a therapeutic benefit, for example, to reduce the restenosis often encountered after angioplasty and stenting. The short half life of stable element S provides negligible radiation when not irradiated, as imposed activity decays in milliseconds after completion of thermal neutron irradiation. [0034] An advantage of the stents constructed in accordance with the present invention is that the stents may be handled without concern for radiation exposure, as they contain only stable nuclides. A still further advantage is that when using, for example, Gd-157 as the neutron capture therapy element, a stent will only produce gamma radiation when subjected to neutron irradiation, as the lifetime of the active gadolinium is very short and decays in microseconds. As already noted, the primary wire mesh metal constituent of the stent will have a very small capture cross-section for neutron irradiation, but will not produce any harmful residual activity. [0035] Irradiation using the present neutron capture therapy may advantageously be limited to when restenosis is observed, and the therapy may be applied repetitively, as needed. This avoids more extensive methods involving rearrangement of existing implanted stents or introduction of new stent devices, due to restenosis. It should be noted that, although stent 20 of FIG. 4 illustrates a device for use in connection with coronary dilatation, a general stent device according to the present invention may also be used in connection with any subcutaneous (or percutaneous) therapy, e.g., in connection with treatment of a tumor. [0036] Radiation sources suitable for use with the stents of the present invention are known. For example, radiation sources capable of delivering a suitable number of thermal neutrons have been developed for boron neutron capture therapy (BNCT) and are expected to be readily applicable to neutron capture therapy in accordance with the present invention. Other sources, such as accelerators and radioactive sources delivering neutrons, may also be used with embodiments of the present invention. [0037] Although preferred illustrative embodiments of the present invention are described above, it will be evident to one skilled in the art that various changes and modifications may be made without departing from the invention. For example, a wide variety of stent designs are known in the art; incorporation of stable nuclides into these designs for the purpose of neutron capture therapy falls within the present invention. It is intended in the appended claims to cover all such changes and modifications that fall within the true spirit and scope of the invention. | Improved method and apparatus for neutron capture therapy are disclosed, which may beneficially be used to counteract restenosis. An improved stent and a method for manufacturing the stent are also presented. The stent comprises a stable nuclide having a large neutron capture cross-section. When a clinical need exists for radiation therapy, the stent is irradiated with thermal neutrons, thereby giving rise to radiation in the proximity of the stent to a therapeutic benefit. Since radiation is applied by an external source, it can be delivered at any time after placement of the stent and easily can be repeated. The stent only contains stable nuclides and therefore can be handled without the precautions needed when handling radioactive matter. | big_patent |
Alexander Litvinenko’s accusation that Vladimir Putin was a paedophile may have been one of the motives for the Russian government to order his assassination, a report into the former Russian spy's death has found. Sir Robert Owen’s inquiry looked at the former FSB agent’s “highly personal attacks” on the Russian President, which culminated with an article on the Chechenpress website in July 2006, four months before he was poisoned. Mr Litvinenko’s article, which was published as evidence in the report, started by recounting a meeting between Mr Putin and a boy “aged four or five” in a square near the Kremlin. Litvinenko widow's statement “Putin kneeled, lifted the boy’s T-shirt and kissed his stomach,” Mr Litvinenko wrote. “Nobody can understand why the Russian president did such a strange thing as kissing the stomach of an unfamiliar small boy.” The former FSB agent claimed there were “blank spots” in Mr Putin’s career that could be explained by his superiors’ alleged knowledge “that he was a paedophile”. Mr Litvinenko claimed the Russian President had himself found “videotapes in the FSB Internal Security directorate, which showed him making sex with some underage boys” that he then hid. Alexander Litvinenko pictured at the Intensive Care Unit of University College Hospital on November 20, 2006 in London Commenting on the extraordinary and unfounded allegations, Sir Robert wrote: “It hardly needs saying that the allegations made by Mr Litvinenko against President Putin in this article were of the most serious nature. Could they have had any connection with his death?” The judge’s 300-page report concludes that Andrei Lugovoi and Dmitri Kovtun poisoned the 43-year-old with radioactive polonium 210 at a Mayfair hotel in 2006. It found that there is a “strong probability” that the Russian secret service directed the killing, and that operation was “probably approved” by Mr Putin. Sir Robert said there were “several reasons” why the Russian state may have wanted to kill Mr Litvinenko by late 2006. The Litvinenko files: Was he really murdered? 8 show all The Litvinenko files: Was he really murdered? 1/8 Alexander Litvinenko in his hospital bed at University College Hospital, London, shortly before he died © PA 2/8 Russian President Vladimir Putin stands accused of ordering the murder of Litvinenko © AP 3/8 Litvinenko’s widow, Marina, wants an official inquiry into her husband’s death © PA 4/8 Former KGB agent Andrei Luguvoi at target practice © AP 5/8 Litvinenko's funeral took place at Highgate Cemetery in north London in December 2006 © EPA 6/8 Marina Litvinenko listens as Alex Goldfarb reads her husband's final statement in London last November © AFP/Getty Images 7/8 Russian exile, multi-millionaire property magnate, and perpetual thorn in Putin's side, Boris Berezovsky was a constant presence behind the scenes © AFP/Getty Images 8/8 In memoriam: a candlelit tribute to Litvinenko in Helsinki the day after his death © AFP/Getty Images “There was undoubtedly a personal dimension to the antagonism between Mr Litvinenko on the one hand and President Putin on the other,” he added. Officials in Moscow have always denied involvement in Mr Litvinenko’s death, with officials previously claiming he was involved in an illicit trade in polonium and poisoned himself. The Russian Foreign Ministry dismissed Sir Robert’s report as “politically motivated” today and warned that it would overshadow relations with the UK. “We need time to study in detail the contents of this document, and then give a detailed assessment,” a spokesperson said. “We would like to note that Russia's position on this issue remains unchanged and is well known…there was no reason to expect the final report of a politically engaged and highly opaque process to be objective and impartial.” LONDON (AP) — President Vladimir Putin probably approved a plan by Russia's FSB security service to kill former agent Alexander Litvinenko, who died three weeks after drinking tea laced with poison at a London hotel, a British judge said Thursday. Marina Litvinenko, widow of former Russian spy Alexander Litvinenko, reads a statement outside the Royal Courts of Justice in London, Thursday, Jan. 21, 2016. President Vladimir Putin probably approved... (Associated Press) Marina Litvinenko, right, widow of former Russian spy Alexander Litvinenko, arrives at the Royal Courts of Justice for the Litvinenko Inquiry statement following publication of the report in London, Thursday,... (Associated Press) FILE - In this July 26, 2010 file photo, Viktor Ivanov, head of the Russian anti-narcotics agency, speaks at a news conference in Moscow. One day in 2006, a former KGB agent who claimed to know dark Kremlin... (Associated Press) FILE - In this Wednesday, June 5, 2013 file photo, Russian President Vladimir Putin, left, shakes hands with Drug Control Agency Chief Viktor Ivanov at the International Anti-Drug Forum in Moscow, Russia.... (Associated Press) FILE - In this Nov. 24, 2006 file photo, Andrei Lugovoi, left, a former KGB officer, speaks to the media as his associate Dmitry Kovtun listens in Moscow, Russia. British police have accused Kovtun and... (Associated Press) FILE - In this Friday, May 10, 2002 file photo Alexander Litvinenko, former KGB spy is photographed at his home in London. On Thursday Jan 21, 2016, British judge Robert Owen will release the long-awaited... (Associated Press) A taxi stops in front of the Millennium Hotel on Grosvenor Square in London, Thursday, Jan. 21, 2016. British judge Robert Owen is set to release Thursday the findings of a lengthy public inquiry into... (Associated Press) A dog walks in front of the Millennium Hotel, center, on Grosvenor Square in London, Thursday, Jan. 21, 2016. British judge Robert Owen is set to release Thursday the findings of a lengthy public inquiry... (Associated Press) FILE - In this Tuesday, March 12, 2013 file photo, former KGB agent Andrei Lugovoi speaks at a news conference in Moscow, Russia, about the 2006 poisoning in London of former Russian agent turned Kremlin... (Associated Press) Marina Litvinenko, second right in black coat, widow of former Russian spy Alexander Litvinenko, arrives at The Royal Courts of Justice for the Litvinenko Inquiry statement following publication of the... (Associated Press) FILE - In this Nov. 1, 2007 file photo, Andrei Lugovoi, left, a former KGB officer, and his associate Dmitry Kovtun agttend a news conference in Moscow, Russia. British police have accused Kovtun and... (Associated Press) FILE - In this Wednesday, June 5, 2013 file photo, Russian President Vladimir Putin, right, meets with Drug Control Agency Chief Viktor Ivanov at the International Anti-Drug Forum in Moscow, Russia. One... (Associated Press) FILE - In this Nov. 1, 2007 file photo, Andrei Lugovoi, left, a former KGB officer, and his associate Dmitry Kovtun attend a news conference in Moscow, Russia. British police have accused Kovtun and Lugovoi,... (Associated Press) FILE - In this Tuesday, March 12, 2013 file photo, former KGB agent Andrei Lugovoi speaks at a news conference in Moscow, Russia, about the 2006 poisoning in London of former Russian agent turned Kremlin... (Associated Press) In a lengthy report, Judge Robert Owen said that he is certain Litvinenko was given tea with a fatal dose of polonium-210, a radioactive isotope that is deadly if ingested even in tiny quantities, in November 2006. He said there is a "strong probability" that the FSB, successor to the Soviet spy agency the KGB, directed the killing, and the operation was "probably approved" by Putin. Before he died, Litvinenko accused Putin of ordering his killing, but this appears to be the first time anyone has officially linked Putin to it. Moscow has always strongly denied involvement in Litvinenko's death, and Russian Foreign Ministry spokeswoman Maria Zhakarova said Thursday that the government does not consider Owen's conclusions to be objective or impartial. "We regret that a purely criminal case has been politicized and has darkened the general atmosphere of bilateral relations," Zhakarova said in a statement. She said Britain's decision to hold a public inquiry on the case was politically motivated and that the process was not transparent for the Russian side or the public. Russia has refused to extradite the two main suspects, Andrei Lugovoi and Dmitry Kovtun. Lugovoi is a member of the Russian parliament, which means he is immune from prosecution. In an interview with the Interfax news agency, he called the charges against him "absurd." "As we expected, there was no sensation," he said. "The results of the investigation that were announced today once again confirm London's anti-Russian position and the blinkered view and unwillingness of the British to establish the true cause of Litvinenko's death." Litvinenko, a former FSB agent, fled to Britain in 2000 and became a vocal critic of Russia's security service and of Putin, whom he accused of links to organized crime. Owen said Litvinenko "was regarded as having betrayed the FSB" with his actions, and that "there were powerful motives for organizations and individuals within the Russian state to take action against Mr. Litvinenko, including killing him." Litvinenko's widow, Marina, said outside the High Court on Thursday that she was "very pleased that the words my husband spoke on his deathbed when he accused Mr. Putin have been proved by an English court." She called for British Prime Minister David Cameron to take urgent steps against Russian agents operating inside Britain in light of the report. "I'm calling immediately for expulsion from the UK of all Russian intelligence operatives... based at the London embassy," she said. "I'm also calling for the imposition of targeted economic sanctions and travel bans against named individuals including Mr (former FSB chief Nikolai) Patrushev and Mr Putin." She said Britain's Home Office had written to her Wednesday night promising action. Home Secretary Theresa May is scheduled to discuss the report in Parliament later Thursday. In his 326-page report, Owen said that based on the evidence he had seen, the operation to kill Litvinenko was "probably" approved by then-FSB head Nikolai Patrushev, now head of Putin's security council, and by Putin. Owen said Litvinenko "had repeatedly targeted President Putin" with "highly personal" public criticism. The British government appointed Owen to head a public inquiry into the slaying, which soured relations between London and Moscow. He heard from dozens of witnesses during months of public hearings last year, and also saw secret British intelligence evidence. Announcing his findings at London's Royal Courts of Justice, Owen said that "there can be no doubt that Alexander Litvinenko was poisoned by Mr. Lugovoi and Mr. Kovtun" in the Pine Bar of London's luxury Millennium Hotel on Nov. 1, 2006. He died three weeks later of acute radiation syndrome. "I have concluded that there is a strong probability that when Mr. Lugovoi poisoned Mr. Litvinenko, he did so under the direction of the FSB... I have further concluded that the FSB operation to kill Mr. Litvinenko was probably approved by My. Patrushev, then head of the FSB, and also by President Putin." Media playback is unsupported on your device Media caption Why would Vladimir Putin want Alexander Litvinenko dead? The murder of ex-Russian spy Alexander Litvinenko in 2006 in the UK was "probably" approved by President Vladimir Putin, an inquiry has found. Mr Putin is likely to have signed off the poisoning of Mr Litvinenko with polonium-210 in part due to personal "antagonism" between the pair, it said. Home Secretary Theresa May said the murder was a "blatant and unacceptable" breach of international law. But the Russian Foreign Ministry said the public inquiry was "politicised". It said: "We regret that the purely criminal case was politicised and overshadowed the general atmosphere of bilateral relations." What Litvinenko report means for UK Key findings of the public inquiry Who was Alexander Litvinenko? Who are the Litvinenko murder suspects? Story of a perplexing murder Dmitry Peskov, Mr Putin's spokesman, said Moscow's official response to the report will happen through "diplomatic channels", the Russian news agency Interfax was quoted as saying. Prime Minister David Cameron said the UK would have to go on having "some sort of relationship with them [Russia]" because of the Syria crisis, but it would be done with "clear eyes and a very cold heart". Media playback is unsupported on your device Media caption David Cameron said the murder of Alexander Litvinenko had been shown to be "state-sponsored" The long-awaited report into Mr Litvinenko's death found that two Russian men - Andrei Lugovoi and Dmitry Kovtun - deliberately poisoned the 43-year-old in London in 2006 by putting the radioactive substance polonium-210 into his drink at a hotel. Sir Robert Owen, the public inquiry chairman, said he was "sure" Mr Litvinenko's murder had been carried out by the two men and that they were probably acting under the direction of Moscow's FSB intelligence service, and approved by the organisation's chief, Nikolai Patrushev, as well as the Russian president. He said Mr Litvinenko's work for British intelligence agencies, his criticism of the FSB and Mr Putin, and his association with other Russian dissidents were possible motives for his killing. 'Send a message' There was also "undoubtedly a personal dimension to the antagonism" between Mr Putin and Mr Litvinenko, he said. The use of polonium-210 was "at the very least a strong indicator of state involvement" as it had to be made in a nuclear reactor, the report said. The inquiry heard evidence that Mr Litvinenko may have been consigned to a slow death from radiation to "send a message". What is polonium-210? Image caption The teapot where traces of polonium-210 were discovered Giving a statement to the House of Commons, Mrs May said Mr Cameron would raise the findings with President Putin at "the next available opportunity". She said the UK would impose asset freezes on Mr Lugovoi and Mr Kovtun and that international arrest warrants for the pair remained in place. They both deny killing Mr Litvinenko. Both men are wanted in the UK for questioning, but Russia has refused to extradite them. Media playback is unsupported on your device Media caption Marina Litvinenko: 'Russian spies must be expelled from UK' Speaking earlier outside the High Court, Mr Litvinenko's widow, Marina, said she was "very happy" that "the words my husband spoke on his deathbed when he accused Mr Putin have been proved by an English court". She urged the UK government to expel all Russian intelligence operatives, impose economic sanctions on Moscow and impose a travel ban on Mr Putin. The view from Moscow Image copyright AP Image caption Andrei Lugovoi, left, and Dmitry Kovtun pictured in Moscow, in 2007 By the BBC's Oleg Boldyrev For years Moscow rejected allegations of high-level involvement in the murder of Alexander Litvinenko. The fact President Putin himself is now associated with this assassination has not changed anything. Taking their lead from Robert Owen's use of the words "high probability", the second tier of the Russian establishment, mainly Kremlin-loyalist MPs, are dismissing the entire report as a politically-based fabrication. Russians on social media are making fun of its conclusions by using the hashtag "PutinPossiblyApproved" in Russian - that is #ПутинВозможноОдобрил - to include all manner of crimes. One Russian MP, Nikolai Kovalev, himself an ex-FSB boss, pointed out relations between Moscow and London would not be harmed by the report as there was no room for making them any worse. How Russian media reported the Litvinenko inquiry Responding to the report, Mr Lugovoi, who is now a politician in Russia, said the accusations against him were "absurd", the Russian news agency Interfax was quoted as saying. "As we expected, there were no surprises," he said. "The results of the investigation made public today yet again confirm London's anti-Russian position, its blinkeredness and the unwillingness of the English to establish the true reason of Litvinenko's death." Mr Kovtun, now a businessman in Russia, said he would not comment on the report until he got more information about its contents, Interfax reported. 'Harm relations' London's Metropolitan Police said the investigation into the "cold and calculated murder" remained ongoing. Alexander Yakovenko, the Russian ambassador in the UK, said Russia would not accept any decisions reached in secret and based on evidence not tested in open court. The length of time taken to come to these conclusions led them to believe it was "a whitewash of British security services' incompetence", he said. Mr Yakovenko said these events "can't help but harm our bilateral relations". White House spokesman Josh Earnest said he did not have "any actions" to announce following the inquiry's findings. "But I certainly wouldn't rule out future steps," he said. Analysis Image copyright Reuters Image caption Alexander Litvinenko at a news conference in Moscow in 1998, when he was an officer of Russia's state security service FSB By BBC security correspondent, Gordon Corera The conclusions of this inquiry are stronger than many expected in pointing the finger at Vladimir Putin personally. The evidence behind that seems to have come from secret intelligence heard in closed session. Saying that Alexander Litvinenko was killed because he was an enemy of the Russian state will raise pressure on the British government to take real action - the steps taken nearly a decade ago were only limited in scope. That may pose difficulties given the importance of Russia's role in the Middle East, but without tough action people may ask if the Russian government has been allowed to get away with what has been described as an act of nuclear terrorism on the streets of London. Mr Litvinenko fled to the UK in 2000, claiming persecution. He was granted asylum and gained British citizenship several years later. In the years before his death, he worked as a writer and journalist, becoming a strong critic of the Kremlin. Image copyright Reuters Image caption Russian President Vladimir Putin "probably" approved the killing, the report says It is believed he also worked as a consultant for MI6, specialising in Russian organised crime. The inquiry heard from 62 witnesses in six months of hearings and was shown secret intelligence evidence about Mr Litvinenko and his links with British intelligence agencies. The Litvinenko case Media playback is unsupported on your device Media caption The son of murdered Russian spy Alexander Litvinenko gives his first television interview 23 November 2006 - Mr Litvinenko dies three weeks after having tea with former agents Andrei Lugovoi and Dmitri Kovtun in London 22 May 2007 - Britain's director of public prosecutions decides Mr Lugovoi should be charged with his murder 5 July 2007 - Russia refuses to extradite Mr Lugovoi, saying its constitution does not allow it May-July 2013 - The inquest into Mr Litvinenko's death is delayed as the coroner decides a public inquiry would be preferable - but ministers rule out the request 11 February 2014 - High Court rules the Home Office was wrong to rule out an inquiry before the outcome of an inquest January 2015 - Public inquiry begins Long road to the truth for Litvinenko family Report Sir Robert Owen, the Inquiry Chairman, published his Report into the death of Alexander Litvinenko on 21 January 2016. Please click on the link below to view the Report. Report (web-optimised PDF) An explanation for the way references are provided is included in paragraph 2.21 in Part 2 of the Report. Where references are to documents, the hyperlink will take you to the complete document. Where references are to transcripts, the hyperlink will take you to the relevant pages. Report cover (print-ready PDF) Report (print-ready PDF) The transcript of Sir Robert Owen’s closing statement is available at the link below. Transcript of statement by the Chairman – 21 January 2016 | A British judge declared a remarkable thing Thursday: Vladimir Putin is "probably" a murderer. The finding came after an inquiry into the high-profile death of former KGB officer Alexander Litvinenko, who was fatally poisoned in 2006. Judge Robert Owen said he's certain that two Russian agents laced Litvinenko's green tea with polonium-210 inside a London hotel-and that the mission "was probably approved" by Putin himself and the head of Russia's FSB spy agency (the successor to the KGB), reports the New York Times. Litvinenko accused Putin from his deathbed, but this appears to be the first time an official inquiry has linked the Russian president to the slaying, notes the AP. Russia, not surprisingly, isn't buying it. "We regret that a purely criminal case has been politicized and has darkened the general atmosphere of bilateral relations," says a rep for the Russian foreign ministry. Britain launched the inquiry at the urging of Litvinenko's widow, who welcomed the findings and called on the UK to issue sanctions on Russia and place a travel ban on Putin, reports the BBC. That seems unlikely, though Britain says it will now freeze the assets of Andrei Lugovoi and Dmitry Kovtun, the two men accused by British police of the killing. Moscow refuses to extradite them. The judge cited a number of possible motives, including Russia's belief that Litvinenko betrayed Moscow by working with British intelligence. He also cited the "highly personal attacks" by Litvinenko on Putin before the agent's death, including Litvinenko's clam that Putin was a pedophile, reports the Independent. "It hardly needs saying that the allegations made by Mr Litvinenko against President Putin in this article were of the most serious nature," wrote the judge. "Could they have had any connection with his death?" Read his full report here. | multi_news |
The large conductance, voltage- and calcium-dependent potassium (BK) channel serves as a major negative feedback regulator of calcium-mediated physiological processes and has been implicated in muscle dysfunction and neurological disorders. In addition to membrane depolarization, activation of the BK channel requires a rise in cytosolic calcium. Localization of the BK channel near calcium channels is therefore critical for its function. In a genetic screen designed to isolate novel regulators of the Caenorhabditis elegans BK channel, SLO-1, we identified ctn-1, which encodes an α-catulin homologue with homology to the cytoskeletal proteins α-catenin and vinculin. ctn-1 mutants resemble slo-1 loss-of-function mutants, as well as mutants with a compromised dystrophin complex. We determined that CTN-1 uses two distinct mechanisms to localize SLO-1 in muscles and neurons. In muscles, CTN-1 utilizes the dystrophin complex to localize SLO-1 channels near L-type calcium channels. In neurons, CTN-1 is involved in localizing SLO-1 to a specific domain independent of the dystrophin complex. Our results demonstrate that CTN-1 ensures the localization of SLO-1 within calcium nanodomains, thereby playing a crucial role in muscles and neurons. Precise control of membrane excitability, largely determined by ion channels, is of utmost importance for neuronal and muscle function. The regulation of ion channel localization, density and gating properties thus provides an effective way to control the excitability within these cells [1]. Indeed, the localization and gating properties of ion channels are often regulated or modified by cytoskeletal and signaling proteins, or auxiliary ion channel subunits expressed in a cell-type specific manner [2]. Potassium channels are critical in determining the excitability of cells, because potassium ions are dominant charge carriers at the cell resting potential. Among potassium channels, the large conductance, voltage- and calcium-dependent potassium BK channels (also called SLO-1 or Maxi-K) are uniquely gated by coincident calcium signaling and membrane depolarization [3], [4]. This feature of BK channels provides a crucial negative feedback mechanism for calcium-induced functions, and plays an important role in determining the duration of action potentials [3]. BK channels are widely expressed in a variety of cell types and are implicated in many physiological processes, including the regulation of blood pressure [5], neuroendocrine signaling [6], smooth muscle tone [7], and neural network excitability [8], [9]. Mounting evidence indicates that BK channels can interact with a variety of proteins that modulate channel function, or control membrane trafficking. For example, the Drosophila BK channel, dSLO, interacts with SLO binding protein (slob), which in turn modulates the channel gating properties [10]. Similarly, mammalian BK channels associate with auxiliary beta subunits that influence channel activation time course and voltage-dependence [11]. In yeast two hybrid screens, the cytoplasmic C-terminal tail of mammalian BK channels has been shown to interact with several proteins, including cytoskeletal elements, such as actin-binding proteins [12], [13] and a microtubule-associated protein [14]. These cytoskeletal proteins are partially co-localized with BK channels, and appear to increase cell surface expression of BK channels in cultured cells [12], [13]. However, it remains to be determined whether these proteins have any role in controlling the localization of BK channels to specific areas of the plasma membrane in vivo. Robust activation of BK channels requires higher intracellular calcium concentrations (>10 µM), which only occur in the immediate vicinity of calcium-permeable channels [4]. Hence, the localization of BK channels to specific areas (i. e. calcium nanodomains) where calcium-permeable ion channels are located is physiologically important for BK channel activation. In C. elegans, loss-of-function mutations in slo-1 partially compensate for the synaptic release defects of C. elegans syntaxin (unc-64) mutants [15] and lead to altered alcohol sensitivity [16]. Recent studies in C. elegans have also implicated SLO-1 in muscle function [17]. slo-1 mutants display an exaggerated anterior body angle, referred to as the head-bending phenotype that is shared by mutants that are defective in the C. elegans dystrophin complex [18]–[20]. Recent evidence that the C. elegans dystrophin complex interacts with SLO-1 channels via SLO-1 interacting protein, ISLO-1, explains this phenotypic overlap [21]. However, C. elegans dystrophin complex mutants do not appear to alter the biophysical properties of BK channels per se [17]. Similarly, ISLO-1 does not modify SLO-1 channel properties [21]. Rather, ISLO-1 tethers SLO-1 near the dense bodies of muscle membranes, where L-type calcium channels (EGL-19) are localized [21]. Consequently, defects in the dystrophin complex or ISLO-1 cause a large reduction in SLO-1 protein levels in muscle membrane, which in turn causes muscle hyper-excitability leading to enhanced intracellular calcium levels. This perturbation of calcium homeostasis has been postulated to be one of the first steps in the degenerative muscle pathogenesis associated with disruption of the dystrophin complex [22]. In this study, we performed a forward genetic screen to identify additional genes responsible for SLO-1 localization and function in C. elegans. We identified ctn-1, an orthologue of α-catulin, as a novel gene that controls SLO-1 localization and function in muscles and neurons. Our analysis showed that ctn-1 uses different strategies to localize SLO-1 in these two cell types. In muscles, CTN-1 utilizes the dystrophin complex to localize SLO-1 near L-type calcium channels via ISLO-1. In neurons, CTN-1 localizes SLO-1 independent of the dystrophin complex. Loss-of-function slo-1 mutants exhibit a jerky locomotion and head bending phenotype [15]. By contrast, gain-of-function slo-1 mutants exhibit sluggish movement combined with low muscle tone [16]. When slo-1 (gf) mutant animals are mechanically stimulated, they fail to make a normal forward movement, and tend to curl ventrally (Video S1). To identify genes that regulate slo-1 function, we performed a forward genetic screen to isolate mutants that suppress the phenotypes of the slo-1 (ky399) gain-of-function mutant. Based on a previous genetic study [21], suppressor genes were expected to encode slo-1, components of the dystrophin complex, as well as novel proteins that control neuronal or muscular function of SLO-1. As expected, several loss-of-function alleles of slo-1 were isolated. In addition to these intragenic suppressors, several mutants could be segregated away from slo-1 (gf) (Figure 1A and Video S1) and exhibited the head bending phenotype. Genetic mapping and complementation testing determined that these extragenic suppressors include dyb-1 and stn-1 which encode two homologous components of the dystrophin complex, dystrobrevin and syntrophin respectively. Additionally we isolated cim6 and eg1167 suppressors that represent novel genes. Compared to slo-1 (ky399) and cim6; slo-1 (ky399) mutants, eg1167; slo-1 (ky399) mutants exhibited a profound improvement in the locomotion speed (Figure 1A). It was previously observed that slo-1 (gf) mutants retain significantly more eggs than wild-type animals due to low activity of the egg-laying muscles [16]. We found that suppressor mutants abolish an egg laying defect of slo-1 (gf) mutants and retain eggs in uteri at levels similar to wild-type animals (Figure 1B). To understand the role of novel genes in slo-1 function, we pursued the identification of genes that mapped to chromosomal locations neither previously implicated in BK channel function, nor encoding known components of the dystrophin complex. Two mutations, cim6 and eg1167, both mapped to the left side of chromosome I and failed to complement each other for head bending, suggesting that these two mutations represent alleles of the same gene. Our quantitative analysis for locomotion and egg laying phenotypes showed that the locomotion speed of eg1167; slo-1 (gf) was higher than that of cim6; slo-1 (gf) whereas egg laying was comparable in both strains (Figure 1A and 1B). We further mapped eg1167 to a 250 kb interval and rescued the phenotype of eg1167 by generating transgenic animals with the fosmid WRM0621cC01 (Figure S1). Next, we rescued the head bending phenotype of eg1167 with a transgene consisting of the ctn-1 gene (Y23H5A. 5) and approximately 4 kb upstream of the translation initiation codon (Figure 2A and 2B). The same transgene caused eg1167; slo-1 (gf) double mutants to revert to the slo-1 (gf) phenotype, displaying sluggish movement and retention of late-staged eggs in uteri (Figure 2C and 2D). These results indicate that a genetic defect in ctn-1 is responsible for suppression of the slo-1 (gf) phenotypes. The ctn-1 gene is orthologous to mammalian α-catulin (39. 4% identity to human α-catulin), and is named on the basis of sequence similarity to both α-catenin and vinculin (Figure 2A) [23]. Vinculin and α-catenin are membrane-associated cytoskeletal proteins found in focal adhesion plaques and cadherens junctions. In C. elegans, vinculin (DEB-1) is localized to the dense bodies of body wall muscle and is essential for attachment of actin thin filaments to the sarcolemma [24], whereas α-catenin (HMP-1) is localized to hypodermal adherens junctions and is essential for proper enclosure and elongation of the embryo [25]. Based on its homology to vinculin/α-catenin and the localization of mammalian α-catulin [26], CTN-1 is likely to interact with other cytoskeletal proteins, which may in turn affect SLO-1 function. Additionally, the ctn-1 gene encodes a predicted coiled-coil domain. Such a coiled-coil domain mediates the interaction between dystrophin and dystrobrevin [27], two components of the dystrophin complex, although we do not know if the coiled-coil domain of CTN-1 is important for the interaction with these proteins (Figure 2A). We determined nucleotide sequence of the predicted exons and exon-intron boundaries of the ctn-1 gene in eg1167 and cim6. The mutation sites found in both alleles create translation-termination codons (R144>STOP in eg1167, Q521>STOP in cim6) (Figure 2A). eg1167 exhibits complete suppression of slo-1 (gf) phenotypes (see below) and is hence considered as a severe loss-of-function or null allele. All subsequent experiments were carried out with eg1167, unless mentioned otherwise. Although both eg1167 and cim6 mutants alone exhibit the head-bending phenotype, they differ with respect to suppression of slo-1 (gf) phenotypes. Whereas ctn-1 (eg1167) suppresses all aspects of the slo-1 (gf) phenotype, ctn-1 (cim6) completely suppresses the egg-laying defect of slo-1 (gf) (Figure 1B), but not the locomotory defect (Figure 1A). These results suggest that the C-terminal third of CTN-1 is required for normal egg laying and head bending, but is not necessary to mediate the locomotion speed defect of slo-1 (gf) mutants. To elucidate the function of CTN-1, we examined the expression pattern of the ctn-1 gene using a ctn-1 promoter-tagged GFP reporter (Figure 2E–2H). We observed GFP fluorescence in body wall muscles, pharyngeal muscle, egg-laying muscle and enteric muscle of transgenic animals as well as in most, if not all, neurons of the nerve ring and ventral nerve cord. Based on the ctn-1 expression pattern and the phenotypic differences between eg1167 and cim6, we investigated whether the head-bending phenotype and the suppression of sluggish movement of slo-1 (gf) mutants are separable by expressing ctn-1 minigenes under the control of either muscle- or neuron-specific promoters in ctn-1 and ctn-1; slo-1 (gf) mutant animals. Muscle, but not neuronal, expression of ctn-1 rescued the head-bending phenotype of the ctn-1 mutant (Figure 2B and Figure S1C). These results are consistent with previous reports that the head-bending phenotype is due to perturbations in muscle function [17]–[19]. Furthermore, muscle expression of ctn-1 in ctn-1; slo-1 (gf) mutants resulted in egg retention to the level observed in slo-1 (gf) mutants, whereas neuronal expression of ctn-1 did not alter the number of eggs retained in the uteri of ctn-1; slo- (gf) mutants (Figure 2C). Conversely, neuronal expression of ctn-1 in ctn-1; slo-1 (gf) mutants reverted the seemingly normal locomotion of ctn-1; slo-1 (gf) to the sluggish, uncoordinated locomotion of the slo-1 (gf) mutant, whereas muscle expression of ctn-1 did not (Figure 2D). These results indicate that the sluggish, uncoordinated locomotory phenotype of slo-1 (gf) mutants comes from presynaptic depression, but not from direct suppression of muscle excitability. Together with the allele specific phenotypic differences indicating different regions of CTN-1 are required for normal locomotory speed and head bending, these results suggest that CTN-1 uses two distinct mechanisms for mediating SLO-1 function in muscle and neurons by interacting with different sets of genes. Most, if not all, of the mutants that exhibit the head bending phenotype have a defect in either a component of the dystrophin complex or proteins that interact with the dystrophin complex [17]–[19]. The dystrophin complex is localized near muscle dense bodies [21]. Because ctn-1 mutants exhibit the head bending phenotype, we determined the subcellular localization of CTN-1 using a GFP-tagged CTN-1 transgene, which rescues the head bending phenotype (data not shown). GFP: : CTN-1 exhibited a punctate expression pattern that resembled that of the dense bodies (Figure 3A). To further define the localization of CTN-1, we stained GFP-tagged CTN-1 transgenic animals with GFP antibodies and vinculin/DEB-1 antibodies that recognize the attachment plaque and dense bodies. CTN-1: : GFP is localized in close proximity to, or partially colocalized with, vinculin/DEB-1 in dense bodies, but not in the attachment plaques, indicating that CTN-1 is localized near dense bodies (Figure 3A). This expression pattern of CTN-1, along with the head bending phenotype of ctn-1 mutants, prompted us to examine whether the ctn-1 mutation disrupts the integrity of the dystrophin complex. We compared the expression pattern of a component of the dystrophin complex, SGCA-1 (an α-sarcoglycan homolog) in wild-type, dys-1, slo-1 and ctn-1 animals using a GFP-tagged SGCA-1 that rescues the head bending phenotype of sgca-1 mutants [21] (Figure 3B). GFP: : SGCA-1 exhibited a punctate expression pattern in the muscle membrane of wild-type and slo-1 mutant animals. By contrast, GFP puncta were greatly diminished in dys-1 and ctn-1 mutants. These results indicate that ctn-1 is critical for maintaining the dystrophin complex near the dense bodies. We previously demonstrated that ISLO-1 interacts with STN-1 through a PDZ domain-mediated interaction, thereby linking SLO-1 to the dystrophin complex [21]. Because we failed to observe a component of the dystrophin complex in the muscle membrane of ctn-1 mutants, we examined mCherry-tagged ISLO-1 in the muscle membrane of wild-type and ctn-1 mutant animals. The punctate mCherry: : ISLO-1 fluorescence was observed in wild-type muscle membranes, but was greatly reduced in ctn-1 mutant (Figure 3C). These results further strengthen the notion that CTN-1 is required for maintaining the integrity of the dystrophin complex. Based on the genetic interaction between ctn-1 and slo-1, and the observation that the integrity of the dystrophin complex and ISLO-1 localization are disrupted in ctn-1 mutants, we hypothesized that CTN-1 regulates the localization of SLO-1 in muscle. To test this hypothesis, we examined the localization of GFP-tagged SLO-1 in muscles of wild-type, dys-1, and ctn-1 animals (Figure 4A and 4B). The punctate SLO-1: : GFP expression pattern in the muscle membrane of wild-type animals was greatly diminished in the muscles of either dys-1 or ctn-1 mutant. Interestingly, the protein levels of SLO-1: : GFP were not significantly different in wild-type, dys-1 and ctn-1 animals (Figure S2B), indicating that mislocalized SLO-1 does not necessarily undergo degradation. The mislocalization of SLO-1 in dys-1 mutants is consistent with the requirement of the dystrophin complex for ISLO-1 localization [21]. These results further indicate that CTN-1 stabilizes or maintains the punctate muscle expression of SLO-1: : GFP in a dystrophin complex-dependent manner. In mammals, BK channels are found in neuronal somata, dendrites and presynaptic terminals [28], [29]. An immunoelectron microscopy study indicates that BK channels are not homogeneously distributed in neurons, but are clustered, presumably near calcium channels [30]. We addressed whether SLO-1 is evenly distributed or clustered in C. elegans neurons by examining SLO-1: : GFP. Wild-type animals displayed patches of fluorescence along the ventral nerve cord or near cell bodies under high magnification (Figure 4C and 4D, Figure S2). Tissue-specific rescue experiments demonstrated that ctn-1 mediates SLO-1 function in neurons independent of the dystrophin complex (Figure 2D). Therefore, we compared neuronal SLO-1: : GFP expression in dys-1 and ctn-1 mutant animals. The clustered GFP expression observed along the ventral cord of both wild-type and dys-1 mutant animals contrasted with the uniform GFP localization in ctn-1 mutants (Figure 4C and 4D). These results indicate that ctn-1 mutation disrupts the neuron-specific clustering of SLO-1: : GFP independent of the dystrophin complex. SLO-1 contributes to the repolarization of the synaptic terminal following neuronal stimulation, thereby terminating neurotransmitter release. Consequently loss-of-function slo-1 mutants are hypersensitive to the paralyzing effects of aldicarb, an acetylcholinesterase inhibitor, a phenotype indicative of enhanced acetylcholine release. Consistent with this interpretation, electrophysiological recordings from neuromuscular junctions of slo-1 loss-of-function mutants exhibit prolonged evoked synaptic responses [15], [16]. If CTN-1 regulates SLO-1 localization in motor neurons and thus slo-1 function, we would expect ctn-1 mutants to exhibit similar pharmacological and synaptic changes. Indeed, we found that ctn-1 mutants were hypersensitive to aldicarb compared to wild-type animals (Figure S3). To confirm this observation directly, we measured synaptic responses from the neuromuscular junctions of dissected wild-type and ctn-1 mutant animals engineered to express channelrhodopsin-2 in motor neurons [31] (Figure 5). Evoked synaptic responses were elicited by blue light activation of channelrhodopsin-2 and recorded from voltage-clamped post-synaptic body wall muscle cells. Consistent with our pharmacological data and localization results, recordings from ctn-1 showed prolonged evoked synaptic responses similar to those of slo-1 (lf) mutants (Figure 5B and 5C). Furthermore, muscular expression of ctn-1 in ctn-1 mutant animals rescued the head-bending phenotype (Figure 2B), but did not rescue prolonged evoked synaptic responses (Figure S3B). These data strongly suggest that altered synaptic responses of ctn-1 mutants result from a neuronal defect. In contrast to the slo-1 (lf) mutants, evoked responses of slo-1 (gf) mutants were short-lived (Figure 5C), and the charge integral, a measure of total ion flux during the evoked response, was significantly reduced (Figure 5E). Our genetic analyses demonstrated that the ctn-1 mutation suppresses the sluggish locomotory phenotype of slo-1 (gf) mutants and disrupts SLO-1 localization (Figure 1A, Figure 4C and 4D). If this is due to loss of neuronal SLO-1 (gf) channels, the ctn-1 mutation should suppress the evoked response defects of slo-1 (gf). Consistent with this prediction, the decay time of the ctn-1; slo-1 (gf) double mutants (t1/2 = 6. 61±0. 53 ms) was significantly longer than slo-1 (gf) (t1/2 = 3. 23±0. 21 ms) (Figure 5D), and the charge integral was restored to wild-type levels (Figure 5E). Interestingly, ctn-1 mutants did not convert the decay time of slo-1 (gf) evoke responses to that of slo-1 (lf), indicating that residual SLO-1 function may be mediated by dispersed SLO-1 channels. In a genetic screen to identify novel regulators of SLO-1, we found two alleles of ctn-1, a gene which encodes an α-catulin orthologue. CTN-1 mediates normal bending of the anterior body through SLO-1 localization near the dense bodies of body wall muscles. CTN-1 also maintains normal locomotory speed through SLO-1 localization within neurons. Based on our data, we propose a model for ctn-1 function in localizing SLO-1 (Figure 6). In muscles, CTN-1 interacts with the dystrophin complex. It is also possible that CTN-1 may influence the stability of another protein that directly interacts with the dystrophin complex. Loss of CTN-1 function disrupts the integrity of the dystrophin complex, thus compromising ISLO-1 and SLO-1 localization near muscle dense bodies, where L-type calcium channels are present. Disruption of SLO-1 localization is expected to uncouple local calcium increases from SLO-1-dependent outward-rectifying currents, resulting in muscle hyper-excitation. Previous studies have shown that the head bending phenotype, shared among mutants that have a defect in the dystrophin complex or its associated proteins, results from muscle hyperexcitability [17]–[19], [32]. Our data further show that this head-bending phenotype does not result from a synaptic transmission defect, but from a muscle excitation and contraction defect. In neurons, SLO-1 localization is not mediated through the dystrophin complex, suggesting that CTN-1 interacts with other proteins to localize SLO-1 to specific neuronal domains. Why does CTN-1 use two distinct mechanisms to localize SLO-1 to subcellular regions of muscles and neurons? BK channels are functionally coupled with several different calcium channels (including voltage-gated L-type and P/Q-type calcium channels and IP3 receptors) that are localized in different subcellular regions [30], [33], [34]. Although it has not been determined whether all of these calcium channels are functionally coupled with SLO-1 in C. elegans, these calcium channels are distributed in different regions of neurons. For example, the L-type calcium channel (EGL-19) is mainly expressed in the cell body and the P/Q type calcium channel (UNC-2) is concentrated at the presynaptic terminals [35], [36]. A distinct set of proteins is perhaps required for SLO-1 channel localization near different calcium channels. How CTN-1 interacts with the dystrophin complex in muscle remains to be determined. It has been suggested that mammalian α-catulin interacts with the hydrophobic C-terminus of dystrophin resulting from alternative splicing [37]. However, the C. elegans dys-1 gene does not encode a hydrophobic C-terminus. Thus, CTN-1 may interact with a different domain of dystrophin, or with another component of the dystrophin complex. In this regard, it is noteworthy that both mammalian dystrophin and C. elegans DYS-1 have multiple spectrin repeat domains, and that the N-terminal region of vinculin which exhibits homology to that of α-catulin (Figure S1) is known to bind the spectrin repeat domain of α-actinin [38]. By extension, we speculate that the N-terminal region of CTN-1 may bind the spectrin repeat domain of DYS-1 directly. Alternatively, the coiled-coil domain of dystrophin, which is known to interact with the coiled-coil domain of dystrobrevin [27], may potentially bind the coiled-coil domain of CTN-1. Interestingly, CTN-1 exhibits high homology to vinculin in both the N-terminal and C-terminal regions (Figure S1B). The C-terminal region of vinculin interacts with cytoskeletal molecules or regulators (F-actin, inositol phospholipids and paxillin) in focal adhesion and adherens junctions [39]. Because the C-terminal region of CTN-1 is also necessary for normal head bending, we speculate that this C-terminal region may be important for tethering the dystrophin complex to other cytoskeletal proteins. In mammalian striated muscle, dystrophin is enriched in costameres [40] which are analogous to C. elegans dense bodies. A costamere is a subsarcolemmal protein assembly that connects Z-disks to the sarcolemma, and is considered to be a muscle-specific elaboration of the focal adhesion in which integrin and vinculin are abundant. Compromised costameres have been postulated to be an underlying cause of several different myopathies [40]. It was recently shown that ankyrin-B and -G recruit the dystrophin complex to costameres [41]. Based on overall high homology of ctn-1 to vinculin and α-catenin, we speculate that CTN-1 similarly interacts with cytoskeletal proteins in the dense bodies, and links the dystrophin complex to the dense bodies. Another intriguing conclusion from our data is that loss of CTN-1 does not completely abolish SLO-1 function. Complete abolishment of SLO-1 function in ctn-1 mutant should alter the decay time for evoked synaptic responses of ctn-1; slo-1 (gf) to the same degree as slo-1 (lf) mutants, rather than to that of wild-type animals (Figure 5D). Mutants including slo-1 (gf), that have defects in neural activation or membrane depolarization, are reported to cause str-2, a candidate odorant receptor gene, to be expressed in both AWC olfactory neurons whereas wild-type animals express str-2 in only one of the AWC pair [42]. We find that ctn-1 mutation does not suppress the misexpression of str-2 in both AWC neurons in slo-1 (gf) mutants, suggesting that ctn-1 mutations do not completely abolish SLO-1 function (unpublished observations, HK). It is thus likely that the defect in SLO-1 localization in ctn-1 mutants makes it less responsive to local calcium nanodomains found at presynaptic terminals and dense bodies, but still able to respond to depolarization-induced global calcium increases, albeit at a lower level. In conclusion, we have identified ctn-1, a gene encoding the C. elegans homolog of α-catulin, and demonstrated that CTN-1 mediates SLO-1 localization in muscles and neurons by dystrophin complex-dependent and -independent mechanisms, respectively. How SLO-1 is localized to certain neuronal domains will require further screening of slo-1 (gf) suppressor mutants. Given that proteins affecting components of the dystrophin complex are likely to contribute to the pathogenesis of muscular dystrophy, α-catulin is a candidate causal gene for a form of muscular dystrophy in humans. The genotypes of animals used in this study are: N2 (wild-type), CB4856, dys-1 (eg33) I, stn-1 (tm795) I, ctn-1 (eg1167) I, ctn-1 (cim6) I, slo-1 (eg142) V, slo-1 (ky399gf) V and sgca-1 (tm1232) X. The following transgenes were used in this study: cimIs1[slo-1a: : GFP, rol-6 (d) ] [21], cimIs5[mCherry: : islo-1, ofm-1: : GFP] [21], zxIs6[unc-17: : chop-2 (H134R) -yfp; lin-15 (+) ] [31], cimIs6[GFP: : sgca-1, rol-6 (d) ], cimEx5[ctn-1, ofm-1: : GFP], cimIs7[GFP: : ctn-1, rol-6 (d) ], cimEx6[Pmyo-3ctn-1, Pmyo-3GFP, ofm-1: : GFP] and cimEx7[PH20ctn-1, PH20GFP, ofm-1: : GFP]. Gain-of-function slo-1 (ky399) mutants were mutagenized by exposure to 50 mM EMS (ethane methyl sulfonate) for 4 h [43]. Suppressors that suppress or ameliorate the sluggish locomotory phenotype of slo-1 (ky399gf) mutants were selected from F2 progeny of the mutagenized animals. We screened approximately 5,000 haploid genome size for suppressor mutants and identified a total of 17 suppressor mutants. Genetic analysis of these suppressor mutants indicates that three of these have a second mutation in the slo-1 gene. In addition, we found that eight have mutations in genes causing head-bending phenotype (2 alleles of dyb-1,3 alleles of stn-1 and 2 alleles of ctn-1). The remaining six mutants do not exhibit distinct locomotory phenotypes when segregated from slo-1 (gf). For genetic mapping, slo-1 (ky399) mutants were outcrossed 12 times to the CB4856 strain. The resulting strain was used for SNP (single nucleotide polymorphism) mapping [44]. Alternatively, we used CB4856 as a mapping strain when mapping is based on the head-bending phenotype. For transgenic rescue, fosmid clones purchased from Gene services Inc. (Cambridge, UK) were injected into the gonad of ctn-1 mutant at 2 ng/µl along with ofm-1: : GFP marker (30 ng/µl). Once we rescued the head bending phenotype of ctn-1 with a single fosmid, we rescued ctn-1 mutant with a genomic DNA fragment encompassing the entire coding sequence of ctn-1 and approximately 4 kb upstream of the putative translation site. To verify the predicted coding sequence of ctn-1, we first performed BLAST search analysis using the genomic sequences of C. briggsae and C. remanei. This analysis suggested that the first and 12th exons are longer than predicted in WormBase (WS208), and that an additional exon (10th exon) is present. Second, we sequenced C. elegans ORF ctn-1 clone (9349620) and confirmed the 10th and the 12th exon sequences. Third, we performed sequence analysis of the DNA fragment obtained from RT-PCR with a primer set (SL1 and an internal primer) and identified the trans-splicing site which is 29 bp upstream of the newly-defined translation initiation site. Our analysis indicates that ctn-1 encodes a predicted protein with 784 amino acids (Figure S1B). The ctn-1 genomic DNA (approximately 4 kb upstream of the promoter and the entire coding sequence) was amplified by the expand long template PCR system (Roche Applied Science) and used directly for rescue. For PH20ctn-1 and Pmyo-3ctn-1 constructs, the neuron-specific H20 [45] or muscle-specific myo-3 promoter sequences were fused to the translation initiation site of the ctn-1 genomic DNA in frame by the overlapping extension PCR (Roche). For localization of CTN-1, we inserted the GFP sequence to the translation initiation site of ctn-1 cDNA, and then the ctn-1 promoter sequence was inserted before the GFP sequence. The resulting construct rescued the head-bending phenotype of ctn-1 mutants and was used for generating integrated transgenic animals. For GFP: : sgca-1 construct, the GFP sequence was inserted in-frame right after the signal sequence of sgca-1 open-reading frame as described previously [21]. Transgenic strains were made as described [46] by injecting DNA constructs (2–10 ng/µl) along with a co-injection marker DNA (pRF4 (rol-6 (d) ) or ofm-1: : GFP) into the gonad of hermaphrodite animals at 100 ng/µl. We obtained at least 3 independent transgenic lines for rescue, and found that all lines show similar results. To remove bacteria attached to animals, approximately fifteen age-matched (30 hr after L4 stage) hermaphrodite animals for each genotype were placed on a NGM (nematode growth medium) agar plate without bacteria for 15 min. The animals were then placed inside one of two copper rings embedded in a NGM plate. We found that age of agar plate influences the speed of animals, probably because the surface tension resulting from the liquid surrounding animals slows down movement. We used approximately one week-old plates for our assay, and compared with the speed of wild-type control animals. Video frames from two different genotypes were simultaneously acquired with a dissecting microscope equipped with Go-3 digital camera (QImaging) for 2 min with a 500 ms interval and 20 ms exposure. We measured the average speed of animals by using Track Objects from ImagePro Plus (Media Cybernetics). The activity of egg laying muscle was measured indirectly by counting eggs retained in uteri. Single age-matched (30 hrs post-L4) animals (total 15 for each genotype) were placed in each well of a 96 well plate that contains 1% alkaline hypochlorite solution. The eggshells protect embryos from dissolution by alkaline hypochlorite. After 15 min incubation, the remaining eggs were counted in each well. Body curvature analysis was previously described [47]. A single animal was transferred to an agar plate and its movement was recorded at 20 frames per second. We limited image acquisition within 15 to 60 seconds after transfer, because the head bending phenotype is prominent when animals are stimulated to move forward rapidly. A custom-written software automatically recognizes the animal and assigns thirteen points spaced equally from the tip of nose to the tail along the midline of the body, and produces the pixel coordinates of thirteen points. First supplementary angles were calculated from the coordinates of the first three points with MATLAB software. First angle data were obtained when the head swing of an animal reached the maximal extension to the dorsoventral side. Mixed stage worms were washed and collected in M9 buffer. Equal volume of 2× Laemmli sample buffer was added to the worm pellets. The resulting worm suspension was heated at 90 °C for 10 min, centrifuged at 20,000 g for 10 min, and then immediately loaded on 7. 5% SDS-PAGE gel. The Western blot analysis was performed using anti-GFP antibody (Clontech, JL-8) and anti-α-tubulin antibody (Developmental hybridoma bank, AA4. 3). Fixation and immunostaining procedures are previously described [21]. Fluorescence images were observed under a Zeiss Axio Observer microscope with 40× objective (water-immersion, NA: 1. 2) or an Olympus Fluoview 300 confocal microscope with a 60× objective (oil-immersion, NA: 1. 4) or 100× objective (oil-immersion, NA: 1. 4). We typically observed more than 50 animals for each genotype. Images for quantification were acquired under an identical exposure time, gains and pinhole diameter. The intensity of puncta from acquired images was analyzed using linescan (Metamorph, Molecular Devices) and presented as values obtained by subtracting background levels from the peak grey levels of puncta. Electrophysiological methods were as previously described [48]. Briefly, animals raised on 80 µM retinal plates, were immobilized with cyanoacrylic glue and a lateral cuticle incision was made to expose the ventral medial body wall muscles. Muscle recordings were made in the whole-cell voltage-clamp configuration (holding potential −60 mV) using an EPC-10 patch-clamp amplifier and digitized at 2. 9 kHz. The extracellular solution consisted of (in mM): NaCl 150; KCl 5; CaCl2 5; MgCl2 4, glucose 10; sucrose 5; HEPES 15 (pH 7. 3, ∼340mOsm). The patch pipette was filled with (in mM): KCl 120; KOH 20; MgCl2 4; (N-tris[Hydroxymethyl] methyl-2-aminoethane-sulfonic acid) 5; CaCl2 0. 25; Na2ATP 4; sucrose 36; EGTA 5 (pH 7. 2, ∼315mOsm). All of the animals carry a transgene (zxIs6) that expresses channelrhodopsin-2 under the control of the cholinergic motor neuron (unc-17) -specific promoter. Evoked currents were recorded in a body-wall muscle after eliciting neurotransmitter release by a 10 ms illumination using a 470 nm LED (Thor labs) triggered with a TTL pulse from the EPC10 pulse generator [31]. Evoked post-synaptic responses were acquired using Pulse software (HEKA) run on a Dell computer. Subsequent analysis and graphing was performed using Pulsefit (HEKA), Mini analysis (Synaptosoft Inc) and Igor Pro (Wavemetrics). The data were analyzed with one-way ANOVA followed by Dunnett' s multiple comparison. | Calcium ions are essential for many physiological processes, including neurosecretion and neuronal and muscle excitation. Paradoxically, abnormal accumulation of calcium ions is associated with cell death and has been documented as an early event in muscle and neural degenerative diseases. One mechanism to avoid detrimental calcium accumulation is to link the calcium increase with activation of calcium-dependent potassium ion channels, thereby reducing cell excitability and preventing further calcium influx. This negative feedback requires these potassium channels to be localized in close proximity to sites of calcium entry. In a Caenorhabditis elegans genetic screen, we identified α-catulin, known as a cytoskeletal regulatory protein in mammals, important for the localization of calcium-dependent potassium channels in both muscles and neurons. In muscle, α-catulin controls the localization of the dystrophin complex, a multimeric protein complex implicated in muscular dystrophy. The dystrophin complex in turn tethers the calcium-dependent potassium channels near calcium channels. In neurons, the α-catulin-mediated localization of the potassium channels is independent of the dystrophin complex. Lack of α-catulin results in mislocalization of the potassium channels, and in turn causes defects in neuromuscular function. Our results support the idea that cytoskeletal proteins function as anchor molecules that localize ion channels to specific cellular domains. | lay_plos |
Insects exposed to pesticides undergo strong natural selection and have developed various adaptive mechanisms to survive. Resistance to pyrethroid insecticides in the malaria vector Anopheles gambiae is receiving increasing attention because it threatens the sustainability of malaria vector control programs in sub-Saharan Africa. An understanding of the molecular mechanisms conferring pyrethroid resistance gives insight into the processes of evolution of adaptive traits and facilitates the development of simple monitoring tools and novel strategies to restore the efficacy of insecticides. For this purpose, it is essential to understand which mechanisms are important in wild mosquitoes. Here, our aim was to identify enzymes that may be important in metabolic resistance to pyrethroids by measuring gene expression for over 250 genes potentially involved in metabolic resistance in phenotyped individuals from a highly resistant, wild A. gambiae population from Ghana. A cytochrome P450, CYP6P3, was significantly overexpressed in the survivors, and we show that the translated enzyme metabolises both alpha-cyano and non–alpha-cyano pyrethroids. This is the first study to demonstrate the capacity of a P450 identified in wild A. gambiae to metabolise insecticides. The findings add to the understanding of the genetic basis of insecticide resistance in wild mosquito populations. Insecticide resistance in disease vectors is one of the greatest challenges to the reduction of the burden caused by vector-borne diseases in developing countries. Beyond their public health importance insect vectors are increasingly regarded as model organisms with insecticide resistance serving as an excellent example of natural selection [1]. In many parts of Africa, the malaria vector, Anopheles gambiae shows high levels of resistance to pyrethroid insecticides which are the mainstay of vector control [2] and there is evidence that this resistance may reduce the efficacy of treated bednets and indoor residual spraying with pyrethroids [3]. Moreover, some pyrethroid resistance mechanisms that confer cross-resistance to DDT are geographically widespread [4]. The stark reality facing control program managers is that there is resistance to the primary compounds used for vector control and that no new active ingredients have become commercially available for public health use in the last 20 years. Studies of resistance mechanisms are key to both understanding the evolution of resistance and to minimising its impact on disease control. At the biochemical level, two classes of mechanism are predominantly associated with insecticide resistance; changes in the sensitivity of insecticide targets in the nervous system and metabolism of insecticides before they reach their target [5]. In A. gambiae, target-site resistance to DDT and pyrethroids is associated with a knock-down resistance (kdr) mutation in the voltage-gated sodium channel gene. The kdr alleles are characterised by two point mutations resulting in either a L1014F [6] or L1014S [7] substitution. The mutations may be present alone or in combination and have arisen from multiple mutation events [1]. While insecticide resistance associated with kdr is well studied at the physiological, behavioural and population level, much less is known about the enzymes associated with metabolic resistance. One route of metabolic resistance is through up-regulation of detoxification enzymes. Overexpression of enzymes related to insecticide resistance is generally assumed to be associated with cytochrome P450-dependent monooxygenases (P450), carboxylesterases (COE), and glutathione-S transferases (GST). Among these three families, evidence suggests that P450s commonly play a primary role in pyrethroid resistance (for reviews see [8] and [9]). To date, out of 111 putative A. gambiae s. s. P450s [10] four have been observed to be overexpressed in adult mosquitoes from colonies characterised as pyrethroid resistant, namely CYP6Z1, CYP6Z2, CYP6M2 and CYP325A3 [11]–[13]. Although the up-regulation of these P450s was associated with resistance, their potential to metabolise pyrethroids remains unclear. While Chiu et al. [14] demonstrated that CYP6Z1 metabolises DDT, to date only CYP6Z2 interacts with pyrethroids. McLaughlin et al. [15] found that CYP6Z2 binds to two pyrethroids, permethrin and cypermethrin. Their data, however, also suggested that the pyrethroids were not metabolised by this P450 highlighting the importance of functionally characterising putative candidates involved in pyrethroid metabolism. These earlier studies have potential confounding effects of colonisation including genetic drift and physiological adaptations to the artificial laboratory environment. The study of immune response in natural mosquito populations has highlighted that mechanisms found in laboratory colonised material may be less relevant in nature [16]. While genetic markers for target site insensitivity are available [6], [7] and widely used, we lack simple screening methods for alleles associated with up-regulation of detoxification enzymes. As a result, the role of metabolic resistance in reducing the efficacy of malaria vector control is unknown. The current study was carried out as part of the Innovative Vector Control Consortium (IVCC) to develop a tool to monitor mosquito field populations for resistance alleles [17]. We set out to identify enzymes that metabolise pyrethroid insecticides by selecting field-caught mosquitoes against the lethal time to kill 50% of the mosquito population (LT50). Genes potentially associated with detoxification of xenobiotics were screened for differential gene expression between survivors and unexposed mosquitoes using the A. gambiae detox chip [11]. We then expressed the most promising candidate in Escherichia coli to examine its pyrethroid metabolising potential. Mosquito collections were carried out in the village of Dodowa, Ghana (05°52. 67′N, 000°06. 36′W) between October and November 2006. A detailed description of the field site can be found in Yawson et al. [18]. Mosquitoes morphologically identified as members of the A. gambiae species complex [19], [20] were sampled from natural breeding sites and raised to adults in an insectary located in Dodowa. Larvae were given ground TetraMin fish food and adults were provided with 10% sugar solution. Newly emerged adults were separated into females and males and kept as cohorts of same age. All bioassays and selections were performed on the third day post-eclosion. In addition to the larval collections blood-fed females were caught using aspirators inside houses and family lines reared as described in Müller et al. [21]. These family lines were used to compare constitutive versus induced gene expression (see below). Before selecting mosquitoes against permethrin we determined the lethal time (LT) of 0. 75% permethrin treated filter paper for 50% mortality (LT50) using World Health Organization (WHO) test kits following standardised conditions [22]. To estimate the LT50 we first established a time-response curve by exposing approximately 100 individuals to one of six different exposure times (12. 5,25,50,100,150 and 200 min). Mortality was recorded 24 h post exposure and data were fitted by a logistic regression model using logit-transformed probabilities [23] to predict the LT50. All analyses were performed using the open source statistical software package R (http: //www. r-project. org). All R-code required to perform these calculations is available from the first author on request. Once the LT50 was determined, cohorts of 3-day old adult females were split into two groups; one group was exposed to 0. 75% permethrin and the other group to a control paper which contained only the insecticide carrier (silicone oil). Both groups were exposed for the LT50 using WHO test tubes and then transferred to holding tubes. In order to examine constitutive differences in gene expression between selected and unselected mosquitoes all individuals were kept in the holding tubes for 48 h before they were killed in 70% ethanol. A recovery time of 48 h was chosen to control for potential permethrin-induced gene expression. Vontas et al. [24] showed that permethrin-induced gene expression regains constitutive levels within 24 h of a non-lethal exposure. To test for permethrin-induced gene expression additional family lines were reared and 3-day old adult females split into two groups. One group was exposed to 0. 75% permethrin for 30 min and the second group served as a control. After a recovery time of 48 h post exposure four to five female mosquitoes from each group were pooled and RNA extracted. Using RT-PCR gene expression levels of exposed and unexposed individuals were compared in a pair wise t-test. For all mosquitoes one hind leg was removed for DNA extraction and the remaining body parts were transferred to RNAlater (Ambion) to prevent RNA degradation. Genomic DNA was extracted from legs using the DNeasy kit (Qiagen) and used to identify each specimen to species and molecular form [25]. The same DNA was used to screen for the presence of the L1014F [6] and L1014S [7] substitutions within the voltage-gated sodium channel protein causing knockdown resistance (kdr) by a heated oligonucleotide ligation assay (HOLA) [26]. Only mosquitoes identified as members of A. gambiae s. s. S form and homozygous for the L1014F kdr allele were included in the microarray study. Total RNA was extracted from pools of 10 mosquitoes which were either selected against 0. 75% permethrin for the LT50 or not exposed to the insecticide. The quality and quantity of all RNA pools was measured by a spectrophotometer (Nanodrop Technologies) and a random subset was also assessed using a 2100 Bioanalyzer (Agilent Technologies). RNA extraction, amplification and labelling protocols followed those described in Müller et al. [21]. Labelled targets were hybridised to an updated version of the A. gambiae detox chip [11], [21] which was printed with a physical rearrangement of the probes (ArrayExpress accession A-MEXP-863). The probes on the microarray include 103 cytochrome P450s, 31 esterases, 35 glutathione S-transferases and 85 additional genes such as peroxidases, reductases, superoxide dismutases, ATP-binding cassette transporters, tissue specific genes and housekeeping genes. The microarray experiment compared RNA pools from selected vs. unselected mosquitoes, comprising six independent replicates with dye-swaps (12 arrays in total). As each probe was spotted in replicates of four and measurements were obtained for both red and green wavelengths in each array, a total of 96 measurements per probe were obtained. After visual inspection of each array, spot and background intensities were calculated from the scanned array images using GenePix Pro 5. 1 software (Axon Instruments). Raw intensities were then analysed with Limma 2. 4 software package [27] running in R. Any spot that showed a median intensity in one or both channels at saturation was excluded from the analysis. For each spot background intensities were subtracted (i. e. method = “subtract”) from the total spot intensities and adjusted intensities were transformed into intensity log-ratios and normalised. For the comparison between the two groups, selected vs. unselected, estimates for technical replicates (dye-swaps) were first averaged and then compared between the two groups. A detailed description of the methods used for normalisation and statistical analysis is given in Müller et al. [12]. All microarray data has been deposited in ArrayExpress (accession E-MTAB-52). In terms of absolute fold change our values are likely to underestimate true fold differences between mosquitoes that would survive an LT50 and those that would not. This is a result of the study design whereby the LT50 survivors were compared with a control group that would be expected to be a mixture of 50% mosquitoes surviving and 50% mosquitoes dying after exposure to 0. 75% permethrin. It was not possible to select a fully susceptible control group due to the expected RNA degradation postmortem. The underestimation of fold changes may occur wherever resistant mosquitoes are compared with their parental line. Details of how this study design limits maximum fold change are given in Figure S1. As a consequence we have chosen to rank our genes by statistical significance (i. e., −log10 P-value) rather than setting an arbitrary fold change cut-off to filter for candidates. Quantitative RT-PCR was used to validate microarray data and for comparisons with the “Kisumu” strain, a susceptible A. gambiae s. s. laboratory colony. An aliquot of 75 ng from each pool of total RNA served as template for making target specific cDNA by reverse transcription in a single multiplex assay using the GenomeLab GeXP Start Kit (Beckman Coulter) and the gene-specific primers in Table 1. The primers were designed using the eXpres Profiler software (Beckman Coulter) based on cDNA sequences retrieved from the sources given in Table 1. The GeXP multiplex system uses a combined primer of target-specific and a universal sequence to reverse transcribe mRNA into cDNA. The reverse transcription step was followed by a PCR step in which during the first three cycles amplification was carried out by chimerical forward and reverse primers (Table 1). For the subsequent cycles (numbers 4 to 35), amplification was carried out using universal forward and universal reverse primers provided by the kit. The PCR conditions were 95°C for 10 min, followed by 35 cycles of 94°C for 30 s, 55°C for 30 s and 68°C for 1 min. Multiplexing primer specificity was confirmed by sequencing the PCR products obtained from single reactions. The universal primers that come with the kit were fluorescently labelled and yielded signals that corresponded to the amount of product in the multiplex reaction. PCR products were quantified with a CEQ 8000 Genetic Analysis System (Beckman Coulter) running a GenomeLab GeXP eXpress analysis program (Beckman Coulter) that computes peak areas for each target. The peak area of a control gene, S7 (VectorBase: AGAP010592) was used to normalise for variation in total mRNA amount. Normalised peak areas were then log2-transformed to approximate a normal distribution. Messenger RNA from the susceptible lab colony A. gambiae “Kisumu” (3-day old adults) was isolated using the PicoPure kit (Arcturus) and cDNA prepared using Superscript III (Invitrogen). Initial efforts to express CYP6P3 using the E. coli OmpA signal peptide as previously described for CYP6Z2 [15] were unsuccessful. Therefore, we used another common strategy for P450 expression, which is to replace the natural P450 amino-terminus with a sequence (MALLLAVF) derived from the bovine steroid 17 α-hydroxylase [28]. To introduce the amino-terminal 17α modification the 5′-end of CYP6P3 cDNA was amplified using KOD DNA polymerase (Novagen) with ECG169 (5′TTTCATATGGCTCTGTTATTAGCAGTTTTTGCCGCGTTCATCTTCGCAGTGTCGATCGTG 3′), introducing a NdeI restriction site at the initiation codon (underlined), and ECG137 (5′-ATGAATTCTACAACTTTTCCACCTTCAAG -3′) complementary to the 3′-end of the CYP6P3 cDNA, and introducing an EcoRI site (underlined). The resulting 17α-CYP6P3 was ligated into pCWompA2 via NdeI and EcoRI to create pCW: : 17α-cyp6p3. The construct was sequenced and compared with the database sequence of CYP6P3 (GenBank: AAL93295). In addition to the four amino acid substitutions to the membrane anchoring sequence as a result of the 17α modification (E2A, I4L, N5L, and L8F – numbering relative to published sequenced), there were two nucleotide changes that encoded amino acid substitutions R154W and L292V. These nucleotides changes were are also present in CYP6P3 amplified from Kisumu genomic DNA and are therefore not due to PCR errors. For functional expression of CYP6P3 and its redox partner cytochrome P450 reductase (CPR), competent E. coli DH5α cells were co-transformed with pCW: : 17α-cyp6p3 and pACYC-AgCPR. This transformant was grown in 0. 4 l of Terrific Broth with ampicillin and chloramphenicol selection at 37°C until the optical density at 595 nm reached 0. 8 units. The culture was then cooled to 25°C, supplemented with 0. 5 mM 5-aminolevulinic acid (Melford, UK) and 1 mM isopropyl β-D-1-thiogalactopyranoside (Melford) before incubation continued at 25°C with orbital shaking at 150 rpm. The cells were harvested and membranes prepared as described previously [15]. P450 function was quantified by CO-difference spectroscopy [29] and CPR activity was estimated by cytochrome c reductase activity [30]. CYP6P3 was expressed at 50–100 nmol of P450 litre of culture. The isolated bacterial membranes contained 0. 5 nmol of CYP6P3 per mg protein and the specific activity of CPR was 61 nmol cytochrome c reduced min−1 mg−1 protein. Total protein concentration was determined by Bradford assay, with bovine serum albumin standards. Deltamethrin and permethrin (Chemservice, West Chester, PA) were incubated with 0. 25 µM CYP6P3 in 0. 2 M Tris. HCl, pH 7. 4,0. 25 mM MgCl2 in the presence or absence of an NADPH generating system (1 mM glucose-6-phosphate (Melford), 0. 1 mM NADP+ (Melford), 1 unit ml−1 glucose-6-phosphate dehydrogenase (G6PDH) in a total volume of 100 µl. Reactions were carried out in triplicate at 30°C with 1,200 rpm shaking. Samples were pre-warmed for 5 min before reactions were initiated by addition of the membrane preparation. Reactions were stopped with 100 µl of acetonitrile and incubated for a further 20 min to ensure that all pyrethroid was dissolved. The quenched reactions were centrifuged at 20,000 g for 10 min before transferring the supernatant to glass HPLC vials. 100 µl of the supernatant was loaded onto a mobile phase with a flow rate of 1 ml min−1 and 23°C for separation on a 250 mm C18 column (Acclaim 120, Dionex). Time-trial reactions were run with a linear gradient from 0% to 90% acetonitrile in water (v/v) over the first 6 min, 90% was then held for 10 min before returning 0% with a linear gradient over 2 min followed by equilibration with 0% for another 4 min. Pyrethroid elution was monitored by absorption at 232 nm and quantified by peak integration (Chromeleon, Dionex). For kinetics of deltamethrin, varying concentrations of substrate (0. 5–16 µM) were used. Deltamethrin concentrations were determined by HPLC as describe above, but using an isocratic mobile phase with 90% acetonitrile in water. Rates of deltamethrin turnover from three independent reactions were plotted versus deltamethrin substrate concentration. Km and Vmax were determined using SigmaPlot v10. 0 (Systat Software, Inc) by fitting to the Michaelis-Menton equation using non-linear regression. Before selecting individuals the LT50 to 0. 75% permethrin was determined by exposing 98 to 110 individuals per time point and sex (Figure 1). Using logistic regression models we estimated an LT50 of 122 min for females and 95 min for males. Mortality rates for a WHO standard 1 h exposure were 16. 8% and 30. 5% for females and males, respectively (Figure 1). Mortality in the controls was 2. 3% for females (N = 171) and 2. 1% for males (N = 116). A total of 333 A. gambiae s. l. females were stored for gene expression studies and identified to species level, molecular form and kdr genotype (Table 2). The majority (99. 4%) of the sampled mosquitoes were A. gambiae s. s. belonging to the molecular S form, and only two individuals in the control group were M form. The L1014S kdr mutation was detected in three individuals, although it was not possible to confirm this result by sequencing. There was no difference in L1014F frequencies between the control and selected groups (Fisher' s exact test, P = 0. 22). This latter mutation was close to fixation with 91. 3% of the screened individuals in the control group being homozygous for the L1014F mutation (Table 2). All specimens included in the microarray analysis were A. gambiae s. s., molecular S form and homozygous for the L1014F kdr mutation to minimise confounding effects. Three P450s were consistently (very low P-values) expressed at higher levels in LT50-selected vs. unexposed mosquitoes; CYP6P3, CYP4H24 and CYP4H19 (Figure 2, Table 3). CYP6P3 and CYP4H19 were 1. 6-fold over-expressed and CYP4H24 was 1. 5-fold over-expressed in specimens surviving the LT50. The same RNA pools used in the microarray analysis were additionally evaluated by multiplex quantitative reverse transcription (RT) PCR for 14 selected genes (Table 1). The transcripts were selected from the pool of genes that were differentially expressed in the microarray analysis. Two genes, CYP4H19 and COEAE2G were removed from the analysis due to missing PCR products for some of the RNA pools. Both methods were in concordance for several genes, though not for all, including CYP6P3, CYP6M2, CYP6AK1, GSTD1-4, ABCC9 and CYP6Z2 (Figure 3A). For all other genes, ABCC11, CYP4D22, CYP4H24, CYP6M3, CYP6N1 and CYP12F4 the lack of concurrence between the two methods is probably related to low levels of fold change [31]. On the basis of having the most consistent gene expression pattern, the catalytic properties of CYP6P3 enzyme was further investigated by heterologous expression in E. coli. The comparison of CYP6P3 expression levels between LT50-selected mosquitoes and a susceptible laboratory (Kisumu, A. gambiae s. s.) strain also showed increased levels in the wild mosquito population (Figure 3B), showing additional evidence for an association between CYP6P3-overexpression and permethrin resistance. A comparison of CYP6P3 expression levels between permethrin-exposed and unexposed female siblings 48 h post exposure showed no sign of gene induction (pair wise t-test, P-value = 0. 49, N = 7 families; data not shown). Hence, overexpression of CYP6P3 may be assumed to be constitutive rather than induced upon permethrin exposure. CYP6P3 was co-expressed with A. gambiae cytochrome P450 reductase (CPR) in E. coli to produce a functional monooxygenase complex, and the ability of CYP6P3 to metabolise permethrin was evaluated from time-dependant elimination of a 10 µM mixture of four isomers. Permethrin eluted with R and S trans-isomers at 16. 1 min and R and S cis-isomers at 17. 4 min in HPLC analysis. In the absence of NADPH there was no significant change in permethrin concentration over the 30 min incubation period (Figures 4A and 5A). With the NADPH regeneration system included, 72% of the total permethrin was eliminated in 30 min (Figure 4B) with a steady rate of elimination (Figure 5A). This indicates a turnover of 0. 59±0. 04 min−1, (slope from linear regression±S. E. M.) for the trans-permethrin isomers and 0. 37±0. 02 min−1 for the cis-permethrin isomers (combined rate of 0. 97±0. 06 min−1). CYP6P3 activity was also tested against an alpha-cyano pyrethroid, deltamethrin, commonly used on insecticide-treated bednets. Deltamethrin eluted at 14. 5 min and like permethrin, NADPH-dependent elimination by CYP6P3 was observed (Figures 4C, 4D, and 5B). The single isomer at 10 µM was turned over slightly slower than permethrin at a constant rate of 0. 86±0. 03 min−1. Deltamethrin metabolism was studied in greater kinetic detail due its availability as a single isomer. Analysis of initial metabolic rate (5 min reactions) in response to deltamethrin concentration revealed Michaelis-Menten kinetics: the Vmax was 1. 8±0. 2 min−1 and the Km was 5. 9±1. 2 µM (±S. E. M., N = 3). In this study, we selected wild-caught mosquitoes from a highly permethrin resistant field population in southern Ghana against the insecticide permethrin. Using a custom made microarray we identified CYP6P3, a P450 that was overexpressed in mosquitoes surviving exposure to 0. 75% permethrin for 2 h, the time that kills 50% of the mosquito population. Heterologous expression of CYP6P3 in E. coli yielded a protein that metabolises permethrin and deltamethrin. This is the first study to identify a gene encoding for an enzyme that mediates pyrethroid detoxification in the malaria vector A. gambiae s. s.. As our findings are based on the study of gene expression in wild-caught, phenotyped mosquitoes, the results are of significant importance in the field context. 3-day old females of the mosquito population under study showed 83% survival rate at the WHO diagnostic to 0. 75% permethrin for 1 h. To our knowledge, this is the highest survival rate reported against permethrin in an A. gambiae field population to date. This population has a high frequency of the L1014F kdr allele, which confers resistance to pyrethroids and DDT [6]. The population is almost fixed with 91% of screened individuals found to be homozygous for the L1014F substitution at the this locus, a 12% increase compared to a survey conducted in 2002 at the same field site [18]. There has been much debate over the extent to which target-site and metabolic resistance mechanisms contribute to the observed phenotype [3], [7], [32]. To exclude any possible effect of known target-site mutations we performed all gene expression analyses only on RNA extracted from specimens homozygous for the L1014F kdr type. This is a considerable improvement over previous expression studies which were potentially confounded by comparing the susceptible Kisumu strain with resistant laboratory strains having either the L1014S [11], [13], [24] or L1014F [12] kdr mutation. Furthermore, in the field population studied L1014F allele was close to fixation and hence the observed variability in resistance phenotype is most likely attributable to an additional mechanism. The time-response, showing mortality as a function of exposure time to 0. 75% permethrin, is represented by a very broad symmetrically shaped sigmoid curve. This implies that the population has a broad distribution of resistant phenotypes [33] which suggests that there are multiple resistance mechanisms present in the population. Three P450s up-regulated in the permethrin-selected specimens showed good accordance between the microarray and RT-PCR data, including CYP6P3, CYP6M2 and CYP6AK1. P450s are an abundant family of enzymes which can mediate resistance to all classes of insecticides and their up-regulation has been documented in a broad range of insect species [8], [9]. Although up-regulation has been identified for a large number of P450s in insecticide resistant insects, studies of catalytic activity are generally limited [9]. To date two A. gambiae P450s (CYP6Z1 and CYP6Z2) have been functionally characterised [14], [15]. While CYP6Z1 is capable of metabolising DDT [14] and CYP6Z2 binds to pyrethroids, a catalytic capacity could not be shown for pyrethroids [15]. The current study focused on the characterisation of CYP6P3 because there was a strong association between gene expression and resistance phenotype. CYP6P3 is the first enzyme with a demonstrated potential for catalytic activity with pyrethroids in A. gambiae. Intriguingly, CYP6P9, the A. funestus ortholog of CYP6P3 [34], is located within a major Quantitative Trait Locus (QTL) conferring pyrethroid resistance [35] and overexpressed in adults of the pyrethroid resistant FUMOZ-R strain [36]. As both the QTL marker and the A. funestus CYP6P9 locus are physically mapped to the same region on chromosome 2R, it has been postulated that up-regulation is mediated via mutations in cis-acting elements [36]. In A. gambiae s. s. the question whether CYP6P3 is cis- or trans-regulated remains unanswered and further studies are needed to identify how up-regulation is controlled. This information will facilitate the development of expression-associated DNA markers that would allow screening of wild populations for the presence of metabolic resistance alleles. The second P450 which showed convincing evidence for association with permethrin-resistance was CYP6M2. Moreover, CYP6M2 has previously been identified in a colonised laboratory strain from the same field site [12]. Enzyme characterisation of CYP6M2 is currently underway. The third P450, CYP6AK1, has not previously been associated with pyrethroid resistance and was down-regulated in the DDT-resistant ZAN/U strain [11]. CYP6AK1 has not been investigated further, but this gene may become an interesting candidate if found in future studies. We expressed the full-length cDNA of CYP6P3 in E. coli along with its cognate redox partner CPR to produce a functional enzyme for characterisation studies. Consistent with a role in detoxification, CYP6P3 was found to metabolise permethrin. Permethrin consists of four isomers: (R) cis, (R) trans, (S) cis, (S) trans, and it is the cis isomers that has greater insecticidal activity, possibly due to slower metabolism [37]. Since two peak mixtures of cis R/S and trans R/S isomers are separated by HPLC chromatography, rates of metabolism of individual isomers could not be determined. However, both (1RS) cis and (1RS) trans isomers were eliminated from enzyme reactions indicating that metabolism of the active form occurs. Moreover the enzyme was efficient in metabolising deltamethrin, which is widely used in agriculture and in the production of insecticide-treated bednets, further emphasising a potentially important role in metabolic resistance. Modest rates of metabolism of the pyrethroids by the heterologously expressed CYP6P3 were observed. Substrate turnover values were in the range 0. 5–2 min−1, which were 5 to 10-fold slower than the rates observed for the in vitro P450 metabolism of pyrethroids reported from other species; the lepidopteran CYP6B8 has a Vmax for α-cypermethrin of 13 min−1 [38] whereas rat CYP3A2 has 14-fold higher turnover than CYP6P3, although the Km for deltamethrin is not significantly different [39]. This could potentially be due to the absence of cytochrome b5 in our system, which is known to enhance the activity of some P450s [8]. Indeed, increased levels of cytochrome b5 are associated with P450 mediated insecticide resistance in some insects and are directly involved in CYP6D1 mediated cypermethrin metabolism in the house fly [40]. Investigations are underway to examine the influence of cytochrome b5 on metabolism and to further define the molecular interactions of pyrethroids and other insecticides with CYP6P3. Our data demonstrates that a P450, CYP6P3 is up-regulated in highly permethrin resistant A. gambiae s. s. mosquitoes in the field and functional characterisation of the enzyme strongly suggest that CYP6P3 metabolises both permethrin and deltamethrin. The overexpression of its ortholog in A. funestus provides further support to the importance of this enzyme for pyrethroid resistance in malaria vectors. CYP6M2 was also overexpressed in this study and a study on a laboratory strain colonised from the same area [12] and thus merits further investigation. Yet, although its origin is from the same locality as the existing population, the previous analysis did not detect the change in CYP6P3. The current study emphasises the importance of studying metabolic resistance in natural mosquito populations. | Malaria, a disease spread by anopheline mosquitoes, is a global health problem with an enormous economic and social impact. Pyrethroid insecticides are critical in reducing malaria transmission, and resistance to these insecticides threatens current control efforts. With a limited number of public health insecticides available for the foreseeable future, it is vital to monitor levels of resistance to facilitate decisions on when new strategies should be implemented before control fails. For monitoring, simple molecular assays are highly desirable, because they can detect resistance at very low frequencies and should identify the presence of single recessive alleles well before bioassays. An understanding of the mechanisms conferring resistance facilitates the development of such tools and may also lead to novel strategies to restore the efficacy of the insecticide, or the development of new compounds. We set out to identify enzymes that may confer metabolic pyrethroid resistance by comparing levels of messenger RNA between insecticide-selected versus unselected mosquitoes. We caught members of the major malaria vector, A. gambiae s. s. from a highly pyrethroid resistant field population. We found increased transcript levels for a cytochrome P450, CYP6P3, and demonstrate that it encodes for an enzyme that metabolises pyrethroids. | lay_plos |
Identifying the targets of broadly neutralizing antibodies to HIV-1 and understanding how these antibodies develop remain important goals in the quest to rationally develop an HIV-1 vaccine. We previously identified a participant in the CAPRISA Acute Infection Cohort (CAP257) whose plasma neutralized 84% of heterologous viruses. In this study we showed that breadth in CAP257 was largely due to the sequential, transient appearance of three distinct broadly neutralizing antibody specificities spanning the first 4. 5 years of infection. The first specificity targeted an epitope in the V2 region of gp120 that was also recognized by strain-specific antibodies 7 weeks earlier. Specificity for the autologous virus was determined largely by a rare N167 antigenic variant of V2, with viral escape to the more common D167 immunotype coinciding with the development of the first wave of broadly neutralizing antibodies. Escape from these broadly neutralizing V2 antibodies through deletion of the glycan at N160 was associated with exposure of an epitope in the CD4 binding site that became the target for a second wave of broadly neutralizing antibodies. Neutralization by these CD4 binding site antibodies was almost entirely dependent on the glycan at position N276. Early viral escape mutations in the CD4 binding site drove an increase in wave two neutralization breadth, as this second wave of heterologous neutralization matured to recognize multiple immunotypes within this site. The third wave targeted a quaternary epitope that did not overlap any of the four known sites of vulnerability on the HIV-1 envelope and remains undefined. Altogether this study showed that the human immune system is capable of generating multiple broadly neutralizing antibodies in response to a constantly evolving viral population that exposes new targets as a consequence of escape from earlier neutralizing antibodies. Neutralizing antibodies are the principal correlate of protection for most preventative vaccines. Designing suitable vaccine immunogens to elicit these types of antibodies has been relatively simple for conserved pathogens such as smallpox and other DNA viruses. For more diverse pathogens like HIV-1, the neutralizing antibodies elicited by vaccination or during natural infection are largely strain-specific and therefore would not be protective against globally circulating viral variants [1]–[5]. The HIV-1 envelope glycoprotein spikes mediate viral entry and are the sole targets for neutralizing antibodies. The spikes are trimeric, made up of three non-covalently associated gp41-gp120 heterodimers, each with a conserved core that mediates infection of CD4+ T-cells. Functionally conserved sites are protected by extensive glycosylation, and large solvent exposed hypervariable structures (the V1–V5 loops, and the α2-helix in C3) [6]. All HIV-1 infected individuals develop strain-specific neutralizing antibodies which target these sequence variable regions, but only a quarter develop broadly neutralizing antibodies [7]–[11], which will likely be needed for a preventative HIV-1 vaccine. To engineer an envelope immunogen that can specifically elicit these antibodies, the HIV-1 vaccine research field has adopted a strategy based largely on rational design: identifying the targets for these broadly cross-reactive antibodies, and elucidating the pathways that promoted their development. Plasma mapping strategies and the isolation of monoclonal antibodies have defined four major targets for broadly neutralizing antibodies on the HIV-1 glycoprotein [7]–[10], [12]–[20]. The CD4 binding site (CD4bs) of gp120 and the membrane proximal external region (MPER) of gp41 are glycan independent epitopes, while the V1/V2 sub-domain and the co-receptor/V3 site on gp120 are sites of vulnerability for glycan binding antibodies (predominantly at positions N156/N160 and N301/N332 respectively) [14]–[16]. Both CD4bs antibodies and co-receptor/V3 antibodies bind well to monomeric gp120, while MPER antibodies bind to a linear peptide in gp41. This makes it possible to adsorb out their neutralization activity from plasma with various recombinant proteins. In contrast the epitope for V2 antibodies (such as PG9/16) consists of two anti-parallel β-sheets (B- and C- strands) of a Greek key motif, and the glycans therein, that is preferentially formed on the native trimer. This region is critically important for the gp120-gp120 interactions that stabilize the envelope glycoprotein spike in its unliganded conformation, and therefore cannot be readily adsorbed [16], [21]. Various sub-epitopes within each of these four major sites of vulnerability have also been identified through subtle differences in the mechanism of neutralization [22]. For instance antibodies targeting the CD4bs can be sub-divided into two groups: those that are sensitive to the D368A and/or E370A mutations in the CD4 binding loop α3 (such as VRC01); and those that are dependent on amino acids D474, M475, and/or R476 in α5 termed CD4bs/DMR (such as HJ16) [23]–[26]. Despite this detailed knowledge, epitope mapping strategies have failed to identify the neutralization targets in a subset of plasma samples [7]–[9], [17], [19], [27], [28]. The antibodies mediating breadth in these samples could target sub-epitopes within one of the four sites of vulnerability or they may target entirely novel epitopes. In the CAPRISA 002 Acute Infection Cohort, we previously identified seven individuals with broadly neutralizing antibodies. In five cases we were able to map the plasma antibody specificities to known epitopes (two targeted N332, two the V2 epitope, and one the MPER) [7]. In this study we have focused on one of the individuals (CAP257) for which the target was undefined. Heterologous and autologous neutralization data as well as viral sequences from longitudinal samples were used to identify the epitopes for CAP257 broadly neutralizing antibodies. We showed that heterologous neutralization in CAP257 was conferred by three distinct, sequentially occurring antibody waves, two of which were mapped to epitopes in V2 and the CD4bs respectively. While individuals with more than one broadly neutralizing antibody specificity have been previously identified [29]–[31], there is little information on how the dynamic relationship between host and pathogen contributed to the development of antibodies targeting multiple epitopes. We have shown previously that escape from strain-specific neutralizing antibodies can drive the formation of epitopes for broadly neutralizing antibodies [32]. Here we found that viral escape from broadly neutralizing antibodies targeting V2 promoted the development of a second broadly neutralizing antibody response targeting a glycan dependent epitope in the CD4bs. We also identified early escape mutations from both the V2 and CD4bs antibodies that drove an increase in the neutralization breadth of CAP257 plasma. These findings have implications for the design of HIV-1 vaccine antigens and sequential immunization strategies. We have previously described the development of neutralization breadth in CAP257 using longitudinal plasma samples from HIV-1 seroconversion to three years post-infection (p. i.) [7]. Here, we extended this analysis until the start of anti-retroviral therapy at four and a half years p. i. (Figure 1A). Longitudinal plasma was tested against the autologous CAP257 virus amplified from the earliest available time point (7 weeks p. i.), the subtype C consensus sequence (ConC) [33], 4 Tier 1b viruses, and 39 Tier 2 viruses [34]. Autologous neutralizing antibodies appeared by 14 weeks of infection with a peak titer at two years of 1∶6,754. This was followed by the neutralization of heterologous viruses 30 weeks after infection. CAP257 neutralized 84% of the heterologous viruses at three years (174 weeks) with neutralization breadth of 100% against subtype A (6/6 viruses), 96% against subtype C (25/26 viruses, including ConC), and 50% against subtype B (6/12 viruses). The titers of these broadly neutralizing antibodies peaked and waned in three separate waves. The first wave of neutralization breadth (typified by CAP63) peaked at 67 weeks p. i. with a maximum titer of 1∶1,493 and exclusively neutralized subtype C viruses (Figures 1A and 1B – red curves). Wave 1 titers dropped to as low as 1∶145 by 149 weeks of infection. As this early heterologous neutralization began to wane, CAP257 plasma gained the capacity to neutralize additional subtype C viruses as well as several subtype A and B viruses (Figures 1A and 1B – blue and green curves). This second wave (typified by Q842) peaked at 122 weeks p. i. with titers as high as 1∶8,565 against RHPA that dropped to 1∶1,254 by 213 weeks of infection. Finally, a third wave of heterologous neutralization (represented by Du156) appeared by 149 weeks p. i. and peaked at 213 weeks p. i. (Figure 1B – brown curve). This third wave was also largely subtype C specific. These data suggested that the neutralization breadth of CAP257 plasma was mediated by at least three distinct antibody specificities. To identify the targets of each of the three broadly neutralizing antibody specificities in CAP257 plasma we first assessed whether they targeted epitopes in monomeric gp120 (such as the CD4bs or N301/N332 glycans). We adsorbed out the gp120 binding antibodies in plasma samples from the peak neutralizing activity of each wave using recombinant ConC gp120 coupled to tosyl-activated magnetic beads (Figure 1C). The adsorbed plasma was compared with untreated plasma for activity against a heterologous virus neutralized by each wave. Neutralization by wave 1 (67 weeks p. i.) and wave 3 (213 weeks p. i.) was not affected by adsorption with monomeric gp120 (Figure 1C – red and brown curves), however neutralization by wave 2 antibodies (122 weeks p. i.) could be partially adsorbed with the ConC gp120 protein (Figure 1C – blue curve). The neutralizing activity of wave 2 could also be equally adsorbed with a core gp120 lacking the hypervariable loops V1/V2 and V3 (Figure 1C – green curve). These data supported our hypothesis that CAP257 heterologous neutralization was mediated by more than one neutralizing antibody specificity, two of which were largely subtype C specific and targeted an epitope not present on monomeric gp120, and a third whose epitope in gp120 was more conserved across clades and did not require the hypervariable loops V1/V2 or V3. The inability of gp120 to adsorb out wave 1 neutralization suggested these antibodies might recognize the trimer specific epitope in V1/V2 defined by PG9/16 [16]. Therefore, we performed mapping studies using ConC, which was neutralized by all three waves of neutralizing antibodies (Figure 2A – red curve). Seven mutations in the V2 region (F159A, N160A, R166A, K168A, K169E, K171A, and I181A) each abrogated wave 1 neutralization, but did not significantly affect the titers of waves 2 or 3 (Figure 2A – purple curves). In contrast, a D167N mutation resulted in enhanced neutralization by wave 1 antibodies, but did not significantly affect the titers of waves 2 or 3 (Figure 2A – orange curve). A second mutation (L165A) also resulted in significant neutralization enhancement at all the time points tested, including those preceding breadth (Figure 2A – grey curve), suggesting that this mutation resulted in general neutralization sensitivity. Overall, these data indicated that wave 1 antibodies (but not waves 2 or 3) targeted residues in the V2 region. To define whether the V2 epitope recognized by CAP257 plasma antibodies overlapped with that of known broadly neutralizing antibodies to this site, we tested the sensitivity of PG9/16, CH01-04, and PGT145 to the same ConC V2 mutations described above and compared them to CAP257 neutralizing antibodies at the peak of wave 1 activity, 67 weeks p. i. (Figure 2B). Of the seven mutations that abrogated CAP257 neutralization only two (N160A and K169E) resulted in complete resistance to all the antibodies tested, consistent with previous data [35], [36]. Neither the monoclonal antibodies nor CAP257 wave 1 antibodies were sensitive to deletion of the N156 glycan (through the S158A mutation) in ConC. Lastly, mutations at two hydrophobic amino acids in V2 (F159A and I181A) that do not form part of the PG9 epitope as defined by the crystal structure [37], had a significant effect on the neutralization of monoclonal antibodies targeting V2, and CAP257 wave 1 antibodies. To define escape from wave 1 neutralizing antibodies, we examined sequences from the V2 region of CAP257 over time. Using single genome amplification (SGA) we obtained 125 full envelope sequences from twelve time points between 7 and 213 weeks p. i., and focused on the N160 glycan and the cationic C-strand in V2 that are the targets of wave 1 antibodies (Figure 3A). Interestingly, the earliest virus (7 weeks p. i.) had an asparagine at position 167. This N167 residue is rare, occurring in only 5. 6% (196 of 3,478) of sequences in the Los Alamos National Laboratory (LANL) HIV sequence database. By the time of the earliest detectable heterologous neutralization (30 weeks p. i., maximum titer of 1∶49) mutations in sites forming part of the wave 1 V2 epitope were already apparent in 6/14 autologous sequences at positions R166, K169, and Q170 (Figure 3A). Of the remaining eight sequences, six exhibited other mutations either in the N160 glycosylation sequon or the V1/V2 C-strand. This rapid selection pressure in the C-strand of V1/V2 was sometimes an N167D mutation (4/14 autologous sequences) that was unlikely to be selected for by wave 1 broadly neutralizing antibodies, as all the heterologous viruses neutralized by wave 1 had a D167 residue. Since the V1/V2 region is a common target of strain-specific neutralizing responses [38]–[41], these data suggested the possibility of an earlier neutralizing response targeting N167 in V2 that preceded the development of broadly neutralizing antibodies. Wave 1 mapping data (Figure 2A) further supported this possibility because the reverse D167N mutation enhanced the neutralization of ConC by wave 1 antibodies only, and resulted in earlier neutralization kinetics (Figure 2A – orange curve). To test this we selected an envelope from 174 weeks p. i. (CAP257 3 yr) that was completely resistant to CAP257 neutralizing antibodies (consistent with ongoing neutralization escape), and back-mutated the V1/V2 region to match the earliest sequence from 7 weeks p. i. (Figure 3B). The neutralization sensitivity of the back-mutated virus, CAP257 3 yr (V1/V2s), was then compared to the parental CAP257 3 yr virus using longitudinal plasma samples. In contrast with the resistant CAP257 3 yr virus, the back-mutated V1/V2 virus (CAP257 3 yr (V1/V2s) ) became sensitive to neutralization at 23 weeks p. i. (Figure 3B – black curve). This suggested the emergence of a strain-specific V1/V2 response 7 weeks prior to the development of wave 1 broadly neutralizing antibodies at 30 weeks p. i. To establish whether these strain-specific V1/V2 neutralizing antibodies targeted the same epitope as wave 1 broadly neutralizing antibodies, we introduced selected escape mutations (N167D, N160D/S and K169E) into the sensitive CAP257 3 yr (V1/V2s) back-mutated envelope. Introduction of the N167D mutation (Figure 3B – orange curve), a common V2 change at 30 weeks p. i., shifted the timing of autologous V1/V2 neutralization to overlap with the emergence of wave 1 broad neutralization. The introduction of N160D/S mutations that deleted the N160 glycan (Figure 3B – purple curves), further shifted autologous neutralization to overlap with the emergence of wave 2 neutralizing antibodies. Finally, when the K169E mutation was introduced (Figure 3B – pink curve), autologous V1/V2 neutralization titers were completely abrogated. As the N160A and K169E mutations in ConC also completely abrogated neutralization by CAP257 wave 1 broadly neutralizing antibodies (Figure 2B) these data suggest that the strain-specific V2 neutralizing antibodies in CAP257 plasma targeted the same site of vulnerability that was later targeted by wave 1 broadly neutralizing antibodies. However the strain-specific V2 antibodies recognized the rare N167 immunotype of V2 present in the CAP257 infecting virus. Following an early N167D escape mutation at this site, V1/V2 neutralization became N167 independent, allowing recognition of the more common D167 immunotype. This switch in the fine specificity of CAP257 V2 antibodies correlated with the emergence of broadly neutralizing wave 1 antibodies. Wave 1 broadly neutralizing antibodies were completely dependent on the glycan at N160 (Figure 2A). Viral escape from wave 1 neutralizing antibodies by deletion of this glycan first occurred at 54 weeks p. i. (in 37. 5% of the sequences), immediately prior to the development of wave 2 neutralizing antibodies (Figure 3A – pie charts). This escape pathway persisted at 93 weeks p. i. (in 30% of sequences), but by 122 weeks p. i., at the peak of wave 2 activity, alternative escape pathways existed, and all sequences contained the N160 glycan. The transient nature of this highly effective escape pathway suggested that deletion of the N160 glycan had a deleterious effect on the virus. A potential mechanism for this came from the observation that deleting the N160 glycan (critical to wave 1 neutralization) in the CAP257 3 yr (V1V2s) virus conferred slight neutralization sensitivity coinciding temporally with wave 2 (Figure 3B – purple curves). These data suggested that mutations at N160 exposed the wave 2 epitope. To examine whether loss of the N160 glycan enhanced CAP257 wave 2 neutralization we selected two viruses, Q842 and RHPA, which were neutralized at high titer by wave 2 but resistant to wave 1, and deleted the N160 glycan in each. The effect of these N160K mutations was assessed longitudinally using CAP257 plasma. While the timing of RHPA N160K and Q842 N160K neutralization by CAP257 was not altered compared to the wild-type viruses, these mutant viruses were neutralized 2–8 fold more potently by wave 2 antibodies (Figure 3C – purple curves). A similar 2 fold increase in titer was shown at the peak of wave 2 activity when the N160 glycan was deleted in ConC (Figure 2A – purple closed circles). As deletion of the N160 glycan in the autologous virus occurred prior to the development of wave 2 neutralizing antibodies, these data suggest that this particular escape pathway from wave 1 neutralizing antibodies may have contributed to the development of wave 2 antibodies, possibly by better exposing the epitope. Therefore, after the development of wave 2, the K169E mutation that also allowed escape from wave 1 broadly neutralizing antibodies, but did not enhance wave 2 neutralization, was preferentially selected over deletion of the N160 glycan (Figure 3A). The V1/V2 sub-domain of gp120 plays an important role in shielding the envelope from neutralization. More specifically, several modifications in V1/V2 (particularly at N-glycosylation sites) have been implicated in either shielding or exposing the CD4bs to neutralizing antibodies [42]–[51]. As escape from wave 1 at N160 enhanced neutralization by wave 2, these data suggested the CD4bs as the target for wave 2 antibodies. This was supported by the adsorption data (Figure 1C), showing that wave 2 neutralizing antibodies bound the core gp120 protein. Therefore we examined envelope sequences from 191 weeks p. i. for selection pressure in the conserved CD4bs (Figure 4). Three mutations in the D-loop (N276D/S, T278A/K and N279D) and one at the base of V5 in the β23 sheet of C4 (R456W) dominated the viral population at this time point. Both the D-loop and V5 have been previously implicated in resistance to CD4bs antibodies [52]–[54]. To assess whether these CD4bs mutations mediated resistance to wave 2 neutralization, we introduced the three most common mutations (T278A, N279D and R456W) simultaneously into two heterologous viruses (Q842 and RHPA) neutralized by wave 2 but not by wave 1 (Figure 1A), and tested them against plasma from the peak of wave 2 activity (122 weeks p. i.). The mutants were at least 20 fold more resistant to neutralization at this time point than the wild-type viruses, confirming the role of CD4bs mutations in escape from wave 2 antibodies (Figure 5A). To further characterize the epitope targeted by wave 2 antibodies, we assessed the dependence of wave 2 binding on the D368 residue in α3 (critical for VRC01-like antibodies), and residues 474/475/476 in α5 (critical for HJ16-like antibodies) by adsorption studies. The D368R or D474A/M475A/R476A mutations were separately introduced into ConC gp120, and compared to the wild-type protein for their ability to adsorb out the neutralizing activity against Q842 and RHPA at peak wave 2 titers. Both mutant gp120s (Figure 5B – yellow and brown bars) adsorbed out a significant fraction of the neutralizing activity against Q842 and RHPA, equivalent to that adsorbed by wild-type gp120 (Figure 5B – white bars). These data suggested that CAP257 antibody binding was not dependent on these residues in the α3 and α5 helices. CAP257 neutralization could not be adsorbed with the RSC3 protein used to isolate VRC01, which binds weakly to the HJ16 class of CD4bs antibodies [55]. Of the three changes identified above as mediating escape from wave 2 antibodies, N279 and R456 make contact with CD4, while T278 forms part of an adjacent glycosylation sequon [6]. This glycosylation sequon is conserved in 96% (n = 3,475) of envelope sequences in the LANL HIV sequence database. Its deletion via N276D/S or T278A/K mutations in later viruses (Figure 4) for wave 2 escape was therefore striking, and suggested a possible role for glycan recognition by CAP257 wave 2 antibodies. To examine wave 2 glycan binding, we expressed an RHPA core gp120 in GnTI (−/−) 293S cells, which allowed for deglycosylation of the protein using Endo-H, and assessed the ability of both glycosylated and deglycosylated proteins to adsorb out wave 2 neutralization activity. While neutralizing activity against RHPA was efficiently adsorbed with the glycosylated RHPA gp120 core (Figure 5C – white bars), the deglycosylated protein only adsorbed out a fraction of that activity (Figure 5C – purple bars) confirming the importance of glycans in wave 2 antibody binding. Similarly HJ16 was not adsorbed by the deglycosylated protein suggesting glycan dependence. In contrast, VRC01 was adsorbed equally effectively by both proteins. To assess in more detail the role of the N276 glycan in wave 2 neutralization, we generated four mutants in RHPA, comparing the effects of two conservative mutations (N276Q, T278S) with the effects of two alanine substitutions (N276A, T278A) in the N276 glycosylation sequon (Figure 5D – boxed in purple). Both alanine mutations significantly affected wave 2 peak titers by 18 and 30 fold respectively. The N276Q mutation which deleted the glycan but retained the amino acid properties at position 276 also affected wave 2 neutralization by 21 fold (a similar effect to the N276A mutation), while the T278S mutation that retained the N276 glycan had no effect on neutralization. These data suggested that sensitivity to wave 2 neutralization was largely dependent on the glycan at position 276, rather than the N276 amino acid side chain. We also assessed the effect of the remaining two autologous mutations in the CD4bs (N279D and R456W) identified above (Figure 5D). The N279D mutation alone had a relatively small 2 fold effect on neutralization, suggesting only a minor role in escape from wave 2. When an alanine was substituted at position 279 instead, wave 2 neutralization was enhanced. The R456W mutation had a more significant 10 fold effect on CAP257 neutralization, but was still less effective than the glycan deleting mutations at positions 276/278 which were the major escape mutations. We next compared the epitope for CAP257 wave 2 neutralizing antibodies with that of HJ16 (CD4bs/DMR) and VRC01 (CD4bs). Both monoclonal antibodies were profoundly affected by the R456W mutation (514 and 30 fold respectively). Like CAP257, HJ16 neutralization was significantly dependent on the glycan at position 276 with glycan deleting mutations (N276Q/A and T278A) resulting in a 74–106 fold increase in IC50 (Figure 5D). This dependence on the N276 glycan distinguished both CAP257 and HJ16 from VRC01, for which neutralization was slightly enhanced (2–3 fold) when the glycan was removed. This effect on VRC01 is consistent with previous studies showing that deleting the N276 glycan exposes the CD4 binding site to neutralization by VRC01 or b12 [52], [56]. Introducing all three CAP257 escape mutations identified above therefore had a compensatory effect on VRC01 resistance (8 fold compared to 30 fold effect for R456W alone), but completely abrogated HJ16 neutralization, confirming the similarities between HJ16 and CAP257 plasma. Despite these overall similarities, some differences were apparent between CAP257 wave 2 antibodies and HJ16, such as the preference of HJ16 for the threonine at position 278 and the asparagine at position 279. Unlike CAP257, the N279A mutation did not enhance HJ16 neutralization but rather resulted in a 4 fold reduction in titer. These data may suggest minor contacts between HJ16 and the amino acid side chain at position 279. The N279A mutation also significantly affected VRC01 neutralization (76 fold), consistent with either asparagine or aspartic acid residues at position 279 being directly contacted by W100B in the CDR3-H3 of VRC01 [52], [57]. To clarify the role of glycan binding we tested three CD4bs neutralizing antibodies (VRC01, b12, and HJ16) in ELISA for binding to either the glycosylated or deglycosylated RHPA gp120 core proteins (Figure 5E). The neutralizing antibody 2G12 has a well-defined glycan epitope and was used as a positive control [58]–[62], while CAP88-3468L (a V3 binding antibody) served as a negative control [63]. Deglycosylation significantly affected the binding of both 2G12 and HJ16 (Figure 5E – blue and purple curves), but did not significantly affect binding of either VRC01 or b12 (Figure 5E – green and yellow curves) to the RHPA gp120 core. These data confirmed the glycan binding properties of HJ16, and suggest that both CAP257 wave 2 neutralizing antibodies and HJ16 have a glycan dependent mechanism of neutralization at the CD4bs. While the simultaneous introduction of T278A, N279D, and R456W mutations into heterologous viruses Q842 and RHPA made them resistant to wave 2 neutralization at 122 weeks p. i. (Figure 5A), when each mutation was introduced individually into RHPA the results were varied (2–30 fold reductions in titer) with no single mutation resulting in complete escape (Figure 5D). These data suggested that escape from wave 2 required a combination of all three mutations. Longitudinal sequence analysis showed that the N279D mutation emerged first at 93 weeks in 5% of the population, followed by N276D/S glycan deleting mutations at 122 weeks in 36% of the population, and then by substitution of the R456 residue with a bulky amino acid side chain (H, Y, or W) at 161 weeks p. i. in 17% of the population (Figure 4). To better characterize their contributions to escape from wave 2 antibodies, each mutation (T278A, N279D, and R456W) was introduced separately into Q842 and RHPA and compared to the wild-type viruses. The N279D mutation (Figure 6 – orange curves) had a significant effect only at the beginning of wave 2 activity against Q842, shifting the earliest heterologous neutralization of Q842 from 80 weeks to 93 weeks p. i. (with a 5 fold effect on titers at 107 weeks p. i.). The mutation also affected the titers against RHPA by 2–3 fold. This suggested an initial dependence on N279, with later wave 2 antibodies being less vulnerable to mutations at this residue. This reduced dependence on N279 coincided with the emergence of the N279D mutation at 93 weeks p. i. (Figure 6 – orange dotted lines) providing a mechanism for maturation of the antibody response. The T278A mutation (Figure 6 – purple curves) that deleted the N276 glycan conferred almost complete resistance in Q842, and shifted the earliest neutralization of RHPA from 67 to 122 weeks p. i. The R456W mutation (Figure 6 – pink curves) did not result in a further right shift in the neutralization curves for RHPA relative to the T278A mutation, but did affect the neutralization kinetics of Q842 relative to the wild-type or N279D mutant viruses. As with the N279D mutation, these changes in the fine specificity of the maturing antibody response reflected the emergence of either T278A or R456W mutations in longitudinal autologous sequences (Figure 6 – purple and pink dotted lines respectively). These data are consistent with accumulating resistance to wave 2, and suggest that after each successive round of escape, new antibody variants emerged that were able to neutralize first the N279D mutant, and then later autologous viruses with additional polymorphisms at positions 276,278, or 456. We wished to determine whether the ability to neutralize escaped viral variants correlated with increased wave 2 neutralization breadth. Heterologous viruses neutralized by wave 2 were divided into two groups, those neutralized at 67 weeks p. i. (early wave 2 neutralization), and those first neutralized at 93 weeks p. i. (late wave 2 neutralization) after the emergence of initial wave 2 escape mutations (Figure 7A). Viruses also neutralized by wave 1 were omitted as the overlapping titers confounded this analysis. Inspection of the envelope sequences (particularly in the D-loop) showed that all of the viruses neutralized by early wave 2 antibodies had the N279 immunotype (Figure 7B – boxed in orange). In contrast 44% of viruses neutralized by later wave 2 antibodies had the D279 immunotype. Furthermore, of the viruses neutralized by later wave 2 antibodies, one (Q259) lacked the N276 glycan and four others also had additional non-conservative mutations at positions 273–275 in the N-terminus of the D-loop (Figure 7B – boxed in blue) that may also affect early wave 2 neutralization. These data suggest that wave 2 escape mutations guided maturation of the CD4bs response, enabling later wave 2 antibodies to neutralize additional heterologous viruses and ultimately resulting in the increased neutralization breadth of CAP257 plasma. Like wave 1, wave 3 neutralization could not be adsorbed with monomeric gp120, suggesting that these antibodies targeted a quaternary epitope, or an epitope in gp41. To assess whether wave 3 was a distinct antibody specificity, or a re-emergence of wave 1 antibodies, we selected a virus (Du156) that was sensitive to waves 1 and 3, and less sensitive to wave 2 (Figure 8A – red curve). Resistance to wave 1 (V2) and wave 2 (CD4bs) neutralizing antibodies was established by introducing the N160K and T278A mutations identified above. The resulting virus (Du156 N160K/T278A) remained sensitive to wave 3 neutralization only (Figure 8A – brown curve), suggesting that wave 3 differed from wave 1 (which was completely abrogated by the N160K mutation). To confirm this, we introduced additional V2 mutations (R166A, K168A, K169E, K171A) known to abolish wave 1 neutralization, into the Du156 double mutant. None of these mutations significantly affected the titers of wave 3 neutralizing antibodies (Figure 8A – yellow curves), confirming that the epitope for wave 3 antibodies did not overlap with the wave 1 V2 epitope. An N332A mutation was also introduced to confirm that wave 3 antibodies did not target this glycan. To test whether CAP257 wave 3 neutralizing antibodies targeted the MPER region of gp41, we coupled MPER peptides to magnetic beads and used them to adsorb out MPER specific binding antibodies in CAP257 plasma. The adsorption of MPER binding antibodies (Figure 8B) did not affect neutralization of Du156 or the double mutant Du156 N160K/T278A (with wave 1 and 2 resistance mutations) when compared to untreated plasma (Figure 8C). Thus, while we cannot exclude the possibility that wave 3 antibodies target V2 or gp41, these neutralizing antibodies appear to target an epitope distinct from any of the four known sites of vulnerability in the HIV-1 envelope. We hypothesized that the selection pressure exerted by these broadly neutralizing antibodies would impact on the overall neutralization phenotype of CAP257 viruses over time. Therefore we tested the sensitivity of envelopes from 7,30,54,93, and 174 weeks p. i. to broadly neutralizing monoclonal antibodies targeting the four major epitopes (Figure 9). The 7 week clone from CAP257 was sensitive to neutralization by anti-V2 antibodies, but following the development of wave 1 V2 neutralizing antibodies, CAP257 viruses became more resistant to PG9 and PGT145 neutralization. The 7 week clone was also highly sensitive to HJ16 (0. 02 µg/mL) and VRC01 (1. 79 µg/mL), however clones isolated after the emergence of wave 2 neutralization were increasingly resistant to neutralization by antibodies targeting the CD4bs. CAP257 viruses from all the time points selected were sensitive to neutralization by antibodies targeting the MPER or N301/N332, neither of which was targeted by broadly neutralizing antibodies in CAP257 plasma. These data confirm that CAP257 developed broadly neutralizing antibodies targeting the CD4bs and V2. Furthermore, the finding that CAP257 viruses remained sensitive to neutralization by antibodies targeting N301/N332 and the MPER supports our hypothesis that wave 3 antibodies target a novel epitope on the HIV-1 envelope. A preventative HIV-1 vaccine remains the most likely way to end the HIV pandemic, but current envelope immunogens have so far failed to elicit broadly neutralizing antibodies. Nonetheless, the development of cross-reactive antibodies in approximately a quarter of HIV-1 infected individuals has confirmed that the human immune system can make such antibodies. Much emphasis has been placed on mapping the targets for these broadly neutralizing antibodies in an attempt to define viral vulnerabilities for immunogen design. Here we analyzed CAP257 heterologous neutralization over a 4. 5 year period, describing the sequential evolution of three distinct broadly neutralizing antibody specificities within a single HIV-1 subtype C infected individual. We further showed how early viral evolution in the context of broadly reactive antibodies may profoundly shape the maturing antibody response towards enhanced neutralization breadth, in a process that may inform immunogen design. These data have been summarized in Figure 10. The CAP257 autologous virus efficiently escaped all three specificities. As a consequence the antigenic stimulus for these broadly neutralizing antibodies declined, and antibody titers dropped at least ten fold within a three year period. The waxing and waning of the broadly neutralizing specificities in CAP257 confounded our previous attempts to map the targets at 174 weeks p. i. [7], when the titers of the three waves overlapped significantly. As most mapping studies are cross-sectional, the number of individuals who mount multiple broadly neutralizing antibody responses may therefore be underestimated, and may make up a significant proportion of those plasma samples that remain undefined. Nonetheless we were able to finely map 2 of the 3 specificities in this study and showed that they targeted known sites of vulnerability on the HIV-1 envelope. Wave 1 antibodies targeted the site defined by PG9/16 and were completely dependent on N160 and K169, consistent with previous data describing PG9/16 dependence on the N160 glycan and the positively charged amino acids in the C-strand of V1/V2 [16], [37]. In general the epitope for wave 1 antibodies showed a larger footprint in V2 when compared to the epitopes of other monoclonal antibodies targeting this site, but behaved most similarly to PGT145. However none of the antibodies or plasma tested was sensitive to removal of the N156 glycan. While the crystal structure of PG9 with V2 showed interactions with the N156 glycan [37], the effect of deleting the N156 glycan is variable [16], [35], [36]. This effect might be explained by recent data suggesting that PG9 recognizes two N160 glycans (from two adjacent gp120 monomers) but only one N156 glycan [64]. The requirement for a lysine at position 169 explains the subtype C specificity of CAP257 wave 1 antibodies, as this residue is less common in subtypes A and B [35]. Wave 2 antibodies targeted a known site of vulnerability, the CD4bs, but these antibodies had an unusual glycan dependent mechanism of neutralization. CAP257 wave 2 and HJ16 neutralization were both highly dependent on interactions with the N276 glycan. N276 is also the recently described target of the broadly neutralizing antibody 8ANC195, but this antibody does not appear to interact with the CD4bs [65]. While glycan dependence for neutralization has not previously been described for CD4bs antibodies, including HJ16, Balla-Jhagjhoorsingh et al. reported that resistance to HJ16 involved an N276D mutation (deleting the glycan) with a hundred fold drop in titer [66], providing support for our observations. The glycan dependence of both HJ16 and CAP257 wave 2 antibodies suggests that they target a similar sub-epitope of the CD4bs that may be better defined as an N276 glycan dependent class of neutralizing antibodies, which is distinct from the VRC01 class. Recently it was shown that related variants of VRC01 do bind the glycan at N276 [67], however this glycan is not a major determinant of neutralization sensitivity to VRC01 [52], [56], [57], [68]. Rather, N276 has been described as a protective shield for the CD4bs, and deleting this glycan enhances the neutralization of CD4bs antibodies VRC01 and b12 [52], [56]. Removing N276 from gp120 also enabled binding to the predicted germline antibody for VRC01, which otherwise did not bind to gp120, and has been suggested as a modification for candidate vaccine immunogens [69], [70]. However, the glycan shield is increasingly recognized as a major site of vulnerability on the HIV-1 envelope [15], [16], and as with the glycans at N156/N160 and N301/N332, conservation of the N276 glycan bordering the CD4bs may make it a promising target for vaccine design. Characterization of viral escape from CAP257 CD4bs antibodies indicated that deletion of the N276 glycan alone did not confer complete resistance. Escape required accumulating mutations in the CD4bs site, consistent with the functional conservation of this epitope. In addition to deletion of the N276 glycan, CAP257 escape occurred through a R456W mutation that also significantly affected neutralization by VRC01 (30 fold) and HJ16 (514 fold). This mutation likely contributed towards the evolution of VRC01 resistant virus by 174 weeks. W456 is extremely rare, occurring in only 0. 78% (27 of 3,481) of sequences in the LANL HIV-1 sequence database. A crystal complex for HJ16 is not available, however the structure of VRC01 bound to its epitope showed that this antibody does not make significant contact with the R456 side chain in gp120, but rather hydrogen bonds with the R456 backbone carbonyl group. This suggests that the R456W mutation provides an indirect mechanism for resistance. The highly conserved R456 side chain can make hydrogen bonds with backbone carbonyl groups of amino acids at position 277 and 278 in the D-loop, as well as hydrogen bonds with E466 side chain in β24, C-terminal to V5 (Figure 11). Loss of these bonds and localized conformational changes to accommodate a bulky tryptophan residue may destabilize this critical component of the CD4bs epitope [52], [56], [71]. This study adds to data showing that the immune system can target multiple conserved epitopes [29]–[31]. It is striking that in three of these four studies, antibodies targeted both V2 and the CD4bs (donors CH219, AC053, and CAP257), suggesting an association between these two epitopes. Indeed, there is a well-documented relationship between V1/V2 and the CD4bs. The V1/V2 region protects the receptor binding sites from neutralization [42]–[51], and also interacts with V3 at the trimerization domain to hold the CD4bs in its pre-liganded conformation [21]. The crystal structures of monoclonal antibodies PG9, CH58, and CH59 bound to their epitopes in V2 show that the conformation of the V1/V2 sub-domain may vary significantly, but the factors that govern these conformational states are not known [37], [72]. In CAP257, deletion of the N160 glycan increased exposure of the CD4bs. It is possible that certain immunotypes of the V1/V2 epitope, such as the rare mutations at N160 or D167 described here, shifted the equilibrium of V1/V2 toward conformations that better exposed the CD4bs to neutralizing antibodies. This is supported by previous observations that introduction of the N160K and D167N mutations simultaneously into JR-FL resulted in a >50 fold increase in neutralization sensitivity to CD4bs antibodies [73]. CAP257 was infected with the less common N167 variant, and therefore following escape from V2 wave 1 antibodies a similar conformational state (D/S160, N167) would have been presented to the immune system. Our data suggest that this escape pattern further exposed the glycan dependent CD4bs sub-epitope. Although vaccination with the K160 and/or N167 immunotype may improve antibody responses to the CD4bs site, antibodies induced to the V2 epitope would be relatively strain-specific, like monoclonal antibody 2909 that recognizes the K160 immunotype and is therefore specific for SF162 [74]. In CAP257, switching from N167 to the more common D167 residue resulted in escape from the strain-specific response to V2, and this coincided with the development of a much broader response targeting the same epitope. Sequential immunization may be a useful strategy to promote the broadening of the B-cell response. Recently Murphy et al. showed that two light chain variants paired with a single heavy chain of a strain-specific neutralizing antibody differentially neutralized early autologous envelopes [75]. While evolution of that strain-specific epitope did not affect the development of broadly neutralizing antibodies in this individual, the data supports the possibility that viral evolution might facilitate the neutralization of amino acid variants within a given epitope. Similarly, we have previously shown in an individual who developed broadly neutralizing antibodies to the V2 region that viral escape drove a maturation of the antibody response towards recognizing multiple V2 variants [35]. Here we show that emergence of an aspartic acid at position 279 preceded a broadening of the B-cell response to the CD4bs. The N279 and D279 amino acid variants of the CD4bs are equally common among sequences in the LANL HIV-1 database (50% and 48% respectively, n = 3,479), and preferential neutralization of either immunotype would halve the neutralization breadth of an antibody. CAP257 wave 2 neutralizing antibodies and HJ16 were somewhat sensitive to the N279D change. However the resistance of D279 containing viruses to CAP257 antibodies was rapidly lost after the emergence of the N279D escape mutation. Therefore, like the N167D mutation in V2 described for wave 1, this change in the fine specificity of CAP257 antibodies coincided with an increase in the neutralization breadth of the CD4bs response. We hypothesize that position N279 was non-critical for antibody binding, and despite temporarily facilitating neutralization escape, the N279D mutation then promoted affinity maturation by reducing the dependence of CAP257 antibodies on this amino acid. This adds to recent data suggesting a major role for viral evolution in the development of neutralization breadth to the CD4bs [76]. These data support the possibility that a sequential immunization strategy would enhance neutralization breadth by systematically presenting common variants in a given epitope. Such residues would have to be non-critical to antibody binding allowing for the evolution of higher affinity variants that would in turn recognize multiple immunotypes. Overall these data highlight how interactions between the host immune system and viral escape mutations shaped the development of broadly neutralizing antibodies. The escape pathways identified here that led to the increased breadth of neutralization for both V2 and CD4bs antibodies provide potential pathways for generating broadly neutralizing antibodies. Further defining these pathways through the isolation of monoclonal antibodies will provide valuable insight into how these types of antibodies could be elicited using sequential immunization in a vaccine setting. The CAPRISA Acute Infection study received ethical approval from the Universities of KwaZulu-Natal (E013/04), Cape Town (025/2004), and the Witwatersrand (MM040202). CAP257 provided written informed consent for study participation. The CAPRISA Acute Infection cohort is comprised of women at high risk of HIV-1 infection in Kwa-Zulu Natal, South Africa [77]. Here we studied one individual (CAP257) from seven weeks p. i. through four and a half years of infection, until she started anti-retroviral therapy. During this time she had an average viral load of 60,784 copies/mL and an average CD4 count of 498 cells/µL. Plasma samples collected at 30 time points were used in this study. The amplification of envelope genes from single HIV-1 RNA genomes has been previously described [78]. Viral RNA was isolated from CAP257 plasma using a Viral RNA Extraction Kit (QIAGEN), and cDNA was synthesized with Superscript III Reverse Transcriptase (Invitrogen). The reaction product was treated with RNase H (Invitrogen) and envelope genes were amplified by nested PCR with Platinum Taq (Invitrogen). Amplicons were purified with a PCR Clean-up Kit (QIAGEN) and single genome amplification confirmed by DNA sequencing using the ABI PRISM Big Dye Terminator Cycle Sequencing Ready Reaction kit (Applied Biosystems) and an ABI 3100 automated genetic analyzer. The full-length env sequences were assembled and edited using Sequencher v. 4. 5 (Genecodes) and Bioedit v. 7. 0. 5. 3. The TZM-bl cell line engineered from CXCR4-positive HeLa cells to express CD4, CCR5, and a firefly luciferase reporter gene (under control of the HIV-1 LTR) was obtained from the NIH AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, NIH (developed by Dr. John C. Kappes, and Dr. Xiaoyun Wu [79], [80]). The 293T cell line was obtained from Dr George Shaw (University of Alabama, Birmingham, AL). Cells were cultured at 37°C, 5% CO2 in DMEM containing 10% heat-inactivated fetal bovine serum (Gibco BRL Life Technologies) with 50 ug/ml gentamicin (Sigma) and disrupted at confluency by treatment with 0. 25% trypsin in 1 mM EDTA (Sigma). Selected envelope sequences were re-amplified from first round nested PCR products (described above) with PfuUltra II (Stratagene), purified by a Gel Extraction Kit (QIAGEN) and cloned into pcDNA3. 1 (Invitrogen). The envelope plasmids were co-transfected into 293T cells using FuGENE 6 (Roche) with the pSG3ΔEnv backbone (obtained from the NIH AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, NIH). Cultures were incubated for 48 hours to produce Env-pseudotyped viral stocks that were filtered through 0. 45 µm and frozen in DMEM supplemented with 20% FBS. Mutant envelopes were generated with the QuikChange Lightning Kit (Stratagene) and confirmed by DNA sequencing (as above). The TZM-bl neutralization assay has been described previously [4], [81]. It measures a reduction in relative light units generated by a single round of infection in TZM-bl cells with Env-pseudotyped viruses after pre-incubation with monoclonal antibodies or a plasma sample of interest. Samples were serially diluted 1∶3 and the ID50 calculated as the dilution at which the infection was reduced by 50%. Plasmids encoding Histidine tagged recombinant envelope proteins were transfected into 293T cells using polyethylenimine 25 kDa (Polysciences). Recombinant proteins were expressed and purified as previously described [9]. Aliquots of 400 µg each were coupled to MyOne Tosyl-activated magnetic Dynabeads (Invitrogen) at 37°C pH 9. 5 overnight, and then blocked with 0. 5% BSA in 0. 05% Tween20 PBS overnight at 37°C. Protein coupled beads were incubated with 200 µL of plasma (diluted 1∶20) for two hours at 37°C, then the beads were removed magnetically and the remaining plasma assessed for binding and neutralizing antibodies using ELISA and neutralization assays respectively. Protein antigens were coated at 4 µg/mL onto high binding 96-well ELISA plates (Corning) overnight at 4°C. All subsequent steps were carried out in 5% fat-free milk, 0. 05% Tween20 in PBS for 1 hour at 37°C. The plates were blocked and then probed with serial dilutions of the adsorbed plasma or specific monoclonal antibodies, biotinylated goat anti-human polyclonal antibodies (KPL), and an anti-biotin monoclonal conjugated to HRP (Calbiochem). Antigen-antibody complexes were detected by incubating with 100 µL 1-Step Ultra TMB-ELISA (Thermoscientific) for five minutes and then the reaction was stopped with 25 µL 1 M H2SO4. Absorbance was read at 450 nm on a VERSAmax tunable microplate reader (Molecular devices). Plasmid encoding the RHPA gp120 core was transfected with polyethylenimine-MAX 40 kDa (Polysciences) into GnTI (−/−) 293S cells and purified using two step lectin chromatography and Ni-NTA affinity chromatography. Protein was assessed for purity and conformation by SDS-PAGE and ELISA. 1 mg of the RHPA core gp120 was deglycosylated overnight at 37°C in 500 mM sodium chloride, 100 mM sodium acetate pH 5. 5 with 0. 5 U of Endo-H. Glycans were removed through buffer exchange into PBS using Vivaspin 20 mL concentrators (Sartorius stedim). Deglycosylation was confirmed by SDS-PAGE and sandwich ELISA (described above) using lectins as a capture protein. | Four sites of vulnerability for broadly neutralizing antibodies to HIV-1 have been identified thus far. How these broadly reactive antibodies arise, and the host-pathogen interactions that drive the affinity maturation necessary for neutralization breadth are poorly understood. This study details the sequential development of three distinct broadly neutralizing antibody responses within a single HIV-1 infected individual over 4. 5 years of infection. We show how escape from the first wave of antibodies targeting V2 exposed a second site that was the stimulus for a new wave of glycan dependent broadly neutralizing antibodies against the CD4 binding site. These data highlight how antibody evolution in response to viral escape mutations served to broaden the host immune response to these two epitopes. Finally, we document a third wave of neutralization that targets an undefined epitope that did not appear to overlap with the four known sites of vulnerability on the HIV-1 envelope. These data support the design of templates for sequential immunization strategies aimed at increasing neutralization breadth through the recognition of multiple epitopes and their immunotypes. | lay_plos |
[PREVIOUSLY_ON]
Fred: "Handsome man. Saved me from the monsters."
Gunn: "Anyone talked Fred lately?"
Angel: "She's been back in this world for three months and she still hasn't gone out into it."
Gavin: "I'm working on a little something aimed at Angel Investigations."
Lilah: "What are you doing? Building code violations."
Gavin: "I'm fighting Angel in my *own* way."
Lilah: "I bet he's really terrified."
Lilah to Angel: "It's just business."
Angel: "Don't you come at me through Cordelia ever again." Night, Hyperion Lobby, Cordy is looking through a magazine, Wes is reading a book and Gunn is playing a hand-held video game. Fred comes down the stairs and walks up to Cordy. Cordy jumps and lets out a scream as Fred leans in to look at the magazine. Wes jumps and looks around at them. Gunn without looking up from his game: "Hey, Fred."
Fred: "Sorry! Did - did I startle you guys?"
Wes: "No."
Cordy: "Only in the sense of shocking and jolting us. - What's up?"
Fred: "Nothing. Just taking a little stroll and... (looks at open magazine) Why would girls wanna look like that? I spent years in a cave starving. What's their excuse?"
Cordy: "Fashion." Fred laughs. Stops, sits down beside Cordy and looks around the lobby.
Fred: "So - everybody's just reading and hanging out?"
Cordy: "Angel's upstairs."
Fred: "Oh. - He's probably reading, too. He's so deep, you know? Thoughtful. I'm guessing "The Brothers Karmazov", Joyce, a little Goethe to round things out." Angel appears on the open hallway overlooking the lobby, holding a newspaper.
Angel: "Am I the only one who read this?"
Wes: "Read what?"
Angel: "Charlton Heston. Double feature! (Comes down the steps) At the Nu-art. "Soylent Green" and - "The Omega Man."
Gunn, eyes on his game: "Wow."
Angel: "It's two for one. Did I mention, Charlton Heston? Who's in?" Fred jumps up and raises her hand: "That sounds great!"
Angel: "Fred. Wesley?"
Wes: "Well, I'm in the middle of translating Fassad's guide from the original Sumerian."
Angel: "Gunn. Cordy?" They don't even look up at him, so he goes to grab his coat.
Angel: "Looks like it's just you and me, Fred. - Well, the worm certainly has turned."
Fred, giggling: "Y-y-yeah. The worm's turning and... (Stops smiling) Am I the worm?"
Angel: "No. You may not know this, Fred, but certain friends and co-workers have been known to accuse *me* of being the quiet, stay at home, sulky one. (All three of them turn to look at Angel) Some people - just don't know how to have fun anymore." Big hotel, young man is in bed with two girls.
1.Girl: "Can we take a breather, stud?"
Woody: "If you need one." He turns to the other girl.
2.Girl: "Hey, tiger. Me too. Just for a minute?" Guy gets out of bed with a sigh and goes to pour himself a martini.
1.Girl: "Pace yourself, sweetheart." Woody turns to admire his body in the mirror.
Woody: "Mmm, it's good to be young. (Walks back towards the bed) So. Ready for round four?" Woody suddenly hunches over in pain.
2.Girl: "You okay, baby?"
Woody: "Oh - it's been fun. - Alli permutat anima kimota. Alli permutat anima kimota. Alli permutat anima kimota." A stream of red-white energy issues forth from his eyes and mouth. The stream cuts off and Woody turns to look at the girls watching wide-eyed from the bed. The girls start to scream as Woody begins to steam and his body begins to deflate into a puddle of skin on the ground. Intro. Wes is sitting behind his desk listening to Fred.
Fred: "And he opened every door for me and he paid for the tickets. And even bought a giant popcorn. And every few minutes he'd go like this (motions like she is tipping a tub of popcorn towards someone, laughs). Because he wanted me to know it was okay for me to have some. (drops in a chair) And he's so lonely because he's the last man on earth."
Wes: "Angel?"
Fred: "No! Charlton Heston. The Omega Man? Omega being the last letter of the Greek alphabet so it's a metaphor. (Jumps back up) And he walks on the street side and not the building side. It's old-fashioned, but kind of chivalrous, you know?"
Wes: "We're back to talking about Angel."
Fred: "Right. And even though he didn't talk a lot, it was still okay. It was comfortable. It wasn't that awkward kind of quiet. You know that awkward kind of quiet?" There is a moment of silence.
Wesley: "No. That's never happened to me." Angel is sitting in the lobby reading the newspaper.
Cordy: "You need to talk to Fred."
Angel: "What about?"
Cordy: "About the big date you guys had last night."
Angel: "Woah! Date? - It was just a movie."
Cordy: "That's what you need to tell *her*. She's in there going on and on about what a super time you guys had."
Angel: "She's just enthusiastic. Don't read too much into it." Cordy pushes his feet off the footstool and sits down on it.
Cordy: "She's got the big puppy love. I mean, who wouldn't? You're handsome, and brave, and heroic, emotionally stunted, erratic, prone to turning evil and, lets face it, a eunuch."
Angel: "Hey, how can you... I'm not a eunuch."
Cordy: "Angel, it's just a figure of speech."
Angel: "Find a better one."
Cordy: "I just mean that s*x is a no-no for you. Because of this whole 'if you know perfect bliss you'll turn evil' curse. Really no cure for that, huh? - Listen, all I'm trying to tell you is, this thing with Fred, it's going to go bad unless it's nipped in the bud."
Angel: "Okay. Maybe just a short talk. - So how soon can you do that?"
Cordy gets up: "Nice try. It's gotta come from you." She takes a hold of Angel and pulls him up out of his chair and into Wes' office.
Cordy: "Angel has something to say." Angel looks from Fred, who smiles at him, to Wes. Holds up his newspaper.
Angel: "Hey, did anybody else see this? (Lays the paper down in front of Wes) Police found the body of a twenty-six year old Woodrow Raglan in a two-bedroom suite at the Elondria Hotel. Unnamed witness said it was as if his insides had just..."
Wes: "...collapsed. You know, there was something else like that - last week."
Cordy: "Uhm, may I just point out that no one is actually hiring us to look into this and that we should be doing more important things?" Wes holds up a newspaper clipping: "Here. Ten days ago, a body - found in another hotel room - under similar conditions."
Angel: "What do you think? Spell, curse - serial demon?"
Wes: "Though to say. Worth a closer look."
Angel: "I'll say. Cordelia, open up a case file. We have to get on this right away." Angel hands Cordy the file with the newspaper clipping as he walks past her out of the office.
Cordy: "Angel!" Cordy hurries after him. Wolfram and Hart, day.
Gavin: "Good morning, Lilah."
Lilah: "Good morning. Well, here we are at *my* office. Bye."
Gavin: "You're a though one. I know I'm gonna have to earn your respect. But give me a little time. You'll see I'm a creative guy."
Lilah: "Oh, like your 'lets torment Angel with building code violations' idea? Uh, so machiavellian! We'll just drown him in red tape."
Gavin: "There are other level's to this, Lilah. Avenues of interest I have... One of them being: does Angel even exist?"
Lilah: "Are you getting metaphysical on me?"
Gavin: "No. The guy has no social security number, no tax payer ID, no last name as far as I know. How can he go down to the building department, or anywhere else in officialdom for that matter? - He's the rat and we're the maze. Don't you wanna see what he'll do next?"
Lilah: "He might just rip out your throat."
Gavin: "Do you think he'd do something that cliched? Gosh. Maybe you don't know him as well as you think." Gavin walks off down the hallway. Lilah looks after him, then, instead of entering her office, goes over to the desk of her secretary.
Lilah: "Get Carter Williams on the phone. (The girl at the desk just looks at her) The graphic artist? (Lilah sighs) Oh, look under 'F' for forger." Lilah turns and walks into her office, closing the door. Hyperion, night. Cordy takes a printout from the printer and carries over to where the others are leaning against the reception counter.
Wes: "There was a third victim five weeks ago. They were all young, healthy males. They all died in expensive hotel suites."
Fred: "Can you imagine shelling out all that money for a snazzy suite and then kerplop, you're a big bag of mush bones? I guess it wouldn't be good wherever that happened. (Wes looks at her) Oh - please continue."
Wes: "Gunn, I was thinking you could interview the staff of these hotels where the guys died. I'm meeting a contact of mine from the coroner's office in thirty minutes. See what I can learn about these bodies."
Cordy: "They were all members of the same health club. The bodies - when they weren't - you know - dead ones." Angel walks over to look at Cordy's printout, pulling out his car keys.
Angel: "Cordy and I'll go check out the gym."
Wes: "My thought exactly."
Cordy grabs the keys: "I'll drive."
Gunn: "What are we waiting for?"
Wes: "Everyone know what they're doing? Good." They all leave - except for Fred, still standing in front of the reception desk.
Fred: "I'll just stay here. (Laughs) Okay. I'll do that." Cordy and Angel are walking into the health club.
Cordy: "You can't just keep ignoring Fred! You have to speak to her. You know, there is your business life and then there is your social life, and everybody knows that you keep those two things sepa..." Cordy trails off as one of the male health club members walks by.
Cordy: "I'm gonna go see if *he* knows anything." Walks away from Angel. Angel spots a club attendant and walks up to him.
Angel: "Hi. I was just wondering,could ask you a few questions? My name is Angel."
Phil shakes Angel's hand: "Angel. Good news, dude, we are running our best offer ever! Okay, I can get you a six months trial membership right now for three hundred and fifty dollars."
Angel: "No. I'm looking into some guys that were members here." Angel pulls out the newspaper clipping and shows it to Phil.
Phil: "Oh, yeah, Woody, right. I heard he like - died."
Angel: "He like did. Along with the others. All members. So, I need to ask you, Phil, does the club condone steroid use?"
Phil: "No. No, no, no a-a-absolutely not."
Angel: "Then we should probably keep this between ourselves, don't you think? I'll just take a look at their records and I'll get out of your hair."
Phil after a beat: "Yeah, yeah, o-okay." He and Angel leave. Cordy is interviewing two guys.
Cordy: "So, did you ever see anyone come in who looked suspicious - or really pale - or maybe green and scaly?" The guys look at each other. Angel and Phil are looking through some papers.
Phil: "You know, I-I-I don't see anything that connects the three of them - except they were all in the evening Pilates class together."
Angel: "Pilates, is that like Tae-bo?"
Phil laughs: "Yeah - if you're living in 1999." Angel walks into the room where a group of people are participating in an exercise class.
Instructor: "Relax your neck and shoulders, using your lower abdominals, bring the spine down to the floor. Take a deep breath in and as your arms come up to the ceiling..." Angel circles the room, he looks out of the big window at the back of the room. He sees light reflecting off a pair of circles in one of the windows of the building across the street. Looks at the sign in front of the other building: Monserrat, retirement community.
Cordy, talking to four guys: "There could be follow-up questions. I'll need some home phone numbers. Why don't we start with you, Benny?" Angel comes up behind her and leans in close to her.
Angel: "There is a retirement home in the street behind us. I'm gonna check something out."
Cordy: "Bye." Angel looks at the four guys, starts to leave then turns back to Cordy.
Angel: "Maybe when you're done with your *work* - here - you can pick me up. - Okay." Angel leaves.
Cordy: "He's just someone I work with. Anyway..." Angel looks up at the window of the retirement home where he saw the reflection. Angel knocks at the door of room 316. The nameplate says Marcus Roscoe. An old man wearing big, round glasses slowly opens the door.
Angel: "Mr. Roscoe. My name is Angel." Angel hands him one of their business cards.
Marcus: "Angel Investigations."
Angel: "Would it be alright if I came inside and asked you a few questions?"
Marcus: "Well, it's ah, pretty late."
Angel: "Shouldn't take long." Marcus turns and walks back into his room, leaving the door open. Angel shifts but remains standing outside. Marcus turns back and motions to him.
Marcus: "Come on if you're coming." Angel walks in, closing the door behind him. He looks around then walks over to the window.
Angel: "Nice to have view. I bet you, ah, spend a lot of time enjoying it."
Marcus: "Not that, uh, much... (Sees Angel holding up the pair of binoculars he found on the window sill) Uh. - Well, I don't see any harm in looking. That's about all I can do anymore. Uh - what is it you want?"
Angel: "Your help. (Angel puts the binoculars down and pulls out some newspaper clippings) I wonder if you've seen either of these men across the way in the gym."
Marcus flips through them: "No. I don't think so. I'm more of a girl watcher. You know what I'm saying? - Jeez - they all died? How?"
Angel: "That's what we're trying to find out."
Marcus: "You work with the police?"
Angel: "I'm a private investigator. I work with a team."
Marcus: "Hmm, sounds nice. I was a salesman. Worked alone for fifty years."
Angel: "Hmm. (Spots a shelf holding various pottery) Nothian herb jar. (Picks it up) That's a - pretty exotic item. Did you, ah, deal in the occult?"
Marcus: "Occult shmuccult. I traveled a lot. Picked up some trinkets." Angel spots some extreme sports magazines lying on the table. He puts the jar back on the shelf.
Angel: "Do a lot of bungee jumping, Mr. Roscoe?"
Marcus: "More than you might think, Mr. Angel."
Angel: "Just Angel." Marcus takes off his glasses and puts them in the breast pocket of his shirt. Takes a couple of steps closer to Angel.
Marcus: "I'll remember that. (Looks at Angel) Alli permutat anmia kimota. Alli permutat anmia kimota."
Angel chuckles: "You might wanna think twice about trying to cast a sp..." Red-white light streams from Marcus into Angel, while blue-white light streams from Angel to Marcus. After the light vanishes, Angel looks around, shrugs his shoulders. Marcus stares at Angel, eyes slightly squinted.
Marcus: "You *are* me." Angel grabs Marcus by the shoulders and head-butts him, then lowers the unconscious old man into a chair.
Angel: "That's gonna smart later." Angel walks out of the gate of the Moserrat and starts to saunter down the sidewalk.
Cordy: "Ah, hello!" Angel turns and sees Cordy sitting at the wheel of the convertible. He walks over, chuckles and leans on the top of the windshield.
Angel: "He-llo."
Cordy: "So, what did you find at the old folks home?"
Angel: "Uh - nothing. Didn't pan out. How about you?"
Cordy: "I got a two month free trial membership, and I made some new friends... - Alright. I got nothing."
Angel: "Pretty clear we're barking up the wrong tree here, huh?"
Cordy after a beat: "Yeah. - Well - get in. I'll take you back to the hotel." Angel grins and gets into the car and puts his arm on the back of Cordy's seat.
Angel: "Alright! You and me - going back to the hotel. Nice, huh?"
Cordy: "Are you alright?"
Angel grinning: "Honey, I've never been better." Break Angel follows Cordy into the lobby of the Hyperion.
Angel: "Nice! (Sees Cordy walking around the reception desk) You supposed to be back there? - (Hits the little bell on the counter) Ding, ding! Paying customers. Hellooo. (Whistles) Slow night, huh?"
Cordy: "Yeah. But maybe Wes or Gunn found out something."
Angel: "Wes or Gunn." Angel notices the stand of 'Angel Investigations' business cards on the counter. There are some with Wesley Wyndam-Pryce, some with Charles Gunn, some with just the business name, and others with Cordelia Chase, Senior Associate on it.
Angel quietly: "They're a great part of our investigating team. Hmm. Working here with us in this old abandoned hotel." Picks up one of Cordy's cards.
Angel: "Cordelia... (Cordy turns to look at him) have I ever told you you are a *very* *beautiful* woman?" Cordy goes back to sorting through the papers on her desk.
Cordy: "Ha, ha. Very funny. I know you never said anything that tacky or overt to Fred. But you're still gonna have that talk - whether you want to or not." Cordy walks around the counter to stand a little ways down from Angel.
Angel: "Talk with Fred."
Cordy: "Yes! Just - keep it simple. One: you're not like other men. Two: there is no room in the workplace for romance."
Angel: "Romance with Fred. - So I'm a... (Looks down at his clothes) Obviously."
Cordy turns to go: "Get some rest. See you tomorrow." Marcus wakes up in his chair. Looks around and tries to get up. Doesn't make it. Tries again. Still can't just get up. Puts his hands on the arms of the chair and levers himself to his feet. He walks over to look in a mirror but only sees a blurry man-shape. Puts on his glasses and watches Marcus reflection become clear. Marcus is sneaking through the lobby of the Monserrat to the receptionist desk. Picks up the phone and dials. Angel is looking through papers back in Wesley's office. The phone rings but he lets the machine get it.
Cordy's voice: "You've reached the offices of Angel Investigations. Please leave a message after the tone." Beep.
Marcus voice: "Cordelia? Are you there? Pick up!"
Angel picks up the phone: "Hey, Angel. How's my head? Hope you put some ice on it. Sweet deal you've got going on here, pal. Love the hotel. And Cordelia - whoh! That's how I spell w-o-m-a-n!"
Marcus: "Where is she?"
Angel: "You don't have to worry about anything except eating some nice soft foods and staying out of Ryan's way."
Marcus: "Ryan?" An orderly takes the phone from Marcus.
Ryan: "You wouldn't think that we just talked about this! (Hangs up the phone) There go your phone privileges for the rest of the month." Angel takes the tape out of the answering machine and smashes it. Ryan is walking Marcus down a hall at the Monserrat.
Ryan: "You know you're not supposed to be out of your room at this hour."
Marcus: "I was stretching my legs." Ryan puts a hand over his name badge: "Who am I?"
Marcus: "You're Ryan."
Ryan chuckles: "At least you're not having an episode. My advice, Marcus: if you start thinking you're a twenty-four year old stud, or a famous skateboarder, keep it to yourself. Unless you *wanna* wake up in iso and restraints again. Copy?"
Marcus points at himself: "I know who *I* am."
Ryan: "Then let's get *you* back to beddy-bye." Hyperion, day. Cordy walks into Wes' office to find Angel slumped asleep on top of the desk with papers littering the whole room.
Cordy: "Angel!" Angel's head jerks up, obscured by the page of paper that he was sleeping on, stiil stuck to his face.
Cordy: "What happened?" Angel pulls the paper off his face and takes a quick glance around.
Angel: "Uh... hey, doll. I ah, (puts some papers back into an open file) was working on the case. I must have dozed off."
Cordy: "You were too tired to go up to your room?"
Angel: "My room, right - which I have upstairs. Well, you know me. Always giving a hundred percent. (Gets up and looks through the mess) Now what did I do with the darn case file..."
Cordy quietly: "You gave it to me yesterday."
Angel: "Ha. Must be getting old." Angel reaches for the file but Cordy puts it behind her back.
Cordy: "Not until you have that talk with Fred." Wes walks in carrying an old English teapot.
Wes: "You know there is something about brewed tea you simply can not replicate with a bag. (Sees the mess) What happened here?"
Angel shuffling papers together: "I was just looking for something. Uh, I'll clean it up!"
Cordy turns to go: "Don't avoid the talk."
Angel: "I know. I know." Wes walks around the desk, picking up some of the papers, and looking over the mess.
Angel: "Hey. How're you doing?"
Wes: "Alright. Well... - you?" Angel pulls up a chair on the other side of Wes' desk and sits down.
Angel: "So, we gotta talk. The thing is, I've got nothing against you personally. It's just..." Wes raises his head to stare at Angel who shifts uncomfortably in his chair.
Angel not looking at Wes: "O-ho, this is gonna be harder than I-I thought. I just don't know how to spit this out." Wes comes around the desk and sits down on it's edge facing Angel.
Wes: "Angel. Whatever it is, you know I'm here for you." Wes stretches out a hand towards Angel, and Angel hurriedly scoots his chair back.
Angel: "Yeah. That may be the problem. (Chuckles uncomfortably) I mean, whatever we - had... - whatever we - did. I just think that we should keep that - behind us. - Start from scratch. You know, two men working side by side. But, you know, none of that - funny stuff." Wes straightens up, a slight frown on his face. Angel offers his hand.
Angel: "Shake on that?"
Wes after a beat: "I guess." Wes takes Angel's hand, who gives it a hearty shake, then pulls Wes into a big hug.
Angel: "Hey, all right. Gimme a hug."
Cordy: "Wesley, food's here."
Wes: "Okay." Angel pulls back from Wes, holding him at arms length.
Angel: "Wesley?"
Wes: "Yes?"
Angel: "Do you know where Fred is?"
Wes: "Uhm - up in her room I'd expect."
Angel: "*Her* room. Right. - Somebody say something about food? I could eat a horse." Angel turns and walks out of Wes' office. Gunn is standing in front of a carton holding cups and fast food.
Gunn: "Breakfast burritos all around." Angel grabs one of the burritos, and sticks some money into the breast pocket of Gunn's jacket.
Angel: "Thanks, bro. Keep the change on that."
Gunn: "O-kay."
Wes: "Get anything from the hotel staff?" Angel sitting at his little folding desk munching on the burrito looks over at Gunn and Wes.
Gunn: "Yeah, I did. All these guys ran up huge service bills, mostly alcohol. Well, at least they went out partying. Oh, and I got copies of their telephone bills, too." Angel talking with his mouth full: "Hey, isn't that illegal? I mean, don't these guys deserve a little privacy?" They all turn to stare at Angel.
Angel: "What?"
Cordy: "Why are you eating?"
Angel: "I'm hungry."
Wes: "Looks like they called the same number."
Gunn: "Yeah, saw that, too. Checked it out. (Pulls out a paper and hands it to Wes) First class escorts, La Brea and sixth." Angel gets up and moves over to them.
Cordy: "Escorts. Oh, you mean hookers?" Gunn takes the paper out of Wes' hand: "I should probably interview them right away - while the trail is hot."
Wes takes the paper back: "Ah, I'll take this one. You interviewed the hotel staff. It's only fair if we divvy it up."
Gunn again taking the paper: "Yeah, but I figured it out."
Cordy snatches the paper: "I'll interview the hookers. Are there any men who aren't just dogs?"
Angel: "Not very many, I'm afraid. (Leans in close to Wes) You know a woman is more than a piece of meat. I'm sorry. That's just how I feel." Wes' beeper goes off and he checks the display.
Wes: "Ah, my contact at the coroner. I can see one of the bodies. I should go."
Cordy: "Gunn can go with you."
Gunn: "That wasn't the kind of body I had in mind to see. (Cordy looks at him) We're going, we're going." Gunn and Wes leave and Cordy follows them.
Angel: "Hey, you know what? That's a great idea. I'll just stay here, hold the fort - keep an eye on the evidence." Crams some more burrito in his mouth. Marcus is sitting on a sofa in the lobby of the Monserrat with an open book on his lap. He sees the security guard at the desk by the doors get up to help an old lady with a walker accompanied by a younger woman through the door. Watches as the guard helps them over to the elevators. He gets up to head for the unguarded doors, but is intercepted by an old, black man being visited by his family.
Jackson: "Marcus, I got someone I want you to meet."
Marcus: "Oh, I can't right now." Jackson takes the baby from his daughters arms and hands her to Marcus.
Jackson: "This is my baby granddaughter Katrina. (To baby) Girl's gonna *rule* the world! (To Marcus) Isn't she something?" Marcus takes the girl in his arm a smile spreading across his face. He looks past Jackson to the unoccupied guard desk, looks back down at the baby.
Marcus: "She's beautiful." Marcus looks over to see that the guard has returned to his post. Angel is in Wes' office, shredding the newspaper clippings of the mysterious dead guys. That done, he sits back with a sigh, puts his feet up on the desk and picks up a martini glass sitting on it to take a sip.
Fred: "What you doing?"
Angel sees Fred and grins: "Well. Hey, sweetheart. Where you've been hiding?"
Fred flustered: "You know, up in my room. Everybody keeps saying 'Fred, you should get out more' so, well..."
Angel: "Fred - mmm." Angel takes a sip from his glass then gets up and walks around the desk to stand in front of Fred.
Angel: "Have I ever told you you are a very *beautiful* woman?
Fred: "Uhm - no?"
Angel: "Do you like olives?" Angel pulls the toothpick with the olive out of his drink and offers it to Fred, who eats it out of his hand.
Angel: "Tell you what, I have some work I have to finish up here. Why don't you go on upstairs and put on something pretty and we'll go out on the town."
Fred: "Really?" Angel tips her on the nose with the toothpick: "And that's just for starters."
Fred: "Okay, I'll just - I'll go and - okay." Runs out of the office.
Angel shakes his head: "Hoo!" He goes to sit back down at the desk, whistling and shredding files. Lilah walks into the office and Angel turns the shredder off.
Angel: "And what can I *do* for *you*?"
Lilah: "Don't go all nightstalker on me. I'm here to do you a favor. (Angel checks Lilah out) We both agree that business with Cordelia was just business, right?"
Angel after a beat: "Sure." Lilah pulls a bundle of folded papers from her briefcase and drops on the desk in front of Angel.
Lilah: "It's all in there. Earthquake safe certification, statement of asbestos level compliance... All of it." Angel picks up the bundle then drops it back on the desk. He looks up at Lilah. Stands up, picking up his martini glass and walks around the desk.
Lilah: "I'm not playing you here. It's not about you. It's about Gavin. He thinks he's so smart. (Angel pour two martini glasses) - You're welcome."
Angel: "I'm sorry. Thanks. (Puts olives in each glass) That was a really - thoughtful favor. - How about a drink? (picks up the two glasses and offers one to Lilah) Have I ever told you you're a very *beautiful* woman?" Lilah hesitantly accepts the glass and Angel clinks his against hers. Marcus is walking down some steps in the Monserrat. Peeks around the corner at the end of the hallway to see the security guard at the desk reading a newspaper. Scoots back away. Notices the fire alarm on the wall. Glances back at the guard station, then trips the alarm. The alarm sounds and the guard jumps up from his desk and hurries away to look for the source. Marcus shuffle-runs towards the desk and the exit. Looks down at his left arm, grabs it with his other hand, starts to huff and puff.
Marcus: "M-my heart..." Slowly collapses to the ground. Lilah is sitting in a chair across from Angel, who is sitting on Wes' desk. She sets down her empty glass.
Angel: "Want another?"
Lilah: "I'm gonna have to call a taxi as it is." Lilah gets up to leave - only to run against Angel's leg, stretched straight out to bar her way.
Angel chuckles: "Oops!"
Lilah: "What do you want?" Angel brushes the hair back from Lilah's face: "You. - Don't tell me you never thought about it." Angel stands up, leans in close and kisses her. Lilah pulls away shaking her head a little. Angel smiles at her then pulls her in for another kiss - which Lilah returns, passionately. They start groping each other. Lilah rips Angel's shirt open. Angel sweeps the stuff on Wes' desk to the side and pushes her down onto it. Fred walks up to the open door of Wes' office, wearing a long dress, her long hair open, with a shy smile on her face. She walks in to see Angel and Lilah groping wildly on top of Wes' desk. Her eyes widen, then she turns and runs away. Angel is nuzzling Lilah's neck. Suddenly vamps out and bites her as Lilah lets out a surprised scream. Break
[SCENE_BREAK]
Lilah pushes Angel off her and gets up off the desk.
Lilah: "You son of a bitch!"
Angel: "Whoa! I'm sorry! It just - felt like the thing to do. (Stares at the cross Lilah is holding up to ward him off) Whoa! What are you born again all of a sudden?"
Lilah: "I don't know what kind of sick game this is, Angel, but I hope you enjoyed it because you're never getting this close to me again." Angel is trying to get closer only to find himself shying back from the cross in her hands. Lilah walks out and Angel lifts a hand to run it through his hair - only to touch the thickened brow of his forehead.
Angel: "What? This is new." Angel explores his face with his fingers, pricks his right thumb on his fangs.
Angel: "Ow!" Angel reflectively sticks his thumb in his mouth and sucks on it, takes it back out to look at it in surprise, then sticks it back in to suck some more.
Angel: "Ah. Hmm..." Still sucking on his thumb he walks into the bathroom - only to *not* see himself in the mirror.
Angel: "What the..." Marcus is lying on a hospital bed, and IV going into the back of his right hand, watching the lights on the heart monitor beside his bed.
Ryan: "You're awake."
Marcus looks up at him: "It's beating." His right hand is resting on his chest over his heart, gently keeping the beat.
Ryan: "That was your fourth heart attack, Marcus. I don't know if you can survive another one. You got lucky this time. Try something like that again, you may not be." Marcus returns to watching the heart monitor display. Hyperion, night. Cordy walks into the dimly lit lobby.
Cordy: "If Julia Roberts ever makes a realistic movie about being an escort, I think it should be called pretty skanky woman." Cordy stops by the counter to look around the deserted lobby.
Cordy: "Angel? - Fred!" Cordy puts her stuff down. Hears some soft sobbing noises. She walks over and opens the elevator doors to reveal Fred hunched up in one corner of it, her arms wrapped tightly around her knees, crying.
Cordy: "Fred. What's wrong? What happened?"
Fred: "I should've knocked. I always forget to knock because, you know, I didn't have a door for so long. (Fred looks over at Cordy, trying to suppress her sobs) He called me a sweetheart. But it's just an expression, isn't it? Like when a waitress calls you honey, it doesn't mean your special or anything. It's just a word, right? Sweetheart."
Cordy softly: "Is this about Angel? (Fred nods) Oh. - He talked to you, didn't he?" Cordy sits down beside Fred and sighs.
Cordy: "This is all my fault. I told him to do that." Fred's turns her head to stare at Cordy.
Fred: "You told him to make out with that woman on the desk?"
Cordy: "What? No. - What woman?" Angel walks into a nightclub, moving with the music. Stops by the bar and turns to survey the room. He spots a pretty, dark haired girl sitting at a table with her boyfriend. The girl looks over at him and their eyes lock for a moment before she turns her attention back to her boyfriend. Angel keeps watching as the boyfriend gets up, taking his empty glass with him. The girl looks back over at Angel still standing by the bar. Some people walks past between them and after they're gone the girl's face falls as Angel is gone as well, only to set a full martini glass down in front of her a moment later. Angel is sitting with the girl at the table, whispering something in her ear. Under the table his hand is on her leg. Flash cut to Angel pulling the laughing girl after him out of the door to the club onto a balcony. The boyfriend and another guy walk up to the table to find it deserted. Angel and the girl are kissing out in front of the club.
Girl with a smile: "My boyfriend is probably looking for me right now. He could catch us at any moment!" Pulls Angel in close for another kiss.
Girl: "Sort of makes it more exciting doesn't it?"
Angel: "Yeah. (Pulls back from nuzzling her neck and the girl screams at his vamp face) It does." Angel leans in and bites her, just as her boyfriend and two other guys walk out onto the balcony behind them.
Boyfriend: "Hey!" Angel lets the girl go and spins around, morphing back into his human face as he does so.
Boyfriend: "What the hell do you think you're doing?" Angel licks the blood from the corner of his lips.
Girl: "He bit me!"
Boyfriend: "Freak!" He hauls back to hit Angel, but Angel catches his fist in his hand, clamping down on it. The guy groans in pain, then flies back as Angel hits him. Angel hold up his fist and looks at it.
Angel: "Nice!" Angel stands there inviting the other two guys to hit him. He takes a kick to the face from the first guy, then grabs him by the throat, head butts him and tosses him aside. Walks right into the fist of the second guy, and returns the hit with a wide swing of his own then tosses that guy to the side as well and turns to face the boyfriend, scooting backwards away from him.
Angel: "Come on! Bring it on. Is that it? Is that all you got?"
Boyfriend: "What are you on?"
Angel: "Well, you know, I'd say I'm high on life only - I ain't alive - which means - I'm never gonna die. I'm gonna be young, handsome and *strong* forever! (Laughs) There is just one thing I gotta do first!" With that Angel turns away, runs over to the railing, jumps on top, then off of it, only to easily land some stories below running down the street laughing.
Wes: "I do not believe it. On my desk?"
Gunn: "Well, it did used to be his. Maybe he was just kinda - reclaiming it."
Wes: "How? By marking it? - This isn't like him." Wes walks around the desk and crouches down to look at some books lying open on the floor.
Cordy: "What? This is totally like him. Doing the mystery dance with some cheap blonde?"
Fred: "Brunette. She was a cheap brunette."
Cordy: "You're right. This isn't like him."
Gunn: "So, who was she?"
Wes: "I don't think it matters who *she* was. The question is, who is *he*?"
Cordy: "Uh-huh. We're all thinking it. - He's Angelus again."
Fred: "Who's Angelus?"
Gunn: "The bad-ass vamp Angel turns into when he gets evil. But then why is there no body here? Wouldn't he've just killed her?"
Wes: "No, that's not what I meant. Why would Angel, or Angelus for that matter, (holds up the open books) need to read about vampires?"
Cordy: "He wouldn't."
Gunn: "Wait. What are you getting at?"
Wes: "This case we've been working on. Each of the victims exhibited wild, uncharacteristic behavior just before they died. They weren't themselves."
Gunn: "Oh! So you think Angel's been infected by whatever got into those gym boys."
Wes: "Not 'whatever' - whomever. Cordelia, when you and him were at the gym did anything unusual happen to Angel?"
Cordy: "No, not really. I was with him pretty much the whole time. - Except for when he went across the street to the..."
Cut to the Monserrat retirement community. Marcus is sneaking down a hallway. He enters a deserted common area off the lobby. Sticking his hands in his pockets, he tries to act casual.
Jackson: "Hey. (Marcus jumps and turns around) What the hell are you doing out of bed? You trying to bust loose again, ain't you? Damn, Marcus - you don't quit, do you? Do want to have another heart attack?"
Marcus: "Look - I can't really explain this, but I *need* to get out of here. (Puts his hands on Jackson's shoulders) Just - don't turn me in."
Jackson: "Not gonna have to. Your kid was signing in at reception when I came down."
Marcus: "My kid?"
Jackson: "M-hm." Jackson walks off. Marcus turns and sees Angel talking to Ryan at the other end of the lobby. Angel turns and looks at Marcus with a smile.
Angel: "Hi, dad." Break Marcus backs out of Angel's view. Angel slowly saunters across the lobby after him. Angel turns the corner down a branch of a deserted hallway. He sniffs the air, then turns around, smiling, to enter the darkened rec-room. Marcus is standing in the shadows besides the door. As Angel turns on the light, Marcus hits him over the head with a shuffleboard staff. Angel is thrown off balance but catches Marcus' next hit and pulls the staff from his hands. Marcus staggers back.
Marcus: "So, I guess you finally found a body that won't burn out, huh, Marcus?"
Angel: "Looks like."
Marcus: "Only one way you can keep it though, right? You got to kill yourself!" Marcus smacks his fist against his chest over his heart.
Angel mimics the gesture: "Mmm! I can live with that!"
Marcus: "You sure? I don't think you *really* know what you're getting into." Angel steps away from the door, walking towards Marcus, standing in the middle of the room.
Angel: "Oh, I know what I'm getting into. *You're* the one that doesn't seem to know what you had. As far as I can tell you were the world's worst vampire. Vampires don't *help* people, you *moron* - they kill 'em! Here, let me show you."
Marcus holds up a hand: "You may have the attitude, and you may have the power - but there is one thing you don't have, and never will: friends. (The shot widens to reveal Cordy, Fred, Wes and Gunn walking in through the door behind Angel) Four of them, standing behind you (Angel spins around to look) with big, heavy things."
Angel: "Guys! It's about time. It's *him* - he's the one who's been casting that spell."
Cordy: "You're Angel? With *that* cologne? I don't think so." Gunn walks up and put his loaded crossbow against Angel's chest.
Marcus: "Don't stake him." Angel bats Gunn's crossbow aside and lifts his staff to push both Gunn and Wes into the wall. Angel drops the staff and turns to run - right into Cordy and Fred and their wooden baseball bats. Gunn uses the shuffleboard staff to swipe Angel's legs out from under him, dropping Angel. Angel jumps back up, wrestles the bat away from Fred and turns with it towards Marcus. Cordy pulls out a tazer and hits Angel with a charge of blue-white light. Angel lands face down, in an unmoving heap at Marcus feet.
Cordy: "God, I love technology. (Hurries over to Marcus) Are you alright?"
Marcus: "I gotta pee."
Wes: "Did you happen to notice a small Algurian conjuring orb? Could have been glowing."
Marcus: "In his room, on a shrine."
Wes: "Then I was right. (Looks down at Angel's body) Algurian body-switching spell. Keep an eye on him." Fred hauls back and hits Angel's body over the head, causing Gunn to jump.
Marcus: "Fred! He's out! He's out!" They have tied Angel's unconscious body to a chair in Marcus' room. Marcus sits down on the chair across from him as Wes hands him a piece of paper.
Wes: "Read this."
Marcus: "Alli permutat anima kimota. Alli permutat anima kimota." Angel's head comes up. A blue light starts to issue from Marcus eyes and nose, a red one from Angel's eyes. The blue light disappears into Angel's mouth, while the red light enters Marcus as soon as the blue light leaves it. Marcus and Angel's head sag onto their chests, but after a moment Angel's head comes back up. He looks up at Gunn, a slightly dazed expression on his face.
Angel: "It's cool, Gunn. It's me." Gunn bends to untie Angel's hands.
Cordy: "I got his conjuring stone." Angel takes it into his hand.
Marcus: "You can't take that!" Angel closes his hand, crushing the stone to powder.
Marcus: "You...! You don't deserve that body!"
Angel: "Funny. I was gonna say the same thing to you. (Stands up) I tell you why you have a weak heart, Marcus. You never use it." Angel starts to leave, with the others falling in behind him.
Marcus: "You're pathetic! (Stands up) You're all pa..."
Angel, turning back: "You should try and keep a lid on that rage, Marcus. It's - not healthy." Angel and the gang walk out as Marcus sinks back into his chair, clutching at his heart.
Marcus moaning weakly: "Help..." Ryan and another attendant see Angel and the gang coming out of Marcus room.
Ryan: "What's going on?"
Angel: "Dad's having a bad night." The Hyperion's garden court, night. Fred is sitting on the edge of the dry fountain, reading a book. Angel comes down the steps behind her.
Angel: "Hi."
Fred: "Hey. - How's your head? S-sorry about all that..." Fred makes some hitting movements.
Angel: "Ah, I - gather I - had it coming."
Fred: "Mmm. - Yes." Angel goes to sit down beside her.
Angel: "Fred, I've been meaning to talk to you about something." Fred puts her book down and smiles at him: "Okay." Angel sits there silently staring into the night for a beat.
Fred: "Is this about how you're not like other men - what with that curse and all... and how you're really fond of me, but that's as far at it goes?"
Angel: "Uhm... - yeah."
Fred: "Cordelia explained it to me. (Gives a little chuckle) She said you'd probably just screw it up."
Angel: "Oh, she did, did she? - And she's probably right." Fred sighs and looks down.
Angel: "What?"
Fred: "It's like something out of Fitzgerald. - The man who can have everything but love. - Well, maybe in some ways you're better off, because love is... - Well, in a way it's everything. - But it's also heartache and disappointment. - And those are good things to avoid." Cordy comes hurrying out and Angel and Fred turn their heads to look at her.
Cordy, a bit breathless: "Angel, Willow's on the phone... She's alive! Buffy's alive!" With that Cordy turns around and hurries back in. Angel and Fred look at each other for a beat then Angel jumps up and runs into the hotel after Cordy.
Fred: "Buffy?" | While investigating some mysterious deaths, Angel switches bodies with a old guy named Marcus. Marcus immediately takes over Angel's life, while Angel is stuck in a retirement home. When Marcus realizes that Angel's body gives him vampire powers, he decides that they only way to keep it is to get rid of Angel once and for all. | summ_screen_fd |
UIW police officer involved in fatal shooting SAN ANTONIO — Just before University of the Incarnate Word senior Robert Cameron Redus was fatally shot in a struggle with a UIW police officer Friday morning, a neighbor overheard what may have been his last words. “I heard (a man) say, 'Oh, you're gonna shoot me?'” in a surprised voice, said Mohammad Haidarasl, 22, who was on his couch in his ground-floor unit at the Treehouse Apartments in Alamo Heights at about 2 a.m. Less than a minute later, Haidarasl heard four to six gunshots. “I jumped up and hid in my closet,” he said. Photo: JOHN DAVENPORT, SAN ANTONIO EXPRESS-NEWS Image 1 of / 12 Caption Close Image 1 of 12 Law enforcement officers investigate a crime scene where a 23-year-old man that was shot to death after 2:00 a.m. by a University of the Incarnate Word police officer after a traffic stop that ended in the parking lot of the Treehouse Apartments at Broadway and Arcadia Place in Alamo Heights. Lt. Cindy Pruitt of the Alamo Heights Police Department said the man struggled with the officer after the stop and was shot. An investigation of the incident is ongoing. less Law enforcement officers investigate a crime scene where a 23-year-old man that was shot to death after 2:00 a.m. by a University of the Incarnate Word police officer after a traffic stop that ended in the... more Photo: JOHN DAVENPORT, SAN ANTONIO EXPRESS-NEWS Image 2 of 12 Law enforcement and Bexar County Medical Examiner's personnel prepare to remove the body of a 23-year-old man that was shot to death after 2:00 a.m. by a University of the Incarnate Word police officer after a traffic stop that ended in the parking lot of the Treehouse Apartments at Broadway and Arcadia Place in Alamo Heights. Lt. Cindy Pruitt of the Alamo Heights Police Department said the man struggled with the officer after the stop and was shot. An investigation of the incident is ongoing. less Law enforcement and Bexar County Medical Examiner's personnel prepare to remove the body of a 23-year-old man that was shot to death after 2:00 a.m. by a University of the Incarnate Word police officer after a... more Photo: JOHN DAVENPORT, SAN ANTONIO EXPRESS-NEWS Image 3 of 12 Law enforcement officers investigate a crime scene where a 23-year-old man that was shot to death after 2:00 a.m. by a University of the Incarnate Word police officer after a traffic stop that ended in the parking lot of the Treehouse Apartments at Broadway and Arcadia Place in Alamo Heights. Lt. Cindy Pruitt of the Alamo Heights Police Department said the man struggled with the officer after the stop and was shot. An investigation of the incident is ongoing. less Law enforcement officers investigate a crime scene where a 23-year-old man that was shot to death after 2:00 a.m. by a University of the Incarnate Word police officer after a traffic stop that ended in the... more Photo: JOHN DAVENPORT, SAN ANTONIO EXPRESS-NEWS Image 4 of 12 Law enforcement and Bexar County Medical Examiner's personnel prepare to remove the body of a 23-year-old man that was shot to death after 2:00 a.m. by a University of the Incarnate Word police officer after a traffic stop that ended in the parking lot of the Treehouse Apartments at Broadway and Arcadia Place in Alamo Heights. Lt. Cindy Pruitt of the Alamo Heights Police Department said the man struggled with the officer after the stop and was shot. An investigation of the incident is ongoing. less Law enforcement and Bexar County Medical Examiner's personnel prepare to remove the body of a 23-year-old man that was shot to death after 2:00 a.m. by a University of the Incarnate Word police officer after a... more Photo: JOHN DAVENPORT, SAN ANTONIO EXPRESS-NEWS Image 5 of 12 Law enforcement officers investigate a crime scene where a 23-year-old man that was shot to death after 2:00 a.m. by a University of the Incarnate Word police officer after a traffic stop that ended in the parking lot of the Treehouse Apartments at Broadway and Arcadia Place in Alamo Heights. Lt. Cindy Pruitt of the Alamo Heights Police Department said the man struggled with the officer after the stop and was shot. An investigation of the incident is ongoing. less Law enforcement officers investigate a crime scene where a 23-year-old man that was shot to death after 2:00 a.m. by a University of the Incarnate Word police officer after a traffic stop that ended in the... more Photo: JOHN DAVENPORT, SAN ANTONIO EXPRESS-NEWS Image 6 of 12 Alamo Heights Police Lt. Cindy Pruitt reads a statement to the media Friday December 6, 2013 about a fatal shooting incident involving a University of Incarnate Word police officer that took place in Alamo Heights on the 100 block of Grandview. The fatal shooting took place about 2:00 a.m. Friday December 6, 2013 at the Treehouse Apartments after a traffic stop. The deceased suspect driver has been identified as Robert Cameron Redus, a 23-year-old University of the Incarnate Word student. The press conference took place at Alamo Heights city offices at 1248 Austin Highway. less Alamo Heights Police Lt. Cindy Pruitt reads a statement to the media Friday December 6, 2013 about a fatal shooting incident involving a University of Incarnate Word police officer that took place in Alamo... more Photo: JOHN DAVENPORT, SAN ANTONIO EXPRESS-NEWS Image 7 of 12 Mohammad Haidarasl, a UIW student, looks out his apartment to the parking lot of the Treehouse Apartments where his upstairs neighbor Robert Cameron Redus was shot to death by UIW campus police officer Chris Carter, following a scuffle. Friday, Dec. 6, 2013. Haidarasl lives directly below Redus and was asleep on his sofa next to the window, center, when he heard a struggle, yelling, and then gunshots. The motorcyle and bike at left, belong to Redus. less Mohammad Haidarasl, a UIW student, looks out his apartment to the parking lot of the Treehouse Apartments where his upstairs neighbor Robert Cameron Redus was shot to death by UIW campus police officer Chris... more Photo: BOB OWEN, San Antonio Express-News Image 8 of 12 Bea Perry, right, reacts after neighbor Mohammad Haidarasl tells her that their neighbor Robert Cameron Redus, a UIW student from Baytown, TX, was shot to death by UIW campus police officer Chris Carter, Friday, Dec. 6, 2013. Haidarasl lives directly below Redus and was asleep on his sofa when he heard a struggle, yelling, and then gunshots from the parking lot. Perry also heard the gunshots early Friday morning. less Bea Perry, right, reacts after neighbor Mohammad Haidarasl tells her that their neighbor Robert Cameron Redus, a UIW student from Baytown, TX, was shot to death by UIW campus police officer Chris Carter,... more Photo: BOB OWEN, San Antonio Express-News Image 9 of 12 Law enforcement officers including some from Alamo Heights Police Department, leave the Treehouse Apartments after inspecting the apartment, above right, of Robert Cameron Redus, a UIW student from Baytown, TX who was shot to death by UIW campus police officer Chris Carter. Friday, Dec. 6, 2013. less Law enforcement officers including some from Alamo Heights Police Department, leave the Treehouse Apartments after inspecting the apartment, above right, of Robert Cameron Redus, a UIW student from Baytown, TX... more Photo: BOB OWEN, San Antonio Express-News Image 10 of 12 A note for the Redus family with a charm and a rose was placed outside the apartment of Robert Cameron Redus, a UIW student from Baytown, TX, who was shot to death by UIW campus police officer Chris Carter. Friday, Dec. 6, 2013. less A note for the Redus family with a charm and a rose was placed outside the apartment of Robert Cameron Redus, a UIW student from Baytown, TX, who was shot to death by UIW campus police officer Chris Carter.... more Photo: BOB OWEN, San Antonio Express-News Image 11 of 12 Robert Cameron Redus was on the dean's list in fall 2012 and spring 2013. Robert Cameron Redus was on the dean's list in fall 2012 and spring 2013. Image 12 of 12 UIW police officer involved in fatal shooting 1 / 12 Back to Gallery Haidarasl didn't look out the window until he heard emergency vehicles converging on the quiet apartment complex. It wasn't until later that he realized the person shot was his upstairs neighbor, whom he described as “the nicest guy.” Redus, 23, would have graduated from UIW in May, university officials said. He was on the dean's list in fall 2012 and spring 2013, the university's website noted. Videos from UIWTV.org, a campus television station operated by students in the Communication Arts Department, showed Redus anchoring news segments last year. He had four brothers and grew up in Baytown, where he graduated as co-valedictorian from Baytown Christian Academy, friends said. A statement released by the family said, “We are understandably devastated by the death of our dear son Cameron and we ask for your prayers as we deal with our tragic loss. We trust that God is faithful and will see us through this most difficult time.” As details of the shooting were slow to emerge Friday, friends had questions about what exactly occurred when UIW police Cpl. Christopher J. Carter tried to pull Redus over for a traffic violation, and whether the use of force was necessary. Sara Davis, 20, and her sister Annie Jones, 22, both of Baytown, described Redus as kind, intelligent, compassionate and well-loved within the community. “He was not an aggressive person at all, so the story doesn't make sense,” Davis said. Alamo Heights police Lt. Cindy Pruitt said Carter, in a marked UIW pickup, told officials he noticed Redus speeding and driving his Ford Ranger pickup erratically on Broadway. He was not on campus, Pruitt said, and officials did not give an exact location where Carter first spotted Redus. Pruitt said both vehicles, with Carter's emergency lights on, drove north on Broadway until they pulled into the parking lot of the Treehouse Apartments at Broadway and Arcadia Place. Once in the parking lot, they both got out of their vehicles and became involved in a struggle, Pruitt said. The officer radioed for help during the struggle and Redus was shot multiple times. He was pronounced dead at the scene. Carter has been placed on paid administrative leave during the investigation, the university announced Friday afternoon. He was described by university officials as having an “extensive law enforcement background.” Over the course of Carter's eight-year law career in Texas, he has held nine jobs at eight agencies, including two stints at the Bexar County Sheriff's Office, files kept by the state agency for licensing peace officers show. He rarely was at an agency more than a year and his shortest job was for seven months as a reserve officer for the San Antonio Municipal Court Marshals Division. So far, the two years and seven months he's spent at UIW is the longest stretch of employment, the files show. The University of the Incarnate Word employs 17 police officers who all are licensed and trained as state-certified peace officers, university spokeswoman Debra Del Toro said. None of the officers carries a Taser. A state-licensed peace officer working for a private institution can enforce state and municipal laws outside the campus jurisdiction in a variety of instances, says the Texas Education Code, which governs the agencies. That includes “whether the officer is on property under the control and jurisdiction of the institution, but provided these duties are consistent with the educational mission of the institution and are being performed within a county in which the institution has land,” the code states. “Our thoughts and prayers go out to the families of the student and officer involved in this incident,” UIW President Dr. Lou Agnese said in a news release. Police were investigating whether Redus had a weapon or was threatening the police officer's life, Pruitt said. The Texas Rangers are assisting in the investigation. “This is really in its preliminary stages,” Pruitt said. “This investigation will go on for days.” Haidarasl said no one from the Alamo Heights Police Department had tried contacting him, other than Friday morning when he went outside and officers told him he wouldn't be able to move his truck for a while. Friday afternoon, he stood and chatted with neighbor Bea Perry, who lives in the same building. She, too, expressed surprise that Redus was the one shot by officers. “He was such a nice kid; he had so much potential,” Perry said, adding that she hoped someone would be around to care for Redus' fluffy black cat. “So did you see anything?” she asked Haidarasl. He launched into his story again, explaining that he heard the officer say more than once, “Stop resisting, stop resisting.” He thought he heard a struggle, he said. “Then the cop said, 'I'm going to shoot,'” Haidarasl said. And that, Haidarasl added, was what garnered the off-handed, sarcastic comment from their neighbor. Then the silence, followed by gunfire. [email protected] Staff Writer Alia Malik contributed to this report. An earlier version of this story mischaracterized a statement from witness Mohammad Haidarasl. Story highlights Student hit police officer with officer's own baton, university says University says Cpl. Christopher Carter now on administrative leave Police: Campus officer said Robert Cameron Redus struggled with him Friends: Redus was co-valedictorian of Christian high school Those who know him call him the "gentlest person," but campus police said Monday that a 23-year-old student at a San Antonio-area Christian college took an officer's police baton and struck him before the officer fatally shot him. There is no dashboard video available, according to the university. The incident began when Cpl. Christopher Carter, a police officer with the University of the Incarnate Word in Alamo Heights, saw Robert Cameron Redus near campus "driving erratically at a high rate of speed" Friday, a university statement said. "Carter was obligated to pull the suspect over to ensure the public's safety," the statement said. Redus pulled into an apartment complex, and Carter followed, mistakenly reporting the wrong street location to police dispatchers, which prompted his call to be routed from the Alamo Heights Police Department to its San Antonio counterparts, the statement said. This caused a delay of several minutes in response time. "During the wait for assistance, the officer tried to restrain the suspect who repeatedly resisted," the statement said. "During the struggle, the officer attempted to subdue the suspect with his baton.... The baton was taken by the suspect who used it to hit the officer. "The officer drew his firearm and was able to knock the baton from the suspect who continued to resist arrest. Shots were fired." Redus did not identify himself, the university said, and there is "no evidence" the officer knew he was a student or where Redus lived. University police vehicles are typically equipped with dashboard cameras, but Carter's vehicle joined the fleet two days before the incident, and its camera fell off the next day when a temperature change prevented the glue from setting, the school said. "Officers had made arrangements to have it remounted," the statement said. Carter, who has "an extensive law enforcement background," has been placed on administrative leave -- standard procedure in these types of incidents, a university statement said, adding that all campus officers "are licensed and trained as certified peace officers by the state of Texas." Friends at the school say the Cameron Redus they know isn't the type to attack police. They knew a student who made the dean's list at the Catholic college and had been co-valedictorian of a Christian high school back home in Baytown, Texas. They knew a fun-loving campus television news anchor who was "the sweetest, kindest, gentlest person," as friend Annie Jones described him to CNN affiliate WOAI-TV. A resident of Redus' apartment complex, 22-year-old Mohammad Haidarasl, told the San Antonio Express-News that Redus was his upstairs neighbor. Haidarasl told the paper he was on his apartment sofa at 2 a.m. when he heard noise outside, and a voice he believes to have been the officer's saying, "Stop resisting, stop resisting." The newspaper quoted Haidarasl as saying he thought he heard a struggle and "Then the cop said, 'I'm going to shoot.' " A male voice replied, " 'Oh, you're gonna shoot me?' like sarcastic almost," Haidarasl said. Less than a minute later, he said, he heard shots. Alamo Heights police acknowledged the officer fired several shots. But they would not discuss other details of the alleged struggle, citing the ongoing investigation. The university said it was awaiting the results of the Alamo Heights police probe, which is being conducted with assistance from the Texas Rangers. Redus' family released a statement to CNN affiliate KENS-TV saying, "We are understandably devastated by the death of our dear son Cameron and we ask for your prayers as we deal with our tragic loss. We trust that God is faithful and will see us through this most difficult time." University President Lou Agnese said in a statement released to WOAI, "Our thoughts and prayers go out to the families of the student and officer involved in this incident." The university released a statement saying it, too, "is deeply upset over the loss of life regardless of the circumstances." It further said this was the first shooting in university history. Hundreds of people, including relatives of Redus, gathered at the university's convocation center Saturday for a vigil. Students brought a slideshow of Redus in happy poses. "It makes me feel better that we've got a lot of support for Cameron," classmate Albert Salinas said outside the event in an interview with CNN affiliate KSAT-TV. But they left with no better idea of what happened to their friend. Shooting leaves 2 high-schoolers dead at house party near Houston | A student at a Christian university was shot dead on Friday for allegedly resisting arrest, but fellow students are struggling to believe the official story, CNN reports. It goes like this: Robert Redus, a student at the University of the Incarnate Word in Alamo Heights, was "driving erratically at a high rate of speed" near campus. When police officer Christopher Carter confronted him in an apartment complex, the student resisted arrest, grabbed his baton, and "used it to hit the officer," according to the university. Carter pulled his gun and knocked the baton away, but when Redus kept resisting arrest, "shots were fired." Carter's police vehicle didn't have the usual dashboard camera because his car was new, and the camera had fallen off when a temperature change stopped the glue from setting, the university said. But Redus' friends say he was unlikely to attack anyone-a fun-loving co-valedictorian at a Christian high school, he was "the sweetest, kindest gentlest person," a friend said. A neighbor told the San Antonio Express-News he heard the struggle downstairs, with one man telling the other to stop resisting. "Then the cop said, 'I'm going to shoot,'" the neighbor said, and a man replied, "'Oh, you're gonna shoot me?' like sarcastic almost." Then shots were fired. Carter is on administrative leave while police investigate. | multi_news |
H2A. Z is a histone H2A variant conserved from yeast to humans, and is found at 63% of promoters in Saccharomyces cerevisiae. This pattern of localization suggests that H2A. Z is somehow important for gene expression or regulation. H2A. Z can be acetylated at up to four lysine residues on its amino-terminal tail, and acetylated-H2A. Z is enriched in chromatin containing promoters of active genes. We investigated whether H2A. Z' s role in GAL1 gene regulation and gene expression depends on H2A. Z acetylation. Our findings suggested that H2A. Z functioned both in gene regulation and in gene expression and that only its role in gene regulation depended upon its acetylation. Our findings provided an alternate explanation for results that were previously interpreted as evidence that H2A. Z plays a role in GAL1 transcriptional memory. Additionally, our findings provided new insights into the phenotypes of htz1Δ mutants: in the absence of H2A. Z, the SWR1 complex, which deposits H2A. Z into chromatin, was deleterious to the cell, and many of the phenotypes of cells lacking H2A. Z were due to the SWR1 complex' s activity rather than to the absence of H2A. Z per se. These results highlight the need to reevaluate all studies on the phenotypes of cells lacking H2A. Z. In addition to their role in genome packaging, histones also play a role in the functional organization of eukaryotic genomes. Clear causal relationships have been established between some specific modifications of histones at specific loci and the subsequent events that occur at these loci. Histones are modified by enzymes that couple acetyl, methyl, phosphoryl, ubiquitin, or sumo moieties to specific locations either on histone tails, which extend outward from the nucleosome core, or at positions in the core, such as acetylation of H3 lysine 56, near where the DNA helix enters and leaves the nucleosome [1]. Modified histone tails serve in some cases as docking sites for protein complexes. Thus, in principle, a particular collection of modifications on the nucleosomes of a locus can recruit specific complexes to that locus to achieve a particular outcome [2]–[7]. In addition to histone modifications, nucleosomes can also be specialized by virtue of the presence of histone variants. Saccharomyces encodes three histone variants: H2A. Z, which is conserved from yeast to humans; a variant of H2B called H2B2, conserved among yeasts; and Cse4, an H3 variant, which functions at the nucleosomes at centromeres [8]. Like Cse4p, H2A. Z is also localized to specific chromosomal locations with specialized functions. In S. cerevisiae, H2A. Z is incorporated into nucleosomes near, but not at, centromeres, at the borders of heterochromatic domains, and near the promoters of 63% of genes [9]–[12]. H2A. Z is incorporated into chromatin by the SWR1 complex (SWR1-Com) a multi-subunit enzyme whose catalytic subunit, Swr1, is a member of the Swi2/Snf2 family of chromatin remodeling enzymes [13]–[15]. H2A. Z' s localization at promoters suggests that it plays an important role in gene expression. Yet genome-wide micro-array analyses indicate that H2A. Z affects the steady-state mRNA levels of only 5% of S. cerevisiae' s genes [16]. Interestingly, most of the genes downregulated in cells lacking H2A. Z were near the boundaries of SIR-silenced heterochromatin. This observation revealed that H2A. Z functions as part of the boundary separating euchromatin and heterochromatin [16]. H2A. Z is acetylated at up to four positions on its N-terminal tail by the NuA4 and SAGA histone-acetyltransferase complexes [17]–[19]. Moreover, H2A. Z' s heterochromatin-boundary function depends on its acetylation [17]. Promoter-proximal H2A. Z is also acetylated and, as measured on a cell population, the level of acetylation correlates with the gene' s expression level [19]. Recent work suggests that acetylated-H2A. Z promotes transcription of adjacent genes. Specifically, H2A. Z at the promoters of the oleate-responsive genes CTA1, POX1, POT1, and FOX2 is acetylated on Lys14. Cells with a mutant form of H2A. Z that cannot be acetylated at this position are defective in induction of these genes [20]. H2A. Z' s contribution to gene-induction was first explored in the context of the GAL1, GAL7, and GAL10 genes [21], [22], which are induced in medium containing galactose, repressed in medium containing glucose, and expressed at a basal uninduced level by cells grown in medium with nonfermentable carbon sources [23]–[26]. Because the galactose regulon is one of only a handful of thoroughly studied regulated genes in yeast, it has provided many fresh insights into gene regulation. Hence results on, and claims about, this regulon take on special importance in the field. Induction of GAL1, GAL7, and GAL10 occurs more rapidly when S. cerevisiae cells are grown in a nonrepressing, noninducing carbon source (such as raffinose) and then shifted to inducing conditions (galactose) than when cells are grown in repressing conditions (glucose) and then transferred into inducing conditions [23]–[26]. The one exception to this pattern involves a phenomenon known as transcriptional memory. S. cerevisiae cells grown in inducing conditions prior to short-term growth in repressing conditions are able to reinduce GAL- gene expression upon induction as rapidly as cells grown continuously in nonrepressing conditions [27]–[29]. This “memory” of recent inducing conditions is reported to be H2A. Z dependent [27], although other explanations have been offered [29]. The role of H2A. Z in galactose induction extends beyond its role in GAL1 transcriptional memory. Cells that are grown in nonrepressing conditions prior to galactose induction require H2A. Z for the rapid induction of GAL1 [21], [22]. H2A. Z promotes the rapid induction of GAL1 by recruiting the Mediator complex to the GAL1 promoter [30], [31]. The work presented in this paper was aimed at testing the potential role of H2A. Z acetylation in gene induction and transcriptional memory. We found no evidence for a role for H2A. Z in GAL1 transcriptional memory, discovered a role for H2A. Z acetylation in gene induction, and discovered a confounding influence of SWR1-Com on gene regulation in cells lacking H2A. Z. Upon galactose induction, cells previously grown long-term in repressing conditions induce GAL1 expression more slowly than cells previously grown in noninducing-nonrepressing conditions. The conclusion that H2A. Z is essential for GAL1 transcriptional memory was based on the following two observations. First, when transferred to inducing conditions from long-term growth in repressing conditions HTZ1 and htz1Δ cells induce GAL1 slowly and at a similar rate [27]. Second, when transferred to inducing conditions from short-term growth in repressing conditions, HTZ1 cells induce GAL1 transcription rapidly, but htz1Δ cells are reported to not induce GAL1 any more rapidly than htz1Δ cells that had been grown long term in repressing conditions prior to galactose induction [27]. We reasoned that if H2A. Z acetylation were required exclusively for transcriptional memory, then cells carrying an unacetylatable allele of HTZ1, htz1-K3,8, 10,14R, would exhibit defective GAL1 induction following short-term growth in glucose, but exhibit normal GAL1 induction following long-term growth in glucose. To determine first whether H2A. Z-acetylation had any role in galactose expression, GAL1 mRNA levels were evaluated by quantitative reverse transcriptase (Q-RT) PCR in HTZ1 (JRY7971), htz1Δ (JRY9001), and htz1-K3,8, 10,14R (JRY7983) cultures grown in long-term repressing conditions prior to galactose induction. Cells grown continuously in glucose medium were transferred to galactose medium and GAL1 induction was evaluated at 2-h intervals for 14 h. One characteristic shared between all three strains' GAL1 induction phenotypes was an approximately 3-h lag period with little to no GAL1 expression. Quantitative analysis suggested that neither htz1Δ nor htz1-K3,8, 10,14R cultures exhibited substantially different lag periods prior to the onset of GAL1 expression than those exhibited by HTZ1 cultures (Figure 1A; Table 1, column A). These results suggested that neither H2A. Z nor its acetylation influenced how rapidly the cultures exited glucose repression and began GAL1 transcription. Other than their lag periods the two mutant cultures exhibited significantly different GAL1 induction phenotypes than those of HTZ1 cultures. Cultures of the two mutant strains had lower steady-state GAL1 expression levels than HTZ1 cultures (Figure 1A; Table 1, column C). Quantitative analysis suggested that htz1Δ and htz1-K3,8, 10,14R cultures required 54. 7% and 60. 2% more time, respectively, than HTZ1 cultures to reach half steady-state GAL1 expression levels (Figure 1A; Table 2, column E; note that half steady-state levels were used instead of half-maximum levels because the level of expression during induction typically overshot the induced steady-state level). These values, however, underplayed the severity of the htz1Δ and htz1-K3,8, 10,14R cultures' GAL1-transcription rate phenotypes because all three strains spent the majority of time that was required to reach half-steady-state levels in the lag period prior to GAL1 activation (Figure 1A; Table 1, columns A and E; note that half steady-state levels were used instead of half-maximum levels because the level of expression during induction typically overshot the induced steady-state level). To accurately compare the GAL1 transcription rates of the three strains it was necessary to determine the amount of time that cultures of these strains required to reach half-steady-state levels of GAL1 expression from the time of GAL1 activation. These values were determined for each culture by subtracting its GAL1 activation time from the time required to reach the half steady-state level of GAL1 expression. This analysis revealed that once they had begun expressing GAL1, htz1Δ and htz1-K3,8, 10,14R cultures required 503% and 625% of the time required for HTZ1 cultures, respectively, to express GAL1 at half-steady-state levels (Table 2, column G). Thus, both H2A. Z and its acetylation contributed to the rate of GAL1 expression in cultures grown under long-term glucose repression prior to galactose induction. Because the expression of GAL1 in htz1Δ and htz1-K3,8, 10,14R strains was similar, the role of H2A. Z in GAL1 expression was presumably dependent upon its acetylation. To determine whether H2A. Z acetylation affected the level of H2A. Z at the GAL1 promoter, chromatin immunoprecipitation experiments were performed with qPCR to quantitate the level of enrichment. Both acetylatable and unacetylatable H2A. Z were present at approximately equal levels at GAL1 (Figure 1b). Therefore, acetylation of H2A. Z was important for GAL1 induction at some point after H2A. Z' s incorporation at the GAL1 promoter. To determine whether H2A. Z acetylation had a role in transcriptional memory, GAL1 mRNA levels were evaluated in HTZ1 (JRY7971), htz1Δ (JRY9001), and htz1-K3,8, 10,14R (JRY7983) cultures that were grown short-term in repressing conditions prior to galactose induction. Cells grown in galactose medium prior to short-term growth in glucose medium (12 h) were transferred to galactose medium and GAL1 induction was evaluated for 14 h in inducing conditions. None of the three strains exhibited a significant lag in GAL1 expression (Figure 1C; Table 1, column B). Quantitative analysis of these data suggested that all three strains, when grown short-term in repressing conditions, expressed GAL1 in half the time, or less, than when the same strains were induced following long-term growth in repressing conditions (Table 3, column D). The combined effect of near-zero onset times and increased GAL1 transcription rates was that all three strains reached half steady-state GAL1 expression levels in 90% less time than was required for the same strains to reach this level when they were grown long-term in repressing conditions prior to galactose induction (Table 3, column C). Thus, all three strains exhibited transcriptional memory with respect to GAL1 transcription. Importantly, relative to the HTZ1 strain, the two mutant strains exhibited less severe phenotypes when they were grown short-term in repressing conditions prior to induction than when they were grown long term in repressing conditions prior to induction (Table 2, compare column G with H). Thus, neither H2A. Z nor its acetylation played an important role in GAL1-transcriptional memory. Because the results described above differed substantially from ostensibly equivalent experiments [27], we obtained the strains used in the previously published experiments, HTZ1 (CRY 1) and htz1Δ (DBY 50), and attempted to reproduce the previously published results. Just as described above, both HTZ1 (CRY 1) and htz1Δ (DBY 50) cultures exhibited a similar lag period before GAL1 mRNA was detectable (Figure 2A; Table 1, column A). As before, when grown under long-term repressing conditions prior to galactose induction, galactose-induced HTZ1 (CRY1) cells had both higher steady-state GAL1 mRNA levels and faster GAL1 transcription rates than htz1Δ (DBY 50) cells (Figure 2A; Table 1, columns D and G). Quantitative analysis suggested that once both cultures had begun expressing GAL1, the htz1Δ (DBY 50) cultures required about 3. 5× more time than HTZ1 (CRY 1) cultures to reach half-steady-state GAL1 expression levels (Table 2, column G). Additionally, as was the case with the other set of strains, both HTZ1 (CRY 1) and htz1Δ (DBY 50) cultures induced GAL1 expression significantly more rapidly when grown short-term (12 h) in repressing conditions prior to galactose induction than when the same cultures were grown long term in repressing conditions prior to galactose induction (Figure 2B; Table 1, column B). Cultures of both strains also required significantly less time to accumulate half-steady state levels of GAL1 mRNA when grown short term rather than long term in repressing conditions prior to galactose induction: HTZ1 (CRY 1) and htz1Δ (DBY 50) cultures required 88% and 69% less time, respectively, under these conditions to accumulate half-steady levels of GAL1 mRNA transcripts (Table 3, column C). Thus, as before, both HTZ1 and htz1Δ cultures exhibited transcriptional memory of prior GAL1 induction. Therefore, H2A. Z was important for GAL1 induction regardless of whether cells were induced from short-term or long-term growth in repressing conditions prior to induction. Two factors contribute to the GAL1 expression level in a culture of cells: the proportion of cells that are expressing GAL1, and the level of GAL1 expression in the fraction of cells in which it is expressed. S. cerevisiae regulates GAL1 expression in response to different growth conditions both by increasing the number of GAL1-expressing cells and by increasing the level of GAL1 expression. Both parameters respond independently to different aspects of growth conditions [32]. To determine whether htz1Δ and htz1-K3,8, 10,14R cultures' GAL1- expression defects were attributable to decreased proportions of GAL1-expressing cells, or to decreased GAL1 expression level per cell, flow cytometry was used to monitor galactose induction of a fusion protein containing the entire GAL1 coding sequence, with a C-terminal fusion to green fluorescent protein (GFP), in htz1-K3,8, 10,14R, htz1Δ, and HTZ1 cells (Figures 3 and S6, S7, S8). If H2A. Z were to contribute to the probability that a cell enters the galactose-induced state per unit of time, but not to the expression level in those induced cells, then htz1Δ cultures should have a smaller proportion of GFP-positive cells at each postinduction time point than HTZ1 cultures, but the GFP-positive cells should have similar fluorescence intensities to those in HTZ1 cultures. However, if H2A. Z were important for achieving high expression levels but did not influence the probability of induction per se, then htz1Δ and HTZ1 cultures should have similar proportions of GFP-positive cells, but the GAL1-GFP-expressing cells from htz1Δ mutant cultures would have lower GFP fluorescence than GAL1-GFP-expressing cells from HTZ1 cultures. The same logic would apply to the possible roles of H2A. Z acetylation. To compare the results of these experiments, a threshold value of GFP-intensity was used to classify cells as either GFP-positive or GFP-negative. This threshold was set so that between 1% and 2% of cells from noninduced HTZ1 cultures were classified as GFP-positive. On average htz1-K3,8, 10,14R cultures had 33% fewer GFP-positive cells than HTZ1 cultures at all postinduction time points (Figures 3 and 4A). Additionally, GFP-positive cells from htz1-K3,8, 10,14R cells had, on average, 17% lower mean-GFP intensity than HTZ1 cultures (Figures 3 and 4B). The simplest interpretation of these findings was that H2A. Z-acetylation influenced both the time required to induce GAL1-GFP expression and the rate at which Gal1-GFP accumulated once induced. Another possibility was that the differences between HTZ1 and htz1-K3,8, 10,14R cells were due exclusively to differences in either the time required for induction or to the rate of Gal1-GFP accumulation. To distinguish between these two possibilities, the GAL1-induction times and Gal1-GFP accumulation rates were determined for both cultures by fitting a simple mathematical model of gene expression to the data for each culture (the model is described in Materials and Methods; Figures 5 and S1, S2, S3, S4). The model simulated the galactose induction phenotype of a culture by estimating the distribution of activation times and expression rates of the measured cells. The model' s parameters were fitted to the observed data for each strain by optimizing the fit to cell-specific measurements of GAL1-GFP levels. Each culture' s average induction time and average accumulation rate are presented in Tables 4 and 5, respectively. This analysis revealed that htz1-K3,8, 10,14R cells induced GAL1-GFP expression 31% (+/−3. 3%) more slowly than did HTZ1 cells (Table 4), and that induced cells in both HTZ1 and htz1-K3,8, 10,14R cultures accumulated Gal1-GFP at similar rates (Table 5). Thus, with respect to GAL1 induction, H2A. Z-acetylation reduced the amount of time required to induce GAL1, but did not influence the rate at which induced cells accumulated Gal1-GFPp. Interestingly, htz1Δ cells had a more severe defect in GAL1-GFP expression phenotypes than did cells with unacetylatable H2A. Z (Figures 3,4A, and 4B; Tables 4 and 5). On average, htz1Δ cultures had 28% fewer GFP-positive cells than htz1-K3,8, 10,14R cultures at the 4-h and 6-h time points. At these same time points, the average GFP-intensity of GFP-positive cells in htz1Δ cultures was 46% lower than that of GFP-positive cells in htz1-K3,8, 10,14R cultures. Moreover, htz1Δ cells induced GAL1-GFP 18. 2% (+/−3. 8%) later and accumulated Gal1-GFP 38. 1% (+/−5. 6%) more slowly than htz1-K3,8, 10,14R cells (Tables 4 and 5). These results were surprising because htz1Δ and htz1-K3,8, 10,14R cultures had similar GAL1 mRNA induction phenotypes (Figure 1A). mRNA analysis revealed that the htz1Δ cultures used in these experiments accumulated GAL1-GFP transcripts, in contrasts to the GAL1 transcripts in Figure 1A, more slowly than either HTZ1 or htz1-K3,8, 10,14R culture (Figure 4C). These results suggested that htz1Δ cells accumulated Gal1-GFP more slowly than htz1-K3,8, 10,14R cells because they produced GAL1-GFP mRNA more slowly than htz1-K3,8, 10,14R cells. All of the mRNA measurements performed in this study were performed on bulk cultures, whereas the flow cytometry measurements were made on single cells within cultures. To determine whether the flow cytometry measurements of Gal1-GFP accumulation in HTZ1, htz1Δ, and htz1-K3,8, 10,14R strains corresponded well with each strain' s GAL1-GFP mRNA accumulation phenotype, the average GFP intensity of each culture was determined (Figure 4D). The galactose-induction phenotypes of all three strains, as measured by average GFP accumulation, were qualitatively similar to their galactose induction phenotypes as measured by GAL1-GFP mRNA accumulation. Thus, the flow-cytometry data in these studies reflected GAL1-GFP mRNA accumulation. At face value, the more severe galactose-induction phenotypes of htz1Δ than of htz1-K3,8, 10,14R cells suggested that H2A. Z' s role in GAL1 induction was only partially dependent on its acetylation. However, as described below, the more severe GAL1-expression defects in htz1Δ cells resulted from secondary complications that arose from the action of SWR1-Com in cells lacking H2A. Z. Acetylation of lys14 on H2A. Z is important for its role in FOX2 and POT1 induction [20]. To determine whether the acetylation of lys14 or other lysine residues of H2A. Z contributed to GAL1 induction, the GAL1 induction phenotypes of diploid cultures each with one null allele and individual lys-to-arg mutations as the other allele (htz1-K3R/htz1Δ, htz1-K8R/htz1Δ, htz1-K10R/htz1Δ, and htz1-K14R/htz1Δ) were determined using flow-cytometry. Surprisingly, none of the single acetylation-site mutants exhibited GAL1-GFP expression defects (Figure 6; example FACS profiles are in Figures. S6, S7, S8). Thus, H2A. Z' s role in GAL1 induction depended on its acetylation, but did not depend exclusively on the acetylation of any single tail-lysine residue. These results were surprising given the focus on the acetylation of H2A. Z lys14 in previous studies in S. cerevisiae [18], [19], but they are consistent with discoveries made in Tetrahymena. In Tetrahymena, acetylation of H2A. Z' s tail lysines contributes to H2A. Z' s function simply by decreasing the positive charge of H2A. Z' s tail and thus all sites of acetylation function equally well in this respect [33]. SWR1-Com deposits H2A. Z into chromatin in a two-step process, removing H2A from nucleosomes and subsequently replacing it with H2A. Z [13]. We hypothesized that if H2A. Z were not available, then SWR1-Com might still perform the first step of this mechanism, disrupting the structure of nucleosomes at those positions at which H2A. Z would normally reside, and that this disruption could affect normal promoter function. Thus, the phenotype of cells lacking H2A. Z might be a composite of two different defects: the lack of H2A. Z' s function per se, and SWR1-Com' s nucleosome-disrupting activity in the absence of H2A. Z. If this hypothesis were correct, then a subset of htz1Δ' s phenotypes should be suppressed in cells lacking SWR1-Com function. Indeed as predicted by this model, strains with the htz1Δ mutation in combination with a null mutation in any gene encoding an important component of the SWR1 complex (SWR1, SWC2, SWC3, SWC5, and SWC6) exhibited less severe mutant phenotypes than htz1Δ single-mutant strains on medium containing compounds that each cause a different type of stress (Figure 7). To determine if the htz1Δ mutant' s galactose-induction was more defective than that of the unacetylatable H2A. Z mutant for a similar reason, the GAL1 expression phenotypes of both swr1Δ HTZ1 (JRY9005) and swr1Δ htz1Δ (JRY9006) double-mutant cultures were determined using flow cytometry. Prior to induction, htz1-K3,8, 10,14R, swr1Δ HTZ1, and swr1Δ htz1Δ cultures had similar proportions of GFP-positive cells, and fewer GFP-positive cells than htz1Δ cultures (Figures 8 and 9A). Thus, the swr1Δ mutation completely suppressed the htz1Δ mutant' s apparent glucose-repression defect. At every postinduction time point, swr1Δ HTZ1 and swr1Δ htz1Δ cultures had similar proportions of GFP-positive cells to htz1-K3,8, 10,14R cultures and significantly higher proportions of GFP-positive cells than htz1Δ cultures (Figures 8 and 9A). The swr1Δ HTZ1 and swr1Δ htz1Δ cells induced GAL1 expression as rapidly as htz1-K3,8, 10,14R cells and significantly earlier than htz1Δ cells (Table 4). Thus, the severity of the htz1Δ mutant' s delayed GAL1-induction phenotype was suppressible by the swr1Δ mutation and therefore likely resulted from the SWR1 complex' s activity in the absence of H2A. Z. Furthermore, because htz1-K3,8, 10,14R cells and htz1Δ swr1Δ cells needed approximately the same amount of time to induce GAL1, H2A. Z' s role in promoting rapid GAL1 activation completely depended on its acetylation. Interestingly, GAL1-expressing cells from swr1Δ HTZ1 and swr1Δ htz1Δ cultures had significantly higher average GFP intensities than those from htz1Δ cultures but they had significantly lower average GFP intensities than those in htz1-K3,8, 10,14R cultures (Figures 8 and 9B). Quantitative analysis revealed that GAL1-expressing cells from both swr1Δ HTZ1 and swr1Δ htz1Δ cultures accumulated Gal1-GFP 18. 8% (+/−5. 3%) more rapidly than htz1Δ cells and 23. 8% (+/−6. 7%) more slowly than htz1-K3,8, 10,14R cells (Table 5). Thus the severity of the htz1Δ mutant' s Gal1-GFP-accumulation-rate phenotype was suppressible by the swr1Δ mutation and therefore likely resulted from the activity of SWR1-Com in H2A. Z' s absence. Moreover, our finding that swr1Δ HTZ1 and swr1Δ htz1Δ cells accumulated Gal1-GFP more slowly than htz1-K3,8, 10,14R cells suggested that H2A. Z has an acetylation-independent role in increasing GAL1-expression rate. In this work, we showed that H2A. Z, through its acetylation, contributed to induction of the GAL1 gene, a paradigmatic example of a highly inducible gene of Saccharomyces. Acetylated H2A. Z contributed to GAL1 induction both by increasing the fraction of cells that induced at each time point, and by increasing the level of expression per induced cell. Earlier work established that GAL1 induction has a property termed transcriptional memory, reflecting the ability of cells that were recently induced to be more easily reinduced following short incubations in repressing conditions than after extended incubations in repressing conditions [27]–[29]. Moreover, H2A. Z has been thought to be a key participant in this transcriptional memory [27]. The conclusion that H2A. Z is important for GAL1 transcriptional memory is based on experiments involving the induction of GAL1 as a function of its expression history: when induced from long-term growth in repressing conditions, both htz1Δ and HTZ1 cultures were reported to have induced GAL1 at similar rates. htz1Δ cultures were reported to have induced GAL1 at a similar rate regardless of whether they had been grown under repressing conditions for either short or long periods of time. However, HTZ1 cultures that were grown in repressing conditions for short periods of time were reported to induce GAL1 expression much more rapidly than those grown in repressing conditions for long periods of time [27]. Our work was originally directed at understanding the importance of H2A. Z acetylation to the role of H2A. Z in GAL1- transcriptional memory. To this end, we determined the GAL1- induction phenotypes of htz1-K3,8, 10,14R cultures, which carry an unacetylatable allele of H2A. Z. Surprisingly, both htz1Δ and htz1-K3,8, 10,14R cultures grown in inducing conditions prior to short-term growth in repressing conditions induced GAL1 expression more rapidly than those grown long-term in repressing conditions prior to galactose induction. Thus, both htz1Δ and htz1-K3,8, 10,14R cells exhibited GAL1 transcriptional memory. Moreover, regardless of whether they were grown long-term or short-term in repressing conditions prior to induction, htz1Δ and htz1-K3,8, 10,14R cultures induced GAL1 more slowly than HTZ1 cultures. These results indicated that H2A. Z was important for GAL1 induction regardless of a cell' s growth conditions prior to induction. Thus, the galactose-induction defects that we observed for htz1Δ and htz1-K3,8, 10,14R strains grown short-term in repressing conditions prior to galactose induction were reflective of H2A. Z and acetylated H2A. Z having a general role in GAL1 induction rather than a specific role in GAL1- transcriptional memory. If H2A. Z or H2A. Z acetylation had a specific role in transcriptional memory, then one would expect cells lacking H2A. Z or containing only an unacetylatable form of H2A. Z to exhibit more severe phenotypes when reinduced than during the primary induction. However, quantitative analysis of the GAL1 induction phenotypes of the htz1Δ and htz1-K3,8, 10,14R strains indicated that the difference between the two mutant strains' GAL1 induction phenotypes were less severe, with respect to the HTZ1 strain' s GAL1 induction phenotypes, when cultures of these strains were reinduced rather than induced. Thus, neither H2A. Z nor acetylated H2A. Z contributed to GAL1-transcriptional memory, other than in the general processes of GAL1 transcription, at least under the conditions of these experiments. To be completely clear, our data did not discount the existence of what has been referred to as transcriptional memory of GAL1 induction. A better explanation for memory has been provided by the discovery that the Gal1p protein itself has both galactokinase activity that is crucial for galactose metabolism, as well as Gal3 activity, which is also encoded by the separate GAL3 gene. Gal3p activates the GAL4-encoded activator of GAL1 induction. Thus GAL1-transcriptional memory can be explained by a positive feedback loop in which GAL1 induction leads to the synthesis of a protein that is both an enzyme and an autoinducer, as shown by others [29], [34]. Our contribution was limited to discounting a role for H2A. Z in this memory. This understanding of GAL1-transcriptional memory suggests a possible explanation for why previously published experiments concluded that htz1Δ cultures lack GAL1- transcriptional memory [27]. The model presented above posits that a cell' s ability to reinduce GAL1 expression rapidly following short-term repression requires the persistence of Gal1p in the cytoplasm. Thus the amount of time that dividing cells retain the ability to rapidly reinduce GAL1 expression following repression is a function of both the stability of Gal1p and its abundance prior to glucose repression. The abundance of Gal1p in a cell prior to glucose repression is important because of its dilution with cell division, and thus at some number of cell divisions, the amount of Gal1p will not meet the threshold level required for its role in GAL1 reinduction. We observed that htz1Δ cultures had a nearly 20% lower steady-state GAL1 expression level than HTZ1 cultures and that when grown long-term in repressing conditions prior to galactose induction htz1Δ cultures did not reach this level of expression until galactose induction had proceeded for more than 14 h. If the previously published experiments did not allow galactose induction to occur for a sufficient period of time, then the htz1Δ and HTZ1 cultures used in these experiments would not be directly comparable with respect to Gal1p levels. Thus cells within htz1Δ cultures would be less likely than those in HTZ1 cultures to have sufficiently high Gal1p levels to allow for the rapid reinduction of GAL1. The discrepancies between the previously published data [27] and those presented here, concerning the role of H2A. Z in primary inductions of GAL1, have a straightforward explanation. The conclusion that H2A. Z was not important for primary galactose inductions was based upon htz1Δ cells having induced GAL1 expression less well than HTZ1 cells after a 2-h induction following short-term growth in repressing conditions, whereas HTZ1 and htz1Δ cells induced GAL1 equally well following long-term growth in repressing conditions. Our observations were quantitatively similar. However, the critical point is that the magnitude of induction at this early time point was negligible in both htz1Δ and HTZ1 cultures. At all longer periods of galactose induction, htz1Δ cells induced GAL1 expression significantly less well than HTZ1 cells. We believe the earlier conclusions were based upon inadequate induction periods in some experiments. The original work implicating H2A. Z in transcriptional memory of GAL1 also reached the same conclusion for INO1. However, the data offered in support of these conclusions are weaker than those offered in support of H2A. Z' s role in GAL1 induction memory. First, these studies fail to establish that S. cerevisiae exhibits transcriptional memory of INO1 in the same way that it exhibits transcriptional memory of GAL1. Unlike GAL1, cells that are grown short term in repressing conditions prior to induction induce INO1 more slowly and at lower levels than cells that had been grown long term in repressing conditions prior to induction [27]. Thus, transcriptional memory of INO1 functions in the opposite way of how it functions in GAL1 transcription—decreasing rather than increasing a cell' s response to inducing conditions. Second, since INO1-transcriptional memory results in slower INO1 reinductions, cells lacking INO1-transcriptional memory should induce INO1 more rapidly than cells that have INO1- transcriptional memory. These studies show that htz1Δ cells both induce and reinduce INO1 more slowly than HTZ1 cells [27]. Therefore, htz1Δ cells do not seem to lack transcriptional memory of INO1, rather they seem to exhibit defective INO1 transcription regardless of whether they had recently induced INO1 expression. Because SWR1-Com catalyzes a two-step reaction removing H2A from nucleosomes and replacing it with H2A. Z, we considered the possibility that SWR1-Com' s function, in the absence of H2A. Z, might leave those nucleosomes normally destined to receive H2A. Z compromised in some way. Thus the overall phenotype of htz1Δ would be a composite of those consequences due to the lack of H2A. Z, and those due to uncoupled H2A removal from nucleosomes. Two lines of evidence supported this hypothesis. First, the severity of htz1Δ cells' sensitivities to various agents with different mechanisms and targets were substantially suppressible by mutations in genes encoding subunits of SWR1-Com. Second, the difference between GAL1 induction in htz1Δ cells and in cells with unacetylatable H2A. Z was largely suppressed by the swr1Δ mutation, creating the less severe phenotype of the unacetylatable H2A. Z mutant. This model is further supported by the observation that htz1Δ cells have chromatin that is in the partially open configuration at the PHO5 promoter under noninducing conditions [21]. We predict that this partially open configuration is a physical manifestation of the mischief wrought by the Swr1-Complex in the absence of H2A. Z. The benomyl-sensitivity phenotype of the swc5Δ htz1Δ double mutant suggests another possible explanation for why SWR1-Com is dangerous for cells that lack H2A. Z. Unlike the swr1Δ, swc2Δ, swc3Δ, and swc6Δ mutations that strongly suppressed the htz1Δ mutant' s benomyl sensitivity phenotype, the swc5Δ mutation only weakly suppressed this phenotype. In vitro studies have shown that SWR1-Com complexes lacking Swc2p, Swc6p, Swc4p, Yaf9, or Arp6 bind nucleosomes less well than complete SWR1-Com complexes. In contrast, SWR1-Com complexes that lack Swc5p bind nucleosomes better than complete SWR1-Com complexes [35]. Since Swc5p is required for SWR1-Com' s function, the simplest model for why the swc5Δ mutation does not strongly suppress the htz1Δ mutant' s benomyl sensitivity is that mutant SWR1-Com complexes lacking Swc5 may persist in chromatin, perhaps removing H2A, but be unable to replace it with H2A. Z. Our observation that swr1Δhtz1Δ cells required more time to induce GAL1 expression, and expressed GAL1 more slowly once induced, suggested that H2A. Z had two distinct roles in GAL1 expression—one allowing efficient induction of GAL1, and another to increase the rate of GAL1 expression. That H2A. Z had a role in GAL1 induction was not surprising given H2A. Z' s enrichment at the GAL1-promoter. However, that H2A. Z had a role in increasing GAL1' s expression rate, as inferred from our model, was unexpected. There are two lines of evidence that H2A. Z may be important for the expression, per se, of actively transcribed genes. First, even though H2A. Z predominantly localizes to promoters, it is not completely absent from open reading frames (ORFs). The ACT1 and PRP8 ORFs, two loci that have been historically considered nonenriched for H2A. Z, are slightly enriched for H2A. Z relative to no-tag controls (Figure S5). Second, the htz1Δ mutant is sensitive to 6-azauracil, a toxic compound that slows the growth rate of cells that are defective in mRNA transcript elongation [36]. Thus, it is possible that H2A. Z plays a direct role in transcript elongation. Recent reports raise that possibility further, showing that H2A. Z may aid expression by suppressing antisense transcripts [37]. In summary, our results established that H2A. Z plays no significant role in GAL1- transcriptional memory. In contrast H2A. Z, and its acetylation contributed to both the induction of the gene and to its expression per se, adding valuable new insights into one of the best-studied examples of eukaryotic gene regulation. In addition, we showed that SWR1-Com caused defects in gene expression and induction in the absence of H2A. Z, presumably due to nucleosome disruption, that force a reevaluation of all previously described phenotypes of cells lacking H2A. Z. All of the strains used in this study are presented in Table 6. All of these strains were from the W303 background. One-step integration of knockout cassettes has been previously described [38]. JRY9001 was constructed by transforming the KanMX cassette into JRY7754. To generate KWY2512, the DNA sequence encoding GFP was inserted before the stop codon of the GAL1 open reading by transforming a HIS3-marked construct encoding the GFP protein. JRY9002, JRY9003, JRY9004 were segregants from crosses of JRY7972, JRY7983, and JRY9001 to KWY2512, respectively. JRY9005 and JRY9006 were segregants from crosses of JRY7752 to JRY9002 and JRY9004, respectively. JRY9011, JRY9012, JRY9013, JRY9014, JRY9015, and JRY9016 were segregants from crosses of JRY9000 to JRY7972, JRY7983, JRY9007, JRY9008, JRY9009, and JRY9010, respectively. MKY1028/MKY1029, MKY1030/MKY1031, MKY1032/MKY1033, MKY1034/MKY1035, and MKY1036/MKY1037 were created by disrupting SWR1, SWC2, SWC3, SWC5, and SWC6 respectively in MKY1038 using a SpHIS5MX knockout cassette that was amplified from pFA6a-His3MX6 [38]. Yeast media were as defined [39]. Seed culture density affected GAL1 induction phenotypes, so precautions were taken to ensure that seed cultures of all strains had similar growth histories. Specifically, seed cultures for all experiments were grown in YP-Dextrose (D-glucose, 2%) except DBY50 and CRY1, which were grown in CSM-Dextrose (D-glucose, 2%). 50 ml seed cultures were inoculated with cells from a single colony and grown overnight with shaking at 30°C to OD 0. 2, and were then harvested by centrifugation at 2,060g for 1 min. The cells were then washed with 25 ml of prewarmed 30°C YP-galactose and resuspended in 50 ml of 30°C YP-galactose, except in experiments performed with DBY50 and CRY1, in which CSM-Galactose was used instead of YP-galactose for both washing and resuspending in order to follow precisely the procedures of others [27]. The volume of culture removed for each time point was replaced with the same volume of 30°C YP-galactose. Both determination of mRNA levels by quantitative reverse-transcriptase (Q-RT) PCR and ChIP were performed as described [17] except that SYBR GreenER (Invitrogen) PCR reagents were used. H2A. Z-3Flag, and H2A. Z-K3,8, 10,14R-3Flag were immunoprecipitated using the αFlag M2 resin (Sigma). Cells were harvested by centrifugation, fixed in a 4% paraformaldehyde/3. 4% sucrose solution for 10 min at room temperature and then stored overnight at 4°C in a 1. 2 M sorbitol solution with KPO4 buffer at pH 7. 5. GFP expression data were collected for each sample using the FC-500 (Beckman-Coulter) flow cytometer and analyzed using the Flow-Jo software package. The GAL1-GFP expression status of individual cells within cultures on a cell-by-cell basis in each culture was determined by plotting flow-cytometry measurements as a histogram of GFP fluorescence (y-axis number of cells; x-axis Log GFP intensity relative to GFP-negative values). The threshold of GFP intensity was set so that between 1% and 2% of glucose-grown HTZ1 cultures would be classified as GFP-positive. Cells that had GFP-intensity greater than this threshold value were counted as GFP positive (GAL1-GFP expressing). The level of GAL1 expression in different populations was calculated by determining the geometric mean GFP intensity. We developed a simple mathematical model to analyze the dynamics of GAL1 mRNA expression levels. This model allowed us to robustly quantify the onset time of GAL1 induction, steady state GAL1 mRNA level, and the time needed to reach half of the steady-state level. The model is based on three parameters, which we optimized to maximize the fit of the model to the measured GAL1 mRNA levels. These include: (1) the time x when induction of GAL1 mRNA begins; (2) the rate α at which GAL1 mRNA is produced; and (3) the rate δ at which GAL1 mRNA molecules are being degraded. According to the model, the relative amount of GAL1 mRNA at time t, M (t), follows the ordinary differential equation (ODE): Namely, GAL1 is not being expressed at all until time point x, from which point it is produced at a fixed rate α, and being degraded at a fixed ratio δ, until it reaches the steady state equilibrium: Given the model parameters, and starting from zero M (0) = 0, we can solve the ordinary differential equation using the Runge-Kutta method (as implemented in MATLAB 7. 6), and estimate the mRNA level of GAL1 at every time point t. We optimized the three parameters x, α, and δ for every culture to minimize the root-mean-square deviation (RMSD) between the experimental measurements and the modeled values. The values that were used in each of the best-fit models are presented in Table S1. We constrained the parameters x, α, and δ to non-negative values, and used the active-set optimization algorithm (FMINCON function in MATLAB 7. 6). For the memory experiments, the optimized values of the GAL1 expression onset times, for all cultures, were very close to zero, and practically below the time resolution of the model and data. We therefore simplified the model, and explicitly set x to zero. Finally, to estimate the half steady-state time point, we used the optimized parameters for each culture to find the steady state level α/δ, and to solve the ordinary differential equation and identify when GAL1 levels reach half of the steady state level. To analyze the flow cytometry data, the time-course measurements of single-cell Gal1-GFPp intensities were transformed into GAL1-GFP induction times and Gal1-GFP accumulation rates. To do this, a simplified model of GAL1-induction was developed, and its six parameters fitted to the measured data for each culture. For every cell, this model assumes that GAL1 is completely repressed until its induction time ti, when cellular Gal1-GFPp begins to accumulate at a fixed rate xi. We therefore model Ei (t), the Gal1-GPFp content of the ith cell at time t as: where: The estimated expression is added to a stochastic noise term εi, drawn from a Normal distribution with parameters (μ, σ2), to simulate a basal level of GAL1 expression. The model was used to simulate a population of 100,000 cells, whose GAL1-GFP-induction times ti' s and accumulation rates xi' s were sampled independently from two Gamma distributions: ti ∼ Gamma (kt, θt), and xi ∼ Gamma (kx, θx), and their stochastic noise terms sampled from a Normal distribution: εi ∼ Normal (μ, σ2). Given a set of six parameters (kt, θt, kx, θx, μ, σ2) this model sampled activation times, accumulation rates, and noise terms for each of the 100,000 cells in the simulation, and computed the cellular Gal1-GFPp levels Ei (t) for each of the four times points that were measured (0,2, 4, and 6 h following induction), which allowed for the simulation of flow-cytometry outputs. Activation times and accumulation rates were sampled from a stochastic distribution rather than being fixed at specific values to account for the natural variability among cells because of biological variables like cell size, position in the cell cycle, cell age, and other factors that were not treated as variables in the model. Gamma distributions were used due to their non-negativity property. The parameters of the model were optimized by minimizing the root-mean-squared deviation between the measured data (average of triplicates) and the model predictions, summed over the four measured time points (0,2, 4, and 6 h.) To optimize these parameters, genetic algorithms were used (as implemented in the GA function in MATLAB 7. 6) followed by a derivative-free optimization using the simplex algorithm (FMINSEARCH function in MATLAB 7. 6). These optimization steps were repeated with 200 random starting points for each strain, and the optimal set of parameters were then selected (Tables S2 and S3). The error in our estimation of each strain' s induction time and accumulation rate was calculated by determining the range of values for each parameter that were used in the top 50 best-fit simulations for each strain. The models that were determined for each strain' s Gal1-GFP expression phenotype were used as a proxy to quantitatively compare the GAL1-activation times and Gal1-GFPp accumulation rates of HTZ1, htz1-K3,8, 10,14R, htz1Δ, swr1Δ HTZ1 and swr1Δ htz1Δ cells. | Transcriptional memory is the well-documented phenomenon by which cells can "remember" prior transcriptional states. A paradigmatic example of transcriptional memory is found in the yeast Saccharomyces. S. cerevisiae remembers prior transcription of the galactose metabolism gene GAL1. When a gene is transcribed, the DNA must first be at least partially relieved of its packaging into chromatin by histone proteins. Previous research had suggested that S. cerevisiae used a chromatin modification, the incorporation of the histone variant H2A. Z into the region surrounding the GAL1 promoter, to remember the previous status of GAL1 transcription. Not all H2A. Z molecules are the same, however. For example, it has recently been discovered that H2A. Z can be acetylated on the four lysine residues in its N-terminal tail region. In an attempt to determine whether H2A. Z acetylation is required for GAL1 transcriptional memory, we unexpectedly discovered that, although both H2A. Z and H2A. Z acetylation are important for strong and rapid GAL1 induction, neither H2A. Z nor H2A. Z acetylation plays an important role in GAL1 transcriptional memory. We propose that the discrepancy between our conclusions and those in prior publications arise from the prior analysis of insufficiently short periods of GAL1 induction or from complications arising from the comparison of the phenotypes of wild-type yeast strains to those of htz1Δ mutants (carrying the null mutation of the gene encoding H2A. Z) mutants. In the current work we show that the htz1Δ mutant' s phenotype does not simply reflect the absence of H2A. Z in chromatin but instead also reflects the pleiotropic effects of the Swr1 chromatin remodeling complex that is responsible for H2A. Z deposition into chromatin. In the absence of H2A. Z the Swr1 complex itself causes cell damage. In this paper we show that swr1Δ htz1Δ double mutants have substantially less severe mutant phenotypes than htz1Δ mutants. Thus, studies using the swr1Δ htz1Δ mutant offer more detailed insight into the consequences of the absence of H2A. Z in chromatin than do studies performed on single htz1Δ mutants, and our results help to clarify the role of H2A. Z in the regulation of GAL1 induction and transcriptional memory. | lay_plos |
Manipulation of sex determination pathways in insects provides the basis for a wide spectrum of strategies to benefit agriculture and public health. Furthermore, insects display a remarkable diversity in the genetic pathways that lead to sex differentiation. The silkworm, Bombyx mori, has been cultivated by humans as a beneficial insect for over two millennia, and more recently as a model system for studying lepidopteran genetics and development. Previous studies have identified the B. mori Fem piRNA as the primary female determining factor and BmMasc as its downstream target, while the genetic scenario for male sex determination was still unclear. In the current study, we exploite the transgenic CRISPR/Cas9 system to generate a comprehensive set of knockout mutations in genes BmSxl, Bmtra2, BmImp, BmImpM, BmPSI and BmMasc, to investigate their roles in silkworm sex determination. Absence of Bmtra2 results in the complete depletion of Bmdsx transcripts, which is the conserved downstream factor in the sex determination pathway, and induces embryonic lethality. Loss of BmImp or BmImpM function does not affect the sexual differentiation. Mutations in BmPSI and BmMasc genes affect the splicing of Bmdsx and the female reproductive apparatus appeared in the male external genital. Intriguingly, we identify that BmPSI regulates expression of BmMasc, BmImpM and Bmdsx, supporting the conclusion that it acts as a key auxiliary factor in silkworm male sex determination. Genetic systems for sex determination in insects show high diversity in different species. Sex determination in the fruit fly, Drosophila melanogaster, is controlled hierarchically by X: A > Sxl > tra/tra2 > dsx and fru [1,2]. The X: A ratio of 1 promotes transcription of Sex-lethal (Sxl) and results in feminization, while 0. 5 to Sxl suppression and male differentiation [3–10]. Sxl proteins control the splicing of female transformer (tra) mRNAs that give rise to functional proteins, while no functional Sxl proteins exist in the male [11,12]. The search of homolog genes in the sex determination of D. melanogaster has been found a conserved relationship among dsx/tra across dipterans [13,14] In another Diptera, Musca domestica, female employs a FD allele which is encoded by the tra gene [15]. The medfly, Ceratitis capitata, has an as yet unidentified dominant male-determining factor on the Y chromosome [16]. The sex determination factors (F or M) in these two insects control the downstream gene doublesex (dsx) to generate sex-specific splicing isoforms. In contrast to Drosophila, Cctra and Mdtra seem to initiate an autoregulatory mechanism in XX embryos that provides continuous tra female-specific function and acts as a cellular memory maintaining the female pathway [17–19]. Many other insect species also exploit tra as the sex determination factor. For example, the honeybee, Apis mellifera, uses the complementary sex determiner gene (csd) to regulate feminization, which activates the feminizer gene (fem) by directing splicing to form the female functional Fem protein [20]. This fem gene is considered an orthologue of Cctra gene [21]. The tra gene in the red flour beetle, Tribolium castaneum, controls female sex determination by regulating dsx sex-specific splicing [22]. Also, Lucilia cuprina and Nasonia vitripennis, are reported to use tra as a female-determining signal [23,24]. Recently, Hall et al. identified Nix a distant homolog of transformer2 (tra2) from Aedes aegypti as the male-determining factor [25]. These data shows that the dsx/tra axis is conserved in many insect species and tra is the key gene around which variation in sex determining mechanisms has evolved in all insect species with the exception of Aedes and Lepidopteran insects. Species of lepidoptera exhibit markedly different sex determination pathways from those seen in the flies, bees and beetles [26]. In the silkworm Bombyx mori, females possess a female-determining W chromosome have heteromorphic sex chromosomes (ZW) and males are homomorphic (ZZ) [27]. No tra ortholog has been identified in this order, possibly as a result of highly divergent sequence [28]. Bioinformatic analyses fail to identify a tra ortholog in B. mori and no dsxRE (dsx cis-regulatory element) binding sites are found in the target gene ortholog, Bmdsx, resulting in the default mode of female-specific splicing of the latter [29,30]. BmPSI (P-element somatic inhibitor) and BmHrp28 (hnRNPA/B-like 28) were reported to regulate Bmdsx splicing through binding CE1 sequences of the female-specific exon 4 of Bmdsx pre-mRNA [30,31]. Another potential regulator, BmImp (IGF-II mRNA binding protein), enhances the male-specific splicing of Bmdsx pre-mRNA by increasing RNA binding activity of BmPSI [32]. The Z-linked BmImp binds to the A-rich sequences in its own pre-mRNA to induce the male-specific splicing of its pre-mRNA, and this splicing pattern is maintained by an autoregulatory mechanism, being BmImpM (the male-specific splicing form of BmImp) able to bind its corresponding pre-mRNA [33]. Since BmPSI or BmImp products do not exhibit any sequence similarities to known Ser/Arg (SR) proteins, such as Tra and Tra2, the regulatory mechanisms of sex-specific alternative splicing of Bmdsx that of exon skipping is distinct from that of Dmdsx that of 3’ alternative splice[34]. Recently, the product of the W chromosome-derived B. mori sex determination factor fem (female-enriched PIWI-interacting RNA) was identified to target the downstream gene BmMasc for controlling Bmdsx sex-specific splicing [35]. This remarkable finding reveals that fem is the primary female sex determinator in the silkworm. However, the genetic relationship among these genes in B. mori sex determination is still in mystery. We use here a binary transgenic CRISPR/Cas9 system to generate somatic mutations in sex determination pathway genes in B. mori. Three genes, BmMasc, BmPSI and BmImp, are involved in sex regulation only in lepidopteran insects, and two, Bmtra2 and BmSxl, are structural orthologs of the key sex regulation factors in Drosophila. We focus on the sexually dimorphic traits of reproductive structures and sex-specific alternative splicing forms of Bmdsx, the bottom gene of the sex determination. The results show that BmPSI and BmMasc affect Bmdsx splicing and the male reproductive tissues, supporting the conclusion that they have roles in sex determination. Disruption of BmImp or Bmtra2 causes severe developmental defects in both sexes, supporting their critical roles other than sex determination. Furthermore, loss-of-function mutations of BmPSI altered transcriptional or post-transcriptional (splicing) of BmMasc, BmImp and Bmdsx in males. These data strongly support the conclusion that BmPSI plays a key auxiliary in male sex determination in B. mori. We established a binary transgenic CRISPR/Cas9 system to make somatic mutagenesis targeting selected genes. This system contains two separated lines, one is to express Cas9 protein under the control of a B. mori nanos promoter (nos-Cas9) and another is to express sequence-specific sgRNAs under the control of a B. mori U6 promoter (U6-sgRNA). Two independent lines of nos-Cas9 and from three to 29 independent lines of each U6-sgRNA construct were obtained following piggyBac-mediated transgenesis (S1 Table). Genomic mutagenesis in F0 animals was confirmed by genomic PCR, and subsequent physiological phenotypes were investigated (Fig 1). The results confirm that the transgenic CRISPR/Cas9 system works effectively (S1–S5 Figs). Bmdsx is the conserved downstream component of the silkworm sex determination pathway and mutations in BmPSI and BmMasc had an effect on its splicing. Mutations in BmSxl, BmImp and BmImpM resulted in the appearance of a larger BmdsxM (the male-specific splicing form of Bmdsx) mRNA isoform while the profile of BmdsxF (the female-specific splicing form of Bmdsx) appeared unaltered (Fig 2, lanes 4,10 and 12). Sequence analysis of the larger BmdsxM isoform revealed an 81base-pairs (bp) length fragment insertion, creating a novel splice variant (S6 Fig). All Bmdsx isoforms were absent in both sexes in individuals carrying mutations in Bmtra2 (Fig 2, lanes 5 and 6). Mutations in BmMasc and BmPSI in males resulted in a decreased accumulation of BmdsxM and the appearance of BmdsxF (Fig 2, lanes 8 and 14), which was consistent with previous report when BmMasc expression was disrupted by using RNAi [35]. RT-PCR-based analysis revealed that mutations in BmSxl, BmMasc, BmImp and BmImpM had no or minor effect on other genes at transcriptional or post-transcriptional (splicing) levels except Bmdsx. The male-specific BmImp (BmImpM) isoform appeared in female Bmtra2 mutants (Fig 2, lanes 5 and 6). This supports the conclusion that Bmtra2 is not only involved in regulating Bmdsx, but also has a role in splicing regulation of BmImpM. Interestingly, the abundance of BmMasc and BmImpM transcripts decreased in BmPSI mutant males (Fig 2, lane 14). Q-RT-PCR analysis showed that BmImpM and BmMasc mRNA levels decreased by 92% and 60%, respectively, in BmPSI mutant males (Fig 3). These results support the conclusion that BmPSI regulates BmMasc and BmImpM at the splicing level. In contrast to the results in males, with the exception of Bmtra2, no effects on transcript profiles were seen in any of the mutant females. The morphology of the external and internal genitalia provides a direct index of sexual differentiation in B. mori. Mutations in BmMasc result in males with degenerative testes similar to that observed with Bmdsx mutants (Fig 4A, lanes 2 and 4). The external genitalia of males exhibit characteristics of the copulatory organs of both males and females including the female-specific ventral chitin plate and genital papillae (Fig 4B, lanes 2 and 4). The testes and external genitalia of mutant BmPSI males are similar phenotypes of mutations in BmdsxM or BmMasc and result in male sterility (Fig 4A, lane 7; Fig 4B, lane 7). The external and internal genitalia appear normal in both males and females with mutations in BmSxl, BmImp, and BmImpM, (Fig 4A, lanes 3,5, 6; Fig 4B, lanes 3,5, 6). The putative dsx target male-specific expression genes in the male olfactory system, pheromone binding protein 1 (BmPBP1), olfactory receptors (BmORs) BmOR1, BmOR3, were significantly down-regulated in the BmMasc and BmPSI male mutants (Fig 5A–5C). In contrast, the female-specific expression genes in the female oogenesis and olfactory system, vitellogenin (BmVg), BmOR19, BmOR30, were significantly up-regulated in the BmMasc and BmPSI male mutants (Fig 5D–5F). These morphological results and corresponding gene expression profiles provide additional support for the conclusion that BmPSI and BmMasc control male sexual differentiation. Mutations in Bmtra2 result in lethality at later embryonic stages (Fig 6A and 6B). This phenotype is similar to that reported in the honeybee in which down-regulation of Amtra2 causes embryonic viability and affects the female-specific splicing of fem and Amdsx transcripts [36]. Also, in T. castaneum, only a few eggs could be produced by animals after the parental RNAi of Tctra-2 and these eggs ultimately failed to hatch [37]. This is different from Dipteran species, in which tra2 has no vital function in embryogenesis [38]. The similarity of these phenotypes supports the hypothesis that Bmtra2 and its orthologs have an essential, ancestrally- and evolutionarily-conserved function in embryogenesis that is not related to sex determination that predates the divergence of the Lepidoptera, Hymenoptera and Coleoptera. Two distinct types of mutations were induced in BmImp, the first one targets all splice variants (BmImp), and the second one is only in the male-specific splice variant (BmImpM) (S4 Fig). However, neither had an effect on the morphology of the genitalia despite the observed effect on BmdsxF. While the growth indices of BmImpM mutant silkworms were normal, the body size and weight of the BmImp mutants was smaller than wild-type animals (Fig 6C and 6D). They failed to molt at each larval instar and the majority died at the later larval and prepupal stages (Fig 6E and 6F). We provide here genetic evidence for the proposed sex determination pathway in B. mori that emphasizes the key roles of the products of the BmPS1 and BmMasc genes in male determination and differentiation (Fig 7). B. mori has the ZZ/ZW sex chromosome system in which the female is ZW (heterogametic) and the male is ZZ (homogametic). The Fem piRNA gene is located on the W chromosome and maintains feminization through downregulating BmMasc expression. Without the BmMasc protein in ZW embryos, the default (full-length) splicing isoform of Bmdsx (BmdsxF) activates downstream gene expression and produces female-specific development. BmPSI is not involved in this pathway, since mutations in it have no observable effects on female differentiation. BmPSI protein in males interacts with the Bmdsx pre-mRNA and generates the male-specific Bmdsx splice variant (BmdsxM) [30]. The BmMasc product might play the role of a recruitment or splicing factor to participate in this event. BmMasc is expressed normally in males due to lack of Fem piRNA, and thus may be controlled by BmPSI. Significant differences are evident in the B. mori sex-determination system when compared to D. melanogaster. Sxl has a major and early role in sex determination in the fruit fly. Its ‘on/off’ status serves as the primary signal to trigger somatic sex differentiation by controlling its own splicing (autocatalytically) and of tra [38]. However, Sxl does not show sex-specific splicing and function in other dipterans [39]. Furthermore, the silkworm ortholog, BmSxl, has two splicing isoforms, neither of which is regulated in a sex-specific manner [40]. Depletion of Bmsxl induced a longer BmdsxM sex-specific splicing form appeared, just as seen in BmImp or BmImpM disruption. Although the potential role of this splicing form was unclear, it did not bring any phenotypic consequence in male sexual dimorphism. The presence of a longer BmdsxM splicing form might because the spliceosome is complex machinery containing up to 300 proteins and loss of additional auxiliary factors having little roles [41,42]. Dmtra2 is a co-binding protein of sex determination key gene Dmtra, and acts on the D. melanogaster sex-determination cascade to regulate both somatic sexual differentiation and male fertility [43]. However, the ortholog encoding the tra appears to not exist in the silkworm, and therefore Bmtra2 might have distinct roles. Bmtra2 could regulate female-specific splicing of Dmdsx and RNAi knockdown of Bmtra2 in the early embryo could cause abnormal testis formation in B. mori [44]. Our data support the conclusion that Bmtra2 regulates Bmdsx in both males and females, and has sex-independently essential roles during embryogenesis although the mechanism of embryonic lethality induced by Bmtra2 mutagenesis is not known. Another phenomena is BmImpM appeared in the Bmtra2 mutants, indicating that Bmtra2 is involved in regulating the upstream transcriptional factors of BmImpM. It is similar with Dmtra2 which regulates many downstream genes [45]. The BmImp gene was firstly identified in the silkworm as a co-binding protein with BmPSI [32]. BmImp produces two splice variants, one of which (BmImpM) is expressed in various tissues only in males and is proposed to be an essential regulator in the B. mori sex determination cascade [33]. BmImpM could bind the Bmdsx pre-mRNA and knock-down of its product induced the female-specific Bmdsx splicing form in male cell lines and embryos [33,46]. We cannot account for the differences in our results, but speculate that they could be a consequence of different methods between RNAi and Cas9-mediated gene silencing, or different materials between cell lines and the intact animal. However, our phenotypic results are consistent with those seen in D. melanogaster and the mouse. In the fruit fly, loss-of-function Imp mutations were zygotic lethal which through imprecise P excision, or mutants die later as pharate adults by two loss-of-function alleles, H44 and H149 [47,48]. Imp1 mutations in mice produced animals that were ~40% smaller than wild-type controls, and these exhibited high perinatal mortality [49]. Our experiments in which we mutate all isoforms of BmImp or only BmImpM did not result in any effects on sexual organs or sex determination genes. We conclude that BmImp and BmImpM are not essential for sex regulation but are needed for development. BmMasc had been identified as a downstream target of Fem-piRNA which could induce BmdsxF in male embryos after siRNA-mediated knocking down [35]. BmMasc also controls dosage compensation in male embryos, and male embryos injected with BmMasc siRNA did not hatch normally [35]. Kiuchi et al. [35] used two siRNAs to knockdown BmMasc and reported detecting BmdsxF in male embryo and the down-regulation of BmImpM. They proposed that BmImpM and Bmdsx are located downstream of BmMasc in the sex determination cascade. In contrast, our genetic analyses show no evidence of an effect of BmMasc on BmImpM. We propose that BmMasc controls male sexual differentiation by regulating Bmdsx but has no regulatory effect on BmImpM. It is still unclear how BmMasc regulates Bmdsx. F or M factors have been identified as the tra or tra2 gene in several insect species, and these factors directly regulated the dsx gene [25,50]. Previous reports concluded that BmPSI could directly bind Bmdsx pre-mRNA and that the BmImpM product increased BmPSI RNA binding activity in vitro [32]. Mutations of DmPSI in D. melanogaster strongly affect 43 splicing events and DmImp is one of the downstream genes targeted by it [51,52]. We found that BmImp and BmImpM had minor effects on BmdsxF splicing in our molecular genetic analyses. Furthermore, BmPSI did regulate expression of Bmdsx, BmImpM and BmMasc in vivo. It is unknown how BmPSI regulates the splicing of Bmdsx and other potential splicing factors involved in this process. Nonetheless, these data support the conclusion that BmPSI is at least a key auxiliary factor in the silkworm male sexual differentiation gene cascade, and provide the basis for the hypothesis that the BmPSI gene has a major initial role in the sex determination cascade. Silkworms of the same genetic background (Nistari, a multivoltine, nondiapausing strain) were used in all experiments. Wild-type (WT) and mutant larvae were reared on fresh mulberry leaves under standard conditions [53]. A binary transgenic CRISPR/Cas9 system was established to target selected genes. The piggyBac-based plasmid, pBac[IE1-EGFP-nos-Cas9] (nos-Cas9), was constructed to express the Cas9 nuclease in germ-line cells under the control of the B. mori nanos (nos) promoter with the EGFP fluorescence marker gene under the control of the IE1 promoter. The plasmid pBac[IE1-DsRed2-U6-sgRNA] (U6-sgRNA) was constructed to express single guide RNAs (sgRNA) under the control of the silkworm U6 promoter and the DsRed fluorescence marker gene also under control of the IE1 promoter. The sgRNAs targeting sequences were designed by manually searching genomic regions that match the 5′-GG-N18-NGG-3′ rule [54]. sgRNA sequences were checked bioinformatically for potential off-target binding using CRISPRdirect (http: //crispr. dbcls. jp/) by performing exhaustive searches against silkworm genomic sequences [55]. All sgRNA and oligonucleotide primer sequences for plasmid construction are listed in S2 Table. U6-dsx-sgRNA line from our previous report [56]. Each nos-cas9 or U6-sgRNA plasmid mixed with a piggyBac helper plasmid [53] was microinjected separately into fertilized eggs at the preblastoderm stage. G0 adults were mated to WT moths, and resulting G1 progeny scored for the presence of the marker gene product using fluorescence microscopy (Nikon AZ100) (S1 Table). Each U6-sgRNA transgenic line was mated individually with the nos-Cas9 line to derive mutated F0 animals. Genomic DNA of mutated animals was extracted at the embryonic or larval stage using standard SDS lysis-phenol treatment after incubation with proteinase K, followed by RNase treatment and purification. The resulting individual DNA samples from mutant animals were separated by sex using gene amplification with primers specific to the W chromosome (S2 Table). For two sgRNA sites, mutation events were detected by amplification using gene-specific primers which set on the upstream or downstream of the each target (S2 Table). Amplified products were visualized by 2% agarose gel electrophoresis running 30 min at 100V. Amplicons were sub-cloned into the pJET-1. 2 vector (Fermentas) and the positive clones of each we pick six were sequenced. For the one sgRNA, a restriction enzyme (HpyAV, New England Biolabs, Ipswich, MA, USA) cutting site adjacent to the AGG (PAM sequence) was selected to analyze the putative mutations by restriction enzyme digestion (RED) assay. The RED assay was carried out as previous report [57]. Phenotypic analysis focused light-microscope examination of the morphology of secondary sexual characteristics including internal and external genitalia. Photographs were taken with NRK-D90 (B) or DS-Ri1 (Nikon, Tokyo, Japan) digital cameras. Testes or ovaries were dissected from fourth-day, fifth-instar larvae and fixed overnight in Qurnah’s solution (anhydrous ethanol: acetic acid: chloroform = 6: 1: 3v/v/v). Tissues were dehydrated, cleared three times using anhydrous ethanol and xylene, respectively, and embedded in the paraffin overnight. Tissue sections (8 μm) were cut (Leica; RM2235) and stained using a mixture of hematoxylin and eosin solution. All pictures were taken under a microscope (Olympus BX51) using differential interference contrast with the appropriate filter. Total RNA was extracted from silkworm at different stages using Trizol reagent (Invitrogen) and treated with RNase-free DNAse I (Ambion). cDNAs were synthesized using the Omniscript Reverse transcriptase kit (Qiagen) in a 20 μl reaction mixture containing 1 μg total RNA. RT-PCR reactions were carried out using KOD plus polymerase (Toyobo) with gene-specific primers (S2 Table). Amplification of the B. mori ribosomal protein 49 (Bmrp49) was used as a positive control. Quantitative real-time RT-PCR (Q-RT-PCR) assays were performed using SYBR Green Realtime PCR Master Mix (Thermo Fisher Scientific) on an Eppendorf Real-time PCR System Mastercycler realplex (Eppendorf). Q-RT-PCR reactions were carried out with gene-specific primers (S2 Table). A 10-fold serial dilution of pooled cDNA was used as the template to make standard curves. Quantitative mRNA measurements were performed in three independent biological replicates and normalized to Bmrp49 mRNA. Experimental data were analyzed with the Student’s t-test. t-test: *, p < 0. 05, **, p < 0. 01, ***, p < 0. 001. At least three independent replicates were used for each treatment and the error bars show means ± S. E. M. | The sex determination system extremely diverse among organisms including insects in which even each order occupy a different manner of sex determination. The silkworm, Bombyx mori, is a lepidopteran model insect with economic importance. The mechanism of the silkworm sex determination has been in mystery for a long time until a Fem piRNA was identified as the primary female sex determinator recently. However, genetic and phenotypic proofs are urgently needed to fully exploit the mechanism, especially of the male sex determination. In the current study, we provided comprehensively genetic evidences by generating CRISPR/Cas9-mediated knockout mutations for those genes BmSxl, Bmtra2, BmImp, BmImpM, BmPSI and BmMasc, which were considered to be involved in insect sex determination. The results showed that mutations of BmSxl, BmImp and BmImpM had no physiological and morphological effects on the sexual development while Bmtra2 depletion caused Bmdsx splicing disappeared and induced embryonic lethality. Importantly, the BmPSI regulates expression of BmMasc, BmImpM and Bmdsx, supporting the conclusion that it acts as a key auxiliary factor to regulate the male sex determination in the silkworm. | lay_plos |
Tyrosine kinases are regarded as excellent targets for chemical drug therapy of carcinomas. However, under strong purifying selection, drug resistance usually occurs in the cancer cells within a short term. Many cases of drug resistance have been found to be associated with secondary mutations in drug target, which lead to the attenuated drug-target interactions. For example, recently, an acquired secondary mutation, G2032R, has been detected in the drug target, ROS1 tyrosine kinase, from a crizotinib-resistant patient, who responded poorly to crizotinib within a very short therapeutic term. It was supposed that the mutation was located at the solvent front and might hinder the drug binding. However, a different fact could be uncovered by the simulations reported in this study. Here, free energy surfaces were characterized by the drug-target distance and the phosphate-binding loop (P-loop) conformational change of the crizotinib-ROS1 complex through advanced molecular dynamics techniques, and it was revealed that the more rigid P-loop region in the G2032R-mutated ROS1 was primarily responsible for the crizotinib resistance, which on one hand, impaired the binding of crizotinib directly, and on the other hand, shortened the residence time induced by the flattened free energy surface. Therefore, both of the binding affinity and the drug residence time should be emphasized in rational drug design to overcome the kinase resistance. The past decade has witnessed the great benefit of the personalized drug therapy in the treatment of non-small-cell lung cancers (NSCLC) [1]–[3], which was designed to target different drug targets, such as KRAS [4], EGFR [5], EML4-ALK [6], the newly found CD74-ROS1 [7], [8], etc. Crizotinib, the latest launched NSCLC drug, was originally designed to competitively inhibit the activity of c-MET [9], whereas has been approved by U. S. Food and Drug Administration (FDA) for the treatment of advanced NSCLC with anaplastic lymphoma kinase (ALK) rearrangements in 2011. And recently, it has also been found with great clinical benefit in the treatment of advanced NSCLC patients with fusion-type CD74-ROS1 tyrosine kinase with the response rate of 57% and a disease control rate at 8 weeks of 79% [10], [11]. Therefore, crizotinib may be the most successful chemical drug for the personalized therapy in NSCLC. Unfortunately, under strong purifying selection, cancer cells can eventually confer resistance to the therapeutic drugs, and they may survive by means of activating other signaling pathways [12]–[16], regulating the expression level of the associated genes or gene products [17]–[19], or more directly, hindering the drugs binding [20], [21], enhancing the substrates binding [22], or re-activating the target [23] with acquired secondary mutations in the drug target. Therefore, it is no surprise that ROS1 was trapped in the crizotinib resistance as well, with very short term of the crizotinib therapy as reported by Awad and colleagues [24]. They had found a de novo secondary mutation G2032R in CD74-ROS1, and this mutation conferred serious resistance to crizotinib. It was supposed that the mutation was located at the solvent front, and might hinder the drug binding. However, it might not be true when one has a view on the crystal structure, where a large binding pocket can be found in the drug-target complex, and actually, a sole mutation may hardly hinder the drug binding as we showed below (the drug could smoothly unbind or rebind to the mutated ROS1 tyrosine kinase). Alternatively, by using advanced molecular dynamics (MD) methodologies (funnel based well-tempered metadynamics and Woo and Roux' s absolute binding free energy calculation scheme), we constructed the free energy surfaces (FESs) along the drug-target distance and the phosphate-binding loop (P-loop) conformational change which is responsible for the binding of competitive inhibitors to tyrosine kinases, and the FESs unrevealed the drug resistance mechanism in detail: the more rigid P-loop region in the G2032R mutant was the main reason for the crizotinib resistance, which on one hand, impairs the binding of crizotinib directly, and on the other hand, shortens the residence time as well. Therefore, considering the importance of the role of kinases in the therapy of carcinomas, we suggests that, besides emphasizing the binding affinity, the residence time should be considered to design potent leads to overcome resistance as well. As shown in Figure S1, all the systems (bound-state WT-ROS1, free-state WT-ROS1, bound-state G2032R-ROS1 and free-state G2032R-ROS1) reached equilibrium after 5 ns simulation, with the RMSDs (A and B) less than 3 Å and RMSFs (C and D) less than 2 Å in most regions. Therefore, the equilibrated trajectories (5∼30 ns) were suitable for the conformational analysis, and the following metadynamics and umbrella sampling simulations. Although no free-state ROS1 tyrosine kinase was crystallized, we could construct a free-state ROS1 by removing the co-crystallized ligand in the crystal structure instead. It is well-known that the binding site of a target could be induced into a suitable conformation when binding with a ligand, the so called induced fit phenomenon, and we detected the conformational difference between the bound-state and unbound-state ROS1 (WT-ROS1 and G2032R-ROS1) in the P-loop region as well. As shown in Figure 1C, the most populated dihedral angle of the P-loop in the bound-state WT-ROS1 is ∼20° smaller than that in the free WT-ROS1, suggesting that a more closed state of the P-loop was prevalent in the bound-state protein, which is consistent with the induced fit theory. The same phenomenon has been observed in G2032R-ROS1 in Figure 1D, where a very different distribution of the dihedral angle of the P-loop region was detected between the bound-state (purple) and free-state (green) G2032R-ROS1, with the middle value 20° larger and more widely distributed in the unbound-state G2032R-ROS1. That is to say, the mutation makes the P-loop region of the free-state G2032R-ROS1 more flexible, and a more opened structure of the P-loop region is dominant in the free G2032R-ROS1. A comparison of the bound-state WT-ROS1 (gray) and G2032R-ROS1 (purple) shows that the P-loop region in G2032R-ROS1 is indeed more opened than that in WT-ROS1 (Figure 1E), and the difference of the dihedral angle is ∼20°, which is close to the angular difference between the bound-state and free WT-ROS1 (Figure 1C). Therefore, a comparison was carried out between the dihedral angle distributions of the unbound-state WT-ROS1 and bound-state G2032R-ROS1. As shown in Figure 1F, interestingly, similar distributions of the dihedral angles were found for the unbound-state WT-ROS1 (orange) and bound-state G2032R-ROS1 (purple), indicating the P-loop regions in the unbound-state WT-ROS1 and bound-state G2032R-ROS1 adopted similar conformations. The averaged structures of the bound-state WT-ROS1 (Figure 1A) and G2032R-ROS1 (Figure 1B) show that the P-loop region (orange in Figure 1A and 1B) in G2032R-ROS1 is indeed upper-moved compared with that in WT-ROS1, and this phenomenon could be attributed to the mutation G2032R directly, which formed a scaffold-like structure (green region in Figure 1B) and supported the P-loop region in G2032R-ROS1. On the contrary, the no-side-chain amino acid glycine in WT-ROS1 (green in Figure 1A) cannot affect the conformation of the P-loop region anymore. Therefore, it could be found (Figure 1A and 1B) that the binding pocket in G2032R-ROS1 was more opened than that in WT-ROS1, and still, the pyridine ring of crizotinib (pink stick model) near the mutated site (green) in G2032R-ROS1 was located more outside of the binding pocket compared with the fragment (green stick model) in WT-ROS1. Awad has supposed that the solvent front mutation G2032R may hinder the drug binding to the mutated ROS1 [24], which seems possible when a small amino acid was replaced by a larger one. However, the fact that the drug has similar binding pose to that of WT-ROS1 (Figure 2A), and most part of the drug is still located in the active pocket of G2032R-ROS1 (Figure 2B). Moreover, no hydrogen bonds were lost (Figure 2C and 2D) or even a new hydrogen bond was formed between the mutated residue R2032 and crizotinib (Figure 2E, green dot line), indicating that the mutation cannot directly hinder the drug binding. Alternatively, it seems that the up-moved P-loop region in G2032R-ROS1 could attribute to the crizotinib resistance by attenuating the interactions between the drug and the enlarged binding pocket, which has also been observed in the C1156Y induced crizotinib resistance in the ALK tyrosine kinase [25]. Therefore, by using advanced free energy calculation approaches, we discussed the drug resistance mechanism in detail in the next two sections. Drug resistances are usually associated with the attenuation in drug binding to its target, which can be mechanically detailed by free energy calculations. Although two-end-state calculations are effective in determining the change of the total binding free energies between the mutated and the wild-type drug-target complexes [26]–[29], it is indeed powerless in describing a physically associated pathway upon how a drug binds to or unbinds from its target, which may be more helpful in understanding the binding or unbinding process of a drug. Alternatively, process-based methods can easily solve the problem by calculating a one-dimensional (1D) free energy profile or a two-dimensional (2D) free energy surface along given reaction coordinates (RCs). Here, funnel based well-tempered metadynamics was employed to construct the free energy surfaces. In the spirit of metadynamics, repulsive Gaussian potentials were periodically added to the selected reaction coordinates, and therefore, the biased molecule could unbind and rebind to the active pocket repeatedly. As illustrated in Figure 3F, the drug was periodically binding (bound-state, red dot line A) to and unbinding (overcome-barrel state, B, and free-moving state, red dot line C) from the active pocket, which represents the convergence of the binding-unbinding process, and the added potentials could be used for the FES construction. As shown in Figure 3D, the 2D free energy surface was plotted with the CoMs distance (the centers of masses between crizotinib and the active pocket of ROS1) as X-axis and the P-loop conformational change (similar path mean-square-deviation of P-loop) as Y-axis. In X-axis, a position near 0 Å denotes the drug bound in the active pocket, while the drug could move freely in bulk at the position of 20 Å. Similar path MSD was employed to detect the conformational change of P-loop when it binds or unbinds the drug. A lower value of the S-path MSD means that the conformation of P-loop is very similar to that of the bound-state P-loop, while a higher value represents the free-state P-loop or much-opened P-loop. In the FES, the area was colored from blue to red where has the lowest and highest ensemble energy, e. g. ligand in the binding site and bulk. The minimum-free-energy pathway (black dot line in Figure 3D) was constructed by connecting the bins with the minimum free energies along the CoM distance (X-axis). Interestingly, an induced fit behavior was observed during the drug binding and unbinding processes from the active pocket, where the P-loop closed when crizotinib binding to the active pocket (lower S-path MSD), and opened when crizotinib freely moved in bulk (higher S-path MSD). More detailed structural descriptions have been illustrated in Figures 3A, 3B, and 3C, which correspond to the stable bound-state (red point A in Figure 3D), overcome-barrier state (red point B in Figure 3D), and free-moving state (red point C in Figure 3D), respectively. Figure 3E represents the longitudinal section of the 2D FES, and a high barrier was found located at 4∼8 Å of the RC (position B), indicating that high energy was needed to overcome the barrier when crizotinib got into or out of the binding site. The overlapped structures of the bound-state (gray cartoon model) and overcome-barrier state (orange cartoon model) WT-ROS1 complexes uncovered the high barrier mechanism (the induced fit phenomenon). As shown in Figure 3G, a large conformational change of the P-loop region was observed in the overcome-barrier state WT-ROS1 (pink cartoon region), which was markedly up-moved compared with that of the bound-state WT-ROS1 (green cartoon region), and this needed much energy to cancel the conformational energy loss of P-loop. Similar behavior of crizotinib was found in the G2032R mutated ROS1 tyrosine kinase. As shown in Figure 4F, the drug periodically bound into and unbound from the active pocket as well, and a same process was observed of the drug unbinding from the target, where the pyridine ring was first getting out of the binding site and followed by the halogenated benzene fragment (Figure 4A, 4B, and 4C). However, the free energy surface was different from that of WT-ROS1 to a certain extent. At first, there was no energy barrier located at 4∼8 Å of the CoM-distance based RC (X-axis) as shown in Figure 4E (point B). Second, the most favorable unbinding pathway (minimum-free-energy pathway) showed a much lower S-path MSD value (∼0. 5 Å2) in G2032R-ROS1compared with that in WT-ROS1 (>1 Å2). Structural observation showed that, unlike WT-ROS1, there was no conformational change of the P-loop region when the drug unbinding form the active pocket as illustrated in Figure 4G, where the bound-state and overcome-barrier state G2032R-ROS1 complexes were shown in purple (yellow in P-loop) and orange (pink in P-loop) cartoon models, respectively. Therefore, no energy was needed to cancel the energy loss associated with the conformational change of P-loop. Besides, as discussed above, the conformation of the P-loop region in the bound-state G2032R-ROS1 is very similar to the free-state P-loop in WT-ROS1, indicating that no much conformational change was needed to bind or unbind the drug in G2032R-ROS1. As a result, slight change of the S-path MSD was observed in the bound-state (point A in Figure 4D) and unbound-state (point C in Figure 4D) G2032R-ROS1. Although metadynamics simulations have been widely used in FES construction, it will be hard to get a convergent result when missing any associated reaction degree of freedom. Therefore, by using the absolute binding free energy calculation based on the umbrella sampling (US) simulations, we validated the consistence of our results, and obtained a more stable prediction of the binding free energy based on the minimum-free-energy pathway derived from the metadynamics simulations. Woo and Roux' s scheme [30] was employed to calculate the absolute binding free energy. The starting structure of each window (a total of 40 windows) of the separation US simulation was derived from the metadynamics simulations, which have constructed the most favorable unbinding pathway of crizotinib [31]. As shown in Figure 5, six points were selected to control the rotational and translational degree of freedoms of the crizotinib unbinding process, where the point PC was substituted by a fictitious atom placed at 5 Å away from LC along X-axis. Therefore, it can be found in Figure 6A that the minimum energy points of WT-ROS1 and G2032R-ROS1 are both located at 5 Å of the RC. In the restrained US simulations, the conformation-dependent PMFs of crizotinib were first calculated in the binding site (Figure 6B1) and bulk (Figure 6B2). As shown in Figure 6B1, the shape of the PMF line in G2032R-ROS1 (orange) has a much broader local minima region compared with that in WT-ROS1, indicating that a more loose binding conformation was employed by G2032R-ROS1. This inference has been validated by our analysis above that a more opened P-loop was dominated in the bound-state G2032R-ROS1, which leads to the loose-binding of crizotinib. Compared with the bound-state RMSD based PMFs, a much broader RMSD change of crizotinib was found in bulk (Figure 6B2). Although different starting conformations of crizotinib were employed for the conformation-restrained simulations in WT-ROS1 and G2032R-ROS1, a very similar shape of the RMSD based PMFs was observed for WT-ROS1 and G2032R-ROS1 (individualized bound-state crizotinib was used as the reference conformation for the restrained US simulation), which means that consistent binding conformations of crizotinib were used inWT-ROS1 and G2032R-ROS1. Due to the existence of the conformational restrains in crizotinib, no significant difference has been observed in the angle based PMFs (α, β, γ, θ, and Θ) between WT-ROS1 (Figure 6C1–6C5) and G2032R-ROS1 (Figure 6D1–6D5). However, large difference has been found between the separation PMFs of WT-ROS1 (blue) and G2032R-ROS1 (orange). As shown in Figure 6A, the separation PMF in G2032R-ROS1 was much lower than that in WT-ROS1, suggesting that serious crizotinib resistance could be induced by the mutation G2032R in the ROS1 tyrosine kinase. The energetic component contributions have been summarized in Table 1, where the conformational restrains (binding site,, and bulk,), rotational restrains (binding site,,, and, and bulk,), and translational restrains (binding site, and) associated energies were calculated by the direct integration of the Boltzmann factor based on the PMFs (,,,,,, and) or numerical integration (). The separation PMFs () were obtained by finding the energy difference between the bound-state and unbound-state ensembles. As listed in Table 1, all the energetic components contributed slightly to the difference of the total energies except the separation PMF. Therefore, the separation PMF difference should be the main contributor for the crizotinib resistance, which is consistent with the analysis shown above that the opened structure of P-loop in G2032R-ROS1 leads to more loose binding of the drug. Although the predicted binding free energies given by the two methodologies (US based binding free energy, ΔGbind-US, and metadynamics based binding free energy using minimized pathway, ΔGbind-Meta) are a bit different, the binding free energies both markedly decreased in G2032R-ROS1, which is consistent with the experimental data of IC50. By using advanced molecular dynamics techniques, namely funnel based well-tempered metadynamics and umbrella sampling based absolute binding free energy calculation approaches, we investigated the drug resistance mechanism of G2032R in ROS1 tyrosine kinase. A more rigid conformation of the P-loop was detected in G2032R-mutated ROS1 tyrosine kinase, which was much opened even in the bound-state and had a similar conformation as that of the free-state wild-type ROS1 tyrosine kinase. The conformational analysis showed that the scaffold-like side chain of the mutation R2032 was responsible for the markedly opened structure of the P-loop in G2032R-ROS1, which supported the P-loop and hindered its closure during the drug binding. Therefore, the P-loop was hard to be induced during the whole binding/unbinding process of crizotinib, and thus, an attenuated binding state was dominated between crizotinib and binding pocket of G2032R-mutated ROS1 tyrosine kinase. In addition, we have analyzed the energetic contribution to crizotinib on residue level as well, which showed that the residue Leu18 (located just in the P-loop region) contributed the most to the attenuated binding of crizotinib to G2032R-ROS1 as shown in Figure S3, therefore well supporting the issue that the P-loop conformation governs crizotinib resistance in G2032R mutated ROS1 tyrosine kinase. It has been discussed above that the up-moved P-loop has directly attenuated the binding of crizotinib to G2032R-ROS1, which corresponds to a substantial loss of the total binding free energy compared with WT-ROS1 (ΔΔG of ΔGbind-Meta and ΔGbind-US). Nevertheless, another reason may still contribute to the drug resistance, namely, the shortened residence time of crizotinib in G2032R-ROS1. As the notion describes, a larger activation free energy of dissociation, ΔGoff, corresponds to a longer residence time:, where 1/koff has been defined as the residence time (t = 1/koff). Therefore, a lead may be more promising to be a drug if it has a longer residence time in the organisms [32]–[35]. It can be found in Figure 3E that a large barrier was located at 4∼8 Å of the RC in WT-ROS1, which, although, might hinder the drug getting into the active pocket, it could indeed significantly increase the residence time with the ΔGoff of ∼12 kcal/mol, which is much larger than that in G2032R-ROS1 (ΔGoff = ∼5. 5 kcal/mol, as shown in Table 1). Taken together, it could be summarized that the up-moved P-loop region, which was supported by the scaffold-like conformation of the large side chain of R2032, has contributed mainly to the crizotinib resistance in G2032R-ROS1, where it, on one hand, decreased the binding affinity of the drug by loosed-binding-state because of the enlarged binding pocket, and on the other hand, shortened the residence time by flattening the free energy surface. The calculated binding free energy was reasonably consistent with the experimental data, suggesting that, besides binding affinity, residence time should be considered as well for rational drug design to overcome drug resistance. The X-ray crystal structure of ROS1 complexed with crizotinib (PDB code 3ZBF [24], resolution 2. 2 Å) was used as the initial structure for the MD simulations. The drug was optimized at HF 6-31G* level of theory using Gaussian 09 program [36], and the electrostatic potentials were calculated at the same method based on the optimized structure. The atomic partial charges were obtained by using the restrained electrostatic potential technique [37] (RESP) in Ambertools 1. 5 [38]. The missing residues, G1954-F1956, which are located at the P-loop region of ROS1 and were renumbered as Gly21, Ala22, and Phe23 in this study, were built with the loop module in SYBYL-X1. 2 simulation package. Although the P-loop region was involved in the imperfect crystallization, the missing residues seemed contribute little to the binding of crizotinib with the energetic contribution ∼0 kcal/mol of the three residues as shown in Figure S3A and S3B. The residue G2032 was mutated into R2032 by using the biopolymer module, and followed by structural adjustment in SYBYL-X1. 2. For convenience, all the residue indexes were renumbered from 1 to 285. The protonation states of residues, such as histidines and cysteines, were determined using PROPKA (version 3. 1) [39]. The Amber ff99SB force field [40] and General AMBER Force Field (GAFF) [41] were used for the protein and ligand, respectively. For the unbound-state systems, crizotinib was directly removed from the crystal structures. 3 Na+ and 2 Na+ were added to neutralize the wild-type (WT) and mutated systems, respectively. Cubic TIP3P [42] water boxes were added to the systems with 10 Å extended from any solute atoms. All the simulations were performed with NAMD version 2. 8 in conjunction with PLUMED 1. 3 [43], [44]. A 10 Å cutoff was used for the short range electrostatic and van der Waals interactions, and the particle mesh Ewald (PME) [45] algorithm was used to handle the long-range electrostatic interactions. All covalent bonds involving hydrogen atoms were constrained with the SHAKE algorithm [46]. Prior to MD simulation, four steps of minimization were preformed to the systems. At the first stage, only hydrogen atoms were able to move freely (2500 steps). Afterward, the heavy atoms of solvent and ions were free as well (2500 steps). Thirdly, heavy atoms in side chain and backbone of protein within 5 Å of the mutation were relaxed (5000 steps). At last, all the atoms were optimized for 20,000 steps. In the MD simulation stage, the time step was set to 2 fs. The systems were gradually heated from 0 K to 310 K in 1 ns with a restrain of 5 kcal/mol·Å2 to the heavy atoms in the backbone in an NVT ensemble. Afterwards, the systems were relaxed for 0. 2 ns with the restrain gradually decreased from 5 to 0 kcal/mol·Å2 in an NPT (P = 1 atm and T = 310 K) ensemble. The Poisson Piston algorithm was used to control the pressure [47]. Finally, 30 ns production runs were performed with the collective interval of 5 ps (200 frame/ns), and a total of 6000 frames were collected for the conformational space analysis. Metadynamics simulation has been widely used in enhanced sampling simulations [48], [49], which was effective in describing free energy surface (FES) in terms of ligand-receptor binding process [50]–[54], protein conformational transition or activation [55]–[60], and protein-protein interaction [61], [62]. Moreover, numerous on-the-fly metadynamics-based techniques have been developed in recent years, such as well-tempered metadynamics [63], reconnaissance metadynamics [64], funnel metadynamics [65], parallel tempering metadynamics [56], and bias-exchange metadynamics [66], which all significantly accelerated the sampling and convergence rates compared with the standard metadynamics. Taken well-tempered metadynamics as an example, it adds a history-dependent Gaussian repulsive potential on the selected collective variables (CVs) as shown in equation (1): (1) where V (s, t) is the history-dependent biasing potential, t′ denotes the deposition time. At each time interval τ, a Gaussian potential, with the height of, will be added on the concurrent position s (t′) of the biased molecule. Here, ΔT was set to 3100 K corresponding to a bias-factor of 10 in well-tempered algorithm. Different from the standard metadynamics that uses an immutable hill height in the simulation, the initial hill height in well-tempered metadynamics (ω) is scaled by the exponential of V (s, t′) /ΔT to accelerate the convergence. Therefore, by using the advanced metadynamics techniques, namely, well-tempered metadynamics and funnel metadynamics, we explored the free energy surfaces of the crizotinib unbinding from WT and G2032R-mutated ROS1 tyrosine kinase, which were designed to move against the ligand-receptor distance and P-loop conformational change (open or close) as described in the previous studies [50], [67]. The averaged structures (derived from the equilibrium trajectories) of WT and G2032R-mutated crizotinib-ROS1 complexes were used as the initial structures for the metadynamics simulations. Prior to the metadynamics simulations, the complexes were immersed in a rectangular water box with the largest pocket direction rotated to X-axis (30 Å out of the solutes in X-axis), which was detected by Caver 2. 0 [68], as we did previously [67], [69]. Then, the systems were minimized with a large restrain (100 kcal/mol·Å2) on the heavy atoms of the complexes. Afterward, the systems were equilibrated for 1 ns with the heavy atoms constrained as well. The final structures were submitted to the metadynamics simulations. In the well-tempered metadynamics, the heavy atoms out of 15 Å of crizotinib in proteins were constrained with 5 kcal/mol·Å2 to prevent drifting issues [70]. The initial hill height (ω) was set to 1, and the deposition rate of the added biasing potential was set to 1 kcal/mol·ps with the bias-factor parameter of 10 at 310 K of the simulation temperature. Two CVs were used for the construction of the free energy surface. The first CV was the distance between center-of-mass (CoM) of heavy atoms in crizotinib and center-of-mass of the binding site (heavy atoms within 5 Å of crizotinib in ROS1). The width of Gaussian hill (σ) was set to 0. 4 Å, and 400 bins were divided from the range of 0 to 24 Å. The second CV was known as the similar path (S-Path) of the P-loop region of the ROS1 tyrosine kinase, which corresponds to the conformational change of the P-loop region from the bound-state conformation to the unbound-state conformation based on the measurement of Mean Square Deviation (MSD) [71]. The conformations of the bound-state and free-state P-loop were derived from the equilibrated conventional MD trajectories by measuring the dihedral angle of Cα in Glu25, Ser20, Ala22 (which are located in the P-loop region), and Arg150 (which is located in the active site with stable conformation). A total of 7 frames were used for the S-Path calculation with the MSD interval of 1 Å2 on average ranged from 0 to 6. The width of the Gaussian hill for the second CV was set to 0. 05 Å2 and 350 bins were collected for the construction of FES. Due to the hardness of convergence of the FES, funnel-based metadynamics was employed in conjunction with the well-tempered algorithm to accelerate the convergence, which adds a harmonic restrain wall around the CVs [65]. Here, a cylinder-shaped restrain funnel was constructed along the first CV (distance between CoMs of the receptor and ligand) to prevent the drug absorbing on the unrelated region of the target. The radius of the cylinder was set to 15 Å to provide enough space for the rotation of the biased molecule (∼10 Å in length of crizotinib). A restrained energy (with the elastic constant of 100 kcal/mol·Å2) will be added to the Hamiltonian of the biased molecule if it goes out of the cylindrical funnel for the purpose of forcing the biased molecule back to the reaction associated sampling space. Therefore, the method significantly enhances the sampling in the associated reaction-space and made the convergence of the FES rapidly. Due to the use of cylindrical restrains in the metadynamics simulations, the absolute binding free energy needs to be adjusted according to Equation (2), and the detailed descriptions could be found in reference [65]. (2) where ΔGbind-Meta represents the absolute binding free energy calculated based on metadynamics simulation, and ΔGbind is the PMF depth corresponding to crizotinib unbounded from binding site to bulk, and is the surface of the cylinder used in the restrains. kB is Boltzmann constant, and C° is the standard concentration corresponding to 1/1661 Å3. To construct one-dimensional free energy profiles from two-dimensional free energy surfaces, bins with lowest energy among the S-path MSD (Y-axis in Figure 3D and 4D) were connected along the CoM distance between crizotinib and the active pocket of ROS1 tyrosine kinase (X-axis in Figure 3D and 4D). Among the enhanced sampling methodologies, umbrella sampling may be the most classic, widely used, and easily accepted method [72]–[81], which adds biasing potentials along a given reaction coordinate (RC) to drive the system from one thermodynamic state to another [82]. In detail, the RCs are usually divided into several parts, named windows, and to make things easy, harmonic potentials are often added in each window for biased sampling as shown in Equation (3). (3) where ki is the elastic constant in window i, and denotes the reference state of window i, which could be a reference conformation (RMSD), angle (α, β, γ, θ, Θ), or position (r) as shown below. After the enhanced sampling simulation, the distribution of biased samples could be unbiased to obtain the free energy change in each window. Finally, the calculated free energy in each window could be integrated by reweighted method, such as weighted histogram analysis method (WHAM) [83], [84], to give a total free energy change along the reaction coordinate. Compared with the probability based method, such as umbrella sampling, another kind of widely used enhanced sampling approach, adaptive biasing force (ABF) [85]–[94], is the interaction-based method, which adds biasing force on the investigated molecule (or fragment) for the purpose of canceling the local barrier acted on the molecule (or fragment) [95], [96]. Therefore, as a result, all the positions of the reaction coordinate can be sampled with equal probability and the biased molecule can go with a free-diffusion-like behavior along the reaction coordinate [67], [94]. Moreover, ABF may be a more convenient method with fewer priori parameters and simulation windows needed to be tested and divided, such as it does not need a well-tested elastic constant added in each window as umbrella sampling simulation does [82], and we can use only one window to sample the orientation-associated PMFs of the biased molecule as shown below, which have a same behavior as Gumbart' s result [97]. Roux and co-workers have developed a well-characterized and well-tested absolute binding free energy calculation scheme by using various restrains, including conformational, rotational, and translational restrains, to the investigated systems, which significantly accelerated the convergence due to the constriction of the relative external degrees of freedom of the systems [30], [70], [97]–[104]. In the spirit of Woo and Roux' s scheme [30], the absolute binding free energy could be obtained by calculating the reaction equilibrium constant (Kbind) with restrains at the biased molecule' s conformation (corresponding to and), orientation (corresponding to,,, and), and translation (corresponding to, and), as shown in equations (4) and (5): (4) (5) where ΔGbind-US is the absolute binding free energy calculated based on umbrella sampling simulation, and S* and I* are associated with the angular and translational restrains in bulk and separation PMF depth, respectively [30]. By using the combination of US, ABF, and Roux' s absolute binding free energy calculation scheme, we accurately characterized the one-dimensional (1D) free energy profile of crizotinib separated from the binding sites of the WT and G2032R-mutated ROS1 to the bulk. Umbrella sampling was used to the conformational restrained simulations (RMSD) and separation simulations (r), while adaptive biasing force was employed for the angular restrained simulations, including rotational (α, β, γ) and translational (θ, Θ) associated simulations. The same well-equilibrated structures of WT and G2032R-mutated crizotinib-ROS1 complexes as those used in metadynamics simulations were employed as the initial structures for the US and ABF simulations. In the phase of conformational restrained simulations, the RMSD change of crizotinib was used as the RC for umbrella sampling (k = 0. 01 kcal/mol·Å2). The RCs were divided into 7 and 11 windows for the bound-state and free crizotinib, respectively, with the size of each window 0. 5 Å, namely, a range of 0∼3 Å and 0∼5 Å of the RMSD for the bound-state and unbound-state crizotinib. Each window was simulated for 3 ns, and the samples of the last 1. 5 ns were used for the construction of the PMFs. For the angular restrained simulations, the five angles (α, β, γ, θ, and Θ) were orderly sampled with crizotinib constrained in the initial state (at the state of RMSD = 0 and a restrain of 1 kcal/mol·Å2 was used for the constrain of the conformation of crizotinib, and followed by the orderly restrains in the angles with 0. 3 kcal/mol·Å2). ABF was employed for the angular restrained sampling. The bin size was set to 0. 2 Å, and only one window was used for 5 ns simulations. Due to the algorithm of ABF in NAMD code, fictitious particles were used to construct an extended and generalized coordinate as proposed by Gumbart et al [97]. As shown in Figure 5, the RC of separation simulations were constructed along the vector of, where the PC point is a dummy atom placed at 5 Å away from the LC point (C12 in crizotinib). 41 windows were used for the US simulation with each window 0. 5 Å across the range of 5∼25 Å of the RC. The elastic constant was set to 5 kcal/mol·Å2 in the middle of each window to drive the drug from binding site to bulk. The initial structure in each window (except for the initial window) was dirived from the trajectory of metadynamics because metadynamics could give a most feasible unbinding pathway of the system [31]. Each window of the separation simulation was preformed with the existence of conformational and angular restrains in crizotinib. 7 ns and 3. 5 ns simulations were preformed for each window involved in large (5∼13 Å) and low (13∼25 Å) barrier regions, respectively. A total of ∼200 ns simulation was preformed for each separation sampling, and the two systems both reached convergence as shown in Figure S2. The detailed simulation time can be found in Table S1. Because of the isotropy of the bulk, the energy associated with orientation in bulk could be obtained by direct numerical integration without actual MD simulation [30]. | Cancers can eventually confer drug resistance to the continued medication. In most cases, mutations occurred in a drug target can attenuate the binding affinity of the drugs. Here, we studied the drug resistance mechanisms of the mutations G2032R in the ROS1 tyrosine kinase in fusion-type NSCLC. It is well known that the phosphate-binding loop (P-loop) plays a vital role in the binding of competitive inhibitors in tyrosine kinases, and numerous mutations have been found occurred around the P-loop, which may affect the binding/unbinding process of a drug. Free energy surfaces were constructed to characterize the impact of the mutation to the binding/unbinding process of a well-known NSCLC drug, crizotinib. Two advanced free energy calculation methods, namely funnel based well-tempered metadynamics and umbrella sampling based absolute binding free energy calculation achieved consistent results with the experimental data, suggesting that the rigid P-loop of the mutated target was mainly responsible for the crizotinib resistance to ROS1 tyrosine kinase. | lay_plos |
The response of a neuronal population over a space of inputs depends on the intrinsic properties of its constituent neurons. Two main modes of single neuron dynamics–integration and resonance–have been distinguished. While resonator cell types exist in a variety of brain areas, few models incorporate this feature and fewer have investigated its effects. To understand better how a resonator’s frequency preference emerges from its intrinsic dynamics and contributes to its local area’s population firing rate dynamics, we analyze the dynamic gain of an analytically solvable two-degree of freedom neuron model. In the Fokker-Planck approach, the dynamic gain is intractable. The alternative Gauss-Rice approach lifts the resetting of the voltage after a spike. This allows us to derive a complete expression for the dynamic gain of a resonator neuron model in terms of a cascade of filters on the input. We find six distinct response types and use them to fully characterize the routes to resonance across all values of the relevant timescales. We find that resonance arises primarily due to slow adaptation with an intrinsic frequency acting to sharpen and adjust the location of the resonant peak. We determine the parameter regions for the existence of an intrinsic frequency and for subthreshold and spiking resonance, finding all possible intersections of the three. The expressions and analysis presented here provide an account of how intrinsic neuron dynamics shape dynamic population response properties and can facilitate the construction of an exact theory of correlations and stability of population activity in networks containing populations of resonator neurons. Integration and resonance are two operational modes of the spiking dynamics of single neurons. These two modes can be distinguished from each other by observing the neuron’s signal transfer properties: how features in its input current transfer to features in its output spiking. The traditional approach to investigating neuronal transfer properties is to measure the stationary response: the time-averaged rate of firing of spikes as a function of the mean input current, or fI-curve. In Hodgkin’s classification [1], Type I membranes can fire at arbitrarily low rates, while the onset of firing in Type II membranes occurs only at a finite rate. This distinction arises naturally from the topology of the bifurcations that a neuron can undergo from resting to repetitive spiking [2]. In many central neurons, it is fluctuations rather than the mean input current that drive spiking, putting them in the so-called fluctuation-driven regime [3]. Many dynamical phenomena are nevertheless tightly linked to excitability type. For example, Type II neurons exhibit rebound spikes, subthreshold oscillations and spiking resonance (e. g. mitral cells, [4–6], respectively). The qualitative explanation for these phenomena is that the dynamical interplay of somatic conductances endow some neurons with a voltage frequency preference, i. e. a subthreshold resonance. This preference can contribute to a superthreshold resonance in the modulation of their output spiking [7]. How dynamic response properties of spiking dynamics such as resonance emerge can be directly assessed by considering the neuron’s dynamic gain. Dynamic gain, first treated by Knight [8], quantifies the amount by which features at specific frequencies in the input current to a neuron are amplified or attenuated in its output spiking. It can accurately distinguish functional types and unveil a large diversity of phenomena shaping the response to dynamic stimuli [9–18]. Dynamic gain and response are also essential ingredients for theoretical studies of network dynamics in recurrent circuits [8,12,13,18–49]. First, they determine the stability of the population firing rate dynamics [21,25,26]. Second, they determine how input correlations between a pair of cells are transferred to output correlations [28,42,44–49], from which self-consistent relations for correlations in recurrent circuits can be obtained. Experimental studies have started over the past years to use dynamic gain measurements to investigate the encoding properties of cortical neuron populations [9–18]. Although theoretical studies have investigated many neuron models, very few models are known for which dynamical response can be explicitly calculated. One basic reason for this lies in the fact that Fokker-Planck equations for neuron models with two or more degrees of freedom are not solvable in general [50]. For Type II neuron models that require at least two degrees of freedom, no solvable model is known. The simplest model capable of subthreshold resonance was introduced by Young [51] in the early theories of excitability. Later, Izhikevich formulated a structurally similar neuron [52]. Richardson and coworkers performed the first calculation of the linear response function of a neuron model capable of resonance, the Generalized Integrate-and-Fire (GIF) neuron [22,29]. Only in the limit of relatively slow intrinsic current time constant can analytical expressions for the GIF response be obtained, however. The distinct transfer properties of resonant vs. non-resonant dynamics leads to different information transfer properties. While this has been demonstrated in the mean-driven regime [53,54], no such results exist for the fluctuation-driven regime, in part due to a lack of exact analytical expressions for even the linear dynamic gain. Type II excitability and dynamic response thus are representative of the more general challenge posed by response properties of neurons with complex intrinsic dynamics. In the current study, we derive and analyze the linear response function in the fluctuation-driven regime of a neuron model capable of resonance. We express it as a filter cascade from current to voltage to spiking. It is valid across all relevant input frequencies and over all relevant values of the intrinsic parameters. In particular, we apply to the GIF neuron model the Gauss-Rice approach in which the voltage reset after a spike is omitted. The methods generalize to additional intrinsic currents and to the full nonlinear response with spike generation. To understand how subthreshold features interact to determine a neuron’s filter characteristics, including resonance, we provide a two-dimensional representation of the response properties that completely characterizes all possible filter types. For this idealized model, we determine analytically and numerically a wide and biologically-relevant regime of validity of the derived expression. The paper begins with the definition of the model and its numerical implementation. We then derive a general expression for the linear response in the mean channel of a Gauss-Rice neuron. In the next section, the analytical results for the response properties of the Gauss-Rice GIF neuron model are obtained. The final section then presents an analysis of the expression. For the sake of mathematical clarity, most calculations appear in the main text; the rest, including an exposition of model assumptions, are contained in the Methods. We consider the most simple hard-threshold, no-reset, GIF-type neuron capable of exhibiting resonator dynamics, whose response properties have been partially studied in [25]. A reset version of this model is treated in [29], where the population spiking response properties were calculated assuming large intrinsic time constant. In the Methods, we present a more detailed exposition of the model assumptions, and justify an additional simplification of the voltage reset after a spike. The feature that distinguishes the GIF model from the classical Leaky Integrate-and-Fire (LIF) model is that the dynamics of the voltage, V, is coupled to an intrinsic activity variable, w, τ V V ˙ = − V − g w + I s y n τ w w ˙ = V − w, (1) where g is a relative conductance and τV and τw are the respective time constants of the dynamics. The notation x ˙ denotes the derivative with respect to time of the variable x. Spikes are emitted at upward crossings of a threshold, θ. Synaptic current modeled by Isyn drives the model whose dynamics are kept stable by keeping g > −1. When g < 0, w is depolarizing. When g > 0, it is hyperpolarizing and can lead to resonant voltage dynamics. Response theory captures the population response to input signals with arbitrary frequency content and so we now turn to it, and linear response theory in particular, in the pursuit of understanding the population firing rate dynamics of the GIF neuron model. The formal, implicit definition of the linear response function, ν1 (ω), arises from a weak oscillatory modulation of amplitude A and frequency ω in the mean input, and an expansion of the response, ν (t), in powers of A, ν (t) = ν 0 + ν 1 (ω) A e i ω t + O (A 2), (9) where ν0 is the stationary response, Eq (6). In the Methods, we restate how the linear response can be obtained (Eq (59) ) directly from the spike times using the complex response vector, r (ω) ≔〈 e−iωtm 〉m. Below, we show the classic formulation that shows that it can also be obtained from the voltage dynamics. In this section, we take the general result of the previous section, Eq (23), and go through its explicit calculation for a population of Gauss-Rice GIF neurons to obtain the result Eq (41). A work taking a similar approach, partly inspired by this work, though with with less intermediate analysis has recently appeared [25]. Our novel findings arise from an exhaustive characterization of the parameter dependence across the phase diagram of the voltage response, Fig 3. We calculate the current-to-voltage filter, expressing it in each of the three representations listed in Table 1, Eqs (25,26 and 27) respectively. We show (Fig 4) how the low pass component of the filter undergoes a qualitative change from second-order low pass to first-order low pass to resonant as QL is increased. We find the voltage resonance condition, ω L τ w > Q L - 2 - 1, where the resonance has a contribution from slow adaptation and from the frequency, Ω. Either can exist without the other (Fig 5). We then compute the voltage correlation function, Eq (37) ), whose envelope depends on the relaxation time, τr (Fig 6). From this, the variances are calculated and an expression for the differential correlation time, τs, Eq (39) is obtained. We show a characteristic dependence on the ratio τw/τI (Fig 7). Finally, we show in Fig 8 how the stationary firing rate has unimodal dependence on the time constants, τV and τI, monotonic rise with input variance, σ I 2, and monotonic decay with intrinsic frequency, Ω. In this section, we characterize the qualitative features of the response function, Eq (41), again with a focus on completeness. We first show that the high and low input frequency limits of the response constrain the parameter sets that can achieve high and low pass behavior and we give an expression, Eq (45), of the critical stationary rate separating these two regions in terms of the other parameters. We then reparametrize the expression for the response, Eq (47), using the height of the response at its center frequency, νωL and high frequency limit, ν∞, both relative to its low frequency limit. The two-dimensional shape parameter space give responses with a peak, dip or step at ωL whose width varies with QL. The additional high or low pass nature of the filter give six classes of filter shape. The constraint of stable voltage dynamics restricts the area accessible to the model to νωL ≥ QL (1 + ν∞). Neuron models with hard-thresholds, such as the LIF and GIF, have been unexpectedly successful in modeling cortical neurons [58]. They are obtained from more complex models by a series of reductions. In Methods, we gave a rationale for the reduction to a no-reset, hard-threshold model, where we state the additional limitations imposed by lifting the voltage reset. First, these models do not apply to mean-driven situations and so do not cover phenomena such as the masking of a subthreshold resonance by a resonance at the firing rate [29]. Second, to avoid extremely bursty spike patterns, we extend previous work [19] and argue that the correlation time of the input, τI, and the correlation time of the voltage statistics, τs, can not be too different. This precludes analysis involving white current noise but implies that satisfaction depends additionally on intrinsic parameters through their dependence on τs. For example, since τs ≤ τV, the rough validity condition 1 ∼ τs/τI ≲ τV/τI so that the timescale of the input fluctuations, τI, should not be much slower than the membrane time constant, τV. Third, for correspondence with threshold models the voltage relaxation time, τr, should fall within the average inter-spike interval, ν0τr ≪ 1. Last, these models should only be considered in the irregular firing regime, ν0τs ≪ 1. We found that τr ≤ τs for τw > τI, so that this last constraint is in fact implied by the combination of the second and third. To verify the validity of the no-reset model within the prescribed range, we made a direct, quantitative comparison to a canonical model with an active-spike generating mechanism. The dynamic gain of the two models coincides up to the high frequency limit, flimit, beyond which the low pass effect of the finite action potential rapidness dominates. Thus, all of the 6 distinct types of response shapes are altered by additional low pass behavior at high frequencies. For a previously used value of the rapidness, the intermediate frequency behavior is affected, while for a higher, and perhaps more accurate value it is not, and the artificially flat high frequency response is brought down by the realistic finite onset rapidness. In summary, these results show that the simplification to a no-reset, hard threshold is an adequate approximation when response features are slower than the speed of action potential onset. A topic of related future work regards the possibility of accelerated kinetics of auxiliary currents during a spike [59]. To study such a scenario, one could numerically compute the gain for a model where the auxiliary current, w, undergoes a jump at spike times. In this study of the Gauss-Rice GIF neuron and a previous on the Gauss-Rice LIF [42], exponentially-correlated Gaussian noise was used as an example of a Gaussian input statistics with non-trivial temporal correlations. These input statistics will not in general produce self-consistent firing statistics. It is therefore important to note that the approach to the linear response taken here admits arbitrary temporal correlations in the input, so long as their effect on the short-delay features of the temporal correlation of the voltage can be calculated, since that is what determines τs and thus the effect of temporal correlations on the response properties. We also note that since the voltage correlation affects the response properties only through τs, there is an equivalence class structure over the space of input correlation functions based on how they influence τs. Excitable membranes are classified by the type of bifurcation that they undergo from resting to spiking, with Type I and II referring to super and sub critical Hopf bifurcation, respectively. The respective set of eigenvalues around the resting state are real and complex, with the imaginary part of the latter providing an intrinsic frequency. In this case, the voltage impulse response exhibits decaying oscillations and the voltage response function can exhibit a resonant peak near the intrinsic frequency. The mean-driven stationary spiking response rises continuously from 0 for Type I while firing in Type II neurons begins only at a finite frequency. The dynamic gain of the spiking response of Type II neurons can exhibit a superthreshold resonance arising from such subthrehsold resonance. Frequency-sweeping ZAP input currents have revealed resonant responses from neurons in the inferior olive [60,61], thalamus [62], hippocampus [63], and cortex [64]. Consistent with the type classification, these cells often display Type II membrane excitability properties such as subthreshold oscillations with power at similar frequencies as the spiking resonance (for a review, see ref. [7]). Type II stationary spiking responses have been measured in cortical interneurons [65]. Direct measurements of the dynamic gain of resonator neurons are lacking, however. Moreover, these existing measurements used the mean input to drive the neurons to spike. Resonator response properties in the in vivo fluctuation-driven regime remain unmeasured. Numerical simulations of resonator models containing the minimally required currents can nevertheless reproduce the peaked voltage and ZAP response and bimodal ISI distributions in both mean and fluctuation-driven regimes [66–68]. Inspired in part by the research presented here, Tchumatchenko and Clopath [25] used similar methods as those used here on excitatory and inhibitory GIF networks where they investigated the role of subthreshold resonance and electrical synapses on the emergence of network oscillations for a particular choice of model parameters, in which they also confirm the correspondence between the response properties with and without voltage reset. The remaining few analytical results for the stationary and linear response have so far been restricted to the long intrinsic time constant limit, τw ≫ 1 [22,29]. In this paper, we are able to obtain exact results for the stationary and linear response for all values of τw, something not possible in ref. [22] due to the difficulty of the analytics of the Fokker-Planck approach used there. For large τw and the fluctuation-driven regime, our results qualitatively match their high noise results, where σI ∼ 0. 1 − 1. Since Gauss-Rice models apply only to the fluctuation-driven regime, there is no meaningful mean-driven, deterministic limit attained in the limit of vanishing noise strength with which to compare to the mean-driven results of [22], such as the shift in the resonant frequency with increasing, small amounts of noise. Their phase diagram of subthreshold behavior is essentially the same as ours, up to reparametrization. We also note that the low frequency limit will differ slightly between the models due to the slightly differing slopes of their fI-curves. These small quantitative discrepancies between idealized models should not, however, be emphasized over their ability to provide a qualitative explanation of the phenomena. Finally, we note the GIF Spike-Triggered Averaged can be obtained from our expression for dynamic gain. It has also been computed through other methods [69]. Explicit expressions for the linear response, such as Eq (41) obtained above, are essential ingredients for the analysis of the collective states in recurrent networks. First, they are the key quantity in the evaluation of population stability [21]. The dynamics of the population firing rate linearized around one of its fixed points is defined by the linear response function. Second, knowledge of the response function additionally reveals the correlation gain in the mapping of input current correlations to output spiking correlations. Recurrent networks exhibiting such gain will generate self-consistent patterns of inter-neuron correlations [47,49,70]. In the Gauss-Rice approach used here, the linear response providing the population stability and correlation gain is tractable for arbitrary Gaussian input current. Many networks generate such input statistics, most prominently balanced networks [71,72]. We expect that the correlation gain and population firing rate stability of these networks can be theoretically investigated using the expressions for the linear response derived here. One target application area is in understanding the connection between circuit oscillations and single cell excitability. Subthreshold resonance is often neglected in modeling studies of the PING and ING mechanisms for population oscillations [73]. This is despite the ample suggestive evidence of phase locking between subthreshold oscillations and gamma band population oscillations [7]. This connection has been studied in the olfactory bulb where mitral cells display a host of resonator properties such as subthreshold oscillations [5,6], rebound spikes [74], and Type II phase resetting curves [75]. The role of this resonance in sustaining the population oscillation has not been directly assessed in detailed network models of resonating mitral cells [76], though it should play a role in either of two existing hypotheses for the origin of the oscillations [77]. Combining subthreshold and PING mechanisms has been studied in other contexts [78]. The demonstrated subthreshold resonance in inhibitory interneurons in cortex likely also contributes to the population oscillation observed there (as suggested by the numerical results of [79] and [78]) and could be investigated using the expression for dynamic gain that we provide. A first of such studies inspired by an unpublished version of the work presented here has already appeared [25], where the Gauss-Rice GIF response gain was also derived. Finally, an ad hoc dynamic response filter of the same form as the one derived here [80] has been successful in modeling responses of cortical neurons (personal communication O. Shriki). The explicit dependence in our derived expression on the parameters of an underlying neuron model can be used to extend those studies, in particular, by inferring from the fitted values the properties of the intrinsic dynamics of the measured cells. The differential correlation time, τs, was used in a variety of ways throughout this paper. First, it appeared in expressions for other important quantities in the theory. It appears most prominently in our expression for the fluctuation-driven voltage autocorrelation function for exponentially-correlated Gaussian input current. The result for a Type II GIF, Eq (37), gives exponentially enveloped, oscillatory decay, with a decay constant equal to the relaxation time of the model and oscillation frequency given by the intrinsic frequency, Ω. Despite these oscillations, we find that the dynamic gain depends only on the initial falloff behavior away from 0-delay, a feature that can be shown to define, τs. From the perspective of the response then, voltage correlation functions differ only insofar as they exhibit different τs. The characteristic time, τc, and thus also the attenuation of the spiking filter scales linearly with τs, influencing the high or low pass nature of the filter accordingly. Second, τs appears in the validity conditions for the model. Namely, the range of valid firing rates for all Gauss-Rice neurons must lie below τ s - 1. Third, model parameters such as the intrinsic time scale, τw, have an effect on dynamic response features, such as the high and low frequency limits, only through τs. The analysis of their effect on τs provides insight as to their role in sculpting the response properties. In Fig 7c for example, τs grows with τw, and for large τw saturates at τs, GIF → τs, LIF = τV, so that τs can only be made shorter, not longer, than the membrane time constant, τV, by intrinsic and synaptic current parameters. The central role of τs could be tested by applying a variety of input correlation functions with significant differences only away from the fall-off at 0-delay so that they provide the same τs. Our model predicts no significant change in the response properties. Such a large number of experiments could be performed by methods of high-throughput electrophysiology currently under development. We re-expressed the response expression, Eq (20), using the center and high frequency response relative to the low frequency response, νωL and ν∞ respectively. We find six qualitatively distinct filter shapes distributed around (1,1) in the (ν∞, νωL) plane, with the value of QL determining which of the six are accessible. Depending on the region there is a peak, dip or step at ωL whose width is determined by QL. We summarize below the constraints on the accessible shapes set by QL. For QL < 1/2, all six filters shapes are possible for fast relative spiking (τc < τw). There are no high pass resonating shapes in the limit of vanishing QL for slow relative spiking (τc > τw). For Q L > (- 1 + 5) / 2 ∼ 0. 7 all accessible shapes have elevated response at the center frequency, νωL > 1. For QL > 1, all allowed filter shapes are resonating, that is νωL > ν∞. There are no low pass resonating filters for slow relative spiking and so a sharp resonance, i. e. a high QL, is only possible when the overall filter is high pass. Neither voltage nor spiking resonance strictly imply the other in this model. First, there can be voltage resonance with no spiking resonance because the spiking high pass pulls up the response in the high input frequency range above the elevated response around the intermediate-range resonant input frequency. The high frequency limitation of the approach (e. g. Fig 12) implies that the elevated response extends up to the speed of the action potential, leaving a broad resonant band at high input frequencies. Second, there can be spiking resonance with no voltage resonance because of a low frequency attenuation by the spiking high pass filter of a low pass current-to-voltage filter. In addition, neither voltage nor intrinsic resonance strictly imply the other. First, the existence of an intrinsic frequency does not imply voltage resonance in general because the response at ωL where Ω becomes finite is QL = 1/2 and is thus still attenuated relative to the response at low input frequencies. This response only becomes resonant at QL = 1. Second, there can be a voltage resonance with no intrinsic resonance for the same reason that a high pass with low characteristic frequency (this time from relatively slow intrinsic dynamics) can sculpt a peak from the low pass component of the full filter. Finally, we found that the strength of the spiking resonance (∼νωL) is composed of a contribution from the intrinsic timescale, τw and from the intrinsic frequency, Ω. Nevertheless, νωL is dominated by the attenuation at low input frequencies associated with the high pass effect of large τw, while the unique effect of Ω is to sharpen this resonance. The effect of spiking in the Gauss-Rice formulation of the response is as an explicit first-order high pass filter of the voltage dynamics (see Eq (20) ). We note that this high pass behavior associated with spiking is distinct from that discussed in the literature as arising from sodium channel inactivation [81]. This has nothing to do with the Gauss-Rice high pass arising in this paper. In this work, we always consider the threshold fixed. Closed form expressions are thus obtained for the low frequency limit and characteristic time of this filter in terms of the parameters of the model. When the characteristic frequency is high, the filter has the effect of flattening an otherwise decaying voltage response. The flattening effect is physiologically meaningful up to frequencies at which the spike-generator cut-off appears. It thus sculpts a plateau of constant response at high frequencies that can be elevated or depressed relative to the low frequency response. On the other hand, when the characteristic frequency is low, the resulting effect is a low frequency attenuation that carves out a resonant peak. The high pass characteristics are then also dependent on the intrinsic timescales. Here, we detail how one arrives at a model like the one used in this paper from simplifications made to the synaptic, subthreshold, spiking, and spiking reset currents of a Hodgkin-Huxley type neuron model for the dynamics of the somatic transmembrane voltage potential, V (here measured in mV), C V ˙ = I m e m + I s y n (48) where C is the membrane capacitance, Imem is the sum of all membrane currents and Isyn is the total synaptic current arriving at the soma. Our exposition of the reductions to synaptic and subthreshold currents is standard. To the exposition of the reductions of spiking currents we add analysis determining the high frequency limit, flimit, below which the approximation to a hard threshold is valid. To the exposition of the reduction of reset currents, we add more detailed consideration of the mechanisms through which the no reset approximation breaks down. When the eigenvalues of the solution to the voltage dynamics are complex, we can re-express the denominator of Eq (25) using the intrinsic frequency, i. e. the imaginary part of the eigenvalues of the voltage solution. We first obtain the eigenvalues. For the linear matrix evolution operator B = - 1 τ V - g τ V 1 τ w - 1 τ w (55) the eigenvalues are obtained via the identity λ ± = tr B 2 ± 1 2 tr B 2 - 4 det B (56) = - 1 ± 1 - (ω L τ r) 2 τ r. (57) where tr B 2 = - τ r - 1 = - 1 2 (1 τ V + 1 τ w) as the negative reciprocal of the harmonic mean of the two time constants, τr, and det B = ω L 2 = 1 + g τ w τ V where ωL is the center frequency of the voltage filter. When ωLτr < 1, the magnitude τ r | λ ± | = | - 1 ± 1 - (ω L τ r) 2 |. When ωLτr > 1, the eigenvalues are complex with r: = - τ r - 1 as the real part. We define the imaginary part that plays the role of the intrinsic frequency, Ω > 0, via λ± = r ± iΩ, so Ω = ω L 2 - r 2 and now the magnitude is | λ ± | = r 2 + Ω 2 = ω L. We can substitute the expression for ωL, obtaining the relation between g and Ω, Eq (3), τ V τ w Ω 2 = g - g c r i t (58) where g > g c r i t = (τ V - τ w) 2 4 τ w τ V is the condition for complex eigenvalues (see Fig 1). Here we rederive the linear relationship between the vector strength and the linear response. ν1 (ω) from Eq (9) can be expressed using the complex response vector, rk (ω) =1nk∑jnke−iωtjk=1nk∫−T2T2∑jnkδ (t−tjk) e−iωtdt≈1ν0T∫−T2T2∑jnkδ (t−tjk) e−iωtdt, where in the last step we use nk ≈ ν0T, good when T is made much larger than ν 0 - 1. Taking the ensemble average, 〈 rk (ω) 〉=1ν0T 〈 ∫−T2T2∑jnkδ (t−tjk) e−iωtdt 〉=1ν0T ∫−T2T2〈 ∑jnkδ (t−tjk) 〉e−iωtdt=1ν0T∫−T2T2ν (t) e−iωtdt≈1ν0T ∫−T2T2 (ν0+ν1 (ω) Aeiωt) e−iωtdt=Aν1 (ω) ν0 ∫−T2T2dtT〈 r (ω) 〉=Aν1 (ω) ν0. Using the decomposition of the response into its gain and phase, ν1 (ω) = |ν1 (ω) |eiΦ (ω), the dynamic gain is thus obtained from the norm of the ensemble-averaged response vector, called the vector strength, |ν1 (ω) |=ν0A| 〈 eiωtm 〉m |, (59) where here we have simplified the notation by having m run over all the spikes from the full ensemble. We computed this expression using the spike times obtained directly from numerical simulations of the stochastic dynamics generated by the neuron model. We use the result to confirm the validity of the analytical gain function derived below, whose utility goes far beyond the numerical result because it provides the explicit dependence on the model parameters. Rearranging the expression for ν0 and then substituting in the σI-dependent expression for the voltage fluctuations, σ, we have ν 0 = 1 2 π τ s e - 1 2 σ - 2 σ V 2 = - θ 2 2 log 2 π ν 0 τ s - 1 J 2 σ I 2 τ V τ I + 1 · 1 + α w τ w τ I τ V τ e f f α I + τ w τ I = log 2 π ν 0 τ s - 1 σ I 2 = - θ 2 2 τ V τ I + 1 J 2 · τ V τ e f f α I + τ w τ I 1 + α w τ w τ I log 2 π ν 0 τ s. (60) When we study the model’s behavior we will use this relation to set the input variance for a chosen output firing rate so that the dimensions of the parameter space to be explored are the four time scales in the problem, (τV, τeff, τw, 1/ν0) and when Ω exists, (τV, 1/Ω, τw, 1/ν0). The firing rate response derived in this paper allows us to compute the response to any weak signal and we demonstrate that in this section where we derive the response to step-like input. The time-domain version of linear frequency response, ν1 (t), is the impulse response function, which when convolved with any input times series gives the corresponding response time series, ν (t) = ν 0 + ∫ ν 1 (t) I (t - t ′) d t ′, where ν 1 (t) = F - 1 [ ν 1 (ω) ] has units of [Time]−2[Current]−1. If there is an accessible frequency representation of the input, the interaction can be made in the frequency domain and then the result transformed back to the time domain, ν (t) = ν 0 + F - 1 [ ν 1 (ω) I (ω) ]. (61) We used this definition to study the response to step-like input, I (t) = AΘ (t), with step height, A, and with frequency domain expression for the Heaviside theta function, Θ (ω) = π δ (ω) - i ω. (62) Applying the inverse Fourier transform to the product of this with the linear frequency response gives the expression for the response. The relative response is then, ν (t) - ν 0 ν 0 = A D sgn (t) | λ ± | 2 + Θ (t) λ + - λ - ∑ j = +, - (1 + τ c λ j) (1 + τ w λ j) λ j e (λ j t) j (i π 2), (63) with D = θ σ V 2 τ V τ w. We can express Eq (63) in terms of r and Ω, ν (t) - ν 0 ν 0 = A D sgn (t) r 2 + Ω 2 + Θ (t) e r t 2 Ω R + e i (Ω t + ϕ + + π 2) + R - e - i (Ω t + ϕ - - π 2), (64) where R ± ∈ R and ϕ ± ∈ R depend on the parameters. Taking the limit t → 0+, the relative instantaneous jump height is A D τ w τ c = A θ σ V - 2 τ c τ V = A θ 3 π 2 τ s 2 τ c 2 τ c τ V, consistent with the notion that higher characteristic cutoff frequencies, i. e. τ c - 1, imply stronger instantaneous transmission. The exponent of the subsequent decay is r = - 2 τ ¯ - 1, providing an envelope that funnels into the relative asymptotic response, A D | λ | 2, attained in the limit t → ∞. Since the oscillation amplitude scales as 1/Ω while the asymptotic response scales with 1/Ω2, there will be a tapering envelope for Ω > 1. Within this envelope the response oscillates at the intrinsic frequency and with a phase that is explicitly dependent on the neuron parameters, as well as implicitly though τc. This function was used to calculate the step response shown in Fig 2. | Dynamic gain, the amount by which features at specific frequencies in the input to a neuron are amplified or attenuated in its output spiking, is fundamental for the encoding of information by neural populations. Most studies of dynamic gain have focused on neurons without intrinsic degrees of freedom exhibiting integrator-type subthreshold dynamics. Many neuron types in the brain, however, exhibit complex subthreshold dynamics such as resonance, found for instance in cortical interneurons, stellate cells, and mitral cells. A resonator neuron has at least two degrees of freedom for which the classical Fokker-Planck approach to calculating the dynamic gain is largely intractable. Here, we lift the voltage-reset rule after a spike, allowing us to derive a complete expression of the dynamic gain of a resonator neuron model. We find the gain can exhibit only six shapes. The resonant ones have peaks that become large due to intrinsic adaptation and become sharp due to an intrinsic frequency. A resonance can nevertheless result from either property. The analysis presented here helps explain how intrinsic neuron dynamics shape population-level response properties and provides a powerful tool for developing theories of inter-neuron correlations and dynamic responses of neural populations. | lay_plos |
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As of August 2006, the Nuclear Suppliers Group had 45 members, including the United States. (See app. II for a list of signatory countries.) IAEA Has Strengthened Its Safeguards Program, but Weaknesses Need to Be Addressed IAEA has taken steps to strengthen safeguards by more aggressively seeking assurances that a country is not pursuing a clandestine nuclear program. In a radical departure from past practices of only verifying the peaceful use of a country’s declared nuclear material at declared facilities, IAEA has begun to develop the capability to independently evaluate all aspects of a country’s nuclear activities. The first strengthened safeguards steps, which began in the early 1990s, increased the agency’s ability to monitor declared and undeclared activities at nuclear facilities. These measures were implemented under the agency’s existing legal authority under comprehensive safeguards agreements and include (1) conducting short notice and unannounced inspections, (2) collecting and analyzing environmental samples to detect traces of nuclear material, and (3) using measurement and surveillance systems that operate unattended and can be used to transmit data about the status of nuclear materials directly to IAEA headquarters. The second series of steps began in 1997 when IAEA’s Board of Governors approved the Additional Protocol. Under the Additional Protocol, IAEA has the right, among other things, to (1) receive more comprehensive information about a country’s nuclear activities, such as research and development activities, and (2) conduct “complementary access,” which enables IAEA to expand its inspection rights for the purpose of ensuring the absence of undeclared nuclear material and activities. Because the Additional Protocol broadens IAEA’s authority and the requirements on countries under existing safeguards agreements, each country must take certain actions to bring it into force. For each country with a safeguards agreement, IAEA independently evaluates all information available about the country’s nuclear activities and draws conclusions regarding a country’s compliance with its safeguards commitments. A major source of information available to the agency is data submitted by countries to IAEA under their safeguards agreements, referred to as state declarations. Countries are required to provide an expanded declaration of their nuclear activities within 180 days of bringing the Additional Protocol into force. Examples of information provided in an Additional Protocol declaration include the manufacturing of key nuclear-related equipment; research and development activities related to the nuclear fuel cycle; the use and contents of buildings on a nuclear site; and the location and operational status of uranium mines. The agency uses the state declarations as a starting point to determine if the information provided by the country is consistent and accurate with all other information available based on its own review. IAEA uses various types of information to verify the state declaration. Inspections of nuclear facilities and other locations with nuclear material are the cornerstone of the agency’s data collection efforts. Under the Additional Protocol, IAEA has the authority to conduct complementary access at any place on a site or other location with nuclear material in order to ensure the absence of undeclared nuclear material and activities, confirm the decommissioned status of facilities where nuclear material was used or stored, and resolve questions or inconsistencies related to the correctness and completeness of the information provided by a country on activities at other declared or undeclared locations. During complementary access, IAEA inspectors may carry out a number of activities, including (1) making visual observations, (2) collecting environmental samples, (3) using radiation detection equipment and measurement devices, and (4) applying seals. In 2004, IAEA conducted 124 complementary access in 27 countries. In addition to its verification activities, IAEA uses other sources of information to evaluate countries’ declarations. These sources include information from the agency’s internal databases, open sources, satellite imagery, and outside groups. The agency established two new offices within the Department of Safeguards to focus primarily on open source and satellite imagery data collection. Analysts use Internet searches to acquire information generally available to the public from open sources, such as scientific literature, trade and export publications, commercial companies, and the news media. In addition, the agency uses commercially available satellite imagery to supplement the information it receives through its open source information. Satellite imagery is used to monitor the status and condition of declared nuclear facilities and verify state declarations of certain sites. The agency also uses its own databases, such as those for nuclear safety, nuclear waste, and technical cooperation, to expand its general knowledge about countries’ nuclear and nuclear- related activities. In some cases, IAEA receives information from third parties, including other countries. IAEA Has Taken Steps to Strengthen Safeguards, but Detection of Clandestine Nuclear Weapons Programs is Not Assured Department of State and IAEA officials told us that strengthened safeguards measures have successfully revealed previously undisclosed nuclear activities in Iran, South Korea, and Egypt. Specifically, IAEA and Department of State officials noted that strengthened safeguards measures, such as collecting and analyzing environmental samples, helped the agency verify some of Iran’s nuclear activities. The measures also allowed IAEA to conclude in September 2005 that Iran was not complying with its safeguards obligations because it failed to report all of its nuclear activities to IAEA. As a result, in July 2006, Iran was referred to the U.N. Security Council, which in turn demanded that Iran suspend its uranium enrichment activities or face possible diplomatic and economic sanctions. In August 2004, as a result of preparations to submit its initial declaration under the Additional Protocol, South Korea notified IAEA that it had not previously disclosed nuclear experiments involving the enrichment of uranium and plutonium separation. IAEA sent a team of inspectors to South Korea to investigate this case. In November 2004, IAEA’s Director General reported to the Board of Governors that although the quantities of nuclear material involved were not significant, the nature of the activities and South Korea’s failure to report these activities in a timely manner posed a serious concern. IAEA is continuing to verify the correctness and completeness of South Korea’s declarations. IAEA inspectors have investigated evidence of past undeclared nuclear activities in Egypt based on the agency’s review of open source information that had been published by current and former Egyptian nuclear officials. Specifically, in late 2004, the agency found evidence that Egypt had engaged in undeclared activities at least 20 years ago by using small amounts of nuclear material to conduct experiments related to producing plutonium and highly enriched uranium. In January 2005, the Egyptian government announced that it was fully cooperating with IAEA and that the matter was limited in scope. IAEA inspectors have made several visits to Egypt to investigate this matter. IAEA’s Secretariat reported these activities to its Board of Governors. Despite these successes, a group of safeguards experts recently cautioned that a determined country can still conceal a nuclear weapons program. IAEA faces a number of limitations that impact its ability to draw conclusions—with absolute assurance—about whether a country is developing a clandestine nuclear weapons program. For example, IAEA does not have unfettered inspection rights and cannot make visits to suspected sites anywhere at any time. According to the Additional Protocol, complementary access to resolve questions related to the correctness and completeness of the information provided by the country or to resolve inconsistencies must usually be arranged with at least 24- hours advanced notice. Complementary access to buildings on sites where IAEA inspectors are already present are usually conducted with a 2-hour advanced notice. Furthermore, IAEA officials told us that there are practical problems that restrict access. For example, inspectors must be issued a visa to visit certain countries, a process which cannot normally be completed in less than 24 hours. In some cases, nuclear sites are in remote locations and IAEA inspectors need to make travel arrangements, such as helicopter transportation, in advance, which requires that the country be notified prior to the visit. A November 2004 study by a group of safeguards experts appointed by IAEA’s Director General evaluated the agency’s safeguards program to examine how effectively and efficiently strengthened safeguards measures were being implemented. Specifically, the group’s mission was to evaluate the progress, effectiveness, and impact of implementing measures to enhance the agency’s ability to draw conclusions about the non-diversion of nuclear material placed under safeguards and, for relevant countries, the absence of undeclared nuclear material and activities. The group concluded that generally IAEA had done a very good job implementing strengthened safeguards despite budgetary and other constraints. However, the group noted that IAEA’s ability to detect undeclared activities remains largely untested. If a country decides to divert nuclear material or conduct undeclared activities, it will deliberately work to prevent IAEA from discovering this. Furthermore, IAEA and member states should be clear that the conclusions drawn by the agency cannot be regarded as absolute. This view has been reinforced by the former Deputy Director General for Safeguards who has stated that even for countries with strengthened safeguards in force, there are limitations on the types of information and locations accessible to IAEA inspectors. A Number of Weaknesses Impede IAEA’s Ability to Effectively Implement Strengthened Safeguards There are a number of weaknesses that hamper IAEA’s ability to effectively implement strengthened safeguards. IAEA has only limited information about the nuclear activities of 4 key countries that are not members of the NPT—India, Israel, North Korea, and Pakistan. India, Israel, and Pakistan have special agreements with IAEA that limit the agency’s activities to monitoring only specific material, equipment, and facilities. However, since these countries are not signatories to the NPT, they do not have comprehensive safeguards agreements with IAEA, and are not required to declare all of their nuclear material to the agency. In addition, these countries are only required to declare exports of nuclear material previously declared to IAEA. With the recent revelations of the illicit international trade in nuclear material and equipment, IAEA officials stated that they need more information on these countries’ nuclear exports. For North Korea, IAEA has even less information, since the country expelled IAEA inspectors and removed surveillance equipment at nuclear facilities in December 2002 and withdrew from the NPT in January 2003. These actions have raised widespread concern that North Korea diverted some of its nuclear material to produce nuclear weapons. Another major weakness is that more than half, or 111 out of 189, of the NPT signatories have not yet brought the Additional Protocol into force, as of August 2006. (App. I lists the status of countries’ safeguards agreements with IAEA). Without the Additional Protocol, IAEA must limit its inspection efforts to declared nuclear material and facilities, making it harder to detect clandestine nuclear programs. Of the 111 countries that have not adopted the Additional Protocol, 21 are engaged in significant nuclear activities, including Egypt, North Korea, and Syria. In addition, safeguards are significantly limited or not applied in about 60 percent, or 112 out of 189, of the NPT signatory countries—either because they have an agreement (known as a small quantities protocol) with IAEA, and are not subject to most safeguards measures, or because they have not concluded a comprehensive safeguards agreement with IAEA. Countries with small quantities of nuclear material make up about 41 percent of the NPT signatories and about one-third of the countries that have the Additional Protocol in force. Since 1971, IAEA’s Board of Governors has authorized the Director General to conclude an agreement, known as a small quantities protocol, with 90 countries and, as of August 2006, 78 of these agreements were in force. IAEA’s Board of Governors has approved the protocols for these countries without having IAEA verify that they met the requirements for it. Even if these countries bring the Additional Protocol into force, IAEA does not have the right to conduct inspections or install surveillance equipment at certain nuclear facilities. According to IAEA and Department of State officials, this is a weakness in the agency’s ability to detect clandestine nuclear activities or transshipments of nuclear material and equipment through the country. In September 2005, the Board of Governors directed IAEA to negotiate with countries to make changes to the protocols, including reinstating the agency’s right to conduct inspections. As of August 2006, IAEA amended the protocols for 4 countries—Ecuador, Mali, Palau, and Tajikistan. The application of safeguards is further limited because 31 countries that have signed the NPT have not brought into force a comprehensive safeguards agreement with IAEA. The NPT requires non-nuclear weapons states to conclude comprehensive safeguards agreements with IAEA within 18 months of becoming a party to the Treaty. However, IAEA’s Director General has stated that these 31 countries have failed to fulfill their legal obligations. Moreover, 27 of the 31 have not yet brought comprehensive safeguards agreements into force more than 10 years after becoming party to the NPT, including Chad, Kenya, and Saudi Arabia. Last, IAEA is facing a looming human capital crisis that may hamper the agency’s ability to meet its safeguards mission. In 2005, we reported that about 51 percent, or 38 out of 75, of IAEA’s senior safeguards inspectors and high-level management officials, such as the head of the Department of Safeguards and the directors responsible for overseeing all inspection activities of nuclear programs, are retiring in the next 5 years. According to U.S. officials, this significant loss of knowledge and expertise could compromise the quality of analysis of countries’ nuclear programs. For example, several inspectors with expertise in uranium enrichment techniques, which is a primary means to produce nuclear weapons material, are retiring at a time when demand for their skills in detecting clandestine nuclear activities is growing. While IAEA has taken a number of steps to address these human capital issues, officials from the Department of State and the U.S. Mission to the U.N. System Organizations in Vienna have expressed concern that IAEA is not adequately planning to replace staff with critical skills needed to fulfill its strengthened safeguards mission. The Nuclear Suppliers Group Has Helped Stem Nuclear Proliferation, but Lack of Information Sharing on Nuclear Exports Between Members Could Undermine Its Efforts The Nuclear Suppliers Group, along with other multilateral export control groups, has helped stop, slow, or raise the costs of nuclear proliferation, according to nonproliferation experts. For example, as we reported in 2002, the Suppliers Group helped convince Argentina and Brazil to accept IAEA safeguards on their nuclear programs in exchange for expanded access to international cooperation for peaceful nuclear purposes. The Suppliers Group, along with other multilateral export control groups, has significantly reduced the availability of technology and equipment available to countries of concern, according to a State Department official. Moreover, nuclear export controls have made it more difficult, more costly, and more time consuming for proliferators to obtain the expertise and material needed to advance their nuclear program. The Nuclear Suppliers Group has also helped IAEA verify compliance with the NPT. In 1978, the Suppliers Group published the first guidelines governing exports of nuclear materials and equipment. These guidelines established several member requirements, including the requirement that members adhere to IAEA safeguards standards at facilities using controlled nuclear-related items. Subsequently, in 1992, the Nuclear Suppliers Group broadened its guidelines by requiring that members insist that non-member states have IAEA safeguards on all nuclear material and facilities as a condition of supply for their nuclear exports. With the revelation of Iraq’s nuclear weapons program, the Suppliers Group also created an export control system for dual-use items that established new controls for items that did not automatically fall under IAEA safeguards requirements. Despite these benefits, there are a number of weaknesses that could limit the Nuclear Suppliers Group’s ability to curb nuclear proliferation. Members of the Suppliers Group do not share complete export licensing information. Specifically, members do not always share information about licenses they have approved or denied for the sale of controversial items to nonmember states. Without this shared information, a member country could inadvertently license a controversial item to a country that has already been denied a license from another Suppliers Group member state. Furthermore, Suppliers Group members did not promptly review and agree upon common lists of items to control and approaches to controlling them. Each member must make changes to its national export control policies after members agree to change items on the control list. If agreed- upon changes to control lists are not adopted at the same time by all members, proliferators could exploit these time lags to obtain sensitive technologies by focusing on members that are slowest to incorporate the changes and sensitive items may still be traded to countries of concern. In addition, there are a number of obstacles to efforts aimed at strengthening the Nuclear Suppliers Group and other multilateral export control regimes. First, efforts to strengthen export controls have been hampered by a requirement that all members reach consensus about every decision made. Under the current process, a single member can block new reforms. U.S. and foreign government officials and nonproliferation experts all stressed that the regimes are consensus-based organizations and depend on the like-mindedness or cohesion of their members to be effective. However, members have found it especially difficult to reach consensus on such issues as making changes to procedures and control lists. The Suppliers Group reliance on consensus decision making will be tested by the United States request to exempt India from the Suppliers Group requirements to accept IAEA safeguards at all nuclear facilities. Second, since membership with the Suppliers Group is voluntary and nonbinding, there are no means to enforce compliance with members’ nonproliferation commitments. For example, the Suppliers Group has no direct means to impede Russia’s export of nuclear fuel to India, an act that the U.S. government said violated Russia’s commitment. Third, the rapid pace of nuclear technological change and the growing trade of sensitive items among proliferators complicate efforts to keep control lists current because these lists need to be updated more frequently. To help strengthen these regimes, GAO recommended in October 2002, that the Secretary of State establish a strategy that includes ways for Nuclear Suppliers Group members to improve information sharing, implement changes to export controls more consistently, and identify organizational changes that could help reform its activities. As of June 2006, the Nuclear Suppliers Group announced that it has revised its guidelines to improve information sharing. However, despite our recommendation, it has not yet agreed to share greater and more detailed information on approved exports of sensitive transfers to nonmember countries. Nevertheless, the Suppliers Group is examining changes to its procedures that assist IAEA’s efforts to strengthen safeguards. For example, at the 2005 Nuclear Suppliers Group plenary meeting, members discussed changing the requirements for exporting nuclear material and equipment by requiring nonmember countries to adopt IAEA’s Additional Protocol as a condition of supply. If approved by the Suppliers Group, the action would complement IAEA’s efforts to verify compliance with the NPT. U.S. Bilateral Assistance Programs Are Working to Secure Nuclear Materials and Warheads, Detect Nuclear Smuggling, Eliminate Excess Nuclear Material, and Halt Production of Plutonium, but Challenges Remain Reducing the formidable proliferation risks posed by former Soviet weapons of mass destruction (WMD) assets is a U.S. national security interest. Since the fall of the Soviet Union, the United States, through a variety of programs, managed by the Departments of Energy, Defense (DOD), and State, has helped Russia and other former Soviet countries to secure nuclear material and warheads, detect illicitly trafficked nuclear material, eliminate excess stockpiles of weapons-usable nuclear material, and halt the continued production of weapons-grade plutonium. From fiscal year 1992 through fiscal year 2006, the Congress appropriated about $7 billion for nuclear nonproliferation efforts. However, U.S. assistance programs have faced a number of challenges, such as a lack of access to key sites and corruption of foreign officials, which could compromise the effectiveness of U.S. assistance. DOE’s Material Protection, Control, and Accounting (MPC&A) program has worked with Russia and other former Soviet countries since 1994 to provide enhanced physical protection systems at sites with weapons- usable nuclear material and warheads, implement material control and accounting upgrades to help keep track of the quantities of nuclear materials at sites, and consolidate material into fewer, more secure buildings. GAO last reported on the MPC&A program in 2003. At that time, a lack of access to many sites in Russia’s nuclear weapons complex had significantly impeded DOE’s progress in helping Russia to secure its nuclear material. We reported that DOE had completed work at only a limited number of buildings in Russia’s nuclear weapons complex, a network of sites involved in the construction of nuclear weapons where most of the nuclear material in Russia is stored. According to DOE, by the end of September 2006, the agency will have helped to secure 175 buildings with weapons-usable nuclear material in Russia and the former Soviet Union and 39 Russian Navy nuclear warhead sites. GAO is currently re-examining DOE’s efforts, including the progress DOE has made since 2003 in securing nuclear material and warheads in Russia and other countries and the challenges DOE faces in completing its work. While securing nuclear materials and warheads where they are stored is considered to be the first layer of defense against nuclear theft, there is no guarantee that such items will not be stolen or lost. Recognizing this fact, DOE, DOD, and State, through seven different programs, have provided radiation detection equipment since 1994 to 36 countries, including many countries of the former Soviet Union. These programs seek to combat nuclear smuggling and are seen as a second line of defense against nuclear theft. The largest and most successful of these efforts is DOE’s Second Line of Defense program (SLD). We reported in March 2006 that, through the SLD program, DOE had provided radiation detection equipment and training at 83 sites in Russia, Greece, and Lithuania since 1998. However, we also noted that U.S. radiation detection assistance efforts faced challenges, including corruption of some foreign border security officials, technical limitations of some radiation detection equipment, and inadequate maintenance of some equipment. To address these challenges, U.S. agencies plan to take a number of steps, including combating corruption by installing communications links between individual border sites and national command centers so that detection alarm data can be simultaneously evaluated by multiple officials. The United States is also helping Russia to eliminate excess stockpiles of nuclear material (highly enriched uranium and plutonium). In February 1993, the United States agreed to purchase from Russia 500 metric tons of highly enriched uranium (HEU) extracted from dismantled Russian nuclear weapons over a 20-year period. Russia agreed to dilute, or blend- down, the material into low enriched uranium (LEU), which is of significantly less proliferation risk, so that it could be made into fuel for commercial nuclear power reactors before shipping it to the United States. As of June 27, 2006, 276 metric tons of Russian HEU—derived from more than 11,000 dismantled nuclear weapons—have been downblended into LEU for use in U.S. commercial nuclear reactors. Similarly, in 2000, the United States and Russia committed to the transparent disposition of 34 metric tons each of weapon-grade plutonium. The plutonium will be converted into a more proliferation-resistant form called mixed-oxide (MOX) fuel that will be used in commercial nuclear power plants. In addition to constructing a MOX fuel fabrication plant at its Savannah River Site, DOE is also assisting Russia in constructing a similar facility for the Russian plutonium. Russia’s continued operation of three plutonium production reactors poses a serious proliferation threat. These reactors produce about 1.2 metric tons of plutonium each year—enough for about 300 nuclear weapons. DOE’s Elimination of Weapons-Grade Plutonium Production program seeks to facilitate the reactors’ closure by building or refurbishing two fossil fuel plants that will replace the heat and electricity that will be lost with the shutdown of Russia’s three plutonium production reactors. DOE plans to complete the first of the two replacement plants in 2008 and the second in 2011. When we reported on this program in June 2004, we noted that DOE faced challenges in implementing its program, including ensuring Russia’s commitment to shutting down the reactors, the rising cost of building the replacement fossil fuel plants, and concerns about the thousands of Russian nuclear workers who will lose their jobs when the reactors are shut down. We made a number of recommendations, which DOE has implemented, including reaching agreement with Russia on the specific steps to be taken to shut down the reactors and development of a plan to work with other U.S. government programs to assist Russia in finding alternate employment for the skilled nuclear workers who will lose their jobs when the reactors are shut down. Mr. Chairman, this concludes my prepared statement. I would be pleased to respond to any questions you or other Members of the Subcommittee may have at this time. Contacts and Staff Acknowledgments For future contacts regarding this testimony, please contact Gene Aloise at (202) 512-3841 or Joseph Christoff at (202) 512-8979. R. Stockton Butler, Miriam A. Carroll, Leland Cogliani, Lynn Cothern, Muriel J. Forster, Jeffrey Phillips, and Jim Shafer made key contributions to this testimony. Beth Hoffman León, Stephen Lord, Audrey Solis, and Pierre Toureille provided technical assistance. Appendix I: Countries’ Safeguards Agreements with IAEA, as of August 2006 Although North Korea concluded a comprehensive safeguards agreement with IAEA in 1992, it announced its withdrawal from the NPT in January 2003. Appendix II: Members of the Nuclear Suppliers Group, as of June 2006 Appendix III: Additional Information on U.S. Nuclear Nonproliferation Programs Secures radiological sources no longer needed in the U.S. and locates, identifies, recovers, consolidates, and enhances the security of radioactive materials outside the U.S. Global Nuclear Material Threat Reduction Eliminates Russia’s use of highly enriched uranium (HEU) in civilian nuclear facilities; returns U.S. and Russian-origin HEU and spent nuclear fuel from research reactors around the world; secures plutonium-bearing spent nuclear fuel from reactors in Kazakhstan; and addresses nuclear and radiological materials at vulnerable locations throughout the world. Provides replacement fossil-fuel energy that will allow Russia to shutdown its three remaining weapons-grade plutonium production reactors. Develops and delivers technology applications to strengthen capabilities to detect and verify undeclared nuclear programs; enhances the physical protection and proper accounting of nuclear material; and assists foreign national partners to meet safeguards commitments. Provides meaningful employment for former weapons of mass destruction weapons scientists. Provides material protection, control, and accounting upgrades to enhance the security of Navy HEU fuel and nuclear material. Provides material protection, control, and accounting upgrades to nuclear weapons, uranium enrichment, and material processing and storage sites. Enhances the security of proliferation-attractive nuclear material in Russia by supporting material protection, control, and accounting upgrade projects at Russian civilian nuclear facilities. Develops national and regional resources in the Russian Federation to help establish and sustain effective operation of upgraded nuclear material protection, control and accounting systems. Negotiates cooperative efforts with the Russian Federation and other key countries to strengthen the capability of enforcement officials to detect and deter illicit trafficking of nuclear and radiological material across international borders. This is accomplished through the detection, location and identification of nuclear and nuclear related materials, the development of response procedures and capabilities, and the establishment of required infrastructure elements to support the control of these materials. HEU Transparency Implementation project Monitors Russia to ensure that low enriched uranium (LEU) sold to the U.S. for civilian nuclear power plants is derived from weapons-usable HEU removed from dismantled Russian nuclear weapons. Disposes of surplus domestic HEU by down-blending it. Surplus U.S. Plutonium Disposition project Disposes of surplus domestic plutonium by fabricating it into mixed oxide (MOX) fuel for irradiation in existing, commercial nuclear reactors. Supports Russia’s efforts to dispose of its weapons-grade plutonium by working with the international community to help pay for Russia’s program. Site Security Enhancements Provides training and equipment to assist Russia in determining the reliability of its guard forces. Enhances the safety and security of Russian nuclear weapons storage sites through the use of vulnerability assessments to determine specific requirements for upgrades. DOD will develop security designs to address those vulnerabilities and install equipment necessary to bring security standards consistent with those at U.S. nuclear weapons storage facilities. Nuclear Weapons Transportation Assists Russia in shipping nuclear warheads to more secure sites or dismantlement locations. Assists Russia in maintaining nuclear weapons cargo railcars. Funds maintenance of railcars until no longer feasible, then purchases replacement railcars to maintain 100 cars in service. DOD will procure 15 guard railcars to replace those retired from service. Guard railcars will be capable of monitoring security systems in the cargo railcars and transporting security force personnel. Provides emergency response vehicles containing hydraulic cutting tools, pneumatic jacks, and safety gear to enhance Russia’s ability to respond to possible accidents in transporting nuclear weapons. Meteorological, radiation detection and monitoring, and communications equipment is also included. Related GAO Products Combating Nuclear Smuggling: Challenges Facing U.S. Efforts to Deploy Radiation Detection Equipment in Other Countries and in the United States. GAO-06-558T. Washington, D.C.: March 28, 2006. Combating Nuclear Smuggling: Corruption, Maintenance, and Coordination Problems Challenge U.S. Efforts to Provide Radiation Detection Equipment to Other Countries. GAO-06-311. Washington, D.C.: March 14, 2006. Nuclear Nonproliferation: IAEA Has Strengthened Its Safeguards and Nuclear Security Programs, but Weaknesses Need to Be Addressed. GAO- 06-93. Washington, D.C.: October 7, 2005. Preventing Nuclear Smuggling: DOE Has Made Limited Progress in Installing Radiation Detection Equipment at Highest Priority Foreign Seaports. GAO-05-375. Washington, D.C.: March 31, 2005. Nuclear Nonproliferation: DOE’s Effort to Close Russia’s Plutonium Production Reactors Faces Challenges, and Final Shutdown is Uncertain. GAO-04-662. Washington, D.C.: June 4, 2004. Weapons of Mass Destruction: Additional Russian Cooperation Needed to Facilitate U.S. Efforts to Improve Security at Russian Sites. GAO-03- 482. Washington, D.C.: March 24, 2003. Nonproliferation: Strategy Needed to Strengthen Multilateral Export Control Regimes. GAO-03-43. Washington, D.C.: October 25, 2002. Nuclear Nonproliferation: U.S. Efforts to Help Other Countries Combat Nuclear Smuggling Need Strengthened Coordination and Planning. GAO-02-426. Washington, D.C.: May 16, 2002. Nuclear Nonproliferation: Implications of the U.S. Purchase of Russian Highly Enriched Uranium. GAO-01-148. Washington, D.C.: December 15, 2000. This is a work of the U.S. government and is not subject to copyright protection in the United States. It may be reproduced and distributed in its entirety without further permission from GAO. However, because this work may contain copyrighted images or other material, permission from the copyright holder may be necessary if you wish to reproduce this material separately. | The International Atomic Energy Agency's (IAEA) safeguards system has been a cornerstone of U.S. efforts to prevent nuclear weapons proliferation since the Treaty on the Non-Proliferation of Nuclear Weapons (NPT) was adopted in 1970. Safeguards allow IAEA to verify countries' compliance with the NPT. Since the discovery in 1991 of a clandestine nuclear weapons program in Iraq, IAEA has strengthened its safeguards system. In addition to IAEA's strengthened safeguards program, there are other U.S. and international efforts that have helped stem the spread of nuclear materials and technology that could be used for nuclear weapons programs. This testimony is based on GAO's report on IAEA safeguards issued in October 2005 (Nuclear Nonproliferation: IAEA Has Strengthened Its Safeguards and Nuclear Security Programs, but Weaknesses Need to Be Addressed, GAO-06-93 [Washington, D.C.: Oct. 7, 2005]). This testimony is also based on previous GAO work related to the Nuclear Suppliers Group--a group of more than 40 countries that have pledged to limit trade in nuclear materials, equipment, and technology to only countries that are engaged in peaceful nuclear activities--and U.S. assistance to Russia and other countries of the former Soviet Union for the destruction, protection, and detection of nuclear material and weapons. IAEA has taken steps to strengthen safeguards, including conducting more intrusive inspections, to seek assurances that countries are not developing clandestine weapons programs. IAEA has begun to develop the capability to independently evaluate all aspects of a country's nuclear activities. This is a radical departure from the past practice of only verifying the peaceful use of a country's declared nuclear material. However, despite successes in uncovering some countries' undeclared nuclear activities, safeguards experts cautioned that a determined country can still conceal a nuclear weapons program. In addition, there are a number of weaknesses that limit IAEA's ability to implement strengthened safeguards. First, IAEA has a limited ability to assess the nuclear activities of 4 key countries that are not NPT members--India, Israel, North Korea, and Pakistan. Second, more than half of the NPT signatories have not yet brought the Additional Protocol, which is designed to give IAEA new authority to search for clandestine nuclear activities, into force. Third, safeguards are significantly limited or not applied to about 60 percent of NPT signatories because they possess small quantities of nuclear material, and are exempt from inspections, or they have not concluded a comprehensive safeguards agreement. Finally, IAEA faces a looming human capital crisis caused by the large number of inspectors and safeguards management personnel expected to retire in the next 5 years. In addition to IAEA's strengthened safeguards program, there are other U.S. and international efforts that have helped stem the spread of nuclear materials and technology. The Nuclear Suppliers Group has helped to constrain trade in nuclear material and technology that could be used to develop nuclear weapons. However, there are a number of weaknesses that could limit the Nuclear Suppliers Group's ability to curb proliferation. For example, members of the Suppliers Group do not always share information about licenses they have approved or denied for the sale of controversial items to nonmember states. Without this shared information, a member country could inadvertently license a controversial item to a country that has already been denied a license from another member state. Since the early 1990s, U.S. nonproliferation programs have helped Russia and other former Soviet countries to, among other things, secure nuclear material and warheads, detect illicitly trafficked nuclear material, and eliminate excess stockpiles of weapons-usable nuclear material. However, these programs face a number of challenges which could compromise their ongoing effectiveness. For example, a lack of access to many sites in Russia's nuclear weapons complex has significantly impeded the Department of Energy's progress in helping Russia secure its nuclear material. U.S. radiation detection assistance efforts also face challenges, including corruption of some foreign border security officials, technical limitations of some radiation detection equipment, and inadequate maintenance of some equipment. | gov_report |
Senate Votes To End U.S. Support For War In Yemen, Rebuking Trump And Saudi Arabia Enlarge this image toggle caption Zach Gibson/Getty Images Zach Gibson/Getty Images Updated Dec. 13 at 5:21 p.m. ET The Senate voted with support from lawmakers in both parties Thursday to end U.S. military support for Saudi Arabia's war in Yemen. The 56-41 vote marks the first time the Senate utilized powers granted under the 1973 War Powers Act, which gives Congress the power to demand an end to military actions. While the House likely won't vote on the measure, the bipartisan vote is a major rebuke to Saudi Arabia, long a key U.S. ally. "Today we tell the despotic regime in Saudi Arabia that we will not be part of their military adventurism," Sen. Bernie Sanders, I-Vt., a key sponsor of the resolution, said shortly before the final vote. Immediately afterward, the Senate unanimously approved a second resolution condemning the killing of Washington Post writer Jamal Khashoggi and pinning blame for the dissident's death on Saudi Crown Prince Mohammed bin Salman. The effort to stop American involvement in Yemen is still a long way from a done deal. The House would have to pass the resolution by year's end and President Trump would have to sign it — two steps that likely will not happen. Still, earlier this week, Sanders called the vote "a profound message." "It says to the country, it says to the world, the United States Senate — hopefully in good numbers today — says we will not be part of this brutal, horrific war in Yemen led by an undemocratic, despotic regime," Sanders told NPR. "That's a profound statement that will reverberate all over the world." The years-long conflict, viewed as a proxy war between Saudi Arabia and Iran, has spiraled into a growing humanitarian disaster. The U.S. military has provided refueling for Saudi aircraft carrying out strikes — assistance the Trump administration ended amid growing criticism — and helped Saudi Arabia with other strategic assistance, as well. Sanders and a handful of other senators, including Sen. Chris Murphy, D-Conn., and Sen. Mike Lee, R-Utah, have been working for more than a year to round up support for the resolution. Only 44 senators voted for it in March. But two weeks ago, more than 60 lawmakers voted to advance debate on the same bill. Sanders and Murphy say two key moments contributed to the push's growing momentum: the Saudi bombing of a school bus filled with Yemeni children in August and what the CIA believes to be the Saudi government-sanctioned killing in October of Khashoggi. "I think that exposed to the world what this regime is about," Sanders said of the Khashoggi killing before Thursday's vote. "And people began to ask, why are we allied with a Saudi war in Yemen which is killing children? Maybe it's time to rethink that." A late November procedural vote on the resolution came hours after Secretary of State Mike Pompeo stonewalled senators in a closed-door bipartisan briefing about the administration's response to the Khashoggi killing, which also likely contributed to the bipartisan support for the Yemen bill. "There is no direct reporting connecting [Saudi Crown Prince Mohammed bin Salman] to the order to murder Jamal Khashoggi," Pompeo told reporters after that briefing. "I don't think there's anybody in that room that doesn't believe he was responsible for it," Sen. Bob Corker, R-Tenn., the chairman of the Foreign Relations Committee, said minutes later. Corker voted to advance the Yemen resolution that day but said he would vote against the measure this week. Senate Majority Leader Mitch McConnell, R-Ky., said before Thursday that he opposed the measure, even though it was expected to pass with bipartisan support. "If the Senate wants to pick a constitutional fight with the executive branch over war powers, I would advise my colleagues to pick a better case," he said on the Senate floor Wednesday. Regardless of the resolution's future, its Senate passage marks a key turning point in the U.S.-Saudi relationships. In addition to the Yemen bill and the resolution condemning Saudi Arabia's role in the Khashoggi killing, a broad sanctions bill is expected in the coming months. And the Yemen resolution's sponsors have made it clear they'll try to pass the measure again next year, when Democrats control the House. The U.S. has long overlooked Saudi Arabia's human rights record and viewed the country as a close trade, military and energy-production ally. After his inauguration, Trump made Saudi Arabia the first country he traveled to on a state visit. But, in the Senate at least, the Khashoggi killing has put a substantial strain on the U.S.-Saudi relationship. "Just because you're our ally, you cannot kill with impunity and believe you can get away with it," Sen. Bob Menendez, D-N.J., said, promising to introduce a bipartisan sanctions bill early next year when the next Congress convenes. Corker said the Yemen war and Khashoggi killing have likely set back the two countries' relationship for years to come. Sen. Lindsey Graham, R-S.C., a longtime booster of both Trump and Saudi Arabia, clearly agrees. "I'm never going to let this go until things change," he said, calling Salman "so toxic, so tainted, and so flawed," despite the Trump administration's close embrace of Saudi Arabia's heir apparent. "Enough is enough," Graham said. "So to our friends in Saudi Arabia, you're never going to have a relationship with the United States Senate unless things change, and it's up to you to figure out what that change should be." The Senate on Thursday delivered back-to-back rebukes of President Trump’s embrace of Saudi Arabia, first voting to end U.S. participation in the Saudi-led war in Yemen and then unanimously approving a measure blaming the kingdom’s crown prince for the ghastly killing of journalist Jamal Khashoggi. Together, the dual actions represent an unambiguous rejection of Trump’s continued defense of Saudi leaders in the face of a CIA assessment that concluded Crown Prince Mohammed bin Salman likely ordered and monitored Khashoggi’s killing Oct. 2 inside the Saudi consulate in Istanbul. It suggests a bipartisan majority of senators will pursue broader punitive measures when Congress regroups next year — including sanctions and a halt to weapons transfers — despite the administration’s objections. “What we showed in this vote today is that Republicans and Democrats are ready to get back in the business of working with a president — and sometimes against a president — to set the foreign policy of this nation,” said Sen. Chris Murphy (D-Conn.) a longtime advocate for checking Saudi Arabia’s regional expansion. “The United States has said, through the Senate, that our support for the Saudi coalition is no longer open-ended.” The unanimous vote to hold Mohammed responsible for Khashoggi’s killing reflects the extent to which senators in both parties have grown tired of Trump’s continued defense of Mohammed’s denials. It also puts significant pressure on leaders in the House — where the president’s Saudi policy is far more divisive — to allow for a similar vote to condemn the crown prince before the end of the year. Earlier this week, House leaders maneuvered to block rank-and-file members from forcing a vote on any Yemen-related resolutions, an attempt to stop the Senate’s effort to curtail U.S. involvement in the Saudis’ military campaign by invoking the War Powers Resolution. Senators voted 56 to 41 on Thursday to support the Yemen resolution, put forward by Sens. Bernie Sanders (I-Vt.) and Mike Lee (R-Utah), after seven Republicans joined all Senate Democrats to back the measure. That figure strongly suggests a majority of the Republican-led Senate will challenge Trump on his Saudi policy next year, alongside a Democratic-led House, whose incoming leaders also have promised to be proactive about demanding changes to the status quo. “The current relationship with Saudi Arabia is not working for America,” said Sen. Lindsey O. Graham (R-S.C.), a Trump ally who, nonetheless, is a driving force behind several efforts to punish Saudi Arabia. He often refers to Mohammed as a “wrecking ball.” Graham did not vote for the Yemen resolution Thursday, citing concerns about using the War Powers Resolution as a vehicle to call out Saudi Arabia, but he said resolutely this week that his past defense of the kingdom had come to an end. “I think you’re wrong about what’s going on up here,” Graham said late Wednesday in comments directed toward Trump. “I’m never going to let this go until things change in Saudi Arabia.” The Senate votes came as the two sides in the Yemen conflict agreed to a cease-fire in Hodeida, a port city that is key to the supply of humanitarian aid, and a prisoner swap that is expected to free thousands. While previous cease-fire agreements in the four-year civil war have crumbled, Thursday’s agreement — following a week of peace talks in Sweden — have been heralded as an important sign of progress. [Warring sides in Yemen agree to halt fighting in key port city, U.N. says] Sen. Bernie Sanders (I-Vt.) is flanked by Sen. Mike Lee (R-Utah), right, and Sen. Chris Murphy (D-Conn.) while briefing the media Thursday after the Senate voted to withdraw support for Saudi Arabia's war in Yemen. (Jim Lo Scalzo/EPA-EFE/REX/Shutterstock) It has been months since the United Nations declared Yemen to be the world’s worst humanitarian crisis, and the situation has only worsened since, as a proxy battle deepens between U.S.-backed Arab nations and Iran, which supports the rebel Houthi government claiming authority in the country. International pressure has been building on the warring sides to take steps to end the conflict, and some senators said Thursday that the momentum surrounding their Yemen resolution played no small role in bringing about the cease-fire. “The concessions that were made by the Saudi side in the negotiations this morning would not have happened if it wasn’t for the pressure the United States Senate put on those negotiations,” Murphy said. But in the House, Yemen’s peace talks have been an excuse for some lawmakers to seek a pause, to see how things play out before committing to a course of action. The Senate vote came just hours after Secretary of State Mike Pompeo and Defense Secretary Jim Mattis provided a closed briefing to House members. Republicans and Democrats emerged from the meeting urging very different responses to Saudi Arabia and its crown prince over Khashoggi’s killing. “They have to be held responsible,” Rep. Eliot L. Engel (D-N.Y.), the incoming chairman of the House Foreign Affairs Committee, said after the briefing, referring to Mohammed and Saudi King Salman, though he did not back a specific means of holding them responsible. Meanwhile, there remain Republicans in the House who defend the crown prince — and even those who criticize him over Khashoggi’s death said that while he should be rebuked, the punishment should stop there. “We recognize killing journalists is absolutely evil and despicable, but to completely realign our interests in the Middle East as a result of this, when for instance the Russians kill journalists... Turkey imprisons journalists?” Rep. Adam Kinzinger (R-Ill.) said. “It’s not a sinless world out there.” That stands in sharp contrast to the Senate, where several Republicans have been encouraging a broad response to Saudi Arabia over not just Khashoggi’s killing and the Yemen war, but also the kingdom’s blockade in Qatar, its detainment of Lebanese Prime Minister Saad Hariri, and a slate of human rights abuses they say have compromised the U.S.-Saudi alliance. [Senators to try formally condemning Saudi crown prince for journalist’s killing] The House will not take up any such measures this year — not even the Senate-passed Yemen resolution. But leaders will still be under considerable pressure to allow members to vote on the measure condemning Mohammed for Khashoggi’s killing before the end of the year. Trump has refused to condemn Mohammed for the killing of Khashoggi, a Saudi national. Pompeo has echoed Trump’s stance in public interviews and behind closed doors, as well, lawmakers said. “All we heard today was more disgraceful ducking and dodging by the secretary,” said Rep. Lloyd Doggett (D-Tex.), following the meeting. House leaders met with CIA Director Gina Haspel on Wednesday to hear the details of Khashoggi’s slaying. They emerged offering few details about the briefing — or about what step House Democrats would take, once they assume the majority in January, to pursue more punitive measures against Saudi Arabia, beyond holding hearings. Kareem Fahim in Riyadh and Missy Ryan in Washington contributed to this report. Read more at PowerPost CIA Director Gina Haspel leaves the Capitol after giving a classified briefing to the House leadership about the murder of journalist Jamal Khashoggi and the involvement by the Saudi crown prince Mohammed... (Associated Press) CIA Director Gina Haspel leaves the Capitol after giving a classified briefing to the House leadership about the murder of journalist Jamal Khashoggi and the involvement by the Saudi crown prince Mohammed bin Salman, on Capitol Hill in Washington, Wednesday, Dec. 12, 2018. T (AP Photo/J. Scott Applewhite) (Associated Press) WASHINGTON (AP) — The Latest on the congressional response to the killing of Saudi journalist Jamal Khashoggi (all times local): 3:35 p.m. The Senate has passed a resolution saying Saudi Crown Prince Mohammed bin Salman is responsible for the slaying of journalist Jamal Khashoggi. Senators unanimously passed a resolution Thursday in a direct rebuke to the crown prince. It calls for the Saudi Arabian government to "ensure appropriate accountability." It's unclear whether the House will consider the measure. Senators voted on it after President Donald Trump equivocated on who is to blame for Khashoggi's death and praised the kingdom. U.S. intelligence officials have concluded that bin Salman must have at least known of the plot. Passage of the resolution came after senators passed a separate measure calling for the end of U.S. aid to the Saudi-led war in Yemen. ___ 3:30 p.m. Senators have voted to recommend that the U.S. stop supporting the Saudi-led war in Yemen, directly challenging both Saudi Arabia and President Donald Trump in the wake of journalist Jamal Khashoggi's slaying. The bipartisan vote Thursday comes two months after the Saudi journalist's killing at the Saudi consulate in Istanbul and after Trump has equivocated over who is to blame. U.S. intelligence officials have concluded that Saudi Crown Prince Mohammed bin Salman must have at least known of the plot, but Trump has repeatedly praised the kingdom. Frustration with the crown prince and the White House prompted several Republicans to support the Yemen resolution, a rebuke to the longtime ally. Others already had concerns about the brutality of the Yemen war. It's unlikely the House will consider the resolution. ___ 2:20 p.m. Senate Foreign Relations Chairman Bob Corker and Senate Majority Leader Mitch McConnell have introduced a resolution rebuking Saudi Arabia for the slaying of Saudi journalist Jamal Khashoggi (jah-MAHL' khahr-SHOHK'-jee). The Senate could vote on the resolution as soon as Thursday, after considering a separate resolution that would recommend pulling U.S. aid from a Saudi-led war in Yemen. The resolution states that the Senate "believes Crown Prince Mohammed bin Salman is responsible for the murder of Jamal Khashoggi" and calls for the Saudi Arabian government to "ensure appropriate accountability" for those responsible. The resolution also calls the war in Yemen a "humanitarian crisis" and demands that all parties seek an immediate cease-fire. It is unclear whether the House would vote on the resolution if it passes the Senate. ___ 12:45 a.m. Senators are expected to vote on a resolution that would call on the U.S. to pull assistance from the Saudi-led war in Yemen, a measure that would rebuke Saudi Arabia after the killing of journalist Jamal Khashoggi (jah-MAHL' khahr-SHOHK'-jee). The Senate may also consider a separate resolution condemning the journalist's killing as senators have wrestled with how to respond to the Saudi journalist's murder. U.S. intelligence officials have concluded that Saudi Crown Prince Mohammed bin Salman must have at least known of the plot, but President Donald Trump has been reluctant to pin the blame. Senators voted 60-39 on Wednesday to open debate on the Yemen resolution, signaling there's enough support to win the 50 votes needed. But it's unclear how amendments could affect a final vote expected to come Thursday. Senators voted 56-41 on the resolution, which would require the president to withdraw any troops in or “affecting” Yemen within 30 days unless they are fighting al Qaeda. ADVERTISEMENT The resolution would still need to be passed by the House before it could be sent to Trump, who has threatened to veto it. The House on Wednesday narrowly approved a rule governing debate on the farm bill that included a provision that would prevent lawmakers from forcing a war powers vote this year. Still, the Senate vote Thursday underscores the depth of frustration with Saudi Arabia on Capitol Hill, as well as the escalating gap between the White House and Congress on the relationship between the U.S. and the kingdom. It’s a dramatic U-turn from less than nine months ago when the chamber pigeonholed the same resolution, refusing to vote it out of committee and on to full Senate. At the time, 10 Democrats joined 45 Republicans in opposing it. The resolution's passage comes two days after Trump maintained that he would stand by the Saudi government and specifically Crown Prince Mohammed bin Salman, whom U.S. intelligence officials reportedly believe ordered Khashoggi's killing inside the Saudi Consulate in Istanbul in early October. A growing number of senators also believe the crown prince is responsible for the death of Khashoggi, who was a vocal critic of Saudi leadership and lived in Virginia while serving as a columnist for The Washington Post. The Senate passed a separate resolution Thursday afternoon specifically naming the crown prince as responsible for Khashoggi's death. ADVERTISEMENT Trump told Reuters on Tuesday that Riyadh has been “a very good ally” and “at this moment” sticking with Saudi Arabia means standing by the crown prince. The Trump administration had led a lobbying effort to try to quash the Senate resolution withdrawing U.S. support for the military campaign in Yemen. And CIA Director Gina Haspel has met with a group of Senate and House lawmakers earlier this month, but she only appeared to solidify the belief among senators that the crown prince is responsible. Backers of the Yemen resolution did lose some GOP support since the initial procedural vote late last month. Some Republicans, including Graham, say they voted initially to advance the resolution because of the message it sent to Saudi Arabia and not because of the substance of the resolution. "[But] we also want to preserve the 70-year partnership between the United States and Saudi Arabia and we want to ensure it continues to serve American interests and stabilizes a dangerous and critical region," McConnell said. He added that the dynamic presents "challenging circumstances" but "the Sanders-Lee resolution is neither precise enough or prudent enough.” Corker, who spearheaded the resolution finding the crown prince responsible for Khashoggi's death, had negotiated for days with Senate leadership on that resolution. "Unanimously the United States Senate has said that Crown Prince Mohammed Bin Salman is responsible for the murder of Jamal Khashoggi. That is a strong statement. I think it speaks to the values that we hold dear.... I'm glad the Senate is speaking with once voice unanimously toward this end," Corker said shortly after the vote Thursday. As a bipartisan coalition of senators prepared to advance a resolution to end the United States’ role in the Yemeni civil war on Wednesday afternoon, Republicans in the House passed a rule to effectively end a similar measure’s chances in that chamber. House Republican leaders inserted a provision to the rule outlining conditions for debate on the farm bill late Tuesday night that would prevent members such as California Democrat Ro Khanna, sponsor of a Yemen war powers resolution, from forcing a floor vote on the matter during the remainder of the 115th Congress. The House approved the rule Wednesday 206-203, with five Democrats supporting it and 18 Republicans dissenting. By a vote of 206 to 203 congress just flushed our War Powers down the toilet. SAD! — Thomas Massie (@RepThomasMassie) December 12, 2018 “It's kind of a chicken move, but you know, sadly it's kind of a characteristic move on the way out the door,” Virginia Democrat Tim Kaine told reporters of the House rule on Wednesday. “[Ryan is] trying to play Saudi Arabia's defense lawyer, and that's stupid.” Supporters of the Senate resolution had already acknowledged that the issue would have to spill into next year given House Speaker Paul Ryan’s opposition. Ryan contends the action is unnecessary, as the Defense Department ended air-to-air refueling support for Saudi coalition aircraft last month. “We are not involved in ‘hostilities’ in Yemen so the War Powers Act does not apply,” Ryan spokeswoman AshLee Strong said. Since 2015, the United States has provided other support for the Saudis, such as expertise and intelligence-sharing for bombing operations in the bloody stalemate against the Houthis, who are occupying parts of Yemen. The White House argues these activities do not constitute participating in the hostilities, and Trump has said he would veto the Sanders-Lee resolution if Congress passes it. Asked whether he had spoken to Democratic leaders in the House about pursuing the matter when Republicans relinquish the gavel in January, Kaine said he expects Democrats will continue to push to address the Yemen conflict and to punish Saudi Arabia for the orchestrated murder of journalist Jamal Khashoggi in October. “We're going to stand up against [Khashoggi’s killing], and if Paul Ryan thinks on his way out the door his last public service gift to humanity is covering up for Saudi Arabia, great,” said Kaine. “He can make that his legacy, but we're going to be around next year and we'll figure out ways that there can be consequences for this.” Connecticut Democrat Chris Murphy, a co-sponsor of the Senate bill, also said a willingness to move the resolution next year exists in the House, but he noted it would be more challenging to pass in the Senate come January, as the Republican majority will grow to 53 members from the current 51. Still, Murphy expressed optimism — because of its privileged status, the bill requires only 51 votes to pass, and Republicans Mike Lee and Rand Paul have long been staunch supporters. “I expect that we'll start this process again at the beginning of next year to try to get it to the president's desk,” said Murphy. The Senate voted 63-37 to allow a debate on the resolution in November. It had previously failed on the same procedural vote in March, 55-44. The 10 Democrats who initially opposed the resolution all voted in favor of it last month, along with 14 Republicans. The surge in support was partly due to backlash over President Donald Trump’s tepid response to the Khashoggi killing. Trump has split with the intelligence community’s assessment that Saudi leader Mohammed bin Salman (MBS) was responsible for the operation and has stood firmly beside the crown prince, provoking criticism from senators of both parties. Yet several Republicans who voted in November to debate the measure may ultimately vote against it, such as Foreign Relations Committee chairman Bob Corker. Corker on Wednesday said he was set to introduce his own bill to punish Saudi leaders, adding to a growing pile of legislative options such as implementing new sanctions, formally assigning responsibility to MBS for the killing, and ending arms sales to Saudi Arabia. Supporters of the Yemen war powers resolution argue it should be viewed as a strong signal to Saudi leaders on its own, however. And despite the House’s action to prevent a vote on the resolution before January, the Senate’s decision on Wednesday will mark a pivotal moment — it may be the first time Congress votes to invoke the War Powers Act, which allows Congress to order the withdrawal of U.S. forces within 90 days of passage. “What we're talking about is truly an historic moment in the Senate,” Bernie Sanders, another leading co-sponsor of the measure, told reporters. “45 years ago, the War Powers Act was passed. I think we stand a chance today for the first time to use that War Powers Act to stop a war.” If the next step, a vote on the motion to proceed, passes, the Senate is likely to agree to limit amendments to those that are germane to the underlying resolution. Murphy indicated a final vote could occur as soon as Wednesday evening. “Once the motion to proceed passes, you can't stop this. So it's on the floor and you're either amending it, or you're voting on final passage,” Murphy said. “It's likely that this will get finished by the end of today.” WASHINGTON—The U.S. Senate ignored appeals by the Trump administration and passed a resolution on Thursday to withdraw U.S. support for the Saudi-led coalition at war in Yemen, delivering a bipartisan setback for the president’s Middle East policy. The measure, which passed in a 56-41 vote, pits a Senate upset by the October killing of journalist Jamal Khashoggi by Saudi agents against the Trump administration, which views Saudi Arabia as a vital strategic ally. Seven Republicans joined with all 49 members of the Democratic... | The Senate on Thursday sent what the Washington Post described as a historic rebuke to the White House over Saudi Arabia. Actually, it delivered two rebukes. First, senators voted 56-41 to invoke the War Powers Act and demand that the US end its military support of the Saudi-led coalition in the Yemen war, per the Hill. Neither the Senate nor the House has ever invoked the 1973 act previously. The Senate also voted-unanimously-to condemn Saudi crown prince Mohammed bin Salman for the murder of Jamal Khashoggi and called on the Saudi government to "ensure appropriate accountability," reports the AP. The details. Only symbolic? Though historic, it's unlikely the war resolution can pass the House this year. That chamber already has moved to effectively make passage impossible in 2018, reports the Weekly Standard. And even if it did pass, President Trump would likely veto it. Democrats could re-introduce it in 2019, however. Why? Mounting reports of war atrocities and the killing of Khashoggi led to bipartisan anger among senators, per NPR. Seven Republicans bucked Trump on the war vote, including co-sponsor Mike Lee of Utah. Among other things, the legislation forbids the American refueling of Saudi jets and orders the US to scale back its military presence in the region, reports the Wall Street Journal. | multi_news |
With her rise in the coming months to chairman and CEO (as Gianopulos, 64, moves into an undefined "strategic role"), Snider, 55, will preside over the second-hottest studio at the 2016 box office (after Disney). Watts, 46, has become the envy of many of her counterparts at rival studios thanks to hits like The Martian ($630 million). BIG WIN Deadpool ($778 million), another Watts film, is the top-grossing R-rated title ever. BIG BET Will the executive shakeup work out? Fox's future is all about the next Avatar films — and filmmaker James Cameron's strongest relationship at the studio is with Gianopulos. *** What's the least powerful thing about your life? SNIDER I don’t think about my life in terms of power. I think about my life in terms of meaning. WATTS The list expands daily. GIANOPULOS Managing my really smart and independent daughters. I can't get through a day without … SNIDER Quiet time to think (and tea!). WATTS Many espressos. GIANOPULOS Sugar-free Red Bull. (I know, don't tell me.) Who's your most important adviser? SNIDER My husband, Gary Jones. WATTS Jonathan Krauss. GIANOPULOS My wife, Ann, who is amazing, beloved by all (and certainly by me) and always tells me the truth while making it easier to accept. In high school, I was … SNIDER A lot like I am today. WATTS Younger. GIANOPULOS In a Rolling Stones cover band (and unsuccessfully pretending to be Keith Richards). What's your hidden talent? SNIDER I can change from work clothes to sweats in record time. WATTS A really loud two-fingered whistle. GIANOPULOS I can discern meaning in half-a-dozen languages I don't speak. If I could have lunch with anyone, living or dead, I'd choose … SNIDER Martin Luther King Jr. WATTS Vanessa Nadal and Lin-Manuel Miranda. GIANOPULOS Ernest Hemingway, and he could pick the place. The first powerful person I ever met was … SNIDER My teachers at Friends' Central School had the greatest influence on my life (after my parents). But, given their Quaker affiliation, they would not have considered themselves powerful. WATTS Diane Von Furstenberg. GIANOPULOS My father, who overcame odds that still humble anything I've done. And also, the Queen of England (but not at the same time). Have you seen Hamilton and how many times? SNIDER Yes, off-Broadway and then on Broadway. WATTS Yes, I love it! I've been twice. GIANOPULOS Yes, I've seen Hamilton, and even managed to close my dropped jaw long enough to think of something to say to Lin-Manuel Miranda. I told him I had never experienced anything so exalted that so surpassed its expectation. With her rise in the coming months to chairman and CEO (as Gianopulos, 64, moves into an undefined "strategic role"), Snider, 55, will preside over the second-hottest studio at the 2016 box office (after Disney). Watts, 46, has become the envy of many of her counterparts at rival studios thanks to hits like The Martian ($630 million). BIG WIN Deadpool ($778 million), another Watts film, is the top-grossing R-rated title ever. BIG BET Will the executive shakeup work out? Fox's future is all about the next Avatar films — and filmmaker James Cameron's strongest relationship at the studio is with Gianopulos. *** What's the least powerful thing about your life? SNIDER I don’t think about my life in terms of power. I think about my life in terms of meaning. WATTS The list expands daily. GIANOPULOS Managing my really smart and independent daughters. I can't get through a day without … SNIDER Quiet time to think (and tea!). WATTS Many espressos. GIANOPULOS Sugar-free Red Bull. (I know, don't tell me.) Who's your most important adviser? SNIDER My husband, Gary Jones. WATTS Jonathan Krauss. GIANOPULOS My wife, Ann, who is amazing, beloved by all (and certainly by me) and always tells me the truth while making it easier to accept. In high school, I was … SNIDER A lot like I am today. WATTS Younger. GIANOPULOS In a Rolling Stones cover band (and unsuccessfully pretending to be Keith Richards). What's your hidden talent? SNIDER I can change from work clothes to sweats in record time. WATTS A really loud two-fingered whistle. GIANOPULOS I can discern meaning in half-a-dozen languages I don't speak. If I could have lunch with anyone, living or dead, I'd choose … SNIDER Martin Luther King Jr. WATTS Vanessa Nadal and Lin-Manuel Miranda. GIANOPULOS Ernest Hemingway, and he could pick the place. The first powerful person I ever met was … SNIDER My teachers at Friends' Central School had the greatest influence on my life (after my parents). But, given their Quaker affiliation, they would not have considered themselves powerful. WATTS Diane Von Furstenberg. GIANOPULOS My father, who overcame odds that still humble anything I've done. And also, the Queen of England (but not at the same time). Have you seen Hamilton and how many times? SNIDER Yes, off-Broadway and then on Broadway. WATTS Yes, I love it! I've been twice. GIANOPULOS Yes, I've seen Hamilton, and even managed to close my dropped jaw long enough to think of something to say to Lin-Manuel Miranda. I told him I had never experienced anything so exalted that so surpassed its expectation. Whose job is it to change the world? Okay, that’s a big question, so let’s start off with a (somewhat) simpler one: Is admitting that you have a problem, truly the first step to recovery? There’s a new trend in conversations about race, diversity and privilege in America. More and more, you’ll see and hear people “checking” their privilege at the start of such conversations. Now that everyone is talking about the fact that Hollywood has a diversity problem, and America has a race problem (problems that intersect with gender and class, among other factors), there seems to now be a general rule in place that you have to acknowledge these problems and your own privilege in the face of them. The good news is that you don’t have to do much else beyond that. In other words, merely acknowledging the issue of diversity as a real thing that exists—and then doing little else beyond that—has somehow become enough. I was reminded of this new trend when I read the introduction to The Hollywood Reporter’s annual Top 100 list, written by Janice Min. And lastly, yes, we know that a lot of this list is white guys. We won’t need social media, thanks anyway, to drop an anvil on our heads to realize this. Out of the 124 names on the list (due to some shared groupings), there are only 19 women and 10 people of color (six African-Americans, two Asians, two Latinos). I’d like to think we cover the issues of gender and diversity that plague Hollywood with a critical eye and will continue to. And I hope and expect that one day in the near future this list has a wholly improved and reflective composition. This attempt to get in front of social media seems incredibly odd. Firstly, it seeks to name “social media” (which, in this case, is basically synonymous with Black Twitter) as the problem in this scenario. Don’t you dare rise up and point out the fact that there are only 19 women and 10 people of color on this list, Black Twitter—in fact I’ll do it right here and beat you to the punch so you can’t blame me for anything! How odd, to villianize the same people who have forced you to, at the very least, be aware of how many people of color are consistently left off of lists like this. How odd to think that such a flippantly delivered warning will keep us from asking questions about yet another list celebrating the accomplishments of—as you yourself admit—”a lot” of white guys. It’s also interesting (read: infuriating) to note a certain attempt at undercutting the accomplishments of those few women on the list. Take for example, the language used in Channing Dungey’s ranking: Since becoming the first African-American woman ever tapped to lead a Big Four broadcast network in February, Dungey, 47, has exercised her power by slashing seven series, including surprises Nashville and Castle, and adding new ones centered on overweight housewives and talking dogs. BIG WIN: Her new gig, especially given her lack of noncreative experience. Sure, Dungey made the Top 100 list (one of only three black women to do so—she’s joined by Shonda Rhimes and Oprah Winfrey), but it doesn’t sound like THR’s especially excited about that. From what I’m reading here, it sounds like she’s spent the last few months ruining all of TV. But let’s say we can put such tone, and Min’s sly remark about social media anvils to the side. Min is saying we must look at the whole of what The Hollywood Reporter covers to understand how they work against the active and ongoing discrimination against women and blacks, and other people of color in the industry. In other coverage, just not here, she seems to say, we are trying to do our part. The thing is, this isn’t the first time THR has made such a claim, and participated in this trendy movement of checking oneself, while taking little-to-no responsibility for that which required checking. Last year Stephen Galloway made a similar attempt to get in front of social media and any other critics, by penning an article titled Why Every Actress on The Hollywood Reporter Roundtable Cover Is White. Galloway, like Min, hoped to beat all those other writers to the punch, and gain some credit for merely acknowledging the problem. However, neither Galloway nor Min seems willing to take any of the responsibility here. They gladly admit that Hollywood has a problem, but they defend their publication as a mere reflection of Hollywood—not to be blamed for the discriminatory and racist practices of the executives and casting directors, and those others in positions of power. It’s not their job to resolve these issues—in fact, they can’t! They, mere Hollywoodreporters are powerless against the real villain: HOLLYWOOD. Galloway explained this very clearly: So who’s responsible? The Academy drew flak for failing to nominate Selma in many categories, but the Academy doesn’t make films, any more than The Hollywood Reporter does: It recognizes work that the industry creates. He also explained how badly he felt about his all-white cover, and it wasn’t until the very end of the piece that he admitted to a mistake, or a regret at least: On our most recent Director Roundtable, forced to choose among three superb filmmakers for one slot, I opted for Ridley Scott rather than Straight Outta Compton director F. Gary Gray, an African-American. The Martian had opened to exceptional acclaim and box office, and Scott looked like the front-runner for the Oscar. Still, I now wish I had added Gray to the mix, and regret that I ignored both his lawyer’s and his agents’ pleas to do so. At least I can take comfort in having three men of color on our upcoming Actor Roundtable. Well, which is it Hollywood Reporter? Are you powerless in this fight? Or, actually quite powerful, but busy making yourself feel better when you fail to fight for diversity in one issue, but “make up for it” in another issue? After all, you can’t be expected to consistently fight for diversity (also known as normalcy) all of the time. Asking who’s responsible for the diversity problem is like playing chicken vs. egg. For example, if more black actresses covered magazines, would they get more work (short answer: yes)? But should more black actresses cover magazines when they’re not getting enough work? Long answer: Yes, because that is one way to combat the problem of their lack of work. But to do such a thing, you’d have to assume that it’s your responsibility to change Hollywood, and to change the world. You’d have to believe that you don’t have to be a studio executive to exact change. I liked that Min’s opening explained the process of choosing the Top 100. I don’t have her job, and she succeeding in convincing me that defining “power” for an entire industry is quite a feat. She put it beautifully—they are “trying to measure something intangible: clout.” But she didn’t succeed in convincing me that her publication is aware that they are complicit in the problem. (In fact, when I read her explanation, the words of one iconic black artist immediately came to mind: “We don’t believe you, you need more people.”) The world is filled with people everywhere, insisting that it’s not their job to address certain cultural and social issues. Diversity in Hollywood isn’t their problem or fault, racism isn’t their problem or fault, Donald Trump isn’t their doing, etc. They are/we are all innocent, because they/we don’t make the rules. It’s the Pontius Pilot syndrome, and without exaggeration I can say that it does real violence to the culture, and to the people. Acknowledging that your publication has a problem (or pretending to acknowledge the problem, but then pivoting to Hollywood and social media as the real villains), is not enough. If THR wants to convince us that it’s a publication concerned with racist Hollywood practices, it’s going to need to do better than the occasional Chris Rock op ed. Its biggest issues—these power rankings and roundtables—need to stop functioning as mere “reflections” of a troubling place and time; they need to be that change they keep insisting must come first. It’s not activism, it’s innovation. And that should be a concept that we can all get behind. Shannon M. Houston is a Staff Writer and the TV Editor for Paste. This New York-based writer probably has more babies than you, but that’s okay; you can still be friends. She welcomes almost all follows on Twitter. | For the first time, the Hollywood Reporter has put together a list of the 100 most powerful people in entertainment, though it's what Paste Magazine describes as a list "celebrating the accomplishments of... white guys." Only 19 women and 10 people of color make the cut. THR acknowledges the stat- "We won't need social media, thanks anyway, to drop an anvil on our heads to realize this"-but says the list reflects currently reality, not what current reality should be. Here's the top 10: Bob Iger, Disney CEO Rupert Murdoch and sons, 21st Century Fox Leslie Moonves, CBS President and CEO Steve Burke, CEO of NBCUniversal Ted Sarandos, Netflix's chief content officer Shari Redstone and Sumner Redstone of Viacom and CBS Jeff Bewkes, Time Warner Chairman and CEO Peter Rice, Chairman and CEO of Fox Alan Horn, Walt Disney Studios Chairman Leonardo DiCaprio, actor and producer The full list is here, or see Hollywood's most overpaid actors. | multi_news |
Their next and second attempt thereat was more deliberately made, though it was begun on the morning following the singular child's arrival at their home. Him they found to be in the habit of sitting silent, his quaint and weird face set, and his eyes resting on things they did not see in the substantial world. "His face is like the tragic mask of Melpomene," said Sue. "What is your name, dear? Did you tell us?" "Little Father Time is what they always called me. It is a nickname; because I look so aged, they say." "And you talk so, too," said Sue tenderly. "It is strange, Jude, that these preternaturally old boys almost always come from new countries. But what were you christened?" "I never was." "Why was that?" "Because, if I died in damnation, 'twould save the expense of a Christian funeral." "Oh--your name is not Jude, then?" said his father with some disappointment. The boy shook his head. "Never heerd on it." "Of course not," said Sue quickly; "since she was hating you all the time!" "We'll have him christened," said Jude; and privately to Sue: "The day we are married." Yet the advent of the child disturbed him. Their position lent them shyness, and having an impression that a marriage at a superintendent registrar's office was more private than an ecclesiastical one, they decided to avoid a church this time. Both Sue and Jude together went to the office of the district to give notice: they had become such companions that they could hardly do anything of importance except in each other's company. Jude Fawley signed the form of notice, Sue looking over his shoulder and watching his hand as it traced the words. As she read the four-square undertaking, never before seen by her, into which her own and Jude's names were inserted, and by which that very volatile essence, their love for each other, was supposed to be made permanent, her face seemed to grow painfully apprehensive. "Names and Surnames of the Parties"--(they were to be parties now, not lovers, she thought). "Condition"--(a horrid idea)--"Rank or Occupation"--"Age"--"Dwelling at"--"Length of Residence"--"Church or Building in which the Marriage is to be solemnized"--"District and County in which the Parties respectively dwell." "It spoils the sentiment, doesn't it!" she said on their way home. "It seems making a more sordid business of it even than signing the contract in a vestry. There is a little poetry in a church. But we'll try to get through with it, dearest, now." "We will. 'For what man is he that hath betrothed a wife and hath not taken her? Let him go and return unto his house, lest he die in the battle, and another man take her.' So said the Jewish law-giver." "How you know the Scriptures, Jude! You really ought to have been a parson. I can only quote profane writers!" During the interval before the issuing of the certificate, Sue, in her housekeeping errands, sometimes walked past the office, and furtively glancing in saw affixed to the wall the notice of the purposed clinch to their union. She could not bear its aspect. Coming after her previous experience of matrimony, all the romance of their attachment seemed to be starved away by placing her present case in the same category. She was usually leading little Father Time by the hand, and fancied that people thought him hers, and regarded the intended ceremony as the patching up of an old error. Meanwhile Jude decided to link his present with his past in some slight degree by inviting to the wedding the only person remaining on earth who was associated with his early life at Marygreen--the aged widow Mrs. Edlin, who had been his great-aunt's friend and nurse in her last illness. He hardly expected that she would come; but she did, bringing singular presents, in the form of apples, jam, brass snuffers, an ancient pewter dish, a warming-pan, and an enormous bag of goose feathers towards a bed. She was allotted the spare room in Jude's house, whither she retired early, and where they could hear her through the ceiling below, honestly saying the Lord's Prayer in a loud voice, as the Rubric directed. As, however, she could not sleep, and discovered that Sue and Jude were still sitting up--it being in fact only ten o'clock--she dressed herself again and came down, and they all sat by the fire till a late hour--Father Time included; though, as he never spoke, they were hardly conscious of him. "Well, I bain't set against marrying as your great-aunt was," said the widow. "And I hope 'twill be a jocund wedding for ye in all respects this time. Nobody can hope it more, knowing what I do of your families, which is more, I suppose, than anybody else now living. For they have been unlucky that way, God knows." Sue breathed uneasily. "They was always good-hearted people, too--wouldn't kill a fly if they knowed it," continued the wedding guest. "But things happened to thwart 'em, and if everything wasn't vitty they were upset. No doubt that's how he that the tale is told of came to do what 'a did--if he WERE one of your family." "What was that?" said Jude. "Well--that tale, ye know; he that was gibbeted just on the brow of the hill by the Brown House--not far from the milestone between Marygreen and Alfredston, where the other road branches off. But Lord, 'twas in my grandfather's time; and it medn' have been one of your folk at all." "I know where the gibbet is said to have stood, very well," murmured Jude. "But I never heard of this. What--did this man--my ancestor and Sue's--kill his wife?" "'Twer not that exactly. She ran away from him, with their child, to her friends; and while she was there the child died. He wanted the body, to bury it where his people lay, but she wouldn't give it up. Her husband then came in the night with a cart, and broke into the house to steal the coffin away; but he was catched, and being obstinate, wouldn't tell what he broke in for. They brought it in burglary, and that's why he was hanged and gibbeted on Brown House Hill. His wife went mad after he was dead. But it medn't be true that he belonged to ye more than to me." A small slow voice rose from the shade of the fireside, as if out of the earth: "If I was you, Mother, I wouldn't marry Father!" It came from little Time, and they started, for they had forgotten him. "Oh, it is only a tale," said Sue cheeringly. After this exhilarating tradition from the widow on the eve of the solemnization they rose, and, wishing their guest good-night, retired. The next morning Sue, whose nervousness intensified with the hours, took Jude privately into the sitting-room before starting. "Jude, I want you to kiss me, as a lover, incorporeally," she said, tremulously nestling up to him, with damp lashes. "It won't be ever like this any more, will it? I wish we hadn't begun the business. But I suppose we must go on. How horrid that story was last night! It spoilt my thoughts of to-day. It makes me feel as if a tragic doom overhung our family, as it did the house of Atreus." "Or the house of Jeroboam," said the quondam theologian. "Yes. And it seems awful temerity in us two to go marrying! I am going to vow to you in the same words I vowed in to my other husband, and you to me in the same as you used to your other wife; regardless of the deterrent lesson we were taught by those experiments!" "If you are uneasy I am made unhappy," said he. "I had hoped you would feel quite joyful. But if you don't, you don't. It is no use pretending. It is a dismal business to you, and that makes it so to me!" "It is unpleasantly like that other morning--that's all," she murmured. "Let us go on now." They started arm in arm for the office aforesaid, no witness accompanying them except the Widow Edlin. The day was chilly and dull, and a clammy fog blew through the town from "Royal-tower'd Thame." On the steps of the office there were the muddy foot-marks of people who had entered, and in the entry were damp umbrellas. Within the office several persons were gathered, and our couple perceived that a marriage between a soldier and a young woman was just in progress. Sue, Jude, and the widow stood in the background while this was going on, Sue reading the notices of marriage on the wall. The room was a dreary place to two of their temperament, though to its usual frequenters it doubtless seemed ordinary enough. Law-books in musty calf covered one wall, and elsewhere were post-office directories, and other books of reference. Papers in packets tied with red tape were pigeon-holed around, and some iron safes filled a recess, while the bare wood floor was, like the door-step, stained by previous visitors. The soldier was sullen and reluctant: the bride sad and timid; she was soon, obviously, to become a mother, and she had a black eye. Their little business was soon done, and the twain and their friends straggled out, one of the witnesses saying casually to Jude and Sue in passing, as if he had known them before: "See the couple just come in? Ha, ha! That fellow is just out of gaol this morning. She met him at the gaol gates, and brought him straight here. She's paying for everything." Sue turned her head and saw an ill-favoured man, closely cropped, with a broad-faced, pock-marked woman on his arm, ruddy with liquor and the satisfaction of being on the brink of a gratified desire. They jocosely saluted the outgoing couple, and went forward in front of Jude and Sue, whose diffidence was increasing. The latter drew back and turned to her lover, her mouth shaping itself like that of a child about to give way to grief: "Jude--I don't like it here! I wish we hadn't come! The place gives me the horrors: it seems so unnatural as the climax of our love! I wish it had been at church, if it had to be at all. It is not so vulgar there!" "Dear little girl," said Jude. "How troubled and pale you look!" "It must be performed here now, I suppose?" "No--perhaps not necessarily." He spoke to the clerk, and came back. "No--we need not marry here or anywhere, unless we like, even now," he said. "We can be married in a church, if not with the same certificate with another he'll give us, I think. Anyhow, let us go out till you are calmer, dear, and I too, and talk it over." They went out stealthily and guiltily, as if they had committed a misdemeanour, closing the door without noise, and telling the widow, who had remained in the entry, to go home and await them; that they would call in any casual passers as witnesses, if necessary. When in the street they turned into an unfrequented side alley where they walked up and down as they had done long ago in the market-house at Melchester. "Now, darling, what shall we do? We are making a mess of it, it strikes me. Still, ANYTHING that pleases you will please me." "But Jude, dearest, I am worrying you! You wanted it to be there, didn't you?" "Well, to tell the truth, when I got inside I felt as if I didn't care much about it. The place depressed me almost as much as it did you--it was ugly. And then I thought of what you had said this morning as to whether we ought." They walked on vaguely, till she paused, and her little voice began anew: "It seems so weak, too, to vacillate like this! And yet how much better than to act rashly a second time... How terrible that scene was to me! The expression in that flabby woman's face, leading her on to give herself to that gaol-bird, not for a few hours, as she would, but for a lifetime, as she must. And the other poor soul--to escape a nominal shame which was owing to the weakness of her character, degrading herself to the real shame of bondage to a tyrant who scorned her--a man whom to avoid for ever was her only chance of salvation... This is our parish church, isn't it? This is where it would have to be, if we did it in the usual way? A service or something seems to be going on." Jude went up and looked in at the door. "Why--it is a wedding here too," he said. "Everybody seems to be on our tack to-day." Sue said she supposed it was because Lent was just over, when there was always a crowd of marriages. "Let us listen," she said, "and find how it feels to us when performed in a church." They stepped in, and entered a back seat, and watched the proceedings at the altar. The contracting couple appeared to belong to the well-to-do middle class, and the wedding altogether was of ordinary prettiness and interest. They could see the flowers tremble in the bride's hand, even at that distance, and could hear her mechanical murmur of words whose meaning her brain seemed to gather not at all under the pressure of her self-consciousness. Sue and Jude listened, and severally saw themselves in time past going through the same form of self-committal. "It is not the same to her, poor thing, as it would be to me doing it over again with my present knowledge," Sue whispered. "You see, they are fresh to it, and take the proceedings as a matter of course. But having been awakened to its awful solemnity as we have, or at least as I have, by experience, and to my own too squeamish feelings perhaps sometimes, it really does seem immoral in me to go and undertake the same thing again with open eyes. Coming in here and seeing this has frightened me from a church wedding as much as the other did from a registry one... We are a weak, tremulous pair, Jude, and what others may feel confident in I feel doubts of--my being proof against the sordid conditions of a business contract again!" Then they tried to laugh, and went on debating in whispers the object-lesson before them. And Jude said he also thought they were both too thin-skinned--that they ought never to have been born--much less have come together for the most preposterous of all joint ventures for THEM--matrimony. His betrothed shuddered; and asked him earnestly if he indeed felt that they ought not to go in cold blood and sign that life-undertaking again? "It is awful if you think we have found ourselves not strong enough for it, and knowing this, are proposing to perjure ourselves," she said. "I fancy I do think it--since you ask me," said Jude. "Remember I'll do it if you wish, own darling." While she hesitated he went on to confess that, though he thought they ought to be able to do it, he felt checked by the dread of incompetency just as she did--from their peculiarities, perhaps, because they were unlike other people. "We are horribly sensitive; that's really what's the matter with us, Sue!" he declared. "I fancy more are like us than we think!" "Well, I don't know. The intention of the contract is good, and right for many, no doubt; but in our case it may defeat its own ends because we are the queer sort of people we are--folk in whom domestic ties of a forced kind snuff out cordiality and spontaneousness." Sue still held that there was not much queer or exceptional in them: that all were so. "Everybody is getting to feel as we do. We are a little beforehand, that's all. In fifty, a hundred, years the descendants of these two will act and feel worse than we. They will see weltering humanity still more vividly than we do now, as Shapes like our own selves hideously multiplied, and will be afraid to reproduce them." "What a terrible line of poetry!... though I have felt it myself about my fellow-creatures, at morbid times." Thus they murmured on, till Sue said more brightly: "Well--the general question is not our business, and why should we plague ourselves about it? However different our reasons are, we come to the same conclusion: that for us particular two, an irrevocable oath is risky. Then, Jude, let us go home without killing our dream! Yes? How good you are, my friend: you give way to all my whims!" "They accord very much with my own." He gave her a little kiss behind a pillar while the attention of everybody present was taken up in observing the bridal procession entering the vestry; and then they came outside the building. By the door they waited till two or three carriages, which had gone away for a while, returned, and the new husband and wife came into the open daylight. Sue sighed. "The flowers in the bride's hand are sadly like the garland which decked the heifers of sacrifice in old times!" "Still, Sue, it is no worse for the woman than for the man. That's what some women fail to see, and instead of protesting against the conditions they protest against the man, the other victim; just as a woman in a crowd will abuse the man who crushes against her, when he is only the helpless transmitter of the pressure put upon him." "Yes--some are like that, instead of uniting with the man against the common enemy, coercion." The bride and bridegroom had by this time driven off, and the two moved away with the rest of the idlers. "No--don't let's do it," she continued. "At least, just now." They reached home, and passing the window arm in arm saw the widow looking out at them. "Well," cried their guest when they entered, "I said to myself when I zeed ye coming so loving up to the door, 'They made up their minds at last, then!'" They briefly hinted that they had not. "What--and ha'n't ye really done it? Chok' it all, that I should have lived to see a good old saying like'marry in haste and repent at leisure' spoiled like this by you two! 'Tis time I got back again to Marygreen--sakes if tidden--if this is what the new notions be leading us to! Nobody thought o' being afeard o' matrimony in my time, nor of much else but a cannon-ball or empty cupboard! Why when I and my poor man were married we thought no more o't than of a game o' dibs!" "Don't tell the child when he comes in," whispered Sue nervously. "He'll think it has all gone on right, and it will be better that he should not be surprised and puzzled. Of course it is only put off for reconsideration. If we are happy as we are, what does it matter to anybody?" | The morning after the boy arrives, Jude and Sue discover he is called Little Father Time because, as he says, he looks so old. Jude and Sue give notice of their wedding and invite Mrs. Edlin, the widow who took care of his aunt, to come. The night before they are to marry, Mrs. Edlin tells a tale about a man hanged near the Brown House, a man who may have been an ancestor of Sue and Jude. The next day they go to marry at the registry office, but after watching other couples, first Sue and then Jude decide the setting is too sordid. They go to a parish church to watch a wedding, but they agree they can't go through with a ceremony like that they both experienced before. They decide that the compulsion of marriage is not for such as they, and Sue says, "If we are happy as we are, what does it matter to anybody?" | booksum |
15:04 More from Cardiff where Theresa May was booed after attending a Brexit meeting with the heads of the devolved assemblies. steven morris (@stevenmorris20) JMC meeting in Cardiff - small demo outside. Protesters worried about Trump and Brexit. pic.twitter.com/cSg4t74Sum The Scottish first minister, Nicola Sturgeon, has said that Donald Trump’s state visit ought to be cancelled while travel bans are in place and has called on the prime minister to speak up more strongly against the values that the president’s policies have exposed, writes Steven Morris. British Prime Minister Theresa May sits alongside members of her Cabinet as she prepares to chair a Joint Ministerial Committee at Cardiff City Hall in Cardiff. Also attending the talks were Outgoing Northern Ireland First Minister, Arlene Foster, Welsh First Minister Carwyn Jones, and Scotland’s First Minister Nicola Sturgeon Photograph: Ben Birchall/AFP/Getty Images Speaking after a one-to-one meeting with Theresa May that took place before the meeting of the joint ministerial committee in Cardiff, Sturgeon said she told the prime minister she should voice concerns about Trump more forcefully. Sturgeon told the Guardian: “I said [to her] that while everybody understands that she wants to build a constructive relationship that relationship has to be based on values. “I think many people would like to hear a stronger view from the UK government about the immigrant and refugee ban that was announced. “I also said that I don’t think it would be appropriate in these circumstances for the state visit to go ahead while these bans are in place given the understandable concern that people have about them and the messages they send and the impact they have.” Asked if May had gone to meet Trump too quickly, Sturgeon replied: “She’s the prime minister of the UK. Everybody would understand she wants to build a positive relationship with the president of the United States. As first minister of Scotland I want to build a constructive relationship with the new administration. I’m not criticising her for that. But relationships have to be based on values. “We’ve all got a duty to speak up for fundamental values. There’s a real concern on the part of many that introducing what is seen by many as a ban on Muslims, banning people because of their origin or faith, is deeply wrong and likely to be counterproductive in terms of the fight we all have an interest in against extremism and terrorism. “In terms of the refugee ban that in my view would go against the international obligations in terms of the Geneva Convention and the moral obligation we all have to deal with the refugee crisis. “The prime minister made the obvious point that these are matters for the US government. But these are issues that start to touch on moral issues that go beyond individual countries policies. And we all have a duty in these instances to speak up when we consider values that we all hold dear to be under threat. As I said to the prime minister I think a lot of people would like to see her say something much more strong along those lines.” She continued: “Morality is something we all have to judge for ourselves. I think there’s a very strong body of opinion across the UK. Nobody is suggesting the president of American can’t come to the UK nor is anyone suggesting a state visit is not appropriate at some stage but while these bans that have caused so much concern are in place it would be inappropriate for the state visit to proceed. It would be better to reconsider the timing of it.” Asked if she would meet Trump in Scotland, Sturgeon said: “The relationship between Scotland and America is important. I’m not going to start getting into refusing meeting people but nor am I going to maintain diplomatic silences over things that are really important in a values and principles sense.” Finding Dory, the long-awaited sequel to Pixar's Finding Nemo, enjoyed rave reviews and the highest-grossing animated film opening ever recorded when it opened last summer. The family film picks up with forgetful Blue Tang fish Dory, who, due to her short-term memory loss, has not only lost her parents, but forgotten most of what she knew about them. Over the course of the next 97 minutes, Dory makes friends with creatures from different backgrounds to make her way back home across the ocean. This was the film that Donald Trump chose for his first White House screening as US president. While the aquatic adventure entertained Trump's guests, thousands of green card holders, visa holders and pre-approved refugees from seven countries in the Middle East and Africa were kept at airports and taken off flights internationally, after his executive order on Friday afternoon. The order inspired thousands to gather and protest in a park across from the White House on Sunday afternoon. As Albert Brooks, who voiced Nemo's worrisome father Marlin in the film, posted on Twitter, the choice of film was an unfortunate one given the circumstances: Immigrant rights groups scored a series of early court victories against President Donald Trump’s terrorism-focused executive order limiting travel from seven Muslim-majority countries, but legal experts and administration officials said the impact of those initial legal victories could prove fleeting. There is little doubt the orders — issued by federal judges in New York, Boston, Alexandria, Virginia, and Seattle — helped bolster the resolve of anti-Trump protesters who flooded airports over the weekend and turned up by the thousands at the White House Sunday. The rulings may also sour public perceptions of Trump’s directive. Story Continued Below However, lawyers pressing the cases acknowledged that their courtroom wins so far may directly benefit no more than a couple of hundred people essentially caught in limbo when Trump signed his order Friday afternoon limiting travel to the U.S. by citizens of seven countries: Iran, Iraq, Libya, Somalia, Sudan, Syria and Yemen. The flurry of legal rulings Saturday and early Sunday do not appear to have disturbed the central thrust of Trump’s order. The directive suspends immigration and tourism from nationals of the seven countries and halts refugee admissions while stricter vetting protocols are implemented. Tens or perhaps even hundreds of thousands of people could be affected by those changes. “Obviously, this initial litigation is not on behalf of everyone who will be ultimately affected by the executive order, but it’s the first step in challenging the executive order,” said Lee Gelernt of the American Civil Liberties Union, who argued and won an order in New York Saturday night blocking deportations of those held at airports nationwide. “By sheer numbers it doesn’t affect as many people as would be affected by a lawsuit for people overseas, but I think it’s critically important because it’s the first challenge to the executive order. I think there will be broader challenges,” Gelernt added. A Trump administration official who briefed reporters on the executive order Sunday night minimized the impact of the early court rulings. “The executive order is prospective not retroactive. The purpose of the executive order is to prevent the issuance of new visas until such time as responsible vetting measures can be put in place,” said the official, who spoke on condition of anonymity. “The specific issue of travelers who were in transit during the EO’s [issuance] is by and large a one-time situation.... It’s a non-sequitur.” One Muslim-rights group, the Council on American Islamic Relations, said it planned a new federal lawsuit Monday charging that Trump’s order is unconstitutional because it amounts to thinly veiled discrimination against Muslims. That suit could face an uphill battle because courts have rarely accorded constitutional rights to foreigners outside the U.S. However, foreign citizens who are permanent U.S. residents generally have a stronger claim to recourse in the courts. In addition, legal experts say U.S. citizen relatives of foreigners could have legal standing to pursue a case charging religious discrimination. Still, presidents have broad discretion over the nation's immigration and refugee policy. A 1952 immigration law gives the chief executive the power to bar "any class" of immigrants from the country if allowing them is deemed "detrimental to the interests of the United States." For his part, Trump insisted in a new statement Sunday that he was not discriminating against those of the Islam faith. “To be clear, this is not a Muslim ban, as the media is falsely reporting,” Trump said. “This is not about religion — this is about terror and keeping our country safe. There are over 40 different countries worldwide that are majority Muslim that are not affected by this order. We will again be issuing visas to all countries once we are sure we have reviewed and implemented the most secure policies over the next 90 days.” The administration also made clear for the first time Sunday night that green card holders would be presumptively exempt from the new policy, unless there was specific reason to suspect them of terrorist links. Carving out U.S. permanent residents could strengthen Trump’s legal hand to defend the policy in the courts. While broad challenges to the Trump orders could take months or even years to play out in the courts, immigration advocates involved in the legal challenges already underway had differing views about the immediate impact of the various injunctions judges issued over the weekend. While the New York judge’s order barred deportation of people held at airports following Trump’s directive, the order issued by two judges in Boston appeared to go further, requiring them to be released if they would have been under the policies in place prior to the new president’s executive action. The discrepancies between different court orders were notable enough for some lawyers to advise green-card holders flying back to the United States to reroute their flights to Boston’s Logan Airport, since the federal ruling there was the most expansive of the injunctions issued Saturday. “It’s broader in substance because it’s a prohibition on relying solely on the Trump executive order as the basis to detain or remove anyone who’s otherwise lawfully entitled to enter the U.S. The detention piece is new and the range of people is a little bit broader than in New York,” said Matthew Segal, a lawyer with the ACLU’s Massachusetts chapter. He said he believes the order issued there against detentions under the Trump order was nationwide in scope. However, the broad federal injunction issued in Boston raised questions about whether airlines would have to allow immigrants, even from the affected countries, to board an airplane to the United States. “Customs and Border Protection shall notify airlines that have flights arriving at Logan Airport of this order and the fact that individuals on these flights will not be detained or returned based solely on the basis of the Executive Order,” District Judge Allison Burroughs and Magistrate Judith Dein wrote. Segal said lawyers arguing on behalf of immigrants urged that language as part of an effort to try to limit the disruption to travelers already on flights, as well as those trying to come to the U.S. “There was some discussion in the hearing last night of making sure this order would be meaningful, so people could not only get off planes, but also get onto the plane in the first place,” Segal said. “We had been hearing from people throughout the day, folks who were essentially turned away at airports around the world … We wanted to make sure people could get onto these planes.” Normally, airlines are reluctant to allow individuals to board flights if they’re likely to be rejected by immigration authorities at the other end. Large fines are sometimes assessed. It was unclear whether the Boston judges’ order would persuade airlines to allow, for example, the boarding of a green-card holder from one of the seven countries listed in the Trump order, even if that person had not gone to a U.S. Embassy or consulate for special additional screening. Department of Homeland Security officials issued a statement Sunday night asserting that they were complying with the court orders and that they were instructing airlines to deny boarding to affected travelers. “Upon issuance of the court orders yesterday, U.S. Customs and Border Protection (CBP) immediately began taking steps to comply with the orders,” the DHS statement said. “We are also working closely with airline partners to prevent travelers who would not be granted entry under the executive orders from boarding international flights to the U.S. Therefore, we do not anticipate that further individuals traveling by air to the United States will be affected.” Two narrower injunctions were also issued Saturday. One order from a judge in Seattle appeared to affect just two immigrants being held at the airport there, barring their removal from the U.S. at least through Friday. The other order, issued by a judge in Alexandria, Virginia, Saturday night, blocked the deportation of green-card holders from Dulles Airport outside Washington. That order also required that Customs officials allow green-card holders to consult with lawyers. Immigration lawyers, Sen. Cory Booker (D-N.J.) and others complained that the access-to-counsel requirement was being ignored. At Dulles Sunday, attorney Sara Dill said lawyers have been barred from meeting with detainees and that Customs agents refused to confirm whether anyone is being held. She said several attorneys have discussed filing a contempt order to compel cooperation. Most green-card holders in detention at Dulles appeared to be released by late Saturday, although there were reports at least one was deported sometime over the weekend. Attorneys said Sunday afternoon that CBP had pledged to give green-card holders a list of pro bono lawyers and an opportunity to contact them. But attorneys and advocates fanned out at airports across the country complained that CBP agents were not complying with the court injunctions — reporting instances of foreigners in San Francisco and New York who faced “imminent deportation." In some cases, Customs reversed course and said it would not remove the travelers from the United States. Top administration and White House officials indicated to lawmakers and reporters that the immigrants ensnared by the executive order were all being properly processed and would be released. Senate Minority Leader Chuck Schumer (D-N.Y.) said during a news conference in New York that DHS Secretary John Kelly assured him that 42 foreigners stuck at airports across the country would be processed and allowed to enter the United States. But advocates said those instructions weren’t being translated to officers on the ground. “The last 48 hours has been really full of chaos and the sense of the federal government completely deciding to not comply with the Constitution and on top of that, not providing guidance to the field with respect to arriving immigrants and refugees,” said Marielena Hincapie, the executive director of the National Immigration Law Center. Kyle Cheney contributed to this report from Chantilly, Virginia, and Tony Romm from New York. Authors: (CNN) Donald Trump first introduced the idea of a Muslim travel ban in December 2015, shortly after Syed Rizwan Farook and his wife, Tashfeen Malik, shot and killed 14 people in San Bernardino, California. But President Trump's executive order temporarily banning all refugees and suspending travelers from seven Muslim-majority countries would not have applied to either Farook or Malik. Nor would the travel ban have affected the perpetrators of any of the major Islamic terrorist attacks on American soil in recent years. No person accepted to the United States as a refugee, Syrian or otherwise, has been implicated in a major fatal terrorist attack since the Refugee Act of 1980 set up systematic procedures for accepting refugees into the United States, according to an analysis of terrorism immigration risks by the Cato Institute Before 1980, three refugees had successfully carried out terrorist attacks; all three were Cuban refugees, and a total of three people were killed. Since the Cato Institute analysis was published in September 2016, a Somalian refugee injured 13 people at Ohio State University in November in what officials investigated as a terrorist attack. No one died. In fact, the primary perpetrators of the major terror attacks have mostly been US-born citizens or permanent legal residents originally from countries not included in the ban. Here's a look at the origin stories of the terrorists who committed major attacks in the name of radical Islam in recent years, including those in San Bernardino, Orlando, Boston and New York. San Bernardino attacks Although San Bernardino inspired Trump's travel ban, neither of the shooters would have been affected by it. Farook, 28, was an American citizen born in Chicago. Malik, 29, was born and raised in Pakistan, and later lived in Saudi Arabia. She arrived in the United States on a K-1 fiancée visa and later became a permanent resident. Trump's executive order bans travel from seven countries -- Libya, Sudan, Yemen, Somalia, Syria, Iraq and Iran -- but it does not ban travel from residents of Pakistan or Saudi Arabia. In addition, the K-1 fiancee program remains in place. New York and New Jersey explosions Ahmad Khan Rahimi faces an array of bombing, weapons and attempted murder charges in two on September 17, 2016, incidents. He is accused of detonating bombs in New Jersey and in New York's Chelsea neighborhood. The explosion in Chelsea injured 29 people. Rahimi was born in Afghanistan and first came to the United States in 1995, following several years after his father arrived seeking asylum. Rahimi became a naturalized US citizen in 2011. He had recently spent time in Afghanistan and Pakistan, officials said. Neither Afghanistan nor Pakistan is on Trump's list of banned countries. Orlando Pulse nightclub shooting Omar Mateen, the man who shot and killed 49 people at a gay nightclub in Orlando, was an American citizen living in Fort Pierce, Florida. He was born in New York, and his parents were from Afghanistan. His widow, Noor Salman, was arrested earlier this month on charges of obstruction of justice and aiding and abetting her husband's material support to ISIS. She grew up in Rodeo, California, and her parents immigrated to the United States from the West Bank in 1985, according to The New York Times Neither Afghanistan nor the West Bank is included on the list of banned countries. Boston Marathon bombings Tamerlan and Dzhokhar Tsarnaev, who carried out the Boston Marathon bombings in 2013, were born in Kyrgyzstan to parents originally from war-torn Chechnya. The Tsarnaev family arrived in the United States when Dzhokhar Tsarnaev was 8 years old, and they applied for and were granted political asylum. The process for applying for political asylum is different from the process of arriving as a refugee. Dzhokhar Tsarnaev, the younger brother, became a naturalized citizen in September 2012. Chechnya and Kyrgyzstan are not included on the list of banned countries. World Trade Center, September 11, 2001 Of the 19 people who hijacked four planes on September 11, 2001, 15 of them hailed from Saudi Arabia. Two were from the United Arab Emirates, one was from Egypt, and one was from Lebanon. None of those countries is included on the list of banned countries. CLOSE Defeating President Donald Trump in court has translated into a big payday for the American Civil Liberties Union. Nathan Rousseau Smith (@fantasticmrnate) has the story. Buzz60 Hundreds of people gathered in Durham, N.C., on Friday to show their support for refugees and immigrants and stand against President Trump's immigrant ban. (Photo: Bernard Thomas, AP) The American Civil Liberties Union shattered fundraising records this weekend after taking the White House to court over President Trump's executive order banning people from seven Muslim-majority countries from entering the United States. The ACLU said it has received more than 350,000 online donations totaling $24 million since Saturday morning. The non-profit organization that aims to protect individuals' rights and liberties guaranteed in the Constitution typically raises about $4 million online in a year, according to Executive Director Anthony Romero. "It's really clear that this is a different type of moment," Romero said. "People want to know what they can do. They want to be deployed as protagonists in this fight. It's not a spectator sport." The ACLU filed a lawsuit on behalf of two men from Iraq who were detained Friday at New York's John F. Kennedy International Airport after Trump signed his executive order. In response to the suit, a federal judge in Brooklyn blocked part of Trump's controversial order late Saturday, barring officials from deporting those detained in U.S. airports because of the ban. WATCH: ACLU Executive Director Anthony D. Romero coming out of the court where the ACLU just argued and won block of Trump's Muslim ban. pic.twitter.com/kvWDgWiUIn — ACLU National (@ACLU) January 29, 2017 Here's a look at the ACLU's fight with the Trump administration, by the numbers: The ACLU now has 1 million members. Its membership has doubled since the election. "People understand the threats the Trump administration poses and they are willing to take action to fight those threats," Romero said. "They don't just want to write a check and be done with it." Its membership has doubled since the election. "People understand the threats the Trump administration poses and they are willing to take action to fight those threats," Romero said. "They don't just want to write a check and be done with it." Nearly 140,000 people signed up for the ACLU's email list since Saturday. signed up for the ACLU's email list since Saturday. Car-service Lyft pledged on Sunday to donate $1 million to the group. Uber went in a different direction. Now #DeleteUber is trending. to the group. Uber went in a different direction. Now #DeleteUber is trending. Singer Sia pledged to donate up to $100,000. So did Rosie O'Donnell. So did Rosie O'Donnell. The ACLU saw a 1,900% increase in the number of gifts received this month compared to January 2016. in the number of gifts received this month compared to January 2016. The donations will be used to increase staffing, initially by 50 to 100 people this year. this year. The organization has a seven-point plan to take on the Trump administration. Romero said of Trump: "He has to understand governing is different than running for office." The executive order didn't consider the "legal or policy implications, or the practical implications of people traveling to the country." He said Trump "is walking down a very dangerous path.... He will be met with resistance every step of the way." Read or Share this story: http://usat.ly/2jGTI9I Tweet with a location You can add location information to your Tweets, such as your city or precise location, from the web and via third-party applications. You always have the option to delete your Tweet location history. Learn more Ann M. Donnelly waited half a year for her chance to convince the Senate that she would make a good federal judge. When the day finally came, she packed as many relatives as she could into the benches. In her opening comments, she named every single one of them. Donnelly introduced the senators to her husband, Michael. Her sister Sarah and brother Thomas. And then their spouses and their four children. And to her mother and late father — “I know he is watching,” she told the Judiciary Committee in the spring of 2015. And her two daughters, and their boyfriends. And then Saturday night, after a year and a week on the federal bench, Donnelly sat in her own courtroom in Brooklyn while families shouted and cried in airports nationwide. ACLU attorney Lee Gelernt announces to a crowd outside a Brooklyn courthouse that a federal judge had stayed deportations nationwide of those detained on entry to the United States following an executive order from President Trump that targeted citizens from seven predominantly Muslim countries. (ACLU Nationwide/Facebook) President Trump’s sudden ban on refugees and visitors from seven Muslim-majority countries had interrupted reunions midflight, leaving aunts, nephews, fathers and daughters stranded on opposite sides of security cordons, while federal officials decided who would be deported by presidential decree. That’s the night the daughter of Mary and Jack Donnelly — whose speeches and rulings had rarely traveled beyond courthouse walls — became known across the world as the first judge to block Trump’s order. Never before in her long legal career had Donnelly gained such attention. Nowhere close. Her old college roommate, Darcy Gibson Berglund, remembers the Ohio-raised English major starring in a campus rendition of “Pippin” in the late 1970s but then quickly leaving the stage for the law. “She’s an intellectual, she was not going to pursue theater,” Berglund said. But she said her friend retained “a facility with language” after graduating from law school in 1984. Donnelly spent the next quarter-century as a New York prosecutor. Her most famous case was against two executives who looted their company — a trial that the New York Times described as “six months of sometimes tedious testimony.” The paper recounted Donnelly’s closing arguments in the Tyco International case, when she “at times seemed like a schoolteacher lecturing her students.” The executives “believed they were above the law, and they believe the rules that apply to other people do not apply to them,” Donnelly told the jury in 2004. The men were convicted. This week, some of Donnelly’s old colleagues praised her demeanor during that trial — one telling the Times she was “the calm center of the spinning wheel,” even then. She made state judge a few years after her victory in the Tyco trial, in 2009, and for years handled mostly criminal trials. [Trump official appears to walk back inclusion of green-card holders in travel ban] Donnelly would later tell senators that sentencing someone to prison “is one of the most difficult tasks a judge faces.” Some of her cases, however, were so horrific it didn’t seem hard. “Not only did you strangle this woman, you then chopped her up,” she told a man in 2010, according to the New York Daily News, before sentencing him to 19 years to life for killing his ex-girlfriend and burying her in concrete. If the killer were ever released, the paper noted, he would be deported because he had come to the United States illegally. Several years later, a populist Republican would begin crafting campaign speeches around violent immigrants. But first, Donnelly had to wait. And wait, and wait, and wait after President Barack Obama nominated her to the federal court in November 2014, promising she would “serve the American people with integrity and an unwavering commitment to justice.” The Senate Judiciary Committee did not hold hearings to approve her for months, a common theme in an era when White House and Congress stood divided. [McConnell: We don’t have religious tests in this country] “I’m thrilled the committee is finally moving forward,” Sen. Charles E. Schumer (D-N.Y.) said in May 2015. “I know Ann well.” He spoke of her parents in Ohio, and her work prosecuting sex crimes. He told his colleagues of an office Donnelly had left long ago, where “her reputation is legendary.” “She is at her core a kind, thoughtful, compassionate person,” Schumer said. He asked her family to stand. “You’ll see, it’s a great sight.” Donnelly’s mother and a dozen-some siblings, children, spouses, nieces and friends all rose in the chamber. One woman wiped tears from her eyes after Donnelly took the table at the front of the room. The senators asked her only two questions, and only about criminal law. “It’s a certain risk a judge takes,” Donnelly told Sen. Al Franken (D-Minn.), speaking of times she had tried to rehabilitate rather than punish a young offender, in hopes to “save someone from what is bound to be a life of crime.” But mostly, Donnelly spoke of her family before ceding the table and waiting for the Senate’s decision. Another half a year passed until the Senate confirmed her, nearly unanimously, with only two nays. [Senate Democrats vow legislation to block Trump’s travel ban] Yet more months went by before Donnelly was sworn in, quoting Abraham Lincoln in Brooklyn, and speaking once again about her family. She made no great news for a full year on the federal bench — until Saturday evening, when protesters thronged major U.S. airports and an executive with the American Civil Liberties Union tweeted directions to Donnelly’s courthouse. “Go right now if you can,” he wrote. It had by then been a full day since Trump signed an executive order he said would “keep radical Islamic terrorists” out of the country — but which turned out to instantly bar people who had spent weeks or years planning journeys to the United States, and in some cases were already here. Thank you Judge Donnelly!! You have upheld the constitution today and America thanks you!! #muslimban #RefugeeBan #RefugeesWelcome pic.twitter.com/KKy9VV3n4h — Guthrie Graves-Fitz. (@GuthrieGF) January 29, 2017 They had names like Labeeb Ali, who told The Washington Post he had sold his business and belongings in Iraq and obtained a U.S. visa before finding out at the airport that he couldn’t board his flight. And Binto Adan, who The Post reported had flown thousands of miles with her 8- and 9-year-old children expecting to see her husband but who ended up being detained all day at Dulles International Airport because her family is Somali. “I am looking for my parents! They are elderly!” a crying woman shouted in the same airport that night. And in cities from Dallas to Seattle, bewildered families sought missing members, and the ACLU’s emergency request to stop the deportations found its way to Donnelly’s courtroom in New York. She had once been a government lawyer, but that night, she showed little patience for their arguments, The Post reported. “Our own government presumably approved their entry to the country,” Donnelly said, weighing the risks of sending unknown numbers of people back across the oceans. An ACLU lawyer interrupted the hearing to warn Donnelly that a flier was about to be deported to war-torn Syria unless she acted immediately. Donnelly asked whether the government could guarantee that person’s safety and, unconvinced by the answer, issued her order just before 9 p.m. Sending travelers back could cause “irreparable harm,” she ruled. She’d turned more eloquent phrases, but this time her written words were photographed and immediately shared across the world. Stay of ban removals pic.twitter.com/jZjtidm2IF — Omar C. Jadwat (@OmarJadwat) January 29, 2017 “Stay is granted,” the executive director of the ACLU Voting Rights Project wrote on Twitter. “Stay is national.” By early Sunday morning, tearful and exhausted people were emerging from security areas across the United States. They had no guarantee for their future in the United States, but they had a reprieve from immediate deportation. And as families filtered out into the cities, the name of a federal judge they’d never heard of was in headlines across the globe. An earlier version of this post referred incorrectly to the “federal circuit.” Donnelly is a federal judge for the Eastern District of New York. 1 of 29 Full Screen Autoplay Close Skip Ad × How people in the District reacted to Trump’s immigration ban View Photos Thousands protest the president’s executive order outside the White House, Trump International Hotel and at Dulles International Airport Caption Thousands protest the president’s executive order outside the White House, Trump International Hotel and at Dulles International Airport Jan. 29, 2017 Protesters rally outside the White House to criticize President Trump’s immigration ban on citizens from seven majority-Muslim countries. Aaron P. Bernstein/Reuters Buy Photo Wait 1 second to continue. More reading: Trump promised disruption. That’s exactly what he’s delivering. Trump’s travel ban could make Rex Tillerson’s potential job harder, a former defense secretary says Image copyright AFP/Getty Image caption Thousands marched through London on 21 January to voice their opposition to President Trump Downing Street has rejected calls to cancel President Donald Trump's proposed state visit to the UK after a US clampdown on immigration. A source said a rejection would be a "populist gesture", adding that the invitation had been accepted and scrapping it would "undo everything". Labour leader Jeremy Corbyn said PM Theresa May would be failing the people if she failed to postpone the visit. A petition to stop it has gathered more than 900,000 signatures since Saturday. Only 100,000 signatures are needed for Parliament to consider debating a petition. The visit was announced during PM May's trip to the US - no date has been set but it is expected to take place later this year. However, on Friday Mr Trump signed an executive order halting the US refugee programme for 120 days, indefinitely banning all Syrian refugees and suspending the entry of all nationals from seven Muslim-majority countries. Moves to implement the measure have triggered anger and protest across the world. Buckingham Palace has declined to comment on the row. The Downing Street source told the BBC: "America is a huge important ally. We have to think long term." But Shadow Attorney-General Shami Chakrabarti said the government's position "sounds like appeasement". Mr Corbyn, who has urged followers to support the petition, tweeted on Sunday: "@Theresa_May would be failing the British people if she does not postpone the state visit & condemn Trump's actions in the clearest terms." "@realDonaldTrump should not be welcomed to Britain while he abuses our shared values with shameful #MuslimBan & attacks on refugees & women," he added. Liberal Democrat leader Tim Farron backed the call. He said: "Any visit by President Trump to Britain should be on hold until his disgraceful ban comes to an end. "Otherwise Theresa May would be placing the Queen in an impossible position of welcoming a man who is banning British citizens purely on grounds of their faith." Image copyright Getty Images Image caption Protesters have gathered at airports across the US to demonstrate against the executive order Alex Salmond, the SNP's foreign affairs spokesman, said he thought the state visit was "a very bad idea". Also appearing on Sky News' Sophy Ridge, he said: "You shouldn't be rushing into a headlong relationship with the President of the United States." Mr Salmond said reports Mr Trump was reluctant to meet Prince Charles during the visit were "an indication of the sort of enormous difficulties you get into when you hold somebody tight who is unpredictable, who has a range of views you find unacceptable." And Mayor of London Sadiq Khan said the visit should not happen while the executive order was in place. He told Sky News: "I am quite clear, this ban is cruel, this ban is shameful, while this ban is in place we should not be rolling out the red carpet for President Trump." Leeds solicitor Graham Guest, who started the petition, said he wanted it to "put the spotlight" on Mr Trump. He told the Press Association: "A state visit legitimises his presidency and he will use the photo opportunities and being seen with the Queen to get re-elected. "The wording in the petition is quite precise as I actually say that he should come here as the head of government to do government-to-government business. "At the end of the day he is still the president and we've just got to live with that. But there's no reason why he should get all the pomp and publicity of a state visit." 'British values first' Former shadow cabinet member Chuka Umunna also backed the calls to cancel the trip. "State visits happen at the instigation of governments and, of course, you have got a prime minister who you want to have a decent working relationship with a US president. "But they need to understand, just as they will put America first, we will put British values first." Ruth Davidson, leader of the Scottish Conservatives, said she hoped Mr Trump would reconsider his position on immigration "immediately". "State visits are designed for both the host, and the head of state who is being hosted, to celebrate and entrench the friendships and shared values between their respective countries," she said. "A state visit from the current president of the United States could not possibly occur in the best traditions of the enterprise while a cruel and divisive policy which discriminates against citizens of the host nation is in place." Paddy Ashdown, former leader of the Liberal Democrats, questioned the state visit on Twitter. He wrote: "Am I alone in finding it impossible to bear that in pursuit of her deeply wrong-headed policies our PM is now forcing THAT MAN on our Queen?" Conservative MP for Totnes, Sarah Wollaston, earlier tweeted that the US President should not be invited to address the Houses of Parliament, saying Westminster Hall "should be reserved for leaders who have made an outstanding positive difference in the world". Washington (CNN) Republican Sens. John McCain of Arizona and Lindsey Graham of South Carolina broke the GOP silence on Capitol Hill on Sunday to issue a scathing condemnation of President Donald Trump's ban on travel to the United States from seven Muslim-majority countries. "We fear this executive order will become a self-inflicted wound in the fight against terrorism," McCain and Graham said in a joint statement, adding that Trump's executive order "may do more to help terrorist recruitment than improve our security." Senate Foreign Relations Chairman Bob Corker said Sunday the administration should immediately make revisions to the executive order. "We all share a desire to protect the American people, but this executive order has been poorly implemented, especially with respect to green card holders," said Corker. "The administration should immediately make appropriate revisions, and it is my hope that following a thorough review and implementation of security enhancements that many of these programs will be improved and reinstated." It was the strongest criticism Trump has faced yet from the right, as congressional leaders largely deflect questions about the ban and aides say Trump is doing exactly what he pledged he'd do on the campaign trail. That could change this week when lawmakers return to Washington and the Senate considers several of Trump's Cabinet nominees' confirmation -- with Democrats determined to force the new administration to backtrack, protests continuing and confusion about the fate of green card holders, including some who have spent years in the United States. Trump responded, tweeting: "The joint statement of former presidential candidates John McCain & Lindsey Graham is wrong - they are sadly weak on immigration. The two... Senators should focus their energies on ISIS, illegal immigration and border security instead of always looking to start World War III." The joint statement of former presidential candidates John McCain & Lindsey Graham is wrong - they are sadly weak on immigration. The two... — Donald J. Trump (@realDonaldTrump) January 29, 2017...Senators should focus their energies on ISIS, illegal immigration and border security instead of always looking to start World War III. — Donald J. Trump (@realDonaldTrump) January 29, 2017 Trump also issued a statement defending the new order, saying: "We will continue to show compassion to those fleeing oppression, but we will do while protecting our own citizens and voters." "This is not a Muslim ban, as the media is falsely reporting. This is not about religion -- this is about terror and keeping our country safe," he said, adding that his first priority "will always be to protect and serve our country, but as President I will find ways to help all of those who are suffering." Senate Democratic leader Chuck Schumer of New York teared up at a Sunday news conference as he called the ban "mean-spirited and un-American." He also heaped pressure on majority Republicans in the House and Senate to play a role in blocking Trump -- noting McCain's denouncement. "We should have other Republicans speaking out against it and maybe we can pass something in the Congress," Schumer said. "We can only do it with Republican support to at least undo this or stay it." On Capitol Hill, many GOP offices were non-responsive in the 24 hours after the executive order was signed, though more are now making statements. The reason, according to GOP sources in both chambers, was two-fold: They were left out of the loop by the White House before the travel ban was announced, and they see political risk coming from both directions. "Support it and get hit, oppose it and get hit," one GOP source said. "There will be time for discussion about this. Right now we'll let the administration take the lead." Only a handful of lawmakers were looped in on the general content of the order, and even then, most on Capitol Hill were operating off a draft of the order that was circulating among reporters and agency sources, one aide said. Committees with jurisdiction reached out to the relevant agencies at various point throughout the week, from the Department of Homeland Security to the Justice Department and even the White House, and were met mostly with silence or an acknowledgment that their contacts were also in the dark, several aides said. One GOP aide noted that the House, in passing a bill to suspend the refugee program for participants in Iraq and Syria in with a veto-proof majority in 2015, had already laid down a similar marker on the issue. The order itself fell into line generally with that bill and was "in no way a Muslim ban." But even so, the confusion that appeared pervasive on the agency side extended to Capitol Hill. The status of green card holders, along those refugees already in transit, was something two GOP aides said they had been told was more clear-cut than originally perceived. Throughout the day Saturday, several senior GOP officials were still attempting to get firm answers on what precisely the exemption and waiver process would be for those programs. Still, a reality several pointed to, separately: Trump is doing what voters elected him to do. "It's not like this was a secret during the campaign," one Senate aide said. "He ran on it. He won." Reflecting the muted reaction, Sen. Todd Young, R-Indiana, said in a statement: "The federal government has no more important responsibility than protecting the American people, and refugees from any country should only be permitted to enter the United States if we are certain they do not represent a threat to our citizens." "I am eager to ensure that the administration's new policy allows Iraqis and Afghanis who faithfully supported our troops and who face threats to their safety -- and who do not represent a terrorist threat -- to come to the United States," Young said. And at least one member offered praise --House Permanent Select Committee on Intelligence Chairman Devin Nunes. "In light of attempts by jihadist groups to infiltrate fighters into refugee flows to the West, along with Europe's tragic experience coping with this problem, the Trump Administration's executive order on refugees is a common-sense security measure to prevent terror attacks on the homeland," he said in a statement. "While accommodations should be made for green card holders and those who've assisted the US armed forces, this is a useful temporary measure on seven nations of concern until we can verify who is entering the United States." GOP criticism Trump famously campaigned on a pledge to indefinitely ban Muslims from entering the United States, something his campaign aides later tried to finesse as a broader policy aimed at implementing "extreme vetting" for immigrants from certain countries. And the White House says its executive action doesn't specifically target Muslims -- instead focuses on countries that are terrorism hotbeds that also happen to be majority Muslim. But McCain and Graham said that, in effect, Trump has created the perception that he is banning Muslims from the United States. "This executive order sends a signal, intended or not, that America does not want Muslims coming into our country," the two said. McCain and Graham aren't the only Republican senators to criticize Trump so far. He said he's glad to see two federal judges temporarily block Trump's executive order, and said Congress should be involved in strengthening the nation's vetting of visa applicants. "We ought to be part of it. We've been working on this," Portman said. Sen. Ben Sasse, R-Nebraska, said in a statement that "while not technically a Muslim ban, this order is too broad." "There are two ways to lose our generational battle against jihadism by losing touch with reality," he said. "The first is to keep pretending that jihadi terrorism has no connection to Islam or to certain countries. That's been a disaster. And here's the second way to fail: If we send a signal to the Middle East that the U.S. sees all Muslims as jihadis, the terrorist recruiters win by telling kids that America is banning Muslims and that this is America versus one religion." Sen. Orrin Hatch, R-Utah, urged Trump's administration to tailor the executive order to be "as narrowly as possible." Hatch said as a Mormon, he is aware that many of his ancestors were refugees himself, as he called on Trump to reduce "unnecessary burdens on the vast majority of visa-seekers that present a promise -- not a threat -- to our nation." Sen. Jeff Flake, R-Arizona, like McCain and Graham, said Trump's executive order appears to target Muslims broadly. "President Trump and his administration are right to be concerned about national security, but it's unacceptable when even legal permanent residents are being detained or turned away at airports and ports of entry," he said in a statement. "Enhancing long term national security requires that we have a clear-eyed view of radical Islamic terrorism without ascribing radical Islamic terrorist views to all Muslims." Republican leaders silent On ABC's "This Week," Senate Majority Leader Mitch McConnell, R-Kentucky, said Trump should have "a lot of latitude" to secure the country by improving vetting of immigrants. He said he opposes "religious tests," but did not specify whether Trump's executive order is one. "The courts are going to determine whether this is too broad," McConnell said. A spokeswoman for House Speaker Paul Ryan, R-Wisconsin, was similarly vague -- telling The Washington Post : "This is not a religious test and it is not a ban on people of any religion." Democrats push back Democrats from liberal havens to conservative states have equally condemned the travel ban. Sen. Kamala Harris, a freshman Democrat from California, wrote to the Department of Homeland Security urging that Customs and Border Protection grant those detained at airports immediate access to lawyers. Customs agents, she wrote, should "be directed to grant individuals detained at ports-of-entry throughout the United States timely and unfettered access to legal counsel." And two red-state Democrats up for re-election in 2018 -- North Dakota Sen. Heidi Heitkamp and Montana Sen. Jon Tester -- both criticized it in statements Sunday. "This executive order is having harmful consequences on children and brave allies who are helping us fight terrorism," Tester said. "We must take strong steps to protect our nation from those who want to harm us, but we cannot sacrifice our religious freedom and our American values." | President Trump's travel ban on citizens of seven countries continued to cause chaos and protests at airports around the country and around the world Sunday, though an administration official insisted it was a "massive success," Reuters reports. The attorneys general of 15 states denounced the Trump order as unconstitutional, and in Canada-where the US Consulate in Toronto will be closed Monday because of a planned protest-officials announced that anybody stranded in the country because of the Trump order will be granted temporary residency. In other coverage: Republican Sens. Lindsey Graham and John McCain issued a statement Sunday warning that the ban could become a "self-inflicted wound in the fight against terrorism," CNN reports. Trump tweeted that the statement was "wrong." He accused the senators of being "sadly weak on immigration" and urged them to focus on the issue "instead of always looking to start World War III." After much confusion and conflicting statements about whether the order would apply to green card holders, the Department of Homeland Security said Sunday evening that legal residents would be presumed exempt from the ban, though there will still be "case-by-case determinations," the New York Times reports. Officials said 392 green card holders had already been issued waivers to travel, though it's not clear how many more are still stuck overseas. Politico looks at early immigrant rights groups' court victories against the ban, and warns that some of them are unlikely to last. The American Civil Liberties Union says it smashed fundraising records in the wake of the ban, raising $24 million in online donations over the weekend, USA Today reports. That's six times what the ACLU raises in a typical year, Executive Director Anthony Romero says. The Washington Post profiles Ann Donnelly, the rookie federal judge who issued the first order against the ban in an effort to save a Syrian refugee at immediate risk of deportation. Trump chose Finding Dory as his first White House screening, drawing criticism from the movie's voice stars, who found it ironic that he picked a movie about tolerance, the Telegraph reports. "Odd that Trump is watching Finding Dory today, a movie about reuniting with family when he's preventing it in real life," tweeted Albert Brooks, who voiced Nemo's father, Marlin. In Britain, a petition to cancel a planned Trump visit received almost a million signatures, though a government source told the BBC that it was considered a "populist gesture" and would not affect the state visit. CNN looks at recent terrorist attacks in the US and notes that the number of fatal attacks carried out by refugees since the 1980 Refugee Act stands at zero. The Guardian reports that one of the biggest airport protests in the US was at LAX, where thousands gathered Sunday night chanting "let them in." | multi_news |
Prologue: A large park. The camera pans along some flowery foliage until it reaches a pair of green scaly legs firmly planted to the ground and a pair of human legs dangling and kicking from someone pinned high against a tree. The monster makes gurgling noises and low-pitched roars. Cut to Buffy being held against the tree by her neck. The monster's face has tentacles coming off of the back of his head as well as the front in place of lips. His teeth are placed vertically between the two center tentacles. His scaly green skin glistens with slime.
Buffy: (yells out desperately) Nnnrrf! Nnnrrf! Near a picnic table Xander is just coming to, apparently having been knocked around by the monster as well. At the table Willow frantically searches through their bag of demon-killing implements.
Willow: Oh, God! Demon! Demon! What kills a demon?! Buffy struggles with the monster's hand at her neck, but can't get it to budge and has a hard time breathing.
Buffy: Nnnrrf! Nnnrrf!
Willow: (still searching) Oh, Nerf! Not Nerf. Knife! She finds a knife and runs with it to Buffy's aid. She tosses the knife to Buffy, who blindly grabs it from behind out of mid-air and stabs the monster in the chest. The monster immediately falls over dead, taking Buffy down with it. Willow and Xander help her up.
Buffy: Okay. That was too close for comfort. Not that slaying is ever comfy, but... you know what I mean? (takes a deep breath) If you guys hadn't been here to help...
Willow: But, we were, and we did, and, and we're all fine. (looks down at the monster) Isn't he gonna go poof?
Buffy: Mm, I guess these guys don't. We'll have to bury him or something. Uhhf... They walk over to the picnic table.
Buffy: Makes you appreciate vamps, though. No fuss, no muss. Buffy sits cross-legged on one end of the table, Xander sits on the other and Willow sits on the bench below and between them and grabs a drink.
Xander: So how come Faith was a no-show? I thought mucus-y demons were her favorites. (munches on a snack)
Buffy: Couldn't reach her... again. She hasn't been hanging out much.
(reaches for a snack)
Xander: I detect worry.
Buffy: A little bit. Slaying's a rough gig. Too much alone time isn't healthy. Stuff gets pent up. (munches the snack)
Willow: We should try to do more socializing with her.
Xander: Well, burial detail aside, does this cap us off for the day?
Buffy: You got plans?
Xander: I cannot stress enough how much I *don't* have plans.
Buffy: No luck reaching Cordelia?
Xander: I've left a few messages. Sixty... Seventy... But you know what really bugs me? (to Willow) Okay, we kissed. It was a mistake. But I
know that was positively the last time we were *ever* gonna kiss.
Willow: Darn tootin'!
Xander: And they burst in, rescuing us, without even knocking? I mean, this is really *all* their fault.
Buffy: Your logic does *not* resemble our Earth logic.
Xander: Mine is much more advanced.
Willow: At least tomorrow's Monday, another school day.
Buffy: Well, that's good. You know, focus on school. That's the strong Willow way to heal.
Willow: Actually, I was more thinking Oz will be there, and I can beg for forgiveness.
Buffy: That works, too.
Willow: I-I wanna be strong Willow. But then I think I may never get to be close to Oz again, and it's like all the air just goes out of the room.
Buffy: I know the feeling.
Xander: Right. I mean, you went through it with Angel, and you're still standing. So tell us, Wise One, how do you deal?
Buffy: I have you guys.
Cut to Cordelia's bedroom. She's sitting on her bed with the lights very low. She has a picture of herself, Xander, Willow and Buffy all with their arms around each other, and is cutting each person off with a straight vertical cut. She lets the pieces fall into a bowl on a breakfast tray. In the background her answering machine plays back her messages.
Machine: Hey, it's Xander. If you get this, call me. The last part of the picture left in her hand is of Xander, and she cuts diagonally right through his face.
Machine: Hi! Xander. I, uh... Well, I'm in if you feel like calling. Bye. Cordelia's eyes and cheeks are heavy with tears. She sighs, takes a match and strikes it. She lights Xander's part of the picture.
Machine: Hi, Cordelia. Um... If you get the chance, if we could talk, I'm here. She drops the lit piece into the bowl and holds the match to the others. They suddenly all burst into flame. The light of the flame shows just how tired, slagged and haggard Cordelia has become.
Machine: Hey again! It's me. I'm here. Again. She watches as the flames consume the image of Xander. Opening credits roll. Buffy's theme plays. ~~~~~~~~~~ Part 1 ~~~~~~~~~~ The halls at Sunnydale High School. Willow waits around the corner from Oz's locker, peeking around every few seconds to see if he's arrived, looking very worried. Buffy comes up behind her.
Buffy: How's it goin'?
Willow: Oz hasn't been to his locker. There may be books in there that he needs, but still, he doesn't come. (looks at the locker)
Buffy: Has Xander seen Cordelia?
Willow: I don't think so. But she is coming in today. Amy saw her last night at the mall. (looks at the locker)
Buffy: How was she?
Willow: I don't know. Amy said she looked pretty... scary.
Cut to the student parking lot. Cordelia has the top down on her convertible. She steps out looking very hot in a brown leather skirt and jacket with matching top, and alligator high heel d'Orsay pumps with matching bag from Prada. She confidently walks into school.
Cut to the breezeway leading to the quad. She walks through, not looking quite as confident anymore since the other students are just passing her by. She stops when she sees Harmony coming her way with some of her friends. When Harmony sees Cordelia, she stops, too, for an instant, but then approaches her, all smiles.
Harmony: Cordelia! You look amazing. Cordelia, confidence restored, exchanges a non-touching hug and kisses on both cheeks.
Harmony: Oh. You have to meet Anya. (pulls her to the front) She just moved here, and her dad just bought -- what was it -- oh. A utility. Or something.
Anya: (to Cordelia) Nice bag. Prada?
Cordelia: Good call! Most people around here can't tell Prada from Payless.
Harmony: God, Cordy, when I heard about... Well, I mean, I couldn't believe it. But it was smart. You know, the injury thing? You take a week off, let everybody forget about the temporary insanity that was Xander Harris.
Cordelia: (raises her eyebrows) Xander who?
Harmony: Oh! They all exchange a little fake nervous laughter.
Cordette: You know what you have to do. Start dating. Get back on the horse.
Cordelia: Oh, absolutely! I am ready to ride!
Harmony: Then I have just the stallion. He's *so* you. She leads her over to the outside stairs where Jonathon is sitting, nursing a soft drink. He is taken aback by the sudden attention, and looks around to see if they didn't really mean someone else, but there is no one else. Cordelia realizes she's been had.
Harmony: (giggles) I'm pretty sure he won't cheat on you. At least not for a while. Plus, he's got a kill moped. She laughs, and she and her group walk off. Jonathon gives her a sympathetic look, knowing what it's like, and goes back to nursing his soda.
Cut to the halls. Oz finally shows up at his locker. As he works his combination, Willow comes around the corner and pretends it's a chance meeting.
Willow: Oz! Wow. He stops opening his locker and slowly turns to face her.
Willow: Look at us, running into each other, as two people who go to the same school are so likely to do now and then.
Oz: Hey. (starts to leave)
Willow: (stops him) Oz, wait. Please? He stops and reluctantly gives her his attention.
Willow: What I did... When I think that I hurt you...
Oz: Yeah. You said all this stuff already.
Willow: Right, but... I wanna make it up to you. I mean, if you let me, I wanna try.
Oz: Just... You can leave me alone. I need to figure things out.
Willow: But maybe if we talk about it, we could...
Oz: Look... I'm sorry this is hard for you. But I told you what I need. So I can't help feeling like the reason you want to talk is so you can feel better about yourself. That's not my problem. Willow is left feeling completely helpless. Oz goes on his way leaving her standing there.
Cut to the stairs by the student lounge. Cordelia walks down and turns down the hall. She sees Xander come out of the cafeteria at the far end. When he spots her he stops. Cordelia looks for a way to turn this to her advantage, and pulls an old boyfriend out of the crowd.
Cordelia: Hey, John Lee. Do I have something caught in my teeth? She smiles to expose her teeth and angles her head up so he can see. She shifts her head back and forth, and John Lee dutifully follows her movements. From a distance it looks to Xander like they are kissing. When he's seen enough, he goes back into the cafeteria. When Cordelia sees that he's gone, she steps back from John Lee.
Cordelia: So... What's new? God, it's been, like, a gazillion years!
John Lee: (smiles) Look, the guys are kinda down on me lately. Coach has cut me back to second string. If anyone saw me hanging with Xander Harris' castoff on top of that... Death, you know, but... maybe... (makes suggestive eyes) If you wanna go someplace private...
Cordelia: (surprised to find the tables turned) What?
John Lee: Think about it. (leaves) Cordelia can't believe what a social leper she's suddenly become. She starts back down the hall, and is startled when Anya bumps into her.
Anya: (smiles) Hey.
Cordelia: Go ahead. Dazzle me with your oh-so-brilliant insults. Just join the club.
Anya: Hardly. Uh, actually, I've been looking for you. Ever since we met this morning, I was, like, thank God there's one other person in this town who actually reads W.
Cordelia: But Harmony...
Anya: Oh, she follows me around. If that girl had an original thought, her head would explode.
Cordelia: (notices Anya's pendant) Is that Gucci?
Anya: Um... no. It's an actual old thing, sort of a, um... good luck charm my dad gave me.
Cordelia: Too bad I didn't have one of those pre-Xander. They start down the hall.
Anya: Can I just say... Men.
Cordelia: Second it.
Anya: Apart from being without class, the guy's obviously blind. Deserves whatever he gets.
Cordelia: I'm not even thinking about him. I am past it. I am living my life.
Anya: Still, I mean... Don't you kinda wish...
Cordelia: I don't wish. I act. Starting now, Xander Harris is gonna get a bellyful of just how over him I am.
Cut to the Bronze that night. "Tired of Being Alone", by The Spies, plays in the background. Cordelia is at the bar, dressed sexily in red, pretending to have great conversation with a guy. Behind her sitting on the couch beneath the stairs, Willow and Buffy look glum, while Xander pretends to be having fun, forcing himself to laugh. He looks back at Cordelia, who seems to be enjoying her conversation. Xander looks back at Willow and Buffy and forces out gales of laughter. Willow and Buffy exchange a look, then Buffy gives Xander a creepy look.
Xander: Excuse me. I need to be both giving *and* receiving of mirth. Is it too much to ask for a little backup?
Buffy: (puts her hand on his knee) I'm here for you, Xand. I'm Support- O-Gal. (takes her hand back) I just... feel a little weird about this us-against-Cordelia thing. She's had a rough time. At the bar Cordelia is still enjoying herself.
Willow: It's true. Cordelia *belongs* to the justified camp. She *should* make us pay. And pay and pay and pay... In fact, there's just not enough pay for what we...
Xander: (interrupts) Look, you want to do guilt-a-palooza, fine, but I'm done with that. Starting this minute, I'm gonna grab ahold of that crazy little thing called life and let it do its magical little heal-y thing. What's done is done. Let's be in the moment. Behold the beauty that is now. (bounces his eyebrows) Who's with me?
Buffy: He's actually making sense. We're young and free in America. How dare we be spun by love or the lack of same?
Willow: Absolutely. I-it's self-indulgent. I-I'm in. I'm on the joy train. (smiles) They all put on bright smiles and radiate them into the Bronze. Slowly their luster fades, and they all end up looking glum again.
Buffy: That didn't work. Who wants chocolate? Willow and Xander both raise their hands.
Buffy: I'm up. She gets up and heads for the cappuccino bar.
Xander: Look at her. (indicates Cordelia) Tears of a clown, baby. Or is it... grins of a sad person? (reaches over to Willow in his old familiar way) Or maybe it's...
Willow: Xander, your hand.
Xander: (jerks back his hand) Oops! Sorry. But why 'oops'? I mean, we always touch digits. It's a friend thing. Comfort. Like chocolate.
Willow: (shrugs) Maybe it used to be, but since we... It's different. (Xander looks away) I-I'm sorry. But if I wanna make things right with Oz, my hands, (Xander looks back) my -- all my stuff -- has to be for him only. Xander understands, but he sure isn't happy about it.
Cut to the cappuccino bar. While waiting in line, Buffy notices Cordelia talk briefly with Anya and say goodbye. As she goes a boy bumps into her, jostling her wound. She puts her hand over it as she walks out. Buffy decides to follow. Cut outside. Buffy catches up with her.
Buffy: Hey, Cordelia, wait a second.
Cordelia: (stops and faces her) Did Xander send you to beg for him? Because if he did...
Buffy: No. I'm a free agent, I promise. I just wanted to see how you are.
Cordelia: Never been better. (starts to go)
Buffy: (follows) Cordelia, I know what it's like to be hurt by someone. (Cordelia faces her) Hurt so much that you don't think you're gonna make it. But I told my friends how I felt, and you know what? It got a little better. Suddenly a vampire jumps down behind Buffy and swings at her head, but she middle blocks it and punches him in the face, which sends him to the pavement on his side. Buffy punches him in the face while he's down, reaches for his shirt and yanks him back up to his feet. She spins him around and lets go of his shirt. He staggers backward a couple of steps, but keeps his footing. He advances and does two roundhouse kicks, which Buffy low blocks. He tries a wide punch to Buffy's head, but she ducks it and rises back up to deliver a roundhouse kick to his side. This sends him stumbling backward right at Cordelia.
Buffy: Cordelia, look... Cordelia has no time to react, and gets knocked into a pile of garbage.
Buffy: ...out. The vampire gets back up to his feet and comes at Buffy, jumping into a half spinning crescent kick, which Buffy easily ducks. She grabs him when he comes at her again and knees him in the stomach, then flips him over onto his back. She pulls out her stake and jams it home. The vampire bursts into ashes. Buffy turns her attention back to Cordelia, who flicks a few pieces of trash from her dress and slowly climbs out of the garbage heap. Buffy looks at her apologetically. Behind her she hears the laughter of a group of girls, so she quickly tosses aside her stake. Harmony and some of her friends walk by, look Cordelia over and keep laughing as they go. After they've gone, Cordelia vents on Buffy.
Cordelia: You know what I've been asking myself a *lot* this last week? Why me? Why do *I* get impaled? Why do *I* get bitten by snakes? Why do *I* fall for incredible losers? And you know, I think I've finally figured it out, what my problem is? It's...
Cut to the quad at school the next day. Cordelia and Anya walk together.
Cordelia: ...Buffy Summers. That's when all my troubles started. (winces in pain and holds her side) When she moved here.
Anya: Are you okay?
Cordelia: Oh, I just pulled some stitches last night. Know why? (looks in Buffy's direction) Surprise. It was Buffy's fault. Anya follows Cordelia's gaze and sees Buffy and company sitting on a bench. Harmony interrupts Cordelia and Anya.
Harmony: Oh, hey, it's Garbage Girl. Loved the look last night, Cor. Dumpster chic for the dumped. She and her troop rudely walk right between Cordelia and Anya, giggling and smiling. Cordelia looks down in embarrassment. Anya takes her pendant off.
Anya: Here. I think you need this more than I do right now. Cordelia lifts her hair away from her neck and lets Anya put it on.
Cordelia: Yeah, I can use some luck. (eyes Buffy) And a stick with pointy, sharp bits. If that Buffy wasn't... I swear. She's a pain.
Anya: But Xander, he's an utter loser. Don't you wish...
Cordelia: I never would've looked twice at Xander if Buffy hadn't made him marginally cooler by hanging with him.
Anya: Really? (looks over at Buffy)
Cordelia: Yeah, I swear! I wish Buffy Summers had never come to Sunnydale. Anya turns back to Cordelia, who gasps to see that her face has suddenly become very wrinkled and raw-looking, the embodiment of Anyanka, Patron Saint of all women scorned.
Anyanka: Done. The picture fades to white. ~~~~~~~~~~ Part 2 ~~~~~~~~~~ The picture fades from white back to the quad at Sunnydale High. Cordelia looks around her. Anya is gone. Buffy, Willow and Xander are not sitting at the bench anymore. There are far fewer students in general. The place is, in fact, rather a mess. Garbage and palm leaves are strewn about the quad.
Cordelia: Anya? Suddenly she notices that she no longer has her injury. Anya's pendant, however, is still around her neck. Slowly she begins to figure it out.
Cordelia: 'I wish Buffy Summers had never come to Sunnydale.' (smiles) She was, like... a good fairy. A scary, veiny... good fairy. (smiles widely) She laughs as she heads into the halls. Cut into the halls. Just like outside, there are far fewer students inside. Everyone is dressed in dark and drab clothing. Cordelia is the standout in her bright turquoise dress. She sees Harmony and her friends at her locker, and hesitates. Harmony closes her locker and sees her.
Harmony: Where have you been?! She approaches Cordelia, and her friends follow. Cordelia gives them a careful smile.
Harmony: Ted Chervin just totally went for third with Ginger in front of everybody.
Cordette: (to Cordelia) Love the dress. It's so daring. Harmony nods in agreement. John Lee walks up to them.
John Lee: Cordelia.
Cordelia: Yeah?
John Lee: (pulls her aside) Look, every guy on campus has probably asked, but if you're not going to the Winter Brunch with anyone, I'd be honored, and we'd have fun.
Cordelia: (considers her response) I'll get back to you.
John Lee: Really?
Cordelia: Yeah. (smiles)
John Lee: Great! He heads down the hall, a happy man. Harmony steps over to her.
Harmony: Cordy, you reign!
Cordelia: I do? I mean, I do. So what's with the Winter Brunch thing?
Cut to class. It is less than half full, but even so most of the students sit toward the back. The bell rings. The teacher hurriedly gathers his things.
Teacher: Alright. Now, don't forget, tomorrow we have our, uh, monthly memorial, so, uh, there's no class. He rushes out of the room. The students also make a point of getting out of there quickly.
Cordelia: What's the rush?
Harmony: Oh, you know, my mom hates it when I'm late.
Cordelia: Since when? Aren't we going out tonight? (gets up)
Cordette: Curfew starts in an hour.
Cordelia: Curfew? Come on, I'm in a really good mood! Let's go to the Bronze! Harmony and her friends all stop and give Cordelia a disbelieving look.
Harmony: Is that a joke?
Cordelia: Oh! The Bronze isn't cool in this reality. I've gotta make these little adjustments. (smiles) Harmony and Cordette exchange a look.
Harmony: Cordy, what's with you? (the others leave) I mean, you wear this come-bite-me outfit, you make jokes about the Bronze, and you're acting a little schizo.
Cordelia: You're right. I just... Well, I bumped my head yesterday, and I keep forgetting stuff. Not that I care, but Xander Harris, he's miserable, right? And that Willow freak he hangs with, not even a blip on the radar screen, right? (smiles)
Harmony: (confused) Well, yeah. They're dead. Cordelia's smile fades, not at all sure how she feels about that. Harmony rolls her eyes and leaves the room.
Cut to what Cordelia thinks is the student parking lot. The lot is completely empty and full of fallen leaves.
Cordelia: Okay. Not funny. (stops a passing janitor) Hey! You! Where did you put my car?
Janitor: Pardon?
Cordelia: My auto! El convertablo?
Janitor: You students aren't allowed to drive, and you know it.
Cordelia: What?!
Janitor: Go on now, Miss. You better get in before the sun sets. The janitor hurries off. Cordelia is now very confused. She starts on her way home.
Cut to a street in town. The Sun Cinema is closed. The last shop pulls a metal gate across its storefront. The street is dirty. A smashed car just sits in the middle of it. In the distance Cordelia can hear sirens and screams as she walks along. Suddenly Xander appears in front of her, wearing only a white T-Shirt and black leather jacket and pants. She startles and stops short.
Xander: Well, whadaya know? Cordelia Chase.
Cordelia: What is this? Some kind of sick joke? Harmony told me you were dead.
Xander: (plays her game) Now, why would she say something like that? Let's think.
Cordelia: Listen to me. We have to find Buffy. She'll figure out a way to save us. She was supposed to be here, and as much as it kills me to admit it... things were better when she was around.
Xander: Buffy? The Slayer?
Cordelia: No! Buffy the dog-faced girl! Duh! Who do you think I'm talking about?
Willow: Bored now. She slowly walks up to them. She is also dressed in black leather. The bodice of her outfit is trimmed in red lace.
Willow: This is the part that's less fun. When there isn't any screaming.
Cordelia: What's up with you two and the leather?
Willow: (to Xander) Play now?
Xander: It's not that I don't appreciate your appetite, Will, but I thought we agreed it was my turn. Willow whines and brushes her hand against Xander's chest.
Cordelia: No. No! No way! I wish us into Bizarro Land, and you guys are still together?! I cannot win!
Xander: Probably not. (vamps out) But I'll give you a head start.
Cordelia: (gasps) No! She drops her bag and begins to run.
Willow: I love this part. They kiss passionately with lots of tongue. Then Xander turns his attention to the chase.
Xander: You love all the parts. Willow follows at a walk as Xander runs after Cordelia. He jumps up, runs over the smashed car and jumps down behind her, grabs her by the neck and throws her down to the street. She rolls to a stop, unconscious.
Willow: No fun. She didn't even hardly fight. Suddenly a van comes screeching around the corner.
Xander: Aw, swell. It's the White Hats. The van screeches to a stop next to Cordelia, and Giles jumps out with a large cross in hand to ward them off. They have to back away. At the driver's seat Oz has the crossbow trained on them. Larry and Nancy jump out of the sliding door, he with a stake held ready, she with another cross.
Giles: I've got them! Get the girl! Larry and Nancy pick Cordelia up and carry her into the van. Xander and Willow growl angrily as they watch their prize being stolen from them. When Larry and Nancy have Cordelia safely inside, Giles hops back in, they slam the doors shut and take off.
Cut to the library. Cordelia is laid out on the large center table.
Oz: How's she doing?
Giles: Her pulse is strong.
Nancy: What was she doing wearing that? Everyone knows that vampires are attracted to bright colors.
Larry: That's Cordelia. It's better to look good than to feel alive.
Giles: Uh, go and, uh, watch the perimeters in case they follow.
Cut to the Bronze. "Dedicated to Pain", by Plastic, blares loudly as Xander and Willow approach the club. A couple of the vampires standing outside feast on fresh victims caught out after dark. The two of them go into the club and check out the happenings inside. There are several cages containing terrified humans suspended a few feet from the floor. Willow reaches in to one and strokes his cheek. They head toward the back of the club, past the pool tables where a vampire has a wayward biker tied to all four corners. Xander runs his hand across the man's chest.
Xander: (to the vampire) Slap my hand, dead soul man. They shake hands, and Xander and Willow continue into the back. A guard vampire holds the curtain aside for them to enter. When he sees them come in, the Master rises from his throne. His two favored vampires come to stand before him.
Master: Ahh. Xander... Willow... Hungry? He grabs a girl by the hair and lifts her by it. The girl remains silent with fear, but keeps her eyes fixed on the Master.
Master: (disgustedly) I've lost my appetite for this one. She keeps looking at me. I'm trying to eat, and she *looks* at me. He notices Willow's desire for a kill, and turns the girl's head toward her.
Master: Go on! Willow smiles up at Xander, who gives her a look of approval. She turns back to the Master and vamps out. The Master shoves the girl over to her. She catches her in her arms with her head laid back and bites her hard. Xander, as always, is impressed with her zest for a fresh drink.
Master: I remember that lust for the kill. (sits back down on his throne) Now... What news on the Rialto?
Xander: Had a prime kill. An old crush, actually, till that wannaslay librarian showed up.
Master: He'll be dealt with soon enough. Willow comes back to Xander's side, licking her fingers.
Xander: Weird thing: girl kept talking about Buffy. 'Gotta get Buffy here.' Isn't that what they called the Slayer?
Willow: (strokes his chest) Hmm. Buffy. Ooo. Scary.
Xander: Someone has to talk to her people. That name is striking fear in nobody's hearts.
Master: (stands up) She talked of summoning the Slayer here, now, at this time, and you didn't kill her?
Willow: Well, they had crosses.
Master: The plant begins operation in less than twenty-four hours. (steps up to them) You will find this girl. (strokes their cheeks) You will kill her before she contacts the Slayer. Or I'll see you two kissing daylight.
Cut to the library. Cordelia groans as she regains consciousness. Giles comes rushing down to her from the stacks and tries to keep her from getting up too fast.
Giles: Hey! Hey...
Cordelia: (frantic) Giles! It's all my fault! I wasn't... I made this *stupid* wish...
Giles: Come on. Please lie...
Cordelia: No! You have to get Buffy. Buffy changes it. (Giles lets go of her) It wasn't like this. It was better. I mean, the clothes alone... (Giles takes off his glasses) But people were happy. Mostly. And... Wait. (slides off of the table) Why are you here and she's not? I mean, y-you were her Watcher. Giles is amazed by what he's hearing.
Giles: H-how do you know I was a Watcher? I've never... They hear a series of thumps and taps outside.
Cordelia: What? Giles looks around carefully and puts his glasses back on.
Cordelia: What?
Giles: I thought I heard something. He goes into the cage and grabs a large cross and a stake from the weapons cabinet.
Giles: Now, I want you to start again and explain everything very carefully. Before he can come out, he finds the cage door slammed shut on him.
Willow: You're in a big cage. She taunts him with the key. Xander has Cordelia pinned against him with his hand over her mouth. Willow looks over at him.
Xander: Not too bright, Book Guy. Willow turns back to Giles, who slams the cross against the cage, forcing Willow back. She growls angrily. Xander forces Cordelia closer.
Xander: So you're a Watcher, huh? (smiles widely) Watch this. He lets go of Cordelia's mouth and sinks his teeth into her neck. Giles rattles the cage hard in protest, helpless to do anything. Willow smiles at him, then turns around and bites Cordelia also on the other side of her neck. Together they suck her dry. Xander reaches his arm around Willow's head and caresses her hair. Giles rages in anger as he is forced to watch. In another moment Cordelia is dead, and Xander pushes her lifeless body aside, letting it fall to the floor. Xander starts out of the library. Willow gives Giles a smile and tosses the key at him as she also leaves. Giles pants heavily as he looks down at the body of the latest victim of these two vampires.
[SCENE_BREAK]
~~~~~~~~~~ Part 3 ~~~~~~~~~~ The library. Giles swings at the cage door with a double-bladed battle- ax. It soon gives way and opens, and he rushes out to check on Cordelia. He feels for a pulse, but he's too late. Larry and Oz come running in through the stacks.
Larry: They hit us right outside.
Giles: Nancy?
Oz: She's dead. Giles takes the news as well as can be expected.
Giles: Um... Would you mind... Could you take her to the incinerator? I have some business to... Larry and Oz set themselves to their grim task. Oz goes around to get her legs, Larry grabs her by the shoulders. Just as they are about to go, Giles notices the pendant around her neck.
Giles: Wait a moment. He takes it off, and the boys carry her body away.
Cut to the Bronze. The Master takes a fresh hot demitasse of blood espresso from his machine and sips it. He blows on it and takes another sip. Behind him Xander and Willow report back from their mission. The Master turns to face them.
Xander: The deed is done.
Master: You killed the girl that sought the Slayer?
Xander: It was too easy.
Willow: I felt cheap.
Master: Excellent. The opening will commence as scheduled. (takes another sip)
Willow: (approaches) So, you're pleased?
Master: Ecstatic.
Willow: Then... can I play with the puppy?
Master: Ooo. (smiles) Be my guest. Willow smiles as the Master hands her the keys.
Cut to Giles' apartment. He's on the phone with Buffy's Watcher.
Giles: Yes, I understand, but it's imperative that I see her. Here. (listens) Well... when will you? (listens) Yeah, well, you are her Watcher. I'd expect her to at least check in to... (listens) Yes, I'm aware that there's a great deal of demonic activity in Cleveland. (listens) It... Well, it happens, you know, that, that Sunnydale is on a Hellmouth. (listens) It, it is so! (listens) Well... Just... Just give her the message, if you ever see her again. (hangs up)
Cut to an external view of Sunnydale by day. The camera pans over the red Spanish roofs typical of most of town. In the distance there is a low haze over the ocean.
Cut to the Bronze. Cut inside. Willow approaches what can only be described as a jail cell in the basement.
Willow: Bored now. She walks over to the wall of whips, chains and other instruments of torture.
Willow: Daytime's the worst. (runs her hand over the leather) Cooped up for hours. Can't hunt. She takes a pair of iron shears and clinks it along the bars of the cell.
Willow: But the Master said I could play. Inside the figure begins to stir.
Willow: Isn't that fun, Puppy? She unlocks the cell door and swings it in.
Willow: Aw... Puppy's being all quiet. Come on. Don't be a spoilsport. The man groans as she straddles him. She grabs him by the hair and jerks his head up. It's Angel, and he moans from the rough treatment. He seems constantly short of breath.
Willow: Guess what today is? She runs the tip of the shears along his chin and down his throat.
Willow: Today the plant opens. It's a big party. She licks him from the base of his ear to his forehead and runs her sharp fingernails along his neck.
Willow: You remember I told you about the plant? All those people you tried to save? It's gonna be quick for them. Not for you, though. It's gonna be slow for you. She flips him over onto his back and straddles his stomach. He lets out a painful moan.
Willow: That's right, Puppy... Willow's gonna make you bark. (smiles) He cries out when she rips open his shirt to reveal several very deep and bloody wounds on his chest. When she touches them he flinches hard.
Willow: Oh... Maybe I went too hard on you last time. Behind her Xander strikes a wooden kitchen match with his thumbnail and tosses it onto Angel's chest. Angel cries out in pain.
Xander: Too hard? No such thing.
Willow: Watch it with those things. You almost got my hair.
Xander: Sorry. Got carried away. He tosses her the large box of matches.
Willow: Don't you want to?
Xander: No, thanks, baby. I just wanna watch you go. Willow smiles and turns her full attention on Angel. She lights another match, and the screen cuts to black. Angel screams in agony.
Cut to the library. Giles is in his office while Oz tunes the crossbow and Larry carves stakes in the main area.
Giles: Here it is! I've found it. (comes out of his office) Look. He sets down a book which is opened to a page with a sketch of Cordelia's pendant.
Giles: It's what, um, Cordelia was wearing. It's the, the, uh, symbol of, of Anyanka.
Oz: I don't think I know her.
Giles: Well, no. Um, Anyanka is a, sort of a Patron Saint of scorned women. (sits on the table)
Larry: What does she do?
Giles: Uh, sh-she grants wishes.
Oz: So Cordelia wished for something? Well, if it was a long, healthy life, she should get her money back.
Giles: She said something about everything being different, that the... the world wasn't supposed to be like this. It was, um, better. Before.
Larry: Okay. The entire world sucks because some dead ditz made a wish? (gets looks from Giles and Oz) I just, I just want it clear.
Giles: She said the, uh... the Slayer was supposed to be here, was, um, meant to have been here already.
Oz: Certainly would've helped.
Giles: Yes. I tried calling her, but, um... (stands up) Look, I'm, I'm, I'm gonna have to... research this Anyanka thing further. Um, I have some more... volumes at, at home. You two, two get some sleep. (goes)
Oz: Watch your back.
Cut to the street. Giles drives along in his ancient Citroen. As he drives by a park he sees a bunch of people being herded into a stepvan. He stops his car, grabs his large cross and rushes over to help. He holds the cross up to the two vampires, who are forced to back away, and yells to the people in the van.
Giles: Run! When the people have all run away, he turns to run back to his car, but a third vampire slams the rear van door into his face, knocking him flat on his back. They try to grab him to load up, but the one at his feet suddenly finds himself flying through the air and landing hard on his back. The other two attack, but meet with similar fates. The first one runs at his attacker again, but gets staked. The attacker grabs another and stakes him. The others flee. Giles looks up at the person standing at his feet.
Giles: Buffy Summers?
Buffy: That's right. Wanna tell me what I'm doing here? ~~~~~~~~~~ Part 4 ~~~~~~~~~~ Giles' apartment. Buffy looks around, bored out of her mind. Giles is on the stairs looking through a book, and finally finds something.
Giles: Ah! Ah! Ah! Yes! (glances at Buffy and stands up) Here. (reads) 'In order to defeat Anyanka, one must destroy her powercenter. (walks down the steps) This should reverse all the wishes she's granted, rendering her mortal and powerless again.' You see? Without her powercenter, she'd j-just be a-a-an ordinary woman again, and all this would be, um... well, different. (gets no reaction) Well, I'd say that my, my Watcher muscles (closes the book) haven't completely atrophied after all. (takes off his glasses)
Buffy: (unimpresssed) Great. What's her powercenter?
Giles: Um, well, um, um... (glances at the book again) It doesn't say.
Buffy: Why don't I just put a stake through her heart? (goes to his kitchen bar)
Giles: She's not a vampire.
Buffy: Mm, well, you'd be surprised how many things that'll kill.
(sniffs a liqueur bottle)
Giles: I don't want to kill her, Miss Summers. I want to reverse whatever effect she's had on this, this... world.
She puts the bottle back down and turns to face him.
Buffy: You're taking an awful lot on faith here, Jeeves.
Giles: Giles.
Buffy: (shrugs) Kill the bad fairy... destroy the bad fairy's powercenter, whatever, and all the troubles go away?
Giles: Yeah, well, I'm sure it's not that simple, but...
Buffy: (interrupts) World is what it is. We fight. We die. Wishing doesn't change that.
Giles: I have to believe in a better world.
Buffy: Go ahead. I have to live in this one. She strolls over to his chess table, lifts her right leg up to set her boot on its edge and spits into her hand to literally give it a spit shine.
Giles: Cordelia said she knew that I was meant to be your Watcher. She said she knew you.
Buffy: (works her spit into the leather) She's probably just a big fan.
Giles: The Master sent his most vicious disciples to kill her. Now, she, she must have posed some threat to him. (puts his glasses back on)
Buffy: (suddenly attentive) The Master?
Giles: Um, supreme vampire around these parts. He, he lives on the outskirts of town in an old club.
Buffy: You know where he lives, and no one's ever tried to take him out?
Giles: People have tried.
Buffy: Well, point the way. I might as well do some good while I'm in this town. (goes to get her weapons)
Giles: You can't just walk in there and...
Buffy: Look, you wanna stay here and play make-believe, fine. (puts her crossbow strap over her shoulder) I'm not gonna be any help to you anyway. There's only one thing I'm good at.
Giles: At least let's muster some kind of force.
Buffy: I don't play well with others. Now, I'm gonna ask you this once, and then I'm gonna get testy. Giles gives in and tosses his book onto his desk.
Buffy: Where's this club?
Cut to the Bronze. Buffy whips aside the curtains and comes out of the Master's sitting area. She strolls through the club and sees the hanging cages, the ropes dangling from the pool tables, and everything for the most part put away as though the place were just closed for the day. Eveything, that is, except for the dead boy in one of the cages.
Cut to the stairs to the basement. Buffy comes down quickly and finds the cell where Angel is chained to the wall. He is shivering hard. She looks around a bit as she walks up to the bars. He looks up at her, and a look of recognition appears on his face. She in turn just gives Angel a blank look, turns and starts to walk away.
Angel: Buffy. She stops in her tracks.
Angel: Buffy Summers. She turns to face Angel and gives him an inquiring look. Angel gets another look at her, and now he's sure.
Angel: (weakly) It's you. I mean... you don't remember. How could you?
Buffy: How did you know my name?
Angel: I waited. I waited here for you. But you never... I was supposed to help you.
Buffy: (huffs) You were gonna help me.
Angel: (weakly) The Master rose. He let me live... to punish me. I kept hoping maybe you'd come. My destiny.
Buffy: (huffs) Is this a get-in-my-pants thing? You guys in Sunnydale talk like I'm the Second Coming.
Angel: I'm sorry. I just meant...
Buffy: (interrupts) Look, I don't have time for stories. Where's the Master?
Angel: They're at his factory. It starts tonight.
Buffy: Factory?
Angel: (tries to move) I (grunts) I can take you there. Buffy is wary of the whole situation, but decides she can at least give him a chance. She kicks in the door to the cell and approaches Angel. She reaches behind him to get at his chains, but in doing so the cross around her neck hangs down in his face, and he flinches from it. Buffy reacts, jerks back and drops the chains.
Buffy: Oh, you gotta be *kidding* me! She stomps out of the cell.
Angel: Wait! I won't hurt you.
Buffy: (faces him) No. You'll leave that to your Master.
Angel: You don't believe I wanna help you? He makes a hard effort to stand up and opens his shirt to show her his wounds.
Angel: Believe I want him dead. She stares at his wounds for a long moment.
Cut to the Master's factory. The camera pans from a control panel across the crowd of gathering vampires, past a wooden cage full of humans and the machine waiting in front of it, and over to the Master up on a stage.
Master: Vampires, come! Behold the technical wonder, which is about to alter the very fabric of our society. Some have argued that such an advancement goes against our nature. They claim that death is our art. I say to them... Well, I don't say anything to them because I kill them. Undeniably we are the world's superior race. (the camera closes in on him) Yet we have always been too parochial, too bound by the mindless routine of the predator. Hunt and kill, hunt and kill. Titillating? Yes. Practical? Hardly. Meanwhile, the humans, with their plebeian minds, have brought us a truly demonic concept: (spreads his arms) mass production!
Vampires: (cheer) Yeah! Yeah!
Xander: We really are living in a golden age. He is visibly moved by the proceedings. Willow tilts her head toward him and smiles.
Cut to Giles' apartment. He has several bags and bowls of various herbs and powders laid out on his chess table. He grabs a couple of them and goes over to his desk with them, where he has a large golden goblet already smoldering. He pulls bits of an herb from a bushel and drops them into the goblet as he recites the ritual to summon Anyanka.
Giles: Oh... Anyanka... I-I beseech thee... (puts on his glasses to read) Um... (turns a page) In the name of all women scorned... (adds more herb to the fire) Come before me. He looks around his apartment to see where she might appear. She does so, but in the shadows under the stairs to his loft, where he doesn't notice. Slowly she walks into the dim light of the room.
Giles: Oh! (lets out a nervous breath)
Anyanka: Do you have any idea what I do to a man who uses that spell to summon me? Giles gazes at her with a look of foreboding.
Cut to the factory.
Master: Bring on the first! At the cage the vampires shove a couple of the humans back from the gate, lift off the crossbar and open it. Oz realizes what's about to happen, but can't do anything. Two vampires go into the cage and choose a victim.
Vampire: You! He points to and grabs Cordette. Xander and Willow look on as she screams and is dragged out.
Cordette: Nooo! No! Please! No! Help me! No! Noooo! Some of the men in the cage attempt to resist and help her, but they are easily knocked aside. Once they have her outside, one of them shocks her with a cattle prod. Her body goes limp. The gates to the cage are closed, and the mortals all gather to watch in horror. The two vampires drag her to the end of the machine. One of them lifts her into a long stainless steel pan like the ones used for autopsies and lays her down in it.
Master: She's still alive, you see, for the freshness. The machine is turned on, and the pan moves along the conveyor to the blood draining station. On either side are four arms that extend over Cordette, each with a very large needle on the end. They all plunge into her body and begin to suck the blood from it. At one end of the contraption is a tap for sampling the blood, and a glass is filled for the Master to taste. In the cage Larry and Oz watch in disbelief. At the back of the factory Buffy and Angel peek around a corner. On the machine Cordette lets out her last few muffled sounds and dies. Xander and Willow watch with anticipation.
Angel: (to Buffy) What's the plan?
Buffy: (holds up her stake) Don't fall on this. The glass with the blood sample is passed up to the Master. Buffy and Angel calmly make their way through the crowd of vampires toward the stage. The Master rubs his fingers in anticipation of the first taste of blood from his new machine. The arms extract themselves from Cordette, and the pan with her body moves along the conveyor for disposal. The glass of blood is handed up to the Master. He holds it up to his subjects for a toast.
Master: Welcome to the future.
Vampires: To the future! To the future! To the future! Buffy raises her crossbow at the Master and fires. Instantly the Master pulls Xander in front of him, and the bolt hits him in the right shoulder. Buffy aims the crossbow at another target, but it gets knocked from her hand. Panic sets in among the vampires. Buffy ducks a wide swing from a vampire. She jumps up and brings her foot down to smash the back of his knee. Angel attacks a vampire by the cage, punches him in the face and shoves him aside. He rushes over to the gate and throws off the crossbar.
Willow: (smiling) Uh-oh. Puppy got out. Angel throws open the cage's gate and starts pulling people out. Buffy twists a vampire's arm around, immobilizing him, and does a jumping roundhouse kick to his gut. The crowd of humans streams into the fray. Oz reaches up and breaks a piece off of one of the wooden cage bars. He immediately jams it into the back of a vampire. All around humans and vampires fight. Xander and Willow finally decide it's time to join in and jump down from the stage.
Cut to Giles' apartment. Anyanka slowly approaches Giles, who bravely stands his ground.
Giles: Cordelia Chase. What did she wish for?
Anyanka: I had no idea her wish would be so exciting! Brave New World. I hope she likes it.
Cut to the factory. Buffy ducks a swing from a vampire and repeats her earlier maneuver of stomping on the back of his knee. She grabs him by his shirt and throws him over the conveyor. He pulls a section of it over with him. Buffy punches another vampire in the face. Yet another one tries to grab her by the neck from behind, and she spins around and elbows him in the face. As the second one comes at her again, she side steps him and sends him barreling into the third one. Gut to Giles' apartment.
Giles: You're gonna change it back. Anyanka finally gets too close, and he takes a couple of steps back.
Giles: I'm not afraid of you. Your only power lies in the wishing. Anyanka makes a sudden and hard grab for his neck.
Anyanka: Wrong! She lifts him and slams him against a wall.
Cut to the factory. Willow swings at Buffy, but misses as Buffy ducks the punch. Buffy backhand punches Willow in the face and follows up with a roundhouse kick to her stomach. Willow falls to the ground. Buffy senses something behind her and turns around in time to backhand punch an incoming vampire. She spins around again, this time to face Xander. She grabs onto his shoulder and yanks his body down to meet her knee, getting him twice in the gut and then in the face. She turns again to find her next target. Xander gets to his feet and comes at Buffy. Angel sees him make his move, and runs to Buffy's aid.
Angel: Buffy, look out! Still unaware of Xander's imminent attack, Buffy roundhouse kicks another vampire while holding onto his arm. She lets go of him as he falls. Angel runs past her and uppercuts Xander in the face. Xander in turn lunges at Angel with the crossbow bolt that he's pulled from his shoulder, and impales Angel. Angel turns to face Buffy and grabs his wound.
Angel: Buffy... He crumbles to ash. Buffy takes it like he's just another dead vampire, and marches over to another fray to continue the fight.
Anyanka: This is the real world now.
Cut to Giles' apartment. She still has him pinned to the wall.
Anyanka: This is the world we made. Isn't it wonderful?
Cut to the factory. Buffy notices a vampire run up behind her and backhand punches him, sending him flying through the air. She turns her attention back to Xander, who is just throwing a man aside. He sees her coming, and advances on her in turn. He swings at Buffy, and she punches his arm away. Taking advantage of the opening, she swings her stake into his chest, and he explodes into ashes. Without a care, Buffy turns back around and starts looking for her next victim. Willow sees her love staked and makes a move toward Buffy, but Larry grabs her by an arm and tries to pull her back. Oz shakes free of a vampire and runs to Larry's assistance, grabbing her by the waist and shoving her back into a broken piece of the cage. She instantly bursts into ashes. Buffy high side kicks a vampire in the face, knocking him to the floor, and turns to face the Master. He slides down the stair railings from the stage and shoves aside the vampire and mortal blocking his way. Buffy begins a determined stride in his direction. The Master shoves more people and vampires aside in his determination to get at the Slayer. Buffy does the same.
Cut to Giles' apartment. He is beginning to choke. Just then he notices the amulet around Anyanka's neck begin to glow green, and makes a grab for it, wresting it from her neck. This causes her to let go of him, and he backhand punches her in the face, sending her staggering across the room.
Cut to the factory. Buffy and the Master finally meet with swings that middle block each other. Buffy tries to wrap her hands around the Master's forearm.
Cut to Giles's apartment. He scrambles to his desk, lays the amulet on it and searches frantically for something to smash it with. He soon has his marble paperweight in his hand. Anyanka gets up from the floor.
Anyanka: You trusting fool! How do you know the other world is any better than this?
Giles: Because it has to be.
Cut to the factory. The Master does a backhand swing, snapping Buffy's head back, dazing her. He grabs her by the shoulders and pulls her to him.
Cut to Giles' apartment. He raises the paperweight and starts to swing it down with full force at Anyanka's amulet.
Anyanka: No!
Cut to the factory. The Master grabs Buffy's head.
Cut to Giles' apartment. He swings the paperweight down.
Cut to the factory. The Master gives Buffy's head a hard twist, snapping her neck.
Cut to Giles' apartment. He swings the paperweight further down.
Cut to the factory. Buffy's expression is blank as she begins to sink to the floor.
Cut to Giles' apartment. He smashes the paperweight onto Anyanka's amulet. It shatters into thousands of pieces and emits a burst of green light.
Cut to the factory. The Master watches as Buffy's body begins to fall, then moves on. The picture fades to bright white. The picture fades from white back to the quad at Sunnydale High School and Cordelia making her wish. She no longer has Anya's pendant around her neck.
Cordelia: I wish Buffy Summers had never come to Sunnydale. Anya turns back to face Cordelia.
Anya: Done. She is taken aback when nothing happens, not yet quite aware that she is no longer immortal.
Cordelia: That would be cool! No, wait. I wish Buffy Summers had never been born.
Anya: Done! She is surprised that nothing's happening. She looks down at herself in confusion.
Cordelia: And I wish that Xander Harris never again knows the touch of a woman. (smiles, starts to walk away) *And* that Willow wakes up tomorrow covered in monkey hair.
Anya: (tries again) Done! The camera follows Cordelia to where the gang is sitting.
Cordelia: In fact, I wish all men except maybe the dumb and the really agreeable kind... Giles walks past the group and makes a point of checking his watch. They all just smile at him and nod.
Cordelia: ...disappear off the face of the earth. That would be *so* cool! Or maybe... The camera pulls in on Xander, Buffy and Willow smiling and talking cheerfully among themselves. | Cordelia's pain over Xander's betrayal summons Anyanka ( Emma Caulfield ), a vengeance demon who grants wishes to scorned women. Cordelia wishes that Buffy had never come to Sunnydale, and constructs an alternate reality where the Master ( Mark Metcalf ) rose from the dead and the Harvest took over Sunnydale. Giles discovers this in the alternate reality and realizes he is able to reverse the events. | summ_screen_fd |
Background Airline Passenger Screening Using Checkpoint Screening Technology Passenger screening is a process by which screeners inspect individuals and their property to deter and prevent an act of violence or air piracy, such as the carrying of any unauthorized explosive, incendiary, weapon, or other prohibited item on board an aircraft or into a sterile area. Screeners inspect individuals for prohibited items at designated screening locations. TSA developed standard operating procedures for screening passengers at airport checkpoints. Primary screening is conducted on all airline passengers before they enter the sterile area of an airport and involves passengers walking through a metal detector and carry-on items being subjected to X-ray screening. Passengers who alarm the walk-through metal detector or are designated as selectees—that is, passengers selected for additional screening—must then undergo secondary screening, as well as passengers whose carry-on items have been identified by the X-ray machine as potentially containing prohibited items. Secondary screening involves additional means for screening passengers, such as by hand- wand; physical pat-down; or, at certain airport locations, an explosives trace portal (ETP), which is used to detect traces of explosives on passengers by using puffs of air to dislodge particles from their bodies and clothing into an analyzer. Selectees’ carry-on items are also physically searched or screened for explosives, such as by using explosives trace detection machines. Assessing Potential Vulnerabilities Related to Not Screening against All Watchlist Records and Ensuring Clear Lines of Authority over the Watchlist Process Would Provide for Its More Effective Use Agencies Rely upon Standards of Reasonableness in Assessing Individuals for Nomination to TSC’s Watchlist, but Did Not Connect Available Information on Mr. Abdulmutallab to Determine Whether a Reasonable Suspicion Existed Federal agencies—particularly NCTC and the FBI—submit to TSC nominations of individuals to be included on the consolidated watchlist. For example, NCTC receives terrorist-related information from executive branch departments and agencies, such as the Department of State, the Central Intelligence Agency, and the FBI, and catalogs this information in its Terrorist Identities Datamart Environment database, commonly known as the TIDE database. This database serves as the U.S. government’s central classified database with information on known or suspected international terrorists. According to NCTC, agencies submit watchlist nomination reports to the center, but are not required to specify individual screening systems that they believe should receive the watchlist record, such as the No Fly list of individuals who are to be denied boarding an aircraft. NCTC is to presume that agency nominations are valid unless it has other information in its possession to rebut that position. To decide if a person poses enough of a threat to be placed on the watchlist, agencies are to follow Homeland Security Presidential Directive (HSPD) 6, which states that the watchlist is to contain information about individuals “known or appropriately suspected to be or have been engaged in conduct constituting, in preparation for, in aid of, or related to terrorism.” HSPD-24 definitively established the “reasonable suspicion” standard for watchlisting by providing that agencies are to make available to other agencies all biometric information associated with “persons for whom there is an articulable and reasonable basis for suspicion that they pose a threat to national security.” NCTC is to consider information from all available sources and databases to determine if there is a reasonable suspicion of links to terrorism that warrants a nomination, which can involve some level of subjectivity. The guidance on determining reasonable suspicion, which TSC most recently updated in February 2009, contains specific examples of the types of terrorism-related conduct that may make an individual appropriate for inclusion on the watchlist. The White House’s review of the December 25 attempted terrorist attack noted that Mr. Abdulmutallab’s father met with U.S. Embassy officers in Abuja, Nigeria, to discuss his concerns that his son may have come under the influence of unidentified extremists and had planned to travel to Yemen. However, according to NCTC, the information in the State Department’s nomination report did not meet the criteria for watchlisting in TSC’s consolidated terrorist screening database per the government’s established and approved nomination standards. NCTC also noted that the State Department cable nominating Mr. Abdulmutallab had no indication that the father was the source of the information. According to the White House review of the December 25 attempted attack, the U.S. government had sufficient information to have uncovered and potentially disrupted the attack—including by placing Mr. Abdulmutallab on the No Fly list—but analysts within the intelligence community failed to connect the dots that could have identified and warned of the specific threat. After receiving the results of the White House’s review of the December 25 attempted attack, the President called for members of the intelligence community to undertake a number of corrective actions—such as clarifying intelligence agency roles, responsibilities, and accountabilities to document, share, and analyze all sources of intelligence and threat threads related to terrorism, and accelerating information technology enhancements that will help with information correlation and analysis. The House Committee on Oversight and Government Reform has asked us, among other things, to assess government efforts to revise the watchlist process, including actions taken related to the December 25 attempted attack. As part of our monitoring of high-risk issues, we also have ongoing work— at the request of the Senate Committee on Homeland Security and Governmental Affairs—that is assessing agency efforts to create the Information Sharing Environment, which is intended to break down barriers to sharing terrorism-related information, especially across federal agencies. Our work is designed to help ensure that federal agencies have a road map that defines roles, responsibilities, actions, and time frames for removing barriers, as well as a system to hold agencies accountable to the Congress and the public for making progress on these efforts. Among other things, this road map can be helpful in removing cultural, technological, and other barriers that lead to agencies maintaining information in stove-piped systems so that it is not easily accessible, similar to those problems that the December 25 attempted attack exposed. We expect to issue the results of this work later this year. By Not Placing Mr. Abdulmutallab on the Consolidated Watchlist or Its Subsets, the Government Missed Opportunities to Use These Counterterrorism Tools Following the December 25 attempted terrorist attack, questions were raised as to what could have happened if Mr. Abdulmutallab had been on TSC’s consolidated terrorist screening database. We created several scenarios to help explain how the watchlist process is intended to work and what opportunities agencies could have had to identify him if he was on the watchlist. For example, according to TSC, if a record from the terrorist screening database is sent to the State Department’s system and the individual in that record holds a valid visa, TSC would compare the identifying information in the watchlist record against identifying information in the visa and forward positive matches to the State Department for possible visa revocation. If an individual’s visa is revoked, under existing procedures, this information is to be entered into the database CBP uses to screen airline passengers prior to their boarding, which we describe below. According to CBP, when the individual checks in for a flight, the on-site CBP Immigration Advisory Program officers already would have been apprised of the visa revocation by CBP and they would have checked the person’s travel documents to verify that the individual was a match to the visa revocation record. Once the positive match was established, the officers would have recommended that he not be allowed to board the flight. Under another scenario, if an individual is on TSC’s terrorist screening database, existing processes provide CBP with the opportunity to identify the subject of a watchlist record as part of the checks CBP is to conduct to see if airline passengers are eligible to be admitted into the country. Specifically, for international flights departing to or from the United States (but not for domestic flights), CBP is to receive information on passengers obtained, for example, when their travel document is swiped. CBP is to check this passenger information against a number of databases to see if there are any persons who have immigration violations, criminal histories, or any other reason for being denied entry to the country, in accordance with the agency’s mission. According to CBP, when it identifies a U.S. bound passenger who is on the watchlist, it coordinates with other federal agencies to evaluate the totality of available information to see what action is appropriate. In foreign airports where there is a CBP Immigration Advisory Program presence, the information on a watchlisted subject is forwarded by CBP to program officers onsite. The officers would then intercept the subject prior to boarding the aircraft and confirm that the individual is watchlisted, and when appropriate based on the derogatory information, request that the passenger be denied boarding. In a third scenario, if an individual is on the watchlist and is also placed on the No Fly or Selectee list, when the person checks in for a flight, the individual’s identifying information is to be checked against these lists. Individuals matched to the No Fly list are to be denied boarding. If the individual is matched to the Selectee list, the person is to be subject to further screening, which could include physical screening, such as a pat- down. The criteria in general that are used to place someone on either of these two lists include the following: Persons who are deemed to be a threat to civil aviation or national security and should be precluded from boarding an aircraft are put on the No Fly list. Persons who are deemed to be a threat to civil aviation or national security but do not meet the criteria of the No Fly list are placed on the Selectee list and are to receive additional security screening prior to being permitted to board an aircraft. The White House Homeland Security Council devised these more stringent sets of criteria for the No Fly and Selectee lists in part because these lists are not intended as investigative or information-gathering tools or tracking mechanisms, and TSA is a screening but not an intelligence agency. Rather, the lists are intended to help ensure the safe transport of passengers and facilitate the flow of commerce. However, the White House’s review of the December 25 attempted terrorist attack raised questions about the effectiveness of the criteria, and the President tasked the FBI and TSC with developing recommendations for any needed changes to the nominations guidance and criteria. Weighing and responding to the potential impacts that changes to the nominations guidance and criteria could have on the traveling public and the airlines will be important considerations in developing such recommendations. In September 2006, we reported that tens of thousands of individuals who had similar names to persons on the watchlist were being misidentified and subjected to additional screening, and in some cases delayed so long as to miss their flights. We also reported that resolving these misidentifications can take time and, therefore, affect air carriers and commerce. If changes in criteria result in more individuals being added to the lists, this could also increase the number of individuals who are misidentified, exacerbating these negative effects. In addition, we explained that individuals who believe that they have been inappropriately matched to the watchlist can petition the government for action and the relevant agencies must conduct research and work to resolve these issues. If more people are misidentified, more people may trigger this redress process, increasing the need for resources. Finally, any changes to the criteria or process would have to ensure that watchlist records are used in a manner that safeguards legal rights, including freedoms, civil liberties, and information privacy guaranteed by federal law. Agencies Do Not Screen Individuals against All Records in the Watchlist, Which Creates Potential Security Vulnerabilities; GAO Continues to Recommend That Agencies Assess and Address These Gaps In reacting to the December 25 attempted terrorist attack, determining whether there were potential vulnerabilities related to the use of watchlist records when screening—not only individuals who fly into the country but also, for example, those who cross land borders—are important considerations. Screening agencies whose missions most frequently and directly involve interactions with travelers generally do not check against all records in the consolidated terrorist watchlist. In our October 2007 report, we noted that this is because screening against certain records may not be needed to support a respective agency’s mission or may not be possible because of computer system limitations, among other things. For example, CBP’s mission is to determine if any traveler is eligible to enter the country or is to be denied entry because of immigration or criminal violations. As such, CBP’s computer system accepts all records from the consolidated watchlist database that have either a first name or a last name and one other identifier, such as a date of birth. Therefore, TSC sends CBP the greatest number of records from the consolidated watchlist database for its screening. In contrast, one of the State Department’s missions is to approve requests for visas. Since only non-U.S. citizens and nonlawful permanent residents apply for visas, TSC does not send the department records on citizens or lawful permanent residents for screening visa applicants. Also, the FBI database that state and local law enforcement agencies use for their missions in checking individuals for criminal histories, for example, also receives a smaller portion of the watchlist. According to the FBI, its computer system requires a full first name, last name, and other identifier, typically a date of birth. The FBI noted that this is because having these identifiers helps to reduce the number of times an individual is misidentified as being someone on the list, and the computer system would not be effective in making matches without this information. Finally, the No Fly and Selectee lists collectively contain the lowest percentage of watchlist records because the remaining ones either do not meet the nominating criteria, as described above, or do not meet system requirements—that is, include full names and dates of birth, which TSA stated are required to minimize misidentifications. TSA is implementing a new screening program that the agency states will have the capability to screen an individual against the entire watchlist. Under this program, called Secure Flight, TSA will assume from air carriers the responsibility of comparing passenger information against the No Fly and Selectee lists. According to the program’s final rule, in general, Secure Flight is to compare passenger information only to the No Fly and Selectee lists. The supplementary information accompanying the rule notes that this will be satisfactory to counter the security threat during normal security circumstances. However, the rule provides that TSA may use the larger set of watchlist records when warranted by security considerations, such as if TSA learns that flights on a particular route may pose increased risks. TSA emphasized that use of the full terrorist screening database is not routine. Rather, TSA noted that its use is limited to circumstances in which there is information concerning an increased risk to transportation security, and the decision to use the full watchlist database will be based on circumstances at the time. According to TSA, as of January 2010, the agency was developing administrative procedures for utilizing the full watchlist when warranted. In late January 2009, TSA began to assume from airlines the watchlist matching function for a limited number of domestic flights, and has since phased in additional flights and airlines. TSA expects to assume the watchlist matching function for all domestic and international flights departing to and from the United States by December 2010. It is important to note that under the Secure Flight program, TSA requires airlines to provide the agency with each passenger’s full name and date of birth to facilitate the watchlist matching process, which should reduce the number of individuals who are misidentified as the subject of a watchlist record. We continue to monitor the Secure Flight program at the Congress’s request. In our October 2007 watchlist report, we recommended that the FBI and DHS assess the extent to which security risks exist by not screening against certain watchlist records and what actions, if any, should be taken in response. The agencies generally agreed with our recommendations but noted that the risks related to not screening against all watchlist records needs to be balanced with the impact of screening against all records, especially those records without a full name and other identifiers. For example, more individuals could be misidentified, law enforcement would be put in the position of detaining more individuals until their identities could be resolved, and administrative costs could increase, without knowing what measurable increase in security is achieved. While we acknowledge these tradeoffs and potential impacts, we maintain that assessing whether vulnerabilities exist by not screening against all watchlist records—and if there are ways to limit impacts—is critical and could be a relevant component of the government’s ongoing review of the watchlist process. Therefore, we believe that our recommendation continues to have merit. Identifying Additional Screening Opportunities and Determining Whether There Are Clear Lines of Authority for and Accountability over the Watchlist Process Would Help Ensure Its Effective Use As we reported in October 2007, the federal government has made progress in using the consolidated terrorist watchlist for screening purposes, but has additional opportunities to use the list. For example, DHS uses the list to screen employees in some critical infrastructure components of the private sector, including certain individuals who have access to vital areas of nuclear power plants or transport hazardous materials. However, many critical infrastructure components are not using watchlist records, and DHS has not finalized guidelines to support such private sector screening, as HSPD-6 mandated and we previously recommended. In that same report, we noted that HSPD-11 tasked the Secretary of Homeland Security with coordinating across other federal departments to develop (1) a strategy for a comprehensive and coordinated watchlisting and screening approach and (2) a prioritized implementation and investment plan that describes the scope, governance, principles, outcomes, milestones, training objectives, metrics, costs, and schedule of necessary activities. We reported that without such a strategy, the government could not provide accountability and a basis for monitoring to ensure that (1) the intended goals for, and expected results of, terrorist screening are being achieved and (2) use of the watchlist is consistent with privacy and civil liberties. We recommended that DHS develop a current interagency strategy and related plans. According to DHS’s Screening Coordination Office, during the fall of 2007, the office led an interagency effort to provide the President with an updated report, entitled, HSPD-11, An Updated Strategy for Comprehensive Terrorist-Related Screening Procedures. The office noted that the report was formally submitted to the Executive Office of the President through the Homeland Security Council and reviewed by the President on January 25, 2008. Further, the office noted that it also provided a sensitive version of the report to the Congress in October 2008. DHS provided us an excerpt of that report to review, stating that it did not have the authority to share excerpts provided by other agencies, and we were unable to obtain a copy of the full report. The information we reviewed only discussed DHS’s own efforts for coordinating watchlist screening across the department. Therefore, we were not able to determine whether the HSPD-11 report submitted to the President addressed all of the components called for in the directive or what action, if any, was taken as a result. We maintain that a comprehensive strategy, as well as related implementation and investment plans, as called for by HSPD-11, continue to be important to ensure effective governmentwide use of the watchlist process. In addition, in our October 2007 report, we noted that establishing an effective governance structure as part of this strategic approach is particularly vital since numerous agencies and components are involved in the development, maintenance, and use of the watchlist process, both within and outside of the federal government. Also, establishing a governance structure with clearly-defined responsibility and authority would help to ensure that agency efforts are coordinated, and that the federal government has the means to monitor and analyze the outcomes of such efforts and to address common problems efficiently and effectively. We determined at the time that no such structure was in place and that no existing entity clearly had the requisite authority for addressing interagency issues. We recommended that the Homeland Security Council ensure that a governance structure was in place, but the council did not comment on our recommendation. At the time of our report, TSC stated that it had a governance board in place, comprised of senior-level agency representatives from numerous departments and agencies. However, we also noted that the board provided guidance concerning issues within TSC’s mission and authority. We also stated that while this governance board could be suited to assume more of a leadership role, its authority at that time was limited to TSC- specific issues, and it would need additional authority to provide effective coordination of terrorist-related screening activities and interagency issues governmentwide. In January 2010, the FBI stated that TSC has a Policy Board in place, with representatives from relevant departments and agencies, that reviews and provides input to the government’s watchlist policy. The FBI also stated that the policies developed are then sent to the National Security Council Deputies Committee (formerly the Homeland Security Council) for ratification. The FBI noted that this process was used for making the most recent additions and changes to watchlist standards and criteria. We have not yet been able to determine, however, whether the Policy Board has the jurisdiction and authority to resolve issues beyond TSC’s purview, such as issues within the intelligence community and in regard to the nominations process, similar to the types of interagency issues the December 25 attempted attack identified. We maintain that a governance structure with the authority for and accountability over the entire watchlist process, from nominations through screening, and across the government is important. On January 7, 2010, the President tasked the National Security Staff with initiating an interagency review of the watchlist process—including the business processes, procedures, and criteria—and the interoperability and sufficiency of supporting information technology systems. This review offers the government an opportunity to develop an updated strategy, related plans, and governance structure that would provide accountability to the administration, the Congress, and the American public that the watchlist process is effective at helping to secure the homeland. Recent Work Highlights the Importance of Conducting Vulnerability Assessments and Operational Testing Prior to Deployment of New Checkpoint Technologies While TSA Has Not Yet Deployed Any New Checkpoint Technologies Nationwide, It Plans to Have Installed Almost 200 AITs by the End of 2010 As we reported in October 2009, in an effort to improve the capability to detect explosives at aviation passenger checkpoints, TSA has 10 passenger screening technologies in various phases of research, development, procurement, and deployment, including the AIT (formerly Whole Body Imager). TSA is evaluating the AIT as an improvement over current screening capabilities of the metal detector and pat-downs specifically to identify nonmetallic threat objects and liquids. The AITs produce an image of a passenger’s body that a screener interprets. The image identifies objects, or anomalies, on the outside of the physical body but does not reveal items beneath the surface of the skin, such as implants. TSA plans to procure two types of AIT units: one type uses millimeter wave and the other type uses backscatter X-ray technology. Millimeter wave technology beams millimeter wave radio frequency energy over the body’s surface at high speed from two antennas simultaneously as they rotate around the body. The energy reflected back from the body or other objects on the body is used to construct a three-dimensional image. Millimeter wave technology produces an image that resembles a fuzzy photo negative. Backscatter X-ray technology uses a low-level X-ray to create a two-sided image of the person. Backscatter technology produces an image that resembles a chalk etching. As we reported in October 2009, TSA has not yet deployed any new technologies nationwide. However, as of December 31, 2009, according to a senior TSA official, the agency has deployed 40 of the millimeter wave AITs, and has procured 150 backscatter X-ray units in fiscal year 2009 and estimates that these units will be installed at airports by the end of calendar year 2010. In addition, TSA plans to procure an additional 300 AIT units in fiscal year 2010, some of which will be purchased with funds from the American Recovery and Reinvestment Act of 2009. TSA plans to procure and deploy a total of 878 units at all category X through category IV airports. Full operating capability is expected in fiscal year 2014. TSA officials stated that the cost of the AIT is about $130,000 to $170,000 per unit, excluding installation costs. In addition, the estimated training costs are $50,000 per unit. While TSA stated that the AIT will enhance its explosives detection capability, because the AIT presents a full body image of a person during the screening process, concerns have been expressed that the image is an invasion of privacy. According to TSA, to protect passenger privacy and ensure anonymity, strict privacy safeguards are built into the procedures for use of the AIT. For example, the officer who assists the passenger never sees the image that the technology produces, and the officer who views the image is remotely located in a secure resolution room and never sees the passenger. Officers evaluating images are not permitted to take cameras, cell phones, or photo-enabled devices into the resolution room. To further protect passengers’ privacy, ways have been introduced to blur the passengers’ images. The millimeter wave technology blurs all facial features, and the backscatter X-ray technology has an algorithm applied to the entire image to protect privacy. Further, TSA has stated that the AIT’s capability to store, print, transmit, or save the image will be disabled at the factory before the machines are delivered to airports, and each image is automatically deleted from the system after it is cleared by the remotely located security officer. Once the remotely located officer determines that threat items are not present, that officer communicates wirelessly to the officer assisting the passenger. The passenger may then continue through the security process. Potential threat items are resolved through a direct physical pat-down before the passenger is cleared to enter the sterile area. In addition to privacy concerns, the AITs are large machines, and adding them to the checkpoint areas will require additional space, especially since the operators are segregated from the checkpoint to help ensure passenger privacy. TSA Reports That It Is Taking Steps to Operationally Test AITs but Has Not Conducted Vulnerability Assessments We previously reported on several challenges TSA faces related to the research, development, and deployment of passenger checkpoint screening technologies and made a number of recommendations to improve this process. Two of these recommendations are particularly relevant today, as TSA moves forward with plans to install a total of 878 additional AITs—completing operational testing of technologies in airports prior to using them in day-to-day operations and assessing whether technologies such as the AIT are vulnerable to terrorist countermeasures, such as hiding threat items on various parts of the body to evade detection. First, in October 2009, we reported that TSA had relied on technologies in day-to-day airport operations that had not been proven to meet their functional requirements through operational testing and evaluation, contrary to TSA’s acquisition guidance and a knowledge-based acquisition approach. We also reported that TSA had not operationally tested the AITs at the time of our review, and we recommended that TSA operationally test and evaluate technologies prior to deploying them. In commenting on our report, TSA agreed with this recommendation. A senior TSA offici stated that although TSA does not yet have a written policy requiring operational testing prior to deployment, TSA is now including in its contracts with vendors that checkpoint screening machines are required to successfully complete laboratory tests as well as operational tests. The test results are then incorporated in the source selection plan. The official also stated that the test results are now required at key decision points by DHS’s Investment Review Board. While recently providing GAO with updated information to our October 2009 report, TSA stated that operational testing for the AIT was completed as of the end of calendar year 2009. We are in the process of verifying that TSA has tested all of the AIT’s functional requirements in an operational environment. Deploying technologies that have not successfully completed operational testing and evaluation can lead to cost overruns and underperformance. TSA’s procurement guidance provides that testing should be conducted in an operational environment to validate that the system meets all functional requirements before deployment. In addition, our reviews have shown that leading commercial firms follow a knowledge-based approach to major acquisitions and do not proceed with large investments unless the product’s design demonstrates its ability to meet functional requirements and be stable. The developer must show that the product can be manufactured within cost, schedule, and quality targets and is reliable before production begins and the system is used in day-to-day operations. TSA’s experience with the ETPs, which the agency uses for secondary screening, demonstrates the importance of testing and evaluation in an operational environment. The ETP detects traces of explosives on a passenger by using puffs of air to dislodge particles from the passenger’s body and clothing that the machine analyzes for traces of explosives. TSA procured 207 ETPs and in 2006 deployed 101 ETPs to 36 airports, the first deployment of a checkpoint technology initiated by the agency. TSA deployed the ETPs even though agency officials were aware that tests conducted during 2004 and 2005 on earlier ETP models suggested that they did not demonstrate reliable performance. Furthermore, the ETP models that were subsequently deployed were not first tested to prove their effective performance in an operational environment, contrary to TSA’s acquisition guidance, which recommends such testing. As a result, TSA procured and deployed ETPs without assurance that they would perform as intended in an operational environment. TSA officials stated that they deployed the machines without resolving these issues to respond quickly to the threat of suicide bombers. In June 2006, TSA halted further deployment of the ETP because of performance, maintenance, and installation issues. According to a senior TSA official, as of December 31, 2009, all but 9 ETPs have been withdrawn from airports and 18 ETPs remain in inventory. TSA estimates that the 9 remaining ETPs will be removed from airports by the end of calendar year 2010. In the future, using validated technologies would enhance TSA’s efforts to improve checkpoint security. Furthermore, retaining existing screening procedures until the effectiveness of future technologies has been validated could provide assurances that use of checkpoint technologies improves aviation security. Second, as we reported in October 2009, TSA does not know whether its explosives detection technologies, such as the AITs, are susceptible to terrorist tactics. Although TSA has obtained information on vulnerabilities at the screening checkpoint, the agency has not assessed vulnerabilities— that is, weaknesses in the system that terrorists could exploit in order to carry out an attack—related to passenger screening technologies, such as AITs, that are currently deployed. According to TSA’s threat assessment, terrorists have various techniques for concealing explosives on their persons, as was evident in Mr. Abdulmutallab’s attempted attack on December 25, where he concealed an explosive in his underwear. However, TSA has not assessed whether these and other tactics that terrorists could use to evade detection by screening technologies, such as AIT, increase the likelihood that the screening equipment would not detect the hidden weapons or explosives. Thus, without an assessment of the vulnerabilities of checkpoint technologies, it is unclear whether the AIT or other technologies would have been able to detect the weapon Mr. Abdulmutallab used in his attempted attack. TSA is in the process of developing a risk assessment for the airport checkpoints, but the agency has not yet completed this effort or clarified the extent to which this effort addresses any specific vulnerabilities in checkpoint technology. TSA officials stated that to identify vulnerabilities at airport checkpoints, the agency analyzes information such as the results from its covert testing program. TSA conducts national and local covert tests, whereby individuals attempt to enter the secure area of an airport through the passenger checkpoint with prohibited items in their carry-on bags or hidden on their persons. However, TSA’s covert testing programs do not systematically test passenger and baggage screening technologies nationwide to ensure that they identify the threat objects and materials the technologies are designed to detect, nor do the covert testing programs identify vulnerabilities related to these technologies. We reported in August 2008 that while TSA’s local covert testing program attempts to identify test failures that may be caused by screening equipment not working properly or caused by screeners and the screening procedures they follow, the agency’s national testing program does not attribute a specific cause of a test failure. We recommended, among other things, that TSA require the documentation of specific causes of all national covert testing failures, including documenting failures related to equipment, in the covert testing database to help TSA better identify areas for improvement. TSA concurred with this recommendation and stated that the agency will expand the covert testing database to document test failures related to screening equipment. In our 2009 report, we also recommended that the Assistant Secretary for TSA, among other actions, conduct a complete risk assessment—including threat, vulnerability, and consequence assessment—for the passenger screening program and incorporate the results into TSA’s program strategy, as appropriate. TSA and DHS concurred with our recommendation, but have not completed these risk assessments or provided documentation to show how they have addressed the concerns raised in our 2009 report regarding the susceptibility of the technology to terrorist tactics. Mr. Chairman, this concludes our statement for the record. Contacts and Acknowledgments For additional information on this statement, please contact Eileen Larence at (202) 512-6510 or [email protected] or Stephen Lord at (202) 512-4379 or [email protected]. In addition to the contacts named above, Kathryn Bernet, Carissa Bryant, Frances Cook, Joe Dewechter, Eric Erdman, Richard Hung, Anne Laffoon, Linda Miller, Victoria Miller, and Michelle Woods made key contributions to this statement. This is a work of the U.S. government and is not subject to copyright protection in the United States. The published product may be reproduced and distributed in its entirety without further permission from GAO. However, because this work may contain copyrighted images or other material, permission from the copyright holder may be necessary if you wish to reproduce this material separately. | The December 25, 2009, attempted bombing of flight 253 raised questions about the federal government's ability to protect the homeland and secure the commercial aviation system. This statement focuses on the government's efforts to use the terrorist watchlist to screen individuals and determine if they pose a threat, and how failures in this process contributed to the December 25 attempted attack. This statement also addresses the Transportation Security Administration's (TSA) planned deployment of technologies for enhanced explosive detection and the challenges associated with this deployment. GAO's comments are based on products issued from September 2006 through October 2009 and selected updates in January 2010. For these updates, GAO reviewed government reports related to the December 25 attempted attack and obtained information from the Department of Homeland Security (DHS) and TSA on use of the watchlist and new technologies for screening airline passengers. The intelligence community uses standards of reasonableness to evaluate individuals for nomination to the consolidated terrorist watchlist. In making these determinations, agencies are to consider information from all available sources. However, for the December 25 subject, the intelligence community did not effectively complete these steps and link available information to the subject before the incident. Therefore, agencies did not nominate the individual to the watchlist or any of the subset lists used during agency screening, such as the "No Fly" list. Weighing and responding to the potential impacts that changes to the nomination criteria would have on the traveling public will be an important consideration in determining what changes may be needed. Also, screening agencies stated that they do not check against all records in the watchlist, partly because screening against certain records may not be needed to support a respective agency's mission or may not be possible because of the requirements of computer programs used to check individuals against watchlist records. In October 2007, GAO reported that not checking against all records may pose a security risk and recommended that DHS and the FBI assess potential vulnerabilities, but they have not completed these assessments. TSA is implementing an advanced airline passenger prescreening program--known as Secure Flight--that could potentially result in the federal government checking passengers against the entire watchlist under certain security conditions. Further, the government lacks an up-to-date strategy and implementation plan--supported by a clearly defined leadership or governance structure--which are needed to enhance the effectiveness of terrorist-related screening and ensure accountability. In the 2007 report, GAO recommended that the Homeland Security Council ensure that a governance structure exists that has the requisite authority over the watchlist process. The council did not comment on this recommendation. As GAO reported in October 2009, since TSA's creation, 10 passenger screening technologies have been in various phases of research, development, procurement, and deployment, including the Advanced Imaging Technology (AIT)--formerly known as the Whole Body Imager. TSA expects to have installed almost 200 AITs in airports by the end of calendar year 2010 and plans to install a total of 878 units by the end of fiscal year 2014. In October 2009, GAO reported that TSA had not yet conducted an assessment of the technology's vulnerabilities to determine the extent to which a terrorist could employ tactics that would evade detection by the AIT. Thus, it is unclear whether the AIT or other technologies would have detected the weapon used in the December 25 attempted attack. GAO's report also noted the problems TSA experienced in deploying another checkpoint technology that had not been tested in the operational environment. Since GAO's October report, TSA stated that it has completed the testing as of the end of 2009. We are currently verifying that all functional requirements of the AIT were tested in an operational environment. Completing these steps should better position TSA to ensure that its costly deployment of AIT machines will enhance passenger checkpoint security. | gov_report |
Molecular evolution is an established technique for inferring gene homology but regulatory DNA turns over so rapidly that inference of ancestral networks is often impossible. In silico evolution is used to compute the most parsimonious path in regulatory space for anterior-posterior patterning linking two Dipterian species. The expression pattern of gap genes has evolved between Drosophila (fly) and Anopheles (mosquito), yet one of their targets, eve, has remained invariant. Our model predicts that stripe 5 in fly disappears and a new posterior stripe is created in mosquito, thus eve stripe modules 3+7 and 4+6 in fly are homologous to 3+6 and 4+5 in mosquito. We can place Clogmia on this evolutionary pathway and it shares the mosquito homologies. To account for the evolution of the other pair-rule genes in the posterior we have to assume that the ancestral Dipterian utilized a dynamic method to phase those genes in relation to eve. Molecular phylogenies based on protein coding genes have greatly enhanced evolutionary theory, and in favorable cases even allow a reconstruction of the last common ancestral gene or even full evolutionary pathways [1]. However regulatory sequence evolves more rapidly than coding sequence and the functional binding sites can move around without impacting the function of a ∼1kb functional regulatory module [2,3]. Thus one is often in the situation where gene homologies are obvious, yet there is no visible sequence homology in the regulatory regions. At the phenotypic level, gene expression domains can be easily mapped by in-situ hybridization yet a molecular understanding is limited outside of model organisms. There is considerable need for a computational tool that can take sparse phenotypic information, e. g., broadly defined space-time gene expression, and construct the simplest phylogenetic relationships consistent with data, thereby highlighting interesting events for molecular follow up. Drosophila segmentation is a paradigmatic example of dynamic developmental network. Positional information propagates from maternal gradients such as bicoid (bcd) and caudal (cad) to gap genes such as hunchback, giant, knirps and Kruppel (respectively hb, gt, kni, Kr), and then to the striped expression of primary pair-rule genes such as even-skipped (eve), hairy (h), runt (run), and partially fushi-tarazu (ftz) [4,5]. The pair-rule genes in turn control the segment polarity genes that are broadly conserved across the arthropods [6]. Mutagenesis and bioinformatics studies have revealed the main DNA motifs controlling the expression of gap and pair-rule genes [7] while systematic quantitative imaging has led to phenomenological models for segmentation dynamics [8,9]. Recent evo-devo studies have started to map the segmentation hierarchy in other dipterans (Anopheles [10], Clogmia [11], Megaselia [12]). Almost all information comes from localizing the relevant mRNA by in-situ hybridization, and knocking down (KD) various transcripts with RNA interference. Information in each of these three species is still very sparse: while we know the position of the gap genes and the single pair-rule gene eve, there is only few information on the phasing of the other pair-rule genes relative to eve. Whether they are positioned by the gap genes or other so-called primary pair-rule genes is not known in those Dipterans. There are no defined gene regulatory modules in these species, so all information about gap gene regulation is inferred from their position and shifts in putative targets under KD. In spite of this sparse information, some interesting questions can be posed. The anterior gap gene pattern appears invariant in all species as do the eve stripes, though there are only six in Clogmia before gastrulation vs 7 in Drosophila and up to 8 in Anopheles. There is more variability in the posterior. The relative positions of the posterior domains of hb and gt are inverted in Anopheles with respect to Drosophila, while in Clogmia, neither of these gap genes are expressed posteriorly before gastrulation. It is reasonable to assume that the primary pair-rule stripes are positioned by gap gene repression, so the evolutionary interchange of the posterior hb and giant domains poses problems for individual eve stripe regulatory modules. For instance, eve 5 in Drosophila is repressed posteriorly by gt so if the posterior gt domain is removed, eve 5 extends broadly posteriorly in Drosophila [13]. So how can gt domain be much more posterior in Anopheles, and virtually nonexistent in Clogmia? Similarly the two nested modules eve3+7 and 4+6 are both defined by kni repression from the interior and hb repression from the exterior [14], which seems less plausible in Anopheles based on the relative positions of the eve stripes and gap genes. How is computational modeling best harnessed to the task of inferring the evolutionary path between fly and mosquito with such sparse information about one endpoint and intermediates? One very general lesson from the machine learning field is to avoid overfitting [15] [16]. More parameters make less predictive, “hairball” models [17] that can always be complexified rather than falsified. The temptation in the present instance is to import into the evolutionary simulation all the molecular details we have accumulated about Drosophila. A realistic model for the AP patterning in Drosophila with multiple factors, short range repression and cooperativity, was formulated in [18], and applied to the evolution of new enhancers in [3]. When guided by strong selection for the correct domain of expression [3], new modules can evolve on the time scale of 107 years [19]. The key point made in these and related papers is that de novo evolution of enhancers is fast because their genotype to phenotype map can be optimized by point mutations and hill climbing. These papers also observe that under the quick and sloppy logic of evolution, the excess of binding sites or the prevalence of generic activators and position specific inhibitors can all be understood as the most quickly realized solutions to the fitness optimization problem. We do not see the creation of new modules in response to strong selection as necessary for the transition from fly and mosquito back to their last common ancestor (LCA). Rather via the logic of evolutionary bricolage [20], organic evolution and thus computation, should seek the most quickly evolved repurposing of existing components that connects the two defined endpoints subject to the constraint of viability for all intermediates. We will show that gap and pair-rule regulation in fly can be continuously adjusted to accommodate the observed changes in the posterior gap gene expression patterns. Given the range of times we have to cover, the high rate of churn in regulatory sequence among the Drosophlids [19] (with little effect on phenotype), and the changes in regulatory factors such as the absence of bicoid in Anopheles, it is thus most practical and informative to simulate the phenotype and ignore the molecular level. Phenotypic models have been informative in other areas [21,22] and in the present context fit quantitative genetic data as to how expression domains shift when upstream factors are altered. Similar approaches are found in [23], and [8]. We then use an evolutionary computation that initializes the network model with Drosophila parameters, and mutates and selects with a ‘fitness’ that directs the model towards Anopheles. Putting aside the specific molecular information we have for Drosophila makes our approach applicable to a wider range of problems. Invariably we find, eve stripe 5 disappears and either (or both) the eve 4+6 or 3+7 modules add a third posterior stripe to compensate. Thus the posterior eve stripes are not homologous in Drosophila and Anopheles. When we consider regulation of the other primary pair rule genes in fly, we conclude that the most plausible common long-germ ancestor of fly and mosquito employed a dynamic patterning system based on a forward shift in the eve pattern as observed in Clogmia and Drosophila [24] to impose phase relationships on the remaining pair rule genes. Thus there should be no homology in the posterior gap gene regulation of run, h, or ftz between fly and mosquito. We emphasize that no computation, no matter how complex, will ever prove one evolutionary scenario over another. Computation is at best a heuristic tool to uncover interesting hypothesis that one could not guess, and buttress those hypothesis by their fidelity with a quantitative phenotypic model for regulation. The computation is like a screen for all solutions to an evolutionary problem given defined rules. To the extent the ingredients of the phenotypic model are plausible and transparent, and the predictions intuitive, they may stimulate experiments. The main lesson of two decades of quantitative analysis of Drosophila segmentation is that positional information of pair-rule stripes is essentially defined by gap-gene repression (see e. g. review in [5]). Gap genes themselves are positioned by a mixture of cross-repression [9,25] and activation provided by maternal gradients. We will build our genetic or phenotypic model for Drosophila by defining an interaction kernel for each gap gene and pair-rule regulatory module. The kernel takes the numerical values of the inputs and outputs the expression. The general functional form is given in the S1 Text, and the specific inputs shown in the next subsection. The evolutionary algorithm that will produce the Anopheles network is allowed to change only the numerical parameters within the kernel functions. Thus the parameters that define the maternal to gap regulation, interactions among the gap genes, and their regulation of the pair rule genes all change. The algorithm does not create new kernels nor add new inputs to existing kernels, but it is important to include from the start all potential regulatory inputs that might play a role during evolution, even if their effect is minor in Drosophila. The output of a kernel is allowed to become 0 signifying its elimination. This conservative choice for the allowed ‘mutations’, was motivated above, and justified here. Firstly we show that the desired conversion from fly to mosquito can be realized without adding new kernels, and merely modifying existing ones. Binding sites turn over rapidly in modules so parameter evolution in existing kernels should be fast, while creating kernels in the absence of directional selection in anticipation of a future need is more speculative, and arguably slower. Secondly the anterior (roughly eve stripe 4 and forward) gap gene pattern in Anopheles and the intermediate species is largely invariant, while several of the pair-rule gene modules control both an anterior and a posterior stripe. Since we will impose that the anterior regulation is invariant, it was most logical to keep the inputs to these two stripe kernels invariant also. Once the allowed mutations are defined, the algorithm proceeds by rounds of mutation-selection. A population of networks is initialized to the Drosophila parameters, each network is mutated and retained if it is more fit than its parent. The most fit half of the population is duplicated and forms the next generation. Details on the code can be found in [26,27], and our code is available upon request. The function (negative fitness) that we want to minimize for each network is a sum of terms measuring (1) deviation of the posterior hb and gt profiles from the Anopheles pattern (2) deviation of the anterior eve profile from Drosophila. In addition there must be at least 7 eve stripes. From [10] we know hb moves forward in mosquito, while posterior gt is weak and probably plays no role in patterning so we assume it’s absent. Note we constrain the expression profiles, so evolution has to find a way to alter the posterior hb kernel to move its expression forward and match Anopheles. When we include a second pair rule gene, ftz, to define the 14 stripe segment polarity pattern, we insist its stripes alternate with those of eve. Nothing about intermediate species such as Clogmia is assumed. Once the evolutionary path to Anopheles is understood, and with it the regulation, the homology of the 6 Clogmia stripes becomes obvious without any further computation as we explain below. We do not impose that the eve stripes be of equal width, though in a number of instances we checked that local parameter optimization can readily satisfy this constraint, see e. g. S4 Video. Normal Drosophila segmentation is known to be extremely precise, [28,29]. However considerable change in eve expression in the blastula is not incompatible with adult viability. An early example was induced by variable bcd dosage [30]. Later examples include loss of parasegments 7 and 11 [31], and even abdominal segment A5 [32,33], with further details left for the discussion. Some variation in phenotype is essential for evolution. Since one can only claim heuristic value for our evolutionary computations, trying to better define the fitness costs of quantitatively imperfect patterns adds more uncertainty than it resolves and encumbers a simple story. The starting point of our simulations is an idealized Drosophila shown in Fig 1. S1 Text details our assumptions, we summarize their main features below. There are maternal input gradients, bcd anterior and cad posterior, repressed by bcd. bcd is frozen throughout the simulation. While there is no bcd in mosquito, we assume some other gene such as otd takes its place [34]. In addition we have fixed profiles of tailless (tll) and huckebein (hkb) in the posterior. Those gradients supply positional information to the gap genes, hb, gt, kni, Kr, which are the only ones we need to follow during the evolution. At the phenotypic level we consider, gap domains look very similar in Drosophila and Anopheles, the main difference being the posterior exchanges between hb and gt. Our description of the kernels defining gap territories incorporates regulatory interactions inferred from genetics, that are presumably conserved in evolution given the observed similarities of the gap patterns (see details in S1 Text). Repression comes from more than the immediately adjacent gap genes, since when these are mutated, expression typically does not extend to the anterior or posterior pole of the embryo. We omit other potential interactions because they do not impact the conserved qualitative gap pattern and would further require detailed molecular data to be fit in a species-specific manner [9,35]. The reader will observe that all gap gene expression patterns along the computed pathways from fly to mosquito remain fixed in size, suggesting we are not omitting any essential interactions as gt and hb interchange. For eve we include only stripes 2 to 7. (We do not simulate eve stripe 1 because we focus on the posterior regulation, and its regulation is decoupled from the other stripes.) and thus have four eve modules to consider eve 2 [36], eve 3+7, eve 4+6 [14,37] and eve 5 [13]. There is good genetic evidence, reinforced by bioinformatic studies [5,7], that their position is largely defined by gap gene repression. We include more than the minimal interactions required to fit the wild type eve and gap gene patterns in the posterior since hb and gt domains interchange as we evolve to mosquito, and mutagenesis experiments in fly suggest stripe regulation by more than the closest gap genes. For instance, in a hb mutant background, neither eve 6 nor eve stripe 7 expand much in the posterior [13] and in a gt mutant, eve 5 stripe only extends posterior to eve 7 stripe [13]. Thus there must be additional repression from the posterior that we assume comes from tll. We allow a uniform activator for stripes 3–7 and 4–6 (supplied by DSTAT [7,37] or Zelda [38,39]), but in our framework no positional information is given by activators. There are a similar set of ftz modules defined by gap gene repression in Fig 1. ftz 4 represents a special case in that there is no stripe specific element and it appears that ftz stripe 4 is only expressed as part of the 7 stripe ‘zebra’ element [5]. Thus ftz has partially the character of a secondary pair rule gene that takes input from other primary genes, a fact that will be important in the following. Simulations begin from the Drosophila network in Fig 1 and target the Anopheles gap pattern as an end point. The number of eve stripes (including 1) must be at least 7, and there is no restriction on their relative size or position. A typical example of such simulation is provided on Fig 3, with intermediate steps pictured on the phylogeny in Fig 2A. As the posterior hb domain moves forward it splits eve stripe 7. The repression from hb that defined the posterior boundaries of eve 6–7 gradually shifts to tll. Stripe 5 transiently fragments into two additional domains, Fig 3B, neither of which emerges as an distinct stripe. But once eve 7 splits in two, the stripe 5 element can disappear while respecting our constraint of at least 7 stripes. After it disappears posterior gt, is superfluous. A variation on this pathway is presented on Fig 4. This time the evolution of the posterior hb domain anterior, splits eve 6 to create a new eve 8. Once a new eve stripe appears in the posterior (Fig 4B), posterior gt first disappears so that eve 5 expands posteriorly, fusing with eve 6 (thus effectively disappearing, (Fig 4C). Thus the eve 5 stripe module is no longer needed and disappears, leading to a final configuration similar to Fig 3D. The evolutionary scenario with creation of a new eve stripe in the posterior and subsequent removal of eve 5 and gt posterior is highly reproducible in our simulations for a variety of conditions that implement the same evolutionary pressures. Thus stripes 4+6 and 3+7 in Drosophila become stripes 4+5 and 3+6 in Anopheles. Furthermore one of these Drosophila modules controls 3 stripes in Anopheles. On Fig 3, Drosophila eve3+7 gives rise to stripes 3,6, 7, while on fig 4, Drosophila eve4+6 gives rise to stripes 4,5, 7. In both cases, one eve stripe is split by hb to give two stripes in the posterior that are symmetrically positioned around the hb domain. The 8 stripes in Anopheles would be most easily explained if both modules grow another stripe with Drosophila eve 4+6 (resp. 3+7) becoming 4,5, 8 (resp. 3,6, 7) in mosquito. Our simulations also predict that intermediate dipterans must have retained this logic where eve 4+5 (resp. 3+6) are homologous to the eve 4+6 (resp. 3+7) module in Drosophila. Both modules are repressed by kni and hb, so in particular stripes 4–5 and 3–6 should be laid symmetrically with respect to kni in those intermediate dipterans. This is a prediction of our computation, that exploits the known gap gene regulation, but was in no way imposed. Strikingly, eve stripes 4+5 and 3+6 in both Clogmia and Anopheles are indeed laid rather symmetrically with respect to kni contrary to the situation in Drosophila. Furthermore Clogmia has only 6 eve stripes prior to gastrulation and consistent with our model, lack the posterior hb domain that generated the two additional posterior stripes in Anopheles. We could not find examples of viable mutant flies missing 2 consecutive segments (corresponding to one full pair-rule period). This suggests that, even if in some mutants (e. g. the hopscotch mutant [32]), when one eve stripe disappears, the embryo needs to keep some polarity information required for the definition of parasegments. Since eve 5 overlaps and defines A4p and A5a, proper parasegment definition means that the polarity of A4a and A5p must be maintained (and subsume cells that were in A4p and A5a). A natural hypothesis is then to assume another pair rule gene, out of phase with eve, must persist when eve stripe 5 disappears to provide input to the segment polarity system. We chose to add ftz to our model. We recognize that the proximate input to the segment polarity genes is not directly from eve and ftz but we have to insist that the model respect the minimal information logically required for the segment polarity pattern. Thus when an eve stripe disappears, the neighboring ftz stripes merge and only one parasegment disappears. (We will consider below how the constraints on evolution imposed by the other primary pair-rule genes in Drosophila [5] can be satisfied, if we insist that the relative phase among the pair rules genes is maintained.) In a first round of simulations where we model ftz as a primary pair-rule gene (and postulate a pure ftz stripe 4 module delimited by hb on its anterior side and gt posteriorly), the evolutionary pathway observed in the previous section dies. There are several reasons for this: first gt controls both eve 5 and the putative ftz 4, so it is very difficult to have it disappear given this dual role while keeping the ftz/eve alternation that we impose. Second, if a new eve stripe appears in the posterior as before, it has no reason to be coupled properly to a corresponding alternating ftz stripe. Essentially, if ftz is primary, simulations fail to evolve new eve posterior stripes without breaking the alternation of ftz/eve. It is thus interesting in this context that ftz stipe 4 appears only together with the 7 stripe zebra element [5]. Thus if we allow repression of ftz 4-zebra by eve and not gt, it becomes slaved to eve in the posterior and functions as a secondary pair rule gene. In the simulation, Fig 5, ftz stripes 5,6, 7 that are positioned by gap genes, gradually disappear in favor of the zebra element. The modules that controlled pairs of stripes 1+5,2+7 and 3+6 now control only the anterior member and can evolve to interdigitate with the eve stripes. With the posterior ftz stripes controlled by eve repression, the pattern can evolve to the Anopheles configuration as before while preserving eve and ftz alternation throughout, Fig 5. When eve 5 disappears ftz 4 and ftz 5 merge (since eve repression is keeping them distinct) Fig 5B and 5C, thus preserving the eve, ftz alternation. The fact that our evolutionary simulations fail when ftz is purely primary and succeed when ftz is more secondary suggests that it will be the same for other primary pair-rule genes such as h and runt. We nevertheless need to ask how the relative phase of the primary pair rule could be conserved in the evolutionary scenarios presented here. We propose, by means of a quantitative model, that pair-rule regulation in the posterior of the LCA is more dynamic than conventionally assumed in Drosophila. This will imply that there is no homology between the posterior regulation of the pair-rule genes by gap genes, other than for eve itself. Specifically both in Drosophila [40] and Clogmia [24]eve stripes move from posterior to anterior prior to gastrulation. Our idea is that suitable combinations of strong and weak repression among run, h, ftz, and eve can read this phase information and stabilize the pair-rule pattern we observe in Drosophila, without direct gap gene input. The model is related to the pair-rule gene oscillator that patterns the posterior of short germ insects, as previously suggested in [41]. (However the model is not capable of intrinsic oscillations since eve is driven by gap genes and not by other pair rule genes, though it is easy to envisage how intrinsic pair rule feedback on eve could be gradually replaced by extrinsic gap regulation during the short to long germ band transition.) Viewed within a single cell, the forward displacement of eve appears as one complete temporal cycle, thus a gene regulatory network derived from a delayed negative feed back oscillator among the pair rule genes can use the same interactions to produce stable phases in space. In certain respects our conjectured LCA resembles Nasonia [42] where the segments posterior to A5 are patterned dynamically as we reconsider in more detail in the Discussion. To implement our model, we control the maternal gradients to move the gap-genes forward and they drag eve along with them. The maternal gradients are adjusted to induce a forward shift of precisely one period in the eve pattern, Fig 6. Then for both the Drosophila and Anopheles gap gene patterns, the interactions shown in Fig 6A will direct an arbitrary expression pattern for the pair-rule genes other than eve to stably assume the relative phases we know for Drosophila, (or any other one by adjusting the strengths of repressions, see S3 and S4 Videos). For demonstration purposes only, we applied our model to the entire anterior-posterior axis, though we expect as in Fig 5 that the anterior gap gene regulation can persist. More detailed models of the gap gene network reproduce directly the anterior shift that we put in by hand [9,35]. However they take as input the dynamic maternal gradients (in particular cad), and further observe that bcd itself is dynamical, which is consistent with what we assumed. A difference is that their models, along with [23], aim to reproduce precise developmental dynamics, while we have sacrificed this level of detail and prefer to reveal parsimonious phenotypic mechanisms that have a greater claim to validity over the large evolutionary distances we cover. In our simulated evolution from Drosophila to Anopheles, eve 5 always disappeared. Thus an important consistency check is to show how eve 5 can appear when evolving from a LCA as appears in Figs 3 and 5C, to Drosophila. The solution was already suggested in Fig 4C, when a weak eve 2 stripe emerged from the stripe 5 module. Indeed these two stripes share the Kr and gt repressors. In Fig 7, we indeed see that when the posterior gt domain moves anterior the LCA-eve 2 module develops a second stripe, (and we imagine a distinct stripe 5 later evolves by drift). The tripartite 3,6, 7 stripe loses its last component as hb moves posterior, and a reasonable Drosophila pattern is restored. Our LCA will generate a Clogmia like pattern if we remove posterior hb, illustrated on??. There also is one eve stripe less than our presumptive LCA and Drosophila, because hb is not here to split ancestral eve 3–6 module in two in the posterior. Both these features qualitatively correspond to the observed pre-gastrulation Clogmia pattern, with only 6 stripes (vs 7 in Drosophila). We have used computational evolution and the observed gap gene patterns in Drosophila and Anopheles to suggest how the gap and pair-rule network evolved between these species and their LCA as well as the relation to Clogmia. Our strongest conclusions are that Anopheles eve stripes 3,6, 7 (resp. stripes 4,5, 8) are derived Drosophila eve modules 3+7 (resp. 4+6) by the elimination of Drosophila eve stripe 5, and the forward shift and renumbering of stripes 6,7, Fig 2B. Stripes 7 and 8 in Anopheles are the reflection of stripes 6 and 5 respectively in the repositioned hb domain. The Clogmia pattern follows Anopheles, except for the elimination of stripes 7,8 which are generated by the posterior hb domain, which is absent in Clogmia. Drosophila eve 5 arises from stripe 2 of the LCA. Our model is formulated entirely within a phenotypic or genetic description of the regulatory network, yet generated surprising, but after the fact, plausible predictions. The homologies between the eve stripe 3+6 and 4+5 modules in Anopheles or Clogmia and Drosophila could have been guessed from their symmetries around the kni domain, but we are not aware of a reference to that effect. But it could not be guessed that the continuous transition from Drosophila to Anopheles could be accomplished merely by adjusting the parameters within the regulatory kernels defined by Drosophila. This is surely the most parsimonious route between fly and mosquito, and perhaps the most rapidly evolved since it only requires mutating binding sites in existing gene regulatory modules which we know to be rapid [19,43]. The complex structure of Drosophila regulation, such as duplicate enhancers, only becomes implicated in the evolutionary transition if they were found to persist in intermediate species. We can never preclude more complex scenarios, such as new modules, but their rate of evolution is uncertain in the absence of positive selection. Thus our computation is a useful heuristic tool to show that the desired transition can be accomplished by reparametrizing existing kernels without creating new ones, via evolutionary bricolage [20]. The most immediate tests of our predictions require identifying pair-rule gene regulatory modules in Anopheles and in fact none has been found in that species or Clogmia to our knowledge. The most expeditious route to their discovery, with modern technology (e. g. [44]) would be CHIP-seq with antibodies against the gap genes. Putative binding sites could be refined computationally and then clusters of them could be matched numerically against the regulatory regions of relevant genes, [45,46]. In Drosophila, computationally defined clusters of binding sites were very successful in reconstructing gap and pair-rule regulation and this approach could in principle be applied to other species. Our first prediction is the existence of eve modules in Clogmia with purely kni hb and tll binding sites and suggestive of their expression as stripes 3+6 and 4+5, and the analogous prediction of putative 3-stripe modules in Anopheles. A more dramatic confirmation of theory would be observing the expression of these modules in Drosophila. This requires sufficient homology between the gap gene proteins, which is not a given, since computational screens of these other genomes with the Drosophila binding site weight matrices has yielded nothing. However the conservation of enhancer function between Drosophila and Tribolium, in several cases gives one hope [47,48]. More speculatively, our model requires a LCA where the posterior pair-rule genes other than eve derive their phasing from the anterior shift of eve observed in Drosophila and Clogmia [24,40]. The connection between an anterior shift of the posterior pair-rule genes and a segmentation clock was made in the discussion of a paper that revealed that mechanism in Tribolium, [41], but is speculative. Our model concerns only long-germ dipterians. Recent work on Nasonia, a long-germ band Hymenoptera, provides a very informative bridge between short and long germ band insects and the state we impute to our LCA. Hymenoptera is an out-group for the order Diptera considered here [42,49]. Nasonia gap/eve pattern is qualitatively very similar to Drosophila in the anterior part of the embryo [50,51], precisely until abdominal segment A5 (corresponding to ftz 5 in fly) [42] which is the position where some strong variability between species in gap/eve is observed in our simulations. Posterior to this segment, Nasonia pair-rule pattern presents all the characteristic of an insect segmentation clock, with eve on the top of the hierarchy controlling waves of expression of odd [42]. (Note eve has the segmental period in the posterior, as also seen in the centipede Strigamia [52].) Other pair-rule genes necessary to set proper segment polarity appear downstream of this clock system [42]. We chose ftz as the second pair-rule gene in the simulations to define the 14 parasegments since it regulates engrailed and has its zebra regulatory element that allowed the simulation to position the posterior ftz stripes by repression from eve. odd might seem a more logical choice to define the 14 parasegments, since it is part of the posterior oscillator in short germ insects, but the simulation would encounter the same difficulty as found for ftz, namely it is impossible to find paths for posterior hb and gt that are compatible with the known gap gene regulatory kernels in Drosophila and preserve the pair-rule gene alternation. If we combine the phylogenic evidence from Nasonia with our inability to evolve multiple pair-rule genes with purely gap gene regulation, then an alternate conceptual distinction between primary and secondary pair-rule genes naturally arises, along the lines already suggested in [53] using data from Strigamia (see also data from Glomeris [54]). Primary pair-rule genes are those involved in the posterior segmentation clock, the secondary genes take input from the primary and control the segment polarity layer. Delayed negative feedback is a natural way to build an oscillator with a stable period. If the segmentation clock operates by phased sequential repression among the primary pair-rule genes then the same repression could operate in space, anterior to the oscillating growth zone, to fix the relative position of these same genes with the same relative phases. (A related conversion of a temporal signal to a static one was derived in a prior study on the evolution of Hox patterning during the short to long germ transition [55].) This is a prediction that could be tested in Tribolium [56]. We have assumed that in the ancestral short to long germ transition (or fly to mosquito), it is eve that first acquires gap gene input and breaks the negative feedback oscillator, based on circumstantial evidence, but this is not a logical necessity of the model. If we are correct that the LCA used the anterior shift of eve to set the relative phase of the other pair-rule genes, then the posterior regulation of these genes by the gap genes would be recent and derived, and it should not be the basis for classifying primary vs secondary. Thus we would not expect any homology between the posterior gap gene input to the pair-rule genes other than eve in fly and mosquito. This proposal is difficult to test since convergent evolution is a real possibility here, since any module will use the gap genes that are appropriately positioned for its regulation. Most evo-devo studies involve close enough species that there is no question that intermediates are viable. However the LCA of fly and mosquito was more than 200 million years ago [57] and we are proposing an evolutionary chain of events in the blastula and presuming viable adults exist along the way! The best evidence we can offer, is the hopscotch mutants [32] (a component of the Jak-Stat pathway). A maternal hypomorph rescued by a wildtype male, loses A5, yet gives rise to fertile flies of both sexes. So in this mutant, no essential part of the anatomy is lost with A5. The gap gene expression is unaffected, but stripe run 5 is absent, and eve 3/5 are suppressed, eve 5 more so than eve 3 [33]. The embryo tolerates other abdominal segment loss, e. g., reduced expression of eve 4/6 results in loss of two abdominal segments but viable adults [31]. If we consider the Hox genes as the basic mediators of segment identity then based on expression, abdominal segments 2–7 are identical [58], but more subtle differences in Hox regulation remain [59] Our modeling differs from earlier work that focused more on the developmental dynamics of gap gene expression in Drosophila, such as [8,9]. As noted in [9], fitting dynamic data will be difficult to scale up for more complex pathways, and these authors did not consider the pair rule genes. Thus it might prove challenging to study significant evolutionary changes with such detailed models. Our coarse-grained description, relying on minimal interaction (in a spirit similar to [23]) allows us to model long evolutionary time-scales, moving from microevolution to mesoevolution [60]. Our approach illustrates the interest of phenotypic models for evolutionary systems biology. It gives a quantitative framework to make qualitative predictions (such as “one stripe appears in this region while one stripe disappears in another region”) using semi-quantitative phenotypic data directly obtained from experiment (here, gap gene positioning and constraints on stripe number/alternation). The ability to generate novel predictions directly from currently available measurements is thus of interest for a broad swath of biological modeling [21,27]. | The last common ancestor of the fruit fly (Drosophila) and mosquito (Anopheles) lived more than 200 Million years ago. Can we use available data on insects alive today to infer what their ancestor looked like? In this manuscript, we focus on early embryonic development, when stripes of genetic expression appear and define the location of insect segments ( "segmentation"). We use an evolutionary algorithm to reconstruct and predict dynamics of genes controlling stripes in the last common ancestor of fly and mosquito. We predict a new and different combinatorial logic of stripe formation in mosquito compared to fly, which is fully consistent with development of intermediate species such as moth-fly (Clogmia). Our simulations further suggest that the dynamics of gene expression in this last common ancestor were similar to other insects, such as wasps (Nasonia). Our method illustrates how computational methods inspired by machine learning and non-linear physics can be used to infer gene dynamics in species that disappeared millions of years ago. | lay_plos |
While sensory neurons carry behaviorally relevant information in responses that often extend over hundreds of milliseconds, the key units of neural information likely consist of much shorter and temporally precise spike patterns. The mechanisms and temporal reference frames by which sensory networks partition responses into these shorter units of information remain unknown. One hypothesis holds that slow oscillations provide a network-intrinsic reference to temporally partitioned spike trains without exploiting the millisecond-precise alignment of spikes to sensory stimuli. We tested this hypothesis on neural responses recorded in visual and auditory cortices of macaque monkeys in response to natural stimuli. Comparing different schemes for response partitioning revealed that theta band oscillations provide a temporal reference that permits extracting significantly more information than can be obtained from spike counts, and sometimes almost as much information as obtained by partitioning spike trains using precisely stimulus-locked time bins. We further tested the robustness of these partitioning schemes to temporal uncertainty in the decoding process and to noise in the sensory input. This revealed that partitioning using an oscillatory reference provides greater robustness than partitioning using precisely stimulus-locked time bins. Overall, these results provide a computational proof of concept for the hypothesis that slow rhythmic network activity may serve as internal reference frame for information coding in sensory cortices and they foster the notion that slow oscillations serve as key elements for the computations underlying perception. Oscillatory activity generated by local cortical networks is considered to be a crucial component of sensory processing [1], [2], [3] and has been implicated in processes such as the temporal binding of neural assemblies, the control of information flow between areas [4], [5], or the multiplexing of information across different time scales [6], [7]. Theoretical work has also proposed a critical role for slow oscillations in temporally organizing the information carried by prolonged neural responses [8], [9], [10], [11], [12], [13]. Sensory information provided by neural firing patterns about naturalistic stimuli is often stretched over periods of several tens to a few hundreds of milliseconds, and the full information provided by such responses can only be extracted when considering the extended firing pattern as a whole [14], [15], [16], [17]. For example, hippocampal place cells encode the current position of the animal in space, yet meaningful trajectories can only be read out when observing the activity of such populations over periods of several hundreds of milliseconds [18], [19]. In addition, natural stimuli such as sounds or movies entrain cortical activity on slow time scales and require the readout of response patterns over several tens to hundreds of milliseconds to correctly decode different scenes [14], [20], [21], [22]. Such extended and time-varying representations can be correctly interpreted only when the decoder is able to partition the full response into smaller chunks of a few tens of milliseconds, and to correctly position these chunks relative to each other within the neural response and relative to the sensory input [16], [17], [23]. For data analysis, such temporal partitioning is usually performed by aligning single trial responses relative to stimulus onset and dividing them into equally-spaced and precisely stimulus-locked time bins. While this is easily done by the experimenter, it makes the assumption that the decoder has access to a highly precise clock. Aligning neural responses to the stimulus requires the decoder to have precise knowledge about the timing of sensory events (e. g. stimulus onset). In addition, the ability to partition longer spike trains into smaller patterns requires either a nearly perfect representation of time intervals (the analogues of “time bins”) or the ability to represent multiple reference time points during a temporally extended stimulus with high precision. Sensory cortical circuits, however, do not have access to the experimenter' s clock and have to rely on intrinsic (either absolute or relative) timing mechanisms [24], [25], [26]. While population responses or the responses of specific subsets of neurons have been suggested as a potential intrinsically available signal of stimulus onset [24], [25], it remains unclear what intrinsic timing signal is exploited to partition longer spike trains. Slow oscillations with cycle lengths of 100 ms or longer (such as delta or theta band rhythms) have been proposed as basis for temporal response partitioning [13], [19]. Oscillation-based partitioning can, for example, be achieved by considering different oscillatory phase angles as partitions of the longer period represented by the full cycle, effectively creating a serial order of partitions within an oscillation cycle. Indeed, work on the hippocampus has suggested that hippocampal theta oscillations can be used as internal temporal reference frame to reconstruct firing assemblies and to decode single neuron' s responses [8], [13], [19],. Importantly, such an oscillatory reference frame is intrinsic to the cortical network and its specific timing parameter, i. e. the oscillatory phase, is likely to be directly accessible to the local network [13], [29]. When considering sensory cortical structures, however, the role of oscillations as a network-intrinsic reference has been mostly treated at a conceptual level and the degree to which slow oscillations are useful for partitioning spike trains into temporal units of information remains to be investigated in a quantitative way [10], [13], [30]. Part of this problem is methodological as long spike trains partitioned into a finely timed pattern constitute a high dimensional neural code, for which it is challenging to estimate sensory information due to dimensionality issues and a lack of viable models of sensory encoding [31], [32]. Because of such difficulties, prior work on the complementarity of stimulus information in spikes and the phase of slow oscillations has concentrated mostly on short time scales of a few tens of milliseconds [12], [20], [21]. As a result, previous studies have succeeded in revealing the complementarity of information in instantaneous and stimulus locked spike patterns and oscillatory phase, but did not address whether the phase might provide an effective temporal reference to partition longer responses into informative units over the scale of a few hundreds of milliseconds. The specific goal of this study is to quantitatively test the hypothesis that slow oscillations can serve as a reference for partitioning spike trains of tens to hundreds of milliseconds into a highly informative code without requiring an external timing reference. Stimulated by the observation that naturalistic stimuli entrain slow cortical activity [6], [33] and that such oscillatory activity likely plays a key role in sensory perception [10], [34] we focus on slow oscillations (<30 Hz) as a putative reference. We systematically compare the information carried by codes that establish temporal relations between spikes either by the binning of spikes into stimulus-locked and equally spaced time bins or by the binning of spikes using the phase of an oscillatory signal. We apply this analysis to evaluate the sensory information carried by single neuron responses recorded in primate auditory and visual cortices during stimulation with long stretches of naturalistic stimuli. We begin by illustrating the concept of partitioning a neural response during a time window T using either a stimulus-locked or an oscillatory reference. The timing of individual spikes, such as in the illustration in Fig. 1A, can be measured using a laboratory-based clock, which registers the precise timing of the stimulus (e. g. at t = 0) and of each spike. This stimulus-locked timing is used, for example, when computing a classical peri-event time-histogram (PETH) using a sub-division of the time window T into smaller and equally-spaced time bins of length Δt (Fig. 1A). Counting the number of spikes per bin defines a neural code based on stimulus-locked partitioning. We denote the so defined single-trial response as the time-partitioned spike train (abbreviated as time-partitioned in the following Fig. 1B). While this temporal partitioning provides a convenient and frequently used representation of neural responses for analysis, it requires a highly accurate representation of time intervals needed to establish the equally spaced time bins Δt. This information is easily available to the experimenter, but it is not likely to be available to sensory cortical networks. One way to partition a spike train using a signal intrinsic to the neural network is to use the timing of spikes relative to the phase (the position within an oscillatory cycle) of periodic network activity [18], [20], [21], [35]. Here we consider slow rhythms (e. g. theta range, 2–6 Hz) in local field potentials (LFP) as an oscillatory reference, as oscillations in this frequency range have been implicated in sensory encoding in previous studies [6], [8], [10], [20], [33]. We use the phase of these LFP oscillations (recorded on the same electrode as the spikes) to construct time dependent responses that preserve the sequential order of spikes within the oscillation cycle. In other words, the phase (i. e. the position within a cycle) of the rhythm is used as temporal reference (i. e. as a virtual ‘time axis’) for the temporal organization of responses into distinct but possibly not equally-spaced epochs. In the example we colored four quadrants of the phase angle and labeled spikes falling within each quadrant with the corresponding color (Fig. 1A). The phase-partitioned spike train code (abbreviated as phase-partitioned) was constructed, within each time window T, as the number of spikes occurring within each phase quadrant (Fig. 1B). This definition of a neural code is similar to previous studies on hippocampal place cells that have explored the role of theta phase precession in providing information about the animal' s location in space [19]. It should be noted that a priori both codes capture distinct and potentially independent aspects of the response. However, if the oscillation is well aligned to the sensory stimulus, both codes will likely carry related patterns of stimulus selective responses. In fact, the main result of our study is that because slow oscillations in auditory and visual cortex are stimulus entrained both codes carry related information and the phase-partitioned code can provide a large proportion of the information that is extracted by time-partitioned responses. However, it does so without relying on a precise and stimulus-locked clock. For comparison, we also quantified the information provided by the total spike count within the same window. This code provides an estimate of the information that can be extracted without temporally partitioning responses within the time window T and serves as a reference to compare the additional information that can be obtained using the two partitioning schemes above the information available in the spike count. We implemented the spike count using a bin-shuffling procedure that preserves the dimensionality of the time- and phase-partitioned codes and using a 1-dimensional representation. Both yielded very comparable results. To quantitatively assess the effectiveness of the oscillatory phase in partitioning spike trains in comparison to other codes we used a framework of single trial decoding. We applied this analysis with a wide range of parameters and to different data sets obtained from primary auditory and visual cortices of non-human primates. We compared the stimulus discrimination performance in the phase-partitioned code to the spike count in order to assess the gain by introducing a phase-based temporal partitioning. And we compared the phase-partitioned to the time-partitioned code to judge the performance of the oscillatory phase in partitioning spike trains against an ideal external observer with independent precise knowledge of the time course of both neural and sensory events. The auditory system is often faced with a stream of sounds and has to represent individual sound objects within a continuously evolving environment [10], [36]. Examples are individual words in a spoken sentence, a melody in a song or individual sounds appearing in a cacophony of environmental noises. To quantify the performance of each of the proposed codes in such a scenario, we analyzed neural responses recorded from primary auditory cortex of awake animals during the presentation of a 52 second continuous sequence of naturalistic sounds, such as animal calls and environmental sounds (40 responsive neurons recorded from 23 recording sites in three animals, from [20]). To quantify the stimulus discriminability afforded by each code we randomly sampled sets of 10 epochs from the long sound sequence and used these epochs as ‘stimuli’ for the decoding analysis (Fig. 2A). Fig. 2B displays the response of one example neuron to one set of stimulus epochs. The raster plots display the spike trains evoked on individual repeats of the sound sequence. To illustrate the temporal organization of the responses with respect to the oscillatory phase we colored individual spikes according to the phase of the theta LFP (2–6 Hz) at the time of spike. To illustrate the stimulus selectivity of different neural codes, the right-hand panel displays the trial-averaged responses to each stimulus for each code. The stimulus dependence of these average responses is visible in the different profiles of the time or phase-partitioned responses, or the difference in overall spike count across stimuli. We systematically compared the decoding performance across a range of time bins, window durations and frequency bands used to extract the phase, every time averaging decoding performance over 100 sets of randomly selected stimulus epochs to ensure the generality of results across a wide range of acoustic inputs. Across this parameter range, decoding performance was consistently highest using the time-partitioned code and lowest using the total spike count. For example, when using 8 bins of a theta band (2–6 Hz) oscillation to divide a 160 ms time window T the decoding performance was 23. 6±2% (correct discriminations, chance level 10%) for the time-partitioned code, 20. 4±1. 5% for the phase-partitioned code and 14. 8±0. 5% for the spike count (mean ± s. e. m., n = 40 units; Fig. 3A). Differences between all codes were statistically significant (paired t-tests, at least p<10−3), showing that temporal response partitioning using the oscillatory phase constitutes a code that affords a higher level of stimulus discrimination than provided by the spike count. To directly assess the difference in decoding performance between partitioning schemes on a neuron by neuron basis we calculated the relative difference in performance of time- and phase-partitioned codes to the spike count. Given that both time- and phase-partitioned codes implicitly include the spike count, the excess information in either partitioning scheme reflects the amount of stimulus discrimination that is made available by each partitioning scheme above and beyond what is available from the spike count [6], [37]. The result (Fig. 3B) demonstrates a consistent increase of decoding performance when using phase-partitioning over the spike count for each individual unit, and demonstrates that the time-partitioned code provides only a little more information than the phase partitioned code. Indeed, the excess performance in the phase-partitioned codes was close to that of the time-partitioned code (91±2%). Importantly, the excess information in either partitioning scheme over the spike count was highly correlated across neurons (spearman-rank correlation r = 0. 87). Good stimulus discrimination afforded by one partitioning scheme hence implies good discrimination performance also in the other. This correlation of performance across neurons already suggests that both codes effectively capture similar aspects of the neural response. This can be understood intuitively by onsidering the fact that, while the two partitioning schemes rely on the precise alignment of spikes to two potentially very different reference frames (one based on an external timing signal, one based on intrinsic brain activity), in practice the two reference frames are correlated because low frequency oscillations are often entrained by the dynamic environment [20], [33]. This makes it possible that both schemes carry largely similar information. To directly quantify the overlap in decoding performance between the two partitioning schemes we performed additional calculations. First, we calculated the information in a dual-code consisting of both time- and phase-partitioned responses. If these codes would characterize the same or similar response features, the information in the dual-code should exceed the information in the best individual code by only a small amount. However, in case both would characterize independent response aspects, performance in the dual code should by far exceed the best individual code. We found that performance in the dual code was only 4±1% above the best individual code (Fig. 3C), demonstrating a high degree of overlap in stimulus selectivity. Second, we directly investigated the impact of oscillatory trial-by-trial phase alignment on the performance of the phase-partitioned code. Oscillatory phase alignment of the theta LFP was measured for each stimulus epoch using the average phase coherence during that epoch; the phase coherence was computed across trials and averaged over time points during the stimulus epoch. This phase coherence value indicates how well the oscillation is locked relative to the sensory stimulus (hence to the time-partitioning reference) within each epoch. For each unit and for each of the three codes we correlated the phase coherence with the decoding performance across stimulus epochs. This revealed a considerable correlation between phase coherence and decoding performance in the phase-partitioned code (median correlation r = 0. 31; Fig. 3D), and (as control) considerably weaker correlations between phase coherence and the performance of the time-partitioned codes (r = 0. 1) and the spike count (r = 0. 07). These finding were robust to the number of bins used to divide the time window or the phase range and to the length of the time window T within which the neural response was considered. We varied both parameters independently (Fig. 3E, Supplemental Fig. S1) and found that the phase-partitioned code provided good discrimination performance regardless of whether the time window T was shorter (e. g. 80 ms) or longer (e. g. 320 ms) than a typical cycle of the slow rhythm used to derive the phase (the median cycle length of the 4–8 Hz band was 182 ms across sites). We also investigated how the information carried by phase-partitioned code depends on the specific frequency band used to derive the phase. Previous work has suggested that stimulus specific information in auditory cortical field potentials is highest for low (<8 Hz) frequency oscillations [20], [33], [38], [39]. We found that this also holds in the present setting, where the oscillatory phase is used to partition longer spike trains into stimulus-specific response patterns. Specifically, we computed the ratio of the decoding performance in the phase-partitioned response relative to the information in the spike count when considering different frequency bands (Fig. 3F). The performance gain in the phase-partitioned code relative to the spike count was largest when deriving the phase from theta-band oscillations (2–6 Hz), mean ratio 2. 58±0. 38 and was significantly smaller when using e. g. beta (14–18 Hz, ratio 1. 34±0. 1, t-test p<10−5) or higher (26–30 Hz, ratio 1. 1±0. 08, p<10−6) frequency bands. This result was independent of the specific choice of filters used to derive the frequency band (Supplemental Fig. S2A). To test the validity of these findings in a different sensory modality, we repeated the above analysis on data recorded in primary visual cortex during the presentation of commercial color movies (37 responsive neurons recorded from 37 recording sites in four animals). The example data in Fig. 4A illustrate the selectivity of each code for a set of scene epochs extracted from the long (several minutes) video presentation. As for the auditory data, we found that partitioning spike trains using theta range (2–6 Hz) oscillation phase provided significantly better decoding performance than obtained from the spike count (T = 160 ms, 8 bins; spike count: 17. 9±0. 4%, phase-partitioned code: 23. 6±0. 7%; paired t-test p<10−8; n = 37 units; Fig. 4B). The difference in decoding performance between partitioning based on phase and stimulus-locked time-bins was quantitatively small, though significant (time-partitioned code: 24. 8±0. 9%; p<0. 05). Still, the excess information in the phase-partitioned code over the spike count was nearly that of the excess information in the time-partitioned code (96±2%). Hence, in this dataset partitioning by the oscillation phase provides a code that is nearly as informative as partitioning using stimulus-locked time bins. As for the auditory dataset we found that performance in both partitioning schemes was highly correlated across neurons (Fig. 4C, median r = 0. 83), and that combining both codes provided only 6±0. 1% higher performance than the best performing individual partitioning scheme (Fig. 4D). Also as for the auditory dataset, the decoding performance using phase-partitioning was correlated with the oscillatory phase-coherence during the stimulus epoch (median r = 0. 26) and similar results were found when using fewer or more bins and when considering time windows of different duration (Fig. 4E). The performance gain in the phase-partitioning relative to the spike count was largest when deriving the phase from low frequency oscillations in the theta frequency range (Fig. 4F). This suggests theta-range rhythms as privileged candidates for intrinsically-available reference frames in sensory cortex. The above analysis has one potential limitation. While within the coding window T spikes are grouped using either partitioning scheme the analysis assumes that the time windows T themselves are perfectly aligned relative to each other across trials. Thereby we assume that the decoder can compare a single trial response with the across-trial distribution of responses at exactly the same position in the stimulus time course (Fig. 5A); i. e. the decoder hence relies on a ‘codebook’ (the set of all single-trial responses) that is sampled at a fixed position relative to the stimulus. This may not be a realistic scenario for actual sensory cortical networks [40], [41]. A downstream decoder might not know the post-stimulus time (neither at the millisecond nor the tens of milliseconds scale) at which a response was emitted and hence may not have access to the ideal codebook used above. We tested the robustness of the different codes to temporal uncertainty about the precise response alignment in the decoding process. Specifically, we simulated temporal uncertainty by incorporating a jitter Δt (randomly drawn in each trial from a uniform distribution between −J/2 and J/2, J being the degree of maximal uncertainty) in the alignment of responses across trials when deriving the codebook (Fig. 5B). We then evaluated the decoding performance for a range of values of the maximal time shift. This directly probes the robustness of each code to errors made by downstream decoders due to temporal uncertainty in the alignment of single trial responses and sensory events, and therefore provides a crucial test for the computational viability of neural codes under these more stringent and probably more realistic conditions. We found that the phase-partitioned code was more robust to temporal uncertainty than the time-partitioned code, both in the visual and auditory datasets (Figs. 5C, 5D, N = 8bins, T = 160 ms). Decoding performance in each code decreased with increasing uncertainty J, but this decrease was largest for the time-partitioned code. For temporal uncertainties of J = 80 ms or larger (i. e. half the coding window T) the phase-partitioned code provided significantly higher stimulus discrimination than the time-partitioned code (J = 80 ms, auditory data: time-partitioned 16. 7±1. 1%, phase-partitioned 18. 3±1. 2%; t-test p<0. 01; visual data: time-partitioned 20. 5±0. 8%, phase-partitioned 22. 4±0. 7%; p<10−4) and this difference was further enhanced for larger temporal uncertainties (Fig. 5C, 5D). This demonstrates that oscillatory phase provides a reference to partition temporal response patterns in a manner that is robust to temporal uncertainty in the decoding process and hence excels under conditions which are likely to be present in real cortical networks. Any neural code that is to operate under realistic conditions must not only provide information about clearly perceivable stimuli but must also be robust to noise in the sensory environment that often compromises stimuli of interest. We therefore performed additional analyses to directly quantify the impact of sensory noise on the different codes. Specifically, we analyzed auditory cortical data recorded using a stimulus set where a naturalistic target sound-sequence was systematically degraded by adding background noise [20]. The background noise consisted of a cacophony of natural sounds and a different noise mixture was added on each trial (Fig. 6A). The same target sound was presented either without noise or with noise of three different levels (labeled ‘low’, ‘medium’, and ‘high’; +6,0 and −6 dB r. m. s. intensity relative to the target sound). Decoding performance for all codes was reduced by the presence of background noise, and the reduction in performance was greater for higher levels of noise (Fig. 6B, n = 43 units; N = 8bins, T = 160 ms). In all conditions the time-partitioned code provided the highest decoding performance. However, the differences between codes reduced with increasing noise level. In the absence of background noise the average decoding performance differed significantly between all codes (pair-wise t-tests, at least p<0. 01). For the highest noise level, however, the phase-partitioned and time-partitioned codes provided comparable levels of decoding (13. 5±0. 5%, 12. 9±0. 3%, p>0. 05), and significantly more than the spike count (11. 2±0. 2%, both p<0. 001). This shows that the phase-partitioned code provides robust and high levels of stimulus discrimination performance in face of external sensory noise, which is ubiquitous in every-day sensory scenarios. We confirmed that the above results were insensitive to the particular choice of decoding algorithm used for single-trial evaluation of the different codes. To this end we repeated the analysis with a range of single-trial classification algorithms (c. f. Materials and Methods). Note that this does not concern the definition of neural codes, but concerns only the specific choice of classification algorithm used to quantify the degree of single-trial stimulus discrimination afforded by each neural code. Supplemental Fig. S2C shows the results for the auditory cortical dataset. While there was some variation in performance level across classifiers for each given code, the relationships between the three neural codes were preserved for each decoding algorithm, and there was no combination of classifiers for which the performance in the spike count was greater than in the phase-partitioned code. In addition, the results regarding robustness of the different codes in the face of temporal uncertainty or sensory noise were also unaffected by the specific choice use of classifier. Overall we found that for the given size of typical neural datasets (here 30–50 trials per stimulus), simpler algorithms with higher bias but lower variance (e. g. nearest mean or Poisson naïve Bayes) performed slightly better than algorithms with more parameters, and hence lower bias but higher variance (e. g. k-nearest neighbors or multinomial naïve Bayes). This suggests that for applications such as the evaluation of neural codes on experimental data simple decoding algorithms such as template matching are among the best choices and have the additional advantage of being extremely efficient computationally. Our work builds on the previous finding that slow cortical rhythms entrain to the dynamics of sensory stimuli [10], [34]. More precisely, previous work studying sound driven low frequency oscillations in auditory cortex using MEG or EEG [33], [38], [39] or intracranial recordings [20], [34] has shown that low frequency oscillations are entrained and phase-locked to repetitive or complex time-varying sounds. As a result of this entrainment the phase of these oscillations becomes reliably time-locked to the stimulus during epochs of dynamic changes in the sensory environment [38], and the phase angle of the oscillation becomes an effective network-intrinsic copy of the stimulus-locked time axis. Previous studies showed that this entrainment is particularly strong in delta and theta bands (i. e. below about 8 Hz) [33], [38], [39]. Our results of highly correlated stimulus decoding performance in time- and phase-partitioned responses and the correlation between oscillatory phase coherence and decoding using phase-partitioned responses show that this alignment of oscillations to sensory inputs is the key component of why a phase-partitioned code successfully recovers a high proportion of the stimulus information available in responses partitioned using a highly precise external clock. Previous work from our group has reported that the instantaneous phase of slow LFP fluctuations carries information largely complementary to that carried by stimulus-locked spike patterns defined on short time scales of few tens of ms [20], [21]. While this previous work already highlighted the potential importance of spike-phase relations for neural coding, there are key differences to the present study. The previous work treated spike-patterns in a strictly stimulus-locked manner, because it quantified spike patterns by measuring inter-spike intervals with millisecond precision and directly relative to the sensory input, i. e. corresponding to the time-partitioned code used here. In contrast, in the present work we consider how the oscillatory phase can be used to partition longer spike trains into short and stimulus-informative patterns without using a millisecond-precise and stimulus locked clock to measure inter-spike intervals or to form equally spaced time bins. The complementary nature of spikes and network oscillations has also been explored in the hippocampus, where the instantaneous phase was used as a complementary signal to enhance the information derived from the combined neural signal about the animal' s position in space [18], [19]. How can one reconcile the previous finding that the instantaneous phase of slow network fluctuations carries information complementary to spike rates computed in short time epochs with the suggestion that the phase of slow rhythms can be used as a network-intrinsic temporal reference frame for partitioning spike patterns extending over hundreds of ms? One possible explanation stems from the fact that the phase of slow oscillations is likely to reflect changes in the local excitation-inhibition balance entrained to slow variations in the sensory environment [10], [53], [55]. Modeling studies show that in recurrent and balanced networks the slow oscillatory phase reflects the network entrainment by slow input dynamics whereas spike rates and spike patterns defined on short time scales reflect the instantaneous strength of the network input [56]. In this view, slow network fluctuations reflect patterns of stimulus dynamics over longer time scales while faster variations in spike patterns encode instantaneous values of specific sensory features. This theoretical framework can provide explanations for both our previous and current findings about the role of slow oscillatory phase in sensory coding. On the one hand, it predicts that the entrainment of low network fluctuations to the stimulus time course makes them a good “time axis surrogate” over scales compatible with the cycle of the slow oscillations (as we found here). On the other hand, it also predicts our previous observation that the oscillatory phase at any given time provides information complementary to that carried by the rate or spike patterns defined on short window [20], [21] because the latter may encode the current value of the stimulus whereas the former reports its temporal position within an excitability cycle. Noteworthy recent work in the olfactory system has shown spikes precisely locked to the rhythmic sniff cycle carry information about different odors [57] and that the animals can discriminate activity patterns occurring at different phases of the sniff cycle [58]. Hence, at least for this system, there is evidence that the relative timing of spikes and oscillatory network activity can be directly exploited to guide behavior. Previous studies also implicated slow rhythms as a mechanism for binding neural ensembles that collectively encode specific sensory attributes at particular instances in time [8], [11], [59], [60]. While this hypothesis differs from the role as an internal temporal reference for response partitioning, it is possible that the same oscillatory signal could serve as a basis for both grouping processes, whether across time or across a population. Multiplexed spatio-temporal sensory representations across populations of neurons are prominent in sensory cortices and emerging evidence highlights the complexity of population-based codes across multiple scales [6], [12], [17], [47], [61]. Future modeling studies could explore the simultaneous role of oscillations in chunking spike trains into informative units and in dynamically binding ensembles for population coding. Sensory coding in natural environments is complicated by several environmental factors, one being sensory noise that can occlude or corrupt stimuli of interest [62]. We analyzed a dataset designed for testing the performance of neural codes in the presence of sensory noise in the auditory domain and found high noise-robustness in the phase-partitioned code. This robustness is likely a direct result of the prominent entrainment of auditory cortical low frequency oscillations by a wide range of complex sounds [38], [55], [63], suggesting that the prominent entrainment of slow oscillatory activity to the dynamic natural environment might be critical in establishing robust mechanisms of sensory coding. Another factor that can reduce the ability of neural systems to discriminate sensory stimuli is uncertainty about the occurrence and timing of sensory stimuli. It may be possible for the brain to infer the stimulus timing under specific conditions, such as following well-defined and isolated stimulus onsets [24], [25], [40]. We have previously shown that a subset of neurons in auditory cortex provide a powerful network-intrinsic latency signal that indicates the onset of an unexpected (sudden) stimulus with high precision and fidelity [25]. This signal can be used to construct, in a period of few hundreds of ms following stimulus onset, informative spike patterns without relying on explicit and external knowledge about stimulus timing [25]. However, it remains unclear whether such a latency signal can be used as a timing reference during prolonged periods of sensory stimulation. In fact, recent work suggests that the spike timing of sensory cortical neurons is very precise immediately after stimulus onset, but their precision degrades after some few tens of ms from stimulus onset [64], [65]. In addition, while deriving an intrinsically defined reference point for stimulus onset, our previous work still relied on the millisecond-precise knowledge about the timing of subsequent spikes relative to this reference point, i. e. we exploited equally-spaced time bins that were derived using a ‘perfect’ clock [25]. However, it is unlikely that the brain can keep a millisecond-precise representation of time intervals for prolonged periods of continuously evolving naturalistic sensory stimuli. As we show here, slow oscillations may provide a temporal reference to overcome this problem on longer scales. To further understand the putative reference frames provided by oscillatory network activity and population spiking responses, modeling studies on coordinated excitability changes in large-scale networks as well as physiological recordings will be required. Knowledge about the relative timing of sensory events and neural responses is not only required for partitioning longer response into informative units, but also when explicitly decoding temporally extended response patterns with regard to potential sensory inputs. A downstream decoder that lacks knowledge about the exact post-stimulus time at which a given response was emitted may not be able to access the perfect codebook used in conventional analysis, but rather may rely on a temporally ‘blurred’ version of it. We investigated the robustness of neural codes to such temporal uncertainty by systematically incorporating a temporal jitter at the single-trial level. We found that slow oscillations provide a reference frame that provides significantly greater robustness to temporal uncertainty than stimulus-locked partitioning. This robustness likely results from the locking of individual spikes to cortical oscillatory rhythms [47], [63], which is independent of the alignment of either signal relative to the stimulus. All in all, our quantitative comparisons highlight key computational advantages provided by partitioning spike trains using oscillatory reference frames that prevail especially during those conditions were sensory evidence is impoverished due to noise and uncertainty. Most existing models exploring the encoding of information in the relative timing of spikes and oscillatory activity were developed to explain the prominent theta phase precession observed in hippocampal data [27], [66], [67]. However, Nadasdy [30], [68] recently proposed a unifying model that uses phase-coding for the transmission and binding of information across the thalamo-cortical and limbic systems. This model highlights the necessity that similar frequencies are used as internal clocks within and across structures in order for phase-based coding to operate efficiently within the brain [30], [68]. While hippocampal and entorhinal neurons lock mainly to theta rhythms, classical studies on sensory cortices have mostly emphasized the locking of sensory neurons to gamma-band oscillations [5], [11], [30]. Our finding that theta band oscillations in primary visual and auditory cortices can be used as reference frame to effectively partition spike trains suggests that slow oscillations may be sufficiently wide-spread to meet the consistency demands for phase-based exchange of information across cortical and sub-cortical structures. Low frequency oscillations serving as temporal reference frames may also form a crucial component for plasticity in down-stream synapses. Recent work has shown that embedding firing patterns within an oscillatory cycle facilitates downstream learning and decoding with spike timing-dependent plasticity (STDP). Simulation studies show that model neurons equipped with STDP robustly detect a pattern of input currents encoded in the phase of a subset of its afferents, even when these patterns are presented at unpredictable intervals [69]. While in principle STPD rules can be adapted to learn sequences of precise inter-spike-intervals [70], learning patterns referenced to the phase of oscillatory activity facilitates learning even when only a fraction of afferents are organized according to the phase [69]. The present results together with such modeling studies underline the flexibility and power of phase-based reference frames, paving the way for a general framework to quantify the performance of neural codes through entire encoding-decoding and learning chains. The data analyzed here was obtained as part of previous studies [20], [71]. Recordings were obtained from the auditory and visual cortices of adult rhesus monkeys (Macaca mulatta) using procedures described below. All procedures were approved by local authorities (Regierungspräsidium Tübingen) and were in full compliance with the guidelines of the European Community (EUVD 86/609/EEC) and were in concordance with the recommendations of the Weatherall report on the use of non-human primates in research [72]. Prior to the experiments a form-fitting headpost and recording chamber were implanted under aseptic surgical conditions and general balanced anesthesia [73]. As a prophylactic measure antibiotics (enrofloxacin, Baytril) and analgesics (flunixin, Finadyne vet.) were administered for 3–5 d post-operatively. The animals were socially (group-) housed in an enriched environment, under daily veterinary supervision and their weight as well as food and water intake were monitored. As described in more detail previously [14], [20], neural activity was recorded from caudal auditory cortex of three alert animals using multiple microelectrodes (1–6 MOhm impedance), high-pass filtered (4 Hz, digital two pole Butterworth filter), amplified (Alpha Omega system) and digitized at 20. 83 kHz. Recordings were performed in a dark and anechoic booth while the animals were passively listening to the acoustic stimuli. Recording sites were assigned to auditory fields (primary field A1 and caudal belt fields CM, CL) based on stereotaxic coordinates, frequency maps constructed for each animal and the responsiveness for tone vs. band-passed stimuli [74]. Spike-sorted activity was extracted using commercial spike-sorting software (Plexon Offline Sorter) after high-pass filtering the raw signal at 500 Hz (3rd order Butterworth filter). For the present study only units with high signal to noise (SNR>8) and less than 2% of spikes with inter-spike intervals shorter than 2 ms were included. Field potentials were extracted from the broad-band signal after sub-sampling the original recordings at 1 ms resolution. Different frequency ranges of the LFP were extracted as described below. Acoustic stimuli (average 65 dB SPL) were delivered from two calibrated free field speakers (JBL Professional) at 70 cm distance. For the present study we analyzed auditory cortical data from two experiments, conducted as part of a previous study [20]. The first set of neurons was recorded during the presentation of a continuous 52 s sequence of natural sounds. This stimulus sequence was created by concatenating 21 snippets, each 1–4 s long, of various naturalistic sounds, without periods of silence in between (animal vocalizations, environmental sounds, conspecific vocalizations and short samples of human speech). In the second experiment a 15 s section of the long natural sound was presented either in its original form or mixed with background noise of three different levels. The background noise was obtained by randomly averaging many snippets of natural sounds, resulting in a cacophony-like noise stimulus with a similar power spectrum as the long natural sound, but devoid of clearly discernible sound objects. To quantify the relative contribution of this background noise to the stimulus on individual trials, we used the relative intensity (root mean square intensity over the full 15 s) of the target stimulus relative to that of the background expressed in units of dB [75], [76]. Specifically, the original sound was mixed with background noise of three relative levels: 6 dB softer than the original sound (‘low noise’), the same level as the original sound (‘medium noise’) and 6 dB louder (‘high noise’). Importantly, to resemble true noise a different background noise was randomly generated for each trial. For both datasets each stimulus was repeated many times (on average about 50 repeats of the same stimulus, range 39 to 70 repeats). As described in more detail previously [71], neural activity was recorded from the opercular region of primary visual cortex of two animals while the animals were anaesthetized (remifentanyl, 1 µg/kg/min), muscle-relaxed (mivacurium, 5 mg/kg/h) and ventilated (end-tidal CO2 33 mmHg, oxygen saturation >95%). Body temperature was kept constant and lactated Ringer' s solution supplied (10 ml/kg/h). Vital signs (SpO2, ECG, blood pressure, endtidal CO2) were continuously monitored. Signals were recorded using microelectrodes (FHC Inc., Bowdoinham, Maine, 300–800 k Ohms), high-pass filtered (1 Hz, digital two pole Butterworth filter), amplified using an Alpha Omega amplifier system (Alpha Omega Engineering) and digitized at 20. 83 kHz. Spike-sorted activity was extracted using the online available Matlab-based spike-sorting software Wave_Clus (http: //www. vis. caltech. edu/~rodri/Wave_clus/Wave_clus_home. htm) after high-pass filtering the raw signal at 500 Hz (3rd order Butterworth filter). Field potentials were extracted from the broad-band signal after sub-sampling the original recordings at 1 ms resolution. Different frequency ranges of the LFP were extracted as described below. Binocular visual stimuli were presented at a resolution of 640×480 pixels (field of view: 30×23 degrees, 24 bit true color, 60 Hz refresh) using a fiber optic system (Avotec, Silent Vision, Florida). Stimuli consisted of ‘naturalistic’ complex and commercially available movies (30 Hz frame rate; (Star Wars Episode 4 and The Last Samurai), from which 240 s long sequences were presented and repeated 30–40 times. For the main analysis we extracted individual frequency bands (4 Hz width) from the broadband signal using 3rd order Butterworth filters. The phase angle of the narrow-band signal was subsequently determined using the Hilbert transform. We systematically tested frequency bands with center frequencies from 4 to 32 Hz. In additional control analysis we made sure that our results do not depend on this specific choice of filter. In particular, we compared the performance of the phase-partitioned code using four different filters to derive the theta range: 1) 3rd order Butterworth filter between 2–6 Hz; 2) the same filter to derive a broader frequency band of 2–10 Hz; 3) Kaiser window FIR filter between 2–6 Hz (1 Hz transition bandwidth, passband ripple of 0. 01 dB and stopband attenuation of 60 dB; 3) Kaiser window FIR filter 2–8 Hz (2 Hz transition bandwidth, passband ripple of 0. 01 dB and stopband attenuation of 30 dB); 5) Morlet wavelet filtering (4 Hz center frequency, standard deviation σf of 0. 6/4 Hz). The stimulus decoding performance of the phase-partitioned code changed only minimally with the filter (Supplemental Fig. S2A). We quantified the level of stimulus discrimination afforded by three hypothetical neural codes, each derived from the same neural response in the same time window. We defined neural codes in time windows of length T, whereby T was chosen to roughly match the duration of one cycle of the considered oscillation, i. e. T = 160 ms for the 2–6 Hz frequency band (Fig. 1). Given the spiking activity and LFP within this time window, we defined the following three codes. 1) The time-binned firing-rate, which was defined by splitting the response window T into N precisely aligned, equally spaced time bins and counting the number of spikes occurring in each bin. Formally, this code can be described as with ri being the number of spikes within the i-th time bin, with the bins being defined as the time intervals ([0, T/N], …[ (N−1) *T/N, T]). 2) The distribution of phase of firing, defined by splitting the phase of the slow reference rhythm into N equally spaced phase bins and allocating each spike to the bin corresponding to the instantaneous phase value at the time of the spike. Formally, this can be described as with xi being the number of spikes within the i-th phase bin, with the bins being defined as the phase intervals ([0,2*pi/N], …[ (N−1) *2*pi/N, 2*pi]). 3) We defined the spike count as the total number of spikes per time window T, regardless of their temporal sequence. Practically, we implemented the spike count using two different procedures, which yielded very similar results. One procedure was based on shuffling (independently for each trial and time window) the time bins of the time-partitioned code. This shuffling effectively destroys the temporal response pattern and preserves only the total spike count. Formally, this code can be described as with ri being the number of spikes within a randomly selected (without replacement) time bin. The decoding performance for the spike count using this shuffled procedure was obtained by averaging performance from several repeated computations, to minimize the effect of shuffling (20 times). This shuffling reproduces the information contained in the total spike count, but preserves the dimensionality of the neural code, which is important to ensure the comparability of results obtained in the decoding analysis [14]. In addition, we also implemented the spike count using a 1-dimensional representation, which yielded very similar results as obtained using the shuffling procedure (Supplemental Fig. S2B). The neural codes defined by the time- and phase-partitioned responses include differences in the spike count. As a consequence, the stimulus discrimination performance afforded by these two codes includes discrimination performance afforded by differences in spike count alone and hence the decoding performance using these two codes is at least as high as from the spike count. The excess decoding performance in each partitioning scheme over that provided by the spike count reflects the information that the respective code carries above and beyond that provided by the spike count [6], [37]. We quantified this excess performance by calculating the difference in decoding performance between each partitioning scheme and the spike count (e. g. Fig. 3B, 4C). We also directly compared the relative excess performance in both partitioning schemes for each neuron by expressing the excess performance in the phase-partitioned code relative to that in the time-partitioned code as percentage. To more directly quantify the overlap of the decoding performance permitted by time- and phase-partitioned codes we also performed an analysis on their decoding redundancy. We created a dual-code based on the joint response of both time- and phase-partitioned response vectors. Formally this code can be represented as, with ri being the number of spikes within the i-th time bin and xi being the number of spikes within the i-th phase bin. The decoding performance of this dual-code should exceed the performance of the individual codes by an amount proportional to the degree of separate stimulus discriminability afforded uniquely by each code. We expressed the relative performance of the dual code to the better of the two individual codes as a percentage. For each dataset we quantified the performance of the different codes for a wide range of parameters for the time window T (80 to 480 ms), the number of bins N (2 to 16) and the frequency band of the local field potential from which the oscillatory phase was extracted (center frequencies from 4 to 32 Hz). To quantify the impact of frequency band on performance of the phase-partitioned code, we computed the ratio of decoding performance between the phase-partitioned code and the spike count after subtracting the chance performance. If not stated otherwise, results refer to a ‘standard’ choice of T = 160 ms, N = 8 and the 2–6 Hz band. We quantified the stimulus discrimination afforded by each code using a single-trial decoding procedure, applied to the responses in ten epochs of duration T. These epochs were randomly sampled (non-overlapping) from within the entire stimulation period (Fig. 2A) and the decoding procedure was averaged over 100 independent sets of epochs. For decoding we used linear template matching in conjunction with a leave-one-out cross-validation procedure [32], [77]. For each individual trial from a given stimulus (say S1) this proceeded as follows: 1) The average responses to all other 9 stimuli were computed by averaging the responses of all repeats of the respective stimuli. 2) For the current stimulus (S1) the mean response was computed by averaging across all trials, excluding the current ‘test’ trial. These average responses to each stimulus then represented the ‘codebook’. 3) The Euclidean distance was computed between the response vector (representing spike counts in time or phase bins) on the test trial and the average responses in the codebook. The test trial was decoded as that stimulus yielding the minimal distance to the test response. Formally, let denote the vector representing one of the neural codes for the presentation of stimulus i on trial j, and let denote the mean response to stimulus i (computed excluding the current leave-one-out test trial). The test trial is decoded as the stimulus index i whose mean distance to the test trial is smallest: (1) This nearest mean or template matching procedure can be shown to be the Bayes optimal decoder in the case where the stimulus conditional response ensembles are independent multivariate normal distributions (constant diagonal covariance). This procedure was repeated for each trial of each of the 10 stimuli and the total percentage of correctly decoded trials and the confusion matrix were determined. To quantify the performance of the decoder we used the percent of correctly identified trials and averaged this measure over 100 sets of independently sampled epochs. To validate that our results do not depend on this specific choice of single trial decoding algorithm we repeated the entire analysis using a wider range of classification algorithms [78]. Specifically, we implemented a range of classifiers that permit an efficient implementation of the above leave-one-out cross validation procedure. These classifiers were: (1) the above described nearest mean template matching procedure, which corresponds to an optimal linear discriminant classifier in the case where the stimulus conditional response distributions are multivariate normal, independent, and with equal variances. These assumptions inherent to the nearest mean classifiers can be progressively relaxed, leading to classifiers estimating the covariance matrix pooled across stimuli (2: linear classifier), estimating the full covariance matrix for each stimulus (3: quadratic classifier). For these classifiers, in order to avoid numerical problems due ill-conditioned matrices we added a small random jitter (normally distributed with standard deviation 0. 001) to the discrete count responses independently for each trial and bin. We verified that this procedure did not affect results for repetitions without numerical problems, and that adding the jitter produced similar results to the more computationally intensive use of the matrix pseudo-inverse. In addition we implemented Naïve Bayes decoders, which assume that response variables (e. g. counts in each time or phase bin) are independent and calculate the most likely stimulus using Bayes theorem. We implemented (4) Poisson naïve Bayes, which assumes the counts are Poisson distributed, and (5) multinomial naïve Bayes, which samples the full discrete stimulus-conditional probability distributions of counts for each response bin. Finally (6) we also implemented a k-nearest neighbor classifier with Euclidean distances. When computing stimulus information in face of temporal uncertainty about the precise alignment of individual time windows T to the sensory stimulus, we added a jitter to the alignment of time windows across trials when calculating the codebook (Fig. 5A, 5B). Specifically, we randomly shifted the window extracted for each trial by a random lag into the future or past that was randomly sampled for each trial and which was uniformly distributed between −J/2 and +J/2, where J corresponds to the (maximal possible) temporal uncertainty. These time-shifted single trial responses were then averaged to obtain the codebook (steps 1 and 2 in the decoding process). Again the entire process was repeated 100 times using different selection of stimulus epochs. We calculated the trial-by-trial coherence of the oscillatory phase using the inter-trial phase coherence index. This index is a measure of phase concentration across trials and is defined as (2) where <. > denotes the trial average, φ (t) the phase at time t and |. | the absolute value of the complex number. To compute the correlation of oscillatory phase coherence and decoding performance (Fig. 3D) we averaged phase coherence across time points within the decoding window T, resulting in a single phase coherence value for each stimulus epoch. This value was then correlated with the decoding performance for each of the considered codes across stimulus epochs. | Neurons in sensory cortices encode objects in our sensory environment by varying the timing and number of action potentials that they emit. Brain networks that 'decode' this information need to partition those spike trains into their individual informative units. Experimenters achieve such partitioning by exploiting their knowledge about the millisecond precise timing of individual spikes relative to externally presented sensory stimuli. The brain, however, does not have access to this information and has to partition and decode spike trains using intrinsically available temporal reference frames. We show that slow (4-8 Hz) oscillatory network activity can provide such an intrinsic temporal reference. Specifically, we analyzed neural responses recorded in primary auditory and visual cortices. This revealed that the oscillatory reference frame performs nearly as well as the precise stimulus-locked reference frame and renders neural encoding robust to sensory noise and temporal uncertainty that naturally occurs during decoding. These findings provide a computational proof-of-concept that slow oscillatory network activity may serve the crucial function as temporal reference frame for sensory coding. | lay_plos |
FIELD OF THE INVENTION The invention relates to a process for enhancing some of the functional properties of denatured proteinaceous material of vegetable origin. BACKGROUND OF THE INVENTION Vegetable protein products obtained from seed and leaf materials are used as replacements and extenders for proteins derived from animal, marine and poultry sources. Such vegetable protein products are usually low fat or defatted materials. They are commonly referred to as "flours" if they contain less than 65% protein, as "concentrates" if they contain from 65 to 90% protein and as "isolates" if they contain above 90% protein. Some methods for manufacturing vegetable protein products customarily include heat treatment and aqueous alcohol extraction, as well as other chemical treatments and applied conditions which denature and substantially reduce the functional properties of the proteinaceous products. These functional properties of such proteins are characterized by their ability to hold oil or fat and water, to emulsify and to form protein-containing products having a firm consistency. Since these functional properties of the protein in a vegetable protein product are desired characteristics in many applications, as for example in food recipes (e.g. in meat, dairy and bakery products) and in industrial applications (e.g. in the paper coating industry), the enhancing of these properties in low functional proteinaceous materials is thus of substantial economic and technological importance. Although one of the functional properties of protein is its solubility, the other properties are not necessarily interdependent on the protein solubility and soluble protein material can have other poor functional properties, while protein material of low solubility can have high functional properties. It is well known that protein material which is normally soluble at pH's other than its isoelectric pH, has a reduced solubility in all pH ranges when it becomes denatured and will not be affected by either simple alkaline or acid treatments which will only change the pH. Likewise, upon denaturing, most of the original functional properties, as hereinbefore stated, will be lost. Prior art teachings indicate that in order to introduce or regenerate some of these functional properties in denatured material, either, high temperatures above the boiling point of water, such as are obtained by steam jet cookers, or high shear forces, as obtained by successive pressure and cavitation cycling, such for example, by using a centrifugal homogenizer, must be employed on an aqueous, slightly alkaline slurry of the denatured proteinaceous material. A patent by Gobi et al, (U.S. Pat. No. 4,113,716) discloses a process designed to obtain improved soy protein fractions from previously denatured soy protein material by cooking slightly alkaline aqueous slurry of the denatured soy protein material at temperatures between 110° C. to 150° in a jet cooker chamber and obtaining from this high temperature treated material soluble fractions with "properties superior to those obtained by using undenatured defatted soy flakes as starting material." A patent by Howard et al (U.S. Pat. No. 4,234,620) discloses a process in which aqueous vegetable protein dispersions are subjected to high shear forces of successive pressure and cavitation cycling obtained by centrifugal homogenization at temperatures below 150° C., under slight alkaline pH's, to provide a high protein solubility, low viscosity product at high solids levels, which may be dried, generally after pH adjustment with acid, to provide a vegetable protein product similar to milk protein. British Patent No. 1,575,052 discloses that the flavor, color, nutritive value and some functional properties of protein material selected from single cell protein material, plant protein material, whey solids and mixtures thereof can be improved by heating aqueous slurries of the protein material to temperatures ranging from 75° to 100° C., adjusting the pH to a pH range of 6.6 to 8, preferably 7.2 to 7.6, by adding a pH adjusting compound, preferably a calcium compound, maintaining the temperature for a period of 1-120 minutes and drying the product. The product obtained may be used as a replacer for egg solids and non-fat dry milk, especially in baked goods, as the process reduces the yeasty off-flavor, releases buffering materials from yeast cell material, saponifies lipid material and reduces available thiol (-SH) groups. The present invention is based on a surprising discovery that some of the functional properties of denatured protein in a vegetable protein product can be enhanced, without causing substantial changes in the protein by a very simple, economical and efficient process. The enhanced properties include the ability to hold oil or fat and water, to emulsify and to form products of firm consistency. The process of the present invention does not require the use of temperatures above 100° C., nor any special equipment or forces. As will be seen from the details described below, the process of the invention provides a simple low-cost vegetable protein product having improved functional properties compared with corresponding untreated denatured proteinaceous material. It is the object of the present invention to enhance the functional properties of denatured vegetable proteinaceous material having poor functional properties. Another object of the invention is to enhance the functional properties of denatured vegetable proteinaceous material without fractionation, without addition or introduction of salts, without super-heating above water boiling temperature, and without the use of high shear forces such as obtained by centrifugal homogenization. Yet another object of the invention is to provide a simple, efficient and economical process to achieve the same. A further object of the invention is to provide a vegetable proteinaceous material having enhanced functional properties. SUMMARY OF THE INVENTION The invention thus provides a process for increasing the functional properties of denatured proteinaceous material of vegetable origin, having relatively poor functional properties, which comprises maintaining such denatured proteinaceous material in warm aqueous ammonia at a temperature between 75° C. to 100° C. and a pH of about 8 to 9.5, wherein the ratio of the aqueous phase to solids in the slurry is from about 3:1 to 15:1. The time can vary from less than 1 minute to several hours depending on the type of denatured protein, its particle size, the temperature and pH. The ammonia may then be stripped off after the treatment to obtain an ammonia-free, salt-free highly functional proteinaceous product. This product is capable of providing a high viscosity product at low solids levels upon addition of water, without the material having undergone any substantial chemical change with the exception of functionality. Also included within the scope of the invention is denatured proteinaceous material of vegetable origin which has been treated in accordance with the process of the invention. DETAILED DESCRIPTION OF THE INVENTION It will be appreciated that, in general, the enhanced functional properties obtained by the process of the invention are measured by an increase in the ability of the proteinaceous material to hold oil or fat and water, to emulsify the same and to form products having a firm consistency upon heating and cooling. The method for determining these functional properties of vegetable protein products is as follows: 7 parts of refined vegetable oil (e.g. corn oil) and 3.5 parts of water are well mixed in a blender at maximum speed for 5 minutes. One part of vegetable protein material and additional 3.5 parts of water are added and mixing is continued for an additional 10 minutes. The mixture is quickly heated to 90° C., poured into cups and cooled down overnight to a temperature of about 5° C. Formation of a homogeneous product having a firm consistency without separation of oil or water is indicative of highly functional vegetable protein product. The weight ratio of the aqueous ammonia phase to the proteinaceous material can vary within the range of about 3:1 to about 15:1. The pH range for operating the process of the invention is from about 8.0 to about 9.5, preferably 8.5 to 9.2. Preferably, the process of the invention is effected at a temperature within the range of about 75° C. to about 100° C. The desired temperature may be attained by utilizing a double jacketed construction in the vessel in which the slurry is being held by injecting steam directly or indirectly, or by other heating means such as heating coils. The time required for the aqueous ammonia to effect the enhancement of the functional properties of the proteinaceous material varies depending on the source, the degree of particle size, pH and temperature. The reaction time can thus vary to a great extent, for example, from one minute or less, to thirty minutes or more. In general the longer the reaction time, within one to thirty minutes, the better the improvement of functional properties. Some improvement, however, is usually observed even after a very short time. The process may be operated under pressure. In such a case the time required is reduced. Subsequent to the treatment with ammonia, the pH of the slurry is lowered to the initial pH of the untreated proteinaceous material by stripping off the ammonia. This can be done by pulling a vacuum on the warm slurry. Preferably further drying is applied to the product in which the residual ammonia and most of the water are simultaneously removed. The ammonia treated product should be dried to a water content of less than about 10%. While any suitable drying method which will not impair the functional properties of the protein product and which would assure total stripping of the ammonia may be utilized, it is presently preferred that the product is dried by spray drying. In this case it is preferred that the inlet temperature not be above 300° C. and the outlet temperature not greater than about 120° C., preferably in the range of 80° C.-100° C. The low functionality proteinaceous starting material may be derived from vegetable proteins which have been denatured such as by heat treatment, treatment with aqueous alcohols and/or other chemical treatment. In particular, there may be used, for example, defatted or low fat vegetable protein products derived from soybeans, lupins, sesame, cotton seeds, peanuts, rape seeds, sunflowers seeds, maize, wheat, barley, oats, lentils, peas, legumes generally, vegetable leaves and the like, including "flours", "concentrates" and "isolates", as described above. The invention will be illustrated by the following non-limitative example: EXAMPLE 1 Aqueous alcohol washed denatured soya protein concentrate of low functionality HAYPRO (Hayes Ashdod Ltd., Israel) 1 kg., having 71% protein on dry basis; 6.5% moisture and pH 6.9, was combined and slurried with 8 liters of water in a double jacketed vessel tank. Concentrated ammonium hydroxide (29% ammonia content) was added to the slurry to provide an initial pH of 9.0. The vessel was tightly closed and steam was injected into the jacket to provide a constant and controlled temperature of 86° C. The walls of the slurry containing vessel were continuously scraped to assure even temperature all through the slurry. After 20 minutes, the warm slurry was vented, transferred from the vessel tank by means of a pump and dried in a spray drier (APV Anhydro) with an inlet temperature of 280° C. and an outlet temperature of 98° C. to a final moisture content of 3%. The spray dried product was found to contain 71% protein on dry basis; 3% moisture and pH 6.9. Samples of untreated and treated soya protein concentrates were tested for functionality by the method hereinbefore described. The low functionality untreated product did not form any firm consistency and did not hold the oil and the water, whereas the treated protein formed a very viscous and firm consistency product and no oil nor water separation were observed. EXAMPLE 2 A comparative test was conducted to measure the functional properties of treated soya protein concentrate of Example 1 with a soya protein concentrate, STA-PRO, 66.5% protein on dry basis, (A. E. Staley Manufacturing Co., Decatur, Ill., U.S.A.) understood to be manufactured according to U.S. Pat. No. 4,234,620. The functional test described above was applied to both samples. The viscosities of 10% solids in water concentration were also measured by a Brookfield Viscosimeter (12 rpm: SPDL 2: 25° C.) after 2 and 10 minutes and the water and fat binding capacities were measured by a modified method of the one disclosed hereinbefore in which the amounts of water and oil were adjusted to give equally firm consistency products without water or oil separation. The results are shown below. ______________________________________Test Results U.S. Pat. No. Example 1 4,234,620______________________________________Viscosity (CPS) 2 minutes 2500 75010 minutes 2200 370Water absorptionWt. Water/wt. protein 6.1:1 4.5:1Water/oil binding capacityWt. protein:wt. water:wt. oil 1:10:10 1:7:7Total Ash 5.8% 7.5%Sodium 0.02% 0.4%______________________________________ The product of Example 1 had far better water absorption, water and fat binding, higher viscosity "body" forming capacity, as well as higher protein content and lower total ash and especially much lower sodium than the STA-PRO product. EXAMPLE 3 Another comparison was conducted wherein an aqueous alcohol denatured soya protein concentrate HAYPRO (Hayes Ashdod Ltd.) was treated according to British Patent No. 1,575,052 Example VII by adding calcium hydroxide and calcium carbonate to an aqueous suspension thereof to give a pH of 7.5, heating at 90° C. for 30 minutes and spray drying the product. The product obtained was tested by the method hereinbefore described. The results are tabulated in Table I. The treated product had no firm consistency and did not hold the oil and water. It behaved similarly to the control of Example 1, i.e. untreated protein. TABLE I______________________________________ U.S. Pat. No. GBProduct Control.sup.(1) Example 1 4,234,620 1,575,052______________________________________Viscosity low high medium-low lowWater low high medium lowabsorptionWater/oil low high medium-high lowholding______________________________________.sup.(1) Denatured untreated vegetable protein (soya protein concentrate) While the invention has been described above with reference to particular embodiments, it will be appreciated by those skilled in the art that many variations and modifications may be made. The invention is accordingly not to be constructed as limited to such embodiments, but is defined only by the claims which follow. | A process for enhancing the functional properties of denatured proteinaceous material of vegetable origin. The enhanced properties include at least one property selected from the group consisting of water absorption, water binding capacity, oil binding capacity, fat binding capacity, and the ability to produce viscous aqueous suspensions. The process includes the steps of obtaining the denatured proteinaceous material by treating undenatured proteinaceous material with aqueous alcohol and maintaining a slurry of the denatured proteinaceous material in warm aqueous ammonia in which the weight ratio of the aqueous phase to solids is between 3:1 and 15:1 at a temperature between 75° C. to 100° C. and within a pH range of from 8.0 to 9.5. | big_patent |
Recent advances in DNA sequencing have enabled mapping of genes for monogenic traits in families with small pedigrees and even in unrelated cases. We report the identification of disease-causing mutations in a rare, severe, skeletal dysplasia, studying a family of two healthy unrelated parents and two affected children using whole-exome sequencing. The two affected daughters have clinical and radiographic features suggestive of anauxetic dysplasia (OMIM 607095), a rare form of dwarfism caused by mutations of RMRP. However, mutations of RMRP were excluded in this family by direct sequencing. Our studies identified two novel compound heterozygous loss-of-function mutations in POP1, which encodes a core component of the RNase mitochondrial RNA processing (RNase MRP) complex that directly interacts with the RMRP RNA domains that are affected in anauxetic dysplasia. We demonstrate that these mutations impair the integrity and activity of this complex and that they impair cell proliferation, providing likely molecular and cellular mechanisms by which POP1 mutations cause this severe skeletal dysplasia. Skeletal dysplasias (SD) are a group of genetic disorders affecting skeletal development that cause deficiencies and deformities of the limbs and spine, dwarfism, or abnormal bone strength. SDs are usually inherited as dominant or recessive monogenic Mendelian traits or occur as a result of de novo mutations. Recent advanced in targeted whole-exome DNA re-sequencing have enabled several groups to identify of causative mutations underlying various Mendelian diseases, in which traditional linkage approaches were not feasible because of the paucity of familial cases in which to perform the mapping [1]–[7]. In this study we report the mapping of mutations causing a severe bone dysplasia in a family of unrelated unaffected parents with two affected siblings. The clinical and radiographic features of the affected siblings showed similarities to anauxetic dysplasia, an autosomal recessive spondylo-epi-metaphyseal dysplasia characterized by extremely short stature [8], [9]. Both siblings had severe growth retardation of prenatal onset, a bone dysplasia affecting the epiphyses and metaphyses of the long bones particularly in the lower limbs, and abnormalities of the spine including irregularly shaped vertebral bodies and marked cervical spine instability (Figure 1). Anauxetic dysplasia is caused by mutations in RMRP, an untranslated intronless gene, mutations of which also cause cartilage-hair hypoplasia (CHH, OMIM 250250), another severe form of dwarfism [10]. As RMRP mutations had been excluded in this family, we sought to identify the disease-causing variants by whole-exome sequencing. We sequenced exomes of both parents and the affected siblings using the Nimblegen SeqCap Ez exome capture protocol and the Illumina Genome Analyser II paired-end sequencing method. High quality sequence reads were aligned to the human reference genome (UCSC assembly hg19); 90% of targeted bases had coverage of fourfold or higher; and 79% of targeted bases had coverage greater than tenfold. We then used SAMtools [11], Genome Analysis Toolkit (GATK) [12] and custom scripts to detect polymorphic sites in the four individual exome sequence data sets. Following quality filtering, we retained approximately 15,000 SNPs per sequenced exome (Table 1). The vast majority of these SNPs (>96%) were reported in the recent NCBI dbSNP 131 release, and were therefore excluded from further analysis as unlikely to cause this severe, rare, phenotype. Following the functional annotation of the remaining novel SNPs, we focused our analyses on a set of 483 unique novel coding non-synonymous SNPs that were detected in at least one sample. Given the affected status of both siblings and unaffected status of the parents we considered autosomal recessive homozygous and autosomal recessive compound heterozygous as the two most likely inheritance models; as the parents are unrelated, a compound heterozygous model was considered most likely. All novel non-synonymous SNPs were analyzed assuming either a homozygous or a compound heterozygous model. We did not find any novel non-synonymous mutations that satisfied a recessive homozygous inheritance model. To be accepted as candidate mutations for a compound heterozygous inheritance model, we sought genes with at least two novel non-synonymous SNPs within the protein coding sequence in both siblings; additionally, the same alleles had to be present in the parents in a mutually exclusive manner. We identified four candidate genes (POP1, PKD1, MICALL2, and COL5A1) that fitted this model (Table S1). To determine the gene likely to be causing the disease we analyzed the effects of the detected missense mutations on protein function using the SIFT algorithm [13]. In all but one gene the detected missense mutations were predicted to be tolerated in terms of effect upon protein function. The single remaining candidate gene carried two novel alleles – one creating a premature stop codon, and the other causing a missense mutation with predicted damaging effect on protein function, thus representing a strong candidate for the disease-causing gene in this family (Figure 2). Remarkably, we found that this gene encodes the human homolog of the yeast POP1 (processing of precursor RNA) protein, one of the core components of the RNase P and RNase MRP complexes [14], [15]. RNase P and RNase MRP are essential ribonucleoprotein complexes present in all eukaryotes [16]. Both enzymatic complexes have an evolutionarily conserved RNA component as a catalytic moiety. Several proteins essential for enzymatic activity form a constitutive part of RNAse P and RNase MRP complexes. POP1 is the largest constitutive component of both complexes (Figure S1) [15], [16]. Importantly, mutations in RMRP, which encodes the RNA moiety of the RNase MRP complex, cause anauxetic dysplasia and cartilage-hair hypoplasia [10]. To validate the identified POP1 mutations we re-sequenced genomic DNA fragments encompassing mutated alleles of the POP1 gene using Sanger sequencing, confirming that the two affected siblings carry both mutated alleles, while each parent carries only one allele (Figure 2A, 2B). While neither of these mutations is located within known protein domains (as determined by PFAM annotation), both amino acids affected by the mutations show high levels of evolutionary conservation, suggesting their importance for the functional integrity of POP1 and/or RNase P/RNase MRP complexes (Figure 2C; Figure S1). The p. Arg513Ter mutation is likely to be particularly damaging; the premature stop codon located in the exon 10 would trigger mRNA degradation via nonsense mediated decay mechanism. Alternatively (though less likely), p. Arg513Ter mutation would result in translation of a truncated protein lacking the evolutionarily conserved POPLD domain (Figure 2C). These results are consistent with findings in yeast. Xiao et al. [16] identified several point mutations in the yeast POP1 protein that affect either the stability or activity of RNase P and RNase MRP complexes. Importantly, some of the yeast mutations were found close to the mutations identified in our study, supporting our conclusion that these are likely to cause significant detrimental effect on RNase P and RNase RMP function. The very low population frequency of the described skeletal dysplasia, and the essential roles of RNase P/RNase MRP enzymatic complexes in cellular functions, suggest that there is strong negative selection pressure on homozygous and compound heterozygous mutations with detrimental effects on the activity of these complexes. Indeed, analysis of mutations that ablate POP1 function in model organisms demonstrate that POP1 is essential for viability in yeast and Drosophila [14], [17]. In this context, it is likely that one or both detected POP1 mutations have only a partial effect on function of either RNAse P or RNase MRP complexes. This conclusion is consistent with the broad spectrum of mutations observed in RMRP in cases of anauxetic dysplasia and cartilage-hair hypoplasia and their correlation with the severity of the disease phenotype [18]. RMRP mutations implicated in anauxetic dysplasia and cartilage-hair hypoplasia have distinct clustering within the RMRP RNA molecule [18]. Importantly, POP1 directly interacts with the same RMRP RNA domains that are affected in anauxetic dysplasia (Figure S1) [18], [19]. This further strengthens the link between our findings and the molecular mechanisms underlying the clinical phenotype in anauxetic dysplasia and our patients. To assess population frequency of the POP1 alleles we screened 186 DNA samples from healthy control subjects. For the NM_001145860. 1: c. 1573C>T (p. Arg513Ter) allele we used direct Sanger sequencing of PCR-amplified DNA fragments. To estimate the frequency of the second POP1 allele, NM_001145860. 1: c. 1748G>A (p. Gly583Glu), we used a restriction fragment length polymorphism (RFLP) assay designed to detect the loss of the BsaJ1 restriction site (CCNNGG) created by c. 1748G>A (p. Gly583Glu) mutation. None of the 186 control samples contained either mutation. Further, these variants are not reported in public SNP databases including dbSNP 131. Therefore we conclude that both mutations represent novel rare alleles. One of the cellular functions of the RNase RMP complex is processing of precursor 5. 8S ribosomal RNA into the mature form [15]. To determine the impact of the mutated alleles on functional integrity and activity of the RNase P/MRP complexes, we measured relative abundance of the RMRP RNA and unprocessed pre-5. 8S rRNA in the affected siblings, non-affected parents, and unrelated controls. We found that the relative abundance of RMRP RNA was markedly reduced in the affected individuals compared with non-affected parents and controls (Figure 3), indicating that the mutated alleles are likely to cause mis-assembly and destabilization of the RNase MRP complex. These results are consistent with the previous study in yeast demonstrating that mutations in POP1 affect integrity of the RNase MRP complex, resulting in destabilization of the complex and reduction of the abundance of NME1 RNA (the yeast homolog of human RMRP) [16]. We also found that affected individuals have higher abundance of the unprocessed pre-5. 8S rRNA compared to non-affected parents and controls (Figure 3), consistent with the established role of RNase P in processing of rRNA [15] and demonstrating that activity of the RNAse MRP complex is impaired in the affected individuals. To assess the cellular impact of this molecular defect, we measured cell proliferation in stimulated peripheral blood mononuclear cells (Figure 4). These studies showed that, in comparison with healthy controls, the two affected cases had markedly diminished cell proliferation in response to proliferation stimulus, as evidenced by the reduced rate of dilution of CFSE fluorescence intensities (Figure 4A). Additionally, individual SKDP-6. 1, carrying the c. 1573C>T (p. Arg513Ter) allele, had noticeable reduction in cell proliferation rate, consistent with the predicted more damaging effect of this mutation (Figure 4). These findings also explain similarities with the anauxetic dysplasia phenotype, as individuals with this disease also have impaired RNase P/RNase MRP activity, and diminished cell turnover [10], [18]. In conclusion, we have identified mutations in POP1 underlying the skeletal dysplasia presented in this study. Involvement of POP1 in the skeletal dysplasia highlighted the existence of a shared molecular pathway in the etiology of skeletal dysplasias involving the RNAse P and RNase MRP complexes [10], [18] and raises the possibility of the involvement of other components of the pathway, or allelic variants of RMRP or POP1, in other as yet unmapped severe short-stature syndromes. Screening of RMRP-negative cases of anauxetic dysplasia or CHH, or patients with similar phenotypic features, may yield additional mutated alleles within POP1, and possibly in other components of the RNase MRP complex. We further hypothesize that the distinct skeletal phenotype associated with POP1 mutations may indicate that RNAse P and RNase MRP complexes may process other yet unidentified target RNAs with regulatory roles in specific developmental pathways such as osteogenesis. This study also provides an example of the feasibility of whole-exome re-sequencing approach in rare familiar monogenic diseases with small pedigrees. This study was approved by the University of Queensland ethics committee (Approval #2007001196). Written, informed, consent was obtained from all subjects or their respective guardians. 5 µg of human genomic DNA extracted from peripheral venous blood samples was used for preparation of each DNA sequencing library. The DNA libraries were prepared using Illumina' s paired-end sequencing DNA sample preparation kit according to the manufacturer' s protocol. Whole-exome sequence capture was performed using “SeqCap EZ Exome Library v1. 0” liquid phase sequence capture kit (Roche/Nimblegen) according to the manufacturer' s recommendations. Sequence capture efficiency was assessed by quantitative real-time PCR using a standard set of control primers recommended in the sequence capture protocol. Individual DNA libraries were quality-checked and quantified on Agilent 2100 Bioanalyzer using DNA1000 kit, and DNA concentration adjusted to 10 nM. Sequencing was performed on the Illumina Genome Analyzer II using a standard 56 cycle paired-end read sequencing protocol and Illumina' s sequencing reagents according to the manufacturer' s recommendations. Each library was sequenced individually on a single flow cell lane. Base calling and sequence reads quality assessment was performed using Illumina' s Data Analysis Pipeline software v. 1. 6. Alignment of sequence reads to the reference human genome (hg19, UCSC assembly, February 2009) was performed using the Burrows-Wheeler alignment (BWA) tool [20]. Sequence alignment files conversions were performed using SAMtools [11]. Only high quality sequence reads with unique mapping positions to the reference human genome were used for calling SNPs and Indels. Identification of SNPs and Indels in the alignment files was performed using Genome Analysis Toolkit (GATK) [12]. Raw SNP calls were filtered using empirically derived cut-offs for the following GATK filter expressions: –filterExpression “QUAL <50. 0 || AB>0. 75 && DP>40 || QD<5. 0 || HRun>5 || SB>−0. 10” –filterName “StandardFilters” –filterExpression “DP<6” –filterName “LOW_DEPTH”, where QUAL – combined recalibrated quality score at the SNP position; AB – allele balance at the SNP position; DP – sequencing depth at the SNP position; QD – QUAL/DP ratio at the SNP position; HRun – maximal length of the homopolymer run; SB- strand bias at the SNP position. Known SNPs were obtained from the NCBI dbSNP build 131 (http: //www. ncbi. nlm. nih. gov/SNP/). Prediction of the effect of non-synonymous amino acid substitutions on protein function was done using SIFT algorithm [13]. Sanger sequencing of the exons 10 and 12 of POP1 was performed on Genetic Analyzer 3130 (Applied Biosystems) using standard sequencing protocol for PCR-amplified DNA. Primers used for PCR amplification of POP1 DNA fragments and Sanger sequencing were as follows: POP1. 10F 5′-CAATGGGAAGAGAGGGAATACATGTTT-3′; POP1. 10R 5′-CTGGAGAGGTGTCAGAGAAAGAACTCTT-3′; POP1. 12F 5′-CATCCTTGAGAAGGTGTCACTTAATTGTT-3′; POP1. 12R 5′-CACCACTCAATTCCTCCTAAGTTGTACAT-3. 186 DNA samples from healthy white European controls were studied to determine the population frequency of the variants identified. The c. 1573C>T (p. Arg513Ter) allele located within exon 10 of POP1 was tested by Sanger sequencing using POP1. 10F and POP1. 10R primers listed above. The c. 1748G>A (p. Gly583Glu) allele located within exon 12 of POP1 was tested by a restriction fragment length polymorphism (RFLP) assay designed to screen for the loss of the BsaJ1 restriction site (CCNNGG) created by the mutation. Following PCR amplification of exon 12 DNA fragments with POP1. 12F and POP1. 12R primers, DNA fragments were digested with BsaJ1, separated by agarose gel electrophoresis, and visualised by ethidium bromide staining and UV transillumination. Total RNA was extracted from peripheral blood mononuclear cell samples using TRIZOL reagent (Invitrogen) following the manufacturer' s protocol. The RNA concentrations and purity were determined using the RNA 6000 Bioanalyzer kit (Agilent). For cDNA synthesis, 500 ng of total RNA was treated with DNase I (Invitrogen) and then utilized as a template for randomly primed reverse transcription using SuperScript III reverse transcriptase (Invitrogen), according to the manufacturer' s instructions. The resulting cDNA was diluted 1∶25 (v/v) with nuclease-free water. A total of 5 µL of the cDNA was used as a template for quantitative real-time PCR. Relative abundance of unprocessed 5. 8S rRNA and RMRP transcripts in affected in non-affected individuals was determined by real-time PCR using SYBR Green RCR Master Mix (Applied Biosystems) according to manufacturer' s protocol. The PCR amplification was performed on the ABI Prism 7900HT sequence-detection system (Applied Biosystems) in a final volume of 12 µL using standard cycling parameters (10 min, 95°C; 30 sec, 95°C; 30 sec 65°C; 30 sec 72°C, with the latter three steps repeated for 45 times). Primers for the real-time PCR reaction were designed using Vector NTI 11. 0 software package (Invitrogen), following primer design guidelines given in the SYBR green PCR Master Mix and RT-PCR protocol (Applied Biosystems). The melting temperatures of all primers were between 65 and 71°C. All primers were purchased from the ITD (Glycon Australia Pty Ltd). The primers used for detection of pre-5. 8S rRNA were as follows: pre5. 8S_F 5′-TGTGAAACCTTCCGACCCCTCT-3′; pre5. 8S_R 5′-CGAGTGATCCACCGCTAAGAGTCGTA-3′. The primers used for detection of the RMRP RNA (GenBank NR_003051) were as follows: RMRP_F 5′-AGGACTCTGTTCCTCCCCTTTCCGCCTA-3′, RMRP_R 5′-TGGAGTGGGAAGCGGGGAATGTCTA-3′. The primers used for detection of human beta actin mRNA (ACTB, GenBank NM_001101) used as an internal normalization standard were as follows: ActF 5′-TCACCATTGGCAATGAGCGGTT-3′; ActR 5′-AGTTTCGTGGATGCCACAGGACT-3′. The final optimized concentration of primers was 250 nM for all DNA amplicons. The absence of inter- and/or intramolecular duplex formation between primers was confirmed in a control real-time PCR reaction lacking template. Relative quantification was performed as described in ABI Prism 7900HT Sequence Detection System User Bulletin #2 (Applied Biosystems) according to Comparative CT Method. In brief, threshold cycle (CT) values of experimental samples were normalized to corresponding CT values of the ACTB mRNA control, and then quantified relative to the sample with the maximal CT value (calibrator). All real-time PCR reactions were done in five technical replicates, and results were confirmed in two independent experiments. Progressive dilution of the fluorescent dye CFSE during cell division was used as a proxy to assess cell division rates using flow cytometry [21]. PBMC from the affected children (SKDP 6. 3 and 6. 4), their parents (SKDP 6. 1 and 6. 2) and healthy controls were labelled with 2. 5 µM CFSE and plated in triplicate at 5×105 cells/well in 48-well plates. PBMC were stimulated with PMA and Ionomycin or left unstimulated for seven days. Proliferation of PBMC in response to stimulation was assessed by examining CFSE dilution on a FACSCanto flow cytometer (BD). Comparative cell division rates were determined by calculating proliferation indices of cells undergoing 2 or more cell divisions using ModFit software (Verity Software House). POP1 amino acid substitutions are given using human POP1 mRNA sequence and annotations with GenBank accession NM_001145860. Human beta actin mRNA, RMRP RNA, and rRNA polycistron sequences have GenBank accession numbers NM_001101, NR_003051, and U13369 respectively. NCBI and FlyBase accession numbers for Drosophila POP1 homolog are NP_572236 and FBgn0026702 respectively. UCSC genome browser http: //genome. ucsc. edu/; NCBI dbSNP http: //www. ncbi. nlm. nih. gov/SNP/; Protein families database (PFAM) http: //pfam. sanger. ac. uk/; SIFT website http: //sift. jcvi. org/; GATK website http: //www. broadinstitute. org/gsa/wiki/index. php/The_Genome_Analysis_Toolkit; Online Mendelian Inheritance in Man (OMIM), http: //www. ncbi. nlm. nih. gov/Omim/. | Skeletal dysplasias are a group of genetic disorders affecting skeletal development that cause deficiencies and deformities of the limbs and spine, dwarfism, or abnormal bone strength. Skeletal dysplasias are usually inherited as monogenic Mendelian traits or occur as a result of de novo mutations. We report identification of mutations in human POP1 gene as the cause of a severe novel skeletal dysplasia. Molecular analyses presented in our work provide an important link between the pathogenesis of the disease and basic cellular processes including RNA processing and the cell cycle. We posit that our work will also have an immediate impact on assessment and counselling of novel cases of severe short stature. | lay_plos |
Iron sulfur (Fe/S) proteins are ubiquitous and participate in multiple biological processes, from photosynthesis to DNA repair. Iron and sulfur are highly reactive chemical species, and the mechanisms allowing the multiprotein systems ISC and SUF to assist Fe/S cluster formation in vivo have attracted considerable attention. Here, A-Type components of these systems (ATCs for A-Type Carriers) are studied by phylogenomic and genetic analyses. ATCs that have emerged in the last common ancestor of bacteria were conserved in most bacteria and were acquired by eukaryotes and few archaea via horizontal gene transfers. Many bacteria contain multiple ATCs, as a result of gene duplication and/or horizontal gene transfer events. Based on evolutionary considerations, we could define three subfamilies: ATC-I, -II and -III. Escherichia coli, which has one ATC-I (ErpA) and two ATC-IIs (IscA and SufA), was used as a model to investigate functional redundancy between ATCs in vivo. Genetic analyses revealed that, under aerobiosis, E. coli IscA and SufA are functionally redundant carriers, as both are potentially able to receive an Fe/S cluster from IscU or the SufBCD complex and transfer it to ErpA. In contrast, under anaerobiosis, redundancy occurs between ErpA and IscA, which are both potentially able to receive Fe/S clusters from IscU and transfer them to an apotarget. Our combined phylogenomic and genetic study indicates that ATCs play a crucial role in conveying ready-made Fe/S clusters from components of the biogenesis systems to apotargets. We propose a model wherein the conserved biochemical function of ATCs provides multiple paths for supplying Fe/S clusters to apotargets. This model predicts the occurrence of a dynamic network, the structure and composition of which vary with the growth conditions. As an illustration, we depict three ways for a given protein to be matured, which appears to be dependent on the demand for Fe/S biogenesis. Fe/S proteins are present in all living cells, where they participate in a wide range of physiological processes including respiration, photosynthesis, DNA repair, metabolism and regulation of gene expression [1], [2]. Since the discovery of a dedicated system for the maturation of nitrogenase in Azotobacter vinelandii [3], [4], the in vivo mechanism of Fe/S cluster formation and insertion into proteins has received increasing attention. A main conclusion is that this process involves several protein factors that are conserved throughout eukaryotes and prokaryotes [5]–[11]. Besides the NIF system dedicated to nitrogenase maturation, general systems involved in Fe/S cluster formation, called ISC and SUF, have been described. Sulfur is provided to these systems by cysteine desulfurases, referred to as NifS, IscS, and SufS [3], [12]–[16]. The role of CsdA, another E. coli cysteine desulfurase, remains to be assessed [17], [18]. In eukaryotes, iron is provided by frataxin [19], and its alteration causes pathological disorders, e. g. Friedreich ataxia in humans [20], [21]. In prokaryotes, frataxin-like, ferritin-like proteins and siderophores were proposed to provide iron for Fe/S biogenesis [22]–[25]. Scaffold proteins bind iron and sulfur and form Fe/S clusters that are eventually transferred to apotargets. Scaffold proteins were first described in the nitrogen fixing bacterium A. vinelandii [26], and subsequently in other bacteria, yeast, plants and humans, where they are referred to as IscU, Isu, NifU or Nfu [5]–[8], [27]–[29]. The SUF and ISC systems also include ATP-hydrolyzing proteins such as SufBCD, a pseudo ABC ATPase complex [24], [30], or HscBA, a DnaJK-like co/chaperone system [31], [32]. Recent results from the Fontecave lab showed that SufB contains an Fe/S cluster and forms a complex with SufC and SufD that can transfer this Fe/S cluster to apotargets ([30], M. Fontecave et al., personal communication). These observations lead to the hypothesis that the SufBCD complex could fulfill the scaffold function within the SUF system. Last, there are the A-type proteins, e. g. IscA, SufA, IscANif, ErpA, ISA1 and ISA2, whose biochemical functions and cellular roles remain unclear despite their presence in most studied systems. They are the objects of the present study, where they will be referred to as A-Type Carrier (ATC). An important issue regarding the role of ATCs pertains to their specificity in vivo. Indeed, genomes of model organisms, such as E. coli or S. cerevisiae, encode several ATC homologs, raising the question of whether they are functionally redundant. For instance, E. coli synthesizes three ATCs, IscA, SufA and ErpA, sharing 30% sequence identity [33]. Whereas the inactivation of iscA or sufA was almost neutral, an erpA mutation was found to be lethal under respiratory growth conditions [33]. Also, work on A. vinelandii revealed that an iscA mutation was lethal at high oxygen concentration [34]. Hence, these studies supported the notion that there are functional differences between the ATCs in vivo. On the other hand, functional redundancy between ATCs was indicated by other experiments: (i) in E. coli, an iscA sufA double mutant was found to exhibit a strongly decreased growth rate whereas single mutants grew almost like a wild type strain [35], [36, see Discussion], (ii) in S. cerevisiae, a double ISA1 ISA2 knockout exhibited a series of mitochondrial phenotypes not shown by single mutants [37]–[39]. Importantly, the perception that ATCs are functionally redundant is consistent with in vitro studies where either of the ATCs tested was found to transfer Fe/S to apotargets with similar efficiency [7], [29]. In this work, we combined phylogenomic and genetic approaches to answer several of the questions raised by the presence of an ATC in each Fe/S biogenesis system studied so far. Phylogenomic studies allowed us to trace the emergence of ATCs in bacteria and to follow their evolution throughout living organisms. This analysis gave insight on the question of when and where genetic redundancy arose and allowed the identification of three subfamilies (-I, -II and -III) of ATC. In this regard, Gamma-Proteobacteria, including E. coli, stood out as being exceptional in containing three ATCs belonging to ATC-I and ATC-II subfamilies. The genetic study showed that these ATCs are potentially interchangeable but that this bacterium has evolved to prevent full redundancy by controlling the synthesis of each ATC and the efficiency of its partnership with other components of the Fe/S biogenesis line. Phylogenomic and genetic data indicate that the evolution of ATC-II members was constrained by their partnership with scaffolds from which they receive Fe/S clusters, whereas the evolution of ATC-I members was mainly driven by their partnership with the apotargets to which they transfer Fe/S clusters. The net result is that the cell exploits the multiple ATCs homologs by positioning them between the cysteine desulfurase/scaffold duo and the apotargets, where they act as connectors between different routes for conveying Fe/S clusters from the scaffold to apotargets. Experimentally characterized ATCs (e. g. ErpA, SufA, IscA, IscANif, ISA1 and ISA2) contain a single domain of ∼100 residues referred to as PF01521 in the Pfam database. By using this domain as a query, we detected 991 proteins within 622 complete prokaryotic genomes. Among these proteins, 192 contained a truncated PF01521 C-terminal domain, e. g. in Firmicutes, or additional domains, e. g. NfuA proteins found in Gamma-Proteobacteria that contained an Nfu domain fused to PF01521 (see “other” category in Table S1). Similar to the well-characterized ATCs, 799 proteins harbored a single complete PF01521 domain, and were thus considered ATC proteins sensu stricto. These 799 ATC proteins were present in a majority of prokaryotes (436 of 622, ∼70%, Table S1 and Figure 1). In contrast, ATC proteins are absent in all Epsilon-Proteobacteria, Spirochaetes, Fusobacteria, Thermotogae and nearly all Archaea and PVC (Planctomycetales-Verrucomicrobia-Chlamydiae) as well as in many Firmicutes, Delta-Proteobacteria and Bacteroidetes/Chlorobi (Table S1 and Figure 1). Except in the amitochondria Entamoeba and the highly divergent amitochondriate Microsporidia, ATC encoding genes have been detected in all eukaryotic genomes. More precisely, amitochondriate anaerobic eukaryotes such as Giardia lamblia or Trichomonas vaginalis were found to possess only one ATC encoding gene whereas all other eukaryotes encoded at least two ATCs. A third copy is present in the genome of photosynthetic eukaryotes (e. g. Viridiplantae, Rhodophyta and Stramenopiles, see Table S1). The high conservation of ATC highlights the functional importance of these proteins in eukaryotes and bacteria. In order to study the origin and the evolution of the ATC genes, we performed a phylogenomic analysis of the ATC proteins. We inferred the presence of an ATC in the ancestor of a phylum if, in the ATC phylogeny, we found a monophyletic group of ATC that corresponded to the phylum and if the corresponding genes were well distributed within that phylum. The exhaustive phylogenomic analysis of the 911 ATCs from complete prokaryotic genomes showed that the bacterial and the few archaeal sequences did not form two distinct clusters (Figure S1). In fact, archaeal homologs emerged from within different bacterial subgroups. For instance, sequences from Methanosarcina are close to Firmicutes whereas those from Halobacteriales and Delta-Proteobacteria group together (Figure S1). This result, combined with the scarcity of ATC sequences in archaea strongly suggested that the Last Archaeal Common Ancestor (LACA) had no ATC and that a few archaea independently acquired their ATC through horizontal gene transfer (HGT) from bacteria (Figure 1, black circle number 3). Next, in order to pinpoint the origin of bacterial and eukaryotic ATCs, we selected a subset of sequences representative of their diversity to perform a more in-depth phylogenomic analysis. The overall topology of the resulting Bayesian tree was in agreement with the exhaustive phylogeny of the ATC family (Figures 2 and S1). This tree showed a number of monophyletic groups, most of them well supported, which corresponded to major bacterial phyla (Figure 2), i. e. Actinobacteria (Posterior Probability = 1. 0), Acidobacteria (PP = 1. 00), Chloroflexi (PP = 0. 91), Cyanobacteria (PP = 0. 75), Deinococcus/Thermus (PP = 1. 0), and two clusters of Alpha-, Beta- and Gamma-Proteobacteria (PP = 1. 0 and PP = 0. 51). The wide distribution of the ATC encoding genes in these phyla suggested the presence of such a gene in their ancestors (Figure 1, pink filled-in diamonds and Figure S1). In contrast, ATC proteins were probably absent in the ancestors of the PVC, Spirochaetes, Thermotogae and Epsilon-Proteobacteria (Figure 1, pink empty diamonds). The situation was less clear for the remaining bacterial groups (Figure 1, diamonds with “? ” inside), in which ATCs did not form monophyletic groups (e. g. Delta-Proteobacteria and Bacteroidetes/Chlorobi, Figure S1) or are too scarce, (e. g. Firmicutes, Table S1) or because too few representatives of the phylum were available (i. e. only one representative for Aquificae and for Fusobacteria, Figure 1). The presence of an ATC-encoding gene in the ancestors of a number of bacterial phyla indicated that the corresponding protein is ancient in Bacteria and may already have been present in the last ancestor of this domain (Last Bacterial Common Ancestor, Figure 1, black circle number 0). Consequently, the absence of any ATC-encoding gene in some bacterial lineages likely reflects secondary losses (empty pink diamonds in Figure 1). Caution should be taken to not extrapolate these observations to other components of the SUF and ISC systems as their own history will require a thorough phylogenomic analysis. Eukaryotic ATC sequences form three distinct groups in the phylogeny. Two of these emerged within each of the two Alpha-, Beta- and Gamma-Proteobacteria groups (PP = 1. 0 and PP = 0. 99), close to Alpha-Proteobacterial sequences (Figure 2). This did not support the hypothesis that genome duplication was responsible for the appearance of the two eucaryotic ATC copies but rather indicated that the Last Eukaryotic Common Ancestor (LECA) acquired two ATC from these Proteobacteria, likely through endosymbiosis (Figure 1, black circle number 4). The third group of eukaryotic sequences corresponded to the additional copy that is present only in photosynthetic eukaryotes and groups with cyanobacterial sequences (PP = 0. 96), confirming its acquisition through the primary chloroplastic endosymbiosis in the ancestor of Plantae (Figure 1, black circle number 5). The presence of this gene in photosynthetic protists such as Stramenopiles indicated that it has been conserved through secondary chloroplastic endosymbioses (Figure 1, black circle number 6). Our phylogenomic analysis showed that ATC likely originated in the Last Bacterial Common Ancestor and were conserved in most bacterial lineages (Figure 1). Surprisingly, the genomes of nearly all Alpha-, Beta- and Gamma-Proteobacteria were found to code for at least two ATCs sensu stricto (Table S1). The phylogenomic analysis of ATCs showed that these sequences formed two distinct monophyletic clusters (PP = 1. 0 and PP = 0. 51, Figure 2). In agreement with the phylogeny of proteobacteria within each cluster, Alpha-, Beta- plus Gamma-Proteobacteria each formed a monophyletic group. These observations indicated that the common ancestor of these Proteobacteria already contained two distinct ATCs that we propose to call ATC-I and ATC-II (Figure 1, blue and yellow filled-in diamonds). ATC-I includes ErpA from E. coli as well as yeast ISA2 whereas ATC-II includes both the SufA and IscA proteins from E. coli and yeast ISA1. ATC-I and ATC-II were conserved during the evolution of these Proteobacteria and in nearly all eukaryotes after their acquisition through the mitochondrial endosymbiosis. The poor resolution of the ATC phylogeny, in particular for the most basal nodes due to the small size of the set of sequences, did not allow us to identify the evolutionary event at the origin of ATC-I and ATC-II in Proteobacteria (Figure 1, black circle number 1). Two hypotheses can be proposed: (i) one of these two ATCs was acquired by the ancestor of Alpha-, Beta- and Gamma-Proteobacteria via a HGT of an ATC gene from a non-proteobacterial donor, or (ii) these two ATCs arose from duplication of the native proteobacterial ATC gene. Few Gamma-Proteobacteria, mainly Enterobacteriales, harbor two ATC-II homologs, suggesting that these two copies emerged recently in this group, possibly through the duplication of an ancestral ATC-II copy (Figure 1, black circle number 2). This led us to divide the Gamma-proteobacterial ATC-II in two groups: ATC-IIa that includes E. coli IscA and ATC-IIb that contains E. coli SufA. Interestingly, the comparison of evolutionary distances within the ATC-I, IIa and IIb subfamilies reveals that the corresponding ATC sequences did not evolve at the same rate. This is highlighted by the fact that the lengths of the branches separating ATC sequences from two organisms are different according to the considered family. For example, the evolutionary distances deduced from the tree of Figure 2 between ATC sequences from E. coli K12 and its close relative Yersinia enterolitica 8081 are 0. 32 substitutions per site for ATC-IIb, 0. 163 for ATC-IIa and 0. 068 for ATC-I. This clearly indicated that ATC-IIb sequences are the fastest evolving sequences whereas ATC-I sequences are the slowest. Finally, the ATC phylogeny showed another well supported subfamily of ATCs that includes sequences annotated as IscANif involved in nitrogenase maturation. We propose classifying them as a third ATC subfamily (ATC-III, PP = 1. 0, Figure 2). These sequences are present in organisms from various bacterial phyla (Supplementary Table S1), suggesting that they spread to various bacteria via HGT. Based on our data, it is not possible to determine in which bacterial phylum this third ATC family originated. The analysis of the genomic context of ATC genes led us to several new considerations. We first observed that the genetic context of non-proteobacterial ATC and proteobacterial ATC-I encoding genes was not conserved and very rarely contained genes encoding ISC or SUF components (Figure 2). In contrast, most of the ATC-II encoding genes were surrounded by genes encoding ISC or SUF components (Figure 2). More precisely, in Beta- and Gamma-Proteobacteria, most ATC-II encoding genes, including E. coli ATC-IIa member iscA, lie close to genes coding for the ISC system. In contrast, ATC-IIb genes are associated with genes coding for components of the SUF system as it is the case for E. coli sufA. This indicated that the association of ATC-II with genes encoding components of the ISC system is likely ancestral in these two proteobacterial subdivisions. Our phylogenomic analysis of the ATCs suggested that the two ATC-II copies observed in some Gamma-Proteobacteria resulted from a recent evolutionary event. It is possible that one of the two resulting copies (i. e. ATC-IIb) was subsequently associated with the SUF system, whereas the other (i. e. ATC-IIa) remained associated with the ISC system. The situation is less clear for the single ATC-II from Alpha-Proteobacteria since the type of association seemed to have changed many times during the evolution of this subdivision. For example, ATC-II encoding genes from Rickettsiales (e. g. from Rickettsia or Wolbachia) are associated with genes from the ISC system, whereas their close relatives (e. g. from Rhizobium, Nitrobacter or Rhodospirilum) are in the neighborhood of the SUF system encoding genes (Figure 2). The genes belonging to the ATC-III family are surrounded by genes annotated as nif (Figure 2). This strongly suggests that all these ATC-III encoding genes are indeed involved in nitrogenase maturation. Because we showed that ATC-III genes spread by HGT among bacteria from different phyla, this suggests that these HGTs may have involved the whole NIF gene cluster. E. coli emerged from the phylogenomic approach as representative of the subset of bacteria that synthesize three ATCs: one type I, ErpA, and two type II, IscA and SufA. We next exploit the genetic amenability of this organism to investigate the redundancy versus specificity of the ATC proteins. Results are presented in the sections thereafter. To investigate a potential redundancy between iscA and sufA, we sought to construct a strain lacking both genes. For this purpose, the ΔiscA: : cat mutation present in strain DV698 was transduced into strain DV701 (ΔsufA). The number of tranductants was surprisingly low, two orders of magnitude less, at best, than with the wild type MG1655 used as recipient (Table 1). Similarly, the ΔsufA: : kan mutation could not be P1-transduced into the ΔiscA mutant strain DV699. These observations suggested that combining the iscA and sufA mutations in the same background was lethal. To test the hypothesis that the iscA and sufA mutations were synthetically lethal, we used a co-transduction strategy, wherein the acquisition of the mutation to be tested is not used as a direct marker in the selection procedure. Hence, we used strain DV1239 (ΔiscA: : cat zfh-208: : Tn10) in which a Tn10 transposon (TetR) was inserted near the ΔiscA: : cat (CamR) mutation. We selected TetR transductants and co-transduction of the ΔiscA: : cat (CamR) was subsequently analyzed. The co-transduction frequency of the ΔiscA: : cat mutation with zfh-208: : Tn10 was about 75% in the wild type and <1% in the ΔsufA DV701 strain (Table 1). This result, in agreement with other data [36], [40], indicated that the presence of a ΔsufA mutation in the recipient strain counterselected the subsequent acquisition of the ΔiscA: : cat mutation. The same conclusion could be drawn from the reciprocal experiment carried out using strain DV1230 (ΔsufA: : kan zdi-925: : Tn10) with a Tn10 located in the vicinity of the sufA gene as a P1 donor, and DV699 (ΔiscA) as recipient (Table 1). We verified that the incompatibility between the iscA and sufA mutations was not due to polar effects on downstream genes in the isc or suf operon (see Materials and Methods). Taken together, these experiments established that iscA and sufA null mutations are synthetically lethal under aerobic conditions, suggesting that the IscA and SufA proteins are redundant for some function (s) essential for cell survival. IspG/H are essential Fe/S enzymes in E. coli because they participate to the isoprenoid (IPP) synthesis pathway. We therefore speculated that an iscA sufA mutant is not viable because IspG/H proteins are not matured. We demonstrated that this is actually the case by introducing eukaryotic genes that allow IPP synthesis from exogenously added mevalonate (hereafter referred to as the MVA pathway). The presence of this pathway allowed us to co-transduce the ΔiscA: : cat mutation with the zfh-208: : Tn10 locus into the ΔsufA MVA+ strain DV731 (Table 2). Moreover, none of the TetR CamR transductants selected was able to grow in the absence of mevalonate. These results indicate that the heterologous MVA pathway suppresses the defects of an iscA sufA mutant and, by inference that the synthetic lethality under aerobiosis of combining the iscA and sufA mutations comes from a lack of IPP. It is important to underscore that we checked that none of the mutants analyzed is altered in the expression of the ispG or ispH cognate structural genes (data not shown). Hence, we can safely conclude that the molecular bases of the defects exhibited by the iscA sufA mutant in aerobiosis are related to a lack of maturation of the 4Fe/4S-containing IspG and/or IspH proteins. The phenotype of an iscA sufA mutant, i. e. lethality under aerobiosis suppressible by the MVA pathway, is identical to that of an erpA mutant [33]. We therefore tested whether an iscA sufA mutant was viable under anaerobiosis, like an erpA mutant. In these conditions, we could co-transduce the ΔsufA: : kan mutation with the zdi-925: : Tn10 marker into either the wild type strain or the ΔiscA mutant (Table 3). Conversely, we could transduce the ΔiscA: : cat mutation into the wild type or the ΔsufA DV701 strains (Table 3). Interestingly, the ΔsufA ΔiscA transductants selected under anaerobiosis were unable to form colonies when subsequently incubated aerobically (data not shown). Similarly, the iscA sufA MVA+ strains were able to grow in the absence of mevalonate only in anaerobiosis (Table 4). These results extended the similarity between the conditional phenotype of erpA and iscA sufA mutants. The above results showed that the double iscA sufA and the single erpA mutants are each unable to grow under aerobiosis but fully viable under anaerobiosis. To test the hypothesis that, under anaerobiosis, the erpA gene compensates for the lack of iscA and sufA, we transduced the erpA: : cat mutation into the sufA iscA strain. This proved to be impossible unless the iscA sufA recipient contained the MVA pathway and the transductants were selected in the presence of mevalonate (data not shown). Moreover, the triple mutant erpA iscA sufA MVA+ could grow aerobically and anaerobically only in the presence of mevalonate (Table 4). This showed that the growth of the iscA sufA mutant under anaerobiosis depended upon the presence of a functional copy of the erpA gene. Conversely, we tested whether iscA and/or sufA was required for the anaerobic growth of the erpA mutant. First, the erpA sufA strain was found to be viable since we could transduce the erpA: : cat mutation into DV701 (ΔsufA). Moreover, an erpA sufA MVA+ strain was able to grow anaerobically even in the absence of mevalonate (Table 4). Second, in contrast to sufA: : kan, the iscA: : cat mutation could not be transduced into the erpA strain unless it contained the MVA pathway and only if transductants were selected in the presence of mevalonate. The growth of the iscA erpA MVA+ strain was totally dependent on the addition of mevalonate in the medium (Table 4). This showed that a functional copy of the iscA gene was absolutely required for the anaerobic growth of the erpA mutant whereas sufA appeared to be dispensable. Because the inactivation of the ispG or the ispH gene is lethal in anaerobiosis, these results showed that ErpA and IscA were both able to ensure enough IspG/H maturation to produce sufficient IPP to sustain growth (Paths 4 and 7 in Figure 3A). Thus, ErpA and IscA are redundant under anaerobiosis. Searching for genetic suppressors has long been a rewarding strategy for revealing functional overlap between different cellular components or pathways. We therefore used the mevalonate dependency of the strains lacking a combination of ATC encoding genes to isolate second site mutations that would render them mevalonate independent for growth. First, we searched for secondary mutations that could suppress the double iscA sufA mutant. We used strain DV1145 (sufA iscA MVA+), which is mevalonate dependent for growth under aerobiosis, to select and analyze four revertants that could grow in the absence of mevalonate. The mutations that rendered strain DV1145 mevalonate independent were first localized by Hfr mapping and, second, by using a series of Tn10 containing strains as donors in P1 transduction experiments (Materials and Methods). One suppressor mutation, referred to as supSI-1, was unexpectedly found within the erpA gene and led to a glycine-to-valine substitution at position 89 in the C-terminal part of the protein. There are several foreseeable possibilities to account for this by-pass, among which a gained (or optimized) ability to acquire Fe/S from natural (Path 3 in Figure 3A) or alternative sources, or, symmetrically, an increased ability to deliver Fe/S to IspG/H (see below, Path 7 in Figure 3A). The biochemical characterization of this variant is under way. Second, we selected suppressors that allowed the iscA erpA: : cat MVA+ strain to grow aerobically in LB in the absence of mevalonate. Three mutations, referred to as supYI-1 to 3, were found to modify the iscR coding sequence. Actually, in two independently selected suppressors (supYI-1, supYI-2), a single mutation changed the highly conserved Ile-37 residue to Val. The supYI-3 mutant contained a His to Asn change at position 107. Interestingly, transducing a sufA but not sufB or sufCD mutation into any of these three iscA erpA iscR (supYI) mutants abolished colony formation on LB plates unless mevalonate was added, indicating that the suppressive effect of the iscR mutant alleles depended on an active sufA gene. Moreover, the expression of a Psuf+: : lacZ gene fusion (see Materials and Methods) was found to be induced in the presence of the supYI suppressor mutation (data not shown). Taken together, these results indicated that suppression of the mevalonate-dependence of the iscA erpA mutant was due to an increased expression of the IscR-activated suf operon, hence an increase in SufA protein synthesis (Path 5 in Figure 3A). A fourth revertant (supYI-4) was found to be a single G-to-T substitution at position −27 in the promoter sequence of sufA. Remarkably, this mutation altered the putative binding site for the Fur repressor. A possibility was that this mutation led to a derepression of the suf operon, thereby increasing SufA protein synthesis (Path 5 in Figure 3A). This hypothesis was confirmed by fusing this mutant promoter sequence to a lacZ reporter gene (see Materials and Methods): the expression level of Psuf (supYI-4): : lacZ in a wild type genetic context was about 150-fold higher than the expression level of the Psuf+: : lacZ fusion. Overall, this hunt for extragenic suppressors fully confirmed that the three genes iscA, sufA and erpA are interchangeable. These data also revealed that, in vivo, one barrier to full functional redundancy lies in gene expression control. The genetic analysis above suggested that increased synthesis of a given ATC protein could create bypasses, compensating for the loss of the others. We therefore investigated the potential redundancies between ErpA, IscA and SufA by a multicopy suppressor approach. Likewise, the iscA and sufA genes were each cloned under the control of the Para promoter in the pBAD vector, and the resulting plasmids were tested for their ability to suppress the lethality caused by the erpA mutation under aerobiosis. The pLAS-A plasmid (SufA overproducer) suppressed the growth defect of strain LL402 (erpA), although not with wild type efficiency (Table 5). In contrast, the pLAI-A plasmid (IscA overproducer) was unable to suppress the erpA mutant' s lethal phenotype (data not shown). A series of additional genetic experiments further strengthened the notion of a difference between IscA and SufA. For instance, transduction experiments showed that the otherwise lethal erpA: : cat mutation could be transduced into MG1655 under aerobiosis if the recipient carried the pLAS-A plasmid, whereas no transductants were obtained if MG1655 carried pLAI-A (data not shown). Also, a genomic E. coli pUC-based library was screened for plasmids able to restore the growth of the ΔerpA mutant under aerobiosis. Of nine suppressing plasmids recovered, five carried the erpA gene, four carried the sufA gene, but none carried iscA (data not shown). These studies indicated that increased sufA gene dosage was able to sustain the aerobic growth of an erpA mutant (Path 5 in Figure 3A). Conversely, we tested whether increased erpA gene dosage was able to suppress the lethality of the iscA sufA mutant. Strain DV731 (iscA sufA MVA+) was grown in the presence of mevalonate and transformed with plasmid pLAE-A (ErpA overproducer). The transformants became able to grow under aerobiosis, even in the absence of added mevalonate (Table 5). Moreover, we were able to transduce the iscA: : cat allele into strain DV701 (ΔsufA) carrying plasmid pLAE-A. Thus, these results indicate that ErpA, when overproduced, compensates for the defect of IPP synthesis due to the simultaneous absence of both SufA and IscA under aerobic conditions (Path 3 or 7 in Figure 3A). S. cerevisiae encodes two ATCs, ISA1 and ISA2. Heterologous complementation was tested using the E. coli strains erpA and sufA iscA mutants. The plasmid p (Isa1) complemented sufA iscA but not erpA, like the pLAI-A plasmid carrying the E. coli iscA gene. The plasmid p (Isa2) complemented both the iscA sufA and the erpA mutants, like the pLAE-A plasmid carrying the E. coli erpA gene. These results were consistent with the classification of ISA1 and ISA2 being an ATC-II and ATC-I, respectively. Both the extragenic and the multicopy suppressor approaches provided data pointing to potential interchangeability among the different ATCs that the cell can use for IspG/H maturation. We were therefore interested in defining the genetic constraints required for these suppressing effects to occur. In particular, it was of great importance to identify which component of the cellular Fe/S biogenesis system (see Introduction) was required in each of the suppression cases described above. We thus first sought to construct a set of strains in which deletions of each suf gene were combined with deletions of each isc gene. In transduction experiments similar to those described above to assess iscA and sufA incompatibility, we found that, under aerobiosis, the ΔiscA mutation could be combined with a deletion of any of the suf genes (sufB, sufCD, sufS and sufE were tested). Conversely, the ΔsufA mutation could be introduced without loss of viability into the iscU, iscS, fdx, hscA and hscB mutants (data not shown). On the contrary, the iscUA deletion inactivating both iscA and iscU could not be combined with any suf mutation. Likewise, the Δsuf mutation deleting the whole suf operon was not compatible with a mutation in any of the isc genes. In addition, as was the case for the combination of iscA and sufA mutations, all combinations of mutations became possible in the presence of the MVA pathway genes in the strain and the addition of mevalonate in the medium. All together, these results indicate that IscA can function with the SUF system and, conversely, SufA can function with the ISC system (Path 2 in Figure 3A), consistent with the association of ATC-II encoding genes with either the isc or the suf operon in Alpha-Proteobacteria (Figure 1). We then tested which elements were required for sufA to act as a multicopy suppressor of the iscA erpA double mutant (Table 6). The ability of pLAS-A to suppress the iscA erpA strain remained possible even in the absence of IscU because pLAS-A suppressed the lethality of the iscUA erpA strain (Table 6). This indicated that sufA could function in the absence of the ISC system. In addition, pLAS-A could suppress the lethality of the iscA erpA strain in the absence of other suf genes, i. e. strain iscA erpA Δsuf/pLAS-A was viable (Path 5 in Figure 3A). In fact, iscA erpA suppression by pLAS-A could be abolished only in strain iscUA Δsuf erpA. These results confirmed that SufA could function, not only with its “natural” partners of the SUF system, but also with IscU in the ISC system. We showed previously that the growth of the iscA sufA strain in the absence of O2 was erpA dependent, because the growth of the triple mutant iscA erpA sufA strictly depended on the presence of mevalonate (Table 3). An interesting observation was that the growth of iscA sufA in the absence of O2 was also IscU-dependent since the iscUA sufA strain (and Δsuf iscUA) was unable to grow in the absence of O2 unless mevalonate was added in the medium (Table 6). Altogether, these results indicated that the growth of the iscA sufA strain in anaerobiosis likely requires an IscU-ErpA connection (Path 3 in Figure 3A). In the absence of the suf operon, plasmid pLAE-A suppressed the lethality associated with the simultaneous inactivation of the iscA and sufA genes (Table 6). This indicated that ErpA-mediated suppression of the iscA sufA strain was Suf independent. On the contrary, ErpA-mediated suppression of iscA sufA was iscU dependent since plasmid pLAE-A lost its suppressive ability with the additional inactivation of iscU, i. e. strains iscUA sufA/pErpA or iscUA Δsuf/pErpA were inviable in the absence of mevalonate. This strengthened the hypothesis that ErpA could work with IscU (Path 3 in Figure 3) and, moreover, strongly suggested that ErpA could not use the SUF system without SufA. The iscA and sufA single mutants have repeatedly been found to cause marginal defects [5], [6]. However, we find here the iscA sufA double mutant to be conditional lethal in aerobiosis, in agreement with recent reports [36], [40]. In contrast, Lu et al. have found the sufA iscA double mutant to be non viable in synthetic medium but viable when aerobically cultivated in rich medium LB [35]. One reason for the discrepancy between these studies might lie in the fact that the sufA iscA strains generate survivors at a relatively high frequency when cultivated aerobically as we showed here (Table 3, Materials and Methods). Alternatively, the discrepancy might be due to differences in the genetic background: the sufA/iscA combination was found conditionally lethal in MG1655 whereas Lu et al. [35] worked with MC4100, a strain which went through multiple mutagenesis protocols [41]. The conditional phenotype of an iscA sufA mutant could formally be due to the general alteration of the SUF and ISC systems. This hypothesis, however, runs against a series of experimental observations reported in the present and previous studies: (i) analysis of the SUF system revealed the existence of various types of complexes such as SufBCD, SufSE, SufBCDSE, none of them containing SufA ([13], [42], our unpublished results); hence, a mutation in sufA is not predicted to destabilize the entire Suf complex and, indeed, mutations in sufA cause phenotypes much less severe than mutations in other suf genes; (ii) IscA was indeed found to interact with HscA and, because HscA was found to form a complex with IscSUHscB, a formal possibility is that a defect in IscA could alter the functioning of the ISC system; however, this is difficult to reconcile with the fact that a mutation in iscA causes minor phenotype, if at all, compared with mutations in other isc genes [12], [43]; (iii) control experiments allowed us to rule out the hypothesis of a polar effect of the iscA or sufA mutations on the expression of downstream isc or suf genes. Rather, we showed unambiguously in this work that insufficient synthesis of IPP is the cause of the lethality of the sufA iscA mutant. A similar lack of IPP had been put forward to account for the conditional phenotype of the erpA mutant [33]. The last steps of the synthesis of these compounds in E. coli involve two essential enzymes, IspG and IspH, which are known to contain 4Fe/4S clusters necessary for their activity [44], [45]. erpA gene expression was not altered in the sufA iscA mutant, and conversely, no decrease of iscA and sufA mRNA was observed in the erpA mutant (data not shown). Along the same lines, ispG and ispH genes expression was not altered by mutations in sufA, iscA, erpA or combinations thereof (data not shown). Therefore, we conclude that a defect in IspG/H Fe/S maturation is likely the cause of the lack of growth of iscA sufA and erpA mutants. Together with our previous characterization of ErpA, these observations reinforce the idea that IspG/H are the only Fe/S enzymes requiring ATCs essential for E. coli to survive under routine laboratory conditions, and that all three ATCs contribute to the maturation of IspG/H. Mettler et al. recently proposed that the ErpA activity was responsible for the viability of iscA sufA double mutant under anaerobiosis [40]. The present work, in which we showed the lethal phenotype of the iscA sufA erpA triple mutant, fully confirmed this interpretation. On the basis of in vitro studies, ATCs were first proposed to act as scaffolds [46], because they were shown to bind and eventually transfer Fe/S clusters to a wide series of apotargets, including 2Fe/2S proteins like Fdx and 4Fe/4S proteins like BioB, IspG and APS reductase [33], [47]–[49]. Later on, this view was challenged by a series of studies reporting the ability of IscA and SufA to bind in vitro iron only, and by the proposal that they acted as iron sources in vivo [35], [50], [51]. In turn, the “iron only” hypothesis was disputed by the discovery that the as-isolated IscA protein from Acidithiobacillus ferrooxidans contains a 4Fe/4S cluster [52] and by a new study on E. coli SufA [53]. Last, an in vitro study showed that IscU could assist Fe/S acquisition by IscA but the reverse was not true, indicating that IscA might intervene downstream of the IscU-controlled scaffolding step [48]. Our genetic analysis in E. coli showed that maturation of IspG/H could not take place in two situations: (i) the complete absence of the three ATCs and (ii) the simultaneous absence of IscU and the SufBCD complex. Conversely, in the presence of one ATC protein and IscU or SufBCD, we were always able to find at least one experimental condition supporting IspG/H maturation. The fact that the inactivation of iscU and sufBCD is lethal is fully consistent with the proposal that SufBCD acts as a scaffold within the SUF system [30]. A remote possibility is that SufBCD is necessary for the functioning of an as yet unidentified scaffold. Furthermore, the viability of the iscU sufA and iscA sufB (or sufCD) mutants indicates that the ATC proteins intervene at a different level than IscU/SufBCD. This suggests that ATCs are unlikely to have a scaffolding function, a conclusion also reached by other authors [54]. Our results are thus in agreement with a role of the ATCs as intermediates between the Fe/S synthesis systems and the apotargets, likely as carriers of ready made clusters (Figure 3). Despite their established involvement in the maturation of Fe/S proteins in yeast, the molecular function of ISA1 and ISA2 remains obscure. In particular, their ability to bind Fe/S clusters appears uncertain [8]. Importantly, we found S. cerevisiae ATCs ISA1 and ISA2 able to complement a lack of ATCs in E. coli, hence to presumably act as Fe/S carriers. Studies are however required to test the tentative idea that ISA1/2 can fulfil in the bacterial context a function that they do not carry on in the yeast mitochondria. A mutant devoid of all three ATCs did not permit maturation of IspG/H. Furthermore, we were able to identify at least one experimental condition under which a single ATC could ensure apo-IspG/H maturation in the absence of the two other ATCs. Taken together, these two observations are best explained by postulating that all three ATCs are intrinsically able to mature IspG/H. The ATC proteins are thus clearly biochemically redundant, in agreement with results from the in vitro assays and the common evolutionary origin of their encoding genes. The question then arises as to why the erpA, erpA sufA and iscA sufA mutants, which all produce at least one ATC, could not mature IspG/H under aerobiosis but could do so under anaerobiosis. One possibility is that under aerobiosis, the cellular need in IPP exceeds that under anaerobiosis and that the concentration of active IspG/H in these mutants is below the critical minimal level necessary for growth. The fact that overproduction of ErpA or SufA is sufficient to restore the growth of the iscA sufA or iscA erpA mutants, respectively, indeed supports the hypothesis that the limiting step for IPP synthesis in these mutants is the activity carried out by ATCs, namely maturation of IspG/H. However, this interpretation does not explain why oversynthesis of IscA does not allow IspG/H maturation (i. e. iscA does not act as a multicopy suppressor of the erpA mutant). This last observation indicates that, in the presence of O2, IscA does require ErpA to make active IspG/H. Hence, this leads us to propose a model in which the environment controls the specificity of partnerships. The molecular bases of this environmental control appear to lie either directly within the transfer process itself or with gene regulation. Under environmental circumstances that are harmless for Fe/S (Figure 3B), such as anaerobiosis, the IscU scaffold transfers its Fe/S to either ErpA or IscA, both of which can transfer the cluster to apo-IspG/H. In addition, ErpA can receive an Fe/S cluster from IscA. Biochemically, SufA is also potentially able to carry out this activity, but it is not synthesized in sufficiently large amounts in these conditions. A direct transfer of an Fe/S cluster from IscU to apo-IspG/H, bypassing IscA and ErpA, does not occur. When conditions are more hazardous for Fe/S cluster synthesis (Figure 3C), such as aerobiosis, IscA and ErpA cooperate to mature apo-IspG/H, presumably because of the O2-sensitivity of the direct transfer from IscA to apo-IspG/H and the poor synthesis of the SUF system. When conditions become stressful (Figure 3D), SufA recruits the whole SUF system for building up a new pathway for maturation of apo-IspG/H. The model above derives from the interpretation of the phenotypes of mutants and of the effects of plasmids overproducing ATCs. Because we did not directly measure the level of each ATC protein, no definitive conclusion can thus be made on how efficient each ATC protein functions in a particular path in a wild type strain. For example, the transfer of Fe/S clusters from IscU to SufA was put forward to account for the viability of the iscA sufB strain. However, in this strain, the sufA gene expression is increased because of the iscA mutation: the transfer might not occur in a wild type strain with “normal” level of IscA and SufA. Moreover, showing that the proteins are present in normal amounts might not be conclusive either. For instance, a transfer from IscU to ErpA was proposed to occur in the iscA sufA strain but, again, such a transfer ought to be demonstrated to occur in the presence of a competing IscA protein. Hence, our model aims at listing all potential paths for transferring Fe/S from and to ATCs. The next challenge will be to identify conditions under which each path operates. The ISC and SUF systems have been proposed for a long time to overlap and we showed here that they indeed share a common substrate, i. e. IspG/H. This overlap accounts for both the lethality of isc suf mutants [12], [36], [40], [55] and for the suppressing effect of overproducing the suf operon in isc mutants [12], [40]. On the other hand, recent data revealed that the maturation of Fnr is under the control of ISC only under aerobiosis [40]. Also, in Azotobacter, IscA is required for survival at high oxygen tension, implying that an Fe/S protein, essential for growth under these conditions, is specifically targeted by IscA [34]. Finally, in Azotobacter, IscU can be substituted under low oxygen tension by NifU, the scaffold of the NIF system [56]. Collectively, these observations indicate that substrate specificity might also intervene in deciding which Fe/S biogenesis pathway should be used for a given apoprotein. Clearly, more work is required to define the extent of the overlap between the Isc and Suf pathways. Solving the issue will require, in particular, the in vivo study of the maturation of a large set of Fe/S proteins, if not all, in various conditions and genetic backgrounds. Interestingly, it was recently suggested that the maturation of the 4Fe/4S proteins and 2Fe/2S proteins might follow different pathways [57]. The present phylogenomic study lends credence to an evolutionary scenario in which ATC originated in bacteria and were conserved in main bacterial phyla (Figure 1, circle number 0 and pink filled-in diamonds). Subsequently, Alpha-, Beta- and Gamma-Proteobacteria acquired a second ATC gene via either gene duplication or horizontal gene transfer, and the two ATCs were later transferred to eukaryotes via the mitochondrial endosymbiosis. In proteobacteria and eukaryotes, ATCs were subdivided in two sub-families: ATC-I that contains the E. coli ErpA and ISA2 proteins, and ATC-II that contains the E. coli IscA and SufA and the S. cerevisiae ISA1 proteins. Based on genetic experiments, we propose that in E. coli at least, ATC-I became specialized in the transfer of Fe/S to apotargets, while the selection pressure exerted on ATC-IIs tended to keep them in close partnership with proteins of the Fe/S synthesis systems from which they receive Fe/S clusters (Figure 3). This proposal is supported by the fact that: (i) functional analysis of the E. coli system showed that Fe/S transfer does not occur between the ATC-I protein ErpA and SufBCD proteins of the SUF system, and is inefficient between ErpA and IscU (Path 3 in Figure 3); on the contrary, both E. coli ATC-II proteins IscA and SufA can function with the two systems, and (ii) the genomic context of ATC-I encoding genes is devoid of genes involved in Fe/S biosynthesis, in contrast to ATC-II encoding genes, always found associated with such genes in proteobacteria. This scenario led us to speculate that ATC-I (ErpA) intervenes at a different step from ATC-II (IscA/SufA) in the IspG/H maturation process in E. coli (Figure 3). Three main conclusions can be drawn from studying E. coli as a model: (i) ATCs are essential intermediates between Fe/S biogenesis systems and targets, (ii) ATCs possess highly related biochemical properties, and (iii) the seemingly redundant repertoire of ATCs is exploited by the cell as a function of environmental conditions and possibly substrate specificity. The phylogenomic analysis uncovered a wide diversity in the distribution of ATCs. Thus, the comparison of the E. coli system with these widely diverging systems yields immediate questions, which E. coli could help to solve both as a reference and as a tool. Hereafter are listed a few situations we think might be of great interest to investigate. First, there are organisms (e. g. some Archaea, PVC, Spirochaetes, Thermotogae) that lack ATC but encode SUF or ISC components, IscU and/or SufB/D proteins, in particular. It is conceivable that the scaffold (s) of these organisms evolved in such a way that they acquired the ability to directly transfer Fe/S clusters to apotargets, or, alternatively, that other proteins carry out the function of ATCs. One way to understand these cases would be to test whether IscU from ATC-less organisms can complement the lethality of an E. coli strain devoid of ATC and identify the determinants differentiating these IscUs and E. coli IscU. Second, there are organisms having only one ATC. This latter might have conserved its ancestral ability to interact with both Fe/S biogenesis systems and apotargets. Firmicutes rank among them and it will be a feasible task to test this prediction by using Bacillus subtilis as a model. Third, there are organisms that possess multiple ATCs, but with a repertoire different from that of E. coli. This is the case of Anaebena, which has 2 ATC-IIIs in addition to two ATCs, or Rhodospirillum, which has one ATC-I, one ATC-II and one ATC-III. This raises the question whether, like E. coli, these organisms have exploited their repertoire of ATCs by dedicating some ATCs to an interaction with apotargets while other ATCs were kept in closer connection with general components such as scaffolds. ATC-IIIs represent an interesting case since they seem to have evolved to become specific for a particular target, nitrogenase. It would be interesting to know whether ATC-III also evolved to receive Fe/S solely from the NIF system. The rich and synthetic media were LB broth and M9, respectively. Glucose (0. 2%), arabinose (0. 2%), casaminoacids (0. 2%), amino acids (0. 005%), thiamine (50 µg/ml), nicotinic acid (12. 5 µg/ml) and mevalonate (1 mM) were added when required. Solid media contained 1. 5% agar. Antibiotics were used at the following concentrations: chloramphenicol (Cam) 25 µg/ml, kanamycin (Kan) 25 µg/ml, tetracycline (Tet) 25 µg/ml, spectinomycin (Spc) 50 µg/ml and ampicillin (Amp) 50 µg/ml. All the strains are E. coli K-12 derivatives; principal strains are listed in Table 7. Strains carrying the Tn10 transposons used for co-transduction experiments were from the Singer collection [58], [59]; strain JW1674 from the Keio collection [60] kindly provided by P. Moreau, was the donor of the ΔsufA: : kan mutation; the same collection provided all the other suf mutations but Δsuf: : cat and all the deletions of the isc genes but iscA: : cat and iscUA: : cat. The iscA: : cat mutation was generated in a one-step inactivation of the iscA gene as described [61]. A DNA fragment containing the cat gene flanked with a 5′ and 3′region bordering the iscA gene was PCR-amplified using pKD3 as a template and oligonucleotides iscAUP and iscADO (Table S2). Strain BW25113, carrying the pKD46 plasmid, was transformed by electroporation with the amplified linear fragment and CamR clones were selected; one of these clones was used to transduce the iscA: : cat mutation into MG1655, giving strain DV698. To generate the iscUA: : cat deletion, we first cloned the iscUA genes by PCR amplification from E. coli MG1655 chromosomal DNA using oligonucleotides iscU and iscA (Table S2) and subsequent insertion of the PCR product in pGEM-T deleted for the HincII restriction site by the T/A cloning method. The two genes were then disrupted by inserting the cat cassette in the coding region between the two HincII sites of iscUA. The resulting iscUA: : cat disruption was then excised by restriction and the linear DNA electroporated into the BW25113/pKD46 strain; one CamR clone was used to transfer the iscUA: : cat deletion into MG1655 creating strain DV1240. All mutations were introduced into strains by P1 vir transduction [62], selecting for the appropriate antibiotic resistance. The antibiotic resistance cassettes were eliminated when needed using plasmid pCP20 as described [63]. To verify that the isc and suf mutations had no polar effects, we checked that the sufE and hscA genes were correctly transcribed in the mutants. We first extracted the RNA from mutant strains grown overnight in LB medium. RNA was extracted using the SV Total RNA Isolation System according to the manufacturer' s recommendations. To remove further DNA contaminations, the eluted RNA was treated with 2 units of RNase-free DNase and kept at −80°C. The amount of RNA was calculated from 260 nm absorption using a Biowave II spectrophotometer. Conversion of RNA to cDNA was performed using the SuperScript RT System from Invitrogen. For all RT reactions, 1 µg RNA was used and 100 ng random primer was added together with diethylpyrocarbonate (DEPC) -treated water to a final volume of 12 µl. The mixture was then incubated at 70°C for 10 min and transferred to room temperature. To continue the reverse transcription reaction, a master mix was prepared according to the manufacturers protocol, added to the RNA tube and incubated at 42°C for 1 hour, followed by a 15 min inactivation step at 70°C in a Robocycler. cDNA was kept at −80°C. PCR were carried out in a standard PCR Master Mix reaction with 1/30 of the reverse transcription reactions and 100 ng of primers hscARevRT and hscAsensRT or sufErevRT and sufEsensRT in 10 µl final volume (Figure S2A and B). Lack of contaminating DNA from the RNA preparations was checked by performing parallel PCR reactions using RNA at a same concentration (Figure S2C). DNA products were analyzed by 1. 5% agarose gel electrophoresis with Tris-acetate-EDTA (TAE) buffer. To construct the Psuf: : lacZ fusions, PCR amplifications of chromosomal DNA from strains iscA erpA MVA+ and iscA erpA MVA+ sup (YI-4) were carried out using primers sufUP and sufDO (Table S2). The PCR products were digested by EcoR1 and BamHI and cloned into pRS415 [64] to construct transcriptional fusions. The fusions were introduced into the bacterial chromosome of strain DV206 (MG1655 ΔlacZ, [65]) using the RS45 phage and we verified that strains DV1294 (MG1655 ΔlacZ Psuf+: : lacZ) and DV1296 (MG1655 ΔlacZ Psuf (YI-4): : lacZ) were monolysogens as described [66]. ß-galactosidase assays were carried out as described [62]. When grown in LB to OD600 about 0. 5, the activity of the Psuf+: : lacZ and Psuf (YI-4): : lacZ fusions was 4+/−10% and 600+/−10%, respectively. Plasmids pLAI-A (IscA), pLAS-A (SufA) and pLAE-A (ErpA) were constructed by, first, PCR amplification from the MG1655 chromosomal DNA using the following primers: EcoIscA and XhoIscA for pLAI-A; EcoSufA and XhoSufA for pLAS-A; EcoErpA and XhoErpA for pLAE-A (Table S2). The PCR products were then digested by EcoRI and XhoI and ligated into the cognate sites of pBAD-I* (AmpR). Plasmids p (ISA1) and p (ISA2) were constructed by, first, PCR amplification from pETDuet-1 (isa1) with primers T7 and XhoIsa1 or PCR amplification from pE-T3a- (isa2) with primers T7 and XhoIsa2, respectively, and subsequent T/A cloning in pGEM-T. XbaI-XhoI fragments carrying the isa1 or isa2 genes were then subcloned from the pGEM-T derivatives into the NheI/SalI sites of pBAD-I*. This allowed the synthesis ISA1 and ISA2 proteins free of mitochondrial targeting signal. In the presence of exogenously added mevalonate, the DV1145 strain (sufA iscA MVA+) forms tiny colonies after 24 h aerobic incubation. However, we noticed that fast growing clones were often seen in the streaks, indicating that suppressor mutations accumulated at high frequency. To select suppressors, we grew the DV1145 strain under anaerobiosis in the presence of MVA, then plated it onto LB plates that were incubated aerobically at 37°C. Plating efficiency in LB compared to mevalonate-supplemented LB was about 10−4 (Table 2). Suppressor mutants were purified twice from the LB plates after two days incubation and four independent mutants (supSI-1 to -4) were retained for further analysis. Four suppressors supYI-1 to -4 of iscA erpA: : cat MVA+, occurring at a lower frequency (10−6, Table 2), were selected in a similar way. Strains iscA erpA: : cat MVA+ supYI and sufA iscA: : cat MVA+ supSI were crossed with various Hfr Tn10 strains from the Wanner collection [67] kindly provided by M. Berlyn and the exconjugants were selected onto LB medium plates supplemented with Cam, Kan Tet arabinose and mevalonate. The exconjugants were then scored for their ability to grow in the absence of mevalonate, which indicated the presence of suppressor mutations. These experiments showed that the supSI-1+ allele could be transferred at a very high frequency by the HfrC strain (76% of the TetR clones were unable to grow on LB but able to grow on LB supplemented with mevalonate) but not by the P801 and B8 Hfr (none of 80 TetR clones were unable to grow on LB) indicating that the suppressor mutation was located between the transfer starts of these two later Hfr, that is 3,19 and 7,8 min on the chromosomal map. More precise localizations were obtained by the transduction of the suppressor strains with P1 stocks made on various donor strains carrying Tn10 insertions [58], selecting TetR transductant on LB plates supplemented with tetracycline and mevalonate and subsequently scoring the clones on plates devoid of mevalonate. Because we obtained TetR clones unable to grow in the absence of mevalonate, we concluded that the supSI allele was about 30% co-transducible with the zad-220: : Tn10 marker (3,2 min). Suppressors supYI-1, -2 and -3 were located in the same way between the injection points of KL98 and KL14 (54. 3 and 68. 5 min, respectively), while supYI-4 was located between the injection points of B7 and PK19 (32 and 43. 5 min, respectively). The supYI-1, -2 and -3 mutations were about 80% co-transducible with zfh-208: : Tn10 (57. 4 min) and supYI-4 was 30% co-transducible with zdi-925: : Tn10 (38. 3 min). Therefore, we PCR-amplified the erpA region from the chromosome of the supSI-1containing strain using the EcoErpA and XhoErpA oligonucleotides, and its nucleotide sequence analyzed. Similarly, the iscR region of supYI-1, -2 and -3 containing strains and the sufA region of the supYI-4 containing strain were amplified and sequenced. As control, we sequenced PCR amplifications of the regions of the parental strain used to select the suppressors. The complete sequences of 622 prokaryotic (581 bacterial and 41 archaeal) genomes available in February 2008 were downloaded from the NCBI FTP website (ftp. ncbi. nih. gov). The HMMER package [68] and self-written scripts were then used to search for ATC homologs in these complete genomes, requiring the presence of Fe-S_biosyn domain (Iron-sulphur cluster biosynthesis, PFAM accession number PF01521, PFAM 22. 0 version) [69], [70]. Alignments E-value with the Fe-S_biosyn profile less than 0. 1 were considered as significant. The corresponding sequences were subsequently analyzed with the same software in order to determine the presence of additional known functional domains. Additional BLASTP/tBLASTN searches [71] were performed in complete genomes to ensure that the ATC family was exhaustively sampled and in the nr database at the NCBI to retrieve eukaryotic sequences. For each homolog, the gene context, defined as the 5 neighboring genes located upstream and downstream, was investigated using self-written scripts. The retrieved homologous sequences were aligned using CLUSTALW 1. 83 [72]. The resulting alignment was then visually inspected and manually refined using ED program from the MUST package [73]. Regions where homology between amino acid positions was doubtful were removed from the phylogenomic analyses. The phylogeny of all the prokaryotic ATC was constructed using the maximum likelihood method implemented in the PHYML software [74] with a WAG model (including an estimated proportion of invariant sites). According to the high number of sequences (911) and the small number of sites (76 positions), bootstrap analysis was not performed. An in-depth phylogenomic analysis using a more restricted sequence sampling representative of the diversity of bacterial and eukaryotic sensu stricto ATCs was performed using the bayesian approach (58 sequences, 90 positions) implemented in program MrBAYES 3. 1. 2 (with a mixed substitution model and a gamma law (4 rate categories) and a proportion of invariant sites to take among-site rate variation into account [75]). The Markov chain Monte Carlo search was run with 4 chains for 1,000,000 generations, with trees being sampled every 100 generations (the first 2,500 trees were discarded as “burnin”). | Iron sulfur (Fe/S) proteins are found in all living organisms where they participate in a wide array of biological processes. Accordingly, genetic defects in Fe/S biogenesis yield pleiotropic phenotypes in bacteria and several syndromes in humans. Multiprotein systems that assist Fe/S cluster formation and insertion into apoproteins have been identified. Most systems include so-called A-type proteins (which we refer to as ATC proteins hereafter), which have an undefined role in Fe/S biogenesis. Phylogenomic analyses presented, here, reveal that the ATC gene is ancient, that it was already present in the last common ancestor of bacteria, and that it subsequently spread to eukaryotes via mitochondria or chloroplastic endosymbioses and to a few archaea via horizontal gene transfers. Proteobacteria are unusual in having multiple ATCs. We show by a genetic approach that the three ATC proteins of E. coli are potentially interchangeable, but that redundancy is limited in vivo, either because of gene expression control or because of inefficient Fe/S transfers between ATCs and other components within the Fe/S biogenesis pathway. The combined phylogenomic and genetic approaches allow us to propose that multiple ATCs enable E. coli to diversify the ways for conveying ready-made Fe/S clusters from components of the biogenesis systems to apotargets, and that environmental conditions influence which pathway is used. | lay_plos |
A fundamental feature of sexual reproduction in plants and animals is the specification of reproductive cells that conduct meiosis to form gametes, and the associated somatic cells that provide nutrition and developmental cues to ensure successful gamete production. The anther, which is the male reproductive organ in seed plants, produces reproductive microsporocytes (pollen mother cells) and surrounding somatic cells. The microsporocytes yield pollen via meiosis, and the somatic cells, particularly the tapetum, are required for the normal development of pollen. It is not known how the reproductive cells affect the differentiation of these somatic cells, and vice versa. Here, we use molecular genetics, cell biological, and biochemical approaches to demonstrate that TPD1 (TAPETUM DETERMINANT1) is a small secreted cysteine-rich protein ligand that interacts with the LRR (Leucine-Rich Repeat) domain of the EMS1 (EXCESS MICROSPOROCYTES1) receptor kinase at two sites. Analyses of the expressions and localizations of TPD1 and EMS1, ectopic expression of TPD1, experimental missorting of TPD1, and ablation of microsporocytes yielded results suggesting that the precursors of microsporocyte/microsporocyte-derived TPD1 and pre-tapetal-cell-localized EMS1 initially promote the periclinal division of secondary parietal cells and then determine one of the two daughter cells as a functional tapetal cell. Our results also indicate that tapetal cells suppress microsporocyte proliferation. Collectively, our findings show that tapetal cell differentiation requires reproductive-cell-secreted TPD1, illuminating a novel mechanism whereby signals from reproductive cells determine somatic cell fate in plant sexual reproduction. Successful sexual reproduction depends on the specification of different types of somatic and reproductive cells that give rise to eggs and sperm in both plants and animals. The anther is where male gametophytes (pollen) are produced in seed plants; it typically has four lobes (microsporangia) organized into two thecae, each of which has one abaxial and one adaxial lobe [1–6]. Within each anther lobe, the central reproductive microsporocytes (or pollen mother cells) are surrounded by four concentrically organized somatic cell layers: the epidermis, endothecium, middle layer, and tapetum (listed from outside to inside). Microsporocytes yield pollen via meiosis, and the somatic cells, particularly the tapetal cells, are essential for the normal development and release of pollen. Microsporocytes produce tetrads, each of which contains four haploid spores that later mature into pollen. The tapetum is required to remodel the callose coat surrounding microsporocytes and tetrads, provide nutritive support of microsporocytes and pollen, and synthesize most components of the pollen wall. All anther cells originate from Layer 1 (L1), L2, and L3 cells. The L1 cells form the epidermis, while the L3 cells contribute to forming the vascular column found at the center of each anther and the connective tissue that links the lobes to the vasculature [1–3,5, 7–10]. Within an anther lobe, all cells except for those of the epidermis trace back to L2 meristem cells, which give rise to archesporial cells (AR) and subepidermal L2 cells. AR form sporogenous cells and eventually differentiate into microsporocytes. The subepidermal L2 cells differentiate into primary parietal cells (PPC), which undergo periclinal division to produce two layers of secondary parietal cells (SPC). The outer SPC (OSPC) form the endothecium adjacent to the epidermis, and the inner SPC (ISPC) undergo a further periclinal division to establish the middle layer and tapetum, which completes the cell fate specification events in the anther lobe. As the anther is centrally important for plant sexual reproduction and breeding, it is imperative that we obtain an in-depth understanding of the molecular mechanisms underlying somatic and reproductive cell differentiation during anther development. In Arabidopsis, the EMS1 (EXCESS MICROSPOROCYTES1) leucine-rich repeat receptor-like kinase (LRR-RLK) and the small protein, TPD1 (TAPETUM DETERMINANT1), play vital roles in anther cell differentiation. Anthers in ems1 [also known as exs (extra sporogenous cells) ] and tpd1 mutants lack tapetal cells but produce excess microsporocytes, which normally enter meiosis [10–12]. In rice, the MSP1 (MULTIPLE SPOROCYTE 1) gene encodes an EMS1-like protein, and the TPD1 orthologs, MAC1 (MULTIPLE ARCHESPORIAL CELLS1) and TDL1A [also known as MIL2 (MICROSPORELESS2) ], have been identified in maize and rice, respectively [13–16]. Disruption of the MSP1, MIL2/TDL1A, and MAC1 genes yield anther phenotypes similar to those of ems1 and tpd1 mutants [13–17]. Elegant studies in maize showed that, in response to hypoxia, the glutaredoxin, MSCA1 (MALE STERILE CONVERTED ANTHER1), activates AR differentiation [18] likely through the TGA transcription factors [19]. Once the AR are specified, AR-derived MAC1 proteins directly induce the neighboring subepidermal L2 cells to differentiate into PPC while also autonomously limiting the proliferation of AR and subepidermal L2 cells [15,18]. Previous studies have suggested models for the functions of the TPD1/MAC1/MIL2-EMS1/MSP1 (for simplicity, only TPD1-EMS1 is used hereafter) signaling system in anther cell differentiation. There is, however, some debate among these models. In tapetal cell differentiation, some reports have proposed that TPD1-EMS1 determines tapetal cell fate [10,15], while others have found that it does not determine tapetal cell fate, but instead exclusively stimulates a small group of tapetal founder cells to proliferate into a monolayer of tapetum [20]. Moreover, in microsporocyte proliferation, some authors have hypothesized that TPD1-EMS1 limits the proliferation of archesporial cells [11,15,18], while others believe that it suppresses surrounding parietal cells, causing them to “transdifferentiate” into microsporocytes [10,13]. Thus, it is not yet clear how TPD1-EMS1 controls tapetal cell differentiation and microsporocyte proliferation. Although LRR-RLKs are known to be involved in a wide range of plant growth, developmental, physiological, and defensive processes, only a few ligands have been identified for them. These include small post-translationally modified peptides and cysteine-rich peptides as two major types of oligopeptide ligands for LRR-RLKs [21], and the hormone ligand, brassinosteroid (BR), which activates BRASSINOSTEROID INSENSITIVE1 (BRI1), BRI1-LIKE1 (BRL1), and BRL3 [22]. The small (usually less than 20 residues) post-translationally modified peptides include: CLAVATA3 (CLV3), which is a 12-residue peptide ligand for CLAVATA1 (CLV1) [23,24]; PHYTOSULFOKINE 1 (PSK1), which is a 5-residue peptide ligand for PSKR1 [25]; tracheary element differentiation inhibitory factor (TDIF), which is a 12-residue peptide ligand for TDIF RECEPTOR (TDR) [26,27]; INFLORESCENCE DEFICIENT IN ABSCISSION (IDA), which is a 12-residue peptide ligand for HAESA (HAS) and HAESA-LIKE2 (HSL2) [28,29]; and GRIM REAPER (GRI), which is an 11-residue peptide ligand for POLLEN-SPECIFIC RECEPTOR-LIKE KINASE 5 (PRK5) [30]. The cysteine-rich peptides usually contain a C-terminal domain with 4–16 cysteine residues [21]. EPIDERMAL PATTERNING FACTORS 2 (EPF2) and STOMAGEN (EPFL9) serve as ligands for competitively binding ERECTA (ER) -family LRR-RLKs [31–33]. LAT52 and STIGMA-SPECIFIC PROTEIN1 (STIG1, a processed 7-kD peptide) act as ligands of the pollen-specific receptor kinase, LePRK2 [34,35]. In Arabidopsis, TPD1 interacts with EMS1 in vitro and in vivo [9]. Among the orthologs of TPD1, immunostaining showed that the maize MAC1 protein is enriched in AR cells [15]. MAC1 was observed in the extracellular space of onion cells in transient assays, suggesting that it is secreted, and Western blotting suggested that MAC1 is not further cleaved besides the removal of the putative signal peptide. So far, however, the receptor of MAC1 has not been identified. Finally, Bimolecular Fluorescence Complementation (BiFC) assay showed that the rice TDL1A interacts with MSP1 in onion-cell cytoplasm [14]. These data suggested that the presumptive ligand, TPD1/MAC1/TDL1A, might be the first identified small protein ligand for an LRR-RLK. However, several key points had not been established biochemically in previous studies. Here, we demonstrate for the first time that TPD1 is a 13-kD small secreted cysteine-rich protein ligand that interacts with the EMS1 LRR domain at two sites. TPD1-EMS1 signaling initially promotes the periclinal division of secondary parietal cells to form a monolayer of tapetal cell precursors, and then determines the fate of functional tapetal cells. Our findings that tapetal cell differentiation requires TPD1 secreted from normal pre-meiotic cells illuminate a novel mechanism by which signals from reproductive cells determine somatic cell fate in plant sexual reproduction. The TPD1 gene encodes a 176-residue small protein (19. 5 kD) that contains a putative 21-residue signal peptide at the N-terminus, a non-conserved N-terminal region, and a conserved cysteine-rich C-terminal domain (Fig 1A and S1 Fig). Our sequence analysis revealed that the TPD1 C-terminal domain contains the dibasic site, K135R136 (Fig 1A and S1 Fig), which is a conserved proteolytic cleavage site for precursor peptide processing in animals and yeast [36]. To investigate whether TPD1 is similarly processed in planta, we generated TPD1: TPD1sp-GFP-ΔTPD1 (ΔTPD1: full-length TPD1 without the putative signal peptide) and TPD1: TPD1sp-ΔTPD1-GFP transgenic plants to produce TPD1 N-terminal and C-terminal GFP fusion proteins (Fig 1B). Compared with wild-type plants (Fig 1C and 1H), 71. 6% (68/95) of TPD1: TPD1sp-GFP-ΔTPD1/tpd1 plants produced normal pollen and anthers (Fig 1D and 1I), indicating complementation, whereas TPD1: TPD1sp-ΔTPD1-GFP (0/42) failed to complement the tpd1 phenotype (Fig 1E and 1J). Therefore, the TPD1 N-terminal GFP fusion protein had a function comparable to that of the native TPD1, but TPD1 fused with GFP at its C-terminus exhibited functional impairment. To test if the putative signal peptide affects the processing of TPD1 and whether TPD1 is cleaved at the predicted K135R136 dibasic site, we generated TPD1: GFP-ΔTPD1 and TPD1: TPD1sp-GFP-ΔTPD1 K135G R136G transgenic plants to produce a TPD1 N-terminal GFP fusion protein without the signal peptide (GFP-ΔTPD1) and a TPD1sp-GFP-ΔTPD1 K135G R136G protein containing mutations in the K135R136 dibasic site (Fig 1B). However, neither TPD1: GFP-ΔTPD1 (Fig 1F and 1K) nor TPD1: TPD1sp-GFP-ΔTPD1 K135G R136G (Fig 1G and 1L) complemented the tpd1 phenotype. When proteins extracted from young buds were subjected to Western blot analysis with an antibody against GFP, we did not detect any signal from the wild-type (Fig 1M, Lane 1 and Fig 1N, Lane 1) or TPD1: TPD1sp-ΔTPD1-GFP/tpd1 (Fig 1M; Lane 2) samples, but we did observe a 41-kD band in the TPD1: TPD1sp-GFP-ΔTPD1/tpd1 sample (Fig 1M, Lane 3 and Fig 1N; Lane 2). We also detected 45-kD and 48-kD bands corresponding to the predicted sizes of the fusion proteins expressed in TPD1: GFP-ΔTPD1/tpd1 (Fig 1N; Lane 3) and TPD1: TPD1sp-GFP-ΔTPD1K135G R136G /tpd1 (Fig 1N; Lane 4) plants, respectively, indicating that ΔTPD1-GFP and TPD1sp-GFP-ΔTPD1K135G R136G did not undergo cleavage. Similar results were obtained using the leaf protoplast transient expression system and Western blotting. 35S: TPD1sp-GFP-ΔTPD1,35S: GFP-ΔTPD1, and 35S: TPD1sp-GFP-ΔTPD1K135G R136G were introduced into leaf protoplasts obtained from 35S: EMS1 plants (S2 Fig). The 41-kD band was detected in the 35S: TPD1sp-GFP-ΔTPD1 sample, whereas 45-kD and 48-kD bands were found in 35S: TPD1sp-GFP-ΔTPD1 and 35S: TPD1sp-GFP-ΔTPD1K135G R136G samples, respectively (S2 Fig). The 41-kD band detected in TPD1: TPD1sp-GFP-ΔTPD1/tpd1-complemented plants and 35S: TPD1sp-GFP-ΔTPD1 35S: EMS1 protoplasts is smaller than the 45 kD predicted for the full-length fusion protein without the putative signal peptide or the 48-kD predicted for the full-length fusion protein. Considering that GFP is 28 kD in size, our results suggest that cleavage occurs around the C-terminal dibasic site, K135R136, yielding an approximately 13-kD TPD1 protein (residues 22 to possibly 134) that includes four cysteines (Fig 1A). In addition, our findings indicate that the putative signal peptide is important for TPD1 cleavage. To test the functionality of the processed TPD1, we performed genetic complementation experiments by generating a series of N-terminal and C-terminal truncations of the conserved C-terminal domain of TPD1 (Fig 2A). We examined at least 40 independent transgenic lines for each truncation, assessing their ability to restore pollen fertility in the tpd1 mutant background (Fig 2B). Same as the full-length TPD1 gene and ΔC1, a short C-terminal TPD1 truncation version ΔC2, which encodes the processed TPD1 protein, complemented the tpd1 phenotype (Fig 2B). However, the further C-terminal TPD1 truncation mutant, ΔC3, did not complement the tpd1 phenotype. Moreover, ΔN1 and ΔN2, which encoded only short C-terminal domains, failed to complement the tpd1 phenotype (Fig 2A and 2B). These results rule out the possibility that TPD1 is processed into a small functional peptide that contains only the far end of its C-terminus. To confirm that the non-complementing constructs yielded actual expression of truncated TPD1 proteins, we generated TPD1: TPD1sp-GFP-ΔN1/tpd1 (n = 36) and TPD1: TPD1sp-GFP-ΔC3/tpd1 (n = 41) transgenic plants. Western blot analysis detected bands of the predicted sizes (38 kD and 39 kD, respectively) (Fig 2E), supporting our contention that truncated TPD1 proteins smaller than the determined mature size were not functional. The C-terminal domain of TPD1 has six cysteine residues (C77, C107, C111, C120, C142, and C175) (Figs 1A and 2C). To define which residues are essential for the normal function of TPD1, we performed single mutations to replace these cysteines with serines (Fig 2C). Complementation experiments demonstrated that C77, C107, C111 and C120, which lie in the mature TPD1, were required for TPD1 function, whereas mutations of C142 and C175, which are excluded from the mature TPD1, did not affect TPD1 function (Fig 2D). In summary, our results suggest that the processed 13-kD form of TPD1 (residues 22 to possibly 134) is a functional mature small protein ligand that contains four essential cysteines. To determine whether the processed TPD1 interacts with EMS1, we performed Bimolecular Fluorescence Complementation (BiFC) using Arabidopsis mesophyll protoplasts. By inserting nEYFP (N-terminal EYFP) after the putative signal peptide of TPD1, we generated constructs that produced full-length TPD1 (nEYFP-TPD1), mature TPD1 (nEYFP-ΔC2), and mutated TPD1 (nEYFP-TPD1K135G R136G) (Fig 3A). We co-transfected each construct plus a cEYFP (C-terminal EYFP) -LRR (includes the full-length LRR domain; Fig 3E) -encoding construct into Arabidopsis protoplasts, and then tested for protein interactions at the plasma membrane. Our results showed that full-length TPD1 (Fig 3B) and mature TPD1 (ΔC2) (Fig 3C) both interacted with the EMS1 LRR, but TPD1K135G R136G did not (Fig 3D). This indicates that mature TPD1 (ΔC2) can interact with EMS1, and cleavage processing is essential for the TPD1-EMS1 interaction at the plasma membrane. To identify where the mature TPD1 interacts with EMS1, we referred to the primary structure of EMS1 and generated a series of EMS1 LRR truncations fused to cEYFP (C-terminal EYFP) (Fig 3E). The nEYFP-ΔC2 (Fig 3A) construct was used to test for interactions between mature TPD1 and truncated EMS1 LRRs (Fig 3E). The full-length EMS1 (cEYFP-EMS1; Fig 3E and 3F) and the full-length EMS1 LRR domain (cEYFP-LRR; Fig 3E and 3G), but not the EMS1 cytoplasmic kinase domain (cEYFP-KD; Fig 3E and 3H), interacted with the mature TPD1 at the plasma membrane. No interaction was detected between the mature TPD1 and cEYFP-BRI1-LRR (Fig 3E and 3I), indicating the specificity of the TPD1-EMS1 interaction. We further tested the EMS1 LRR domain by separating it to two parts and generated cEYFP-ΔLRR-I and cEYFP-ΔLRR-II constructs (Fig 3E). We detected an interaction between TPD1 and cEYFP-ΔLRR-I (Fig 3J), but not between TPD1and cEYFP-ΔLRR-II (Fig 3K). Within the LRR-I truncation, we further determined that the first three LRRs (cEYFP-ΔLRR-III) were sufficient to interact with TPD1 (Fig 3E and 3L). Moreover, the site-directed mutation of K104N in LRR2 (the genetic allele exs-2), which causes a phenotype similar to that of the null mutant, ems1 [10,11], impaired the TPD1-EMS1 interaction (Fig 3E and 3M). Using the LRR-II truncation, we also detected a weak interaction between cEYFP-ΔLRR-IV and TPD1 (Fig 3E and 3N), but no interaction was observed between cEYFP-ΔLRR-V and TPD1 (Fig 3E and 3O). Our previously identified TPD1 interaction region (TIR) [9] lies in cEYFP-ΔLRR-IV. Although ectopic expression of TPD1 (35S: TPD1) reportedly caused anther development defects and wide siliques [37], our 35S: EMS1 plants had no visible phenotype (S3 Fig). The vast majority of 35S: TPD1 35S: EMS1 plants (87. 5%; 35/40) exhibited significant defects, including short stature and twisted leaves, stems, and inflorescences (S3 Fig). In contrast, almost all 35S: TPD1 35S: EMS1K104N plants (95%; 57/60) resembled 35S: TPD1 plants (S3 Fig), indicating that the function of TPD1/EMS1 signaling depends on the ability of TPD1 to interact with EMS1 in the first three LRRs. Taken together, our results suggest that the mature TPD1 interacts with the EMS1 LRR domain at two sites, and that this is required for the normal function of TPD1. If TPD1 acts as a ligand of EMS1, it should be secreted to the extracellular space and bind to the EMS1 LRR region. To test this, we first analyzed the effect of the putative signal peptide of TPD1 on its function in anther cell differentiation. Using the TPD1 promoter, we created constructs in which the putative signal peptide of TPD1 was replaced with the known signal peptides, CLV3 and PAP1 [38,39] (Fig 4A). The resulting constructs were tested for complementation effects on fertility, pollen viability, and anther cell differentiation in the tpd1 mutant background. Compared with wild-type plants (Fig 4B), the tpd1 mutant was completely sterile (Fig 4C). Eighty percent (96/120) of TPD1: TPD1sp-ΔTPD1/tpd1 plants exhibited normal fertility (Fig 4D), but no TPD1: ΔTPD1/tpd1 plant (0/88) showed restoration of fertility (Fig 4E). Wild-type and TPD1: TPD1sp-ΔTPD1/tpd1 anthers produced viable pollen grains (Fig 4H and 4I), whereas, similar to tpd1 plants (Fig 4J), TPD1: ΔTPD1/tpd1 plants did not produce any pollen (Fig 4K). Stage-5 anthers of wild-type (Fig 4N) and TPD1: TPD1sp-ΔTPD1/tpd1 plants had normal microsporocytes surrounded by tapetum (Fig 1O), whereas tpd1 (Fig 1P) and TPD1: ΔTPD1/tpd1 anthers exhibited a total lack of tapetum but had excess microsporocytes (Fig 1Q). Seventy two percent (36/50) of TPD1: CLV3sp-ΔTPD/tpd1 and 70% (28/40) of TPD1: PAP1sp-ΔTPD1/tpd1 plants exhibited rescued fertility (Fig 4F and 4G), produced viable pollen grains (Fig 4L and 4M), and formed normal anthers (Fig 4R and 4S). Together, our results suggest that the putative signal peptide of TPD1 and the N-terminal signal peptide-directed secretion of TPD1 are required for its function in anther cell fate determination. To test whether TPD1 is a secreted protein, we generated 35S: TPD1sp-GFP-ΔTPD1 transgenic plants and analyzed the subcellular localization of TPD1 in root cells. We observed TPD1 proteins in trafficking vesicles but not at the plasma membrane (Fig 5A–5C). Treatment with brefeldin A (BFA), which blocks the formation of Golgi-derived vesicles [40], caused aggregation of TPD1 proteins (Fig 5D and 5E), suggesting that the TPD1 protein is contained within trafficking vesicles. Since EMS1 is not expressed in the root [10], we generated 35S: TPD1sp-GFP-ΔTPD1 35S: EMS1 double transgenic plants. In the presence of EMS1, we observed TPD1 at the plasma membrane of root cells (Fig 5F–5H). We obtained similar results using the Arabidopsis leaf protoplast system. TPD1 was observed in vesicle-like compartments of protoplasts transfected with 35S: TPD1sp-GFP-ΔTPD1 alone, and at the plasma membrane in the presence of the full-length EMS1 and the EMS1 LRR domain, but not the EMS1 kinase domain (S4 Fig). TPD1 proteins lacking the signal peptide (GFP-ΔTPD1) or with site mutations in the K135R136 dibasic site (TPD1sp-GFP-ΔTPD1 K135G R136G) failed to localize to the plasma membrane regardless of the presence of EMS1 (S4 Fig). Together, these results suggest that TPD1 is a secreted protein that localizes to the plasma membrane in an EMS1-dependent fashion. Analysis of TPD1: mGFP5er [modified endoplasmic reticulum (ER) -localized GFP] plants showed that the TPD1 promoter was active in precursors of microsporocytes (PM) at stage 4 (Fig 6A) and in microsporocytes (M) at stage 5 (Fig 6B). In TPD1: TPD1sp-GFP-ΔTPD1/tpd1 plants, which showed normal anther development (Fig 1D and 1I), the TPD1 protein was present in PM at stage 4 (Fig 6C and 6D). At stage 5, TPD1 was observed not only in M but also at the surface of tapetal cells (T) (Fig 6E and 6F). Similar to our observations in root and leaf cells (Fig 5A–5C; S4 Fig), TPD1 was found in vesicle-like compartments of isolated M (Fig 6G and 6H). In the ems1 mutant anther, the TPD1 localization domain was expanded, although TPD1 proteins were evenly observed only in microsporocytes because the ems1 anther has excess microsporocytes but no tapetal cells (Fig 6I and 6J; S5 Fig). In TPD1: GFP-ΔTPD1 and TPD1: TPD1sp-GFP-ΔTPD1K135G R136G anthers, TPD1 proteins were restricted to M and were not found in T (Fig 6K and 6L), indicating that the secretion and cleavage of TPD1 are required for its localization in T. As described above, GFP-ΔTPD1 (lacking the signal peptide) and TPD1sp-GFP-ΔTPD1K135G R136G (harboring site mutations in the K135R136 cleavage site) were also trapped in the cytoplasm of protoplasts, regardless of the presence of EMS1 (S4 Fig). Our findings suggest that TPD1 is synthesized in PM/M and secreted to their surrounding tapetal cells. The EMS1 promoter was active in both outer secondary parietal cells (OSPC) and inner secondary parietal cells (ISPC) at stage 4 (Fig 6M and 6N). Following the periclinal division of ISPC and their subsequent differentiation, the EMS1 promoter was active in the middle layer (ML) and the precursors of tapetal cells (PT), which are derived from ISPC early in stage 5 (Fig 6O and 6P). Later in stage 5, EMS1 promoter activity was restricted to T (Fig 6Q and 6R). The EMS1 protein was observed at the surface of T in stage-5 anthers (Fig 6S and 6T), suggesting that it is localized at the plasma membrane of these cells. After the GFP signals were analyzed, the anthers were stained with FM4-64 dye, and anther stages and cell types were determined from optical sections under confocal microscopy (S5 Fig). To further examine the subcellular localizations of TPD1 and EMS1, we carried out EM-immunolabeling experiments. Our results showed that the TPD1 and EMS1 proteins were localized to the plasma membrane of PT in early stage-5 anthers (Fig 6U and 6V; S6 Fig). Together, these results suggest that TPD1 is synthesized in PM/M, secreted from these cells to PT/T, and stabilized by interacting with EMS1 at the plasma membrane of PT/T. The activated TPD1-EMS1 signaling may initially promote parietal cell division and then determine the fate of functional tapetal cells. To test whether TPD1 acts as a secreted protein ligand to control anther cell division and tapetal cell fate determination, we manipulated the expression of TPD1 using the Arabidopsis MERISTEM LAYER1 (ML1) promoter, which is specifically active in the epidermis of various organs [41]. In ML1: GFP anthers, the ML1 promoter was specifically active in anther epidermis (Fig 7A–7C). In ML1: TPD1sp-GFP-ΔTPD1 anthers, in contrast, we observed GFP signals in both anther epidermis and subepidermal cell layers (Fig 7D–7F). In ML1: TPD1sp-GFP-ΔTPD1/ems1 anthers, the GFP signal was only observed in anther epidermis (Fig 7G and 7H). Our data suggest that the TPD1 protein is capable of traveling through cell layers from epidermis to inner cells in the presence of EMS1. Wild-type anthers had four somatic cell layers at stage 5 (Fig 8A). Three cell layers were visible at stage 7, reflecting degeneration of the middle layer (Fig 8B). Two cell layers remained at stage 13, reflecting degeneration of the tapetal cells (Fig 8C). In contrast, tpd1 anthers contained just three somatic cell layers (the epidermis, endothecium, and middle layer) throughout their development (Fig 8D–8F). Ectopic expression of TPD1 (ML1: TPD1) caused production of more than four somatic cell layers in both wild-type (Fig 8G and 8H) and tpd1 (Fig 8J and 8K) plants. Interestingly, at stage 13, three cell layers were observed in ML1: TPD1 anthers (Fig 8I), whereas all subepidermal anther wall cells were dead at this point in ML1: TPD1/tpd1 anthers (Fig 8L). Previous in situ hybridization results showed that the ectopic expression of TPD1 triggered expression of the tapetal cell marker gene, A9 [20], in both the tapetum and its neighboring cells in the wild-type background (Fig 8M and 8N). A9 was not expressed in the tpd1 anther (Fig 8O), while in the ML1: TPD1/tpd1 anther, it was expressed in a few cells adjacent to microsporocytes (Fig 8P). Our qRT-PCR results further confirmed that the expression of TPD1 in epidermis significantly increased the expression of the tapetal cell marker genes, A9, A6, and ATA7 (Fig 8Q and 8R), suggesting that TPD1 promotes tapetal cell differentiation in the presence of EMS1. The number of microsporocytes in ML1: TPD1/tpd1 anthers was similar to that in tpd1 anthers (Fig 8S), indicating that the increase in somatic cell layers did not suppress microsporocyte proliferation in the absence of the tapetum. Taken together, our results suggest that TPD1 first promotes the periclinal division of anther wall cells and subsequently determines tapetal cell fate in the presence of EMS1. To test whether TPD1 mediates communication between microsporocytes and tapetal cells, we generated a protein in which TPD1 was fused with the ctVSS C-terminal vacuolar sorting signal (Fig 9A), which directs proteins into vacuoles [39]. The addition of two glycines to the ctVSS sequence disables this sorting function (Fig 9A) [39]. Seventy five percent (30/40) of TPD1: TPD1/tpd1 (Fig 9B and 9E) and 68% (38/56) of TPD1: TPD1-ctVSS-GG/tpd1 (Fig 9D and 9E) plants exhibited normal fertility, whereas 75% (45/60) of TPD1: TPD1-ctVSS/tpd1 plants were sterile (Fig 9C and 9E). Although viable pollen was observed in TPD1: TPD1/tpd1 (S7 Fig) and TPD1: TPD1-ctVSS-GG/tpd1 anthers, no pollen was found in the TPD1: TPD1-ctVSS/tpd1 anther (S7 Fig). Further analyses showed that TPD1: TPD1-ctVSS/tpd1 anthers exhibited various phenotypes, such as degenerated microsporocytes surrounded by a monolayer of vacuolated tapetal-like cells (Fig 9F and 9G), degenerated microsporocytes adjacent to delaminated vacuolated tapetal-like cells (Fig 9H), or a lack of recognizable tapetal cells but the presence of excess microsporocytes (Fig 9I). Moreover, our qRT-PCR results showed that the expression levels of TPD1 were similar in TPD1: TPD1/tpd, TPD1: TPD1-ctVSS/tpd1, and TPD1: TPD1-ctVSS-GG/tpd1 anthers (S7 Fig), supporting the idea that the observed anther defects were caused by abnormal sorting of TPD1 proteins. From these results, we suggest that the TPD1-mediated communication between precursors of microsporocytes/microsporocytes and early anther wall cells is required for tapetal cell differentiation and the maintenance of cell integrity. Although the lack of tapetal cells ultimately causes failure of pollen formation, it does not reportedly affect microsporocyte differentiation in the tpd1, mil2, mac1, ems1, msp1, serk1 serk2, or bam1 bam2 mutants [10,12,13,16,42–44], suggesting that microsporocyte specification is independent of anther wall development. The SOLO DANCERS (SDS) gene is specifically expressed in microsporocytes [45,46]. To test how reproductive cells (e. g. precursors of microsporocytes/microsporocytes) affect somatic wall cell differentiation in anthers, we generated SDS: SDS-BARNASE plants, in which microsporocyte differentiation was specifically impaired by the expression of the toxic gene, BARNASE, in these cells [45,46]. All SDS: SDS-BARNASE plants were male-sterile due to a lack of pollen [46]. Based on semi-thin sectioning of 60 SDS: SDS-BARNASE T1 plants, however, we were able to divide the plants into three classes based on their anther phenotypes at stage 5. In class I plants (16. 7%, 10/60), stage-5 anthers harbored four somatic cell layers that resembled those of the wild-type anther (Fig 10A and 10B). In class II plants (70. 0%; 42/60), stage-5 anthers harbored four somatic cell layers, including either a monolayer of vacuolated tapetal-like cells (Fig 10C) or three somatic cell layers with delaminated vacuolated tapetal-like cells (Fig 10D). In addition, degenerating microsporocytes were observed (Fig 10C and 10D). In class III plants (13. 3%; 8/60), stage-5 anthers lacked tapetal cells but exhibited the presence of excess microsporocyte-like cells; in this, they resembled ems1 and tpd1 mutant anthers, with the addition of early microsporocyte degeneration (Fig 10E). We further performed in situ hybridization to examine the expressions of the A9 and SDS genes. Compared with the strong expression of the tapetum marker gene, A9, in the tapetum of the wild-type stage-5 anther (Fig 10F), A9 expression was progressively decreased in SDS: SDS-BARNASE Class-I to Class-II anthers (Fig 10G–10I) at stage 5, and no A9 expression was detected in the Class-III anther (Fig 10J). Our qRT-PCR results agreed with the in situ hybridization results (Fig 10P). In wild-type anthers, SDS expression was weak in precursors of microsporocytes at stage 4 (Fig 10K) but strong in microsporocytes at stage 5 (Fig 10L). Stage-5 SDS: SDS-BARNASE anthers exhibited decreased SDS expression relative to wild-type anthers (Fig 10M–10O). The expression domain of SDS was similar in wild-type and Class-I anthers (Fig 10M), but relatively expanded in Class-II (Fig 10N) and Class-III (Fig 10O) anthers. Our qRT-PCR results showed that the severity of tapetal cell defects corresponded to the expression level of BARNASE (Fig 10Q). Our results indicate that a monolayer of tapetal cells cannot be fully differentiated when microsporocytes are affected by SDS-BARNASE. Weak SDS-BARNASE function does not affect ISPC division but still leads to abnormal differentiation of tapetal cells. In the presence of moderate SDS-BARNASE function, there are two possibilities: if ISPC division occurs normally, a monolayer of vacuolated tapetal-like cells is formed; if ISPC division is not complete, however, delaminated vacuolated tapetal-like cells are produced. Finally, strong and early SDS-BARNASE function bars ISPC division, resulting in a lack of tapetal-like/tapetal cells but the presence of excess microsporocytes. In summary, our results suggest that the formation of a monolayer of PT and the complete acquisition of tapetal cell fate depend on normal microsporocyte development, while tapetal cells in turn suppress microsporocyte proliferation. In this report, we provide several lines of strong evidence supporting our contention that TPD1 is a small secreted cysteine-rich protein ligand for the EMS1 LRR-RLK, thereby expanding the repertoire of this receptor class. Cysteine-rich peptides/proteins, such as EPF1, EPF2 [31], EPFL9 [32,33,47], LAT52 [35], STIG1 [34], S-locus cysteine rich protein (SCR) /S-locus protein 11 (SP11) [48], RALF [49], LURE1, LURE2 [50], and EC1 [51], serve as ligands for a large group of receptor kinases. The LAT52, LURE1, LURE2, and EC1 peptides remain intact [35,50,51], whereas RALF and STIG1 are cleaved [34,49]. In Arabidopsis, 10 out of 34 AtRALFs contain RR dibasic sites (RRXL). Mutations in these dibasic sites impair the cleavage of AtRALF1 and AtRALF23 [36,52], the latter of which is known to be cleaved at the dibasic site by the AtS1P protease (subtilase) [52]. A 7-kD C-terminal STIG1 peptide, possibly arising from cleavage at the dibasic site, was identified by electrospray ionization–mass spectrometry in tomato [34]. Our present results add to this knowledge by showing that the TPD1 C terminus, which contains the dibasic sites, might be important for the stability or translation of this small protein ligand. TPD1: TPD1sp-GFP-ΔTPD1 functioned as well as the native TPD1 gene. In contrast, TPD1: TPD1sp-ΔTPD1 -GFP did not complement the tpd1 phenotype; it was transcribed (S8 Fig), but no TPD1-GFP protein was detected. Similar results were also obtained from the TPD1: TPD1-GUS gene (S9 Fig), suggesting that the addition of a large protein to the TPD1 C terminus may abolish its function, possibly via protein synthesis failure or misfolding-induced degradation. In planta, the MAC1 protein is an 18-kD intact protein that does not seem to be processed beyond removal of the putative signal peptide [15]. Our results suggest that TPD1 differs from MAC1 in that it is cleaved at the C terminal K135R136 site, resulting in a 13-kD small protein ligand. Our data showed that TPD1 was not cleaved in the absence of the signal peptide or when the K135R136 cleavage site is mutated. In addition, the uncleaved TPD1 was not localized at the plasma membrane or in trafficking-like vesicles even in the presence of EMS1, suggesting that TPD1 cannot enter the secretion secretory pathway unless it has undergone normal processing. LRR is a 20-30-residue motif that has been identified in thousands of proteins across all organisms [53]. Recent crystal structure studies on plant LRRs have demonstrated the molecular mechanisms underlying the activation of LRR-RLK complexes by brassinolide (BL), which is the most active brassinosteroid ligand, and by the 22-residue oligopeptide ligand, flg22 [54–57]. The binding of BL to the BRI1 island domain induces the formation of an interface that subsequently activates the SERK1-BRI1 (co-receptor and receptor) complex. flg22 binds to the concave surface of the FLS2 LRR domain across 14 LRRs and is sandwiched between FLS2 and BAK1 (SERK3) LRR ectodomains. The gluing effect of flg22 causes full activation of the FLS2-BAK1 complex. Our present and previous [9] studies identified two TPD1 binding sites in the EMS1 LRR ectodomain. Here, we hypothesize that the binding of TPD1 to the EMS1 LRR2-constituted domain results in a conformational rearrangement of the interface between EMS1 and its unknown co-receptor, which then becomes more competent for additional binding of TPD1, leading to the full activation of EMS1 signaling. As TPD1 represents a novel small protein ligand for LRR-RLKs, future studies on the structures and dynamic interactions of TPD1 and EMS1 could support the development of a new paradigm for the small-protein-ligand-induced activation of LRR-RLK signaling complexes. The tapetum, which is essential for pollen development, consists of a monolayer of polynucleate cells [58–60] that comes to surround successive stages of microsporocytes, tetrads, microspores, and developing pollen grains as anther development progresses [1,2, 5,8]. In Arabidopsis, a monolayer of precursors of tapetal cells (PT) is established early in stage 5, and the differentiation of functional tapetal cells is completed late in stage 5 [7,10]. Our present results show that the earliest expression of EMS1 is observed in outer and inner secondary parietal cell layers (OSPC and ISPC) at stage 4, when TPD1 also becomes detectable in precursors of microsporocytes (PM) (Fig 11A). We propose that the presence of sufficient TPD1-EMS1 in ISPC initially promotes their periclinal division to generate two cell layers, the middle layer (ML) and PT. Thereafter, sustained TPD1-EMS1 signaling in PT directs the complete differentiation of functional tapetal cells at late stage 5 (Fig 11A). In ems1 and tpd1 anthers, the loss of TPD1 or EMS1 leads to failure of ISPC division, with the result that such anthers exhibit no tapetal cells but an excess of microsporocytes (Fig 11A). Our model is further supported by the observation that manipulation of TPD1 expression and secretion affects the formation of anther wall cell layers and the fate determination of tapetal cells. Epidermis-expressed TPD1 can travel to regions beneath OSPC and ISPC, promoting the periclinal division of these cells and eventually triggering the formation of extra anther wall layers (Fig 11B). Interestingly, in the tpd1 mutant background, epidermis-expressed TPD1 is sufficient to stimulate periclinal division of OSPC and ISPC but insufficient to complete the differentiation of functional tapetal cells. This may explain why epidermis-expressed TPD1 in tpd1 plants was found to increase the expression of tapetal cell marker genes, but such plants experience precocious death of such cells (Fig 8L and 8R). In contrast, the inhibition of TPD1 by the engineered missorting of TPD1 or the ablation of microsporocytes was associated with incomplete tapetal cell differentiation or a lack of tapetal cells (Fig 11C). A monolayer of vacuolated tapetal cells was often observed in TPD1: TPD1-ctVSS/tpd1 and SDS: SDS-BARNASE anthers, suggesting that PT formed but failed to differentiate into functional tapetal cells in the absence of sufficient TPD1. When most TPD1 expression was blocked, PT failed to form and no tapetal cells were observed. Our results collectively support the idea that TPD1-EMS1 signaling is initially required to promote the periclinal division of ISPC to form PT, and then subsequently to determine the final tapetal cell fate. A previous report found that the expression of EMS1 under the control of the tapetum-specific A9 promoter could rescue the tapetum in ems1 anthers, suggesting that TPD1-EMS1 stimulates the proliferation of a small population of tapetal founder cells (or primary tapetal cells) to form a monolayer of tapetal cells via anticlinal cell division [20]. This did not support the idea that TPD1-EMS1 determines tapetal cell fate. If tapetal founder cells exist, wild-type and ems1 anthers should have a similar number of tapetal founder cells. The previous study, however, found that each ems1 anther lobe contained an average of three tapetal founder cells, while wild-type anthers contained a greater number of tapetal founder cells that were organized as a monolayer [20]. Although tapetal founder cells possess some features of differentiated tapetal cells, they are precociously degenerated, indicating that tapetal founder cells are not fully differentiated tapetal cells. On the other hand, anatomically, a monolayer of tapetal cells is hardly established by a small number of tapetal founder cells in one location, since plant cells do not move. The A9 promoter becomes active in the tapetum at stage 5 when EMS1 is expressed. Here, the high-level A9-promoter-driven expression of EMS1 could be sufficient to promote the periclinal division of quiescent ISPC and the final differentiation of PT into functional tapetal cells. In some circumstances, ISPC might be directly differentiated into tapetal cells without the periclinal division step. Together, these results support the notion that TPD1-EMS1 is required for tapetal cell fate determination. Various studies have proposed different interpretations regarding the control of microsporocyte proliferation. In maize, MAC1 represses the excessive proliferation of archesporial cells (AR), either by binding an unknown receptor kinase in AR or by indirectly inducing neighboring somatic cells to produce a repressor of AR proliferation [15,18]. Our results show that EMS1 is not present in AR or microsporocyte/microsporocyte precursors, which rules out the possibility that TPD1-EMS1 signaling directly represses microsporocyte proliferation. If TPD1 activates a different receptor complex in microsporocytes, these cells would not overproliferate in ems1 anthers. If the involved receptor complexes share components, however, then the lack of EMS1 could impair receptors in both somatic and germinal cells. In Arabidopsis, disruption of the RPK2 LRR-RLK gene results in the absence of the middle layer but does not affect the number of microsporocytes [61]. Conversely, loss-of-function mutants of the EMS1, SERK1/2, and BAM1/2 LRR-RLK genes produce anthers that possess excess microsporocytes but lack either the tapetum or all anther wall cell layers except the epidermis [42,43,62]. Our present results show that although ML1: TPD1 tpd1 anthers produce extra somatic cell layers, they still form excess microsporocytes in the absence of the tapetum monolayer (Figs 8J, 8K, 8S and 11B). Moreover, when TPD1 expression is largely blocked via missorting of TPD1 or ablation of microsporocytes, the anthers do not produce tapetum, but instead form excess microsporocytes (Figs 9I, 10E and 11C). Taken together, these results show that the tapetum is required to limit the proliferation of microsporocytes but not other cells of the anther wall. Monocots and dicots exhibit conservation of regulators, but may differ in their sexual reproduction strategies. For example, disruption of SPOROCYTELESS (SPL) /NOZZLE (NZZ), which encodes a novel transcription repressor, blocks the differentiation of primary sporogenous cells, resulting in an absence of microsporocytes in Arabidopsis [63,64]. SPL/NZZ orthologs exist in monocots, such as maize and rice, but their functions in sexual reproduction have not yet been identified [65]. Loss of function in the ROXY1 and ROXY2 glutaredoxins causes sporogenous cell differentiation to fail in the two adaxial anther lobes of Arabidopsis [66]. In contrast, the MSCA1 glutaredoxin acts earlier in maize, as shown by the observation that the archesporial cells of msca1 anthers differentiate into vasculature instead of sporogenous cells [18]. In maize, MAC1 activates the periclinal division of PPC to form endothecium and SPC. In Arabidopsis, however, our present results show that TPD1 is not required for PPC division, but rather functions later to promote the periclinal division of ISPC to form ML and the tapetum. Loss-of-function mutants in the MSP1, MIL2, and MAC1 genes exhibit an anther phenotype similar to those of tpd1 and ems1 plants and additionally produce extra megaspore mother cells (MMC) in their ovules, suggesting that MSP1, MIL2, and MAC1 suppress the proliferations of both microsporocytes and MMC in monocots [13,14,17]. Although neither tpd1 nor ems1 plants exhibited developmental defects in their ovules [10,12], our recent report showed that ectopic ML1-promoter-driven expression of TPD1 affects ovule cell division by regulating auxin signaling and cell-cycle-related genes [67]. It is worth noting that in the anthers of monocots such as maize, the cells of the anther wall (e. g., PPC, SPC, and PT) are established as monolayers at or near the time they are first formed, whereas the corresponding cells in Arabidopsis are not observed as monolayers until tapetal cell differentiation is almost complete [1,2, 5,7, 8,10,18]. Cytologically, reproductive AR and sporogenous cells are more recognizable in monocot anthers than in Arabidopsis anthers. Overall, the differences in anther development between monocots and dicots might be associated with functional differences in the identified regulators. Seed plants have evolved a sophisticated anther wall structure to ensure the success of sexual reproduction. In animals, signals from somatic cells influence the fate determination of reproductive cells, and vice versa [68]. In plant anthers, somatic cells of the anther wall are not required for the differentiation of reproductive microsporocytes [4,10–12,42,43,61,62], but signals from reproductive cells do appear to determine the complexity of anther wall cells. LRR-RLK-linked signals, including TPD1-EMS1, are essential for the communication of reproductive and somatic cells during anther development [4,9–11,13,42,43,61]. In the future, additional studies on LRR-RLK-linked signaling should provide new insights into the molecular mechanisms underlying somatic and reproductive cell fate determination in plants. Arabidopsis thaliana [Landsberg erecta (Ler) ] plants were grown in Metro-Mix 360 soil (Sun-Gro Horticulture City State) in growth chambers under a 16-hour light/8-hour dark photoperiod, at 22°C and 50% humidity. For genetic analyses and protein localization studies, the TPD1, EMS1, and ML1 promoters [10,12,67] were individually introduced into the pENTR/D-TOPO vector (Invitrogen), and the corresponding genes were cloned and inserted. Overlapping PCR was used to replace the TPD1 signal peptide with the CLV3 or PAP1 signal peptide [38,39], and to generate site mutations. To generate the TPD1-GFP fusion protein, GFP was inserted right after the TPD1 signal peptide to generate TPD1-GFP-ΔTPD1. All constructs in pENTR/D-TOPO vectors were sub-cloned into Gateway Binary vectors (pGBWs or pEARLEYs) using the Gateway LR recombinase II enzyme mix (Invitrogen). The resulting constructs were then transformed into Agrobacterium strain GV3101. Transformations were performed using the floral dip method [69]. The transformants were screened on 50 μg/mL kanamycin and 25 μg/mL hygromycin or 1% Basta (PlantMedia). pSAT vectors [70] were used to generate constructs for subcellular localization and BiFC assays in the protoplast system. To generate the BiFC constructs, nEYFP (the N-terminal part of EYFP) was fused to full-length TPD1, mature TPD1, or mutated TPD1 at the cleavage site following the TPD1 signal peptide (Fig 3A). Similarly, cEYFP (the C-terminal part of EYFP) was fused to full-length EMS1 and a series of mutants that were truncated after the EMS1 signal peptide (Fig 3E). Each construct contained the EMS1 signal peptide and transmembrane domain. Phusion High-fidelity DNA polymerase (New England Biolabs) was used for all PCRs. Detailed information for all constructs and the primers used to generate them is presented in S1 and S2 Tables. Alexander pollen staining and anther semi-thin sectioning were conducted as described previously [9,10]. Transient expression of proteins in Arabidopsis protoplasts was performed as described previously [71]. The 35S: EYFP plasmid was used to monitor transfection efficiencies. At least three replicates were performed for each assay. Young buds were collected from wild-type plants, transgenic plants, and transfected protoplasts, proteins were prepared, and Western blotting was performed as described previously [9], utilizing rabbit anti-GFP (Torrey Pines Biolabs) as the primary antibody and anti-rabbit IgG-HRP (Cell Signaling Technology) as the secondary antibody. RNA in situ hybridization was performed as previously described [10,72] using anthers from wild-type, tpd1 mutant, and transgenic plants. The SP6/T7 DIG RNA Labeling Kit (Roche) was used to generate sense and antisense probes against A9 and SDS. RNA was extracted from young buds of wide-type, tpd1 mutant, and transgenic plants using an RNeasy Plant Mini Kit (Qiagen). RNA quantification, reverse transcription, real-time PCR (DNA Engine Opticon 2 system), and data analysis were performed as described previously [72] to examine the expression levels of the anther-specific genes, A9, A6, and ATA7, and the BARNASE gene (for primers, see S2 Table). The constitutively expressed ACTIN2 gene was examined as a control. Three independent experiments were performed. The EM-immunogold-labeling was performed as described previously [73]. Young anthers from TPD1: TPD1sp-GFP-ΔTPD1/tpd1 and EMS1: EMS1-3xGFP/ems1 transgenic plants were dissected and processed by the high-pressure freezing procedure. Rabbit anti-GFP (Torrey Pines Biolabs) was used as the primary antibody, while anti-rabbit IgG conjugated to 15-nm gold particles (Ted Pella) was used as the secondary antibody. Controls were run in parallel without the primary antibody. Images of anther semi-thin sections were visualized and photographed using an Olympus BX51 microscope equipped with an Olympus DP 70 digital camera. For confocal microscopy, samples were observed with a Leica TCS SP2 laser scanning confocal microscope using a 63×/1. 4 water immersion objective lens. A 488-nm laser was used to excite GFP, EYFP, chlorophyll, and FM4-64. Emission were captured using PMTs set at 505–530 nm, 500–550 nm, and 660–760,644–719 nm, respectively. For visualization of cell membranes and trafficking vesicles, roots were treated with 20 μM FM4-64 (Invitrogen) or 30 μM BFA (Sigma-Aldrich) for 5–30 min, washed, and then observed. For localization of TPD1 and EMS1, anthers were dissected from young buds and mounted in water. For immunogold-labeling, images were obtained with an FEI CM 120 electron microscope. | The differentiation of distinct somatic and reproductive cells in flowers is required for the successful sexual reproduction of plants. The anther produces reproductive microsporocytes (pollen mother cells) that give rise to pollen (male gametophytes), as well as surrounding somatic cells (particularly the tapetal cells) that support the normal development of pollen. In animals, signals from somatic cells are known to influence reproductive cell fate determination, and vice versa. However, little is known about the molecular mechanisms underlying somatic and reproductive cell fate determination in plants. In this paper, we demonstrate that TPD1 (TAPETUM DETERMINANT1) is processed into a small secreted cysteine-rich protein ligand for the EMS1 (EXCESS MICROSPOROCYTES1) leucine-rich repeat receptor-like kinase (LRR-RLK). TPD1 is secreted from reproductive cells to the plasma membrane of somatic cells, where activated TPD1-EMS1 signaling first promotes periclinal cell division and then determines tapetal cell fate. Moreover, tapetal cells suppress microsporocyte proliferation. Our findings illuminate a novel mechanism by which reproductive cells determine somatic cell fate, and somatic cells in turn limit reproductive cell proliferation. Plants extensively employ LRR-RLKs to control growth, development, and defense. Our identification of TPD1 as the first small protein ligand for all LRR-RLKs characterized to date will provide a valuable system for studying how small protein ligands activate LRR-RLK signaling complexes. | lay_plos |
Worldwide, amoebic liver abscess (ALA) can be found in individuals in non-endemic areas, especially in foreign-born travelers. We performed a retrospective analysis of ALA in patients admitted to French hospitals between 2002 and 2006. We compared imported ALA cases in European and foreign-born patients and assessed the factors associated with abscess size using a logistic regression model. We investigated 90 ALA cases. Patient median age was 41. The male: female ratio was 3. 5∶1. We were able to determine the origin for 75 patients: 38 were European-born and 37 foreign-born. With respect to clinical characteristics, no significant difference was observed between European and foreign-born patients except a longer lag time between the return to France after traveling abroad and the onset of symptoms for foreign-born. Factors associated with an abscess size of more than 69 mm were being male (OR = 11. 25, p<0. 01), aged more than 41 years old (OR = 3. 63, p = 0. 02) and being an immigrant (OR = 11. 56, p = 0. 03). Percutaneous aspiration was not based on initial abscess size but was carried out significantly more often on patients who were admitted to surgical units (OR = 10, p<0. 01). The median time to abscess disappearance for 24 ALA was 7. 5 months. In this study on imported ALA was one of the largest worldwide in terms of the number of cases included males, older patients and foreign-born patients presented with larger abscesses, suggesting that hormonal and immunological factors may be involved in ALA physiopathology. The long lag time before developing ALA after returning to a non-endemic area must be highlighted to clinicians so that they will consider Entamoeba histolytica as a possible pathogen of liver abscesses more often. Amœbiasis is caused by Entamoeba histolytica (E. histolytica), a protozoan specific to humans. Amœbiasis is present throughout the world, but is endemic in tropical countries where the risk of faeco-oral transmission is high [1]. The main clinical manifestations of amœbiasis are colitis and liver abscess (ALA) [1]. Pleuropulmonar and pericarditis forms also exist but ALA is the most common extraintestinal manifestation of the disease and is one of the etiologies of febrile returning travelers. In Europe, ALA is observed in European-born travelers visiting tropical countries, and in foreign-born patients living in European countries. Complications of ALA are rupture of the abscess into the neighborly cavities of the pleura, pericardium, peritoneum and distant embolic dissemination [2]. ALA was historically responsible for a high number of fatal cases, but since the introduction of medical treatment, the mortality rate is dropped to around 1 to 3%. The management of ALA is still debated, the indications for percutaneous aspiration being a source of controversies. However most authors recognize that liver abscess puncture or drainage is generally indicated in severely ill patients, patients with large abscess, and if there is a immediate risk of rupture [1]. In France, one single study assessed treatment options for imported ALA and proposed an algorithm for drainage procedures [3]. Size of ALA is determined by radiology and is in most cases between 5 and 10 cm [2], [3], [4], [5]. Nevertheless, no factor has been identified as showing a correlation with the size of the abscess up to date. It is critical than clinicians should be aware of the presentation, evolution and management of imported ALA. The aim of this study was to describe the presentation of ALA in the early 2000' s in Paris area where there is a high proportion of travelers and foreign born (i. e. born outside the European Union) individuals. We compared the presentation, treatment and outcome of ALA in these two populations. In addition we aimed to identify risk factors associated with abscess size at diagnosis and with percutaneous aspiration. We also aimed to describe evolution of the abscesses when follow up allowed it. Diagnosis of ALA relied on three criteria: travel associated exposure, presence of at least one liver abscess detected by abdominal ultrasound and/or CT scan in a symptomatic patient, and a positive serology for amœbiasis. Positive serology had to be confirmed by two of the following tests: ELISA, indirect immunoflourescence assay (IFA), indirect haemagglutination (IHA), Latex and counterimmunoelectrophoresis (CIE). Positive serology was defined when titres were above 1∶200 for ELISA, 1∶100 for IFA, 1∶320 for IHA and when there was at least one precipitation arc for CIE. We distinguished two populations and four patient profiles. The first population was represented by European-born patients defined as patients born and living in France who traveled to tropical areas (European travelers) and patients born in France living for more than 6 months in an endemic region (expatriates). The second population consisted of foreign-born patients defined as patients born in an endemic region, having immigrated to France who traveled back to their country of origin to visit friends and relatives (VFRs), and patients born in an endemic region who had not returned to their country of origin since they emigrated to the EU (immigrants). We estimated time of apyrexia and biological normalization for patients who had assessment monitoring data. Abdominal ultrasounds and/or CT-scans, for those patients who had them, were reviewed to estimate the disappearance of the abscess. We analyzed the risk of large ALA at diagnosis, defined as a diameter greater than 69 mm, corresponding to the median abscess size in our patients, using a logistic regression model. Factors investigated were: sex of patients, age (divided into two categories defined by the median age), birthplace (France, Africa and other), patient profile (European travelers, European expatriates, Immigrants and VFR), place of contamination (Africa, Asia and other), HIV status (positive/negative), absence or presence of abdominal pain, laboratory findings such as clinically relevant haemotological and biochemical parameters: anemia defined by haemoglobin lower than 12 g/dL, a White Blood Cells count higher or lower than 10 000 per mm3, a level of C Reactive Protein higher or lower than 200, and cytolysis defined by elevated liver transaminase levels up to the normal, location of the abscess (left or right liver or unknown), number of abscesses (1,2, 3 and 4 or more), lag time between return from an endemic area and date of the onset of symptoms, in categories based on the median (more or less than 128 days) and lag time between onset and treatment in categories based on the median (more or less than 10 days). For all of these factors, missing values were defined as “unknown”. A descriptive analysis of the dependant and independent variables was performed. A univariate analysis was then conducted, and all factors associated with large ALA having a p-value<0. 20 were entered into the multivariate model. A backward stepwise selection procedure was then applied to identify significant (p<0. 05) independent variables. Logistic regression was also used to identify factors associated with the use of percutaneous aspiration. The same factors were investigated, as well as the size of the ALA (≤69 or >69 mm), the treatment and the type of care unit where the patient was admitted (i. e. medical or surgical department). Variables “place of birth” and “patient profile” are correlated. In our model we preferred the variable “patient profile”, which was more relevant to us because the consideration of the place of birth and reason of travelling are more appropriate for clinical practice. Clinical profile, as well as laboratory and radiological findings were also compared between foreign-born patients (i. e., VFRs and immigrants) and European-born patients (i. e., European travelers and expatriates) using a Student t-test, chi-square test or Fischer' s exact test, when required. Using ultrasound and CT scans, we monitored the time to the disappearance of the abscess using Kaplan-Meier estimates. The Kaplan-Meier curves were compared across the different groups using the log-rank test. Factors associated with the disappearance of an abscess were identified using a Cox proportional hazard model. The factors investigated were the same as those used in the logistic regression model, and, for each factor, the proportional hazard assumption was tested based on Schoenfeld' s residuals. All analyses were performed using STATA 10. 0 (Stata statistical software, Stata Corporation, College Station, Texas, USA), and a p-value≤0. 05 was considered as significant. The study received the approval of the French National Commission and Informatics and Liberties under the number 165 29 66 and all data were anonymized. 331 positive amœbiasis serologies were reported. 229 positive amœbiasis serologies were without liver abscess 102 Amoebic Liver Abscesses were diagnosed during the period but 12 medical records were unaivailable (Figure 1). A total of 90 patients with ALA were identified during the study period. The male/female ratio was 3. 5 and the median (inter quartile range (IQR) ) age at diagnosis was 41 (33–53) years. Age distribution was not different between men and women (t-test, p = 0. 46). All the patients had traveled in a region where amœbiasis is endemic: Africa (56%), Asia (19%), and South/North America (4%). There was 38 (51%) European-born patients and 37 (49%) foreign-born patients. We could not determine origin for 15 subjects. The median (IQR) time between the return from the endemic area and the first symptoms was 128 days (61–563). The median time between the first symptoms and diagnosis was 10 days (5–20) and was longer for foreign-born than European-born patients (t-test, p<0. 01). Of the 79 patients tested for HIV, 4 (5%) were HIV-positive. Clinical and laboratory findings are summarized in Table 1. Stool sample examination was performed in 58 patients (64%) (one sample for 34 (38%) and two or more for 23 (26%) ). Stool samples were performed before treatment in 11 patients and were found to be positive for Entamoeba cysts in five patients. Diagnosis of liver abscess relied on ultrasound in 30%, CT scan in 25%, and both in 45% of cases. A single abscess was detected in 77% of cases whereas two abscesses were present in 9% of cases and three or more in the remaining 14%. The median (IQR) diameter at diagnosis was 69 mm (50–290). The main abscess location was the right lobe of the liver (78%). Pleural effusion was found in 12 patients. Reported complications included portal thrombosis, biliary tract rupture and colectomy due to amoeboma. Metronidazole was the most commonly used anti-amoebic agent (94. 5% of the cases) but others, such as tinidazole (n = 3) and ornidazole (n = 2), were also used. The median (IQR) time between first symptoms and treatment, known for 62 subjects, was 9 days (5–17). Treatment duration was longer than 14 days in 39 subjects (43%). Treatment was complemented by a non-absorbed anti-amoebic luminal agent (i. e., tilbroquinol-tiliquinol) in 80% of the patients. At least one antibiotic was additionally prescribed in 59 patients (72%). Third generation cephalosporin (n = 34), or amoxicillin alone (n = 4) or amoxicillin-clavulanate (n = 22) were used. Percutaneous aspiration was associated with the medical treatment in 27 patients. No one in this population had surgical treatment. All the patients were hospitalized. The median (IQR) time to apyrexia after treatment initiation was three days (1–4). Median time for normalization of WBC count and CRP was 5. 5 days (1–30) and 15 days (9–30), respectively. Two relapses of ALA were observed, one and three years after initial treatment, respectively. One patient had not received any anti-luminal agent during the first episode and another patient received tilbroquinol-tiliquinol. There was no fatal case. Our comparison (See Table S1 in appendixes) showed that there were no significant differences between the two populations except that a longer time between return from the endemic area and the onset of the first symptoms was observed for foreign-born patients: 17% of foreign-born patients presented with an ALA more than one year after returning from a tropical area where the disease is endemic, while for European-born patients, all cases of ALA were diagnosed within a year of their return (p<0. 01). The maximum lag-time between the time of return from the endemic area and the onset of symptoms was 14 years in foreign-born patients and 12 months in their European-born counterparts. Data were available for 86 abscesses. Table 2 shows the results of the logistic regression analysis of factors associated with abscesses larger than 69 mm at the time of diagnosis. In multivariate analysis, men compared to women and patient older than the median age of 41 were more likely to present with a larger abscess (odds ratio (OR) = 11. 25, [95% confidence interval (CI): 2. 42–52. 29] and OR = 3. 63 [95% CI: 1. 26–10. 48] respectively), as were immigrants when compared with the European-born travelers' group (OR = 11. 56, [95% CI: 2. 10–63. 41]). The estimated specificity and sensibility of the model were 71% and 70% respectively. The only factor significantly associated with percutaneous aspiration in multivariate analysis was the type of department where the patient was admitted: those initially admitted to a surgical unit were more likely to have percutaneous aspiration than those admitted to a medical unit (OR = 10. 0, [95% CI: 2. 70–37. 03]) (See Table S2 in appendixes). We couldn' t identify any statistical interactions which would be clinically relevant in our models. CT-Scan and/or ultrasound post-treatment monitoring was available in 24 cases. Using Kaplan Meier estimates, the observed median time to abscess disappearance was 7. 5 months (Figure 2). Using a Cox proportional hazard model (See Table S3 in appendixes), the factor most strongly associated with abscess disappearance was its initial size. Larger abscesses were less likely to disappear than smaller ones: hazard ratio (HR) = 0. 40 [95% CI: 0. 15–1. 03]; p = 0. 06). Treatment (duration of antibiotherapy and percutaneous aspiration) was not associated with the probability of disappearance. We identified 90 cases of ALA in the Paris area during the five-year study period. We showed that men were more likely to present with larger abscesses than women. This is the second largest series of imported ALA. In 1984, Laverdant et al. reported in France 152 cases collected over 15 years in two French military hospitals [6]. In this historical and descriptive cohort, the clinical presentation was the same as in our study, with a mean abscess size of 65 mm. Nevertheless the population was different (military personnel) and percutaneous aspiration was less frequently performed than in our study (12% vs. 30%). As in other studies, ALA mainly concerns middle-aged men, presenting with fever and abdominal pain as well as elevated WBC count and CRP rate [3], [7], [8], [9], [10], [11]. ALA was much more frequently diagnosed in men than in women as in several other studies [3], [6], [8], [9], [11], [12], [13], [14]. This is unlikely that the higher proportion of men in foreign-born population living in Western countries is a relevant explanation for this. Indeed the male predominance is also found in the subgroup of European travelers (75. 7% of foreign-born and 81. 6% of European are male). In addition this gender tendency has already been reported for other infectious diseases [15]. For instance, analysis of pyogenic liver abscesses in Denmark over a 30-year period, showed that men were more likely to be affected [16]. This finding is also consistent with the animal model, as Lotter et al. demonstrated that male mice developed larger ALA than female mice [17], [18]. Hormonal factors have been suggested to explain this difference [15]. A recent study in hamsters highlights the role of sex hormones in ALA development [19]. Male hamsters, who were gonadectomized, developed either no or smaller ALAs, suggesting that testosterone could be a host factor favoring the development of ALAs. Other studies strongly support the hypothesis that immunological factors explain this gender difference. Indeed in humans, Snow et al. showed that female serum was more effective in killing E. histolytica trophozoïtes than a male one, thanks to the complement system [20]. In the animal model, female mice recovered more rapidly than their male counterparts due to a higher production of Interferon-γ [17] secreted by Natural Killer T Cells [18]. The absence of association with HIV infection and ALA size in our study, is contradictory to this immunological hypothesis, but can be attributed once again to the low number of subjects. In a study involving more than 2000 ALA cases from an endemic region, Blessmann et al. showed a peak incidence at 40–49 of age as in our cohort [8]. Regarding the positive role of testosterone on susceptibility of ALA, we could expect to observe a decrease of incidence of ALA with age. In our study, we referred to the abscess size as being the size at diagnosis, as opposed to the susceptibility to amœbiasis. For elderly patients, alteration of immunity could be determined as a factor to develop a bigger abscess than for younger patients. This could be explained by the effects of immunosenescence [21]. We have also shown that laboratory findings, especially regarding leucocytosis, C Reactive Protein and hemoglobin, were identical for large and small abscesses. This fact is to be brought to the attention to clinicians who might underestimate ALA size when expecting higher White Blood Cells count, C Reactive Protein level and more severe anemia for bigger abscess. Besides, since no significant difference was observed between the two abscess size groups regarding pleural effusion -which is one of the main complications of ALA-, it seems that localization is more to be linked to pleural complication than size itself in this case. By comparing patients of European origin and foreign-born patients, we found a longer time between the patient' s return from an endemic area and the onset of symptoms and larger abscesses in foreign-born patients. In contrast with this latter result, a study performed in the US showed that travelers born in the US were more likely to have larger abscesses (and chronic illness) compared with patients born in endemic countries [11]. In that study, immigrants came from South America and Asia whereas our patients mainly came from Africa. This longer time to develop ALA and larger ALA may both be explained by immunological reasons due to previous contact with Entamoeba histolytica, possibly leading to a certain protective immunity providing a less acute evolution of the disease. Although few data support this hypothesis, some studies explaining the low rate of relapse by anti-lectin IgA antibody mediated mucosal immunity [22], [23], [24] have suggested that a possible strong protective immunity develops after an episode of intestinal or liver amœbiasis. Another explanation could be that foreign-born patients have delayed access to care as this has already been described for patients infected with HIV [25], [26], a fact which led to their consulting a medical practitioner at a later disease stage. It is worth noting that among foreign-born subjects, immigrants but not VFR, seemed more likely to present with a larger abscess when compared with European-born travelers. This finding, which has not been reported in any other study may be due to better access to care for VFRs as compared to immigrants that are facing economic problems not encountered by VFRs which remain able to travel. Nevertheless, the long delay before developing the disease, sometimes years for foreign born individuals and months for their European-born counterparts, is of clinical interest and should be highlighted. A few data are available about incubation because most of studies about ALA are performed in endemic area, where exposition is permanent. Some authors have ever suggested a long incubation, easily evaluated in case of imported ALA [5]. The problem in these studies, as in ours, is that only the last travel declared by patients is taken into account, omitting the fact they may have been contaminated during previous travel. Clinicians should also remember that, when patients initiate treatment, apyrexia is quickly achieved, a fact which should help them to evaluate the efficiency of their treatment. In addition we were able to estimate to a median time of 7. 5 months the disappearance of ALA in a quarter of the patients. This highlights the fact that recovery is a long process and that the persistence of lesions, as highlighted by radiology techniques, should not alarm clinicians during follow-up in patients free of symptoms. In addition, based on the admittedly small number of subjects in our Cox model, we found that treatment duration was not associated with any decrease in abscess size. Percutaneous aspiration is not as effective as prolonged liver drainage but it is difficult to explain why percutaneous aspiration was not associated with any decrease in abscess size. The small number of subjects who had percutaneous aspiration monitored in our study (8 individuals) could explain these findings. Percutaneous aspiration is usually recommended for ALA bigger than 10 cm, but it is still unclear if it is really beneficial for the patient [4], [27]. In our study, patients admitted to a surgical unit were more likely to receive percutaneous aspiration. We can hypothesize that patients with a larger abscess were more likely to be admitted to a surgical unit, but our data showed that there was no difference between surgical or medical units in terms of the number of large abscesses. The greater susceptibility of surgeons for surgical treatment compared to medical treatment could explain the highest rate of percutaneous aspiration in their units. Guidelines for the use of percutaneous aspiration do not exist. Some authors suggest that percutaneous aspiration be used for abscesses with a diameter greater than 10 cm [3], while others suggest it be performed depending on clinical evolution. Our study was not designed to answer this question. The present study has limitations. First, the retrospective design gives rise to missing data, especially for the time between return in Europe and first symptoms. Place of contamination were unknown for 18 subjects. It could miss some autochthonous case of contamination but, comparing other series of ALA in France, this mode of contamination is exceptional. On the same hand, unknown origin (European or foreign-born) involved 15 subjects. Sensitive analyses, taking account or not these missing subjects, about factors associated to initial size of ALA had similar results. Variables with many missing values were not present in our final models. The small number of subjects in our study is certainly responsible of a low statistical relevance. In the worst situations, an effect could have been masked in our analysis, but any false association has been shown. The sensibility and specificity of our model about abscess size were around 70%. Our study provides trends in presentation of ALA, but has been proved insufficient to bring irrefutable results. The low number of subjects with biological and radiological data explains the low number of cases observed for the Cox multivariate model, but this original analysis is at our knowledge the first for imported cases of ALA. Secondly, our definition of ALA was based on a positive amœbiasis serology associated to a non-bacterial liver abscess. Entamoeba histolytica PCR of the pus aspirate of the abscess is a very useful tool to diagnose ALA. According to case reports, serodiagnostic may lacks sensitivity when compared to PCR, as false positive serologies have been reported in endemic area [5], [28]. PCR has been evaluated in a few studies for imported ALA and should become a gold standard to diagnose ALA when invasive explorations are performed. At least, our study collected data from 13 hospitals with different types of healthcare management. Longitudinal follow-up studies could better describe recovery and also evaluate factors associated with abscess persistence. Similarly, clinical trials are needed to assess percutaneous aspiration and to perform guidelines for its use in the treatment of ALA, something which can often be adequately treated using drugs alone. The clinical presentation and outcomes for ALA were similar in European and foreign-born patients. However foreign-born patients presented with ALA later than their Europeans-born counterparts, sometimes several years after travel. Moreover, we showed that men were more likely to present with larger abscesses than women, something which has already been observed in animal model and patients from other countries. These data support the need for further studies on amoebic liver abscess physiopathology, including the impact of specific immunity and sexual hormones. New tools for an easier and more reliable diagnosis of ALA are also expected as in countries of imported ALA as well as in countries endemic for amœbiasis. | Amœbiasis is caused by Entamoeba histolytica (E. histolytica), a protozoan specific to humans which infects humans by ingestion of contaminated food and water. According to some authors, amœbiasis could be the second leading cause of death from parasitic disease worldwide. It is endemic in tropical countries but can also be diagnosed in industrialized countries, essentially in travelers. One of its main clinical manifestations is amoebic liver abscess (ALA). Abscess can be medically treated by metronidazole, but percutaneous aspiration of the abscess is sometimes performed. Few studies have been performed so far regarding the existence of cases of ALA in the industrialized world. The results of our study reported the existence of 90 cases of imported ALA in France which should raise interest among physicians as well as tourists traveling to endemic areas. Male adults and foreign-born patients presented larger abscesses, suggesting that hormonal and immunological factors could be involved in ALA physiopathology. Foreign-born patients had a longer lag time between the return to France after traveling abroad and the onset of symptoms than European-born ones. This must be highlighted to clinicians who should think about this diagnostic even if a recent travel in a tropical area is not notified by patients. | lay_plos |
We evaluated the fraction of variation in HIV-1 set point viral load attributable to viral or human genetic factors by using joint host/pathogen genetic data from 541 HIV infected individuals. We show that viral genetic diversity explains 29% of the variation in viral load while host factors explain 8. 4%. Using a joint model including both host and viral effects, we estimate a total of 30% heritability, indicating that most of the host effects are reflected in viral sequence variation. There are differences in the rate of disease progression among individuals infected with HIV. An easy to measure and reliable correlate of disease progression is the mean log viral load (HIV RNA copies per ml of plasma). The viral load measured during the chronic phase of infection (referred to as setpoint viral load, spVL) exhibits large variation in a population. Several studies have been carried out to elucidate whether this variation is primarily driven by host genetics [1–4], viral genetics [5–9], or environmental effects [7]. Genome-wide association studies consistently show that amino acid polymorphisms in the peptide binding groove of the HLA-A and HLA–B proteins are associated with the viral load of an individual. Furthermore, variants in the HLA-C and CCR5 genes have also been shown to impact spVL. However, those host factors explain less than 15% of the observed phenotypic variance [4]. In contrast, viral genetic studies and studies of donor-recipient transmission pairs established that 33% of the phenotypic variance is attributable to the transmitted virus itself [5,10–13]. HIV is an extremely variable and adaptive organism with a rapid replication time, and high rates of mutation. Within-host evolution of the viral population occurs during the chronic phase of infection in which the pathogen adapts to its host environment. Several studies showed that a major proportion of the viral sequence is under selective pressure in the host environment, and several viral amino acid changes are associated with host genetic variants in the Human Leukocyte Antigen (HLA) genes [14,15]. Viral strains harbor epitope sequences that can be presented by HLA class I proteins of the infected host, which allows the detection and killing of infected cells. The viral population evades detection through escape mutations that modify the epitope sequence but may incur a fitness cost. Compensatory mutations may follow until the viral population reaches its optimal place in a sequence space constrained by the host immune system [16]. There are two main different approaches to viral heritability estimation in the literature. The first one is based on the regression of phenotypic values in donor-recipient transmission pairs, while the other quantifies the difference between the observed phenotypic variance-covariance structure and the phylogenetic variance-covariance structure. Because our study population did not include donor-recipient data, we used the latter strategy. In particular we used linear mixed models (LMMs) to explain inter-patient differences in spVL while taking into account host and viral genetic relatedness. LMMs use the pairwise relatedness of individuals with respect to a large set of features (rather than the individual data points) to estimate the fraction of phenotypic variance attributable to those features. Such models have been successfully applied to estimate narrow-sense heritability from genome-wide genotype data [17]. Concurrently, LMMs were proposed to incorporate phylogenetic relatedness between samples in comparative analyses [18], a technique that was further developed to estimate the viral genetic contribution to spVL [6,8]. To estimate the respective contribution of host and viral genetics to the variation in spontaneous HIV control, we collected paired viral/host genotypes along with spVL measurements from 541 chronically infected individuals enrolled in two prospective cohort studies in Switzerland and in Canada. We estimated the respective contributions of host and viral genetics to spVL by defining two relatedness measures, one with respect to the host genotypes, the other with respect to the viral genotypes, and used these jointly in a linear mixed model. On the host side, we focused on amino acid variations in the HLA-A, B and C genes due to their established associations with HIV control [1]. In particular, we used 33 amino acid polymorphisms selected by L1 regularized regression [19] to represent the genetic relatedness of the host (S1 Table). Principal component analysis based on host genome-wide genotype data confirmed the lack of major population stratification in the host sample. We built three LMMs, one containing human variants, one derived from phylogenetic trees, and one including both host and virus information (Fig 1). The genetic relatedness matrix created from 33 amino acid polymorphisms of the human class I HLA genes explained 8. 4% (SD = 4%) of the observed variance in spVL. In contrast, 28. 8% (SD = 11%) of phenotypic variation was attributable to the viral phylogenetic tree. Combining the two relatedness matrices in one model yielded a total variance explained of 29. 9% (SD = 12%), less than the sum of the latter two models. Thus, we show that HLA polymorphisms do not explain additional phenotypic variance beyond viral sequence variation. We next assessed the contribution of viral variants most likely to have an impact on spVL. These included amino acids in known CTL epitopes [20] and those positions whose variation is associated with host polymorphisms [14] (82%, 60% and 84% of gag, pol, nef codons respectively, S2 Table). We used phylogenetic trees built from those codons to show that viral variation in epitopes or other HLA-associated positions explain 23. 6% (SD = 11%) of phenotypic variance. However, this explained fraction might be overestimated due to linkage disequilibrium on the viral haplotype. We therefore repeated the analysis after randomly picking 70% of variable viral positions, and obtained very similar results. We thus cannot conclude that viral variants in known epitopes contribute disproportionately to variance in spVL. Additional evidence for the existence of substantial linkage disequilibrium on the viral haplotype comes from the analysis of the smaller, complementary set of variable viral positions (located in non-epitope regions), which explained 18. 5% (SD = 10%) of the phenotypic variance. This leads to lower bounds of 11. 4% and 6. 3% of variance in spVL explained by variation in epitope and non-epitope regions, respectively, leaving 12. 2% of variance unresolved due to linkage disequilibrium. By jointly analyzing host and viral genetic relatedness, we here provide estimates of the total and respective contributions of human and viral genetic variation to HIV control. Our results do not challenge the current consensus estimates of the host or viral contributions to spVL. Nevertheless, our combined analysis demonstrates that human HLA polymorphisms do not explain additional variance in spVL once viral genetic diversity is taken into account. The difference between the variance explained by viral phylogeny and the variance explained by HLA polymorphisms may be attributed to two effects. First, selected viral variants might provide a better surrogate of the impact of the host genotype than the imputed host amino acid variants we used. Rare host genetic factors outside of the major histocompatibility complex region (e. g. the CCR5 deletion), as well as environmental interactions may influence viral fitness, and these effects are not accounted for in our estimate of host heritability. Thus some host effects might be missed from the host partition, while their footprint in the virus is still detected in the viral partition. Second, the difference could partly be due to the effect of viral variation independent of the current host, including transmitted escape mutations, i. e. viral sequence variation carried over from the previous host, rather than induced by the current host. Indeed, a recent study showed that spVL is dependent on the degree of pre-adaptation of the viral strain to the HLA class I genotype of the current host [21]. In particular, an increase in the frequency of pre-existing escape mutations, at the population level, led to higher viral heritability estimates. This indicates that both host and viral estimates of heritability depend on the amount of pre-adaptation in the sample population, which varies based on the level of HLA diversity. It has also been shown that reversion of some fitness reducing escape variants is very slow, potentially allowing for a transitory but measurable effect on viral load at the population level [15,22]. A limitation of our study is the fact that study participants were collected from two cohorts. To reduce batch effect, we included a cohort-specific variable in all our models. Still, differences in inclusion criteria, health system, geographical exposure and other factors are very likely to increase environmental variance, thus negatively impacting our heritability estimates. Another potential shortcoming is our implicit assumption of the absence of selection on spVL, which might be incorrect, as suggested by recent studies [23,24], and might thus lead to over- or under-estimation of heritability due to model misspecification. Still, because our estimates are comparable to results obtained in donor-recipient transmission studies and in host-genetic studies, we conclude that they are useful for the purpose of delineating the respective amounts of host and viral contributions to phenotypic variation of HIV spVL. In conclusion, our results suggest that host genetic association studies not taking the virus into account underestimate the population level effect of host genetic variation. Combining host and pathogen data provides additional insight into the genetic determinants of the clinical outcome of HIV infection, which can serve as a model for other chronic infectious diseases. All participants were HIV-1-infected adults, and written informed consent for genetic testing was obtained from all individuals as part of the original study in which they were enrolled. Ethical approval was obtained from institutional review boards for each of the respective contributing centers. Bulk sequences of the HIV-1 gag, pol and nef genes, human genome-wide genotyping data and viral load measurements were obtained for 541 individuals of Western European ancestry infected with HIV-1 Subtype B, and followed in the Swiss HIV Cohort Study (SHCS, www. shcs. ch) or in the HAART Observational Medical Evaluation and Research study in Canada (HOMER, www. cfenet. ubc. ca/our-work/initiatives/homer) [14]. Viral sequences data were generated from samples collected two to five years after infection (for SHCS) or during chronic infection (for HOMER) but prior to the initiation of antiretroviral therapy. Thus, the viral genotypes reflect the result of natural adaptation of the pathogen to the host environment. The viral sequences for 1262,2187 and 548 nucleotides of the gag, pol and nef genes were available for at least 80% of samples studied. The analysis was limited to these three genes because sequences of the rest of the retroviral genome were not available for the majority of study samples. Overlapping viral genomic regions were excluded from gag, to avoid duplicated sequences in the analysis. Human DNA samples were genotyped in the context of previous genome-wide association studies. High-resolution HLA class I typing (4 digits; HLA-A, HLA-B, and HLA-C) was imputed from the genome-wide genotyping data as described previously [14]. Set point viral load (spVL) was defined as the average of the log10-transformed numbers of HIV-1 RNA copies per ml of plasma obtained in the absence of antiretroviral therapy, excluding VL measured in the first 6 months after seroconversion and during periods of advanced immunosuppression (i. e., with <100 CD4+ T cells per ul of blood). The distributions of spVL in the two cohorts are shown in S1 Fig. The pairwise genetic relatedness of the dominant viral strains observed in the samples was calculated from phylogenetic trees similarly to [6]. Nucleotide sequences were translated to amino acid sequences, which were in turn aligned with MUSCLE [25] and used to derive the correct codon-aware nucleotide alignment. The phylogenetic tree was built from the aligned nucleotide sequences using RAxML [26] with the following command line: “raxml -w {PATH} -s {PATH} -m GTRCAT -f a -N 30 -k -n {NAME} -T {NUMBER} -x 1234 -p 1234”. Individual sequences were then rooted to the HIV-1 group M ancestral sequence, downloaded from the Los Alamos sequence database. Using an HIV-1 subtype C sequence as outgroup led to similar results. The whole tree was scaled with the inverse of the median height of the branches. We followed the method of Hodcroft et al, to create a relatedness matrix from a phylogenetic tree [6]. The genetic relatedness of two samples in a given phylogenetic tree is the amount of shared ancestry, i. e. the distance from the root of the tree (excluding the outgroup) to their most recent common ancestor [27]. We selected 33 amino acid variants with L1-regularized regression (LASSO) out of all polymorphisms in the HLA-A, B and C genes and used them to generate a genetic relatedness matrix as described in [17]. Our relatively small sample size made it necessary to use a small subset of selected markers rather than genome-wide variant information to create the genetic relatedness matrix. Doing otherwise would have resulted in very large errors of the estimates. To estimate heritability, we used the gcta software as a generic implementation of the linear mixed model [17]. In such a framework, a multivariate Gaussian distribution models HIV viral load with a variance-covariance matrix consisting of the linear combination of the sample-sample genetic relatedness matrices (one for the host and one for the virus) and the identity matrix (representing sample-specific noise). The total heritability estimate is the fraction of variance explained by the genetic relatedness matrices over the total variance. All models included a binary variable indicating cohort as a fixed effect. Variance components were estimated by restricted maximum likelihood. | Viral loads of Human Immunodeficiency Virus infections are correlated between the donor and the recipient of the transmission pair. Similarly, human genetic factors may modulate viral load. In this study we estimate the extents to which viral load is heritable either via the viral genotype (from donor to recipient) or via the host's Human Leukocyte Antigen (HLA) genotype. We find that a major fraction of inter individual variability is explained by the similarity of the viral genotypes, and that human genetic variation in the HLA region provide little additional explanatory power. | lay_plos |
BERKELEY Cocaine can speedily rewire high-level brain circuits that support learning, memory and decision-making, according to new research from UC Berkeley and UCSF. The findings shed new light on the frontal brain’s role in drug-seeking behavior and may be key to tackling addiction. Looking into the frontal lobes of live mice at a cellular level, researchers found that, after just one dose of cocaine, the rodents showed fast and robust growth of dendritic spines, which are tiny, twig-like structures that connect neurons and form the nodes of the brain’s circuit wiring. “Our images provide clear evidence that cocaine induces rapid gains in new spines, and the more spines the mice gain, the more they show they learned about the drug,” said Linda Wilbrecht, assistant professor of psychology and neuroscience at UC Berkeley and lead author of the paper published today (August 25) in the journal Nature Neuroscience. For mice, “learning about the drug” can mean seeking it out to the exclusion of meeting other needs, which may explain how addiction in humans can override other considerations that are necessary for a balanced life: “The downside is, you might be learning too well about drugs at the expense of other things,” Wilbrecht said. Using a technology known as 2-photon laser scanning microscopy, researchers made images of nerve cell connections in the frontal cortices of live mice before and after the mice received their first dose of cocaine and, within just two hours, observed the formation of new dendritic spines. “The number of new, robust spines gained correlated with how much the individual mice learned to prefer the context in which they received the drug,” Wilbrecht said. The findings provide clues to behavioral and environmental factors in drug addiction. Previous analyses of postmortem mouse brains have shown that repeated cocaine use and withdrawal changes dendritic spine density after weeks of exposure. But this is the first time that 2-photon laser scanning microscopy has been used to make images of spines in the frontal cortex before and after the first cocaine exposure in living mice, Wilbrecht said. Over the course of a few days, male mice were moved from their cages to a “conditioning box” comprised of two adjoining compartments. Each chamber – one smelled of cinnamon and the other of vanilla – was decorated with different patterns and textures so the mice could differentiate between the two. Initially, the mice were free to explore both chambers, which were connected by a small doorway. Researchers recorded which side each mouse preferred. The next day, the mice were injected with saline, which has no stimulant effect on mice, and were placed for 15 minutes in the compartment for which they had shown a preference. The door between the two chambers was shut. The following day, they were injected with cocaine and placed in the chamber that was not their preferred side. Again, the door between the two chambers was shut and they were inside for 15 minutes. On the fourth day, the door between the two chambers was open, yet the mice overwhelmingly picked the chamber where they had received and presumably enjoyed the cocaine. “When given a choice, most of the mice preferred to explore the side where they had the cocaine, which indicated that they were looking for more cocaine,” Wilbrecht said. “Their change in preference for the cocaine side correlated with gains in new persistent spines that appeared on the day they experienced cocaine.” According to Wilbrecht, “These drug-induced changes in the brain may explain how drug related cues come to dominate decision-making in a human drug user, leaving more mundane tasks and cues with relatively less power to activate the brain’s decision-making centers.” Much of the research was conducted at the Ernest Gallo Clinic and Research Center at UCSF. Other co-authors of the study are Francisco Javier Munoz-Cuevas and Jegath Athilingam from the Ernest Gallo Clinic and Research Center and Denise Piscopo of the University of Oregon. Image caption The researchers looked for tiny protrusions from brain cells called dendritic spines Taking cocaine can change the structure of the brain within hours in what could be the first steps of drug addiction, according to US researchers. Animal tests, reported in the journal Nature Neuroscience, showed new structures linked to learning and memory began to grow soon after the drug was taken. Mice with the most brain changes showed a greater preference for cocaine. Experts described it as the brain "learning addiction". The team at University of California, Berkeley and UC San Francisco looked for tiny protrusions from brain cells called dendritic spines. They are heavily implicated in memory formation. Cocaine hunting The place or environment that drugs are taken plays an important role in addiction. This study gives us a solid understanding of how addiction occurs - it shows us how addiction is learned by the brain Dr Gerome Breen, Institute of Psychiatry In the experiments, the mice were allowed to explore freely two very different chambers - each with a different smell and surface texture. Once they had picked a favourite they were injected with cocaine in the other chamber. A type of laser microscopy was used to look inside the brains of living mice to hunt for the dendritic spines. More new spines were produced when the mice were injected with cocaine than with water, suggesting new memories being formed around drug use. The difference could be detected two hours after the first dose. Researcher Linda Wilbrecht, assistant professor of psychology and neuroscience at UC Berkeley, said: "Our images provide clear evidence that cocaine induces rapid gains in new spines, and the more spines the mice gain, the more they show they learned about the drug. "This gives us a possible mechanism for how drug use fuels further drug-seeking behaviour. "These drug-induced changes in the brain may explain how drug-related cues come to dominate decision making in a human drug user." Commenting on the research, Dr Gerome Breen, from the Institute of Psychiatry at King's College London, told the BBC: "Dendritic spine development is particularly important in learning and memory. "This study gives us a solid understanding of how addiction occurs - it shows us how addiction is learned by the brain. "But it is not immediately apparent how useful this would be in developing a therapy." | Just one dose of cocaine may physically change the brain as the body begins "learning addiction," scientists say. They investigated the effect of the drug on mice, and noted that within two hours of being injected with the drug, brain changes were visible, the BBC reports. After two hours, scans showed that mice on the drug developed more dendritic spines-key to forming memories-than did mice injected with water. "Our images provide clear evidence that cocaine induces rapid gains in new spines, and the more spines the mice gain, the more they show they learned about the drug," says a researcher. At the beginning of the experiment, the mice were placed in a "conditioning box" comprised of two very different rooms linked by a door. After their room preference became clear, they were injected in the opposite room. Afterward, mice tended to hang out on the side where they'd been injected, suggesting "they were looking for more cocaine," says the researcher, per the UC Berkeley News Center. As for us humans, the brain changes could show how "drug-related cues come to dominate decision-making... leaving more mundane tasks and cues with relatively less power to activate the brain's decision-making centers." | multi_news |
Comedian Bill Cosby adjusts his Presidential Medal of Freedom as baseball great Hank Aaron, left, looks on in a ceremony in the East Room of the White House in 2002. A victims' group called on President Obama to revoke the award to Cosby, which was awarded by President George W. Bush. (Photo: H. DARR BEISER, USA TODAY) WASHINGTON — A sexual assault awareness group called on President Obama to revoke Bill Cosby's Presidential Medal of Freedom after the 77-year-old comedian's recently revealed admission that he obtained drugs to give to women with whom he wanted to have sex. The request raises thorny legal and political questions for a White House that has long championed sexual assault prevention. Though the president can change the criteria for granting the nation's highest civilian honor with the stroke of a pen, no president has ever revoked the award. "Certainly it would be an unprecedented event to take this away," said Angela Rose, founder of Promoting Awareness, Victim Empowerment, a decade-old sexual assault education and prevention group. "But it's also an unprecedented moment in our nation's history that such an iconic figure, a legend, be accused by 40 women of sexual violence. We cannot yet give his accusers their day in court, but we can fight back in the court of public opinion." PAVE submitted a petition to the White House on Wednesday. If it gets 100,000 electronic signatures in the next month, the White House has promised a formal response. White House spokesman Josh Earnest said he was unaware of the calls to revoke the medal. "I haven't, at this point, heard any discussion of taking that step," he said. "I don't know whether or not it's legally possible to do so." Unlike the Medal of Honor for military valor, the Medal of Freedom isn't authorized by Congress. President Truman established it by executive order in 1945 to recognize civilians who helped the war effort. President Kennedy expanded it in 1963 to include people who have made contributions through "cultural or other significant public or private endeavors." President George W. Bush awarded the medal to Cosby in 2002 in a class that included baseball great Hank Aaron, children's television pioneer Fred Rogers and former first lady Nancy Reagan. "Bill Cosby is a gifted comedian who has used the power of laughter to heal wounds and to build bridges, " Bush said in awarding the medal. "By focusing on our common humanity, Bill Cosby is helping to create a truly united America." In a court deposition unsealed this week, Cosby admitted to getting Quaaludes to give to women he wanted to sleep with, and dozens of women have alleged he assaulted them, going back five decades. The medal is symbolic and carries no benefits, so it's unclear how it would be revoked. "There's a huge amount of uncertainty here," said Kenneth Mayer, a presidential scholar at the University of Wisconsin-Madison. Though he said it's probably within the president's power to disavow the honor, getting the physical medal back might be another matter now that Cosby owns it. Revoking Cosby's medal would create a precedent that could have repercussions for future presidents and honorees, so it's not something the White House would do lightly, Mayer said. "Even if there's a legal ability to do this, it's not a road that people want to start going down," he said. "There are titanic issues of presidential power that have arisen by what seemed at the time like trivial circumstances." Follow @gregorykorte on Twitter Read or Share this story: http://usat.ly/1IJZL3L Bill Cosby is facing even more backlash following the revelation that the comedian admitted to obtaining Quaaludes for young women he wanted to have sex with. An ET source confirms that Walt Disney World's Hollywood Studios theme park near Orlando, Florida removed a statue of The Cosby Show star after the park closed on Tuesday night. The statue's removal comes after 2005 court documents were unsealed on Monday. This follows sexual misconduct allegations made by more than two dozen women that span several decades. Many of the alleged victims contend that Cosby drugged them before raping or assaulting them, though the comedian has maintained his innocence. WATCH: EXCLUSIVE -- Janice Dickinson Reacts to Bill Cosby's Quaaludes Admission Two more networks have also ceased airing reruns of Cosby's sitcoms. Bounce announced that "effective immediately," it will not broadcast Cosby, the comedian's CBS show from 1996 to 2000. AdWeek also reports that BET's channel Centric will no longer air episodes of The Cosby Show "until further notice." NEWS: Judd Apatow, Jill Scott React to New Bill Cosby Development But not everyone is distancing themselves from the 77-year-old comedian. The Smithsonian's National Museum of African Art is standing behind their exhibition that includes, in part, Cosby and his wife Camille's art collection. On Tuesday, the museum said in a statement to The Associated Press that it's "aware of the recent revelations about Bill Cosby's behavior," and "in no way condones this behavior." That being said, the Smithsonian says the exhibit itself -- which debuted in November -- is about the artwork and the artists and not about the owner of the pieces. NEWS: Whoopi Goldberg Defends Bill Cosby After Quaaludes Admission On Tuesday, the Los Angeles Police Department told ET that they are conducting at least one current criminal investigation into allegations of sexual assault against Cosby. They will also look into any other assault claims made against the comedian, including those past the statute of limitations. Bill Cosby was quietly dumped by CAA, which represented him since 2012, and he is now without talent representation in Hollywood. Cosby, accused of drugging and raping more than two dozen women, was dropped by the talent agency late last year per insiders at the agency – long before the latest revelation surfaced that he’d admitted in a deposition to having obtained Quaaludes with the intent of giving them to young women he wanted to have sex with. “We do not represent him at this time,” a CAA official told Deadline. Cosby is still listed as a CAA client on IMDBpro.com and he was on the list of clients in play when a dozen agents from the comedy department defected to UTA (though he was already in the midst of scandal and was probably the only one not being fought over at the time). Cosby made a splash when he moved to CAA in 2012 after being represented by the William Morris Agency for 48 years. When NBC’s The Cosby Show was at the peak of popularity, he was probably the most important client in the WMA fold, because of all the money that agency made from packaging fees. That is a distant memory, and at present, the only one sticking by him is David Brokaw, his longtime publicist who is the son of Norman Brokaw, the former WMA chairman who was Cosby’s longtime rep. David Brokaw declined comment here. Unless Cosby can somehow prove that the allegations are false, it seems unlikely he will need an agent for he’ll probably never work in this town again. As he turns 78 on Sunday, the latest development in his stunning fall from grace is Disney’s decision to remove a bronze statue from Walt Disney World’s Hollywood Studios in Orlando, Florida. Cosby isn’t the first star to be dumped by his agent after becoming toxic in Hollywood. OJ Simpson was dumped by ICM, which had represented him for 20 years, shortly after he was acquitted of murdering his ex-wife, Nicole Brown Simpson, and Ron Goldman. And Mel Gibson was dropped by WME five years ago after a secretly taped conversation was made public, in which he made racist comments. Today in news that is only surprising because it hadn’t happened already: Deadline is reporting that Bill Cosby has been dropped by his agent, leaving him “without talent representation in Hollywood.” This comes only a day after Bounce TV (finally) pulled its Cosby reruns and Disney World (finally) took down its Bill Cosby bust, both of which are things that also should’ve happened already. Oh, and this is only two days after unsealed court documents revealed that Cosby admitted to drugging multiple women so that he could have sex with them. So, yeah, he’s probably having a fun week of covering his ears and pretending that all of this isn’t going on. Now, Cosby losing his agent will obviously put a huge damper on the future of his acting career, but we can’t imagine it’s any more than the damage that admitting he drugged women so he could have sex with them has already caused. Or, you know, the over 40 accusations from women who claim to have been raped by him. Basically, Cosby’s talent agency dropping him was probably just a formality at this point, like the way you might throw away a burned-out lightbulb, an empty bottle of ketchup, or a formerly beloved comedian who turned out to be a huge creep. Send your Newswire tips to [email protected] PHILADELPHIA (AP) — Here are the latest developments from the release of court documents indicating Bill Cosby admitted in 2005 that he obtained quaaludes with the intent of giving them to women with whom he wanted to have sex (all times local): FILE - In this April 24, 2015 file photo, Whoopi Goldberg attends Variety's Power of Women Luncheon at Cipriani Midtown, in New York. As a chorus of sexual assault accusations against Bill Cosby resounded... (Associated Press) ___ 5 p.m. Philadelphia's Mural Arts Program is considering removing a work featuring entertainer Bill Cosby, the latest fallout from allegations he drugged and sexually assaulted women. An organization spokeswoman said Wednesday "recent headlines" factored into its decision to move the mural much higher on a list of works up for decommissioning. The North Philadelphia mural celebrating Father's Day features Cosby in a trademark purple sweater between South African leader Nelson Mandela and Archbishop Desmond Tutu. It went up in 2008 but had already been considered for decommissioning because the wall under a train bridge where it's painted is in bad shape. The spokeswoman says no one's called to complain. In newly unsealed documents, Cosby is shown testifying in 2005 that he'd obtained quaaludes with the intent of giving them to women before sex. A judge unsealed excerpts from Cosby's deposition after a request by The Associated Press. ___ 2:25 p.m. Bill Cosby's first accuser says his full deposition in her sexual assault lawsuit should be released because he broke the confidentiality pledge sealing their court settlement. The sanctions motion Wednesday comes after a judge unsealed excerpts from Cosby's deposition this week in response to an Associated Press request. Former Temple University employee Andrea Constand's 2005 lawsuit accuses Cosby of drugging and molesting her at his home. Her lawyer argues that Cosby's camp has commented over the years and again this week on the case while Constand has been forced to remain silent. She says Cosby lawyer Patrick O'Connor said this past year: "If he's innocent and the relations were consensual — wow." O'Connor did not immediately return a message seeking comment. ___ 1:20 p.m. Whoopi Goldberg has emerged as Bill Cosby's most prominent public defender this week and she has an angry message for her critics: Back off! Goldberg says Wednesday on ABC's "The View" that she's gotten threats since she expressed support for Cosby on the show the previous day. Goldberg says she takes rape and sexual assault very seriously but also believes in the principle of innocent until proven guilty. She says: "He has not been taken to jail or tried on anything." She adds: "So back off me." When sexual assault allegations against Cosby resurfaced last winter, Goldberg said she would be reserving judgment on him. Documents unsealed Monday show Cosby admitted in 2005 to obtaining powerful sedatives with the intent of giving them to women he wanted to have sex with. ___ 12:55 p.m. A supermodel who accused Bill Cosby of drugging her during an audition for his 1980s sitcom says testimony excerpts unsealed this week give accusers a sense of peace and validation. Beverly Johnson says Wednesday on NBC's "Today" show that she knew that after so many women came forward and told their story that the truth would come to light. Cosby admitted in 2005 to obtaining quaaludes to give to young women he wanted to have sex with. The entertainer acknowledged giving the now-banned sedative to people, including a 19-year-old woman before they had sex in Las Vegas in 1976. Johnson says she managed to get Cosby to back off. She doesn't feel vindication but is encouraged by accusers speaking out. ___ 12:40 p.m. Los Angeles police say they are still investigating a model's allegations that Bill Cosby drugged and sexually abused her at the Playboy Mansion in 2008. Officer Jane Kim said Wednesday that its inquiry into model Chloe Goins' allegations against Cosby is ongoing. No further details were available about the case, which is the only one the department is actively investigating against the 77-year-old comedian. Police opened their investigation in January after Goins met with detectives at the department's downtown Los Angeles headquarters. Goins says Cosby drugged and accosted her in a bedroom of the mansion in August 2008. Cosby's attorney, however, released a statement denying his client was in Los Angeles at the time. An email message to Cosby's attorney wasn't immediately returned Wednesday. | Bill Cosby's acting career is incredibly unlikely to survive the news that he admitted drugging women before having sex with them, and it looks like his talent agency saw the writing on the wall months ago. CAA, which lured Cosby away from the William Morris agency in 2012, "quietly dumped" him late last year, leaving him with no representation in Hollywood, insiders tell Deadline. But with dozens of women accusing Cosby of sexual misconduct-and with the possible release of more Cosby testimony-him losing representation at this point is probably a formality, "like the way you might throw away a burned-out light bulb, an empty bottle of ketchup, or a formerly beloved comedian who turned out to be a huge creep," writes Sam Barsanti at the AV Club. Cosby stands to lose more than Hollywood representation: A sexual assault awareness group wants to see him stripped of the Presidential Medal of Freedom he was awarded by George W. Bush in 2002, USA Today reports. "Certainly it would be an unprecedented event to take this away," the founder of Promoting Awareness, Victim Empowerment says. "But it's also an unprecedented moment in our nation's history that such an iconic figure, a legend, be accused by 40 women of sexual violence." In Philadelphia, a Father's Day mural from 2008 featuring Bill Cosby was already on a list of works for decommissioning, and authorities tell the AP that "recent headlines" have moved it a lot further up the list. | multi_news |
Haploinsufficiency, wherein a single functional copy of a gene is insufficient to maintain normal function, is a major cause of dominant disease. Human disease studies have identified several hundred haploinsufficient (HI) genes. We have compiled a map of 1,079 haplosufficient (HS) genes by systematic identification of genes unambiguously and repeatedly compromised by copy number variation among 8,458 apparently healthy individuals and contrasted the genomic, evolutionary, functional, and network properties between these HS genes and known HI genes. We found that HI genes are typically longer and have more conserved coding sequences and promoters than HS genes. HI genes exhibit higher levels of expression during early development and greater tissue specificity. Moreover, within a probabilistic human functional interaction network HI genes have more interaction partners and greater network proximity to other known HI genes. We built a predictive model on the basis of these differences and annotated 12,443 genes with their predicted probability of being haploinsufficient. We validated these predictions of haploinsufficiency by demonstrating that genes with a high predicted probability of exhibiting haploinsufficiency are enriched among genes implicated in human dominant diseases and among genes causing abnormal phenotypes in heterozygous knockout mice. We have transformed these gene-based haploinsufficiency predictions into haploinsufficiency scores for genic deletions, which we demonstrate to better discriminate between pathogenic and benign deletions than consideration of the deletion size or numbers of genes deleted. These robust predictions of haploinsufficiency support clinical interpretation of novel loss-of-function variants and prioritization of variants and genes for follow-up studies. With array-based copy number detection and the current generation of sequencing technologies, our ability to discover genetic variation is running far ahead of our ability to interpret the functional impact of that variation. Several software tools exist for predicting the phenotypic impact of mutations that change the amino acid sequence of an encoded protein [1]. These tools are essentially proteomic and genomic, rather than genetic, in perspective; no distinction is made between mutations that are dominant or recessive in action. By contrast, there is a lack of tools that predict the phenotypic impact at the organismal level of unambiguous loss-of-function mutations of an encoded protein (e. g. truncating mutations and whole gene deletions). Not all loss-of-function mutations are deleterious, especially when heterozygous. It is generally considered that recessivity is the norm for diploid genomes [2]. Some loss-of-function mutations even confer selective advantages [3]. It is clear from resequenced exomes [4] and genomes [5] and CNV surveys [6] that every genome harbours tens of unambiguous loss-of-function mutations. A pressing clinical need for interpreting genetic variation is in distinguishing between pathogenic and benign copy number variants (CNVs) revealed by array-based profiling of patients [7]. With the current resolution of microarrays in clinical practice, these variants are typically large, rare deletions, often encompassing multiple genes. The most obvious pathogenic mechanism for heterozygous loss-of-function mutations (such as large rare deletions) is haploinsufficiency (HI), wherein a single functional copy of a gene is insufficient to maintain normal function. Only a few hundred genes have been reported haploinsufficient so far [8], [9]. Previous studies have shown that gene sets related to haploinsufficiency, such as genes implicated in dominant diseases and genes overlapped by CNVs, have biased evolutionary and functional properties [10]–[12]. However, such investigations often compare those gene sets to the genome average and have been descriptive rather than predictive in scope. We sought to explore further systematic biases in the properties of known HI genes, and to develop a predictive model to assess for each gene in the genome the probability that it exhibits haploinsufficiency with respect to the severe developmental disorders that are the mainstay of clinical genetic practice. As it is not known for most genes in the genome whether or not they exhibit haploinsufficiency, we maximized the power of this predictive approach by assembling a large training set of ‘haplosufficient’ (HS) genes that do not exhibit haploinsufficiency resulting in severe developmental anomalies. We reasoned that currently the most effective way of screening for HS genes is use robust CNV discovery to identify genes that are wholly or partially deleted among thousands of adults recruited as controls for genome-wide association studies. We take advantage of the fact that the impact of large deletions on coding sequence is more unambiguously interpretable than other types of genetic variation, such as point mutations or small insertion/deletions. We investigated how our gene-based predictions of haploinsufficiency might be used to discriminate between benign and pathogenic genic deletions. We considered that a natural way to score the probability of a deletion causing a haploinsufficiency phenotype is to generate a LOD (log-odds) score comparing the probability that none of the genes covered by the deletion will cause haploinsufficiency with the probability that at least one of the genes will cause haploinsufficiency, as shown schematically in Figure 7. This LOD score is calculated using the formula below: Higher LOD scores indicate deletions that are more likely to be pathogenic as a result of haploinsufficiency. Note that this score assumes that there is no statistical interaction between the genes. We then considered how these deletion-based haploinsufficiency scores might be used to assess whether a genic deletion observed in a patient might cause their disease. One way of framing probabilistically this intuitively simple question is to estimate the opposing probability, that the deletion is unrelated to the patient' s disease status. We can relate this to the probability of drawing an individual at random from a healthy control population with a deletion at least as pathogenic as the deletion in the patient. We can estimate this probability empirically as the proportion of healthy controls with a genic deletion having the same or greater haploinsufficiency score. To test this approach, and to avoid circular reasoning, we generated a set of gene haploinsufficiency predictions using a slightly smaller set of HS genes identified in a large subset of the GWAS controls. The performance of the HI predictions using the slightly smaller HS gene training data was very similar to that of the full predictive model described in the previous section, as assessed by ten-fold cross validation (Text S1). We then identified LOF deletions in the remainder (N = 2,322) of the GWAS controls [17], which had not been used to train the predictive model, and determined the distribution of the maximal deletion haploinsufficiency score (based on the new gene haploinsufficiency predictions) observed in each control individual. We investigated whether the distribution of maximal LOD scores is significantly different between European-American (E-A) and African-American (A-A) GWAS controls. We observed that there was not a significant difference in median haploinsufficiency scores in E-A and A-A populations (p = 0. 71, Mann-Whitney U test), although the E-A controls have a slightly longer tail of more pathogenic deletions (e. g. a higher proportion of E-A controls have deletions with LOD scores greater than 3, Table S4). The 50%, 90%, 95% and 99% percentiles of the distribution for maximal LOD score for E-A and A-A controls cohorts are listed in Table 1. We calculated a LOD score for each of 487 pathogenic de novo deletions submitted by clinical geneticists to the DECIPHER database [18]. We focused exclusively on deletions known to be de novo variants, as we infer that their pathogenicity has been ascribed primarily on the basis of their inheritance status. The distributions of maximal LOD scores in GWAS controls and LOD scores of pathogenic DECIPHER deletions are shown in Figure 7. The pathogenic deletions have strikingly significantly higher LOD scores than deletions observed in GWAS controls (p<1e-30, Mann-Whitney U test). We observed that for 92% of the pathogenic deletions there was a probability of less than 5% of drawing an individual at random from our control population with a genic deletion of equal or greater LOD score, and for 83% of pathogenic deletions there was a less than 1% probability. We computed ROC curves to compare three different approaches for discriminating between pathogenic deletions and deletions seen in controls: (i) our LOD scores, (ii) the length of the deletion, and (iii) the number of genes in the deletion (Figure 8). These ROC curves clearly show that the haploinsufficiency LOD score is the best metric for discriminating between pathogenic deletions in patients and deletions seen in controls. We provide a script and input files to calculate LOD scores and make comparisons with control data (Protocol S1). We investigated whether the gene-based probabilities of haploinsufficiency that we have generated are of general utility across different forms of genetic variation. If this is indeed the case then we should expect that genes harbouring loss-of-function substitutions or small indels in apparently healthy individuals should not have a high p (HI). We identified 349 genes as having LOF substitutions and indels in 12 recently sequenced exomes [4], of these, we could estimate p (HI) for 176 that were not also in the HS training set (and thus represent a fair set for independent comparisons). These genes are highly significantly enriched among genes with low probabilities of exhibiting haploinsufficiency (p = 1. 06e-20 when comparing to the genome, and p<1e-30 when comparing to known HI genes, Mann-Whitney U test). This result implies that there are not substantial differences between genes that tolerate whole gene deletions and those that tolerate smaller loss-of-function variants. Moreover, by studying the allele frequency spectrum apparent in a large gene-resequencing dataset that has been extensively studied for patterns of selective constraint [19]–[21] we observed that nonsynonymous variants in genes more likely to exhibit haploinsufficiency are highly significantly skewed towards rarer variants than nonsynonymous variants in genes less likely to exhibit haploinsufficiency, in both African Americans and European Americans (p = 2. 9e-7 and 4. 0e-3 respectively, one-sided Mann-Whitney U test, see also Figure S9) reflecting greater, on average, selective constraint on genes we predict to exhibit haploinsufficiency (Text S1). We have undertaken a systematic characterization of human haploinsufficient genes by contrasting them with a set of haplosufficient genes derived from non-pathogenic CNVs, developed a prediction model on the basis of the most significant differences, and assigned predicted probability of being haploinsufficient to more than half of the protein-coding genome. Our finding that functional interaction with known HI genes was the single most predictive property of HI genes probably reflects the modularity of the interaction network, suggesting certain pathways or biological processes, such as early development morphogenesis, being more sensitive to dosage changes than others. However, it is also possibly influenced by an ascertainment bias with which HI genes are discovered. The accuracy of our haploinsufficiency probabilties is limited by a number of factors, such as imperfect training data, although we have taken considerable steps to limit false positives, and missing data among predictor variables in our model. As network proximity to known HI genes is the single most predictive variable we eagerly await the construction of networks with increasing coverage and completeness, in the expectation that it should improve our prediction power. The gene coverage of our method could potentially be increased by using multiple imputation approaches to impute missing data [22]. To trial this approach, we imputed missing predictor variables using the predictive mean matching method, which allowed us to increase considerably the number of genes for which we could predict haploinsufficiency from 12,443 to 17,456 (Text S1, Table S5, Dataset S2). The resultant increase in size of training data also led to a slight improvement in prediction accuracy (AUC increased from 0. 81 to 0. 83, Figure S10), and we observed similar levels of enrichment of known dominant genes and mouse haploinsufficient genes pre- and post-imputation (Figure S11 and Figure S12), suggesting that multiple imputation is a reliable method to increase genome coverage. Another limitation of our method is the broad phenotypic outcome being predicted. Essentially, we are contrasting HI genes that cause a broad range of developmental disorders, with HS genes for which haploidy does not majorly impair an individual' s ability to participate as a control in a genome-wide association study. We note that this broad phenotype is nevertheless considerably more constrained than that being considered by prediction algorithms based on evolutionary conservation, which are essentially integrating any deleterious phenotype manifested among any of the environments encountered during millions of years of evolution, across all possible modes of inheritance and genetic backgrounds. Despite the breadth of the phenotype implicit within conservation-based predictions, this class of algorithms has been demonstrated many times to be of appreciable utility [19], [23]. To support clinical interpretation of deletions seen in patients, we have transformed our gene-based predictions of haploinsufficiency into haploinsufficiency scores for individual deletions. Currently, clinicians typically use the length of a deletion or the number of genes deleted to assess the pathogenicity of a deletion. We have shown that our pathogenicity scores represent a superior metric to these existing approaches for classifying pathogenic deletions. We believe that the most appropriate use of these deletion-based haploinsufficiency scores is to compare deletions seen in patients with those seen in controls, and that quantifying the fraction of control individuals with a deletion at least as pathogenic as that seen in a patient provides a rational basis to classify pathogenic deletions. This fraction represents the probability of observing such a deletion by chance and thus the probability that a deletion will have been misclassified as pathogenic. A clinician can therefore set a particular haploinsufficiency score threshold to define pathogenic deletions through considering the misclassification rate with which they are comfortable. We have provided the necessary software tools to allow these haploinsufficiency scores to be calculated for any genic deletion, and automated calculation of these LOD scores and comparison with control deletions will be integrated into the forthcoming release (v5) of the DECIPHER database (personal communication: Helen Firth, Nigel Carter, Manual Corpas), which is used by over 170 clinical centres worldwide to interpret chromosomal rearrangements seen in patients (the gene-based predictions (p (HI) ) are already available in the current release of DECIPHER, Figure S8). We only observed subtle differences in the distribution of haploinsufficiency scores seen in European-American and African-American populations (Table S4), which might reflect a higher fraction of deleterious alleles in populations with non-African ancestry. Further investigation of these differences is warranted to see whether the haploinsufficiency scores observed in a patient ought to be compared with controls from a matched population. It has recently been suggested that some developmental disorders result from the presence of two independent deletions in the same genome, the ‘two-hit’ hypothesis [24]. This hypothesis suggests a subtly different assessment of a patient' s CNV data is required, through considering the question: ‘is the SET of deletions observed in my patient causal of their disease’. Another way of viewing this important question is that it requires consideration of the genome-wide haploinsufficiency ‘burden’ rather than the haploinsufficiency scores of individual deletions. The probabilistic framework we have established for assessing pathogenicity of individual deletions naturally extends to this situation. Rather than combining the haploinsufficiency probabilities for individual genes within a deletion to calculate a haploinsufficiency score for that variant, we can combine the haploinsufficiency probabilities for all deleted genes in the genome to calculate a haploinsufficiency LOD score for that genome, and compare this genome-wide haploinsufficiency score with those observed in healthy controls to assess the probability of sampling a healthy individual with a genome with a haploinsufficiency burden at least as high as that in the patient. This approach also naturally extends to an assessment of the genome-wide haploinsufficiency burden from other classes of LOF mutation. One requirement of the haploinsufficiency score approach for assessing pathogenicity of individual deletions is that data quality between patients and controls is similar. If there are systematic differences between the sensitivity and specificity of CNV ascertainment in patients and controls then this may lead to biased comparisons of haploinsufficiency scores. This potential limitation is largely mitigated by an inherent focus on the largest deletions in a genome, which are typically long enough for many different technology platforms to have essentially complete sensitivity at very high specificities. In addition to the use of the deletion-based and genome-wide haploinsufficiency scores described above, we envisage that our gene-based predictions of haploinsufficiency have two additional applications: (i) prioritization of variants for follow-up studies and (ii) integration into association testing to increase power; and we consider each of these these in turn. First, our predictions of haploinsufficiency provide a rational basis for prioritizing heterozygous variants for follow-up genetic and/or functional studies. The burden of having to validate increasing numbers of benign variants is an appreciable barrier to the translation into clinical practice of genomic technologies of ever-increasing resolution. A method that accurately hones in on potential causal variants could alleviate this burden considerably. The prioritization of variants need not be restricted solely to unambiguous loss-of-function variants. Most rare functional variants in any given genome are heterozygous nonsynonymous substitutions, many of which result in a complete or partial loss-of-function of the encoded gene. We contend that the prediction power of popular methods for predicting the functional impact of nonsynonymous substitutions from structural information and evolutionary conservation, such as SIFT [25] and POLYPHEN [26], is limited by an inability to discern from cross-species alignments whether purifying selection at a given site is acting in a recessive, additive or dominant manner. Combining these genotype-oblivious predictions with our predictions of haploinsufficiency, should enable rational, genotype-aware prioritization of heterozygous nonsynonymous variants. The second application of these predictions is to integrate them directly into association testing. It has been suggested that weighting variants by their probability of having a functional impact should improve power in resequencing studies to detect functional units (e. g. genes, pathways) enriched for functional variants [27]. As noted above, most of the rare variants considered in these studies are only observed in the heterozygous state, thus, if functional, they have to be exerting a dominant or semidominant effect, and predictions of haploinsufficiency, because haploinsufficiency is a major mechanism underlying dominance, are a highly relevant weighting factor. The framework that we have developed that integrates functional, evolutionary and genomic properties of genes, could, by judicious selection of different training datasets be easily and broadly extended to include other classes of variant (e. g. duplications, gain of function mutations), different genetic models (e. g. recessive effects) and different, and potentially more specific, phenotypic outcomes (e. g. disease-specific). We compiled a set of CNVs from three genotyping datasets generated from Affymetrix 6. 0 platform, 210 unrelated HapMap individuals [28], 2,421 control individuals used in GWAS studies of bipolar and schizophrenia [17] and 6,000 individuals participating WTCCC2 as common controls, using Birdsuite [29]. All these CNVs were annotated against EnsEBML protein-coding gene annotation build 50 [30]. Genes with all transcripts satisfying one of the following criteria: deletion of over half of the coding sequence, deletion of the start codon, deletion of the first exon, deletion of splice-signal and deletion that causes frame-shift, were considered loss-of-function (Figure S1) and for the gene to be included as haplosufficient, such events are required to occur in at least two apparently healthy individuals. For continuous variables, the two-tailed Mann-Whitney U test was performed to assess if positive (haploinsufficient) and negative (haplosufficient) training data have the same median value for potential predictor variables. For two-class categorical features, Fisher' s exact tests were performed. Statistical tests were performed using R (http: //www. r-project. org). We assessed different potential sets of predictor variables for input into the predictive model using the following criteria: (i) they allow prediction for at least half the genes in the genome, (ii) the Spearman correlation between all pairs of predictor variables is less than 0. 3, (iii) they are drawn from different broad categories (genomic, evolutionary, functional and network) if possible, iv) achieve best performance in model assessment (see below). The sensitivity of the prediction was plotted against (1 - specificity) and the area under the ROC curve (AUC) [44] was used as quantitative measure of the performance of the model, where sensitivity =, and specificity =. The other measure used is the Matthews correlation coefficients (MCC) [45], defined as: To avoid over-fitting, the sensitivity and specificity were calculated using 10-fold cross-validation. To overcome the variability caused by random partition involved in 10-fold cross-validation, each such assessment was repeated 30 times and the mean values were reported. | Humans, like most complex organisms, have two copies of most genes in their genome, one from the mother and one from the father. This redundancy provides a back-up copy for most genes, should one copy be lost through mutation. For a minority of genes, one functional copy is not enough to sustain normal human function, and mutations causing the loss of function of one of the copies of such genes are a major cause of childhood developmental diseases. Over the past 20 years medical geneticists have identified over 300 such genes, but it is not known how many of the 22,000 genes in our genome may also be sensitive to gene loss. By comparing these ∼300 genes known to be sensitive to gene loss with over 1,000 genes where loss of a single copy does not result in disease, we have identified some key evolutionary and functional similarities between genes sensitive to loss of a single copy. We have used these similarities to predict for most genes in the genome, whether loss of a single copy is likely to result in disease. These predictions will help in the interpretation of mutations seen in patients. | lay_plos |
The functional effects of most amino acid replacements accumulated during molecular evolution are unknown, because most are not observed naturally and the possible combinations are too numerous. We created 168 single mutations in wild-type Escherichia coli isopropymalate dehydrogenase (IMDH) that match the differences found in wild-type Pseudomonas aeruginosa IMDH. 104 mutant enzymes performed similarly to E. coli wild-type IMDH, one was functionally enhanced, and 63 were functionally compromised. The transition from E. coli IMDH, or an ancestral form, to the functional wild-type P. aeruginosa IMDH requires extensive epistasis to ameliorate the combined effects of the deleterious mutations. This result stands in marked contrast with a basic assumption of molecular phylogenetics, that sites in sequences evolve independently of each other. Residues that affect function are scattered haphazardly throughout the IMDH structure. We screened for compensatory mutations at three sites, all of which lie near the active site and all of which are among the least active mutants. No compensatory mutations were found at two sites indicating that a single site may engage in compound epistatic interactions. One complete and three partial compensatory mutations of the third site are remote and lie in a different domain. This demonstrates that epistatic interactions can occur between distant (>20Å) sites. Phylogenetic analysis shows that incompatible mutations were fixed in different lineages. In a half century of molecular phylogenetics there never has been a systematic investigation of the functional and fitness effects of amino acid replacements in evolution. Experimental studies focus on those few mutations that change protein function [1]. Of the remaining thousands of replacements nothing is said – they may or may not be of functional consequence. Sequence analyses use statistical approaches to explore modes of evolution [2], [3]. Rarely does fitting alternative evolutionary models to observed data disallow alternative explanations. For example, to some [4]–[6] an elevated ratio of amino acid replacements to silent substitutions between species (dn/ds) suggests evidence for the action of positive selection. To others [7] it suggests relaxed selection against slightly deleterious amino acid replacements during population bottlenecks. Both interpretations are viable. Non-additive interactions among mutations (epistasis) are critical to protein structure and function [1], [8] and consequently to speciation [9], the evolution of sex [10], recombination [11], dominance [12], robustness [13] and human disease [14]. Non-additive interactions force sites to functionally co-vary during evolution [15], [16]. Computational methods that ignore phylogenetic structure [17]–[29] fail to distinguish between co-variation arising from functional causes and co-variation arising though shared common ancestry. The latter, an ineluctable product of shared history, is reflected in the bifurcating hierarchy of a phylogenetic tree (a tree must collapse to a star burst if no sites co-vary). Computational methods that account for phylogenetic structure [30]–[38] have identified sites likely to functionally co-evolve [32], [36]. The relative scarcity of such sites accords with the observation that most amino acid replacements occur at the surfaces of proteins where solvent exposed side chains are less likely to interact [39], [40]. On the other hand it may simply reflect a lack of statistical power in many, though not all [32], of computational methods used. An alternative approach identifies pathogenic missense mutations in one species that have no obvious detrimental effect in a related species [41], [42]. This approach does not detect deleterious mutations of minor phenotypic effect. Computationally derived predictions need empirical verification [1]. Gloor et al. [43] used site directed mutagenesis to confirm the epistasis predicted between co-evolving residues in yeast phosphoglycerate kinase. Experiments with yeast iso-2-cytochrome c [44] also identified epistatic interactions between sites. However, two other studies, one with game bird lysozymes [45], [46] and one with vertebrate p53 domains [47], failed to find any evidence of epistasis. Several other site directed mutagenesis studies identified epistatic interactions among positively selected replacements in TEM-1 β-lacatamase [48], vertebrate steroid receptors [49] and visual pigments [50] and in coral red fluorescent proteins [51]. In no case, however, have experiments been designed to explore the prevalence of epistasis in molecular evolution in general. Here, we explore the prevalence of epistasis in molecular evolution from the distribution of functional effects caused by individual mutations introduced to one sequence from a homologue in another species. We studied the leuB encoded β-isopropylmalate dehydrogenase (IMDH) because: 1) the enzyme has a conserved well defined role in leucine biosynthesis [52], [53]; 2) high resolution x-ray crystallography of divergent IMDHs (<35% identical) reveals a conserved protein fold [54]–[57]; sequence alignments show that divergent IMDHs rarely differ by more than a few insertions and deletions; and 4) the relationship between enzyme performance (kcat/Km. NAD) and fitness has been determined using Escherichia coli as a model system [58]. The IMDHs from two mesophyles, E. coli and P. aeruginosa, differ at 168 of 365 sites including six small indels (Figure S1) located in flexible loops external to the core structure. Conserved in fold and function, E. coli and P. aeruginosa IMDHs provide excellent material with which to investigate protein evolution arising through sequence divergence in the absence of major changes in structure and function. We constructed 168 site directed mutants of E. coli leuB (each with a single mutation from P. aeruginosa leuB) and then expressed and purified each enzyme and determined its kinetic parameters. The resulting distribution of enzyme performances (kcat/Km. NAD) is strongly skewed to the left and only a single outlier with increased performance lies on the right (Figure 1A). The error distribution, obtained by repeatedly assaying wild-type E. coli IMDH, is Gaussian,, (Figure 1B). This same distribution is expected of amino acid replacements that do not affect function. The 52 mutants with relative performances above E. coli wild-type form a half-Gaussian distribution,,, similar to the error distribution. This suggests mutations have no detectable effect on enzyme performance, 63 reduce it, and one increases it. Pair-wise t-tests (for unequal replication and unequal variances [59]) combine with a 5% false discovery rate [60] to identify 61 mutants of changed performance: 56 have decreased performance and 5 have increased performance, with only the single outlier having performance increased by more than 15%. The assumption that mutations act independently, and hence additively, leads to a predicted performance for P. aeruginosa IMDH that is clearly wrong. The sum of the individual mutational effects and the E. coli kcat/Km. NAD is negative:. This is a physical impossibility. The assumption that mutations act multiplicatively is also wrong. In simple transition state theory, where ΔG′ is the difference in free energy between the ground state and the transition state, R is the gas constant and T is °Kelvin [61]. The difference in free energy between the transition states of the mutant and wild-type enzymes is. With five mutants completely inactive the sum of the ΔΔG′ and is minus infinity and the predicted performance is. In fact P. aeruginosa IMDH has a,, slightly lower than that of E. coli IMDH which has a,. The inescapable conclusion is that amino acid replacements at many sites interact. IMDH evolution is characterized by rampant epistasis that remains cryptic until revealed by experiment. That only 64 of the 168 sites affect function certainly underestimates the number that interact epistatically. To understand this, consider two cases in which two sites are involved in a simple pair-wise interaction (Figure 2). In each case, only one of the two sites reduces function when an amino acid in one species is mutated to that found in another. Mutating the second site restores the ancestral functional state. Mutations at both sites may reduce function if three or more amino acid replacements arose during the course of evolution (Figure S2A) – although this is not guaranteed. In a simple network of pair-wise interactions only two of three sites might be identified (Figure S2B). We expect some of the remaining 104 sites to engage in epistatic interactions. Zuckerkandl [15] first proposed that amino acid replacements at one site in a protein might influence the acceptability of amino acid replacements at other sites. Fitch and Markowitz [16] suggested that as species diverge from a common ancestor their sets of variable sites also diverge to explore different regions of sequence space. Mutating a currently invariant site in one species by introducing an amino acid from a homologous protein in another species risks producing a loss-of-function mutant. The many functionally compromised mutants in this study amply confirm the insight of these early pioneers. Mutations affecting function (<80% wild-type performance) are scattered throughout the IMDH structure (Figure S3). Solvent accessibility, distance to the catalytic center (Asp 251), secondary structure and rate of amino acid replacement per site do not correlate significantly with performance for the mutants analyzed here. Only one mutation, F73L, likely affects catalysis by contacting a substrate directly (Figure S4). We screened for compensatory mutations of F73L, A94D and A284C, all of which lie near the active site and all of which are among the least active mutants. We combined each deleterious mutation with each of the 167 remaining mutations, expressed and purified each double mutant, and assayed their activities. Four mutations compensate the F73L mutation (Table 1). F120A, identified in the original screen because it produces a 50% increase in wild-type performance, now produces a 6-fold increase in performance so that the F73L, F120A double mutant, while not as active as the E. coli wild-type enzyme, is as active as the P. aeruginosa enzyme. Three other compensatory mutants, F132L C136I and I179V, do not individually affect wild-type performance. Performance is completely restored to E. coli wild-type levels in the F73L, I179V double mutant. Performance is partly restored in the F73L, F132L and F73L, C136I double mutants. No mutations were found to restore function to A94D and A284C. Our results are compatible with several types of interactions. Using E. coli IMDH performance as a standard suggests a simple pair-wise interaction between L73 and I179 because only L73F and I179V are fully compensatory. Using the lower P. aeruginosa IMDH performance as a standard suggests a high-order interaction with function compromised only when residues L73, F120, C136, I179 are combined. Replacing any one amino acid, L73F, F120A, C136I, or I179V destroys the 4-way interaction to restore full performance. That no mutation restores function to A94D and A284C demonstrates several compensatory mutations are essential; at least two sites (A and B) must each interact with each original mutation (X) to form a simple chain (A-X-B). Whereas previous experimental studies [41]–[45] assumed interacting residues would be in close physical contact, all compensatory mutations for F73L in the large domain lie more than 20 Å distant in the small domain, close to a hinge in the b-sheet on which the two domains swivel (Figure 3). This suggests a common mode of action, possibly related to repositioning the F73L-shifted nicotinamide ring for catalysis. Our experimental strategy, of moving replacements from one homologue into another and screening for compensatory mutations, is useful in that it provides a general means to identify interacting sites regardless of the mechanisms involved. The predicted fitness effects of most mutations are tiny. Previous work [56] established that wild-type E. coli IMDH lies on a fitness plateau (Figure 4A). In this limit of adaptation [62], increases in performance do not improve fitness and even large reductions in IMDH performance produce small fitness effects (Figure 4B). Indeed, 65% of the selection coefficients are predicted to be less than 10−5/generation. Selection during starvation growth with glucose as the sole limiting resource is far greater than in nature where leucine is both widely available and abundant [63]. Many mutations, including the one with increased performance, are likely selectively neutral, or very nearly so. The cryptic epistasis we revealed is consistent with two modes of neutral evolution: the covarion process [64] and the nearly neutral process [65]. In the covarion process, neutral and/or beneficial mutations are fixed in different lineages that, when brought together in the same protein, are deleterious (Figures 2, Figure S2). In the nearly neutral process successive slightly deleterious alleles are fixed by random genetic drift (particularly during population bottlenecks) until a compensatory mutation arises that, on restoring full activity, is fixed by positive selection (particularly after a population expands). The concave fitness function for E. coli IMDH (Figure S4), typical of dominance curves [12], provides the fitness plateau on which fitness could gradually drift downwards as slightly deleterious mutations sequentially fix before a beneficial compensatory mutation restores full activity. The two processes can be distinguished by determining the order in which mutations arise during the course of evolution. Phylogenetic analysis suggests that the most recent common ancestor (MRCA) of E. coli and P. aeruginosa had amino acids FAFVV at sites 73,120,132,136 and 179 (Figure 5). On the lineage leading to E. coli mutations V136C and V179I arose first (the order is indeterminate) before mutation A120F. Each mutation is compatible with F73. On the lineage leading to P. aeruginosa mutations F73L and F132V arose (the order is indeterminate) before mutation V132L and finally mutation V136I. The presence of V179 is expected to compensate the potentially deleterious interaction between L73 and F132 in the event that the F73L mutation arose first. Hence, the pattern of replacements supports the nearly neutral process because a potentially deleterious mutation never arose before a compensatory mutation in the same lineage. Our demonstration of rampant cryptic epistasis in IMDH is entirely in accord with a recent insightful analysis of protein evolution that invoked extensive epistasis to account for the retarded divergence seen in ancient proteins [66]. There the case was made for a rugged fitness landscape characterized by multidimensional sign epistasis that forces sites to be conserved for billions of years until the right combination of amino acids at other sites to allows them to evolve. Our failure to identify compensatory mutations for A94D and A284C is indicative of multidimensional sign epistasis. That a single replacement is sufficient to compensate the F73L mutation demonstrates that epistasis need not always be multidimensional, however. In an earlier study [67], a mutant library in which 52 natural amino acid replacements from 15 subtilisin orthologues had been recombined was screened for function. Sequence comparisons of the unscreened and the screened libraries suggested that almost all possible pair-wise combinations of amino acids can coexist and that functional co-dependencies are rare. These conclusions seemingly stand in contradiction to ours. The subtilisin experiment suggests that 7 of pairs compromise function for. In other words about a half percent of pair-wise interactions are deleterious. For E. coli IMDH, the probability, f, that introducing an amino acid from an orthologue has no effect on function is, where D is the number of residues that differ between the two sequences. For E. coli IMDH we have, and hence. The two-fold difference between the two estimates of p is small considering the differences between the enzymes and the experimental methods employed. The take-home lesson is that epistatic interactions may be rare individually, but their cumulative impact on evolution rapidly increases with divergence, D. The phenomenon is akin to the snowball effect describing the accumulation of Dobzhanski-Muller incompatibilities during speciation [68]. The simplest model of sequence evolution is a Poisson process in which each site accumulates mutations at a constant rate λ. The expected number of mutations accumulated at time t is simply λt and the variance in the number of substitutions at time t is also λt. This gives the Poisson molecular clock a characteristic variance to mean ratio of. However, sequence analyses show that the molecular clock is the over-dispersed with [69]–[75]. Various hypotheses have been proposed to explain this over-dispersion including episodic bursts of selection, increased rates of fixation of deleterious alleles during population bottlenecks, fluctuating neutral spaces and variable mutation rates [73], [76]–[84]. Cryptic epistasis, in causing constraints at sites to vary and hence substitution rates at sites to vary, undoubtedly contributes to over-dispersion in the molecular clock. Simulations show that ignoring changes in substitution rates (heterotachy) can induce systematic errors in phylogenetic reconstruction, including topological inaccuracies, long-branch biases and other effects [85]–[91]. Simulations also show that ignoring co-dependencies among sites causes the amount of evolution to be underestimated, particularly on branches deep in a tree [92]. The resulting impression of rapid ancient radiations with an indeterminate branching order makes identifying the origins of some taxonomic groups difficult [93], [94]. Taking explicit account of co-dependencies within data has been shown to aid phylogenetic inference [92], [95]. While recent advances accommodate temporal variability in substitution rates within sites [87], even going so far as to model pair-wise interactions between sites in close proximity using predefined statistical potentials calculated from structural data [88], general phylogenetic practice does not [89]. The extensive cryptic epistasis we have revealed suggests that the usual practice of ignoring co-dependencies among sites needs reconsidering. Ancestral sequence resurrection is a popular experimental approach to explore ancient phenotypes and adaptations [1]. Accurately inferred ancestral sequences are essential, otherwise there can be little confidence in the experimental results. Caution is warranted when interpreting functional patterns that mimic in silico reconstruction biases [96]. Current methods ignore functional co-dependencies among sites; the consequences for the accuracy of inferred ancestral sequences is largely unexplored. On the one hand, coupling between sites represents a loss of degrees of freedom (knowing the residue at one site allows inferences to be made about the residues at coupled sites) that leads to overconfidence in reconstructed trees [97]. This is particularly problematic if attempting to reconstruct ancestral sequences during a supposed rapid ancient radiation. On the other hand, the same loss of degrees of freedom means that fewer inferences are made, which should improve accuracy. Simulations suggest that the conditions producing phylogenetic uncertainty also make the ancestral state identical across plausible trees [98]. This helps make ancestral sequence reconstructions robust to phylogenetic uncertainty. Our dissection of epistatic interactions with site 73 shows that amino acid replacements accumulated during evolution can interact without affecting protein function. Nevertheless, cryptic epistasis may impact functional evolution. Reconstructing ancestral proteins on either side of an ancient functional change neglects epistatic interactions that earlier prevented the change and that later prevented the new function reverting [99] or changing in response to a new selective pressure. Such canalizing epistasis both retards functional evolution and thwarts attempts to engineer enzymes rationally [100]. Protein breeding experiments commonly use mutant libraries, generated either by recombining related sequences [101] or by allowing sequences to accumulate ‘neutral drift’ mutations [102], to circumvent the canalizing effects of cryptic epistasis. We speculate that the rampant cryptic epistasis, inferred by computational methods [66] and detected in experiments on IMDH, might be sufficiently extensive to resist functional changes on evolutionary time scales. Only when rare neutral mutations relieve its canalizing effects can new functions evolve. This model potentially explains why protein evolution is characterized by long periods of functional stasis punctuated by rapid functional shifts. E. coli K12 strains MG1655, JW5807 (Keio Collection) [103], MM294D and BL21-gold-DleuB: : kanr have been previously described [53], [58]. A derivative of E. coli strain BL21-gold (Stratagene) was constructed by P1 transduction [104] of the ΔleuB-leuC: : kanr construct from strain JW5807. LB medium was supplemented with 15 g/l Bacto agar for plates [104]. TALON Superflow metal affinity resin and TALON xTractor Buffer were purchased from Takara Bio USA (Madison, WI). Unless specified otherwise, chemicals were purchased from Sigma-Aldrich (St. Louis) and restriction enzymes were purchased from New England Biolabs (Ipswich, MA) and Fermentas (Canada). dl-threo-3-isopropylmalic acid was purchased from Wako Pure Chemical Industries (Japan). All mutants sequenced at the BioMedical Genomics Center, University of Minnesota. The leu operon, from mid leuA through leuC, was acquired by genomic PCR from MG1655. The genomic PCR product and pMML22KBA-KYVY [58] were digested with restriction enzymes RsrII and SphI. The vector and insert were ligated by quick ligation (Fermentas), to create pLeuB7. The construct was transformed by RbCl transformation [105] into MM294D and selected on LB/Amp (100 µg/ml), overnight at 37°C. The 5′-primer is designed with 15–20 bases, then the bases to be mutated, followed by a minimum of 12 bases at the 3′ end. The 3′-primer is complementary to the first 15–20 bases 5′-primer. Thus, the primers are staggered and only the 5′-primer encodes the mutations to be introduced. Cytosine residues in plasmid pLeuB7 were methylated by the CpG Methyltransferase M. SssI (New England Biolabs) according to the manufacture' s instructions [106]. Methylated DNA and a nonmethylated control were diluted 1∶25, and 2 µl transformed into E. coli strain MM294D by using the RbCl/CaCl2 method [105]. After transformation cells were plated on LB/ampicillin (100 µg/ml) and incubated overnight at 37°C. Restriction sites (Figure S5) and the 168 single mutants were introduced into wild-type E. coli leuB using the protocol in Table 2 [107]. Five microliters of each finished reaction was run on a 1% agarose gel to verify the PCR worked, and 2 µl was transformed [105] into MM294D, plated on LB/ampicillin (100 µg/ml) and incubated overnight at 37°C. The presence of mutations was confirmed by sequencing. Mutant enzymes with kinetic characteristics different from wild-type had their entire leuB gene re-sequenced to confirm that no other mutations had inadvertently been introduced. Double mutants incorporating F73L, A94D and A284C with other mutations were constructed by restriction digestion and ligation using strain MM294D as a host. F73L, A94D, A284C were restriction digested and inserts with L24V, S156E, and Y360A ligated to form parent vectors F73L, L24V, F73L, S156E, A94D, L24V, A94D, S156E, A284C, S156E, and A284C, Y360A. L24V removes the AflII site, S156E removes the BamHI site, and Y360A removes the SnaBI site. Parent vectors were then restriction digested and inserts, obtained from restriction digests of other single mutants, were ligated in. After transformation [105] cells were plated on LB/ampicillin (100 µg/ml) and incubated overnight at 37°C. Colonies were grown in LB/ampicillin (100 µg/ml) and the plasmids purified. Double mutants were identified by the presence of a restored AflII, BamHI or SnaBI restriction site. Those remaining mutations close to F73L, A94D and A284C that could not be introduced by restriction digestion and ligation were introduced by PCR mutagenesis and the entire gene sequenced. In all 694 mutants were constructed: 17 restriction sites were introduced into pLeuB7,170 single mutants were made (the exact position of one single amino acid deletion could not be reliably identified and so three mutates deleting residues 150,151 and 152, were constructed), and 3×169 double mutants were made. Mutant IMDHs were over-expressed from plasmids in a derivative of E. coli strain BL21-gold (Stratagene) formed by P1 transduction [104] of the ΔleuB-leuC: : kanr construct from strain JW5807. Transformed cells were grown overnight at 37°C in 5 mL of LB containing ampicillin (100 µg/ml) and 0. 2 mM IPTG. Following centrifugation, cells were resuspended in 1 mL of BD TALON xTractor Buffer (Becton-Dickenson). After 10 min rocking at room temperature, the sample was then centrifuged for 20 min at 11,200×g and the supernatant transferred to a TALON 2 mL disposable gravity column containing 2 mL of equilibrated BD TALON Superflow metal affinity resin. The protein was then eluted following the manufacture' s protocol with the exception that potassium salts were substituted for sodium salts. All enzymes were purified to homogeneity as judged using Coomassie stained SDS-PAGE gels. Double mutants were screened for compensatory mutations at 37°C in 25 mM MOPS, 100 mM KCl, 1 mM DTT, pH 7. 3 in the presence of fixed concentrations of 0. 2 mM dl-threo-3-isopropylmalic acid and 5 mM MgCl2 and 0. 1 mM NAD. The concentration of NAD lies far below the Kms of the single mutants (459±51 mM for F73L, 687±35 mM for A94D, 777±34 mM for A284C). With each mutant unsaturated the rate of the reaction is proportional to kcat/Km making improvements in performance readily detectable. Kinetics were performed at 37°C in 25 mM MOPS, 100 mM KCl, 1 mM DTT, pH 7. 3 in the presence of fixed concentrations of 0. 2 mM dl-threo-3-isopropylmalic acid and 5 mM MgCl2, and with concentrations of NAD varied from 1/4 to 10× the apparent Km. Reactions were initiated by adding 10 µL of mutant IMDH (diluted in 50 mM potassium phosphate, 300 mM KCl, 150 mM imidazole, 10 mM β-mercaptoethanol, pH 7. 0) to 1 ml of the reaction mix in a 1 cm semi-UV (methylacrylate) cuvette (Fisher Scientific). Reaction rates were determined spectrophotometrically by measuring the production of NADH at 340 nm using a molar extinction coefficient of 6220 M−1 cm−1, in a thermostated Cary 300 Bio with a 6×6 Peltier block (Varian). Inhibition constants were determined in the presence of varying fixed concentrations of reduced coenzyme. Kinetic parameters Vmax, Km and Vmax/Km were determined using nonlinear regression as implemented in JMP (SAS Institute). Maximum turnover rates,, were calculated with enzyme concentrations, [E], determined spectrophotometrically by Bradford assay [108] (Bio-Rad) using bovine IgG as the standard. Each single mutant was independently expressed, purified and kinetically characterized twice. A total of 537 amino acid sequences (downloaded from GenBank via the NCBI web site http: //www. ncbi. nlm. nih. gov/) were aligned using ClustalW software [109]. X-ray structures (IMDHs 1HEX, 1CNZ, 1CM7, IV53,1W0D, 1WPW, 1VLC, and 1A05) were downloaded from the PDB web site (http: //www. pdb. org/pdb/home/home. do) and superimposed using Swiss-Pdb Viewer software [110]. Superpositioned structures were used as a guide to adjust the alignments of highly divergent sequences. A bootstrapped neighbor joining tree was constructed with PHYLIP [111] using a JTT [112] substitution matrix with deep branches swapped and assessed by maximum likelihood. A consensus tree was generated with Mr. Bayes [113] based on a gamma distributed, mixed model of amino acid evolution. The MCMC was run for 75000 generations sampling every 50 generations with a burn-in of 500. Both trees produced similar results when ancestral sites were reconstructed by fastml [114]. With Bayesian posterior probabilities <0. 9 accounting for <15% of sites (mostly in flexible loops), the amino acid identities at most sites in the deduced sequence of the most recent common ancestor are reliably inferred. | Many bioinformatics and functional genomics predictions are derived from evolutionary patterns of amino acid replacement in protein sequence alignments. Most computational methods assume that replacements in one sequence will be tolerated in all related sequences. Here, we evaluate-by direct experiment-the functional impact of amino acid replacements accumulated during the course of evolution. Our initial results show that cryptic interactions among amino acid replacements are common and that most are deleterious. This result has implications not only for the evolution of function, recombination, sex, dominance, robustness, disease, and even speciation, but also for practical applications-in conservation biology (e. g. to decide which organisms to preserve) and in vaccine design (e. g. using consensus or reconstructed ancestral sequences). Analyzing one interaction in detail, we find that compensatory mutations need not lie in close proximity to the original mutation as generally supposed. This result suggests that unsuspected structure-function relationships can be revealed by analyzing patterns of site-to-site interactions among amino acid replacements in evolution. | lay_plos |
The tapeworm Taenia solium is the parasite responsible for neurocysticercosis, a neglected tropical disease of public health importance, thought to cause approximately 1/3 of epilepsy cases across endemic regions. The consumption of undercooked infected pork perpetuates the parasite’s life-cycle through the establishment of adult tapeworm infections in the community. Reducing the risk associated with pork consumption in the developing world is therefore a public health priority. The aim of this study was to estimate the risk of any one pork meal in western Kenya containing a potentially infective T. solium cysticercus at the point of consumption, an aspect of the parasite transmission that has not been estimated before. To estimate this, we used a quantitative food chain risk assessment model built in the @RISK add-on to Microsoft Excel. This model indicates that any one pork meal consumed in western Kenya has a 0. 006 (99% Uncertainty Interval (U. I). 0. 0002–0. 0164) probability of containing at least one viable T. solium cysticercus at the point of consumption and therefore being potentially infectious to humans. This equates to 22,282 (99% U. I. 622–64,134) potentially infective pork meals consumed in the course of one year within Busia District alone. This model indicates a high risk of T. solium infection associated with pork consumption in western Kenya and the work presented here can be built upon to investigate the efficacy of various mitigation strategies for this locality. The zoonotic tapeworm Taenia solium, has a two host life cycle, with humans as the definitive host, and pigs as an intermediate host. Humans are infected after consumption of viable cysticerci in under-cooked pork and harbour the adult tapeworm, a condition known as taeniosis. Gravid proglottids, containing thousands of infective eggs, detach from the adult tapeworm and are excreted in faeces in an intermittent fashion [1]. Ingestion of these eggs, by either pigs or humans, results in the larval stage penetrating the intestinal wall, moving through the lymph and blood vessels to encyst in muscle, eyes or the central nervous system (CNS) as cysticerci [2]. Infection of the central nervous system, neurocysticercosis (NCC), manifests predominately as epileptic seizures and is thought to be responsible for 29. 0% (95% C. I. 22. 9–35. 5%) of epilepsy cases across endemic regions [3]. Due to the highly clustered nature of the parasite the proportion of people with epilepsy (PWE) suffering from NCC will likely be highly variable both between and within individual countries as illustrated by data from Tanzania suggesting that between 7. 7% (95% C. I. 1–25) and 23% (95% C. I. 15–31) of epilepsy cases are NCC-associated depending on the geographical location [4]. Understanding the burden of NCC related epilepsy in individual countries requires data on the prevalence of both epilepsy and NCC within those countries, data which is currently lacking in many instances [3,5]. A recent meta-analysis estimated the overall prevalence of circulating T. solium antigens in humans of 7. 30% (95% CI [4. 23–12. 31]) for sub-Saharan Africa [6] and it has been estimated that 0. 95–3. 08 million people in sub-Saharan Africa may suffer from NCC-related epilepsy [7]. T. solium cysticercosis has been identified as an important disease predominately in Latin America [8], Asia [9] and across much of Africa [6,10] although the nature of global travel and migration puts individuals from all countries at risk of infection. This is highlighted by cases of NCC being diagnosed in the United States, predominately in immigrants from Latin America with histories indicating that infection was acquired from endemic areas [11–13]. People travelling from endemic areas harbouring T. solium taeniosis, can in turn expose many other people to T. solium eggs, who may develop NCC. A well-known example of this was the detection of NCC in members of an Orthodox Jewish community in NYC, the likely source of infection in these cases was believed to be domestic staff originating from endemic areas [14]. In 2012 T. solium was ranked by FAO as the most important parasitic food safety issue globally [15] and the WHO Foodborne Disease Epidemiology Reference Group (FERG) estimates that it is the foodborne parasite with highest global burden [16]. Better understanding the risk of transmission in the food chain is therefore a priority. The estimated global burden of cysticercosis has recently been revised, and the parasite is thought to be responsible for a global total of 2,788,426 (95% C. I. 2,137,613–3,606,582) disability adjusted life years (DALYs), annually [16]. The burden of this disease lies disproportionally on developing nations, and even more so those with large rural populations and in which treatment for NCC may be lacking; in Cameroon, for example, the burden was estimated to be 45,838 (95% C. I. 14,108–103,469), equating to 9 DALYs per 1000 person years [5]. It is unclear whether this is due to high NCC-associated mortality in Cameroon compared to that in other countries or represents an over-estimation by the authors. [17]. In Tanzania, it was recently estimated that 0. 7 DALYs are lost per 1000 person-years [4] in comparison with an estimate 0. 25 DALYs lost per 1000 person-years in Mexico where NCC patients are five times more likely to receive treatment [17]. In Nepal it is estimated that 0. 543 (95% C. I. 0. 207–1. 0543) DALYs are lost per 1000 person years [18]. In the majority of cases, the only NCC-associated sequela considered in DALY calculations to date has been epilepsy, while severe headache was also included in Mexico [4,5, 17,18]. Despite the inclusion of headaches in the Mexico study, other manifestations such as: headache, visual disturbances, other signs of increased intra-cranial pressure, cranial nerve palsy, gait abnormality, various focal neurological deficits, altered mental state and pyramidal (upper motor neuron damage) signs [19] have not yet been widely included in DALY calculations due to the lack of good estimates of the proportion of these specific manifestations attributable to NCC. All DALY calculations performed for NCC are therefore likely to be underestimations. The consumption of undercooked, infected pork is a major risk factor for acquiring taeniosis. Taeniosis, in turn represents an important public health hazard, with an adult T. solium carrier becoming a focus of infection for both porcine and human cysticercosis [20]. Indeed the consumption of pork [21] and the inability to recognise infected meat [22] are two risk factors which have been significantly associated with human cysticercosis. Other statistically significant risk factors identified in a systematic review from endemic zones (Africa, Latin America and Asia) were; insufficient latrines, history of taeniosis or proximity to carriers, being male and of increased age, lack of potable water, poor personal and house hygiene including washing hands by ‘dipping’, earthen floor, pig owning and/or the presence of infected pigs and low education [6]. Addressing porcine infection and reducing the volume of infected meat entering the food chain is therefore an important issue for public health practitioners in order to reduce the burden of this neglected tropical disease. A process of risk analysis, whereby risks are identified and described, qualitatively and/or quantitatively assessed and then communicated and mitigated, can achieve understanding of the current risks posed by pork consumption in developing countries. The principal of risk analysis allows scientific, justifiable and transparent decisions to be made regarding the risks associated with food products and is a key component of the Codex Alimentarius framework. Codex Alimentarius is a joint FAO/WHO Commission whose role is to protect consumer safety in its member states in such a way that trade can be conducted in an environment where consistent food safety standards are enforced for all countries, removing the potential for non-tariff barriers to trade [23]. A stochastic, quantitative risk assessment, as part of a risk analysis process, allows us to incorporate quantitative data and the uncertainty and variability that surrounds these data, in order to establish a quantitative estimate of risk and a probability interval around that estimate. The aim of the work presented here was to estimate the risk to humans of exposure to T. solium from pork currently entering the food chain in rural western Kenya. We specifically sought to estimate the probability of any one pork meal consumed in western Kenya containing at least one viable, and therefore potentially infective, T. solium cyst. To this end a stochastic risk assessment model with Monte Carlo simulation was built and informed by data gathered in the field in western Kenya and supplemented by data available in the literature Ethical approval for aspects of field data collection pertaining to humans was granted in March 2010 by the Kenya Medical Research Institute (KEMRI) Ethical Review Committee (SSC No. 1701) and all activities were undertaken in accordance with the approved protocols. Once entered into the study every participant was identified by a unique identifying number and never by name, hence ensuring anonymity of all data. Prior to any data collection each human participant in the study was required to sign, or mark with a thumb print, an informed consent document. This document, including the provision of a thumb print in place of a signature, and its administration by trained staff, was approved by the KEMRI Ethical Review Committee. The steps before signing the informed consent document involved ascertaining the appropriate language for communication, an explanation of the project, the sampling procedure and emphasising that participation was entirely voluntary. One copy of the completed form was retained by the project and one copy was provided to the participant. Ethical approval for sample collection from animals was granted by the Animal Welfare and Ethical Review Body (AWERB) at The Roslin Institute, University of Edinburgh (approval number AWA004 Bronsvoort). Sampling of privately owned domestic pigs presented to slaughter houses was carried out by trained veterinarians or animal health assistants after obtaining verbal informed consent from the owners of those pigs. Blood sampling from the cranial vena cava was undertaken according to the guidelines provided by the National Centre for the Replacement, Refinement and Reduction of Animals in Research (http: //www. nc3rs. org. uk/bloodsamplingmicrosite/page. asp? id=346). A stochastic risk assessment model was built to answer the following question: “What is the risk that any one pork meal consumed in western Kenya contains at least one viable cysticercus of Taenia solium? ” A stochastic risk assessment model using Monte Carlo simulation was built using the @Risk (Palisade, Newfield, NY, USA) add-on for Excel (Microsoft corp. USA) which can be found in supporting information S1 and is illustrated in Fig 1. The probability of any one pork meal being infective at the point of consumption was estimated using a decision tree method which comprehensively included 15 possible field situations, described as conditional probabilities) through which a pork meal could ‘move through’ the food chain from pig to plate. Where pigs are informally slaughtered the probability of the situation is defined as the distribution of infection (including no-infection) conditional on the pig being informally slaughtered. Where the pigs are slaughtered formally the probability of detection of infection at meat inspection is included. The 15 situations are defined as follows; Situation 1 = Pig is not detected at meat inspection |* pig being lightly infected | Pig is formally slaughtered Situation 2 = Pig is detected and condemned at meat inspection | lightly infected | Pig is formally slaughtered Situation 3 = Pig is not detected at meat inspection | moderately infected | Pig is formally slaughtered Situation 4 = Pig is detected and condemned at meat inspection | moderately infected | Pig is formally slaughtered Situation 5 = Pig is not detected at meat inspection | heavily infected |Pig is formally slaughtered Situation 6 = Pig is detected and condemned at meat inspection | heavily infected | Pig is formally slaughtered Situation 7 = Pig is not detected at meat inspection | very heavily infected |Pig is formally slaughtered Situation 8 = Pig is detected at meat inspection and condemned | very heavily infected | Pig is formally slaughtered Situation 9 = Pig is not detected at meat inspection | uninfected | Pig is formally slaughtered Situation 10 = Pig is detected and condemned at meat inspection (false positive) | uninfected | Pig is formally slaughtered Situation 11 = Pig is lightly infected | Pig is informally slaughtered Situation 12 = Pig is moderately infected | Pig is informally slaughtered Situation 13 = Pig is heavily infected | Pig is informally slaughtered Situation 14 = Pig is very heavily infected | Pig is informally slaughtered Situation 15 = Pig is uninfected | Pig is informally slaughtered * conditional on With the probability (Pr) of each situation being calculated as; Pr (situation x) = (Pr (slaughter status) *Pr (Infection status) *Pr (intensity of infection) *Pr (detection status at meat inspection) ) The risk of any one pork meal being potentially infective at consumption is expressed as; Pr (any one pork meal is infective at consumption) = ( (Pr (pork meal contains a cyst | Situation 1) *Pr (Situation 1) + Pr (pork meal contains a cyst | Situation 2) *Pr (Situation 2) + Pr (pork meal contains a cyst | Situation 3) *Pr (Situation 3) …… + Pr (pork meal contains a cyst | Situation 15) *Pr (Situation 15) ) *Pr (any one cyst is viable prior to cooking) ) * Pr (Meal undercooked) Briefly this illustrates that the overall probability that any one pork meal is infective at the point of consumption is calculated through the probability of a meal containing a cyst (viable or degraded) given any of the 15 situations described is multiplied by the probability of that situation. These calculations are repeated for each of the 15 situations. The sum of these probabilities is then multiplied by the probability that any one cyst in any meal is viable prior to cooking to give the probability of any one pork meal being infective prior to cooking. This probability is then multiplied again by the probability of a meal being undercooked in the western Kenya context to give an overall probability of any one pork meal being infective at the point of consumption. We used the ‘Auto’ function in @Risk, a function which runs sufficient iterations, to a maximum of 50,000, until all input parameters have converged, using the default settings of 3% tolerance and 95% confidence, i. e. when there is a 95% probability that the mean of the tested output is within +/-3% of its “true” expected value, based upon the accumulated data from the iterations already run. To estimate the total number of potentially infective pork meals consumed in the course of one year within Busia District the following equation was included in the model: Number of potentially infective pork meals consumed per annum (Busia County) = Pr (meal infective) * (number of pork meals consumed per year) Where: Number of pork meals consumed per year = (number of people consuming pork daily *365) + (number of people consuming pork weekly *52) + (number of people consuming pork monthly*12) + (number of people consuming pork yearly *1) + (number of people consuming pork on special occasions *0. 5) Where the number of people consuming pork (daily/weekly/monthly/yearly/special occasions) = population of Busia county (Model parameter P20) * proportion of population consuming pork (daily/weekly/monthly/yearly or on special occasions) (Model parameter P21-25). The parameters of each model input are described fully in Table 1. Beta-PERT distributions were used for the prevalence of porcine cysticercosis in the porcine population, the proportion of pigs slaughtered informally, the proportion of pigs suffering from infections of varying intensity and the proportion of pork meals eaten undercooked. Beta-PERT distributions have been recommended for providing a natural distribution from expert opinion of the minimum, maximum and most likely values of an input and are bound between 0 and 1 [24]. PERT stands for ‘Program Evaluation and Review Technique’ and was a distribution first used for assessing the development schedule and costs of the Polaris weapons system [25]. The distribution is unimodal, continuous and has two non-negative x-axis intercepts which make it suitable for the data being modelled in this study [26]. The distribution was determined using the ‘Beta-PERT’ function, method = ‘Vose’ in the package “Prevalence” [27] for R [28]. The Beta-PERT methodology allows one to parametrize a generalized Beta distribution based on expert opinion regarding a pessimistic estimate (minimum value), a most likely estimate (mode), and an optimistic estimate (maximum value). The maximum and minimum limits of the distributions were set using the 99. 9% confidence intervals from field data as we felt that ‘true life’ data from the field would be a more accurate reflection of reality than an ‘expert’ opinion. Uniform distributions were used for the probability of any one pork meal containing a cyst and the probability of any one cysticercus being viable reflecting the high degree of uncertainty surrounding these values. Each parameter in the model was informed either by field data or from the literature where field data were lacking. Literature used is referenced against the appropriate parameter in Table 1. The Kenyan specific field data were obtained from two complementary studies, both undertaken in the same area of western Kenya, which is representative of the Lake Victoria Basin ecosystem. The first study was a community based cross-sectional study of humans and their livestock from 416 randomly selected homesteads between July 2010 and July 2012, during which questionnaire data were collected on a wide range of homestead and individual level risk factors for zoonotic disease, including meat preparation [29]. The second study investigated the prevalence of T. solium cysticercosis in 343 pigs slaughtered at registered slaughter premises within the same study site[39]. This study used the HP10 Antigen-ELISA which detects circulating antigen from the parasite with an estimated sensitivity of 89. 5% (95% C. I. 82. 3–94. 2%) and specificity of 74% (95% C. I. 56. 6–87. 6%) [30]. Apparent prevalence of porcine cysticercosis was used to estimate the true prevalence after adjustment for an imperfect test using the ‘epi. prev’ function. This function uses apparent prevalence, test sensitivity and test specificity to estimate true prevalence. Confidence intervals for all other variables were determined using the ‘epi. conf’ function, which calculates the confidence interval for proportions using the method first proposed by Wilson [31]. Both functions are found in the package ‘EpiR’ [32] within the ‘R’ environment for statistical computing [33]. A sensitivity analysis was performed to determine the influence of the input parameters on the main output; the probability that pork meal contains at least one viable cysticercus at consumption. The sensitivity analysis was performed in two stages. Spearman rank order correlation coefficients (ρ values) were calculated and a tornado graph produced. This illustrates the relationship between each input and the output of interest, with ρ values near 0 illustrating the input has little effect on the output through to a value at -1 or +1 illustrating that the output is fully dependent on this input. Key inputs (those with ρ >0. 1) were then selected to include in an advanced sensitivity analysis. An advanced sensitivity analysis was then performed with 35simulations of 500 iterations, monitoring the effect of a range of nth percentiles (1%, 5%, 25%, 50%, 75%, 95%, 99%) of the probability distributions of each selected input on the mean of the outcome. A sensitivity tornado graph was plotted illustrating the effect of the key inputs on the mean of the output. After 5,600 iterations all parameters in the model had converged. The model predicted that under the current conditions, any one pork meal consumed (after cooking) in western Kenya has a probability of 0. 006 (99% Uncertainty Interval (U. I). 0. 0002–0. 0164) of containing at least one viable T. solium cysticercus, and therefore being potentially infective to humans (Fig 2). This equates to 22,282 (99% U. I. 622–64,134 potentially infective pork meals consumed in the course of one year within Busia District alone with a human population of 230,253 [34]. Meat inspection, as is currently practised in western Kenya is responsible, according to the model, for avoiding only 1,397 (99% U. I. 5–8,368) potentially infective meals a year. The probability of each of the 15 situations described within the model (e. g. Situation 1 = Pig is not detected at meat inspection | lightly infected | Pig is formally slaughtered etc) is reported in Table 2. A tornado graph illustrating the Spearman rank order correlation coefficients can be seen in Fig 3. The most influential input was the probability that any one cysticercus is viable (ρ = 0. 91), followed by the probability a pig is infected at formally slaughtered (ρ = 0. 16). The 5 inputs with (ρ >0. 1) were selected for inclusion in the advanced sensitivity analysis. This analysis illustrates the range of potential outputs which could be produced by this model based upon the cumulative probability of the selected input distributions. The analysis suggested that with all other parameters fixed, the probability that any one cysticercus is viable (prior to cooking) has the largest effect on the mean output, from a probability of a meal consumed containing a viable cysticercus of 0. 0002 when the input was fixed at the 1st percentile to a probability of 0. 011 when the input was fixed at the 99th percentile. The effect upon the outcome of fixing the most influential 5 input variables monitored in the sensitivity analysis at the 1st and 99th percentile can be found in Table 3 and expressed graphically in Fig 4. The full sensitivity analysis report can be found in supporting information S1. This stochastic risk model has enabled us to express, quantitatively, the risk that pork entering the food chain in western Kenya poses to consumers in terms of potential for infection with the zoonotic tapeworm T. solium. It allows us to better understand the risk of exposure to T. solium in this setting and provides a tool with which the impact and cost-effectiveness of potential mitigation strategies can be explored. We have attempted to build a simple model using transparent parameters and drawn from either our own field data or published literature. Pork consumed in western Kenya presents a risk to consumers of exposure to T. solium. With the current input parameters, there is 0. 006 (99% U. I. 0. 0002–0. 0167) probability that any one pork meal consumed in western Kenya is infected with a viable T. solium cysticercus, and is therefore potentially infectious to humans. This equates to approximately 22,000 potentially infectious meals being consumed in Busia district alone in any one year, among a human population of over 230,000. It must be noted however, that not each potentially infectious meal consumed will lead to a case of taeniosis. The probability of infection after exposure is not yet understood and may not be high, if we consider the generally low prevalence of T. solium taeniosis even in areas known to be endemic for porcine cysticercosis [35]. It is known that the risk of acquiring NCC is increased not only in those who have a history of an adult T. solium infection [36] but also in those living within the vicinity of a taeniosis case [13,20] or coming into contact with infective eggs through food prepared by a T. solium carrier who fails to adhere to good hygiene practices. The implication of this is that any one person acquiring an adult T. solium infection has the potential to expose many more people to the parasite, putting them at risk of NCC. Moreover, individuals with taeniosis provide a source of infective material that can be consumed by pigs, propagating the parasitic life-cycle. Consumption of infectious pork products therefore not only puts the consumer at risk for NCC, but also people around them, therefore it is imperative that the consumption of pork containing viable cysticerci is prevented. There are several strategies that have been suggested for the control of T. solium although evidence for their efficacy is still scarce [37]. Work is ongoing to investigate the cost-effectiveness of several of these strategies in reducing infection risk utilising the model described here. While the input parameters for this model were defined using the best data available at the time, it is important to be explicit about some of the assumptions made. One key assumption relates to the probability of any one meal containing a viable cyst within the different categories of infection intensity. It was assumed in this analysis that cysticerci are distributed evenly throughout the musculature of a pig and therefore the probability of any one meal containing a cyst was calculated by dividing the range of cyst numbers for each infection category by the average number of ‘meals’ that one pig can produce (in terms of kg of meat). The reality is more likely to reflect a more un-even distribution of cysts throughout a carcass, but the ability to model this was beyond the scope of the data used in this analysis. The proportion of pigs falling into each infection intensity category was based upon the results of a random selection of pigs from Zambia (Southern and Eastern Provinces) and we cannot be sure that this can be translated to this Kenyan population. We do know, however, that these pigs were randomly selected from a population of slaughter age pigs (1–5 years) in an endemic sub-Saharan country and we therefore have no reason to believe the infection intensity proportions should be different. The probability of any one cysticercus being viable (prior to cooking) was identified as being the most influential variable in this model through the sensitivity analysis and this is predominately due to the wide distribution used to parameterise the variable. The variable was informed by two studies which indicated a large range (1–100%) of viable cysts in carcasses. There is as yet no data which could be used to better inform this distribution and although dissection of larger numbers of pigs may help, differences between pigs in time of age at exposure or in host-parasite immune-response, mean it may be very difficult to produce a more precise estimate. The results of this model should therefore be considered with these assumptions in mind. A quantitative risk assessment such as that presented here provides a transparent and reproducible way of assessing the current state of risk from a food product. The presence of T. solium in the porcine population combined with the poor risk mitigation shown by the pork industry as presently structured in western Kenya poses a significant public health hazard and requires a concerted effort by policy makers and other stakeholders to address. | Taenia solium is a serious zoonotic helminth which is thought to be responsible for approximately 1/3rd of epilepsy cases in the developing world. The work presented in this paper aimed to understand what the risk is of acquiring T. solium taeniosis from pork slaughtered and consumed in western Kenya. In order to do this we built a stochastic risk assessment model to investigate the safety of pork reaching the consumer in terms of the risk of having viable T. solium cysts in any one portion of meat consumed. We found that pork represents a high risk product in this study area and therefore control strategies are urgently needed to reduce the public health risk posed by this product. | lay_plos |
Ribosomal RNA gene (rDNA) copy number variation modulates heterochromatin formation and influences the expression of a large fraction of the Drosophila ge-nome. This discovery, along with the link between rDNA, aging, and disease, high-lights the importance of understanding how natural rDNA copy number variation arises. Pursuing the relationship between rDNA expression and stability, we have discovered that increased dietary yeast concentration, emulating periods of dietary excess during life, results in somatic rDNA instability and copy number reduction. Modulation of Insulin/TOR signaling produces similar results, indicating a role for known nutrient sensing signaling pathways in this process. Furthermore, adults fed elevated dietary yeast concentrations produce offspring with fewer rDNA copies demonstrating that these effects also occur in the germline, and are transgenera-tionally heritable. This finding explains one source of natural rDNA copy number variation revealing a clear long-term consequence of diet. It is clear that an organism’s gene expression patterns are responsive to environmental input. Often, this influence is not limited to short-term regulatory changes, but can persist through multiple cell divisions and can, in some cases, be transmitted to offspring. Typically, such changes are identified as “epigenetic” and are thought to be mediated by a variety of chromatin modifications [1–8]. However, because genome stability, particularly of highly-repetitive (e. g., pentameric repeat) sequences or middle-repetitive transposable elements, is modified by silencing involving repressive histone modifications, “epigenetic” perturbations may have both direct and long-term consequences: the former caused by disruption of silencing leading to “epigenetic instability, ” and the latter by creating transmissible changes to chromosomes that themselves may affect gene regulation in subsequent generations. This consideration significantly adds to models of epigenetic inheritance that often overlook the ease with which histones and DNA methylation can be modified and the rapid rate at which they are turned over in non-dividing cells [9,10]. Recent [11–14] and previous [15] findings show epigenetic silencing is unstable even in non-dividing cells, making it a particularly difficult challenge to reconcile models of chromatin (e. g., histone) mediated epigenetic silencing with transgenerational (i. e., mitotic and/or meiotic) inheritance. 35S Ribosomal RNA gene (rDNA) transcription has been a powerful model for understanding the regulatory effects of chromatin modification because evidence suggests identical genes may adopt different stable activity states (expressed versus repressed) even when immediately juxtaposed [16–19]. Transcription from tandem repeated rDNA arrays accounts for approximately 50% of total transcription [20] and is regulated such that only a subset of the redundant copies are active in a given cell type, while the remainder are inactive and are accompanied by chromatin structure consistent with silencing [21,22]. Consequences of misregulation are severe, in part due to the tandem repeat of identical sequence. Mutations in silencing factors (e. g., gene products of the dcr-2, Su (var) 3-9, HP1/Su (var) 205, PARP, CTCF, and sir2/sirt loci) result in supernumerary mini- or micro-nucleoli and rDNA copy number reduction [23–27], possibly through increased frequency of intrachromosomal recombination resulting from the repair of transcription-induced damage [28,29]. The tendency for natural loss and the ability of some rDNA arrays to expand through unknown processes [30–32] contribute to striking variation in rDNA copy number in both wild and laboratory strains [33–36]. This variation, in turn, is a potent genetic modifier of a number of phenomena, including the regulation of ecologically- and metabolically-relevant gene networks, the stability of genome structure and heterochromatin silencing, stress responses, and potentially metabolic function [24,26,37–46]. The relationship between rDNA transcriptional activity and rDNA array stability suggests a non-epigenetic mechanism through which the environment might induce heritable and consequential changes in the genome through long-term (i. e., permanent) modulation of genetic variation and epigenetic stability. Although the change may bear the hallmarks of epigenetic regulation (i. e., inducible, heritable, consequential), it may not technically be considered epigenetic because it involves chromosome changes [47]. Nonetheless, because rDNA copy number modulates the stability of epigenetic silencing [24,26,43], the origin of rDNA copy number variation is a significant concern to studies of “hidden” regulatory variation, heterochromatin, and epigenetics. The aim of this study was to identify a natural, ecologically-common, and human health-relevant source of rDNA copy number variation. Expanding on previous work suggesting that an increase in rDNA expression may lead to its loss, we hypothesized that rDNA copy number changes can be induced by modulating diet. In support of our hypothesis, we found that flies raised on high dietary yeast concentration had increased supernumerary nucleoli as larvae, and somatic rDNA copy-number reductions as adults. Similar results were observed in flies expressing a hypermorphic insulin receptor allele, and were reproduced pharmacologically using human insulin in vitro, suggesting that diet-induced rDNA instability is mediated by known nutrient signaling pathways. Drugs that inhibit expression of the rDNA mitigated instability and loss, suggesting the effects were a consequence of expression. Furthermore, adult males fed high-yeast diets produced offspring with fewer Y-linked rDNA copies demonstrating that the effect was transgenerationally heritable. These findings identify diet as a potential source for rDNA variation observed in natural populations and suggest a mechanism through which environmental conditions might result in induced “transgenerational” genome changes. Dietary composition is an easily modified environmental variable in Drosophila laboratory studies and has been shown to influence a variety of complex phenotypes including rRNA transcription and ribosome biogenesis [48–51]; we therefore considered it as a potential source of “natural” rDNA instability and variability. In this study we used two experimental media based on those used in dietary restriction studies [52], consisting of a constant carbon source (5% sucrose) and altered concentrations of nutritional yeast (10%, and 30% w/v). Throughout the study we refer to these as SY10 and SY30 respectively (Sugar-Yeast-w/v). Larvae raised on SY30 developed to pupation approximately 9 hours earlier than their SY10 raised counterparts. Apart from this observation, there were no obvious differences in terms of body mass or survival to adulthood between the two media. To obviate secondary effects on culture conditions based on crowding, and to assure similarity in conditions as much as possible, we controlled density of eggs, larvae, and adults in vials by collecting eggs from petri dishes containing agar made from apple juice, suspending eggs in PBS, and pipetting identical volumes of eggs to SY10, SY30, or standard cornmeal medium. We fist confirmed that altered diet affected rRNA expression. We could discriminate accumulated mature rRNA products (18S, 28S, 5. 8S), for instance bound in ribosomes, from actively-transcribed pre-rRNAs (35S, or 45S in some organisms) by detecting the quantity of cDNA derived from the pre-processed 5’-most sequence of the 35S primary transcript containing the External Transcribed Spacer (ETS). The ETS is constitutively processed during maturation of the pre-rRNA 35S transcript and quickly degraded, and is therefore used to measure de novo rDNA expression [26,53,54]. We compared male flies of genotype yellow1 white67c23/Dp (1; Y) y+, P{w = RS5}10B (henceforth Y, 10B), upon which we have performed other studies of the ribosomal rDNA [24,41,55–58]. We detected an approximately 50% increase in pre-rRNA levels in populations of second instar larvae raised on SY30 compared to Standard media (Fig 1A), confirming the suitability of these media for this study. We addressed whether an increase in dietary yeast concentration during development would result in rDNA loss. In interphase cells, the rDNA is the genetic location of the cytogenetic Nucleolus Organizing Region (NOR), and thus the foundation of the nucleolus. Even single rRNA genes are capable of forming tiny supernumerary nucleoli [59]. Consequently, nucleolar morphology is sensitive to the overall size, activity level, and integrity of the rDNA arrays. In Drosophila, rDNA damage is readily observed in larval salivary glands. Damage and repair are thought to lead to extrachromosomal circles, which coalesce mini- or micro-nucleoli in non-dividing or post-mitotic cells, forming supernumerary nucleoli. Such “fragmentation” has been observed in flies mutant for “heterochromatin components” [23–26], where it is thought to stem from aberrant intrachromosomal recombination resulting in the formation of extrachromosomal rDNA circles [28,60]. We performed immunofluorescence on larval salivary gland cells with an anti-fibrillarin antibody to detect the nucleolar dense fibrillar component which, in Drosophila, typically forms a single focus containing both X-linked and Y-linked rDNA arrays. We observed an elevated frequency of multiple nucleoli in SY30-fed larvae compared to SY10-fed larvae (Fig 1B and 1C). Multiple nucleoli (defined as more than one discrete separate fibrillarin focus) were present in 40% ± 24% of the nuclei within single salivary glands dissected from SY30-fed larvae; in contrast multiple nucleoli were observed in 7% ± 6% of salivary gland nuclei from from SY10-fed larvae. The ranges are standard deviations of nucleolar instability rates from independently grown and dissected salivary glands, indicating that populations of genetically-identical individuals raised on different food sources show variation that can be discriminated by their averages. In dividing cells, acentric extrachromosomal rDNA circles are lost, effectively reducing rDNA copy number and shortening the rDNA array through development. To quantify rDNA loss stemming from diet-influenced extrachromosomal circle formation during development, we used real-time PCR to measure the rDNA abundance of flies raised as larvae on either SY10 or SY30. Relative copy number was quantified using genomic copy number of a tRNA gene as normalization resulting in a DNA-to-DNA proportion [55]. rDNA copy number differences were monitored by comparing the rDNA copy number in freshly eclosed F1 males to that of males taken from the “F0” parental stock (i. e., treated males compared to their fathers and uncles). Male adults were collected within hours of eclosion, thus any diet-influenced changes to rDNA copy number were the result of physiological effects initiated prior to metamorphosis. The rDNA copy number of flies raised on SY10 was indistinguishable from that of their sires, while those raised on SY30 exhibited an average copy number reduction of ~20% (Fig 1D). The standard deviation for these data are derived from independent biological replicate vials from sibling males. The standard deviation of the F0 (not presented on graph, but whose average is defined as 100%) is pooled into the SY10 and SY30 populations. It has been known that culture conditions affect the extent of position effect variegation (PEV), the somaclonal variation that occurs at new juxtapositions of euchromatin and heterochromatin. Specifically, richer or more abundant food leads to reduced heterochromatin-induced gene silencing and thus higher levels of variegating gene expression [61,62]. To address whether SY10 and SY30 alter the extent of PEV, we raised isogenic whitemottled-4 (wm4/Y, 10B) males on SY10 or SY30. The only source of white+ expression in this genotype is from the whitemottled-4 allele. If SY30 reduced silencing, we expected to see increased eye pigmentation. Three days after eclosion, we decapitated males and extracted pigment from their heads in acidified alcohol. The extent of the suppression of variegation, observed as increased pigment extraction (Fig 1E), accords well with the increased expression as a result of reducing rDNA copy number through molecular means [24,55] or from wild-caught strains [43,63]. The quantified loss of rDNA due to SY30 is within the range of natural Y-linked rDNA variation [35,55,64], and within the experimental range used to demonstrate heterochromatin changes and gene regulatory variability by us and others [24,64] on the Y, 10B chromosome specifically. Therefore, altered diet could in principle be responsible in entirety or in part for the natural rDNA copy number variance observed in natural populations isolated from the wild. In Saccharomyces, it is thought that rDNA damage and loss is a consequence of a collision between the replication fork and transcriptional machinery at the rDNA array [29]. This model accounts for the transcription-dependent nature of rDNA recombination as well as the relationship between rDNA stability, cell division, calorie-restriction, and aging [27,50,65]. Much less is known about the regulation of rDNA stability in multicellular organisms and, therefore, the precise mechanism underlying our observations is unknown. There is no known analogous system for specific loss in Drosophila (or any of the metazoa), however the unique retrotransposable elements in arthropods provides one possibility. It is possible that loss of rDNA in Drosophila is a direct consequence of activity, for instance derepression of the rDNA leading to mobilization of R1 or R2 retrotransposons, whose mobilization is known to cause single-stranded DNA breaks. This possibility makes two predictions that we tested. First, we investigated whether consumption of SY30 lead to pronounced derepression of R1 or R2, the retrotransposable elements resident in the Drosophila rDNA. We could not detect significant increases in expression of either when raised on SY10 or SY30 (Fig 2A). Second, we investigated whether R1- or R2-interrupted 35S rRNA genes were preferentially lost after growth on SY30. We observed decreased R1-interrupted and R2-interrupted rDNA copies, as well as loss of uninterrupted 35S rDNA copies, but neither R1 nor R2 reductions were strongly biased over decreases as a result of I-CreI-induced damage (Fig 2B) [55]. It should be noted that absolute numbers of R1- and R2- containing 35S genes are not possible to reliably calculate, nor are our “R1” and “no R1, ” or “R2” and “no R2, ” numbers necessarily additive, because these data are derived from different priming real-time PCR oligonucleotides, thus do not have the same kinetics of amplification [55]. Nonetheless, comparisons between conditions are valid, and in these cases we see no strong biased loss of R1 or R2 sequences (judged by either R1- and R2-containing or R1- and R2-lacking rRNA genes). We cannot specifically quantify rRNA genes with both insertions (although these likely represent a vast minority of rRNA genes [66,67]), or that are free from either retrotransposable element insertions (likely the vast majority). Together these data argue against an R1- or R2-related mechanism of chromosome damage and rDNA loss. Work by Cohen and colleagues additionally showed that repetitious gene arrays that do not house such retrotransposons show progressive loss through the lifespan of Drosophila [28], similar to the rDNA and similar to what we observe in SY30 conditions. However it requires extreme caution in interpreting these results. We know that SY30 causes about 20% reduction in total rDNA copy number, and the rate, extent, and cytological manifestation of this loss indicate that rDNA loss is not of individual rRNA genes. Instead the best inference is that large contiguous blocks of rRNA genes—containing 35S genes interrupted and uninterrupted by R1, R2, or both—are lost as groups. The arrangements of the inserted rDNA genes is thought to vary considerably [18], so recombination, damage-and-repair, or loss events that occur between non-juxtaposed rDNA genes may remove a considerable number of rRNA subtypes (uninterrupted, R1-interrupted, R2-interrupted, and double-interrupted) that themselves were not part of the mechanism of loss. If instability is a consequence of changes in nutrient availability, then we reasoned that modulating known nutrient signaling pathways should produce similar results. In Drosophila, as in Cænorhabditis elegans, mouse, and human, the insulin/insulin-like growth factor signaling (IIS) and TOR signaling networks mediate many cellular responses to nutrient availability, including ribosome biogenesis and rDNA expression [49,68,69]. We expressed a constitutively active form of the insulin receptor (InR. R418P) either generally in larvae (using a Ubiquitin promoter to drive GAL4 expression in all cells) or specifically in salivary glands (using the Sgs3 promoter to drive GAL4 expression in larval salivary glands). Because these cells do not undergo cytokinesis, rDNA removed from the chromosomal rDNA array (e. g., as extrachromosomal circles) would not be measurably lost using real-time PCR, thus we used the formation of supernumerary nucleoli as proxy for rDNA instability [23,25]. In both cases we observed elevated levels of multiple nucleoli (~41% and 28% respectively) (Fig 3A and 3B), indicating that activating the Insulin Receptor was sufficient to induce instability. Raising flies expressing constitutive InR. R418P on food laced with rapamycin mitigated the effect (Fig 3C), suggesting inhibition of rDNA transcription suppressed the nucleolar instability. Expression of an antimorphic allele (InR. K1409A) had no apparent effect (Fig 3D), indicating that either antimorphic receptor expression levels were insufficient to reduce rDNA expression and affect loss rate, or the basal level of nucleolar instability we observe is independent of IIS-mediated rDNA expression. In order to limit our view to acute cell-autonomous effects of nutrient signaling perturbation, we next opted to modulate the activity of these pathways pharmacologically in cultured larval salivary glands, a fully developed, post-mitotic tissue. In this way we could separate developmental defects (as a result of prolonged expression of hypermorphic or antimorphic alleles) from acute defects caused by altered cell physiology. We dissected larval salivary glands from flies expressing a Fibrillarin-RFP fusion protein [70] whose expression was controlled by a heat shock responsive hsp70 promoter. We did not induce expression with heat shock because sufficient nucleolar RFP was detectable without heat shock. Salivary glands were cultured in Drosophila cell/tissue culture medium for 22–24 hours in the presence (or absence) of recombinant human insulin. Treatment with insulin resulted in increased supernumerary nucleoli in live salivary glands (Fig 4A and 4B). Exposure to either actinomycin D or rapamycin (two drugs which block RNA Polymerase I activity, the former directly and the latter by inhibiting TOR) for two hours prior to insulin addition abrogated the multiple nucleolar morphology (Fig 4C and 4E). This pharmacological suppression of supernumerary nucleoli could not be reproduced if the drug was administered in the last two hours of insulin exposure (Fig 4D and 4F), supporting the interpretation that drug-induced stability of the nucleolus was a consequence of reduced rDNA transcription, rather than a reorganization of the nucleolus as a result of drug exposure. We confirmed that drug treatment reduced active rRNA expression by culturing eviscerated whole wild-type larvae in tissue culture medium for 24 hours in the presence of rapamycin or actinomycin D alone. Real-time PCR quantification of cDNAs of pre-processed rRNA junctions (as in Fig 1A) were reduced to 80% (+19. 8%/-15. 9%) and 69% (+13. 1%/-11%) (N = 10 larvae for each condition), respectively, compared to control larvae cultured without any drug. That nucleolar instability is enhanced by insulin and mitigated by rapamycin and actinomycin D suggests that the consequential effect on rDNA stability occurs downstream of the convergence of the activities of these pharmacological agents. Wild-caught Drosophila strains exhibit a wide variance in rDNA copy number [34,35]; the source of this variance, however, is unknown. For this variability to be explained by environmentally induced instability during the life history of these chromosomes, germline rDNA would have to be susceptible to environmental influence. To look for possible germline effects of diet, we used a genetic strategy to specifically measure copy number of Y-linked rDNA. We chose to focus our attention on the Y-linked array because it is preferentially active in males [43,71] and because of the ease with which the Y chromosome is manipulated genetically [55]. We genetically-isolated Y-linked rDNA arrays by crossing adult males to females bearing an rDNA-deficient compound X chromosome (C (1) DX) (Fig 5A). Female progeny of this cross were viable and carried the patroclinous Y-linked rDNA as their sole source of rRNA genes; any differences between rDNA in daughters were due to permanent germline changes to the chromosomes occurring in the fathers. When male flies were raised as larvae on either “Standard” cornmeal medium (“Standard” or “Std”), or SY10 or SY30 (Fig 5A, symbol (1) ) then moved to Standard food and outcrossed to C (1) DX virgin females, the progeny ( (2) ) had no detectable difference in the rDNA copy number (Fig 5B). Thus while the soma was undergoing diet-induced rDNA loss at this stage (Fig 1D), the germline was not susceptible to diet induced loss of rDNA; this was not unexpected because the germline cells are relatively quiescent in larvae. Conversely, the adult germline was found to be sensitive to dietary conditions. Adult males (Fig 5C, (3) ), raised from eggs on Standard medium, were collected 1–4 days after eclosion and allowed to mate with C (1) DX virgin females on Standard food for one day; the female progeny ( (4) ) of this cross served as “Control” (i. e., time-zero) datum for subsequent comparisons. The next day, the males were transferred to experimental conditions (Std, SY10, or SY30), with or without rapamycin, and were allowed to feed. Males ( (5) ) were crossed with fresh C (1) DX virgins after 20 days. In this way, we were able to sample the germline of the same group of males before and after treatment (Fig 5C). Female progeny (“Treated” in Fig 5C, (6) ) had no fewer Y-linked rDNA than their half-sisters if their fathers had spent 20 days eating Standard food (Fig 5D, compare “Control” and “Std”). In stark contrast, daughters of fathers who ate SY10 or SY30 for 20 days exhibited a reduced Y-rDNA copy number, which was different from daughters both of young fathers and fathers who had fed on Standard food for 20 days. rDNA copy number reduction was greater from SY30-fed fathers than from SY10-fed fathers. Thus the father’s germline underwent loss of rDNA in pre-meiotic mitotic (stem-cell) divisions during the 20 day period that they were exposed to SY10 or SY30, and the amount of loss was proportional to dietary largess. Loss was mitigated when 10 µM rapamycin was included in the SY10 and SY30 food. It has previously been confirmed that rapamycin concentrations up to 200 μM have no effect on Drosophila feeding rates [72] suggesting that the effects of rapamycin on germline rDNA loss are pharmacological in origin as opposed to behavioral. Spermatogenesis and germline stem cell proliferation in adults are regulated by both diet and nutrient sensing pathways [73,74]; it is likely that germline rDNA instability in response to increased dietary yeast concentration is a result of the modulation of these pathways, along with any subsequent changes in rDNA regulation. In order for a population to maintain a steady-state rDNA size, natural loss must be balanced by expansion. In Drosophila, rDNA magnification may serve this purpose, although magnification is not wide-spread and is only observed on some chromosomes under certain conditions [30–32,75]; the determinative characteristics of chromosomes that exhibit magnification remain unknown. To test for this sort of reversion of diet-induced rDNA loss, we established independent lines from SY30-fed males and kept them on Standard food as with any Drosophila strain (Fig 6A). This allowed us to monitor transgenerational rDNA copy number for reversion or continued instability. We tested pooled males from three such independent lines that had been kept for two generations on Standard food after being raised for one generation and 20 days as adult on SY10 or SY30 and found that lost rDNA remained lost. Data shown in Fig 6B (Lines 1,2, and 3) show standard error of the mean (S. E. M.) to report the average rDNA copy number of individuals line founded by single males. This observation is consistent with published findings (as well as our anecdotal observations) that while some engineered rDNA deletions lines exhibited moderate (~5%) expansion shortly after production [55], they have been otherwise stable, without selection, over many subsequent generations. Indeed, we tested one such line and found that in relation to the progenitor stock, changes—magnification or continued loss—had not occurred after six years on Standard food, corresponding to no fewer than sixty generations (Fig 6B, bb-183). Loss of rDNA was also achieved by passaging the Y, 10B in a mutant heterozygous for Su (var) 205, which encodes the Heterochromatin Protein 1 gene product. This mutation has been shown to cause rDNA instability [23] and rDNA loss [57], but when the Y, 10B “tempered” by the mutation (10Bt205) was returned to a genetic background wild-type for Su (var) 205, and passaged for a year (approximately 25 generations) the rDNA loss was not reverted (Fig 6C). These observations suggest that, just like any other polymorphism, rDNA deletions (naturally-occurring or otherwise) persist over multiple generations and that magnification, in contrast, is relatively rare. In this work we have established that ribosomal DNA (rDNA) copy number polymorphisms can be created by manipulating the diet of wild-type flies. By directly altering insulin-like signaling and phenocopying nucleolar instability in culture using recombinant insulin, we showed that normal IIS signaling can be a significant source of rDNA copy number variation in the soma. Diet-induced rDNA copy number changes occur in both the soma and germline. As a result, they are both permanent within an organism and are capable of being transmitted to subsequent generations, hence may act as a codex for dietary history of an individual or for a population. Dietary modulation can account for loss of rDNA, but some unknown factor must be responsible for establishing some limit to the loss. We do not know the mechanism for this maintenance, although it could be an as-yet unobserved intentional regulated processes that assures minimal rDNA copy number, or it could be by normal selective pressures exerted by the Minute or bobbed phenotypes that result from very low ribosome number. Alternatively, loss may be balanced by gain of rDNA through unequal sister chromatid exchange, gene conversion, re-replication, or cycles of excision, rolling-circle replication, and re-integration. Meiotic magnification and somatic pseudo-magnification at the rDNA have long been known in Drosophila, although the identification of a mechanism has eluded researchers for over 40 years [32,76]. Part of the asymptotic limit to loss may be the natural ecology of Drosophila, wherein older males (with greater loss, Fig 5) may be less likely to mate, produce fewer offspring, or produce an altered sex ratio; ecological experiments would be needed to address these possible contributions. The rDNA is the major site of nuclear energy utilization—transcription, processing, packaging, and export—and was known to be responsive to the energy status of the cell. This response by rDNA to diet, and its fortuitous cleavage by I-CreI, allowed us to identify rDNA copy number as a factor which stabilizes the genome. This observation is now confirmed in Drosophila [26,43] and similar hypotheses have been proposed for yeast rDNA [40,77]. However it seems unlikely that the rDNA is alone in this ability. Half of the genome of Drosophila is composed of interspersed or tandem repeats—the transposable elements, highly-repetitive DNAs, expressed repeat gene clusters—and these sequences may account for some of the remaining regulatory variation that as yet has been unmapped [63]. It will require the ability to alter and measure copy numbers of the other repeated DNAs of the genome to ascertain if complex or quantitative traits map to these large blocks of “junk. ” Our observation of diet-induced rDNA loss integrates with previous results which indicate that rDNA copy number polymorphisms account for a large fraction of Y-linked gene regulatory variation (termed “YRV”) [43,56,63,78], including the ability of heterochromatin to induce gene silencing (position effect variegation). Ecological phenotypic variation implied by gene expression differences may be quite significant in competitive, food-rich or food-scarce natural environments. Our observations may directly explain why food and culture conditions alter the extent of position effect variegation, and may further explain why chromosomes from different strains—natural isolates or mutant stocks—differ in their ability to suppress position effect variegation [43]. We believe that the diet- and IIS-induced rDNA instability we observe is a general, or at least common, feature of Y-linked rDNA because it has been measurable in males of many strains used in our work. For instance, we specifically tested two other Y chromosomes: a wild-type male from a laboratory Canton-S stock and a freshly wild-caught (“Texas-B”) male by backcrossing males from these strains to a y1; bw1; e1; eyR strain to genetically isolate the Y chromosome [63]. rDNA copy number of flies raised on SY10 or SY30 was compared and otherwise-isogenic males bearing the Canton-S exhibited a 38% decrease in rDNA copy number, while the Texas-B chromosome exhibited an 8% decrease. Thus, while diet-induced loss appears to be a common feature of Y-linked rDNA genes, there are likely other genetic factors that influence the rate or bounds of loss. Additionally, the two presumably-unrelated transgenic lines (the Y from the UAS-InR strain presented in Fig 3 and the Y from the Fibrillarin-RFP strain presented in Fig 4) both showed nucleolar instability under conditions with increased IIS signaling. The same phenomenon of rDNA loss was less clear in females, who appeared to exhibit small amounts of loss that was not statistically robust. Because the biology of X-linked rDNA arrays differs from that of the Y-linked arrays [71,79,80], and the consequence of X-X exchange at the rDNA is very different from that of X-Y exchange, we had no reason to believe that the phenomenon was related and pursued it no further. rDNA instability is observed in a number of eukaryotes and is associated with a variety of complex phenotypes including position effect variegation in Drosophila [24,43], replicative lifespan in yeast [65], plant size in flax [81], cancer progression in humans [82–84], and the aforementioned “hidden variation” of Y-linked Regulatory Variation. Our findings provide a mechanism for the influence of diet on all of these processes. Our findings are likely generally relevant to many organisms due to the conserved structure of ribosomal DNA arrays, the common copy number polymorphisms at that locus [34], and the common modes of rDNA regulation [85]. While we focused on diet, other processes that influence rRNA transcription (e. g. cell proliferation, DNA damage, determination and differentiation, stress, aging, temperature, etc. [86]) would presumably also affect rDNA stability via similar mechanisms, and thus, the rDNA may be a common mediator of induced and heritable effects. We do not expect that induced changes to the genome are limited to the rDNA, in fact satellite sequences show copy number polymorphisms that are only now being investigated [57,87]. In terms of epigenetic inheritance, it is unclear whether diet-induced rDNA copy number polymorphisms may act as an inducible and heritable modifying mutation that subsequently destabilizes epigenetic silencing. y1 w67c23/Dp (1; Y) y+, P{w = RS5}10B was described previously [55,88], and was used for all feeding experiments. Constitutively active insulin receptor expression was performed using the following stocks: w*; P{Ubi-GAL4}2/CyO, w1118; P{Sgs3-GAL4. PD}TP1, y1 w1118; P{UAS-InR. R418P}2, and y1 w1118; P{w[+mC] = UAS-InR. K1409A}2 [89], all of which were obtained from the Bloomington Drosophila Stock Center. w1118; P{w[+mC] = UAS-mRFP-Fib} was used for in vitro salivary gland culturing experiments to visualize nucleolar fibrillarin [70], and was a gift from Dr. Patrick DiMario. Expression was under control of the core hsp70 promoter on the pUAST backbone, which provided ample expression to visualize without a GAL4 driver. C (1) DX, y1 f 1 bb0 was used to genetically isolate Y chromosomes for real-time PCR analysis. The rDNA deletion strain used in Fig 2 and 6 for comparison was y1 w67c23/Dp (1; Y) y+, P{w = RS5}10B, Df (rDNA) bb-183 [55], and the 10Bt205 strain in Fig 6 is described in Aldrich and Maggert [57]. All crosses were performed at 25°C and 80% humidity. Unless otherwise noted in the experiment all stocks were maintained on standard cornmeal molasses medium. We used two experimental media for our feeding experiments. The first was based on SYA media used in dietary restriction studies [52] and contained 5% sucrose, 10% hydrolyzed yeast, 5% cornmeal (w/v), and 1% agar (w/v). These ingredients were boiled in deionized water and mixed until fully dissolved. Media was allowed to cool to 55°C before the addition of propionic acid and tegosept to 0. 3% each. We refer to this diet as SY10. SY30 was identical except it contained 30% yeast (w/v). One-third of the required yeast was added in increments during heating to allow easy dissolution. Standard (cornmeal) medium is 5% cornmeal (w/v), 3% yeast (w/v), 1% agar (w/v), 7% molasses (v/v), and supplemented with proprionic acid and tegosept as above. For experiments testing the effects of larval diet, we collected embryos overnight on apple juice agar plates covered with yeast paste. Embryos were washed off plates using 1X PBS and transferred to experimental media at a uniform density [90]. To measure adult germline effects, males raised on Standard medium were collected 1–4 days post eclosion and crossed in groups of five to C (1) DX/Y virgins on Standard medium. After 24 hours, the males were removed and placed on either Standard medium, or Experimental media with or without 10 μM Rapamycin (LKT Laboratories), while females were maintained on Standard medium and allowed to lay eggs. Progeny from these females served as the baseline for subsequent comparisons. Males were transferred to fresh experimental media every 3 days for 20 days, after which they were again crossed to virgin C (1) DX females on Standard medium. Additional adult males fed in the above manner were crossed to y1 w67c23 females to establish stocks for subsequent analysis. Expression of the rRNA faces three problems. First, it is very stable (by RNA standards), with a half-life of at least two days [91], so steady-state rRNA levels are insufficient to detect changes in rRNA transcription. Second, it is very abundant, accounting for approximately 50% of transcription and 80–90% of steady-state RNA [20], so small differences in loading result in large variance in apparent rRNA concentration. Additionally, selection of any mRNA as a comparison (“denominator” in relative-abundance calculations) introduces even more variance as the quantification of differences in rRNA is more sensitive than differences in mRNAs with lower abundance (i. e., a 10% difference of 1000 is easier to detect than a 10% difference of 10). Third, the a priori assumption that any mRNA may not change in conditions in which rRNA expression changes may not be valid. For these reasons, we measured active pre-rRNA by detecting cDNAs derived from the ETS-18S junction, using reverse transcription primer GGAGGACGAGAAAATTGACAGACGCTTTGAGACAAGCATATAA. This primer was designed to be complementary to the 18S at the junction of ETS and 18S, and have a novel sequence at the 5’ end for use in subsequent real time PCR. RNA and DNA were co-purified to satisfy the need for a stable (non-regulable) denominator for rRNA transcription levels. Total nucleic acids were purified from pools of either one hundred second instar larvae (for Fig 1A) or ten dissected and everted third instar larvae (for measuring effects of rapamycin and actinomycinD). Tissue was homogenized in a solution containing 50 mM EDTA, 100 mM Tris pH 7. 0, and 1% SDS. Homogenate was extracted twice in equal volumes phenol: chloroform: isoamyl alcohol (25: 24: 1, buffered with 1 M Tris pH 7. 0). Under these conditions, all nucleic acids partition to the aqueous phase [92], which was further extracted with chloroform followed by diethyl ether. Nucleic acids were precipitated with 0. 8 volumes propanol, washed with RNAse-free 70% ethanol, and resuspended in DNAse- and RNAse-free water. Reverse transcription was performed in 20 µL reactions with 2 µg nucleic acid, 2 units M-MuLV Reverse Transcriptase (New England Biolabs), 1X Reverse Transcriptase Buffer, 2 µM of each dNTP, and 4 µM primer for 1 hour at 42°C followed by 10 minutes at 90°C. Samples were diluted 1: 250 for subsequent real time PCR reactions, as described below and before [55,57]. Melt-curve analysis was used to assure single melt peaks, and reaction efficiencies were determined using LinRegPCR [93] (average efficiency for the tRNA gene was 92% and for the ETS-18S was 82%). Efficiency correction and fold changes were calculated as before [94]. For detection of retrotransposon R1 and R2 expression, RNA was extracted as previously described [95] from three pools of 20 male wandering third instar larvae. Reverse-Transcriptase reactions for R1, R2, and rho1 cDNAs were individually primed using CCAGCATACGTATGCTCGCTG (for R1), GGGAGTGATTGGAGTTGTTTCCG (for R2), or CTAGCGAATCGGGTGAATCCACTG (for rho1). Real-time PCR reactions were then performed as below using those same primers and additionally GGGACAGCTTAGTGCACTCTAC (for R1), CCCCGGAGTTGCTAATCTAACC (for R2), and GTGGAGCTGGCCTTGTGGG (for rho1). Negative controls for R1 and R2 used the rho1 cDNA template reaction and R1 or R2 real time primer pairs. Whole mount salivary gland immunofluorescence was performed as in (11). Glands were dissected from 3rd instar larvae in 1X PBS and were then transferred to PBT (PBS containing 0. 1% Triton X-100) and fixed in PBT containing 3. 7% formaldehyde and blocked for 1 hour in PBT supplemented with 1% BSA. Glands were washed and incubated overnight at 4°C with a mouse anti-Fibrillarin primary antibody (Abcam) diluted 1: 1000 in PNBT (PBT containing 1% BSA and 500 mM NaCl). Goat anti-mouse conjugated to rhodamine was used as a secondary antibody (1: 1000). DNA was counterstained with 1 ng/mL DAPI (MP Biomedicals). All images were obtained using a Zeiss Axioskop 2 epifluorescence microscope running AxioVision (v. 4. 6. 3. 0) with a 20X objective (numerical aperature = 0. 5). Sequential excitation was performed at 543 nm (for Rhodamine and RFP) and 405 nm (for DAPI). Larvae containing the homozygous P{w[+mC] = UAS-mRFP-Fib} were reared in bottles and aged to the early wandering stage of the third instar. Males were dissected in 1X PBS and salivary glands were moved to Schneider media supplemented with 50 mg/mL streptomycin, 50 mg/mL penicillin, and 10% heat-inactivated fetal bovine serum (Gibco). Five to ten glands per condition were co-cultured and treated with 5 µM human insulin (Sigma) for 22–24 hours, then stained with 1 ng/mL DAPI for one hour prior to visualization. For “pre-treatment, ” salivary glands were treated with 10 µM rapamycin or 0. 6 µM actinomycin D (Acros) for two hours prior to insulin addition. For “post-treatment, ” glands were treated with rapamycin or actinomycin D two hours prior to DAPI addition. Images were taken of all stained salivary gland lobes near the anterior end at 20X, post-processed for bright/contrast, and scored for nucleolar structure. A nucleus was determined to contain “multiple” nucleoli if more than one separate focus of fibrillarin fluorescence was discernible. Glands that had no or poor DAPI staining were assumed to be damaged by treatment and excluded from analysis. The entire dissection and culture experiment was performed eight times over the course of two weeks and all data pooled to calculate frequencies presented in Fig 4. Real-time PCR analysis was performed as described in [55]. DNA was extracted from newly eclosed adult male flies in pools of three or more and quantified using a NanoDrop ND-1000. Real-time PCR was performed with a StepOne Real-time PCR system and Power SYBR Green reagents (Applied Biosciences) using 4 ng DNA per reaction. 18S rDNA was amplified using primers AGCCTGAGAAACGGCTACCA and AGCTGGGAGTGGGTAATTTAC, while the endogenous loading control, tRNAK-CTT, was amplified using CTAGCTCAGTCGGTAGAGCATGA and CCAACGTGGGGCTCGAAC. Relative differences were calculated using the “ΔΔCT” method. Each data point presented consists of at least three independent biological samples quantified in triplicated or quadruplicated technical replicates. Error bars generally represent the standard deviation of biological replicates, as justified in the text [96]; error bars are generally asymmetric around the mean (“+” values are higher than “-”values”) because we calculate error on CT values prior to exponential transformation (2CT) for presentation. P-values were calculated from ΔΔCT values prior to exponential transformation (to get “fold” values) using StatPlus: mac v. 5. 8 (AnalystSoft) or Apple Numbers. | We show that altering the nutritional medium of Drosophila cultures, emulating dietary largess in the wild, increases expression of the high copy-number ribosomal RNA genes and results in rDNA instability and loss. The reduction in gene copy number occurs both in somatic and germ cells, such that altered copy numbers are transmitted to the next generation. Our findings have clear ecological and disease relevance. The reduction of supernumerary ribosomal RNA gene copy number has been previously shown to compromise both epigenetic and genomic stability and alter the expression of hundreds of genes, while rDNA instability itself is a key progression hallmark of some cancers. This study links diet and Insulin/Insulin-like Signaling modulation to changes in the genome and provides an accounting for natural copy number variation of rRNA genes. | lay_plos |
We previously reported that the G allele of rs3853839 at 3′untranslated region (UTR) of Toll-like receptor 7 (TLR7) was associated with elevated transcript expression and increased risk for systemic lupus erythematosus (SLE) in 9,274 Eastern Asians [P = 6. 5×10−10, odds ratio (OR) (95%CI) = 1. 27 (1. 17–1. 36) ]. Here, we conducted trans-ancestral fine-mapping in 13,339 subjects including European Americans, African Americans, and Amerindian/Hispanics and confirmed rs3853839 as the only variant within the TLR7-TLR8 region exhibiting consistent and independent association with SLE (Pmeta = 7. 5×10−11, OR = 1. 24 [1. 18–1. 34]). The risk G allele was associated with significantly increased levels of TLR7 mRNA and protein in peripheral blood mononuclear cells (PBMCs) and elevated luciferase activity of reporter gene in transfected cells. TLR7 3′UTR sequence bearing the non-risk C allele of rs3853839 matches a predicted binding site of microRNA-3148 (miR-3148), suggesting that this microRNA may regulate TLR7 expression. Indeed, miR-3148 levels were inversely correlated with TLR7 transcript levels in PBMCs from SLE patients and controls (R2 = 0. 255, P = 0. 001). Overexpression of miR-3148 in HEK-293 cells led to significant dose-dependent decrease in luciferase activity for construct driven by TLR7 3′UTR segment bearing the C allele (P = 0. 0003). Compared with the G-allele construct, the C-allele construct showed greater than two-fold reduction of luciferase activity in the presence of miR-3148. Reduced modulation by miR-3148 conferred slower degradation of the risk G-allele containing TLR7 transcripts, resulting in elevated levels of gene products. These data establish rs3853839 of TLR7 as a shared risk variant of SLE in 22,613 subjects of Asian, EA, AA, and Amerindian/Hispanic ancestries (Pmeta = 2. 0×10−19, OR = 1. 25 [1. 20–1. 32]), which confers allelic effect on transcript turnover via differential binding to the epigenetic factor miR-3148. Systemic lupus erythematosus (SLE [OMIM 152700]) is a complex and heterogeneous autoimmune disease with a strong genetic component that is modified by environmental exposures. Although the detailed etiopathogenesis of SLE remains unknown, excessive innate immune activation involving toll-like receptors (TLRs, particularly TLR7/8/9) and type I interferon (IFN) has been recognized as an important pathogenic mechanism in the disease [1]. Therapeutics targeting the TLR/IFN pathway are in development for the treatment of SLE, with ongoing clinical trials investigating monoclonal antibodies against IFN-α and inhibitors for TLR7/TLR9 (reviewed in [2]). Recent genome-wide association (GWA) and follow-up studies have revealed the association of a number of polymorphic variants in genes encoding components of the TLR/type I IFN pathway with susceptibility to SLE (reviewed in [3], [4]), providing insights at the molecular level to refine our understanding of this dysregulated pathway in the predisposition to SLE. Our previous study identified a single nucleotide polymorphism (SNP), rs3853839, in the 3′ UTR of an X-linked gene TLR7 to be associated with SLE in 4,334 cases and 4,940 controls of Eastern Asian descent [5], providing the first convincing evidence for the genetic contribution of TLR7 to human SLE. Individuals carrying the risk G allele exhibited increased TLR7 transcripts and a more robust IFN signature than non-risk C allele carriers [5]. In this study, by fine mapping the TLR7-TLR8 region, we confirmed that the previously reported functional SNP rs3853839, located within a predicted binding site of miR-3148, was most likely responsible for observed association with SLE in three populations of non-Asian ancestry. We demonstrated a differential miR-3148 modulation explaining the effect of allelic variation at rs3853839 on TLR7 expression. We conducted genotyping and imputation for genetic variants covering ∼80 kb of the TLR7-TLR8 region on Xp22. 2. After applying quality control measures, 41 genotyped SNPs and 57–75 imputed SNPs/INDELs (insertion-deletion) (varying among different ancestries) were assessed for association with SLE in unrelated cases and healthy controls of European American (EA, 3,936 cases vs. 3,491 controls), African American (AA, 1,679 vs. 1,934) and Hispanic enriched for the Amerindian-European admixture (HS, 1,492 vs. 807) descent (Figure 1A). The strongest association signal was consistently detected at rs3853839 in the three ancestries, including EA (minor allele frequency of 20. 3% in cases vs. 17. 2% in controls, P = 6. 5×10−6, OR [95%CI] = 1. 23 [1. 13–1. 35]), AA (19. 8% vs. 16. 7%, P = 1. 1×10−3, OR = 1. 24 [1. 09–1. 41]) and HS (44. 8% vs. 37. 3%, P = 7. 5×10−4, OR = 1. 26 [1. 10–1. 43]) (Figure 1B, Table 1). After Bonferroni correction for multiple comparisons, the association of rs3853839 with SLE remained significant in EA and HS, and reached a nominal significance in AA. Combining the EA, AA and HS datasets, the meta-analysis P value of rs3853839 (Pmeta = 7. 5×10−11, OR = 1. 24 [1. 18–1. 34]) exceeded the commonly used threshold of 5×10−8 for genome-wide significance (Figure 1C, Table 1). Thus, the association of rs3853839 with SLE previously identified in Eastern Asians was confirmed in three non-Asian ancestries. Only six other SNPs within a relatively small interval of 5 kb spanning from TLR7 3′downstream to TLR8 intron 1 were consistently associated with SLE (P<0. 05) in EA, AA and HS (Table S1), and remained significant trans-ancestral meta-analysis P values after Bonferroni correction (5. 5×10−6≤Pmeta≤1. 3×10−6, Table S1). Linkage disequilibrium (LD) analysis revealed low LD strength between rs3853839 and these SNPs across non-Asian ancestries (r2<0. 26,0. 37, and 0. 51 in EA, AA and HS, respectively), but these 6 SNPs are in strong LD with each other and could be defined as a block (Figure S1). Among them, non-synonymous SNP rs3764880 (Met1Val) located at TLR8 exon1 exhibited the strongest association (Pmeta = 1. 3×10−6, OR = 1. 15; Table S1). To distinguish whether the associations of these 6 SNPs with SLE were independent of rs3853839, we performed conditional haplotype-based association test. After conditioning on rs3853839, association signals detected at these 6 loci were completely eliminated in EA, AA and HS (Figure S1). In contrast, conditioning on rs3764880, a consistent association signal was detected at rs3853839 in EA and HS (Figure S1), indicating that the association signals detected at these 6 SNPs might be attributed to that of rs3853839. Taken together, we confirmed rs3853839 as the only SNP in the TLR7-TLR8 region showing an independent association with SLE across all three non-Asian ancestries. A meta-analysis by combining all datasets of Asian and non-Asian ancestries showed compelling evidence of association with SLE at rs3853839 (Pmeta = 2. 0×10−19, OR = 1. 25 [1. 20–1. 32], Table 1). Given the location of TLR7 at X chromosome, we examined the allelic association of rs3853839 separately by gender. Of note, the sex-specific association of rs3853839 with SLE previously detected in Asian men [5] was not replicated in non-Asian ancestries (Table 1). Given the convincing evidence for the trans-ancestral association of rs3853839 with SLE susceptibility, we then evaluated its effect on regulation of TLR7/8 expression. Messenger RNA (mRNA) levels of TLR7 and the two alternative TLR8 isoforms were measured by real-time PCR in PBMCs from healthy EA individuals (n = 62). TLR7 mRNA levels were significantly different among women (n = 41) carrying different genotypes of rs3853839 [P = 0. 003, one-way analysis of variance (ANOVA) ], in which the GG and GC carriers exhibited notably increased TLR7 mRNA levels compared with the CC carriers [P = 0. 02 for GG (n = 5) vs. CC (n = 18) and 0. 02 for GC (n = 18) vs. CC, respectively, Student' s t test; Figure 2A) and the number of rs3853839 risk G allele was significantly correlated with increased TLR7 mRNA levels (R2 = 0. 26, P = 8×10−4, linear regression test). Consistently, male G allele carriers (n = 5) also had significantly higher TLR7 mRNA expression than male C allele carriers (n = 16) (P = 0. 01, Figure 2A). There was no significant association of rs3853839 genotypes with mRNA levels of two TLR8 isoforms in either women or men (Figure 2A). No sex differences in TLR7 or TLR8 mRNA levels were observed between individuals carrying the same genotype [GG women vs. G men: P = 0. 41 (TLR7), 0. 63 (TLR8a) and 0. 50 (TLR8b); CC women vs. C men: P = 0. 10 (TLR7), 0. 91 (TLR8a) and 0. 65 (TLR8b) ]. These results were in accordance with our previous observations in Chinese [5], supporting the importance of rs3853839 in regulating TLR7 rather than TLR8 gene expression. We assessed the intracellular expression of TLR7 and TLR8 proteins by flow cytometry in PBMCs from 7 pairs of healthy women (GG vs. CC) and men (G vs. C), respectively. Of the 7 pairs of individuals in each gender, 4 pairs were of EA descent and 3 pairs were Asians. Compared with C allele carriers, G allele carriers had significantly higher TLR7 protein levels in PBMCs (P = 0. 038 and 0. 009 in women and men, respectively; Figure 2B), especially in CD19+ B cells and CD14+ monocytes (Figure S2). No significant association between rs3853839 genotypes and TLR8 protein levels was observed in either total PBMCs or in specific cell subsets (Figure S3). We next performed luciferase reporter assays to further confirm the functional effect of rs3853839 on TLR7 expression. PCR-amplified TLR7 3′UTR fragments with either the G or C allele of rs3853839 were cloned downstream of an SV40 promoter-driven Renilla luciferase gene in the psiCHECK-2 vector, which also contained a firefly luciferase gene to serve as an internal transfection normalization control (Figure 2C). Constructs were then transiently transfected into either HEK-293 or differentiated HL-60 (dHL-60, neutrophil-like cells) cells. After 24 hours, cell lysates transfected with the G-allele construct showed significantly higher luciferase activity than those transfected with the C-allele construct in both HEK-293 and dHL-60 cells (P = 0. 026 and 0. 009, respectively; Figure 2C). Taken together, consistent results from ex vivo and in vitro studies indicated that the SLE-risk G allele of rs3853839 conferred elevated TLR7 expression at the both mRNA and protein level. To explore the mechanism of rs3853839 in regulating TLR7 mRNA turnover, we assessed allelic difference in TLR7 mRNA degradation by pyrosequencing. We first determined the rs3853839 G/C allele ratio in genomic DNA (gDNA) and cDNA from healthy EA women (n = 7) carrying the GC genotype. The mean G/C allele ratio in cDNA was significantly higher than the theoretical ratio of 1 as detected in gDNAs (P = 0. 02, Figure S4), indicating a higher expression of the G- than the C-allele containing TLR7 transcripts in heterozygous PBMCs. The allelic specific expression analysis in EA was similar to our previous findings in Chinese [5], and confirmed the result of real-time PCR that the G allele of rs3853839 is associated with increased TLR7 mRNA expression. Then, PBMCs were cultured in the absence or presence of the transcriptional inhibitor actinomycin D (ActD), and the G/C allele ratio in cDNA (normalized to that measured in gDNA) was determined after 0,2, 4,6, and 24 hours, respectively. As shown in Figure 2D and 2E, the G/C ratio in cDNA appeared to change over time when PBMCs were incubated with ActD and exhibited a statistical difference at the 4 hour point (P = 0. 04), implicating slower degradation of the G allele- than the C allele-containing TLR7 transcript in heterozygous PBMCs. The inhibitory effect of ActD on RNA synthesis was corroborated by a decrease in total TLR7 mRNA level at increasing time points after the addition of ActD in PBMC aliquots measured by real-time PCR (Figure S5). MicroRNAs (miRNAs) that bind to target sequences located within the 3′UTR of mRNAs by base pairing have been shown to result in accelerated mRNA turnover or translation repression [6]. Single nucleotide change either within or around the sequence of miRNA target sites can potentially alter the base-pairing patterns and affect miRNA-mediated regulation [7], [8]. The updated TargetScan database (Release 6. 2; http: //www. targetscan. org) indicates that rs3853839 is located within a binding site of miR-3148, where the non-risk allele (C), but not the risk allele (G), is predicted to match miR-3148 at the second position (Figure 3A). We hypothesized that the C to G variation of rs3853839 could reduce the binding and regulation incurred by miR-3148, therefore, leading to dysregulated TLR7 expression. We first showed that transcript levels of miR-3148 and TLR7 were inversely correlated in PBMCs from 16 patients with SLE and 21 healthy controls (R2 = 0. 255, P = 0. 001; Figure 3B), suggesting the possible regulation of TLR7 expression by miR-3148. Next, to verify whether allelic variation of rs3853839 affects the interaction of miR-3148 with TLR7 3′UTR, psiCHECK-2 vectors containing TLR7 3′UTR segment with either the C or G allele of rs3853839 were cotransfected with various doses of miR-3148 or nontarget control mimic into HEK-293 cells. As shown in Figure 3C, we observed significant dose-dependent miR-3148-mediated decrease in luciferase activity for the C-allele construct (P = 0. 0003 over all miR-3148-treated C-allele vector groups, ANOVA test), but not for the G-allele construct (P = 0. 14). Cotransfection with miR-3148 at a concentration of 6,12, and 48 nM, respectively, led to greater than two-fold reduction of luciferase activity in the C-allele than the G-allele construct [reduction in C-allele vs. G-allele construct: 13. 2% vs. 4. 8%, P = 0. 023 (6 nM); 22. 5% vs. 9. 9%, P = 0. 0012 (12 nM); 21. 4% vs. 8. 5%, P = 0. 0031 (48 nM) ]. These data supported the bioinformatic prediction that miR-3148 directly targets TLR7 3′UTR and the C to G variation of rs3853839 within the binding site alters the inhibitory effect of miR-3148 on modulating TLR7 expression. Fine-mapping of the TLR7-TLR8 region with high-density genetic markers based on large scale genotyping and imputation confirmed SNP rs3853839 at TLR7 3′UTR as the most likely causal variant responsible for the association of TLR7-TLR8 region with SLE in populations of EA, AA and HS ancestry. In accordance with our previous observation in Asians [5], we detected elevated TLR7 expression at both mRNA and protein levels in PBMCs from EA homozygous risk G allele carriers, as well as a higher level of the risk than the non-risk allele-containing TLR7 transcripts in EA heterozygous PBMCs. The fact that two distinct ancestries share the same genotype-phenotype association implicates an important regulatory effect of rs3853839 on TLR7 expression. Toward this end, we have extended functional studies showing slower degradation of the risk allele-containing TLR7 transcripts in heterozygous PBMCs and regulation of TLR7 expression by miRNA-3148 that targets 3′UTR at the position of rs3853839. Finally, we showed that the presence of the risk G allele resulted in reduced suppression by miRNA-3148, suggesting a likely mechanism for increased TLR7 expression in risk-allele carriers. The importance of TLR7 upregulation on mediating autoimmune responses has been addressed in murine models of SLE. The Y-linked autoimmune accelerator (Yaa) modifier, suggested mainly due to Tlr7 gene duplication, provides a prime example of TLR7 dysregulation leading to autoreactivity and inflammatory pathology [9]–[11]. Increasing Tlr7 gene dosage via generation of transgenic mice results in development of systemic autoimmunity, the severity of which directly correlates with the degree of Tlr7 overexpression [12]. Increased Tlr7 gene dosage promotes autoreactive lymphocytes activation, dendritic cells proliferation, and secretion of proinflammatory cytokines and IFN-α [12], which in turn upregulates TLR7 expression, leading to a feedback loop exacerbating autoimmunity [13]. In patients affected with SLE, up-regulated expression of TLR7 mRNA has been reported in PBMCs and B cells [14], [15]. Although a copy number variation (CNV) study in Mexican population showed increased TLR7 copies in childhood-onset SLE patients [16], no evidence for common CNVs at the TLR7-TLR8 region has been identified in individuals of diverse ancestries through our previous study by three independent methods including quantitative real-time PCR, PmeI pulsed-field gel electrophoresis and Southern blot [5], two recent studies using customized CGH platforms [17], [18] as well as other studies listed in the Database of Genomic Variants (http: //projects. tcag. ca/variation; the latest version released in November 2010), suggesting that mutations similar to Yaa are not a frequent feature of human SLE. The current study identifying genetic variations conferred by a regulatory SNP in TLR7 expression and SLE susceptibility suggests that murine models provide profound clues to human genetics if we look beyond the specific mutations identified in the relevant pathways. Unlike our findings in Asians that both sexes showed association [5], the impact of rs3853839 on risk for SLE was only observed for women in the non-Asian datasets (Table 1). Given the low prevalence of SLE in men, it is often challenging to collect a large enough number of affected men in a given population. Under the assumption that the associated G allele confers genetic risk with an odds ratio of 1. 26 in EA, 1. 22 in AA and 1. 29 in HS subjects (ORs were determined in female datasets), and considering P<0. 05 as the threshold of significance, the power estimate for female samples in each ancestry reaches more than 85%, whereas for male samples it is only 50% in EA, 19% in AA and 25% in HS dataset. Thus, there was clearly inadequate power to evaluate this association in AA and HS men. Despite a relatively robust sample size of EA men (344 SLE vs. 1,151 controls), a significantly higher G allele frequency was observed in male than female controls (20. 0% vs. 16. 5%, P = 0. 005), contributing to the difficulty in assessing association with SLE in EA male subjects. To our knowledge, the association of rs3853839 (or its tag SNP) with SLE has not been reported in four SLE GWA studies in European-derived populations [19]–[22] and three GWA studies in Asians [23]–[25]. According to the 1000 Genomes Project data, rs3853839 locates in a region with poor LD structure and cannot be tagged by any known SNP at the TLR7-TLR8 region with r2>0. 65. The SNP rs850632, located at TLR7 3′downstream, shows the strongest LD with rs3853839 in Europeans (r2 = 0. 38) and Asians (r2 = 0. 65). However, neither rs3853839 nor rs850632 has been included in predesigned commercial genotyping arrays of those GWA studies, resulting in the absence of associations. Even if rs3853839 was genotyped, the published GWA studies might have inadequate statistical power to capture its association in the initial discovery analyses [5]. Evidence of other TLR7 polymorphisms associated with SLE has been reported, including two intronic SNPs (rs179019 and rs179010) found in Japanese population [26] and an exonic SNP (rs179008) in individuals from Southern Brazil [27]. The reported associations were modest due to limited sample size of these studies (less than 400 cases and 450 controls), and none of them have been confirmed by the current fine-mapping study using a large collection of EA, AA and HS cases-controls (Table S1). TLR8 polymorphisms have been described in infectious diseases [28], [29] with a genetic effect localized to a functional variant at exon 1 (rs3764880, Met1Val). The G allele of rs3764880, which abolishes a putative start codon within the alternative TLR8 transcript isoform a (Figure 1A), conferred a protective effect on susceptibility to pulmonary tuberculosis in Indonesian and Russian men [28], as well as on HIV disease progression in Germans [29]. Our data showed a significantly increased frequency of rs3764880-G allele in SLE than healthy controls in the three non-Asian datasets; however, its association with SLE was dependent on that of TLR7 SNP rs3853839. Other variants at the TLR7-TLR8 region showed either weaker association than rs3853839 in trans-ancestral meta-analysis or association uniquely in EA or HS. Taken together, these data support rs3853839 as the most likely polymorphism associated with SLE shared by multiple ancestries. Although imputation facilitated our ability to capture common variants (MAF>1%), further refinement in genetic effects of rare variants (MAF<1%) is needed by deep sequencing of this locus, especially the intergenic region between TLR7 and TLR8 that was not well imputed in this study. Variations in 3′UTR regions may be important in gene regulation. To date, expression quantitative trait loci (eQTL) mapping has been widely used for characterization of SNPs that affect gene expression [30]. Although the TLR7 expression has been measured in previous whole-genome eQTL studies, currently only those using EBV-transformed lymphoblastoid cell lines of 1000 Genomes Project individuals provide publically available genotyping data of rs3853839. Based on the study by Stranger et al [31], we found that CG carriers of rs3853839 showed elevated TLR7 expression compared with CC carriers in YRI women (P = 0. 012). In male individuals, the G allele of rs3853839 showed a trend of association with elevated TLR7 expression in CHB+JPT, CEU and YRI men, and the association was significant when combining all male data (P = 0. 014). These findings are consistent with our results that rs3853839 alleles are associated with differential TLR7 expression. An important finding of this study is that the SLE-associated variant rs3853839 confers a genetic effect on modulation of TLR7 expression by an epigenetic factor miR-3148. Accumulating evidence suggests that miRNAs are fine tuners of TLR signaling pathways [32]. Regulation by miRNA may occur at various levels of TLR pathways by targeting adaptor molecules, downstream regulators and cytokines (reviewed in [32], [33]). However, few studies point to TLR themselves (e. g. TLR2 and TLR4) being directly targeted by miRNAs [34], [35]. Using algorithms from TargetScan, only the newly identified human miR-3148 [36], which is not evolutionarily conserved among mammals, is predicted to bind TLR7 3′UTR sequences at the position of rs3853839. The inverse correlation of miR-3148 and TLR7 levels in PBMCs, along with functional validation by reporter gene assay, confirms an inhibitory effect of miR-3148 on regulating TLR7 expression and allelic variation of rs3853839 affecting miRNA-mRNA interactions. Further study will focus on investigating miR-3148 expression patterns in specific immune cell types, assessing biological impacts of changes in miR3148-mediated TLR7 expression on downstream immune responses, and evaluating roles of other miRNAs that target sequences in the vicinity of rs3853839. Of interest, an unconventional role for miRNAs has been identified as endogenous activators for RNA-sensing receptors (TLR7/8) in a cell- or tissue-type specific manner [37], [38]. Therefore, miRNA regulation in TLR7 signaling is more complicated than we expected and further functional studies showing the exact effects of miRNAs on TLR7 responses are warranted. In summary, we have advanced our previous study by showing rs3853839 (at TLR7 3′UTR) as the most likely polymorphism responsible for the association of TLR7-TLR8 region with SLE in individuals of EA, AA and HS ancestry, and have characterized a differential miR-3148 modulation which explains the effect of allelic variation of rs3853839 on TLR7 expression. Our study highlights the importance of TLR7 as a shared genetic contributor to SLE in multiple ancestries, and provides evidence that microRNA acts as a negative regulator to control TLR7 expression, suggesting the possibility of miRNA-based therapies for amelioration of autoimmune diseases such as SLE where excessive TLR7 activation exists. Written informed consent was obtained from all study participants and each participating institution had Institutional Review Board (IRB) approval to recruit samples. The overall study was approved by the IRB of the Oklahoma Medical Research Foundation (OMRF). To test the association of TLR7-TLR8 with SLE, we used a large collection of case-control subjects from the collaborative Large Lupus Association Study 2 (LLAS2), including European American (4,248 cases vs. 3,818 controls), African American (1,724 cases vs. 2,024 controls), and Hispanic enriched for the Amerindian-European admixture (1,622 cases vs. 887 controls). African Americans included 286 Gullahs (155 cases vs. 131 controls), who are subjects with African ancestry. Cases were defined by meeting at least four of the 1997 American College of Rheumatology (ACR) revised criteria for the classification of SLE [39]. DNA samples were processed at the Lupus Genetics Studies Unit of OMRF. SNP genotyping was performed using an Illumina custom bead array on the iSCAN instrument for 47 SNPs covering the TLR7-TLR8 region on Xp22. 2 and 347 admixture informative markers (AIMs). SNPs meeting the following criteria were included in the association analysis: well-defined cluster scatter plots, SNP call rate >90%, minor allele frequency >1%, total proportion missing <5%, P>0. 05 for differential missing rate between cases and controls, and Hardy-Weinberg proportion (HWP) test with a P>0. 01 in controls and P>0. 0001 in cases. Subjects with genotype missing rate >10% (due to low quality), shared identical by descent >0. 4 or showing mismatch between the reported and estimated gender were removed. The global ancestry of each subject was estimated based on genotype of AIMs using principal components analysis [40] and ADMIXMAP [41], as described in another LLAS2 study [42], and then genetic outliers were removed. Finally, a total of 13,339 unrelated subjects, including European Americans (EA: 3,936 cases vs. 3,491 controls), African Americans (AA: 1,679 vs. 1,934; composed of 92. 5% of African Americans and 7. 5% Gullahs) and Hispanics enriched for the Amerindian-European admixture (HS: 1,492 vs. 807), were analyzed for 41 genotyped SNPs of TLR7-TLR8. Imputation was performed at 12. 86–12. 95 Mb on Xp22. 2 using IMPUTE 2. 1. 2 [43], with SNP/INDEL genotypes of 381 Europeans, 246 Africans and 181 Americans from the 1000 Genomes Project (“version 3” of the Phase 1 integrated data, March 2012 release) as references in imputation for our EA, AA and HS subjects, respectively. Imputed genotypes had to meet information score of >0. 9, as well as the quality control criteria as described above. After imputation, we obtained an additional 75 variants for EA, 57 for AA and 63 for HS (the number varied based on LD structure) for further analysis. Total RNA was purified with TRIzol reagent (Invitrogen) from PBMCs and reverse-transcribed into cDNA with Superscript II Reverse Transcription kit (Invitrogen). The mRNA levels of TLR7 (NM016562. 3) and TLR8 (isoform a: NM138636. 4 and isoform b: AF246971. 1) were measured by quantitative real-time PCR using TaqMan assays (TLR7 probe: Hs00152971_m1; TLR8 isoform a probe: Hs00607866_mH; TLR8 isoform b probe: Hs00152972_ml, Applied Biosystems). All samples were run in triplicate. Relative expression levels of TLR7 and TLR8 were normalized to the level of RPLP0, calculated by the 2−ΔΔCt method and Log10 transformed. The association of rs3853839 with mRNA levels of TLR7 or TLR8 was evaluated using ANOVA, Student' s t and linear regression test. To examine the correlation of miR-3148 and TLR7 mRNA levels, total RNA enriched in small RNAs were isolated from PBMCs using mirVanaTM miRNA isolation kit (Invitrogen), followed by reverse transcription with TaqMan MicroRNA Reverse Transcription kit (Applied Biosystems; for detecting miR-3148) and Superscript II Reverse Transcription kit (Invitrogen; for detecting TLR7), respectively. The miR-3148 level was quantified using Taqman MicroRNA Expression assay (Applied Biosystems), and the TLR7 level was measured using the same probe as described above. All samples were run in triplicate. Relative expression levels of miR-3148 and TLR7 were normalized to the level of snRNA U6 and RPLP0, respectively, calculated by the 2−ΔΔCt method and Log10 transformed. Association between transcript levels of TLR7 and miR-3148 was evaluated using linear regression test. Four-color flow cytometry was performed to investigate intracellular expression of TLR7 and TLR8 in PBMCs from healthy EA and Asian individuals who were homozygous for rs3853839 (7 pairs of G-allele vs. C-allele carriers in each gender group). Freshly isolated PBMCs were incubated with 2% pooled human serum to block nonspecific binding to Fcγ receptors and then incubated with peridinin chlorophyll protein (PerCP) -conjugated anti-human CD3, allophycocyanin (APC) -conjugated anti-human CD19 and phycoerythrin (PE) -conjugated or fluorescein isothiocyanate (FITC) -conjugated anti-human CD14 (Miltenyi Biotec) to identify T cell, B cell and monocyte subpopulations, respectively. For intracellular staining, PBMCs were fixed in Fixation buffer (R&D Systems) for 10 minutes at room temperature, washed twice in Permeabilization/Wash buffer (R&D Systems) and stained with PE-conjugated mouse anti-human TLR7 mAb (R&D Systems) and FITC-conjugated mouse anti-human TLR8 mAb (Imgenex) for 1 hour at room temperature. Background fluorescence was assessed using appropriate isotype- and fluorochrome-matched control antibodies. Cells were collected and analyzed by FACSCalibur flow cytometer equipped with the manufacturer' s software (CellQuest; BD Biosciences). Student' s t test was used to compare protein levels of TLR7 or TLR8 in PBMCs from individuals of different genotypes. The fragment of TLR7 3′-UTR bearing the G or C allele of rs3853839 was amplified by PCR from genomic DNA of subjects homozygous for the G or C allele using the following primers: 5′-TGTCTCGAGCCCTTCTTTGCAAAAC-3′ (forward) and 5′-AGAGCGGCCGCTAGTTGGCTCCAGCAAT-3′ (reverse). The PCR products were inserted into the downstream of the Renilla luciferase gene in the reporter vector psiCHECK-2 (Promega) by digestion using the restriction enzymes Not I and Xho I. The psiCHECK-2 vector also contained a firefly luciferase gene to serve as an internal transfection normalization control. All constructs were sequenced to assure proper orientation and authenticity in the vector. HEK-293 (human embryonic kidney cell line) and HL-60 (human leukemic cell line) cells were obtained from the American Type Culture Collection (ATCC). HEK-293 cells were maintained in Dulbecco' s modified Eagle' s medium supplemented with 10% FBS, seeded on a 24-well plate at a concentration of 2×105 cells/well, and transiently transfected using Lipofectamine 2000 (Invitrogen) with 1 µg of either rs3853839 G or C reporter construct. HL-60 cells are predominantly a neutrophilic promyelocyte (precursor) and can be induced to differentiate to neutrophil-like cells when grown in RPMI 1640 medium with 15% FBS plus 2 mM L-glutamine, 25 mM HEPES and 1. 25% DMSO [44]. Differentiated HL-60 cells seeded on 24-well plates (2×106 cells/well) were electroporated with 3 µg of report construct on a nucleofector device (Amaxa). The luciferase activity in total cell lysates was measured after 24 hours using a dual luciferase reporter assay system (Promega). Renilla luciferase activities were normalized to firefly luciferase activities. Each transfection was performed in quadruplicates and triplicates for HEK-293 and HL-60 cells respectively, and luciferase assays were repeated four times. MicroRNA hsa-miR-3148 and nontarget control (NC) mimics were synthesized by Thermo Fisher Scientific. To test the effect of miR-3148, HEK-293 cells plated in 96-well plates were transiently cotransfected with 100 ng of each reporter construct (psiCHECK-2 empty vector, rs3853839-G or -C allele constructs) and increasing concentrations (1,6, 12 and 48 nM) of miR-3148 or nontarget control mimic using Lipofectamine 2000 reagent (Invitrogen), and luminescence was measured 24 hours after transfection. Each transfection was performed in quadruplicates and repeated three times. Luciferase activity of reporter vectors was compared using Student' s t test. PBMCs isolated from EA healthy women with the GC genotype of rs3853839 (n = 7) were cultured in the absence or presence of 5 µg/mL ActD for 0,2, 4,6 and 24 hours. Using real-time PCR, we detected a decrease in total TLR7 mRNA levels over time with ActD incubation, which confirmed the transcriptional inhibition by ActD and allowed for detection of allelic differences in mRNA degradation. The G/C allelic ratio in the cDNA and gDNA after treatment of PBMCs with or without ActD were determined by pyrosequencing and calculated using software PSQMA 2. 1 (Biotage) as previously described [5]. The G/C allele ratio obtained in TLR7 transcripts was normalized to that measured from gDNA of the same sample. A paired t test was used to compare the mean G/C allele ratio in TLR7 transcripts in PBMCs treated with ActD or vehicle control at each time point. Associations of SNPs with SLE were assessed in each ancestral group under a logistic regression model adjusted for gender and the first three principal components estimated using AIMs. Conditional haplotype-based association tests were also performed by adjusting for gender and the first three principal components. The trans-ancestral meta-analysis was conducted on 40 genotyped and 14 imputed SNPs that were shared by the three ancestries with both a fixed and random-effects model. Homogeneity of odds ratios was evaluated using Cochrane' s Q test. For each SNP, if the Cochran' s Q test showed no evidence of genetic heterogeneity (P>0. 05), a fixed-effects model was implemented; otherwise, a random-effects model was used. The Bonferroni corrected P-value threshold was adjusted to P<9. 1×10−4 on the basis of the maximum number of tests across all populations (55 independent variants with r2<0. 8). All analyses described above were performed using PLINK v1. 07. Pairwised LD values shown in Figure 1 and Figure S1 were calculated using Haploview 4. 2. Other data were analyzed using GraphPad Prism 4. 0 software. A P value<0. 05 was considered to be statistically significant. | Systemic lupus erythematosus (SLE) is a debilitating autoimmune disease contributed to by excessive innate immune activation involving toll-like receptors (TLRs, particularly TLR7/8/9) and type I interferon (IFN) signaling pathways. TLR7 responds against RNA-containing nuclear antigens and activates IFN-α pathway, playing a pivotal role in the development of SLE. While a genomic duplication of Tlr7 promotes lupus-like disease in the Y-linked autoimmune accelerator (Yaa) murine model, the lack of common copy number variations at TLR7 in humans led us to identify a functional single nucleotide polymorphism (SNP), rs3853839 at 3′ UTR of the TLR7 gene, associated with SLE susceptibility in Eastern Asians. In this study, we fine-mapped the TLR7-TLR8 region and confirmed rs3853839 exhibiting the strongest association with SLE in European Americans, African Americans, and Amerindian/Hispanics. Individuals carrying the risk G allele of rs3853839 exhibited increased TLR7 expression at the both mRNA and protein level and decreased transcript degradation. MicroRNA-3148 (miR-3148) downregulated the expression of non-risk allele (C) containing transcripts preferentially, suggesting a likely mechanism for increased TLR7 levels in risk-allele carriers. This trans-ancestral mapping provides evidence for the global association with SLE risk at rs3853839, which resides in a microRNA-gene regulatory site affecting TLR7 expression. | lay_plos |
An important open problem of computational neuroscience is the generic organization of computations in networks of neurons in the brain. We show here through rigorous theoretical analysis that inherent stochastic features of spiking neurons, in combination with simple nonlinear computational operations in specific network motifs and dendritic arbors, enable networks of spiking neurons to carry out probabilistic inference through sampling in general graphical models. In particular, it enables them to carry out probabilistic inference in Bayesian networks with converging arrows (“explaining away”) and with undirected loops, that occur in many real-world tasks. Ubiquitous stochastic features of networks of spiking neurons, such as trial-to-trial variability and spontaneous activity, are necessary ingredients of the underlying computational organization. We demonstrate through computer simulations that this approach can be scaled up to neural emulations of probabilistic inference in fairly large graphical models, yielding some of the most complex computations that have been carried out so far in networks of spiking neurons. We show in this article that noisy networks of spiking neurons are in principle able to carry out a quite demanding class of computations: probabilistic inference in general graphical models. More precisely, they are able to carry out probabilistic inference for arbitrary probability distributions over discrete random variables (RVs) through sampling. Spikes are viewed here as signals which inform other neurons that a certain RV has been assigned a particular value for a certain time period during the sampling process. This approach had been introduced under the name “neural sampling” in [1]. This article extends the results of [1], where the validity of this neural sampling process had been established for the special case of distributions with at most order dependencies between RVs, to distributions with dependencies of arbitrary order. Such higher order dependencies, which may cause for example the explaining away effect [2], have been shown to arise in various computational tasks related to perception and reasoning. Our approach provides an alternative to other proposed neural emulations of probabilistic inference in graphical models, that rely on arithmetical methods such as belief propagation. The two approaches make completely different demands on the underlying neural circuits: the belief propagation approach emulates a deterministic arithmetical computation of probabilities, and is therefore optimally supported by noise-free deterministic networks of neurons. In contrast, our sampling based approach shows how an internal model of an arbitrary target distribution can be implemented by a network of stochastically firing neurons (such internal model for a distribution, that reflects the statistics of natural stimuli, has been found to emerge in primary visual cortex [3]). This approach requires the presence of stochasticity (noise), and is inherently compatible with experimentally found phenomena such as the ubiquitous trial-to-trial variability of responses of biological networks of neurons. Given a network of spiking neurons that implements an internal model for a distribution, probabilistic inference for, for example the computation of marginal probabilities for specific RVs, can be reduced to counting the number of spikes of specific neurons for a behaviorally relevant time span of a few hundred ms, similarly as in previously proposed mechanisms for evidence accumulation in neural systems [4]. Nevertheless, in this neural emulation of probabilistic inference through sampling, every single spike conveys information, as well as the relative timing among spikes of different neurons. The reason is that for many of the neurons in the model (the so-called principal neurons) each spike represents a tentative value for a specific RV, whose consistency with tentative values of other RVs, and with the available evidence (e. g., an external stimulus), is explored during the sampling process. In contrast, currently known neural emulations of belief propagation in general graphical models are based on firing rate coding. The underlying mathematical theory of our proposed new method provides a rigorous proof that the spiking activity in a network of neurons can in principle provide an internal model for an arbitrary distribution. It builds on the general theory of Markov chains and their stationary distribution (see e. g. [5]), the general theory of MCMC (Markov chain Monte Carlo) sampling (see e. g. [6], [7]), and the theory of sampling in stochastic networks of spiking neurons - modelled by a non-reversible Markov chain [1]. It requires further theoretical analysis for elucidating under what conditions higher order factors of p can be emulated in networks of spiking neurons, which is provided in the Methods section of this article. Whereas the underlying mathematical theory only guarantees convergence of the spiking activity to the target distribution, it does not provide tight bounds for the convergence speed to (the so-called burn–in time in MCMC sampling). Hence we complement our theoretical analysis by computer simulations for three Bayesian networks of increasing size and complexity. We also address in these simulations the question to what extent the speed or precision of the probabilistic inference degrades when one moves from a spiking neuron model that is optimal from the perspective of the underlying theory to a biologically more realistic neuron model. The results show, that in all cases quite good probabilistic inference results can be achieved within a time span of a few hundreds ms. In the remainder of this section we sketch the conceptual and scientific background for our approach. An additional discussion of related work can be found in the discussion section. Probabilistic inference in Bayesian networks [2] and other graphical models [8], [9] is an abstract description of a large class of computational tasks, that subsumes in particular many types of computational tasks that the brain has to solve: The formation of coherent interpretations of incomplete and ambiguous sensory stimuli, integration of previously acquired knowledge with new information, movement planning, reasoning and decision making in the presence of uncertainty [10]–[13]. The computational tasks become special cases of probabilistic inference if one assumes that the previously acquired knowledge (facts, rules, constraints, successful responses) is encoded in a joint distribution over numerous RVs, that represent features of sensory stimuli, aspects of internal models for the environment, environmental and behavioral context, values of carrying out particular actions in particular situations [14], goals, etc. If the values of some of these RVs assume concrete values (e. g. because of observations, or because a particular goal has been set), the distribution of the remaining variables changes in general (to the conditional distribution given the values). A typical computation that needs to be carried out for probabilistic inference for some joint distribution involves in addition marginalization, and requires for example the evaluation of an expression of the form (1) where concrete values (the “evidence”or “observations” have been inserted for the RVs,. These variables are then often called observable variables, and the others latent variables. Note that the term “evidence” is somewhat misleading, since the assignment represents some arbitrary input to a probabilistic inference computation, without any connotation that it represents correct observations or memories. The computation of the resulting marginal distribution requires a summation over all possible values for the RVs that are currently not of interest for this probabilistic inference. This computation is in general quite complex (in fact, it is NP-complete [9]) because in the worst case exponentially in many terms need to be evaluated and summed up. There exist two completely different approaches for solving probabilistic inference tasks of type (1), to which we will refer in the following as the arithmetical and the sampling approach. In the arithmetical approach one exploits particular features of a graphical model, that captures conditional independence properties of the distribution, for organizing the order of summation steps and multiplication steps for the arithmetical calculation of the r. h. s. of (1) in an efficient manner. Belief propagation and message passing algorithms are special cases of this arithmetical approach. All previously proposed neural emulations of probabilistic inference in general graphical models have pursued this arithmetical approach. In the sampling approach, which we pursue in this article, one constructs a method for drawing samples from the distribution (with fixed values for some of the RVs, see (1) ). One can then approximate the l. h. s. of (1), i. e., the desired value of the probability, by counting how often each possible value for the RV occurs among the samples. More precisely, we identify conditions under which each current firing state (which records which neuron has fired within some time window) of a network of stochastically firing neurons can be viewed as a sample from a probability distribution that converges to the target distribution. For this purpose the temporal dynamics of the network is interpreted as a (non-reversible) Markov chain. We show that a suitable network architecture and parameter choice of the network of spiking neurons can make sure that this Markov chain has the target distribution as its stationary distribution, and therefore produces after some “burn–in time”samples (i. e., firing states) from a distribution that converges to. This general strategy for sampling is commonly referred to as Markov chain Monte Carlo (MCMC) sampling [6], [7], [9]. Before the first use of this strategy in networks of spiking neurons in [1], MCMC sampling had already been studied in the context of artificial neural networks, so-called Boltzmann machines [15]. A Boltzmann machine consists of stochastic binary neurons in discrete time, where the output of each neuron has the value or at each discrete time step. The probability of each value depends on the output values of neurons at the preceding discrete time step. For a Boltzmann machine a standard way of sampling is Gibbs sampling. The Markov chain that describes Gibbs sampling is reversible, i. e., stochastic transitions between states do not have a preferred direction in time. This sampling method works well in artificial neural networks, where the effect of each neural activity lasts for exactly one discrete time step. But it is in conflict with basic features of networks of spiking neurons, where each action potential (spike) of a neuron triggers inherent temporal processes in the neuron itself (e. g. refractory processes), and postsynaptic potentials of specific durations in other neurons to which it is synaptically connected. These inherent temporal processes of specific durations are non-reversible, and are therefore inconsistent with the mathematical model (Gibbs sampling) that underlies probabilistic inference in Boltzmann machines. [1] proposed a somewhat different mathematical model (sampling in non-reversible Markov chains) as an alternative framework for sampling, that is compatible with these basic features of the dynamics of networks of spiking neurons. We consider in this article two types of models for spiking neurons (see Methods for details): A key step for interpreting the firing activity of networks of neurons as sampling from a probability distribution (as proposed in [3]) in a rigorous manner is to define a formal relationship between spikes and samples. As in [1] we relate the firing activity in a network of spiking neurons to sampling from a distribution over binary variables by setting (2) (we restrict our attention here to binary RVs; multinomial RVs could in principle be represented by WTA circuits –see Discussion). The constant models the average length of the effect of a spike on the firing probability of other neurons or of the same neuron, and can be set for example to. However with this definition of its internal state () the dynamics of the neural network can not be modelled by a Markov chain, since knowledge of this current state does not suffice for determining the distribution of states at future time points, say at time. This distribution requires knowledge about when exactly a neuron with had fired. Therefore auxiliary RVs with multinomial or analog values were introduced in [1], that keep track of when exactly in the preceding time interval of length a neuron had fired, and thereby restore the Markov property for a Markov chain that is defined over an enlarged state set consisting of all possible values of and. However the introduction of these hidden variables, that keep track of inherent temporal processes in the network of spiking neurons, comes at the price that the resulting Markov chain is no longer reversible (because these temporal processes are not reversible). But it was shown in [1] that one can prove nevertheless for any distribution for which the so-called neural computability condition (NCC), see below, can be satisfied by a network of spiking neurons, that defines a non-reversible Markov chain whose stationary distribution is an expanded distribution, whose marginal distribution over (which results when one ignores the values of the hidden variables) is the desired distribution. Hence a network of spiking neurons can sample from any distribution for which the NCC can be satisfied. This implies that any neural system that contains such network can carry out the probabilistic inference task (1): The evidence could be implemented through external inputs that force neuron to fire at a high rate if in, and not to fire if in. In order to estimate, it suffices that some readout neuron estimates (after some initial transient phase) the resulting firing rate of the neuron that represents RV. In contrast to most of the other neural implementations of probabilistic inference (with some exceptions, see for example [17] and [18]) where information is encoded in the firing rate of the neurons, in this approach the spike times, rather than the firing rate, of the neuron carry relevant information as they define the value of the RV at a particular moment in time according to (2). In this spike-time based coding scheme, the relative timing of spikes (which neuron fires simultaneously with whom) receives a direct functional interpretation since it determines the correlation between the corresponding RVs. The NCC requires that for each RV the firing probability density of its corresponding neuron at time satisfies, if the neuron is not in a refractory period, (3) where denotes the current value of all other RVs, i. e., all with. We use in this article the same model for a stochastic neuron as in [1] (continuous time case), which can be matched quite well to biological data according to [19]. In the simpler version of this neuron model one assumes that it has an absolute refractory period of length, and that the instantaneous firing probability satisfies outside of its refractory period, where is its membrane potential (see Methods for an account of the more complex neuron model with a relative refractory period from [1], that we have also tested in our simulations). The NCC from (3) can then be reformulated as a condition on the membrane potential of the neuron (4) Let us consider a Boltzmann distribution of the form (5) with symmetric weights (i. e.,) that vanish on the diagonal (i. e.,). In this case the NCC can be satisfied by a that is linear in the postsynaptic potentials that neuron receives from the neurons that represent other RVs: (6) where is the bias of neuron (which regulates its excitability), is the strength of the synaptic connection from neuron to, and approximates the time course of the postsynaptic potential caused by a firing of neuron at some time (assumes value 1 during the time interval, otherwise it has value). However, it is well known that probabilistic inference for distributions of the form (5) is too weak to model various important computational tasks that the brain is obviously able to solve, at least without auxiliary variables. While (5) only allows pairwise interactions between RVs, numerous real world probabilistic inference tasks require inference for distributions with higher order terms. For example, it has been shown that human visual perception involves “explaining away”, a well known effect in probabilistic inference, where a change in the probability of one competing hypothesis for explaining some observation affects the probability of another competing hypothesis [20]. Such effects can usually only be captured with terms of order at least 3, since 3 RVs (for 2 hypotheses and 1 observation) may interact in complex ways. A well known example from visual perception is shown in Fig. 1, for a probability distribution over 4 RVs, where is defined by the perceived relative reflectance of two abutting 2D areas, by the perceived 3D shape of the observed object, by the observed shading of the object, and by the contour of the 2D image. The difference in shading of the two abutting surfaces in Fig. 1A could be explained either by a difference in reflectance of the two surfaces, or by an underlying curved 3D shape. The two different contours (RV) in the upper and lower part of Fig. 1A influence the likelihood of a curved 3D shape (RV). In particular, a perceived curved 3D shape “explains away” the difference in shading, thereby making a uniform reflectance more likely. The results of [21] and numerous related results suggest that the brain is able to carry out probabilistic inference for more complex distributions than the order Boltzmann distribution (5). We show in this article that the neural sampling method of [1] can be extended to any probability distribution over binary RVs, in particular to distributions with higher order dependencies among RVs, by using auxiliary spiking neurons in that do not directly represent RVs, or by using nonlinear computational processes in multi-compartment neuron models. As one can expect, the number of required auxiliary neurons or dendritic branches increases with the complexity of the probability distribution for which the resulting network of spiking neurons has to carry out probabilistic inference. Various types of graphical models [9] have emerged as convenient frameworks for characterizing the complexity of distributions from the perspective of probabilistic inference for. We will focus in this article on Bayesian networks, a common type of graphical model for probability distributions. But our results can also be applied for other types of graphical models. A Bayesian network is a directed graph (without directed cycles), whose nodes represent RVs. Its graph structure indicates that admits a factorization of the form (7) where is the set of all (direct) parents of the node indexed by. For example, the Bayesian network in Fig. 1B implies that the factorization is possible. We show that the complexity of the resulting network of spiking neurons for carrying out probabilistic inference for can be bounded in terms of the graph complexity of the Bayesian network that gives rise to the factorization (7). More precisely, we present three different approaches for constructing such networks of spiking neurons: We will show that there exist two different neural implementation options for each of the last two approaches, using either specific network motifs or dendritic processing for nonlinear computation steps. This yields altogether 5 different options for emulating probabilistic inference in Bayesian networks through sampling via the inherent stochastic dynamics of networks of spiking neurons. We will exhibit characteristic differences in the complexity and performance of the resulting networks, and relate these to the complexity of the underlying Bayesian network. All 5 of these neural implementation options can readily be applied to Bayesian networks where several arcs converge to a node (giving rise to the “explaining away” effect), and to Bayesian networks with undirected cycles (“loops”). All methods for probabilistic inference from general graphical models that we propose in this article are from the mathematical perspective special cases of MCMC sampling. However in view of the fact that they expand the neural sampling approach of [1], we will refer to them more specifically as neural sampling. We show through computer simulations for three different Bayesian networks of different sizes and complexities that neural sampling can be carried quite fast with the help of the second and third approach, providing good inference results within a behaviorally relevant time span of a few hundred ms. One of these Bayesian networks addresses the previously described classical “explaining away” effect in visual perception from Fig. 1. The other two Bayesian networks not only contain numerous “explaining away” effects, but also undirected cycles. Altogether, our computer simulations and our theoretical analyses demonstrate that networks of spiking neurons can emulate probabilistic inference for general Bayesian networks. Hence we propose to view probabilistic inference in graphical models as a generic computational paradigm, that can help us to understand the computational organization of networks of neurons in the brain, and in particular the computational role of precisely structured cortical microcircuit motifs. It is well known [15] that any probability distribution, with arbitrarily large factors in a factorization such as (7), can be represented as marginal distribution (9) of an extended distribution with auxiliary RVs, that can be factorized into factors of degrees at most. This can be seen as follows. Let be an arbitrary probability distribution over binary variables with higher order factors). Thus (10) where is a vector composed of the RVs that the factor depends on and is a normalization constant. We additionally assume that is non-zero for each value of. The simple idea is to introduce for each possible assignment to the RVs in a higher order factor a new RV, that has value 1 only if is the current assignment of values to the RVs in. We will illustrate this idea through the concrete example of Fig. 1. Since there is only one factor that contains more than 2 RVs in the probability distribution of this example (see caption of Fig. 1), the conditional probability, there will be 8 auxiliary RVs,, …, for this factor, one for each of the 8 possible assignments to the 3 RVs in. Let us consider a particular auxiliary RV, e. g.. It assumes value 1 only if,, and. This constraint for can be enforced through second order factors between and each of the RVs and. For example, the second order factor that relates and has a value of 0 if and (i. e., if is not compatible with the assignment), and value 1 otherwise. The individual values of the factor for different assignments to, and are introduced in the extended distribution through first order factors, one for each auxiliary RV. Specifically, the first order factor that depends on has value (where is a constant that rescales the values of the factors such that for all assignments to, and) if, and value 1 otherwise. Further details of the construction method for are given in the Methods section, together with a proof of (9). The resulting extended probability distribution has the property that, in spite of deterministic dependencies between the RVs and, the state set of the resulting Markov chain realized through a network of spiking neurons according to [1] (that consists of all non-forbidden value assignments to and) is connected. In the previous example a non-forbidden value assignment is and. But is also a non-forbidden value assignment. Such non-forbidden value assignments to the auxiliary RVs corresponding to one higher order factor, where all of them assume value of 0 regardless of the values of the RVs provide transition points for paths of probability that connect any two non-forbidden value assignments (without requiring that 2 or more RVs switch their values simultaneously). The resulting connectivity of all non-forbidden states (see Methods for a proof) implies that this Markov chain has as its unique stationary distribution. The given distribution arises as marginal distribution of this stationary distribution of, hence one can use to sample from (just ignore the firing activity of neurons that correspond to auxiliary RVs). Since the number of RVs in the extended probability distribution can be much larger than the number of RVs in, the corresponding spiking neural network samples from a much larger probability space. This, as well as the presence of deterministic relations between the auxiliary and the main RVs in the expanded probability distribution, slow down the convergence of the resulting Markov chain to its stationary distribution. We show however in the following, that there are several alternatives for sampling from an arbitrary distribution through a network of spiking neurons. These alternative methods do not introduce auxiliary RVs, but rather aim at directly satisfying the NCC (4) in a network of spiking neurons. Note that the principal neurons in the neural network that implements neural sampling through introduction of auxiliary RVs also satisfy the NCC, but in the extended probability distribution with second order relations, whereas in the neural implementations introduced in the following the principal neurons satisfy the NCC in the original distribution. In Computer Simulation I we have compared the convergence speed of the methods that satisfy the NCC with that of the previously described method via auxiliary RVs. It turns out that the alternative strategy provides an about fold speed-up for the Bayesian network of Fig. 1B. Assume that the distribution for which we want to carry out probabilistic inference is given by some arbitrary Bayesian network. There are two different options for satisfying the NCC for, which differ in the way by which the term on the r. h. s. of the NCC (4) is expanded. The option that we will analyze first uses from the structure of the Bayesian network only the information about which RVs are in the Markov blanket of each RV. The Markov blanket of the corresponding node in (which consists of the parents, children and co-parents of this node) has the property that is independent from all other RVs once any assignment of values to the RVs in the Markov blanket has been fixed. Hence =, and the term on the r. h. s. of the NCC (4) can be expanded as follows: (11) where (12) The sum indexed by runs over the set of all possible assignments of values to, and denotes a predicate which has value 1 if the condition in the brackets is true, and to 0 otherwise. Hence, for satisfying the NCC it suffices if there are auxiliary neurons, or dendritic branches, for each of these, that become active if and only if the variables currently assume the value. The current values of the variables are encoded in the firing activity of their corresponding principal neurons. The corresponding term can be implemented with the help of the bias (see (8) ) of the auxiliary neuron that corresponds to the assignment, resulting in a value of its membrane potential equal to the r. h. s. of the NCC (4). We will discuss this implementation option below as Implementation 2. In the subsequently discussed implementation option (Implementation 3) all principal neurons will be multi-compartment neurons, and no auxiliary neurons are needed. In this case scales the amplitude of the signal from a specific dendritic branch to the soma of the multi-compartment principal neuron. The second strategy to expand the log-odd ratio on the r. h. s. of the NCC (4) uses the factorized form (10) of the probability distribution. This form allows us to rewrite the log-odd ratio in (4) as a sum of log terms, one for each factor,, that contains the RV (we write for this set of factors). One can write each of these terms as a sum over all possible assignments of values of the variables the factor depends on (except). This yields (13) where is a vector composed of the RVs that the factor depends on –without, and is the current value of this vector at time. denotes the set of all possible assignments to the RVs. The parameters are set to (14) The factorized expansion in (13) is similar to (11), but with the difference that we have another sum running over all factors that depend on. Consequently, in the resulting Implementation 4 with auxiliary neurons and dendritic branches there will be several groups of auxiliary neurons that connect to, where each group implements the expansion of one factor in (13). The alternative model that only uses dendritic computation (Implementation 5) will have groups of dendritic branches corresponding to the different factors. The number of auxiliary neurons that connect to in Implementation 4 (and the corresponding number of dendritic branches in Implementation 5) is equal to the sum of the exponents of the sizes of factors that depend on:, where denotes the number of RVs in the vector. This number is never larger than (where is the size of the Markov blanket of), which gives the corresponding number of auxiliary neurons or dendritic branches that are required in the Implementation 2 and 3. These two numbers can considerably differ in graphical models where the RVs participate in many factors, but the size of the factors is small. Therefore one advantage of this approach is that it requires in general fewer resources. On the other hand, it introduces a more complex connectivity between the auxiliary neurons and the principal neuron (compare Fig. 5 with Fig. 2). We have tested the viability of the previously described approach for neural sampling by satisfying the NCC also on two larger and more complex Bayesian networks: the well-known ASIA-network [24], and an even larger randomly generated Bayesian network. The primary question is in both cases, whether the convergence speed of neural sampling is in a range where a reasonable approximation to probabilistic inference can be provided within the typical range of biological reaction times of a few 100 ms. In addition, we examine for the ASIA-network the question to what extent more complex and biologically more realistic shapes of EPSPs affect the performance. For the larger random Bayesian network we examine what difference in performance is caused by neuron models with absolute versus relative refractory periods. There are a number of studies proposing neural network architectures that implement probabilistic inference [15], [17], [18], [35]–[48]. Most of these models propose neural emulations of the belief propagation algorithm, where the activity of neurons or populations of neurons encodes intermediate values (called messages or beliefs) needed in the arithmetical calculation of the posterior probability distribution. With some exceptions [17], most of the approaches assume rate-based coding of information and use rate-based neuron models or mean-field approximations. In particular, in [37] a spiking neural network model was developed that performs the max-product message passing algorithm, a variant of belief propagation, where the necessary maximization and product operations were implemented by specialized neural circuits. Another spiking neural implementation of the sum-product belief propagation algorithm was proposed in [36], where the calculation and passing of the messages was achieved in a recurrent network of interconnected liquid state machines [49]. In these studies, that implemented probabilistic inference with spiking neurons through emulation of the belief propagation algorithm on tree factor graphs, the beliefs or the messages during the calculation of the posterior distributions were encoded in an average firing rate of a population of neurons. Regarding the complexity of these neural models, as the number of required computational operations in belief propagation is exponential in the size of the largest factor in the probability distribution, in the neural implementations this translates to a number of neurons in the network that scales exponentially with the size of the largest factor. This complexity corresponds to the required number of neurons (or dendritic branches) in implementations 1,3 and 5 in our approach, whereas implementations 2 and 4 require a larger number of neurons that scales exponentially with the size of the largest Markov blanket in the distribution. Additionally, note that the time of convergence to the correct posterior differs in both approaches: in the belief propagation based models it scales in the worst case linearly with the number of RVs in the probability distribution, whereas in our approach it can vary depending on the probability distribution. Although the belief propagation algorithm can be applied to graphical models with undirected loops (a variant called loopy belief propagation), it is not always guaranteed to work, which limits the applicability of the neural implementations based on this algorithm. The computation and the passing of messages in belief propagation uses, however, equivalent computations as the junction tree algorithm [24], [50], a message passing algorithm that operates on a junction tree, a tree structure derived from the graphical model. The junction tree algorithm performs exact probabilistic inference in general graphical models, including those that have loops. Hence, the neural implementations of belief propagation could in principle be adapted to work on junction trees as well. This however comes at a computational cost manifested in a larger required size of the neural network, since the number of required operations for the junction tree algorithm scales exponentially with the width of the junction tree, and the width of the junction tree can be larger than the size of the largest factor for graphical models that have loops (see [9], chap. 10 for a discussion). The analysis of the complexity and performance of resulting emulations in networks of spiking neurons is an interesting topic for future research. Another interesting approach, that adopts an alternative spike-time based coding scheme, was described in [17]. In this study a spiking neuron model estimates the log-odd ratio of a hidden binary state in a hidden Markov model, and it outputs a spike only when it receives new evidence from the inputs that causes a shift in the estimated log-odd ratio that exceeds a certain threshold, that is, only when new information about a change in the log-odd ratio is presented that cannot be predicted by the preceding spikes of the neuron. However, this study considers only a very restricted class of graphical models: Bayesian networks that are trees (where for example no explaining away can occur). The ideas in [17] have been extended in [18], where the neural model is capable of integration of evidence from multiple simultaneous cues (the underlying graphical model is a hidden Markov model with multiple observations). It uses a population code for encoding the log-posterior estimation of the time varying hidden stimulus, which is modeled as a continuous RV instead of the binary hidden state used in [17]. In these studies, as in ours, spikes times carry relevant information, although there the spikes are generated deterministically and signal a prediction error used to update and correct the estimated log-posterior, whereas in our approach the spikes are generated by a stochastic neuron model and define the current values of the RVs during the sampling. The idea that nonlinear dendritic mechanisms could account for the nonlinear processing that is required in neural models that perform probabilistic inference has been proposed previously in [39] and [41], albeit for the belief propagation algorithm. In [39] the authors introduce a neural model that implements probabilistic inference in hidden Markov models via the belief propagation algorithm, and suggest that the nonlinear functions that arise in the model can be mapped to the nonlinear dendritic filtering. In [41] another rate-based neural model that implements the loopy belief propagation algorithm in general graphical models was described, where the required multiplication operations in the algorithm were proposed to be implemented by the nonlinear processing in individual dendritic trees. While there exist several different spiking neural network models in the literature that perform probabilistic inference based on the belief propagation algorithm, there is a lack of spiking neural network models that implement probabilistic inference through Markov chain Monte Carlo (MCMC sampling). To the best of our knowledge, the neural implementations proposed in this article are the only spiking neural networks for probabilistic inference via MCMC in general graphical models. In [35] a non-spiking neural network composed of stochastic binary neurons was introduced called Boltzmann machine, that performs probabilistic inference via Gibbs sampling. The neural network in [35] performs inference via sampling in probability distributions that have only pairwise couplings between the RVs. An extension was proposed in [51], that can perform Gibbs sampling in probability distributions with higher order dependencies between the variables, which corresponds to the class of probability distributions that we consider in this article. A spiking neural network model based on the results in [35] had been proposed in [52], for a restricted class of probability distributions that only have second order factors, and which satisfy some additional constraints on the conditional independencies between the variables. To the best of our knowledge, this approach had not been extended to more general probability distributions. A recent study [53] showed that as the noise in the neurons increases and their reliability drops, the optimal couplings between the neurons that maximize the information that the network conveys about the inputs become larger in magnitude, creating a redundant code that reduces the impact of noise. Effectively, the network learns the input distribution in its couplings, and uses this knowledge to compensate for errors due to the unreliable neurons. These findings are consistent with our models, and although we did not consider learning in this article, we expect that the introduction of learning mechanisms that optimize a mutual information measure in our neural implementations would yield optimal couplings that obey the same principles as the ones reported in [53]. While stochasticity in the neurons represents a crucial property that neural implementations of probabilistic inference through sampling rely on, this study elucidates an important additional effect it has in learning paradigms that use optimality principles like information maximization: it induces redundant representation of information in a population of neurons. The existing gap between abstract computational models of information processing in the brain that use MCMC algorithms for probabilistic inference on one hand, and neuroscientific data about neural structures and neural processes on the other hand, has been pointed out and emphasized by several studies [12], [13], [54], [55]. The results in [1] and in this article propose neural circuit models that aim to bridge this gap, and thereby suggest new means for analyzing data from spike recordings in experimental neuroscience, and for evaluating the more abstract computational models in light of these data. For instance, perceptual multistability in ambiguous visual stimuli and several of its related phenomena were explained through abstract computational models that employ sequential sampling with the Metropolis MCMC algorithm [55]. In our simulations (see Fig. 10) we showed that a spiking neural network can exhibit multistability, where the state changes from one mode of the posterior distribution to another, even though the Markov chain defined by the neural network does not satisfy the detailed balance property (i. e. it is not a reversible Markov chain) like the Metropolis algorithm. Our models postulate that knowledge is encoded in the brain in the form of probability distributions, that are not required to be of the restricted form of order Boltzmann distributions (5). Furthermore they postulate that these distributions are encoded through synaptic weights and neuronal excitabilities, and possibly also through the strength of dendritic branches. Finally, our approach postulates that these learnt and stored probability distributions are activated through the inherent stochastic dynamics of networks of spiking neurons, using nonlinear features of network motifs and neurons to represent higher order dependencies between RVs. It also predicts that (in contrast to the model of [1]) synaptic connections between neurons are in general not symmetric, because this enables the network to encode higher order factors of. The postulate that knowledge is stored in the brain in the form of probability distributions, sampled from by the stochastic dynamics of neural circuits, is consistent with the ubiquitous trial-to-trial variability found in experimental data [56], [57]. It has been partially confirmed through more detailed analyses, which show that spontaneous brain activity shows many characteristic features of brain responses to natural external stimuli ([3], [58], [59]). Further analysis of spontaneous activity is needed in order to verify this prediction. Beyond this prediction regarding spontaneous activity, our approach proposes that fluctuating neuronal responses to external stimuli (or internal goals) represent samples from a conditional marginal distribution, that results from entering evidence for a subset of RVs of the stored distribution (see (1) ). A verification of this prediction requires an analysis of the distributions of network responses –rather than just averaging –for repeated presentations of the same sensory stimulus or task. Similar analyses of human responses to repeated questions have already been carried out in cognitive science [60]–[62], and have been interpreted as evidence that humans respond to queries by sampling from internally stored probability distributions. Our resulting model for neural emulations of probabilistic inference predicts, that even strong firing of a single neuron (provided it represents a RV whose value has a strong impact on many other RVs) may drastically change the activity pattern of many other neurons (see the change of network activity after 3 s in Fig. 8, which results from a change in value of the RV that represents “x-ray”). One experimental result of this type had been reported in [63]. Fig. 8 also suggests that different neurons may have drastically different firing rates, where a few neurons fire a lot, and many others fire rarely. This is a consequence both of different marginal probabilities for different RVs, but also of the quite different computational role and dynamics of neurons that represent RVs (“principal neurons”), and auxiliary neurons that support the realization of the NCC, and which are only activated by a very specific activation patterns of other presynaptic neurons. Such strong differences in the firing activity of neurons has already been found in some experimental studies, see [64], [65]. In addition, Fig. 10 predicts that recordings from multiple neurons can typically be partitioned into time intervals, where a different firing pattern dominates during each time interval, see [28], [29] for some related experimental data. Apart from these more detailed predictions, a central prediction of our model is, that a subset of cortical neurons (the “principal neurons”) represent through their firing activity the current value of different salient RVs. This could be tested, for example, through simultaneous recordings from large numbers of neurons during experiments, where the values of several RVs that are relevant for the subject, and that could potentially be stored in the cortical area from which one records, are changed in a systematic manner. It might potentially be more difficult to test, which of the concrete implementations of computational preprocessing for satisfying the NCC that we have proposed, are implemented in some neural tissue. Both the underlying theoretical framework and our computer simulations (see Fig. 8) predict that the auxiliary neurons involved in these local computations are rarely active. More specifically, the model predicts that they only become active when some specific set of presynaptic neurons (whose firing state represents the current value of the RVs in) assumes a specific pattern of firing and non-firing. Implementation 3 and 5 make corresponding predictions for the activity of different dendritic branches of pyramidal neurons, that could potentially be tested through -imaging. We have proposed a new modelling framework for brain computations, based on probabilistic inference through sampling. We have shown through computer simulations, that stochastic networks of spiking neurons can carry out demanding computational tasks within this modelling framework. This framework predicts specific functional roles for nonlinear computations in network motifs and dendritic computation: they support representation of higher order dependencies between salient random variables. On the micro level this framework proposes that local computational operations of neurons superficially resemble logical operations like AND and OR, but that these atomic computational operations are embedded into a stochastic network dynamics. Our framework proposes that the functional role of this stochastic network dynamics can be understood from the perspective of probabilistic inference through sampling from complex learnt probability distributions, that represent the knowledge base of the brain. A Markov chain in discrete time is defined by a set of states (we consider for discrete time only the case where has a finite size, denoted by) together with a transition operator. is a conditional probability distribution for the next state of, given its preceding state. The Markov chain is started in some initial state, and moves through a trajectory of states via iterated application of the stochastic transition operator (more precisely, if is the state at time, then the next state is drawn from the conditional probability distribution. A powerful theorem from probability theory (see e. g. p. 232 in [5]) states that if is irreducible (i. e., any state in can be reached from any other state in in finitely many steps with probability) and aperiodic (i. e., its state transitions cannot be trapped in deterministic cycles), then the probability was the initial state) converges for to a probability that does not depend on. This state distribution is called the stationary distribution of. The irreducibility of implies that is the only distribution over the states that is invariant under the transition operator, i. e. (15) Thus, in order to generate samples from a given distribution, it suffices to construct an irreducible and aperiodic Markov chain that leaves invariant, i. e., satisfies (15). This Markov chain can then be used to carry out probabilistic inference of posterior distributions of given an evidence for some of the variables in the state. Analogous results hold for Markov chains in continuous time [5], on which we will focus in this article. We use two types of neurons, a stochastic point neuron model as in [1], and a multi-compartment neuron model. Let be a probability distribution (23) that contains higher order factors, where is a vector of binary RVs. are the factors that depend on one or two RVs, and are the higher order factors that depend on more than 2 RVs. is the vector of the RVs in the factor, is the vector of RVs that the factor depends on, and is the normalization constant. is the number of first and second order factors, and is the total number of factors of order 3 or higher. To simplify the notation, in the following we set, since this set of factors in will not be changed in the extended probability distribution. Auxiliary RVs are introduced for each of the higher order factors. Specifically, the higher order relation of factor is represented by a set of auxiliary binary RVs, where we have a RV for each possible assignment to the RVs in (is the domain of values of the vector). With the additional sets of RVs we define a probability distribution as (24) We denote the ordered set of indices of the RVs that compose the vector as, i. e. (25) where denotes the number of indices in. The second order factors are defined as (26) where denotes the component of the assignment to that corresponds to the variable, and is the Kronecker-delta function. The factors represent a constraint that if the auxiliary RV has value 1, then the values of the RVs in the corresponding factor must be equal to the assignment that corresponds to. If all components of are zero, then there is not any constraint on the variables. This implies another property: at most one of the RVs in the vector, the one that corresponds to the state of, can have value 1. Hence, the vector can have two different states. Either all its RVs are zero, or exactly one component is equal to 1, in which case one has. The probability for values of and that do not satisfy these constraints is. The values of the factors in for various assignments of are represented in by first order factors that depend on a single one of the RVs. For each we have a new factor with value if, and otherwise. We assume that the original factors are first rescaled, such that for all values of and. We had to modify the values of the new factors by subtracting 1 from the original value, because we introduced an additional zero state for that is consistent with any of the possible assignments of. The resulting probability distribution consists of first and second order factors. In this neural implementation each principal neuron has a dedicated preprocessing layer of auxiliary neurons with lateral inhibition. All neurons in the network are stochastic point neuron models. The auxiliary neurons for the principal neuron receive as inputs the outputs of the principal neurons corresponding to all RVs in the Markov blanket of. The number of auxiliary excitatory neurons that connect to the principal neuron is (is the number of elements of), and we index these neurons with all possible assignments of values to the RVs in the vector. Thus, for each state of values at the inputs we have a corresponding auxiliary neuron. The realization of the NCC is achieved by a specific connectivity between the inputs and the auxiliary neurons and appropriate values for the intrinsic excitabilities of the auxiliary neurons, such that at each moment in time only the auxiliary neuron corresponding to the current state of the inputs, if it is not inhibited by the lateral inhibition due to a recent spike from another auxiliary neuron, fires with a probability density as demanded by the NCC (3): (31) During the time when the state of the inputs is active, the other auxiliary neurons are either strongly inhibited, or do not receive enough excitatory input to reach a significant firing probability. The inputs connect to the auxiliary neuron either with a direct strong excitatory connection, or through an inhibitory interneuron that connects to the auxiliary neuron. The inhibitory interneuron fires whenever any of the principal neurons of the RVs that connect to it fires. The auxiliary neuron receives synaptic connections according to the following rule: if the assignment assigns a value of 1 to the RV in the Markov blanket, then the principal neuron connects to the neuron with a strong excitatory synaptic efficacy, whereas if assigns a value of 0 to then the principal neuron connects to the inhibitory interneuron. Thus, whenever fires, the inhibitory interneuron fires and prevents the auxiliary neuron to fire for a time period. We will assume that the synaptic efficacy is much larger than the log-odd ratio value of the RV given according to the r. h. s. of (3). We set the bias of the auxiliary neuron equal to (32) where gives the number of components of the vector that are 1. If the value of the inputs at time is, and none of the neurons fired in the time interval, then for an auxiliary neuron such that there are two possibilities. Either there exists a component of that is and its corresponding input, in which case the principal neuron of the RV connects to the inhibitory interneuron and inhibits. Or one has in which case the number of active inputs that connect to neuron do not provide enough excitatory input to reach the high threshold for firing. In this case the firing probability of the neuron is (33) and because of the strong synaptic efficacies of the excitatory connections equal to, which are by definition much larger than the log-odd ratio of the RV, it is approximately equal to 0. Hence, only the neuron with has a non-vanishing firing probability equal to (31). The lateral inhibition between the auxiliary neurons is implemented through a common inhibitory circuit to which they all connect. The role of the lateral inhibition is to enforce the necessary refractory period of after any of the auxiliary neurons fires. When an auxiliary neuron fires, the inhibitory circuit is active during the duration of the EPSP (equal to), and strongly inhibits the other neurons, preventing them from firing. The auxiliary neurons connect to the principal neuron with an excitatory connection strong enough to drive it to fire a spike whenever any one of them fires. During the time when the state of the input variables satisfies, the firing probability of the auxiliary neuron satisfies the NCC (3). This implies that the principal neuron satisfies the NCC as well. Introducing an evidence of a known value of a RV in this model is achieved by driving the principal neuron with an external excitatory input to fire a spike train with a high firing rate when the observed value of the RV is 1, or by inhibiting the principal neuron with an external inhibitory input so that it remains silent when the observed value of the RV is 0. We assume that the principal neuron has a separate dendritic branch for each possible assignment of values to the RVs, and that the principal neurons corresponding to the RVs in the Markov blanket connect to these dendritic branches. It is well known that synchronous activation of several synapses at one branch, if it exceeds a certain threshold, causes the membrane voltage at the branch to exhibit a sudden jump resulting from a dendritic spike. Furthermore the amplitude of such dendritic spike is subject to plasticity [22]. We use a neuron model according to [23], that is based on these experimental data. The details of this multi-compartment neuron model were presented in the preceding subsection of Methods on Neuron Models. We assume in this model that the contribution of each dendritic branch to the soma membrane voltage is predominantly due to dendritic spikes, and that the passive conductance to the soma can be neglected. Thus, according to (22), the membrane potential at the soma is equal to the sum of the nonlinear active components contributed from each of the branches: (34) where is the nonlinear contribution from branch, and is the strength of branch (see [22] for experimental data on branch strengths). is the target value of the membrane potential in the absence of any synaptic input. The nonlinear active component (dendritic spike) is assumed to be equal to (35) where denotes the Heaviside step function, is the local activation, and is the threshold of branch. The amplitude of the total contribution of branch to the membrane potential at the soma is then. As can be seen in Fig. 4, the connectivity from the inputs to the dendritic branches is analogous as in Implementation 2 with auxiliary neurons: from each principal neuron such that is in the Markov blanket of there is a direct synaptic connection to the dendritic branch if the assignment assigns to the value, or a connection to the inhibitory interneuron in case assigns the value 0 to. The inhibitory interneuron connects to its corresponding branch, and fires whenever any of the principal neurons that connect to it fire. The synaptic efficacies of the direct synaptic connections are assumed to satisfy the condition (36) where is the set of indices of principal neurons that directly connect to the dendritic branch, is the efficacy of the synaptic connection to the branch from, and is the threshold at the dendritic branch for triggering a dendritic spike. Additionally, each synaptic weight should also satisfy the condition (37) The same condition applies also for the efficacy of the synaptic connection from inhibitory interneuron to the dendritic branch. These conditions ensure that if the current state of the inputs is, then the dendritic branch will have an active dendritic spike, whereas all other dendritic branches will not receive enough total synaptic input to trigger a dendritic spike. The amplitude of the dendritic spike from branch at the soma is (38) where is a positive constant that is larger than all possible negative values of the log-odd ratio. If the steady value of the membrane potential is equal to, then we have at each moment a membrane potential that is equal to the sum of the amplitude of the nonlinear contribution of the single active dendritic branch and the steady value of the membrane potential, which yields the expression for the NCC (4). In this implementation a principal neuron has a separate group of auxiliary neurons for each factor that depends on the variable. The group of auxiliary neurons for the factor receives inputs from the principal neurons that correspond to the set of the RVs that factor depends on, but without. For each possible assignment of values to the inputs, there is an auxiliary neuron in the group for the factor, which we will denote with. The neuron spikes immediately when the state of the inputs switches to from another state, i. e. the spike marks the moment of the state change. This can be achieved by setting the bias of the neuron similarly as in (32) to where is the number of components of the vector that are equal to 1, is the efficacy of the direct synaptic connections from the principal neurons to and is a constant that ensures high firing probability of this neuron when the current value of the inputs is. The connectivity from the auxiliary neurons to the principal neuron keeps the soma membrane voltage of the principal neuron equal to the log-odd ratio of (= r. h. s. of (4) ). From each auxiliary neuron there is one excitatory connection to the principal neuron, terminating at a separate dendritic branch. The efficacy of this synaptic connection is, where is the parameter from (13), and is a constant that shifts all these synaptic efficacies into the positive range. Additionally, there is an inhibitory interneuron connecting to the same dendritic branch. The inhibitory interneuron receives input from all other auxiliary neurons in the same sub-circuit as the auxiliary neuron, but not from. The purpose of this inhibitory neuron is to shunt the active EPSP when the inputs change their state from to another state. Namely, at the time moment when the inputs change to state, the corresponding auxiliary neuron will fire, and this will cause firing of the inhibitory interneuron. A spike of the inhibitory interneuron should have just a local effect: to shunt the active EPSP caused by the previous state at the dendritic branch. If there is not any active EPSP, this spike of the inhibitory interneuron should not affect the membrane potential at the soma of the principal neuron. At any time, the group of auxiliary neurons for the factor contributes one EPSP to the principal neuron, through the synaptic input originating from the auxiliary neuron that corresponds to the current state of the inputs. The amplitude of the EPSP from the sub-circuit that corresponds to the factor is equal to. If we assume that the bias of the soma membrane potential is, then the total membrane potential at the soma of the principal neuron is equal to: (39) which is equal to the expression on the r. h. s. of (13) when one assumes that. Hence, the principal neuron satisfies the NCC. In this implementation each principal neuron is a multi-compartment neuron of the same type as in Implementation 3, with a separate group of dendritic branches for each factor in the probability distribution that depends on. In the group (corresponding to factor) there is a dendritic branch for each assignment to the variables that the factor depends on (without). The dendritic branches in group receive synaptic inputs from the principal neurons that correspond to the RVs. Each dendritic branch can contribute a component to the soma membrane voltage (where is like in Implementation 3 the branch strength of this branch), but only if the local activation in the branch exceeds the threshold for triggering a dendritic spike. The connectivity from the principal neurons corresponding to the RVs to the dendritic branches of in the group is such so that at time only the dendritic branch corresponding to the current state of the inputs receives total synaptic input that crosses the local threshold for generating a dendritic spike and initiates a dendritic spike. This is realized with the same connectivity pattern from the inputs to the branches as in Implementation 3 depicted in Fig. 4. The amplitude of the dendritic spike of branch at the soma should be where is the parameter from (13) and is chosen as in Implementation 3. The membrane voltage at the soma of the principal neuron is then equal to the sum of the dendritic spikes from the active dendritic branches. At time there is exactly one active branch in each group of dendritic branches, the one which corresponds to the current state of the inputs. If we additionally assume that the bias of neuron is, then the membrane voltage at the soma has the desired value (39). | Experimental data from neuroscience have provided substantial knowledge about the intricate structure of cortical microcircuits, but their functional role, i. e. the computational calculus that they employ in order to interpret ambiguous stimuli, produce predictions, and derive movement plans has remained largely unknown. Earlier assumptions that these circuits implement a logic-like calculus have run into problems, because logical inference has turned out to be inadequate to solve inference problems in the real world which often exhibits substantial degrees of uncertainty. In this article we propose an alternative theoretical framework for examining the functional role of precisely structured motifs of cortical microcircuits and dendritic computations in complex neurons, based on probabilistic inference through sampling. We show that these structural details endow cortical columns and areas with the capability to represent complex knowledge about their environment in the form of higher order dependencies among salient variables. We show that it also enables them to use this knowledge for probabilistic inference that is capable to deal with uncertainty in stored knowledge and current observations. We demonstrate in computer simulations that the precisely structured neuronal microcircuits enable networks of spiking neurons to solve through their inherent stochastic dynamics a variety of complex probabilistic inference tasks. | lay_plos |
The seven-year-old British girl shot, bludgeoned over the head and left for dead by an assailant who murdered her parents has regained consciousness in a French hospital. Doctors had placed Zainab al-Hilli, who police hope holds the key to the mysterious assassination, in a medically induced coma to aid her recovery. Relatives from Britain were reported to be at the orphaned girl's bedside when she awoke. Iraqi-born aeronautics engineer Saad al-Hilli, 50, his wife Ikbal, 47, a 77-year-old female relative and a French cyclist, who was passing by the scene, were shot several times at an Alpine beauty spot near Lake Annecy on Wednesday. The killer – or killers – put two bullets in each victim's head, leading to suggestions it could have been a professional assassination. Zainab and her four-year-old sister Zeena, who was the only member of the family to escape unscathed, have been under armed guard since. Eric Maillaud, the public prosecutor in Annecy, confirmed Zainab was now conscious. "She has come out of the artificial coma and is under sedation," he said on Sunday. Asked when the girl, whom he has described as the "key witness", would be interviewed, he said: "I cannot say." Earlier Maillaud had said the French investigation team would wait for doctors to decide whether the child was well enough to be questioned. He described her survival as a miracle, and said it was "out of the question" for investigators to interview her until her condition improved. "We cannot go in a precipitate manner to interview someone who has been injured and traumatised," he said. He added: "We feel desperately sorry for her [Zainab], and it is terrible that a victim, especially a child, has to be a key witness and has to be asked questions that will inevitably cause her even more suffering. We hope she will be able to tell us something, but it will be difficult." Zeena returned to Britain on Sunday after two family members, a British social worker and a police liaison officer travelled to Annecy to collect her. She hid in the rear footwell of the car when the shooting started. The petrified child was saved after taking refuge under her dead mother's legs, where she lay undiscovered for eight hours after gendarmes sealed off the scene, believing there was nobody alive in the car. Zeena has been in the care of a nurse, a child psychologist and has had round-the-clock care from a team from the British embassy. She has been "gently questioned at length" by French investigators over the past few days, but was unable to tell them anything. Apart from hearing "noises and cries", the public prosecutor said Zeena had seen nothing and was unable to advance the inquiry. "The most important thing is to get her back to her family," he said. "She has been interviewed, but we have tried to avoid causing her any further suffering." The victims were each shot twice in the head with an automatic pistol, suggesting an assassination. French investigators say they are still trying to establish if there was more than one killer and how many weapons were used. Around 25 bullet casings were found in and around the family's car. A source said initial ballistic tests suggested the shots had been fired from outside the vehicle at close range. Maillaud has refused to speculate or give details of DNA found on the bullets or the results of the postmortem examinations, for fear of aiding those responsible for the murders. He said all lines of inquiry are being followed. "The investigation has to be the priority now. We are not going to release any details or information that might enable the perpetrator or perpetrators of this savage attack to escape. My only aim is to see whoever did this caught and jailed." He admitted that hopes of solving the case now rest with Zainab. The position of a child's car seat indicated that the elder girl was travelling in the front passenger seat when the family drove up La Route de Combe d'Ire near the village of Chevaline.Zainab was outside the vehicle, parked at the start of a mountain hiking trail, and was shot in the shoulder and subjected to a violent beating about the head, which fractured her skull. The girl collapsed at the feet of a British cyclist, who arrived shortly after the killings. French investigators praised his "exemplary" reaction in placing the critically injured child in the recovery position and calling the emergency services. The Briton, a former RAF pilot, who has a house in the region, was also profoundly shocked to discover the body of a French cyclist nearby. Sylvain Mollier, a 45-year-old father-of-three, had overtaken him on the hill leading to the beauty spot just moments before. Mollier, who police said appeared to have been "in the wrong place at the wrong time", was gunned down after apparently witnessing the bloodshed. Zainab has been under police protection in intensive care at Grenoble university hospital since Wednesday and has undergone operations. Doctors said her life was no longer in danger, but they had put her into a medically induced coma to help her recovery. On Sunday, detectives were going through the holiday caravan the family had stayed in since 3 September, which had been removed from Le Solitaire du Lac campsite on the banks of Lake Annecy on Saturday. French investigators are also looking at the contents of a laptop found in the caravan and two mobile phones found in the Hillis' BMW estate car. They are also communicating with police forces in Italy and Switzerland, where the killer or killers may have fled. Police continued to search the Hillis' Surrey home and contacted Swedish authorities to confirm the identity of the 77-year-old woman, who had a Swedish passport. Seven-year-old girl who nearly died with her family in French Alps slaughter comes out of coma as her sister, four, returns to Britain under tight security Zeena Al-Hilli, 4, was orphaned in Wednesday's attack in eastern France Father Saad Al-Hilli, 50, died with wife Ikbal and his 'Swedish mother-in-law' Meanwhile sister Zainab, 7, has been brought out of coma and is conscious By Peter Allen The four-year-old-girl who escaped the Alps massacre in which her parents were murdered arrived back in Britain today - as her seven-year-old sister was brought out of a coma. Zeena Al-Hilli, 4, who cowered under her mother’s skirt as bullets riddled through the windows of her family’s BMW, was orphaned in Wednesday’s attack in eastern France. Her father, aerospace engineer Saad Al-Hilli, 50, died alongside his dentist wife Ikbal, 47, and another woman believed to be his 74-year-old Swedish mother-in-law. Scroll down for video Murder scene: Zaina is likely to be shown pictures of the car which may prompt her to recall the murders All died in a blaze of semi-automatic gunfire which lasted around 30 seconds and also killed 45-year-old cyclist Sylvain Mollier, a father-of-three who was hit by five bullets. Zeena remained unharmed and was so well hidden next to her mother and grandmother's body that it took police eight hours to find her. Annecy prosecutor Eric Maillaud has now confirmed that Zeena ‘is back in Britain’. He said that she had returned with an ‘aunt and uncle’, believed to be on her late mother Ikbal’s side of the family. Mr Maillaud said Zeena’s return to Britain had been carried out under conditions of ‘utmost secrecy’ for ‘security and safety reasons.’ Saad Al-Hilli who was shot dead in the French Alps alongside his wife and woman believed to be his stepmother ‘She will be far better off back in her home country,’ said a French judicial source. 'Zeena has given us all the information she is capable of providing, and there is no further benefit from keeping her in France.' It has also emerged that Zeena’s seven-year-old sister, Za inab, has been brought out of her medically induced coma and is conscious. P olice had been awaiting permission to question her. Zainab’s uncle and closest living relative, Zaid Al-Hilli, 53, will today be questioned in London over an alleged feud between him and the girls’ murdered father over an inheritance rumoured to be worth anything up to £1million. French prosecutor Eric Maillaud said Zeena had identified her family members, and described what he called the 'fury' and 'terror' of the attack to French police. But he added that because she hid behind her mother and grandmother's skirts when the gunfire began she did not see anything important. 'This is a little girl who must be protected,' he said. 'She should go back to the UK soon so that she can try and forget this nightmare.' The prosecutor said: 'We asked her, “Who were you with?”, and she said first, “With my dad”, and she gave a name, “With my sister”, she gave a name, “My mother”. 'The little girl said, talking like a little girl does, she didn't know [the Swedish woman] very well. We have to assess very clearly, who was that lady with the Swedish passport?' Two French gendarmerie vehicules parked in front of the CHU Hospital, in Grenoble, where one of the two daughters are staying following the shooting Emotional friends of the Al-Hilli family leave flowers at their home in Claygate, Surrey French and British police are waiting for the go-ahead from medical staff at the hospital to talk to Zainab, who was hit with 'tremendous ferocity'. Mr Maillaud said detectives hope the girl, who is in a stable condition, will recover sufficiently from the trauma to speak to them and that her memory was not damaged. He said: 'We are waiting for the ballistic team's report and, when possible, a hearing with the eldest girl. 'Maybe she can give us information on the number of people present for example, or the colour of their skin, and other elements of description that might allow us to consider a bit more seriously a first lead.' Today some 12 gendarmes stood guard at the hospital alongside, with one outside Zainab’s room being changed every four hours. Concerns have been raised about who will now look after the two girls full-time, although Mr Mauillaud said ‘two family members’ arrived in France on Saturday. The family home of Saad al-Hilli in Claygate, Surrey, has become part of the murder investigation ‘I don’t know exactly when they will see the girls but it will be in the presence of an investigator,’ said Mr Maillaud. He said Zeena would not be interviewed as an official witness in the future, but Zainab will be. ‘When she’s recovered, and medics give the green light, she will be interviewed,’ said Mr Maillaud. ‘Her injuries are still extremely serious. As long as we can’t talk to her, we won’t talk to her.’ Two gendarmes – both women – were given responsibility for establishing a dialogue with the two girls. Both women are trained psychologists, specialising in working with children. They are French women from Chambery, but have a translator with them at all times. Flowers were laid at the scene where the massacre took place in the French Alps The gendarmes have interviewed Zeena in a room furnished in bright colours and small armchairs and tables, to make her feel as comfortable as possible. They spoke to her in soft voices and did everything to ‘diffuse the situation so as not to induce a second trauma,’ said a police source. He added: ‘The aim is to create a fluid dialogue – to get the child talking on her own accord.’ The gendarme psychiatrists have been using the so-called ‘Melanie Method’ to interview Zeena, and will do the same with Zaina. It entails placing the child in a comfortable, relaxed environment with soft toys places around the room etc. Then the children are formally asked anything – they are simply ‘coaxed’ in a relaxed, informal manner. Photographs can be shown to the children. When Zeena was shown a picture of her mother, for example, she exclaimed ‘Mummy!’ Zaina is likely to be shown pictures of the car, and other aide memories which may prompt her to recall the chilling events which led to the four murders. VIDEO: Forensics officers search house of Saad Al-Hilli and floral tributes are left by friends and relatives 'He was a control freak, I wanted him wiped out of my life': Astonishing letter shows level of animosity between French Alps murder victim and his brother Childhood friend reveals note sent to her in row over father's assets Did his secret work on satellite contract make him prime assassination target? French investigators to quiz work colleagues at Surrey firm All four victims were shot twice in the head and may have been followed by their killers Four-year-old survivor of French assassination shown a photo of murdered mother and exclaimed'mummy' Police say they are looking at whether murderer may have lured family to killing spot An astonishing letter showing the degree of animosity between two brothers has been revealed today in the baffling murder in France of Briton Saad al-Hilli. As French detectives prepared to question his work colleagues, it emerged that there was a dispute between him and his brother Zaid. Mae Faisal El-Wailly, a childhood friend of the brothers, made available a letter written to her by Saad who described his brother as a 'control freak.' He also said he wanted him 'wiped out' of his life. Scroll down for video The inquiry into the murder of Saad Al-Hilli will now switch to Surrey Satellites Technology Limited (SSTL) near Guildford, pictured Mr Al-Hilli worked on map-making satellites, such as this one, police have discovered that the engineer was working on a secret contract for one of Europe's biggest defence companies The letter, quoted in the Sunday Independent, alludes to a dispute over their father's estate and was dated 16 September 2011. Saad wrote: 'Zaid and I do not communicate any more as he is another control freak and tried a lot of underhanded things even when my father was alive. 'He tried to take control of father's assets and demanded control. Anyway it is a long story and now I have just had to wipe him out of my life. 'Sad but I need to concentrate now on my wife and two lovely girls...' Zaid al-Hilli has yet to give his response to the letter, but on Friday he went to British police and told them: 'I have no conflict with my brother.' And a source close to the family insisted yesterday it was nothing more than a'minor squabble'. Victim: A newly released picture of Saad Al-Hilli who was gunned down in the French Alps French detectives will quiz Mr Al-Hilli's work colleagues after they discovered he was killed while on a secret contract for one of Europe's biggest defence companies. The inquiry's focus will switch to Surrey Satellites Technology Limited (SSTL) near Guildford this week when gendarmes will question the workforce about whether Mr Al-Hilli's job may have made him a target for assassination. As fears grew that Mr Al-Hilli and his family were the victims of contract killers, it emerged that: All four of the adults who died were shot twice in the head – the hallmark of professionals. Two mobile phones found within the Al-Hillis' car could provide vital clues for police. Police are investigating a theory the killers'shadowed' the al-Hillis as they travelled through France. British and French police yesterday conducted a forensic search of the family's £1.5 million home in Claygate, Surrey, as two members of the extended family arrived in France to comfort the orphaned daughters, Zeena, four, and seven-year-old Zainab, who remains in an induced coma after suffering a fractured skull during a suspected pistol-whipping. Iraqi-born aerospace engineer Mr Al-Hilli, 50, his 47-year-old wife Iqbal, and his Swedish mother-in-law were killed in a hail of about 25 bullets outside Chevaline in the French Alps on Wednesday. Local cyclist Sylvain Mollier, 45, was also murdered after disturbing the killers. The Mail on Sunday can reveal that Mr al-Hilli was part of a team involved in an undisclosed project linked to European Aeronautic Defence and Space (EADS) – a pan-European defence giant which has contracts with Russia, China and the Foreign Office. A British police crime scene investigator examines the interior of a window inside the Surrey home of Saad Al-Hilli This evening police remained a presence outside the Al-Hilli home where forensic officers have been searching for clues to following the murder of aerospace engineer Saad Al-Hilli SSTL, which was acquired by EADS in 2008, has raised security levels at its glass-fronted offices since the murders last week, with grieving staff barred from speaking about the tragedy. EADS lists bodies including NASA, the European Space Agency, and MoD defence contractor Thales as clients. A key partner in the Eurofighter project, the company also designs and launches satellites for clients who want an 'eye in the sky' for commercial, civil or security purposes. The Mail on Sunday has discovered that in December Mr al-Hilli visited a sub-division of SSTL called DMC International Imaging, which has recently signed a contract with the Chinese to help map the country via satellite imagery. DMC also has a lucrative satellite-mapping deal with Russia and is working with the Foreign Office in Afghanistan to monitor illicit opium poppy cultivation. Mr al-Hilli, a civilian contractor, has worked as a mechanical design engineer at SSTL for the past two years and was popular with colleagues. Several are listed as his friends on Facebook, but mysteriously none posted any comments on a tribute page set up on the social networking site last week. Now, as the murder inquiry broadens, French and British police are to probe his business dealings and the possibility that the sensitive nature of his work may be linked to the shootings. Investigation: French and British police arrive at the Al-Hillis home in Claygate, Surrey, yesterday One line of inquiry is that Mr Al-Hilli had access to information that would have been valuable to a commercial competitor – or that he had become a victim of blackmail. Yesterday Claude Moniquet, director of the Brussels-based European Strategic Intelligence and Security Centre (ESISC), said: 'Satellite technology, along with drone technology, is the new frontier of science and a market which is worth billions of pounds. 'Competition in a corporate sense is intense, as there is the potential to make a lot of money with each development ahead of your rivals. 'Mr al-Hilli's company was also a renowned leader in satellite mapping, and if it was secretly doing this in countries which would not welcome such an intrusion, then we have a possible motive.' Forensic officers searched the home of the Al-Hilli family yesterday. French investigators are working with British colleagues to find the killers He said another area French police may explore is whether a Middle Eastern group may have been involved. 'The Iranians, for example, are desperate to acquire cutting-edge technology which they cannot legally obtain. If somehow they were either getting it from Mr al-Hilli, or hoped to get it from him and he refused, they would not think twice about killing him. 'Also bear in mind that the Iranian intelligence service works in tandem with many private companies and this is a very lucrative market.' None of Mr al-Hilli's businesses appear to have been making much money, something which has raised questions about how he financed his lifestyle. Shtech, the aeronautical business he ran with his wife Iqbal and which had sub-contracted with SSTL, registered profits of just £8,330 last year. Grief: Family friends outside the Al-Hilli home in Claygate, Surrey, yesterday Mr al-Hilli's brother, who has a background in public relations, was the company secretary since its formation but was abruptly removed in favour of Iqbal in January last year. His Swindon-registered aerial survey company, AMS1087, generated just £2,118 in profits according to its latest accounts. RAF CYCLIST SAYS HE IS 'TERRIFIED OF BEING IDENTIFIED' The RAF veteran with 'nerves of steel' who discovered the bloodbath is terrified of being identified, French police revealed last night. Colonel Bertrand Francois, head of the local gendarmerie, praised the English cyclist for the way he acted after coming across the BMW with three dead bodies in it. He not only put seven-year-old Zainab Al-Hilli in the recovery position, which could have saved her life, he also turned off the car's engine and then called police. But 'he has been unable to sleep' since, said Col Francois, who added: 'His private life is over for the coming months. We are doing everything to protect his anonymity.' There have been fears the killers might target witnesses. Prosecutor Eric Maillaud also praised the cyclist, saying: 'His extraordinary conduct, testimony and bravery should be saluted. He's someone with nerves of steel.' Last night an SSTL spokesman pointedly refused to disclose the precise nature of Mr al-Hilli's work, citing commercial confidentiality. In a statement issued to the media, SSTL chief executive Matt Perkins paid tribute to his dead colleague, saying his murder had left him deeply shocked and saddened. 'Saad's colleagues will remember him as an experienced and committed engineer who worked as part of a tight-knit team,' he said. Yesterday, there was a strong security presence outside Mr al-Hilli's workplace in Guildford. Police and security staff, as well as two senior executives, were stopping all visitors entering the headquarters. Staff declined to answer questions about Mr al-Hilli and it was not until late on Friday that they finally confirmed his link to the company. The Mail on Sunday can reveal that Mr al-Hilli subscribed to the Google Circles social networking website, where he recorded his own movements.This showed that on December 14 and 15 last year he had been working at DMC Imaging, a subsidiary of SSTL. On December 16 he left the Brittany Ferries dock in Portsmouth Harbour on a business visit to France. Senior security sources said last night that the murders in France had no link to Britain's national security, but declined to speculate on whether Mr al-Hilli may have been caught up in industrial espionage. French investigators have said they would look at all aspects of his work. But Surrey Police, who confirmed that they are working with a team of French police, declined to say when officers would be speaking to Mr al-Hilli's work colleagues. Two mobile phones found in bullet-riddled BMW in which the three family members were shot may hold 'crucial clues to the murder. Detectives hope details of the victims' last calls and text messages – along with phone network records showing their movements – will help explain why they were targeted on a remote forest road. A French police source said: 'The phones are crucial to the enquiry. They are now being analysed by specialist officers.' It may also help build a better picture of exactly which route the family took on their 600-mile, 12-hour drive through France. If the family were not the victims of a random shooting, it is possible their killer or killers followed them. Police tape seals the caravan and tent used by Saad al-Hilli and his family while on holiday at the Le Solitaire du Lac campsite on Lake Annecy in the Haute-Savoie region of south-eastern France Delayed reaction: Several hours after police arrived on scene, Mr Al-Hilli's four-year-old daughter Zeena was found alive huddling under her mother's legs inside the car In Claygate yesterday, a procession of Surrey Police officers in scene-of-crime suits was seen entering and leaving the al-Hillis' home. French prosecutor Eric Maillaud said there was still a great deal of suspicion about the relationship between 50-year-old Saad and his brother Zaid, 53. Mr Maillaud said: 'We have raised the conflict between the two brothers as we try to find as much as possible about the companies that he [Saad] worked for and the assets he owned to see if there was any conflict between the two. 'Everyone talks of a dispute, but we have to try to establish that this is true.' But acquaintances of Saad in Mijas, Southern Spain, where his father Kadhim owned a small £40,000 flat above an expat bar, yesterday played down reports of a family feud over money. Saad put a block on his late father's will after he died in Spain in August last year, halting his brother Zaid's claim to any inheritance. Zaid has denied any feud after going to a police station near his home in Kingston, Surrey, to speak to detectives. Ali Al-Hilli, cousin of Zaid and Saad, said Zaid was distraught following the death of his brother and cannot understand why his brother would be targetted by gunmen. He told The Sunday Telegraph: 'When I spoke to him he was clearly devastated. he kept saying, 'why, why, why? How did this happen?' Police are also looking into the possibility that the Al-Hilli family may have been lured into an ambush by the murderers. Asked whether an ambush was a possible scenario, prosecutor Eric Maillaud said: 'Yes. It is something we would like to look at of course.' Little Zeena sees picture and cries 'Mummy!' The four-year-old survivor of the French assassinations has been shown a photograph of her murdered mother, and immediately exclaimed: 'Mummy!' Police psychologists used the picture to gently prompt Zeena Al-Hilli's memory without bringing back the full trauma of the killings. Meanwhile, two relatives have arrived in France with a British social worker to visit Zeena and her seven-year-old sister Zainab, who is in a medically induced coma at the CHU hospital in Grenoble. Police guard: Armed officers outside the children's hospital where Zainab al-Hilli remains in a coma Treatment: The CHU hospital in Grenoble where Zainab al-Hilli is being cared for after the shooting of her family Vigilance: French police keep guard at the hospital where the sisters are being treated Public prosecutor Eric Maillaud said the relatives would not be named for'security reasons'. Two female gendarmes trained in child psychology are coaxing evidence out of Zeena. They have already spoken to her via an interpreter in a room decorated in bright colours designed to make her feel as comfortable as possible. A source said: 'The aim is to create a fluid dialogue – to get the child talking of her own accord.' Mr Maillaud, one of the leaders of the inquiry, said of Zeena: 'The little girl was terrorised. She rushed under her mother's legs. She heard, but she didn't see anything.' He said Zainab's injuries – a fractured skull and a bullet in the shoulder – were still 'extremely serious', adding police were hoping she would talk 'at length' when she is finally able to speak. 'It's awful for a little girl to be a key witness, because she will have to talk about her own suffering,' he said. 'But she's seven and seven is the age of reason – sometimes. She can tell the colour of skin, the colour of clothes and other information we need.' | A new detail in this week's multiple murder in the French Alps: Saad al-Hilli, who was gunned down with his wife and mother, had written a letter calling his brother a "control freak" who should be wiped "out of my life." Saad and brother Zaid clashed over their father's assets, and Zaid and "demanded control," wrote Saad. "Sad but I need to concentrate now on my wife and two lovely girls..." The Daily Mail reports that Zaid is being questioned by police today but dismissed the conflict as a "minor squabble." As for Saad's daughters, the 4-year-old who was unhurt in the attack has returned to Britain to stay with an aunt and uncle, an official tells the Daily Mail. And 7-year-old Zainab, who was shot and bludgeoned, emerged from a medically induced coma today in a French hospital, the Guardian reports. Eric Maillaud, the prosecutor running the investigation, has called her a "key witness" but says there is no rush to question her: "It is terrible that a victim, especially a child... has to be asked questions, that will inevitably cause her even more suffering." | multi_news |
This is the shocking moment three armed raiders threatened Domino's Pizza staff with a knife and guns before making off with cash - in a raid which lasted less than 60 seconds. The men stormed into the takeaway in Corby, Northamptonshire, after pulling up outside the entrance in a red Saab. CCTV footage shows the masked trio sprinting into the store before diving head first over the counter and rounding up the frightened staff inside. Scroll down for video. CCTV footage captured the moment a masked gang of three men burst into the Domino's Pizza in Corby, Northamptonshire. One of the robbers can be seen holding a worker at knife point (right) while another rifles through the tills. The footage shows the robbers storming into the takeaway after pulling up outside the entrance in a red Saab. The gang fled from the takeaway in their getaway car just 55 seconds after they had entered the outlet. One of the men, wearing a red jacket, then grabs a worker around the neck and threatens her with a knife while another rifles through a till in a back room. The gang - who appear to be holding black handguns - can then be seen running back out of the store and vaulting over the counter before getting back into the waiting Saab. Northamptonshire Police have released footage of the raid which shows the robbers fleeing in their getaway car just 55 seconds after bursting into the takeaway last Thursday night. A robber appears to hold a gun as he issues instructions to a terrified worker kneeling on the floor. In the background, another member of staff is held at knifepoint. A robber wearing a red hooded top could be seen brandishing a knife during the 55-second raid last Thursday. Officers believe the red Saab - which could have been a 9-3 model - was stolen from a property in Barton Seagrave, Northamptonshire, overnight on September 22 and 23. A police spokesperson said: 'At about 10.55pm, three offenders entered the shop in Rockingham Park and threatened staff with a knife and what appeared to be handguns, forcing them to hand over cash. 'Another person remained outside in a vehicle - a red Saab, possibly a 9-3 model. CCTV footage shows the masked trio sprinting into the store before diving head first over the counter and rounding up the frightened staff inside. Terrifying: Two hooded robbers, one appearing to hold a handgun, stand over a frightened member of staff during the raid. 'The offenders then left the shop and drove off in the Saab, which had been parked directly outside the premises.' Two of the three robbers are wearing dark clothing with hoods pulled up while the third burglar has a bright red hooded top on. The trio, who are all wearing black gloves, were armed with knives and what appeared to be black handguns during the raid. Anyone who recognises the offenders or saw the red Saab in the area at the time are asked to call Northamptonshire Police on 101 or Crimestoppers on 0800 555111 | CCTV footage shows three robbers entering pizza outlet in Corby, Northamptonshire. The masked men are seen pulling up outside Domino's Pizza in red Saab. They burst into the store before diving over counter and rounding up staff. One terrified worker was grabbed around neck and threatened with a knife. Another raider then rifles through a till in a back room at the food outlet. Gang then seen fleeing in their getaway car 55 seconds after bursting in. | cnn_dailymail |
In the city of Brotherly Love, the home of Rocky Balboa, the place where disgruntled Eagles fans once booed Santa Claus, the increasingly personal and bitter fight for the Democratic nomination took a sharp pugilistic turn Wednesday night. The issue: whether either candidate is even fit for the White House. Hillary Clinton is not qualified to be president, Bernie Sanders told a crowd of supporters packed into Temple University's arena, delivering his fiercest jab yet to the struggling Democratic front-runner. Story Continued Below "Now the other day, I think, Secretary Clinton appeared to be getting a little bit nervous," he began. "We have won, we have won seven out of eight of the recent primaries and caucuses. And she has been saying lately that she thinks that I am, quote unquote, not qualified to be president. "Well let me, let me just say in response to Secretary Clinton: I don't believe that she is qualified if she is, if she is, through her super PAC, taking tens of millions of dollars in special interest funds," he said. "I don't think you are qualified if you get $15 million from Wall Street through your super PAC." Sanders pivoted to her record on foreign policy, saying, "I don't think you are qualified if you have voted for the disastrous war in Iraq. I don't think you are qualified if you've supported virtually every disastrous trade agreement, which has cost us millions of decent-paying jobs. I don't think you are qualified if you supported the Panama free trade agreement, something I very strongly opposed and which, as all of you know, has allowed corporations and wealthy people all over the world to avoid paying their taxes to their countries." Clinton's camp fired back almost immediately — with great umbrage taken — as the Sanders operation shot out new fundraising appeals keying off the senator's comments. Her campaign spokesman, Brian Fallon, denied that she had said the Vermont senator wasn't qualified to be president. "Hillary Clinton did not say Bernie Sanders was 'not qualified.' But he has now — absurdly — said it about her. This is a new low," he tweeted. "Bernie Sanders, take back your words about Hillary Clinton," he said in a follow-up tweet. Michael Nutter, a Clinton backer and former mayor of Philadelphia, where Sanders was speaking, tweeted his disdain for the remarks. "Tonight @BernieSanders said.@HillaryClinton wasn't qualified to be President. THIS is LOW and crosses the line. Take it back, Senator," Nutter wrote. Fallon retweeted the message with the hashtag #TakeItBackBernie. Another hashtag, #HillarySoQualified, became a top trend within hours of Sanders' remarks. Appearing on CNN's "New Day" on Thursday, Clinton supporter Rep. Adam Schiff (D-Calif.) called Sanders' qualification remark a "pretty extraordinary claim." "She’s probably among the very best qualified candidates to run for this office," said Schiff, the ranking member on the House Intelligence Committee. "She was just an extraordinary secretary of state. She has broad experience. I think she’ll make a tremendous commander in chief. I don’t think people will take that very seriously.” "Frankly," he said, "I’m much more concerned this week with what I heard Sen. Sanders say on the gun issue." Sanders told a newspaper recently that shooting victims should not be able to sue gun manufacturers. The Sanders camp, meanwhile, responded that Clinton had started the fight by declining three times on Wednesday to say that Sanders is qualified. When asked point-blank by "Morning Joe" host Joe Scarborough whether Sanders was ready for the Oval Office, Clinton raised the senator's recent interview with the New York Daily News. "Well, I think the interview raised a lot of serious questions," Clinton said. "I think of it this way: The core of his campaign has been 'break up the banks,' and it it didn't seem in reading his answers that he understood exactly how that would work under Dodd-Frank." Asked again whether Sanders is qualified, Clinton dodged. "Well, I think he hadn't done his homework, and he'd been talking for more than a year about doing things that he obviously hadn't really studied or understood, and that raises a lot of questions," she said. Asked a third time, Clinton said she would "leave it to voters to decide who of us can do the job the country needs." By Wednesday afternoon, the Sanders campaign was blasting out two fundraising emails reacting to Clinton’s comments on “Morning Joe,” as well as to an anonymously sourced CNN report claiming her campaign was planning to "disqualify him, defeat him and then they can unify the party later." "The Clinton campaign has been watching these Wisconsin results come in, and the delegate race of course is tight there, but the reality is they're running out of patience. So they're going to begin deploying a new strategy, it’s going to be called disqualify him, defeat him and then they can unify the party later,” one email said, emphasizing the need for cash to help mount a defense. Bernie Sanders addresses supporters during a campaign rally at Temple University on April 6, 2016, in Philadelphia, Pennsylvania. Getty “Polls in Wisconsin haven't even been closed for 24 hours, and we're already seeing the start of the Clinton campaign's full-on attack before the New York primary. We knew they were getting nervous, but candidly, we didn't think they would go this negative so quickly. We have to be ready for what comes next,” another Sanders campaign email read. A third email, sent to reporters after Sanders' comments had exploded online, quoted his attack on Clinton and all but accused her of complicity in Panamanian tax-evasion schemes. The Sanders campaign's more aggressive tone comes along with a newfound swagger. As the Vermont senator has racked up wins — though in states where the demographic mix favored him over Clinton — his aides have spoken more confidently about the once-unthinkable possibility of him becoming the nominee and have speculated about challenging Clinton at an open convention in July. Sanders has also outraised Clinton for three months in a row, pulling in more than $44 million in March alone. Before the evening rally at Temple, Sanders met with the editorial board of the Philadelphia Inquirer and Daily News. “I honestly believe we have the possibility of pulling off one of the greatest upsets in history,” he said, according to Philly.com. By Thursday morning, the Clinton campaign was punching back more aggressively and mobilizing its allies to denounce Sanders — and donate money. In a fundraising appeal to supporters, campaign deputy communications director Christina Reynolds called it a "ridiculous and irresponsible attack for someone to make — not just against the person who is almost certainly going to be the nominee of their party this November, but against someone who is one of the most qualified people to run for the presidency in the HISTORY OF THE UNITED STATES." "Show Bernie Sanders there are consequences for this kind of attack," she wrote, before asking recipients to donate $1. Sanders doubled down on his remarks speaking to reporters Thursday morning ahead of a speech to the AFL-CIO in Philadelphia, slamming the media fortheir perceived lack of interest "about why the middle class declines, about wage and income disparity." Referring to The Washington Post headline that first stoked his ire ("Clinton questions whether Sanders is qualified to be president"), Sanders remarked that he would stick to his message of economic populism but would not take Clinton's words lying down. “If Secretary Clinton thinks that just because I’m from a small state in Vermont and we’re gonna come here to New York and go to Pennsylvania and they’re gonna beat us up and they’re gonna go after us in some kind of really uncalled for way, that we’re not gonna fight back, well we got another — you know, they can guess again because that’s not the case,” Sanders said. “This campaign will fight back.” As Sanders spoke to reporters in Philadelphia, Clinton held an impromptu news conference in the Bronx during which she shrugged off Sanders' comments. “I don’t know why he’s saying that, but I will take Bernie Sanders over Donald Trump or Ted Cruz anytime," she remarked. "So, let’s keep our eye on what’s really at stake in this election. We have Republicans whose values are so antithetical to what’s right for New York or right for America." Hillary Clinton is seen aboard the campaign bus in Cleveland on the third day of a bus tour through Pennsylvania and Ohio. July 31, 2016 Hillary Clinton is seen aboard the campaign bus in Cleveland on the third day of a bus tour through Pennsylvania and Ohio. Melina Mara/The Washington Post The former secretary of state, senator and first lady is the Democratic nominee for president. Former secretary of state Hillary Rodham Clinton has hit the ground running, visiting several states on her quest to become the Democratic nominee for president. Former secretary of state Hillary Rodham Clinton has hit the ground running, visiting several states on her quest to become the Democratic nominee for president. Democratic presidential hopeful Bernie Sanders on Wednesday said that he does not believe Hillary Clinton is qualified to be president based on her acceptance of special-interest money, her support of free trade and her vote for the Iraq War. Sanders’s blunt assessment at a raucous rally here came at the end of a day of testy exchanges between the two White House contenders in a race that Sanders has prolonged by continuing to win nominating contests, despite Clinton’s formidable lead in the delegate count. Earlier Wednesday, Clinton launched a fierce two-pronged attack on Sanders, questioning her persistent challenger’s qualifications as a Democrat and for the presidency — although she stopped short of saying he was unqualified for the job. Appearing at a rally at Temple University, Sanders told supporters that “Secretary Clinton appears to be getting a little bit nervous.” “She has been saying lately that she thinks I am quote-unquote not qualified to be president,” Sanders said. “Let me just say in response to Secretary Clinton, I don’t believe that she is qualified if she is through her super PAC taking tens of millions of dollars in special-interest money. I don’t think you are qualified if you get $15 million through Wall Street for your super PAC.” How Bernie Sanders won Wisconsin, in less than 60 seconds. (Peter Stevenson/The Washington Post) “I don’t think you are qualified if you have voted for the disastrous war in Iraq,” he continued, referring to Clinton’s 2002 vote as a U.S. senator from New York. Sanders also criticized Clinton’s past support of trade deals, suggesting that that also undermines her ability to be president. Responding late Wednesday night on Twitter, Clinton spokesman Brian Fallon said Sanders had reached “a new low.” Counting down to what has become a make-or-break Democratic primary in New York on April 19, the two campaigns traded other zingers Wednesday via speeches, interviews and social media. “If you want to vote for me, I think you should know what I want to do, not just a lot of arm-waving and hot rhetoric,” Clinton said during a visit to a job-training program here. The former secretary of state spoke with new urgency, reflecting both the shrinking window for underdog Sanders to overtake her in the nominating contest and a growing grudge match over which candidate can rightfully claim leadership of a restless Democratic electorate. Sanders also threw some elbows Wednesday when he was asked during a CBS News interview whether he should apologize to victims of the Sandy Hook school massacre for voting for legislation that provided immunity to gun manufacturers — a position Clinton has continued to criticize. This summer's political conventions could get heated – but it certainly wouldn't be the first time. (Peter Stevenson/The Washington Post) “Maybe Secretary Clinton might want to apologize to the families who lost their loved ones in Iraq, or to the massive levels of destabilization we’re now seeing in that region,” said Sanders. Earlier in the day, Clinton did not try to disguise her frustration with Sanders, which bordered on scorn. “Like a lot of people, I am concerned that some of his ideas just won’t work, because the numbers don’t add up,” she told a union audience. “Others won’t even pass Congress, or they rely on Republican governors suddenly having a conversion experience and becoming progressives,” she asserted to laughter. “In a number of important areas, he doesn’t have a plan at all.” Sanders was set to address the same Pennsylvania AFL-CIO convention on Thursday, and both candidates were scheduled to return to New York after that. Although Pennsylvania offers a rich trove of delegates on April 26, it is the rough-and-tumble New York contest that both campaigns have cast as an essential test. Sanders plans a news conference Thursday in Philadelphia to highlight his opposition to a series of “disastrous” trade deals that Clinton supported. He has pressed that issue, with some success, in industrial Midwestern states. Sanders campaign manager Jeff Weaver said that among the deals that Sanders will talk about is the Panama free-trade agreement. In a statement this week, Sanders blasted the deal, saying it had enabled thousands of corporations to evade U.S. taxes by using a law firm in Panama. While Sanders was opposed to the deal from “Day One,” he criticized Clinton for reversing her position on the deal from opposition as a presidential candidate in 2008 to support while she was President Obama’s secretary of state. That put Clinton on the defensive before union workers Wednesday, and she devoted a large section of her speech to a defense of her approach to trade. Clinton has not been able to put the primary phase of the presidential campaign behind her despite holding a lead in overall votes and convention delegates from nearly the start of the contest. Sanders’s easy double-digit victory Tuesday night in Wisconsin was only the latest example of his staying power, while a fierce back-and-forth between campaign aides showed the increasing willingness to attack qualifications and character on both sides. “D-E-L-U-S-I-O-N-A-L,” Fallon tweeted about post-victory comments from Weaver. Clinton’s campaign never formally acknowledged the Wisconsin result. She spent Tuesday evening raising money in New York instead of holding a primary-night party.Her campaign ignored reporters’ requests for information about her plans ahead of time. On Wednesday, the Clinton campaign gloated over Twitter at the New York Daily News front-page critique of Sanders’s comments and record on gun control. Clinton aides also made hay out of Sanders’s stumble in an editorial board interview with the newspaper over his signature promise to break up big banks. “Let’s see how she does before the same editorial board,” Sanders spokesman Michael Briggs said of Clinton. Also Wednesday, Clinton implied that Sanders, a self- described democratic socialist, is not a full Democrat and might not feel the same fealty to the party and its other candidates. The senator has always caucused with Democrats in Congress but is an independent. “I think he himself doesn’t consider himself to be a Democrat,” Clinton said in an interview with MSNBC. “You know, look, he’s raised a lot of important issues that the Democratic Party agrees with, income inequality first and foremost. But it’s up to the Democratic primary voters to make that assessment.” A Clinton loss in New York would bolster Sanders’s claim that he can still catch up to her and become the nominee, perhaps in part by convincing Clinton delegates that she no longer deserves their support. The argument over who is or is not a Democrat is aimed primarily at elected Democrats, party leaders and activists, many of whom are already backing Clinton. As Sanders’s campaign has started talking about “flipping” Clinton delegates, she and her surrogates have begun to question Sanders’s commitment to the Democratic Party and to other elected leaders. Clinton supporters note that Sanders has not raised money for the party. Her campaign has recently emphasized how she and her husband, former president Bill Clinton, have worked for decades to support Democratic candidates.The point has become less subtle as Sanders’s recent string of caucus and primary victories — he has won six out of the last seven state contests — has eroded Clinton’s still-large lead among pledged convention delegates. “I’ve been in the trenches for a long time, and I believe in electing Democrats up and down the ticket,” Clinton said in the MSNBC interview. Weaver disputed Clinton’s contention that Sanders hasn’t helped Democrats in the past, saying he has both campaigned for them and helped raised money for them. Weaver cited fundraising letters Sanders had written for the arms of the Democratic Party that try to get members elected to the House and Senate. Watch CNN and NY1's Democratic debate, moderated by Wolf Blitzer, Thursday, April 14 at 9 p.m. ET. Washington (CNN) Bernie Sanders is standing by his criticism that Hillary Clinton isn't "qualified" to be president as the Democratic presidential candidates engage in an increasingly heated battle ahead of the New York primary. But by Thursday night, the Vermont senator said he didn't want to engage in a tit-for-tat with Clinton, telling CBS News they should be "debating the issues facing the American people." The controversy began late Wednesday when Sanders discussed Clinton's suitability for the White House. He cited a headline in The Washington Post as evidence that Clinton's campaign was questioning his qualifications, warranting his response. Sanders brings a giant printout of one of Donald Trump's tweets, in which the President-elect promised not to cut Social Security, Medicare and Medicaid, to a debate at the Senate in Washington on January 4. Sanders addresses delegates on the first day of the Democratic National Convention in Philadelphia, Pennsylvania, on July 25. Sanders speaks at a rally in Santa Monica, California, on June 7. He pledged to stay in the Democratic race even though Hillary Clinton secured the delegates she needed to become the presumptive nominee. Sanders speaks at a rally in Santa Monica, California, on June 7. He pledged to stay in the Democratic race even though Hillary Clinton secured the delegates she needed to become the presumptive nominee. Sanders speaks at a campaign rally in Ann Arbor, Michigan, on March 7. Sanders won the state's primary the next day, an upset that delivered a sharp blow to Clinton's hopes of quickly securing the nomination. Sanders and his wife, Jane, wave to the crowd during a primary night rally in Concord, New Hampshire, on February 9. Sanders defeated Clinton in the New Hampshire primary with 60% of the vote, becoming the first Jewish candidate to win a presidential primary. Comedian Larry David and Sanders appear together on "Saturday Night Live" on February 6. David has taken the role of Sanders in a series of sketches throughout the campaign season. Sanders sits with Killer Mike at the Busy Bee Cafe in Atlanta in November 2015. That evening, the rapper and activist introduced Sanders at a campaign event in the city. "I'm talking about a revolutionary," Killer Mike told supporters. "In my heart of hearts, I truly believe that Sen. Bernie Sanders is the right man to lead this country." Sanders embraces Remaz Abdelgader, a Muslim student, during an October 2015 event at George Mason University in Fairfax, Virginia. Asked what he would do about Islamophobia in the United States, Sanders said he was determined to fight racism and "build a nation in which we all stand together as one people." Sanders embraces Remaz Abdelgader, a Muslim student, during an October 2015 event at George Mason University in Fairfax, Virginia. Asked what he would do about Islamophobia in the United States, Sanders said he was determined to fight racism and "build a nation in which we all stand together as one people." Seconds after Sanders took the stage for a campaign rally in August 2015, a dozen protesters from Seattle's Black Lives Matter chapter jumped barricades and grabbed the microphone from the senator. Holding a banner that said "Smash Racism," two of the protesters -- Marissa Johnson, left, and Mara Jacqueline Willaford -- began to address the crowd. In July 2015, two months after announcing he would be seeking the Democratic Party's nomination for President, Sanders spoke to nearly 10,000 supporters in Madison, Wisconsin. "Tonight we have made a little bit of history," he said. "You may know that some 25 candidates are running for President of the United States, but tonight we have more people at a meeting for a candidate for President of the United States than any other candidate has." In March 2015, Sanders speaks in front of letters and petitions asking Congress to reject proposed cuts to Social Security and Medicare. In March 2015, Sanders speaks in front of letters and petitions asking Congress to reject proposed cuts to Social Security and Medicare. Sanders and U.S. Rep. Jeff Miller, chairman of the House Committee on Veterans' Affairs, walk to a news conference on Capitol Hill in 2014. Sanders was chairman of the Senate Committee on Veterans' Affairs. Sanders and U.S. Rep. Jeff Miller, chairman of the House Committee on Veterans' Affairs, walk to a news conference on Capitol Hill in 2014. Sanders was chairman of the Senate Committee on Veterans' Affairs. Sanders speaks to reporters in 2010 about the Obama administration's push to extend Bush-era tax cuts. Three days later, Sanders held a filibuster against the reinstatement of the tax cuts. His speech, which lasted more than eight hours, was published in book form in 2011. It is called "The Speech: A Historic Filibuster on Corporate Greed and the Decline of Our Middle Class." Sanders speaks to reporters in 2010 about the Obama administration's push to extend Bush-era tax cuts. Three days later, Sanders held a filibuster against the reinstatement of the tax cuts. His speech, which lasted more than eight hours, was published in book form in 2011. It is called "The Speech: A Historic Filibuster on Corporate Greed and the Decline of Our Middle Class." Sanders chats with Dr. John Matthew, director of The Health Center in Plainfield, Vermont, in May 2007. Sanders was in Plainfield to celebrate a new source of federal funding for The Health Center. Sanders chats with Dr. John Matthew, director of The Health Center in Plainfield, Vermont, in May 2007. Sanders was in Plainfield to celebrate a new source of federal funding for The Health Center. Sanders takes part in a swearing-in ceremony at the U.S. Capitol in January 2007. He won his Senate seat with 65% of the vote. Sanders takes part in a swearing-in ceremony at the U.S. Capitol in January 2007. He won his Senate seat with 65% of the vote. Sanders sits next to President Bill Clinton in 1993 before the Congressional Progressive Caucus held a meeting at the White House. Sanders co-founded the caucus in 1991 and served as its first chairman. Sanders sits next to President Bill Clinton in 1993 before the Congressional Progressive Caucus held a meeting at the White House. Sanders co-founded the caucus in 1991 and served as its first chairman. In 1990, Sanders defeated U.S. Rep. Peter Smith in the race for Vermont's lone House seat. He won by 16 percentage points. In 1990, Sanders defeated U.S. Rep. Peter Smith in the race for Vermont's lone House seat. He won by 16 percentage points. Sanders reads mail at his campaign office in Burlington in 1990. He was running for the U.S. House of Representatives after an unsuccessful bid in 1988. Sanders reads mail at his campaign office in Burlington in 1990. He was running for the U.S. House of Representatives after an unsuccessful bid in 1988. In 1987, Sanders and a group of Vermont musicians recorded a spoken-word folk album. "We Shall Overcome" was first released as a cassette that sold about 600 copies. When Sanders entered the U.S. presidential race in 2015, the album surged in online sales. But at a CNN town hall, Sanders said, "It's the worst album ever recorded." Sanders, right, tosses a baseball before a minor-league game in Vermont in 1984. U.S. Sen. Patrick Leahy, center, was also on hand. Sanders, right, tosses a baseball before a minor-league game in Vermont in 1984. U.S. Sen. Patrick Leahy, center, was also on hand. Sanders takes the oath of office to become the mayor of Burlington, Vermont, in 1981. He ran as an independent and won the race by 10 votes. Sanders takes the oath of office to become the mayor of Burlington, Vermont, in 1981. He ran as an independent and won the race by 10 votes. Sanders, right, leads a sit-in organized by the Congress of Racial Equality in 1962. The demonstration was staged to oppose housing segregation at the University of Chicago. It was Chicago's first civil rights sit-in. Sanders, right, leads a sit-in organized by the Congress of Racial Equality in 1962. The demonstration was staged to oppose housing segregation at the University of Chicago. It was Chicago's first civil rights sit-in. U.S. Sen. Bernie Sanders, an independent from Vermont, is the longest-serving independent in the history of Congress. U.S. Sen. Bernie Sanders, an independent from Vermont, is the longest-serving independent in the history of Congress. "The Washington Post had a headline that said 'Clinton questions whether Sanders is qualified to be president.' That was what was thrown at me." He also cited a CNN report, "Clinton strategy: Defeat Sanders, unify party" as further confirmation that the Clinton campaign started the attacks over qualifications. "I believe the Clinton campaign told CNN that their strategy is, 'we go into New York and Pennsylvania. Disqualify him, defeat him and unify the party later,'" he said. Speaking at a news conference in Philadelphia on Thursday, Sanders said, "They're going to question my qualifications, well I'm going to question theirs." Clinton herself has never said Sanders isn't qualified to be president. When asked Wednesday on MSNBC if she thought Sanders was "ready to be president," she said: "I think he hadn't done his homework and he'd been talking for more than a year about doing things that he obviously hadn't really studied or understood, and that does raise a lot of questions." "Really what that goes to is for voters to ask themselves can he deliver what he's talking about," she had said, referring to an extensive interview Sanders had given with the New York Daily News during which he struggled to answer policy questions related to his signature issue -- reforming Wall Street -- and other topics such as gun control and foreign policy. JUST WATCHED Hillary Clinton, Bernie Sanders brace for New York Replay More Videos... MUST WATCH Hillary Clinton, Bernie Sanders brace for New York 02:16 By Thursday morning, Clinton laughed off questions about Sanders' assertion that she isn't "qualified" for the presidency. "It's kind of a silly thing to say," she told reporters in New York. "But I'm going to trust the voters of New York who know me and have voted for me three times." At his news conference, Sanders said he was trying to run an "issue-oriented campaign" and blamed "the media" for taking things off course. "As we can see, the media is not very interested in wage decline in the middle class, that's not what you're interested in, but we have tried in rally after rally to talk about the most important issues facing the American people," he said. He then repeated on Thursday the same swipes on Clinton he cited the night before. "My response is if you want to question my qualifications, then maybe the American people might wonder about your qualifications Madame Secretary," he said. Sanders' remarks rankled Clinton's aides, with many arguing it shows Sanders' campaign growing desperate in the face of growing odds to win the Democratic nomination. Clinton's aides were outraged late on Wednesday night, when they gathered for a conference call about the change in tone. "Hillary Clinton did not say Bernie Sanders was 'not qualified.' But he has now - absurdly - said it about her. This is a new low," campaign spokesman Brian Fallon tweeted. Asked by CBS News' Charlie Rose Thursday night if he was getting into a "tit-for-tat" with Clinton, Sanders defended the latest round of blasts. "We should not get into this tit-for-tat. We should be debating the issues facing the American people," Sanders said. "All I am saying, if the people are gonna attack us, if they're gonna distort our record, as has been the case time and time again, we're gonna respond." Rose asked Sanders if it was going too far to suggest Clinton bears responsibility for deaths in the Iraq war. "Do I bear responsibility for the tragedy and the horrors of Sandy Hook? So, you know, let's get off of that," Sanders replied. "Of course she doesn't bear responsibility. She voted for the war in Iraq. That was a very bad vote, in my view. Do I hold her accountable? No." Hillary Clinton did not say Bernie Sanders was 'not qualified.' But he has now - absurdly - said it about her. This is a new low. — Brian Fallon (@brianefallon) April 7, 2016 In addition to a trove of delegates New York is an important symbolic contest. Sanders was born in the Empire State, and New York City has been at the center of the national political battle over income inequality -- a signature issue for the Vermont senator. But Clinton represented the state in the Senate, and her campaign headquarters is based in Brooklyn. | Suddenly, it's the Democratic race making headlines for fireworks between the candidates. On Wednesday, Bernie Sanders said Hillary Clinton isn't "qualified" to be president-and ticked off several reasons to make his case, reports CNN. In response, the Clinton campaign said he had reached a "new low." In a speech at Temple University, Sanders said his string of primary wins was apparently making Clinton nervous. "She has been saying lately that she thinks I am quote-unquote not qualified to be president," he said. "Let me just say in response to Secretary Clinton, I don't believe that she is qualified if she is through her super PAC taking tens of millions of dollars in special-interest money. I don't think you are qualified if you get $15 million through Wall Street for your super PAC." And he didn't stop there: "I don't think you are qualified if you have voted for the disastrous war in Iraq. I don't think you are qualified if you have supported virtually every disastrous trade agreement which has cost us millions of decent paying jobs." Clinton herself didn't respond to the attack, but campaign spokesman Brian Fallon did in a tweet: "Hillary Clinton did not say Bernie Sanders was 'not qualified.' But he has now - absurdly - said it about her. This is a new low." Sanders' remarks came after Clinton criticized Sanders for providing what she deemed to be shallow answers to questions about breaking up big banks and other policy issues. "He'd been talking for more than a year about doing things that he obviously hadn't really studied or understood, and that raises a lot of questions," she said. Clinton also reiterated that she thinks Sanders is big on "arm-waving and hot rhetoric" instead of practical reform. But as Politico notes, she didn't quite call Sanders unqualified to be president, though she avoided a question about whether he was qualified three times. Instead, she said she would "leave it to the voters to decide." All of it is leading up to what the Washington Post calls New York's "make-or-break" primary on April 19. | multi_news |
[School Gym]
(Annual Boy Toy Auction)
WHITEY: Come on, let's get those bids up, it's the Annual Boy Toy Auction. Alright I've got $25, do I hear $40? Oh come on people, this is for charity. $40?
GIRL1: $40
WHITEY: $40. Coming up right there. Anybody got $50? How about $50? $50?
GIRL2: $50
WHITEY: $50. And remember for the next five hours, till midnight tonight, these boys are at your call. How about $55?
GIRL3: $55.
WHITEY: $55. Going once. Going twice. Sold for $55. Pay your money and get your boy.
[At the "Pick up" table; Brooke is standing with the boy she bid on]
BROOKE: Look, I have credit cards, okay? Gold? Platinum, for crap sake.
WOMAN: I'm sorry. Auction rules say cash only. Okay?
BROOKE: Here's the thing. I'm kind of coming out of a dark place right now and I could really use the distraction. I need this boy and I need him tonight.
WOMAN: I understand. But by rule, I have to give him to the next highest bidder if she has the money to give. (A girl sticks money in their faces and the woman takes it. Brooke grabs the boys name tag that is being handed over to the girl)
BROOKE: Oh no. No, no.
WOMAN: Brooke.
BROOKE: Uh uh.
WOMAN: Brooke.
BROOKE: No
WOMAN: Brooke. (She slaps her hand and Brooke lets go of the name tag. She gives it to the girl) Thank you.
GIRL: Thank you. (She and the boy leave)
BROOKE: Okay that did not just happen because I had an entire evening planed, so what am I supposed to do now?
WOMAN: Well, there are four boys still up for auction, and a cash machine right down the street.
BROOKE: Okay. (she leaves)
[At the Auction]
(Haley meets up with Peyton and all the cheerleaders)
PEYTON: Hey.
HALEY: See anything you like?
PEYTON: I'm seeing everything I like.
HALEY: Really?
PEYTON: But I think I'm going to bid on Jake.
HALEY: You guys are really hitting it off, huh?
PEYTON: Just friends, really. What about you? Ready to fight off these rabid skanks for Nathan?
HALEY: Actually, I think I'm going to bid on Lucas.
PEYTON: Really?
HALEY: Yeah, I haven't gotten a chance to spend time with him much lately and I get Nathan for free. (Peyton laughs and nudges her)
WHITEY: Alright folks, here we go.
HALEY: Who is up next? (They look at the program) Oh no.
ALL THE GIRLS: Tim. (Tim comes on stage dancing and taking off his jacket. He swirls it around his head then shakes his butt in front of the girls. Peyton and Haley cover their eyes. Deb and a woman are laughing)
WHITEY: Do I hear $30? (Tim does some really bad dance moves and the girls are laughing at him) How about $20?
WOMAN: That boys going to pull something.
WHITEY: Can I get a ten spot?
DEB: I suppose I have some chores around the house. $8?
WHITEY: Sold! (Tim smiles at her and Peyton and Haley laugh)
HALEY: Oh Jake's up next. How much money do you have?
PEYTON: $87.53
HALEY: Okay. (Jake comes out in the Ravens mascot outfit with his jersey over it. Everyone cheers and laughs. He takes off the head and gives it to Whitey.)
PEYTON: $20.
WHITEY: I have a bid for $20. Do I hear $30?
VOICE: $30. (Jake starts taking off parts of the costume)
HALEY: Take it off!
WHITEY: $40?
VOICE: $40.
WHITEY: $40. Right over there.
PEYTON: $50.
WHITEY: $50. Going, going.
VOICE: $75.
WHITEY: $75.
HALEY: Bet it all.
PEYTON: $87.53
WHITEY: Going once.
VOICE: $100. (The girls look around for the girl)
WHITEY: $100.Going once.
HALEY: Who's bidding on Jake?
WHITEY: Going twice. Sold! For $100 American. The highest man of the night, Good job Jaglieski.
[Back Stage]
LUCAS: You dirty bird. Who bought you?
JAKE: I don't know. But whoever it is it will be fun. It's for charity, right?
LUCAS: Yeah man.
JAKE: Go get 'em.
WHITEY: Okay let's have our next boy.
LUCAS: Well, here goes. (He steps out on stage looking nervous)
HALEY: Yeah! I got you.
WHITEY: Alright, let's start the bid at $20.
GIRL: $25.
GIRL2: $35.
GIRL3: $50.
GIRL: $75.
GIRL2: $100.
GIRL3: I'll go $101.
HALEY: $105. $110. Oh what the hell. $115.
WHITEY: Sold!
HALEY: Cafe savings. No way he's worth it though. (Peyton laughs and Lucas leaves the stage)
WHITEY: Alright, we're down to our last boy toy, so loosen up those purse strings, pucker up those lips and remember, this is for a good cause. (Nathan comes on stage wearing sunglasses and everyone cheers) You're on, Nathan. (He tosses his glasses to Mouth who is the DJ and walks downstage. He pulls off his pants and Haley looks surprised) Okay, do I have a first bid?
GIRL: $80.
PEYTON: Wow. (Nathan takes off his shirt and his chest says "BOYTOY")
GIRL2: $85.
PEYTON: It smells like s*x in here.
GIRL3: $90.
HALEY: $91.
PEYTON: That's a creepy threesome.
WHITEY: Do I hear $92?
GIRL: $92.
HALEY: How much money do you have?
PEYTON: Well with the five you gave me, $92.53.
HALEY: Okay bid it.
PEYTON: What?
HALEY: You have to bid it, I want you to buy Nathan.
PEYTON: Why?
HALEY: To keep him away from them.
WHITEY: Going twice.
HALEY: Please, Peyton, please.
PEYTON: $92.53
GIRL: $109 and 40. (Haley looks angry and searches through her purse)
WHITEY: Going once.
HALEY: Where's that emergency 20. Come on. Where are you?
WHITEY: Going twice.
HALEY: Ah. (She pulls out her money and gives it to Peyton)
PEYTON: Oh! $112.53.
HALEY: Ha!
WHITEY: Sold!
HALEY: Yes! (she hugs Peyton) Thank you. (Brooke comes running in with money)
BROOKE: Wait, wait.
WHITEY: I'm sorry we're fresh out of flesh. I want to thank you all for coming this evening supporting this good cause. Now all these young men belong to the highest bidders till that midnight kiss. Let's try to keep things legal this year. (Mouth climbs on stage to collect all the microphones)
GIRL: I'll give $5 for the microphone boy.
GIRL2: I'll go 10.
WHITEY: Do I hear 20?
GIRL3: I will.
GIRL2: $25.
BROOKE: Oh no, I've got $200.
WHITEY: Sold! (He laughs and pats Mouth on the cheek) All sales are final.
[Karen's Cafe]
(Karen is cleaning and Larry comes in)
LARRY: I didn't see you in detention.
KAREN: Can I get you something to eat?
LARRY: Actually I was wondering if I could get you something to eat. Unless you've got plans after work.
KAREN: No.
LARRY: Great. We could go to my place. (Karen doesn't answer) Karen. I'm not a serial killer. It's just a couple of new friends eating food.
KAREN: Sure. Okay.
LARRY: Good, then it's a date.
[Auction]
(Brooke is walking around with Mouth talking out loud to whoever they pass)
BROOKE: Okay, I've got $50 cash on the Mouth-Boy for trade. Who wants him? (Mouth looks at her confused)
(They pass and Deb is left standing in front of Tim)
DEB: Well, Timmy. Looks like you're all mine tonight. How about you grab a change of clothes and I'll meet you at the house.
TIM: Change of clothes?
DEB: Well you'll probably want to take a shower after I get through with you. I plan on getting dirty. So I'll leave the door unlocked. Just come in and get me. (He watches her leave. Jake is walking around looking for the girl who won him. Nikki comes up behind him)
NIKKI: Well, well, Jake. Once again, looks like I own you. (Jake starts walking outside and she's following) Come on, Jake, wait up.
JAKE: I'm not kidding, Nikki. I'm not falling for your crap.
NIKKI: At least talk to me? (He stops and faces her)
JAKE: About what? Where you've been for the last 8 months or how my daughter doesn't have a mother?
NIKKI: I understand you're upset, but there are things you don't know. Please. Jake, come on. I don't want to mess with your head. But, the least you could do is hear me out. After that you can ditch me if you want.
JAKE: What, like you did me?
[Outside]
(Mouth is standing through the sunroof of a limo talking to Brooke who is by the car)
MOUTH: This things is awesome. Where are we going first?
BROOKE: Here's the thing, Lips.
MOUTH: Mouth.
BROOKE: I know. And I know I owe you for the whole cheerleading competition, but I had this whole night kind of perfectly planned out and it's a waste of a Brazilian wax. (He looks upset)
MOUTH: Okay. That's cool. I can take off. (He climbs out of the car) At least let me give you some of your money back.
BROOKE: Oh, no, no, no. I don't want that. Please.
MOUTH: No, no I understand. I just thought the car was cool. Um, I'll see you later. (He starts to walk away)
BROOKE: Wait. Fine! I'll take you to once place.
MOUTH: Really?
BROOKE: What the hell. It's for charity, right? With the night I have planned I might need you to carry me home anyway.
MOUTH: Sweet. (He gets back in the car and Brooke gives the limo driver a look)
[Outside]
(Haley walks up to Peyton)
HALEY: I am so excited to have Lucas all to myself for an evening. We have not had a night to just hang out in forever. (Nathan walks up to them)
PEYTON: Yeah, same goes for me and Nathan.
NATHAN: I know you're still into me, but $112? (Haley laughs and hits him with the papers) Hey you.
HALEY: Hey.
NATHAN: How about you come see me around midnight?
HALEY: Sounds good. (They kiss) Don't have fun.
NATHAN: I wont. (Peyton jokingly looks hurt) Alright, come on. Let's get this over with. (He walks away)
PEYTON: Funny. That's what he'd say before we used to have s*x. (Haley fake laughs and Peyton walks away and jumps on Nathan's back. Haley watches them looking a little hurt)
[Nathan's Apartment]
(They come in and everything is covered with plastic and there is hardly any furniture. Peyton looks around)
PEYTON: So you're planning to kill me?
NATHAN: The painters must have left it. So what do you think of the new place?
PEYTON: You really moved out, huh?
NATHAN: Yeah. Judge declared me emancipated.
PEYTON: Good for you, Nate.
NATHAN: Come on. Check it out. (They walk into the back bedroom)
PEYTON: So what, you got your big cool apartment but you don't believe in furniture? Where are we supposed to eat?
NATHAN: We got the bed. (He sits down and pulls out food from the bag)
[Checkers Restaurant]
(Jake and Nikki are outside eating)
JAKE: Still doing that whole desert before the meal thing, huh?
NIKKI: You used to think it was cute. (She tries to feed him something but he doesn't take it)
JAKE: Yeah. That was before you abandoned my child.
NIKKI: Okay. I guess we'll do this now. I made a mistake. I didn't know what kind of mother I'd be. All my friends were going off to college, my parents...well you know they felt. I just wasn't ready, Jake. Is it that hard to understand?
JAKE: Yeah, Nikki, it is. She was a part of you. You held her in your arms. I spent every night asking myself how you could just leave her behind.
NIKKI: I couldn't. I hated myself for leaving, that's why I came back. I want to be in her life, Jake. And yours too. I missed you, you know? I wanted to call you a thousand times over the last 8 months.
JAKE: Yeah but you didn't. Did you?
[Sawyer House]
(Larry and Karen are having dinner)
KAREN: Well, that was a great dinner, Larry.
LARRY: Thanks. Single parent cooking class. I'm glad you decided to come. Not too painful?
KAREN: No, not at all.
LARRY: But you don't date much?
KAREN: I don't date at all, really. At night I work or I'm at home with Lucas. Sometimes with Keith.
LARRY: Keith seems like a good man.
KAREN: He is. (clears throat) He's a good friend. Well...(She starts to clean up)
LARRY: Oh no. Absolutely not. You're off duty and not allowed to touch anything but your wine glass. Tour the house, make a long distance phone call, whatever, it's called relaxing. (He gets up and takes the dishes away)
KAREN: Well you know I do have some friends in Florence. Maybe I should give them a call.
LARRY: Uh oh.
[On the Roof]
LUCAS: Haley, now this was a great idea.
HALEY: I know.
LUCAS: Did you fill any with milk like we used to?
HALEY: Yeah I did. Some of them. I can't do this stuff with Nathan. It just seems, I don't know, does it seem childish?
LUCAS: Well, yeah.
HALEY: Great!
LUCAS: But in a good way.
HALEY: So, what's your situation now?
LUCAS: What do you mean?
HALEY: With Peyton? Or Brooke. Or bar-slut that I heard about.
LUCAS: Okay. What'd you hear and who'd you hear it from?
HALEY: Just stuff...from people.
LUCAS: I just want to play ball again. You know? Get over this damn drama. You know it wasn't long ago I was happy playing hoops with the guys and hanging out with you.
HALEY: Yes, life was much simpler then, wasn't it? I think I'm going to go call Nathan. (She starts to walk away and Lucas throws a water balloon at her) Ohhh. You are so dead.
LUCAS: I guess that one was a milk balloon. (Haley grabs one and throws it back at him)
[A Club]
(Mouth and Brooke are sitting at a table with a girl dancing above them)
BROOKE: I think she likes you. (Mouth laughs)
MOUTH: So thanks for bringing me out tonight. I've never been to a real club before.
BROOKE: I'm glad I brought you.
MOUTH: Yeah?
BROOKE: Yeah. I enjoy corrupting America's youth. It's kind of one of my hobbies.
MOUTH: So was Lucas one of your test subjects?
BROOKE: Can we not go there tonight?
MOUTH: Okay. Sorry. That girls practically naked up there.
BROOKE: So you don't like naked girls?
MOUTH: Well I've never actually seen one up close. But from what I've found online I'm thinking they're okay. (Brooke grins at him) What?
BROOKE: Want to find out for sure? (She gets up and takes his hand) Let's do some damage.
[Sawyer's House]
(Karen is flipping through Peyton's sketches)
LARRY: Kind of severe, huh? (She sees the one of her, Brooke and Lucas shooting the #3 heart.)
KAREN: Kind of familiar actually. You know when I was a cheerleader we buried a time capsule midfield of the football stadium. I bet if you dug it up you'd find a version of the same thing our kinds are going through now.
LARRY: Well, I got a couple of shovels in the garage. Want to find out?
KAREN: Yeah.
[Pool]
PEYTON: Sweet. You got a pool?
NATHAN: Yeah. You want to go swimming?
PEYTON: No, you are the boy toy you do what I say tonight.
NATHAN: Oh just like old times.
PEYTON: Please. You did whatever you wanted and most of the time it was either sucky or mean.
NATHAN: I know. But you let me.
PEYTON: Well maybe I kept thinking you'd change. Live and learn, right? Is this thing heated?
NATHAN: I don't know, why don't you check it out? (She bends down to touch the water and he grabs her and they fall in. Nathan is laughing)
PEYTON: Oh my god!
NATHAN: I slipped.
PEYTON: What?!
NATHAN: I slipped. Hey, at least it's heated. (Peyton pushes him under)
[Rooftop]
(Haley and Lucas are throwing water balloons at each other.)
[Pool]
(Nathan and Peyton are still splashing each other in the pool)
[Club]
(Brooke and Mouth are lying on a bed in a back room with two strippers dancing on top of them)
BROOKE: Real thing sure beats the internet, huh?
MOUTH: The internet sucks. (Brooke laughs)
[Skipping back and forth between Peyton and Nathan having a good time in the pool and Lucas and Haley on the roof with water balloons, showing the two couples on their dates]
[Deb's House]
(Tim slowly opens the front door)
TIM: Hello? Deb? Miss Deborah?
DEB: Come on in, Timmy, I'm in the bath. (He smiles and puts his bag down. He gets to the bathroom)
TIM: Ready or not, here I come. (Deb is sitting by the bathtub cleaning the hair out of the drain. Tim comes in wearing zebra stripped underwear. They start screaming as they see each other.) Sorry!
DEB: Timmy! (Tim is running around gathering his clothes) I am so sorry. I don't know what gave you the impression that-
TIM: It's okay. I'm fine. (He is trying to put his pants on and falls. Deb comes over to help him)
DEB: It's just that you startled me and -
TIM: Where's my shirt? (Deb bends down to pick it up. Dan walks in and sees Deb on her knees in front of Tim who has his pants half way down. Dan laughs)
DAN: The lawyers are gonna love this one. (He walks out)
[Rooftop]
(Haley is hiding behind a tree)
HALEY: Luke? I'm all out of balloons. Can we please call a truce?
LUCAS: Is it a real truce or a trick truce?
HALEY: It's a real truce, I promise.
LUCAS: Okay.
HALEY: Okay. (She comes out from behind the tree) Or not! (She pulls out one last balloon but Lucas throws his at her first. He grabs her) No Luke, you're going to hurt your shoulder. (She drops her balloon and it breaks) Oh you are so lucky. (Lucas notices something on her back) What?
LUCAS: What's on your back?
HALEY: Nothing.
LUACS: Haley, is that a tattoo?
HALEY: No. It's nothing.
LUCAS: Haley. (He turns her around and there is a small 23 tattooed on her lower back) 23. That's great, Hales. You see, that's why I don't like the guy.
HALEY: Lucas.
LUCAS: No, that's just like him! To get you branded with his jersey number right above your @#%$?
HALEY: He doesn't even know about it. I just, I just did it.
LUCAS: By yourself?
HALEY: Yeah, by myself.
LUCAS: Haley, why would you do that?
HALEY: Because I'm in love with him. (She walks away)
[Karen's Cafe]
(Haley is drying her hair then throws the towel to Lucas and she gets out two mugs and hot chocolate)
LUCAS: Look, I didn't mean to freak out on you up there, okay? But, a tattoo?
HALEY: You got one. You got one with a girl you're not even dating anymore.
LUCAS: I know. How stupid do I look?
HALEY: You hold me to a higher standard than everybody else, Luke, and it's not fair.
LUCAS: Look, I know it's not fair, okay? But that's because I've seen you be better than most people. Let me see it again. (She turns around and lifts up her shirt) How long ago did you do it?
HALEY: A few days ago.
LUCAS: And Nathan had nothing to do with it?
HALEY: No, I told you, Nathan doesn't know. Ugh, God, what am I going to do, Luke? I'm so, I hate being away from him, I think about him constantly. I was in the middle of a history quiz yesterday and I just totally zoned out on him. Maybe we're not going to be together for the rest of our lives, but right now I'm in love for the first time and if I look at this tattoo 20 years from now, and it reminds me of how I feel today, I think I'll be okay with that.
LUCAS: Then why didn't you buy Nathan at the auction? Why hide out with me?
HALEY: Because, I wanted to remember for a night the way that things were. Everything was so much simpler when it was just you and me. And I'm used to being self-confident, and sensible and, I just really feel like a mess right now.
LUCAS: You're not a mess. You're just in love.
HALEY: And I'm not sure if he is.
[SCENE_BREAK]
[Pool]
NATHAN: What happened to us, Peyton? We used to be good together.
PEYTON: No, we weren't. We just had s*x a lot.
NATHAN: You sure about that?
PEYTON: Trust me. You're the only guy I've been with. You knew that.
NATHAN: Yeah, I know, I just, I figured since we broke up, maybe-
PEYTON: No.
NATHAN: Yeah, me neither. It's just s*x, right?
PEYTON: Okay, you know what, Nathan? Haley really deserves better than that. She really, really likes you and she's good for you and more than that, she trusts you-
NATHAN: I know that.
PEYTON: And okay, fine. We could do it, right here in the pool, and nobody would know.
NATHAN: Peyton...
PEYTON: But I would know, and you would know, but I wouldn't do that to Haley. Or myself. Or even you for that matter. Cause you know what? If you screw things up with your relationship with her, than you're a bigger jackass than even I thought.
NATHAN: I know it would hurt Haley. The only reason I mentioned it, was to let you know that I'm not pressuring her.
PEYTON: Right. I knew that.
NATHAN: You said I was good in bed.
PEYTON: No, I didn't.
NATHAN: Oh yes you did.
PEYTON: Oh, God, kill me.
[Checkers]
(Jake and Nikki are walking to the car)
NIKKI: So I left school for good by the way. Thought I might transfer here. Will you at least tell me how she's doing? (He gets in the car) Right. (She gets in) So I guess asking you how you're doing is out of the question. (She takes the keys out of the ignition) You look good.
JAKE: Give me the keys, Nikki. (She is leaning over onto his shoulder) Stop it.
NIKKI: Come on. I missed you, Jake.
NIKKI: Remember how good we were? (She has her face against his neck) I know it's been hard for you, I do. Let me make it easier. (She kisses him and he returns it.) Come back to me. We could be a family. (They kiss for a few seconds then Jake throws her off)
JAKE: Damn it. Damn it, Nikki. You almost had me again. You want to impress me with your Maternal instincts? Get out. (They both get out) Why don't you go buy all the things you think Jenny might need. I'll wait.
NIKKI: Okay.
JAKE: Hey, by the way. She's 9 months old, just incase you forgot.
NIKKI: I'll be fine.
[Whitey's Office]
KEITH: Hey.
WHITEY: Hey, Keith, come on in.
KEITH: I saw your car. So how did pimp duty go?
WHITEY: That's charity coordinator.
KEITH: Right. Assuming you'd raffle yourself off, huh?
WHITEY: Nobody could afford me. I'm glad you came by, Keith. I've been wanting to ask you a question. (he pours them drinks) When do you plan to start living?
KEITH: Well, I am living, coach.
WHITEY: No you're not. You're dying. I for one think it's a damn shame. You know what I'd change in my life if I could? I'd have Camilla back. Just to spend one more day with her. When we were young and in love. You love Karen, don't you?
KEITH: Yeah, I do. And I plan on telling her that it's just, things have been kind of messy since the accident, you know?
WHITEY: I understand. There's something you need to understand. Everyday you wait, is another day you'll never get back again. Trust me on that, son. I know.
[Brooks and Mouth's Limo]
BROOKE: So why don't you have a girlfriend? You're a nice guy.
MOUTH: Well that's the problem. I'm too nice. Girls like jerks.
BROOKE: Yeah, tell me about it.
MOUTH: You mean Lucas?
BROOKE: I thought we weren't going to talk about Lucas tonight, but I could just throw your @#%$ out at the next light.
MOUTH: Okay. So let me ask you a question. What do girls want?
DRIVER: Half your paycheck. (Brooke rolls up the window between them)
BROOKE: Here's my philosophy on dating. It's important to have somebody that can make you laugh. Somebody you can trust. Somebody that, you know, turns you on. And it's really, really important that these three people don't know each other. (They laugh)
[Jake's Car]
(He's waiting for Nikki to come back. She comes walking up and takes things out of the bag)
NIKKI: I got the cutest little stuffed animal.
JAKE: She has a purple monkey that she can't sleep without. Anything else she ignores. (He throws the stuffed animal onto the car. He takes things out of the bag) You got the wrong formula. She needs a special kind because, well she wasn't breastfed. The alcohol in these wipes are bad for her skin. I buy her a special vitamin because she was a little underweight at her six month checkup. She doesn't read. She sure as hell doesn't smoke. Oh well look, at least you got the most expensive kind of ice cream.
NIKKI: I got that for you. Cause on our third date you said that all you needed for life to be good was a pint of this ice cream. I want your life to be good, Jake. I want to be with the boy that told me those things. Where did he go?
JAKE: You left him. Damn it, Nikki, it's not fair for you to come back here and do this. It's not fair to me, and it's not fair to Jenny.
NIKKI: I still love you, you know?
JAKE: It's funny. I can't tell you how many times I spent wondering when I was going to hear you say that again. Just hoping the next time the phone rang it'd be you, calling to say those words.
NIKKI: Jake.
JAKE: And now that you're here, I, I cant even remember why I needed to hear them. You should recognize this next move, Nikki. You perfected it. It called turning my back and leaving you behind. (He get in the car. Nikki has a tear go down her face)
[Football Field]
(Larry and Karen are walking on the field with flashlights)
KAREN: I don't know about this, Larry. We're trespassing.
LARRY: No we're not. Our taxes paid for this place.
KAREN: Oh, okay, let's try vandalism, theft...
LARRY: We loosen dirt, on a field we paid for, get property that belongs to you, and we put the dirt back. What could they charge for us?
KAREN: Immaturity. (She hands him her beer)
LARRY: There's a difference between growing up and growing old, Karen.
KAREN: Give me the shovel. (She starts to dig)
[Another Club]
(Mouth and Brooke are dancing together)
MOUTH: I can't believe you do this every night. You have the greatest life. (She looks around and makes eye contact with a guy at the bar. She walks over to him. Mouth continues to dance with other girls)
BROOKE: Hi.
GUY: You look hot tonight.
BROOKE: Thanks. (She kisses him) So, what's your name?
GUY: You don't remember the last time we did this. You were pretty wasted. You're name is Brooke, right? (Brooke realizes what she had done and looks around the room at all the people hooking up) So, Brooke. Are we going to do this again, or what? (She looks upset and walks away slowly. Mouth notices her leaving and follows.)
MOUTH: You okay?
BROOKE: I need to go home.
[Limo]
(Brooke looks in scared and sad. They are sitting in silence with Mouth watching her)
MOUTH: Brooke, did something happen? (She nods, almost crying) Do you want to talk about it?
BROOKE: How long have you known Lucas?
MOUTH: Since 4th grade. I transferred in.
BROOKE: And you think he's a good guy?
MOUTH: I think he's a great guy. Why?
BROOKE: Remember when I told you what girls want? Girls just want somebody to want them back. At least I do. (She starts crying and leans on his shoulder)
[Football Field]
(Larry is digging and Karen is drinking and doing a cheer next to him)
KAREN: If it's action that you're craving, go and get yourself a Raven. We said go. Ravens. Go mighty Ravens. If you really want to score, gotta dig a little more. We say go, Ravens, go mighty Ravens. (Larry hits something with the shovel)
LARRY: Oh. We got treasure. (They pull out a big box) Oh yeah. Check it out.
KAREN: Oh! (She pulls out a yellow shirt)
LARRY: Bon Jovi. I saw that tour. Scorpions opened for them.
KAREN: Yeah. Oh no!
LARRY: Check out the hair. (They have a picture of Karen and Dan together)
KAREN: Me and Dan.
LARRY: Is that Keith?
KAREN: Yeah. He was always very protective. (Keith is standing in the background behind them) You know I don't think I ever noticed Keith in the picture before.
LARRY: Well the kid in the background is definitely in love with the girl in the foreground.
WHITEY: What in the sam hill is going on here?
KAREN: Oh my God.
WHITEY: Karen?
KAREN: Hi, Whitey.
LARRY: Offer you a beer, coach?
KAREN: We were, um, having dinner and - (Keith comes up behind Whitey) Oh hey, Keith. (He turns around and leaves. Whitey follows him. Karen watches them and Larry laughs.)
[Nathan's Apartment]
PEYTON: It's nearly midnight. Looks like our date's almost over.
NATHAN: Yeah. I think I was hitting on you in the pool.
PEYTON: You think you were?
NATHAN: I don't know anymore. This whole good guy thing, it's new to me. I guess I'll always have feelings for you, Peyton. But I owe it to Haley to be a better guy than I've been. I just don't want to be the kind of guy that cheats no her.
PEYTON: Then don't be. Nathan, I'm proud of you. You know, standing up to your dad, and being a good guy for Haley. You're turning into the kind of guy I always knew you could be.
[Outside the apartment]
(Lucas and Haley are coming up the stairs)
LUCAS: So Nathan got his own place.
HALEY: Yeah this is it. Oh crap. I left some cds for him in the car. I'll be right back.
LUCAS: No, I'll come.
HALEY: No, it's alright. Um, why don't you go ahead and go on in, apartment 11. Hey, ask him if he's in love with me, and if he says no, break up with him for me, okay?
LUCAS: Okay.
HALEY: Great. Hey. Seriously. Say something nice, okay? I mean, he's really a different person.
LUCAS: Okay.
HALEY: Okay. (She goes back down and he goes to the apartment)
[Inside]
PEYTON: I better get going.
NATHAN: Okay. I guess by rule, I owe you a kiss.
PEYTON: I guess so. (Lucas walks up to the door and sees in. Peyton and Nathan kiss for a second then smile at each other. Lucas backs away from the door so they don't see him)
[Outside the apartment]
(Lucas came down to Haley before she could go up)
LUCAS: He wasn't there.
HALEY: Are you sure? Apartment 11? (She calls his cell phone)
LUCAS: Yeah.
HALEY: Voice mail. Great. I guess he's not done yet. I told you Peyton was easy.
LUCAS: Come on, I'll take you home.
HALEY: Okay.
[Limo]
(The driver opens the door for them and they get out)
MOUTH: I guess I should have told my parents I'd be out late. I'm not sure this was a good idea.
BROOKE: Mouth, you've got to live a little.
MOUTH: No, it's not that. I mean, a night with you is like flying first class. My life is coach. It's going to be hard going back to it.
BROOKE: Thanks, but this gets old, pretty quick. Trust me.
MOUTH: Listen, Brooke. I don't know what's going on with you and Lucas but he's a really good guy. I've never been really good at sports, I mean, I'm little, you know. But when I decided I wanted to be a sports announcer, Lucas introduced me to the guys at the river court and it made me feel like I belonged. He has a good heart. And as far as I can tell, you do too. I'd be really surprised if you two couldn't work things out.
BROOKE: Thank you.
MOUTH: Well, I should get home. That stripper might booty call me. (He starts to walk away)
BROOKE: Mouth. (She gives him a kiss)
MOUTH: Oh right. For the charity thing.
BROOKE: Nah. Just cause.
MOUTH: Hey, Brooke. Thanks. This was the greatest night of my life. (he leaves and she gets in the car)
[Haley's House]
(Lucas pulls up in front)
HALEY: Well, did you have fun tonight, slave boy? Thanks for playing along.
LUCAS: Look, Haley. I know we've grown apart a bit lately. And I know we have a lot a head of us. But, I just want you to know I'll always be there for you. And if Nathan doesn't see how special you really are, well, then he's an idiot. Cause I think you're amazing.
HALEY: Thanks, Luke. Oh, technically you owe me a goodnight kiss. (She doesn't look happy about it)
LUCAS: Mm. Rules are rules, I guess.
HALEY: Yeah, I guess so. (They start to lean in but she stops them) Here's the thing, though, if your tongue comes anywhere near my mouth I'm just never speaking to you again. (Lucas laughs) (They have a quick peck on the lips then hug)
LUCAS: Hey, Haley. You're going to be okay. I promise you that. (She smiles and gets out of the car)
[Jake's House]
(Nikki is at the door waiting for him. He open it.)
JAKE: Nikki, don't do this. (He tries to close the door)
NIKKI: Jake, please. Keeping Jenny away from me because I hurt you is wrong. (She tries to look in but he comes out and closes the door behind him) Can't you just forgive me?
JAKE: It was a Wednesday.
NIKKI: What? What was?
JAKE: The day I realized that you weren't coming back. I loved you. You knew I loved you. I would have done anything for you. But you betrayed me. So it was that same Wednesday that I stopped loving you. And I promised myself that my daughter would never feel the pain that you caused me. You want to hear me say it? Fine. You broke my heart, Nikki. But you will never break my daughters heart because you will never, ever have the chance to.
NIKKI: I'm going to be in her life, Jake. With you or without you.
[Lucas' House]
(Karen walks in with Larry)
KAREN: So would you like some coffee?
LARRY: It's getting late, I should probably go.
KAREN: I had a lot of fun tonight. I didn't do much of this in high school.
LARRY: Well maybe next time we could do something less juvenile.
KAREN: Oh well, if we have to.
LARRY: Karen, I know you have some history with Keith. I hope I didn't cause any problems tonight.
KAREN: No.
LARRY: But I like you. For what it's worth.
KAREN: Well I like you too, Larry. And you're right. I do have history with Keith. I'm just not sure we have a future. (He kisses her)
LARRY: Goodnight.
KAREN: Goodnight. (he leaves then the doorbell rings. Keith is there when she answers it) Keith.
KEITH: It's not too late is it?
KAREN: No. Is everything okay?
KEITH: Not really. These last few weeks have been the hardest of my life. Being without you and Lucas, on the outside of your lives. I felt, I felt like a shadow of someone I used to be. And I know I let you down. And , I put Lucas at risk, but you have to know, I love Lucas. And I've been carrying this around with me since the night of the accident. (he pulls out a ring box) I love you Karen.
KAREN: Keith.
KEITH: I always have. Marry me.
[River Court]
(Lucas is sitting on the picnic table. Brooke comes over to him)
BROOKE: I need to talk to you.
LUCAS: Okay.
BROOKE: I spent the entire night trying to avoid this or ignore it, but I cant so I've just got to say it
LUCAS: Brooke what's up?
BROOKE: I think I'm pregnant. (he looks at her shocked) | Lucas, Nathan and Jake are auctioned off to the highest bidder in the annual "Boy Toy" charity auction and the night leads to surprising developments with old flames. Meanwhile, having missed out on the auction, Brooke ends up spending the night with Mouth and he has the time of his life. This episode is named after a song by The Cure . | summ_screen_fd |
Virus-like particles (VLPs) have not been observed in Caenorhabditis germ cells, although nematode genomes contain low numbers of retrotransposon and retroviral sequences. We used electron microscopy to search for VLPs in various wild strains of Caenorhabditis, and observed very rare candidate VLPs in some strains, including the standard laboratory strain of C. elegans, N2. We identified the N2 VLPs as capsids produced by Cer1, a retrotransposon in the Gypsy/Ty3 family of retroviruses/retrotransposons. Cer1 expression is age and temperature dependent, with abundant expression at 15°C and no detectable expression at 25°C, explaining how VLPs escaped detection in previous studies. Similar age and temperature-dependent expression of Cer1 retrotransposons was observed for several other wild strains, indicating that these properties are common, if not integral, features of this retroelement. Retrotransposons, in contrast to DNA transposons, have a cytoplasmic stage in replication, and those that infect non-dividing cells must pass their genomic material through nuclear pores. In most C. elegans germ cells, nuclear pores are largely covered by germline-specific organelles called P granules. Our results suggest that Cer1 capsids target meiotic germ cells exiting pachytene, when free nuclear pores are added to the nuclear envelope and existing P granules begin to be removed. In pachytene germ cells, Cer1 capsids concentrate away from nuclei on a subset of microtubules that are exceptionally resistant to microtubule inhibitors; the capsids can aggregate these stable microtubules in older adults, which exhibit a temperature-dependent decrease in egg viability. When germ cells exit pachytene, the stable microtubules disappear and capsids redistribute close to nuclei that have P granule-free nuclear pores. This redistribution is microtubule dependent, suggesting that capsids that are released from stable microtubules transfer onto new, dynamic microtubules to track toward nuclei. These studies introduce C. elegans as a model to study the interplay between retroelements and germ cell biology. DNA transposons, retrotransposons, and retroviruses that are expressed in germ cells have tremendous potential to damage the genome by creating novel insertions that are transmitted vertically to host progeny. Because DNA transposons replicate by an excision and reintegration mechanism (“cut and paste”), replication of an endogenous element does not necessarily increase copy number [1]. Retrotransposons, however, replicate by first transcribing genomic RNAs that are later reverse transcribed for integration (“copy-and paste”), and endogenous elements thus have the potential to increase their copy numbers exponentially if left unchecked [1], [2]. Accordingly, animal and plant genomes typically contain far more copies of retrotransposons than of DNA transposons. For example, retrotransposons constitute about 40% and 75% of the human and Zea mays genomes, respectively, while DNA transposons contribute 3% and 8. 6% [3], [4]. In striking contrast, retrotransposons constitute only 0. 6% of the C. elegans genome, while DNA transposons make up about 12% 5–7. Although the C. elegans genome contains about 20 distinct families of long terminal repeat (LTR) retrotransposons and retroviruses, each family consists of only one or a few members [8]. Thus, retroelements enter the nematode genome at some frequency, but show comparatively little expansion. No viruses or Virus-Like Particles (VLPs) have ever been reported in the germ cells of C. elegans or other Caenorhabditis species, and experiments designed to identify spontaneous germline mutations in C. elegans yielded only novel insertions by DNA transposons [9], [10]. Indeed, C. elegans has long been considered an inadequate model organism to study natural virus-host interactions, although recent studies have shown that it is possible to artificially infect nematodes or nematode cell lines with promiscuous viruses, and a natural virus that infects intestinal cells has been identified [11]–[15]. A fundamental difference between DNA transposons and retroelements is that DNA transposons undergo their replication cycle entirely within the nucleus, while retrotransposons and retroviruses that infect non-dividing cells must transport their genetic material across nuclear pores. Non-LTR retrotransposons replicate by first producing a genomic RNA that is exported to the cytoplasm. This mRNA forms a ribonucleoprotein particle (RNP) that is imported back to the nucleus for reverse transcription and integration [16]. LTR retrotransposons closely resemble retroviruses, and encode similar GAG proteins (capsid, nucleocapsid) and POL proteins (protease, reverse transcriptase, ribonuclease H, integrase). Like retroviruses, LTR retrotransposons synthesize and export a genomic RNA that combines with capsid and nucleocapsid proteins to form VLPs. Some retroviruses require nuclear envelope breakdown to target host chromosomes, while others such as human immunodeficiency virus (HIV) infect non-dividing cells via entry through nuclear pores [17]. For example, nuclear entry of the HIV pre-integration complex involves the host nucleoporins NUP153 and RANBP2, and the karyopherin Transportin-3 [18], [19]. The HIV Vpr protein can interact directly with FG-repeat (phenylalanine glycine) nucleoporins, and these interactions appear necessary for the preintegration complex to be imported across nuclear pores and into the nucleus. In Caenorhabditis germ cells, nuclear pores are intimately connected with large, germline-specific granules called P granules [20]. Many animals from nematodes to mammals contain distinctive granules in their germ cells that are not found in somatic cells; these granules can be either cytoplasmic or associated with nuclei, and their presence, localization, and morphology can change during germ cell development [21], [22]. However, in contrast to most examples of germ granules in other animals, P granules are found in Caenorhabditis germ cells throughout the life cycle, and are nearly always perinuclear [21], [22]. Moreover P granules cover at least 75% of the nuclear pores on most germ nuclei in the gonad [20]. Like nuclear pores, P granules contain multiple FG-repeat proteins, and can block the passive diffusion of molecules above 40 kDa [23], [24]. Thus, most molecules entering nematode germ nuclei must first traffic through 200–400 nm P granules before they reach the 50 nm nuclear pores. Specific import and export pathways involving FG-repeat nucleoporins are required to move most materials through nuclear pores, and we have proposed that related pathways involving FG-repeat P granule proteins might be necessary to traffic materials through the much larger P granules [24], [25]. C. elegans P granules consist of diverse molecules involved in mRNA metabolism [22], and have been shown to transiently intercept nascent mRNA in adult germ cells [24]. Thus, P granule localization over nuclear pores likely is important for regulatory proteins and RNAs within P granules to survey nascent mRNA before that mRNA is released into the large, syncytial cytoplasm of the gonad. However, we are interested in the possibility that P granule localization might secondarily restrict retroelements that, unlike DNA transposons, must access nuclear pores to complete their replication cycle. If so, nematode retroelements might require strategies to penetrate or bypass P granules. To begin a study of the cell biology of retroelements in nematode germ cells, we first searched for wild strains with VLPs in germ cells. We found VLPs in the germ cells of C. japonica, where they appear to form, or accumulate, directly on P granules. We also discovered that several strains of C. elegans have VLPs, including the standard laboratory strain N2, but only when adults are cultured at temperatures below 25°C. We show that the C. elegans VLPs are products of an endogenous, Gypsy/Ty3 class retrotransposon called Cer1 (C. elegans retrotransposon 1) Cer1 capsids are first detected near meiotic germ cells in early pachytene, but accumulate around nuclei only in late pachytene/diplotene when new nuclear pores are added that are not associated with P granules. We propose that Cer1 capsids target this specific population of germ nuclei by exploiting changes in the stability of germ cell microtubules. Each arm of the C. elegans adult hermaphrodite gonad resembles a U-shaped cylinder, with approximately 1000 germ nuclei near the periphery (Figure 1A) [26]. Most germ nuclei are incompletely cellularized by plasma membranes; the apical plasma membrane has a small opening, called the ring channel, that connects with a large, shared region of gonad cytoplasm called the core (Figure 1B). All images shown in this study are either longitudinal, optical sections taken through the center of the core (central plane) or taken slightly interior to the apical surface (superapical plane; Figure 1A, B). The basal plasma membrane contacts either extracellular matrix, or somatic sheath cells that partially surround the gonad. Although most of the gonad is a syncytium, by convention a nucleus and the surrounding cytoplasm extending up to the ring channel is referred to as a germ “cell”. Cells at the distal end of the gonad divide mitotically, and their descendants enter successive stages of meiosis and oogenesis as they progress toward the opposite, or proximal, end of the gonad. Actin-dependent, cytoplasmic flow transports materials along the gonad core, toward and into enlarging oogonia [27]. When oogonia reach their full size, their ring channels close to form cellularized oocytes. Dense populations of microtubules are present throughout the gonad, both within germ cells and in the gonad core. Pachytene nuclei are surrounded by a basket of microtubules (“basket microtubules”) that extend from the basal and lateral membranes, through the ring channel, and into the gonad core (Figure 1B) [27], [28]. By electron microscopy, other microtubules we call core microtubules appear to extend from the core plasma membrane into the superapical zone, or into deeper regions of the core, without entering germ cells (data not shown). Additional anatomical and physiological features of the gonad that are noteworthy as potential obstacles, or opportunities, for retroelements targeting germ cell chromosomes are summarized in Figure 1C. Electron microscopy was used to screen for candidate VLPs in the adult gonads of 21 Caenorhabditis strains. These included both elegans and non-elegans species, and both hermaphroditic and male/female strains (Table 1, and Materials and Methods). Representative germ cells were analyzed from mitosis through all stages of meiosis, up to the formation of cellularized oocytes; over 17,000 germ cells were scored in the initial survey, and all candidate VLPs were photographed. In these studies, we observed multiple examples of two types of atypical, spherical bodies we considered possible VLPs; two additional nuclear and cytoplasmic bodies were observed in some strains, but these occurred in less than 0. 1% of germ cells and were not analyzed further. The first type of VLP was observed only in C. japonica. These VLPs are about 80 nm in diameter, covered by fine spikes, and have variable, highly electron-dense cores (Figure 1D). The C. japonica VLPs were found in all female and male gonads examined (n>30 for each), and were present in 16–19% of total germ cells beginning in the mid-pachytene region (Table 1). From this region through early oogenesis, essentially all VLPs were adjacent to P granules (Figure 1E and Supplemental Figure S1A, S1B, S1C). The VLPs were present in grape-like clusters that generally increased in size with germ cell development. In oogonia, where P granules detach from the nucleus, the VLP clusters were in the cytoplasm and appeared separated from P granules (data not shown). Very few or no VLPs were detected in mature oocytes or in early embryonic cells. However, morphologically similar VLPs appeared about midway through embryogenesis in several types of differentiating somatic cells, including hypodermis (skin), muscles, and intestinal cells, where the VLPs often were closely associated with microtubules (Supplemental Figure S1D, S1E, S1F). The second type of VLP was found in germ cells of four wild strains of C. elegans and, surprisingly, the standard laboratory strain, N2 (Table 1). We confirmed the presence of N2 VLPs in (1) worms derived from bleached eggs, indicating that the VLPs are endogenous, (2) in a newly obtained stock of the N2 reference strain from the Caenorhabditis Genetics Center, and (3) in a low passage “ancestral” stock of the original N2 isolate (Table 1, http: //www. cbs. umn. edu/CGC). Similar to the C. japonica VLPs, the C. elegans VLPs are about 80 nm in diameter and covered with fine spikes. The interiors of some VLPs contain one or two electron-dense bodies that resemble curved rods and likely correspond to the genetic material (Figure 1F). More commonly, small electron-dense foci are visible in the interior that might represent sectional views of the rods, or distinct stages of maturation or compaction. In contrast to C. japonica, the C. elegans VLPs were not observed in adult male gonads or in fourth larval stage (L4) hermaphrodite gonads, and were not observed in any embryonic cells (Table 1 and data not shown). The C. elegans VLPs were present at much lower abundance than the C. japonica VLPs: More than 95% of the C. elegans germ cells examined lacked VLPs, and the few positive cells typically contained only one or two VLPs. We began our analysis on the C. elegans VLPs, despite their low abundance, because of the numerous advantages of C. elegans for genetic and molecular experiments. To facilitate the analysis, we first asked whether we could increase the number of VLPs through physiological stress (heat, starvation), or by eliminating various pathways (RNAi, cell death) and cell types (coelomocytes) that might have the potential to repress or remove VLPs (Table 2). None of these conditions caused a marked increase in VLPs, although there was a modest and reproducible increase in alg-1 (RNAi); alg-2 (ok304) animals defective in the microRNA pathway (Table 2). We next attempted to use RNAi to identify the source of the VLPs, beginning with the several families of Cer elements in the C. elegans genome (Table 2). We observed no decrease in VLP numbers with dsRNA directed against the retrotransposons Cer2, Cer3, Cer4, Cer5, Cer6, or against the endogenous retrovirus Cer13. In contrast, dsRNA against the retrotransposon Cer1 eliminated all VLPs, strongly suggesting that the VLPs are Cer1 capsids. Cer1 is an 8. 8 kb LTR retrotransposon in the Gypsy/Ty3 family of retroviruses/retrotransposons (Figure 2A) [29]. Cer1 has a single, long open reading frame with the potential to encode a GAG- and POL-containing polyprotein [30]. The N2, laboratory strain of C. elegans contains one, apparently intact, copy of Cer1, and three LTR fragments [8]. The Cer1 insertion in N2 disrupts plg-1, a mucin-like gene that is required for the formation of the copulatory plug in mated adults; wild C. elegans strains are polymorphic for this insertion [31]. The 5′ and 3′ LTRs of Cer1 are identical, which can indicate a recent insertion as both LTRs are generated from a single template during retrotransposon replication [32]. However, there is no evidence that Cer1 remains active in N2: No spontaneous mutations have been shown to result from the transposition of Cer1, or of any other Cer element [9], [10], and no mRNAs that can encode essential GAG or RT proteins from Cer1 have been identified in any of several C. elegans cDNA and EST (Expressed Sequence Tag) projects (compiled at the Wormbase web site, http: //www. wormbase. org, release WS227,2011). Finally, Cer1 was reported to lack an obvious Primer Binding Site (PBS) [8]. A PBS is critical for replication, and typically is found near the 5′ LTR; the complementary 3′ end of a specific host tRNA binds the PBS to prime minus-strand DNA synthesis. However, in a search of the tRNA database GtRNAdb [33] we found that the sequence TGGGGGCCGAACCG, which is directly adjacent to the Cer1 5′LTR, is complementary to the 3′ end of C. elegans tRNA-Pro (TGG) (Supplemental Figure S2). The identical sequence was conserved in a group of C. japonica LTR retrotransposons we identified independently in best reciprocal BLAST searches with the Cer1 GAG sequence (Supplemental Figure S2). In a search of the C. elegans genome for additional copies of the presumptive PBS, we discovered LTRs and protein coding fragments from a larger, now extinct family of Cer1-like retrotransposons (Supplemental Figure S2). Although those LTRs and the C. japonica LTRs are highly diverged from Cer1, each contains one or more predicted binding sites for the C. elegans E2F transcription factor, EFL-1 (Supplemental Figure S2) [34]. In preliminary studies, we found that a transgene containing the 5′LTR of Cer1 and partial coding sequences linked to GFP (Green Fluorescent Protein) was expressed in germ cells, but was expressed in somatic cells instead of germ cells when the predicted E2F binding site GGCGCGAA was mutated to GGGCCGAA (our unpublished results). Because GAG proteins are the main structural components of retroviral and retrotransposon capsids, we generated a monoclonal antibody (αGAG) against the predicted Cer1 GAG protein. αGAG stained small, uniform puncta in N2 gonads (Figure 2B–D), but showed no staining in Cer1 (RNAi) gonads, or in gonads from the CB4856 Hawaiian strain of C. elegans that lacks the Cer1 sequence [31] and that lacks VLPs by electron microscopy (Table 1, Table 3, and data not shown). Similarly, αGAG did not stain gonads from N2 adult males or L4 hermaphrodites that lack VLPs by electron microscopy (Table 3). On Western blots of total worm proteins, αGAG stained a single band of about 82 kDa, compared to a predicted GAG size of 83–86 kDa (Figure 2E). This protein is much smaller than the size of the predicted Cer1 polyprotein, suggesting that the polyprotein is cleaved after synthesis. Unexpectedly, in the course of these experiments we discovered that GAG expression was strongly dependent on culture temperature and adult age. Worms grown at 25°C showed no detectable GAG expression by immunostaining or by Western analysis (Figure 2B, 2E and Table 3). GAG was expressed at low and variable levels in adults cultured at 20–23°C, but was expressed at much higher levels in 15°C adults. Moreover, GAG expression could be either induced, or repressed, by shifting adult animals between 15°C (ON) and 25°C (OFF; Figure 2B–D, Table 3). Because our initial survey of Caenorhabditis wild strains, and of N2-derived mutant strains, was performed on 1 day old (day 1) adults raised at standard culture temperatures of 20–23°C, we repeated electron microscopy on day 1 and day 3 N2 adults grown at 15°C. We found that VLPs (hereafter designated as Cer1 capsids) were much more abundant at 15°C than observed previously at higher temperatures (see below). We have not yet been able to determine if the C. japonica VLPs have similar temperature sensitivity: Abundant VLPs were observed in four separate C. japonica cultures grown over a 4 month interval in the first stages of this study. However, we observed only a few VLPs in more recent preparations, suggesting that expression is subject to silencing. Culture temperatures of 27°C and above are stressful or lethal for C. elegans, and even 25°C can activate variable, low-level expression of some heat shock reporters in some somatic tissues (our unpublished results). To determine whether the lack of Cer1 expression at 25°C resulted from activation of the heat shock pathway, we examined hsf-1 (sy441) mutants defective in the heat shock regulator HSF-1 [35]. Similar to wild-type adults, the hsf-1 (sy441) mutants expressed GAG at 15°C, but not 25°C. Conversely, osmotic stress from culture media containing 350 mM NaCl is sufficient to activate a heat shock response at low temperature (see [36] and references therein), but was unable to repress GAG expression at 15°C (Table 3). To determine whether temperature affected Cer1 mRNA expression, Northern analysis was performed on poly (A) -selected RNA from adult hermaphrodites cultured at either 15°C or 25°C, using probes specific for three regions spanning the Cer1 coding region (Figure 2A and 2F). These results showed that a Cer1 transcript of about 2. 4 kb is produced at both 15°C and 25°C, but that a large transcript of about 8 kb is detectable only at 15°C. We next sequenced RNAs that were amplified by RT-PCR using primers for various Cer1-specific sequences, and an additional primer specific for SL1 (Spliced Leader 1); SL1 is a 22 nt trans-spliced leader sequence found on about 60% of C. elegans mRNAs (Figure 2A and Materials and Methods) [37]. We found that the 8 and 2. 4 kb mRNAs correspond to the sequences diagrammed in Figure 2A; the 2. 4 kb message is spliced and is identical to the sole Cer1 mRNA identified previously in C. elegans EST and cDNA projects (Wormbase web site, http: //www. wormbase. org, release WS227,2011). These results show that Cer1 capsids are not detectable at 25°C because the 8 kb mRNA that encodes the GAG and POL proteins is not detectable at that temperature. The spliced 2. 4 kb mRNA can encode a novel, approximately 60 kDa protein that includes discontinuous, but in-frame, peptide sequences shared with the N-terminus of GAG (Figure 2A). The spliced mRNA utilizes Cer1 sequences that conform to consensus acceptor and donor intron splice sites for C. elegans (Supplemental Figure S3) [38]. This splicing pattern is likely to be a general feature of the Cer1 family, as Cer1-related retrotransposons in both C. remanei and C. japonica contain consensus splice acceptor and donor sequences at similar positions (our unpublished results). We detected the spliced mRNA with, and without, the SL1 leader at both 15°C and 25°C (Figure 2A, 2F and data not shown). In contrast, the 8 kb message did not appear to be spliced, and was not trans-spliced at the site utilized by SL1; it could not be amplified with an SL1-specific primer, but was amplified with primers either 5′ or 3′ of the SL1 site (see legend to Figure 2A). Thus, capsid production at 15°C requires that multiple, consensus splice sites within Cer1 are skipped in order to generate the 8 kb mRNA that encodes GAG and POL. We next performed in situ hybridization on fixed gonads to examine the spatial and temporal expression of the 8 kb mRNA, using probes specific for either rt or gag (diagrammed in Figure 2A). At 15°C, gag-containing RNA was first detectable in the early pachytene region of the gonad, but was not detected in somatic tissues such as the intestine (Figure 2G, 2I). The gonad expression correlates with the onset of GAG immunostaining (Figure 2D), with the first appearance of VLPs by electron microscopy (Supplemental Figure S1G), and is similar to the onset of EFL-1/E2F expression [39]. Fluorescence In Situ Hybridization (FISH) further showed that the RNA was enriched in the superapical zone of the gonad core, with relatively little RNA in the cytoplasm surrounding germ nuclei (Figure 2J). Interestingly, both alkaline phosphatase and FISH detection protocols showed gag- and rt-containing RNA concentrated in a pair of nuclear dots (Figure 2G, 2J and Figure 2K). No nuclear dots were observed in CB4856 worms that lack Cer1, and only one nuclear dot was observed in N2/CB4856 heterozygotes that have one copy of Cer1 (Figure 2L and data not shown). These results suggest that the double dots in N2 represent RNA at or near the site of Cer1 insertion on the paired, homologous chromosomes. Similar nuclear dots were detected in N2 gonads at 25°C that otherwise lack the gag-containing mRNA (Figure 2H). These results suggest that gag-containing RNA is transcribed at 25°C, but fails to accumulate in the cytoplasm. Finally, we wanted to determine whether the temperature-dependence of GAG expression was unique to the N2 copy of Cer1, or was a more general characteristic of Cer1 retrotransposons. Using available data on the pattern of Cer1 insertion in various wild C. elegans strains [31], we immunostained 15 wild isolates that have the N2 pattern of Cer1 insertion in the plg-1 gene, and 15 strains with Cer1 inserted elsewhere in the genome. 21 of the 30 strains contained immunoreactive GAG particles at 15°C that closely resembled the N2 staining pattern, but none contained GAG particles at 25°C (Table 4). Essentially all of the immunostaining results for GAG-containing particles described here correlate with Cer1 capsid localization determined by electron microscopy. Therefore, we refer to the immunostained, GAG-containing particles simply as Cer1 capsids. In support of this designation, biochemical studies to be described elsewhere show that the GAG protein detected on Western blots of worm extracts purifies with a sedimentation profile typical of viruses or capsids (our unpublished results). In overview, Cer1 capsids first appear in early- to mid-pachytene, where they localize almost exclusively in the superapical region of the gonad core, outside of the germ cell proper and away from nuclei (Figure 3A). By electron microscopy, capsids are present in small groups that are closely associated with rough endoplasmic reticulum (Supplemental Figure S1G), but capsids later appear more dispersed and associated with microtubules (Figure 4A, B). Capsids remain largely confined to the gonad core throughout the mid-pachytene region, but during late pachytene/diplotene most capsids accumulate around, or at the base of, germ nuclei (Figure 3A and Figure 4C–4E; see also Figure 5E). In oogonia and early oocytes, most capsids are concentrated in large, crescent-shaped aggregates that are adjacent to the nuclear envelope (Figure 3A and Figure 4F). In mature oocytes, capsids disperse throughout the cytoplasm and most disappear (data not shown). The small population of capsids that persist in newly fertilized embryos exhibit two additional localization patterns: When fertilization initiates the first of two meiotic divisions of the maternal chromosomes, capsids localize equally to both poles of the meiosis I spindle (Figure 3B, 3C). This spindle rotates perpendicular to the anterior surface of the embryo, and during division half of the maternal chromosomes are extruded from of the embryo as the first polar body; capsids at the anterior spindle pole are similarly extruded into the polar body (Figure 3D). During these stages, additional capsids localize in small foci distributed across the periphery of the embryo (arrowheads in Figure 3C). This peripheral localization occurs at about the same time as much larger cortical granules move to the surface and are exocytosed to form an extra-embryonic layer [40]. The peripheral capsids are not visible at later stages, suggesting that some of these are externalized and/or degraded. Very few or no capsids persist into cleavage-stage embryos, and any remaining capsids show no enrichment in the embryonic germ cell precursors (asterisk in Figure 3D). Previous studies showed that cytoplasmic dynein and dynein-associated proteins localize to both poles of the meiosis I spindle and to foci at the periphery of the newly fertilized embryo [41], similar to the localization of Cer1 capsids. We found that each of two temperature-sensitive alleles of dhc-1/dynein resulted in variable and markedly reduced levels of capsid staining throughout the gonad, even at the permissive temperature for both mutations (Figure 3E, quantified in Table 3). We do not yet know from biochemical experiments whether dynein is directly associated with capsids, but we did not observe significant colocalization of DHC-1/dynein and Cer1 capsids in gonads by immunostaining (data not shown). We found that capsid accumulation requires oogenesis but not spermatogenesis (Table 3), and is strongly dependent on adult age. For the latter analysis, we cultured hermaphrodites in the constant presence of males, as oogenesis arrests in the absence of sperm. By the second day of adulthood (day 2 adults) and increasing thereafter, many gonads contain huge numbers of capsids organized into linear arrays that resemble wavy lines, or tangles of wavy lines (Figure 5A–5D, and Supplemental Figure S4A, S4B). Costaining for HIM-4/hemicentin, which localizes by the apical membrane [42], showed that the tips of the wavy lines occasionally and partially enter germ cells through the ring channels (Figure 5B). However, the vast majority of capsids localize in the core, outside of germ cells and away from nuclei, similar to capsids in younger adult gonads (Figure 4B and Figure 5A–5D). The abundance of capsids, and the presence and size of the lines and tangles, varies considerably for germ cells that are at the same developmental stage, but at different positions in the gonad core (double arrow in Figure 5D). Both young and older adults show a marked shift in capsid distribution as germ cells begin to exit pachytene, in the pachytene/diplotene region: Nearly all of the lines and tangles of capsids disappear from the core, and large numbers of capsids redistribute into germ cells and around nuclei (Figure 4E, Figure 5E–5G, and Supplemental Video S1). In normal development, the late pachytene/diplotene nuclei enlarge and add new nuclear pores that are not covered by P granules; all P granules are eventually shed from germ nuclei during subsequent oogenesis [20], [21]. Thus, capsids in both young and older adults only accumulate around germ nuclei with available, P granule-free nuclear pores. Electron microscopy, and co-staining for capsids and P granules, showed that most capsids do not accumulate directly on P granules (Figure 4D, and Figure 5H), although a few capsids are found within P granules at this stage (Supplemental Figure S1H). Instead, most capsids are located near and between P granules, and appear directly associated with microtubules (Figure 4C, 4E and Figure 5H). Several observations suggested that microtubules are involved in capsid localization. First, we found that about 30% of capsids scored in randomly selected electron micrographs appeared to contact one or more microtubules (n = 180/560 capsids, Figure 4A, 4C). As microtubules that don' t present clear cross-sectional or longitudinal profiles in micrographs are difficult to score, this percentage likely underestimates the association. Second, aggregates of large numbers of capsids and microtubules were visible in electron micrographs of day 3 and older adults that resembled the wavy lines and tangles detected by immunostaining (Supplemental Figure S4A, S4B). Finally, the localization of capsids in newly fertilized embryos closely resembles that of cytoplasmic dynein, as described above. Despite the above correlations, the distribution of most capsids in immunostained gonads bears little resemblance to the distribution of microtubules: Microtubules are highly abundant throughout the gonad (Figure 6A), while capsids are concentrated in much smaller regions (Figure 6B, 6C). To determine whether capsid localization was dependent on microtubules, we treated gonads with the inhibitors nocodazole or colcemid. Cytoplasm normally flows proximally through the gonad core, transporting materials toward and into enlarging oocytes, but bypasses the late-pachytene/diplotene germ cells where capsids accumulate [27]. Flow is actin-, but not microtubule- dependent, and can carry transport large, inert beads a distance of nearly half the length of the gonad in only 25–30 minutes [27]. Thus, we anticipated that if microtubules normally anchor the capsids against flow, the capsids would flush proximally in inhibitor-treated gonads. Instead, the inhibitors caused little if any change in the distribution of capsids throughout the early- to mid-pachytene region, including the wavy lines and tangles of capsids present in older adults (data not shown and see below). The nocodazole concentration used for those experiments (10 µg/ml; 1 hr) was the same used in previous studies on gonad microtubules [28]. However, we discovered that a large subset of microtubules was not depolymerized by that treatment, or even higher doses of nocodazole (40 µg/ml; 2 hrs). For our subsequent analysis, we chose a standard dose of 10 µg/ml nocodazole for 30 minutes, and we refer to microtubules that resist depolymerization under these conditions as stable microtubules. We found that this dose of nocodazole effectively depolymerized dynamic microtubules in the spindles of mitotic germ cells in the distal gonad (Figure 6E, 6F), and depolymerized dynamic microtubules in the most mature oocytes (Figure 6D, 6H) [43]. In contrast, large numbers of stable microtubules persisted in the early- to mid-pachytene region, where they were concentrated along the superapical zone of the gonad core (Figure 6D, 6G). A subset of the stable microtubules could be traced into germ cells, indicating that some of these are basket microtubules (Figure 1B, Figure 6G, and data not shown). Cer1 was not the source of microtubule stability, as gonads in each of the following types of animals that lack Cer1 capsids contained stable microtubules: L4 hermaphrodites, adult males, adult hermaphrodites at 25°C, and adult hermaphrodites treated with Cer1 (dsRNA) at 15°C (n = 30–75 gonads scored for each, data not shown). In addition, stable microtubules were observed in the gonads of a hybrid strain we constructed where the intact plg-1 gene from CB4856 was introgressed into N2 to replace Cer1 (see Figure 7B below); we refer to these N2-derived animals that lack Cer1 as Cer1 (-). To determine the relationship with Cer1 capsids, we examined stable microtubules (after nocodazole treatment) in wild-type and Cer1 (-) animals that were grown as adults for 1 to 6 days at 15°C in the presence of males. We found a striking correspondence between the localization of capsids and the stable subset of microtubules in adults at all ages, and in all regions of the gonad. The following describes our results for the mid-pachytene region, where nearly all capsids localize to the core (Figure 7A–7E). In control experiments, the pattern of total microtubules (stable plus labile) in untreated, day 1–6 wild-type gonads appeared identical to the pattern in untreated, age-matched Cer1 (-) gonads (Figure 6B, Figure 7A, and data not shown). The pattern of stable microtubules in day 1–2 wild-type gonads (Figure 6G) closely resembled that in day 1–6 Cer1 (-) gonads (Figure 7B, and data not shown); in both types of gonads, the stable microtubules were enriched in a uniform layer along the superapical zone of the core. Beginning on day 3, however, the wild-type gonads often contained aggregates of stable microtubules (Figure 7C, 7F). The percentage of gonads with aggregates, and the number of aggregates per gonad, both increased with adult age, such that most wild-type gonads contained at least one aggregate by day 6 (Figure 7H). Remarkably, essentially all capsids throughout the mid-pachytene region were localized on stable microtubules, and capsids were particularly concentrated in the aggregates (Figure 7C, 7F). While the stable microtubules in Cer1 (-) gonads had a relatively uniform thickness (Fig. 7D), the capsid-bearing stable microtubules in wild-type gonads varied markedly in thickness, suggesting they were bundled (Fig. 7E). Together, these several results show that stable microtubules exist independent of Cer1 capsids, but that capsids appear to cause an age-dependent aggregation of the stable microtubules. The microtubule aggregates are not apparent in untreated wild-type gonads immunostained for tubulin, presumably because of the high density of total microtubules. However, the presence of microtubule aggregates in untreated gonads is confirmed directly by electron microscopy (Supplemental Figure S4A, S4B), and indirectly by the localization of capsids (Figure 5C, 5D). The largest aggregates of stable microtubules and capsids appear to bridge across the entire diameter of the gonad core, and are often adjacent to regions nearly devoid of stable microtubules (arrowhead in Figure 7C). Thus, the aggregates might grow by stripping stable microtubules from neighboring regions. If the function of the stable microtubules is to maintain gonad architecture, as we consider likely, we wondered whether the aggregates might impact fertility. We compared age-matched wild-type and Cer1 (-) adults over each of 7 days at 15°C, but did not observe a significant difference in the number of dead eggs laid (n>20,000 total eggs for each strain; Figure 7G). We next cultured both strains at 15°C until day 3, to pre-load the wild-type gonads with capsids, then shifted the adults to 25°C and examined the eggs laid over the next 24 hour period. The wild-type, shifted adults produced significantly more dead eggs, and had slightly less total progeny, than the Cer1 (-) adults (Figure 7I, 7J). Thus, under the moderate stress of 25°C, wild-type animals pre-loaded with capsids appear to be less fertile than Cer1 (-) adults. Stable microtubules are present in the gonad core throughout the early to mid-pachytene region, but disappear abruptly as germ cells exit pachytene (Figure 6D, 6G, and Figure 8A). Similarly, the giant aggregates of stable microtubules in older adults nearly always disappear by late pachytene/diplotene: From over 160 nocodazole-treated gonads examined, only one contained a giant aggregate of stable microtubules and capsids in this region (arrow in Figure 8B). Thus, stable microtubules appear to break down, or become destabilized, at the same stage that capsids move out of the core and into germ cells (Figure 5E, and Supplemental Video S1). We found that nocodazole treatment sharply reduced the number of capsids in late- pachytene/diplotene germ cells (compare Figure 8A with Figure 5G). Thus, the movement of capsids into germ cells is dependent on microtubules, but these microtubules are not resistant to nocodazole. The stable microtubules disappear in late pachytene/diplotene germ cells, but reappear in late oogonia and immature oocytes where they are concentrated in a focus near the nuclear envelope (arrow in Figure 6H and in Figure 8D; compare with the untreated oocyte in Figure 8C). This focus of stable microtubules colocalizes with the nuclear-associated crescent of capsids (Figure 8D, see also Figure 4F). Similar crescents of capsids exist in untreated, earlier oogonia that contain few or no stable microtubules (Figure 3A, and Figure 6D), and these latter crescents are disrupted by microtubule inhibitors (arrowhead in Figure 8B). Together, these results suggest that (1) a focus of labile microtubules develops on or near the nuclear envelope after germ cells exit pachytene, (2) that microtubules in the focus gradually stabilize as oogonia develop into immature oocytes, and (3) that capsids accumulate at the focus before and after microtubule stabilization. Finally, the stable microtubules disappear and the capsids disperse in mature oocytes (Figure 6D, 6H). Most capsids eventually disappear in mature oocytes, but at fertilization the remaining capsids aggregate at the meiosis I spindle as described above. We conclude that capsids are associated with microtubules throughout the gonad and in fertilized eggs, and that the various patterns of capsid localization reflect changes in microtubule stability and patterning. Nematode germ cells, like those in other animals, presumably have multiple layers of defense against transposons. For example, studies primarily from Drosophila and mice have provided an elegant model for how germ cells recognize and silence transposons: If replicating transposons integrate into chromosomal traps called piRNA clusters, it primes the synthesis of small, anti-sense RNAs (piRNAs) that can target both the original transposon and closely-related transposons [44], [45]. Thus, novel transposons entering a naïve host have limited windows of opportunity to replicate before the silencing machinery identifies them. In exploit this window, nematode retrotransposons likely require strategies to traffic through, or bypass, both nuclear pores and P granules. VLPs have not been reported previously in C. elegans germ cells, and only a few were observed in our initial electron microscopic survey of several wild strains. However, we showed that the expression of Cer1, a Gypsy/Ty3 retrotransposon, is temperature sensitive, such that very few VLPs are present at typical culture temperatures of 20°C to 23°C. Because previous analyses of spontaneous mutations in C. elegans were done at 20°C, it will be important in future studies to re-evaluate whether Cer1 is capable of transposition in the N2 strain using mated, older adults cultured at 15°C. Temperature-dependent expression appears to be a widespread feature of Cer1 retrotransposons, as a similar dependence was seen in multiple wild strains with different Cer1 insertion sites. There are several possible rationales why Cer1 expression is elevated at low temperatures, the simplest being that 15°C best approximates the average temperature of C. elegans in its natural environment. For example, average annual soil temperatures recorded 10 cm below the surface at sites in Florida, Montana, Oregon, Michigan, Tennessee, Arizona, Alaska were 22. 5,12. 1,12. 8,8. 2,15. 7,19. 5, and 0. 8°C, respectively [46]. In addition, our temperature-shift experiments on older N2 adults suggest that expression at 25°C might exacerbate phenotypes associated with capsid accumulation. Cer1 capsids cannot form at 25°C because GAG and POL proteins are not expressed at that temperature. Cer1 produces an apparently unspliced 8 kb mRNA that can encode GAG and POL proteins as a single polypeptide, and a spliced 2. 4 kb mRNA that encodes a novel protein. The 2. 4 kb mRNA is abundant at both 15°C and 25°C, however, the 8 kb mRNA only accumulates in the cytoplasm at 15°C. It does not appear that small RNA pathways prevent the cytoplasmic accumulation the 8 kb mRNA, as all of the mutants tested to date lack capsids at 25°C, similar to wild-type animals. Splicing is essential for most cellular mRNAs to be exported from the nucleus. However, retrotransposon and retroviral replication requires export of a non-spliced, genomic RNA. Retroviruses can achieve this by structural motifs in the genomic RNA that bind host export factors directly, or by first producing proteins from spliced mRNAs that facilitate the subsequent export of non-spliced, genomic RNA. For example, the HIV Rev protein is the product of a spliced mRNA, and binds to the host export factor CRM-1 as well as to a structured motif (the Rev-response element) in the HIV genomic RNA [47], [48]. The 8 kb Cer1 mRNA is about the same size as the predicted Cer1 genomic RNA, and thus might serve a dual function as genomic RNA, or at least face similar challenges in nuclear export. Because the novel protein encoded by Cer1 shares some peptide sequences with GAG, and one of the expected functions of GAG is to bind and package the genomic RNA, we speculate that this novel protein might similarly bind the 8 kb RNA and regulate its splicing or export. The localization pattern of Cer1 capsids described here for the N2 strain appears very similar to that in other wild strains that express Cer1. Thus, this pattern is likely to be relevant for considering how Cer1 accesses germ cell chromatin. Cer1 does not appear to bypass P granules and the nuclear envelope by targeting dividing cells in larvae or adults: Cer1 is not expressed in L4 or earlier larvae, where all germ cells divide mitotically, and Cer1 is not expressed in the mitotic niche of adult hermaphrodite or male gonads. We found that Cer1 and Cer1-related retrotransposons conserve at least one predicted E2F binding site in their LTRs, and that this site appears important for germline expression. E2F activator proteins have well-studied roles in promoting the mitotic cell cycle in mammals and Drosophila, and DNA tumor viruses de-repress E2F protein complexes to drive S-phase DNA synthesis [49]. However, C. elegans EFL-1/E2F does not appear to function in mitosis, and instead regulates germ cell expression of numerous genes that control oogenesis and early embryogenesis [34]. Indeed, the present network of EFL-1/E2F-regulated genes in C. elegans germ cells might have expanded by the past transposition of Cer1 family members, much as endogenous retroviruses appear to have seeded p53 binding sites in the human genome [50]. If Cer1 has not evolved to target mitotic germ cells in adult or larval gonads, it might instead target the mitotic germ cell precursors in early embryos; these precursors have rapid cycle times of only 15–25 minutes, compared to 16–24 hours for adult germ cells [51], [52]. For this strategy, however, it would seem counterproductive for Cer1 capsids to bind microtubules. If capsids simply did not bind microtubules, they would be transported passively into oocytes by actin-dependent cytoplasmic streaming, and at fertilization would be further transported directly into the embryonic germ cell precursors by a second wave of actin-dependent cytoplasmic flow [27], [53]. Instead, the anchoring of Cer1 capsids to stable microtubules in the gonad core prevents many, if not most, capsids from ever reaching embryos produced by self-fertilization: Each arm of the hermaphrodite gonad can produce about 1000 oocytes, but only produces about 150 sperm. Thus, adults run out of sperm and can no longer self-fertilize at about the same time that peak numbers of capsids accumulate in the gonad core. Most capsids in both C. elegans and C. japonica disappear in mature oocytes, suggestive of host defenses that prevent capsids from entering embryonic cells. For example, oocyte cytoplasm might contain specific proteases that have evolved to recognize, and degrade, capsids. As second possibility is that oocyte cytoplasm triggers the disassembly of capsids. The oocyte cytoplasm contains a very high level of FG-repeat nucleoporins such as NPP-9/RanBP2/Nup358 [24], which presumably is necessary to support the assembly of nuclear pores during the rapid proliferation of early embryonic cells. In addition, oocytes contain high levels of FG-repeat P granule proteins that likely contribute to the dynamic growth, shrinkage and fusion behaviors of detached, cytoplasmic P granules [54]. If Cer1 capsids, like HIV capsids, target FG-repeat proteins on the nuclear pores, large quantities of such proteins in the cytoplasm might effectively become decoys that trigger premature capsid disintegration. While Cer1 capsids are not enriched in any mitotic germ cell population, they are expressed abundantly throughout the pachytene stage of meiosis. However, the vast majority of capsids remain in the gonad core until germ cells begin to exit pachytene, when most capsids transfer into germ cells and near nuclei. As germ cells exit pachytene, they begin to increase in size and eventually become large oocytes. There is a concomitant increase in nuclear size, with the addition of nuclear pores that lack P granules and the gradual removal of existing P granules. Thus, capsids converge on germ nuclei when large numbers of P granule-free nuclear pores first become available. Such a strategy would likely provide only a narrow window of time for nuclear entry and integration, as the post-pachytene germ cells cease transcription and hypercompact their chromatin as they differentiate into oocytes (Figure 1C). By electron microscopy, we detected C. japonica VLPs in the early- to mid-pachytene region, similar to Cer1 capsids in C. elegans gonads. However, the C. japonica VLPs first appear, and then remain, on P granules. As newly exported mRNA traffics directly through P granules [24], this localization might be an efficient strategy for capsid proteins to capture and package viral RNA. In addition, P granule localization maintains capsids by the nuclear envelope, potentially allowing a constant surveillance for free nuclear pores. In contrast, Cer1 capsids in C. elegans appear to traffic toward nuclei from initial positions in the gonad core. Particles the size of the 80 nm Cer1 capsids are not expected to move appreciably without some type of force-generating machinery; studies in other systems have shown that dextran particles greater than 20 nm are essentially immobile in cytoplasm (see [55] and references therein). Actin-dependent streaming moves cytoplasm through the core of the gonad and into late oogonia, but does not appear to transport cytoplasm into the earlier germ cells that show perinuclear accumulation of capsids [27]. We found that Cer1 capsids are associated with microtubules in all regions of the gonad, suggesting that changes in the microtubule cytoskeleton underlie changes in capsid localization. For example, Cer1 capsids are dispersed uniformly throughout the cytoplasm of mature oocytes just prior to fertilization. At fertilization, however, the microtubule cytoskeleton reorganizes to form the meiotic spindle and capsids converge at both poles of the spindle. Viruses often exploit the microtubule cytoskeleton to locate host nuclei [56], [57]. In many cell types, microtubule minus ends are anchored by the centrosome, which functions as the major microtubule organizing center (MTOC). Because the centrosome is often adjacent to the nucleus, viruses can traffic from the cell periphery toward the nucleus by hijacking minus-end directed microtubule motor proteins; upon release from the microtubule, the virus has a high probability of encountering a nuclear pore. For example, herpes simplex virus, HIV-1, and adenovirus each use the minus-end directed motor protein dynein to navigate host cells [56], [57]. C. elegans gonads contain dense populations of microtubules that include subpopulations we term basket microtubules and core microtubules. However, the centrosome appears to lose its MTOC function once germ cells exit mitosis, and the centrosome itself is eliminated during oogenesis [28]. Instead, the germ cell plasma membrane appears to function as an MTOC for basket microtubules [28], and we consider it likely that the core plasma membrane acts as an MTOC for core microtubules. Animal cells typically have dynamic microtubules (t1/2 = ∼10 minutes) as well as long-lived, stable microtubules (t1/2>1 hour) [58]. In the C. elegans gonad, we showed that some basket and core microtubules appear exceptionally resistant to concentrations of microtubule inhibitors that rapidly depolymerize known, dynamic populations of microtubules, such as those in mitotic germ cells and in the most mature oocytes. Because capsids appear to accumulate over time on the stable microtubules, we argue that these same microtubules are long-lived in vivo. We propose a “load-release-transfer” mechanism as a working model for how Cer1 capsids concentrate near post-pachytene germ nuclei (Figure 9). First, capsids that form in the early- to mid-pachytene region load onto surrounding microtubules in the core. Capsids might initially associate equally with dynamic and stable microtubules, but over time would accumulate on the long-lived, stable microtubules. Early to mid-pachytene germ cells are small with relatively little cytoplasm, but microtubules extending from these cells into the core represent a much larger, cognate surface for capsid accumulation. Cytoplasmic dynein is required for normal levels of capsid accumulation in the core, but we do not yet know whether dynein mediates capsid binding to microtubules, or whether capsids can traffic on the stable microtubules. The number of capsids loaded onto stable microtubules increases as germ cells progress through pachytene, often resulting in the bundling or aggregation of stable microtubules in older adults. We consider it likely that the stable microtubules provide the architectural framework for the gonad core throughout the pachytene region, and must be destabilized for germ cells to increase in size as they exit pachytene. The abrupt change in microtubule stability releases the accumulated capsids into a relatively small region of core cytoplasm. There, many capsids transfer to dynamic microtubules, and move into germ cells, before being swept proximally by flow. The transferred capsids might traffic toward nuclei in association with microtubule motor proteins, or by microtubule dynamics. Although we have not yet determined the polarity of microtubules in this region, it seems likely that many are nucleated at or near the nuclear envelope and could concentrate capsids: Microtubules in general are enriched by the nuclear envelope of those cells, and a focus of stable microtubules develops close to the envelope where capsids accumulate. Thus, we hypothesize that a new, nuclear-associated MTOC forms as cells exit pachytene, and microtubules associated with this site gradually stabilize as germ cells mature into oogonia. This MTOC is not associated with the centrosome per se, which is eliminated during oogenesis, but might be associated with a remnant of the centrosome or with centrosome-derived proteins. The present study demonstrates that the genetics and cell biology of C. elegans, one of the best understood models for animal development, can be used to study host-pathogen interactions between retroelements and germ cells. Because viruses necessarily rely heavily on host functions, their analysis has provided significant insights into host biology in many systems. Here, our analysis uncovered stage-dependent changes in the microtubule cytoskeleton of C. elegans germ cells, posed new questions about how microtubule stability is established and regulated, and showed that capsid localization can be used as a proxy for the distribution of stable microtubules. Further dissection of Cer1 expression should provide insights into RNA splicing and export in germ cells, EFL-1/E2F-regulated transcription, and inform strategies for building Cer1-based vectors for temperature-inducible, germ cell-specific gene expression. Although the various small RNA pathways do not silence Cer1 expression in the N2 strain at 15°C, we anticipate that analysis of Cer1-containing wild strains that lack GAG expression should reveal how this element is targeted for silencing in wild populations. For Northern analysis, synchronous hermaphrodites grown from bleached eggs were grown at 20°C until the L4 stage, then either shifted to 15°C or 25°C for 48 hours. About 1 ml of packed adults from each culture was rinsed in PBS twice, then resuspended and homogenized in Trizol (Invitrogen), followed by treatment with DNAse and RNA precipitation as per the manufacturer' s instructions. The RNA was checked for purity by running samples on agarose gels. PolyA-containing RNA was selected using an Oligotex mRNA midi Kit (Qiagen) following the kit protocol. Probes were prepared using Amersham Ready-to-Go DNA labeling beads-dCTP (Amersham), and unincorporated nucleotides were removed with NucAway Spin Columns (Ambion), both following product protocols. RNA blotting and hybridization followed published protocols [59], except with the suggested alternate buffer [Prehybridization/hybridization: 7% SDS, 0. 5 M NaHPO45,1 mM EDTA; wash #1: 5% SDS, 40 mM NaHPO4,1 mM EDTA; wash #2: 1%SDS, 40 mM NaHPO4,1 mM EDTA]. Nylon membrane was used for blotting (BioRad) as per the manufacturer' s protocol. For RT PCR, cDNAs were generated using SuperScript One-Step RT PCR (Invitrogen) and Platinum Taq (Invitrogen). Products were gel purified using QiaQuick Gel Extraction Kit (Qiagen) and sequenced by the FHCRC Genomics Resource. The following primer sequences were used for the analysis summarized in Figure 2A: SL1 (5′-GTTTAATTACCCAAGTTTGAG-3′), KE409F (5′-CGCCTTTGAGTTTGCAATCGTTT-3′), KE402F (5′-AAATCTGAAGAATGGAGGTGAACGAGGGACAGGATA-3′), KE411F (5′-TTGGATAGTGGTGCTAGTATTTC-3′), KE413F (5′-GACTTCGTCATTGACACCTTATC-3′), KE416R (5′-TCCGATTTCACCATTAGCAAAC-3′), KE401BR (5′-ATAACACCTCAAAGCCTCCTG-3′), KE415R (5′-GATAAGGTGTCAATGACGAAGTC-3′), KE402CR (5′-TTCAAAGAAACAGGCAGAGCG-3′). The in situ procedure was modified from the protocol of G. Broitman-Maduro and M. Maduro (http: //www. faculty. ucr. edu/~mmaduro/) as previously described [24]. DIG-labeled ssRNA probes were made by in vitro transcription using DIG RNA Labeling Kit (Roche), using as template a PCR product amplified from genomic DNA with the following primer sequences: rt probe (5′-AGGTGATTGGGAAAGGAGAG-3′, 5′-GCGATTGTAGCGTTGCTTCA-3′), gag probe (5′-TCCCAGGTCCAGACGAATAG-3′, 5′-GCTCTCTCGAGATATTCTGAG-3′) Unless noted otherwise, RNAi experiments used worms cultured at 20–23°C and dsRNA feeding strains from the Ahringer library as described [60]. Third larval stage (L3) alg-2 (ok304) animals were fed on alg-1 (F48F7. 1 dsRNA) until adulthood. For analysis of Cer elements, N2 worms were fed on dsRNA-expressing bacteria for 3 generations before analysis of adults using the following library strains: Cer1 (F44E2. 2 dsRNA), Cer4 (T23E7. 1 dsRNA), and Cer5 (T03F1. 4 dsRNA). Sequences from the remaining Cer elements were PCR amplified from genomic DNA using the following primer pairs then cloned into feeding vector pPD129. 36 as described [61]: Cer2 (5′-GGACGAGCTTATCGTGTTCC-3′, 5′-CAACTCAACGAGCCATCTCC-3′), Cer3 (5′-ATGAGAGAGCTCAGTTACAAGC-3′, 5′-GGTTCTACTAGTATAAGCTATCG-3′), Cer6 (5′-ATGACGGTTAAGGGTCGAGTT-3′, 5′-CGATGAATCCCAGAAACGAGAT-3′), Cer13 (5′-TCTTTCAAGGCATCTCAGCG-3′, 5′-AACCTCGGTCCAATTGATCC-3′ plus 5′-TTCTCAACAGCGTCTCCATCG-3′, 5′-CAGCTGTCCGATCTGTTTCAC-3′. The following antibodies were used: αTubulin (1∶1 YL1/2 and YOL3/4; Abcam), αPGL-1 [62], αHIM-4 [63]. αGAG was generated in the FHCRC Hybridoma Production Facility using bacterially expressed, GST-tagged GAG following published protocols [64]. GAG protein-coding sequences from nucleotide positions 1789–2748 were fused in frame at the C-terminus of GST using pGEX-6P-1 (Amersham), and purified from Rosetta (DE3) pLyS cells (Novagen). Approximately 500 hybridoma colonies were identified that screened positively against GST-GAG by the ELISA screening test. Cell line P3E9 (αGAG) was obtained by re-screening all of the positive hybridoma cell supernatants by indirect immunofluorescence microscopy of fixed wild-type and Cer1 (RNAi) gonads. Worms were collected in drops of M9 buffer [65] on a taped microscope slide and rinsed in three or more changes of M9 to clear bacteria, then rinsed in two changes of gonad culture media (GCM: 50% Leibovitz L-15 medium (GIBCO), 10% Fetal Calf Serum (GIBCO), 5. 6% sucrose, 2 mM MgCL2). Slides containing 20–30 worms in a drop of GCM were placed on a precooled dissecting microscope stage at 8–10°C; cooling was used to slow animal movements to facilitate dissection. Worms were cut immediately with a scalpel blade, just behind the pharynx to release the gonad, and the slides were removed to room temperature; the elapsed time on the cooled stage did not exceed 2 minutes. An equal volume of 15°C GCM was added that contained 2× nocodazole (Sigma) plus 1 mM levamisole (Sigma) in GCM. Nocodazole was diluted fresh each time from a 5 mg/ml stock in dimethylsulfoxide kept at −20°C. The levamisole was added after dissection to prevent damage to the gonad from the contraction of attached muscles during incubation with the inhibitor. The slide with gonads plus inhibitor was placed on a pre-cooled block at 15°C, and transferred to a 15°C incubator for the designated time. MeOH fixation was used for images shown in Figure 3 and for experiments listed in Tables 3 and 4 as follows. Worms were dissected on microscope slides in a small drop of M9 buffer and covered with a coverslip. The slide was frozen on dry ice, the coverslips removed, and the slide immersed in −20°C MeOH (5 minutes) before rinsing in three changes of PBS for 5 minutes each. Slides were incubated with antibody overnight at 4°C. A two step formaldehyde fixation [66] was modified for experiments to simultaneously localize Cer1 GAG and microtubules as follows. Worms were dissected as for the nocodazole experiments above, except that gonad buffer (GB: 75 mM HEPES (6. 9), 1. 2% sucrose, 40 mM NaCl, 5 mM KCL, 2 mM MgCL2,1 mM EGTA) was substituted for GCM. After dissection, an equal volume of fixation buffer #1 [6% formaldehyde, 75 mM HEPES (6. 9), 40 mM NaCl, 5 mM KCL, 2 mM MgCL2,1 mM EGTA] was added for 2 minutes, then two equal volumes of fixation buffer #2 [3% formaldehyde, 30 mM Sodium Borate (11), 2 mM MgCl2] were added briefly before removing the combined fixative and adding fixation buffer #2 for 7 minutes. The gonads were rinsed briefly with PBS, permeabilized in PBS plus 0. 3% Triton X-100 (Sigma) for 10 minutes, then rinsed several times in PBS and immunostained overnight at 4°C. Widefield images (Figure 2B–2D, Figure 3A–3E) were acquired with a Nikon Eclipse 90i upright microscope using 40× (Plan Fluor, 1. 4 NA) and 60× (Plan Apo VC, 1. 4 NA) lenses and a Retiga-4000DC camera (QImaging). Images were exported to Adobe Photoshop for cropping, reorientation, and contrast and brightness adjustments. All other images were acquired with a DeltaVision microscope and processed using deconvolution software (Applied Precision). All images shown are single plane, except for Figure 5A and Figure 5E, which are two plane (0. 2 µm) projections to visualize ring channels. For electron microscopy, L4 hermaphrodites grown at 20°C–23°C were selected and allowed to develop to young adults for 24–48 hours. For male/female strains, L4 females were collected and mated with males. Gonads were dissected from 40–60 adults, then fixed with glutaraldehdye and osmium tetroxide and processed for electron microscopy as described [20]. Grids were viewed with a JEOL JEM-1230 transmission electron microscope. To avoid scoring individual germ cells and gonads more than once in the initial survey, each gonad was numbered and scored in a single, representative thin section through the middle of the core. Germ cells typically were examined at 4,000× to 6,000×, and candidate VLPs were photographed at 8,000× with a Gatan UltraScan 1000 CCD camera. For each sample, germ cells were examined in the distal, mitotic region, and at all stages of meiosis through oogenesis. The gonad core contains large numbers of vesicles of various shapes that might be confused with VLPs, however the cytoplasm around germ nuclei is relatively devoid of organelles. Thus, quantitation was performed only on germ “cells” up to the ring channel, although the core was inspected in many gonads. | Retrotransposons and retroviruses pose enormous threats to animal and plants because of their ability to insert into host genes. Retroelements that replicate in germ cells can, if left unchecked, expand exponentially in the host genome. C. elegans has proven to be an exceptional model system for studying many facets of cell and molecular biology, and the genome contains both retrotransposon and retroviral sequences. However, no virus-like particles have been observed in C. elegans germ cells. We show here that Cer1, an endogenous Gypsy/Ty3 class retrotransposon, is expressed at very high levels in C. elegans germ cells, but escaped detection in previous studies because its expression is both temperature and age dependent. These studies reveal new aspects of microtubule regulation in C. elegans that the retroelement appears to exploit to navigate the germ cell cytoplasm, and demonstrate the power of C. elegans for studying host/pathogen interactions in germ cell biology. | lay_plos |
Recent experimental evidence suggests that coordinated expression of ion channels plays a role in constraining neuronal electrical activity. In particular, each neuronal cell type of the crustacean stomatogastric ganglion exhibits a unique set of positive linear correlations between ionic membrane conductances. These data suggest a causal relationship between expressed conductance correlations and features of cellular identity, namely electrical activity type. To test this idea, we used an existing database of conductance-based model neurons. We partitioned this database based on various measures of intrinsic activity, to approximate distinctions between biological cell types. We then tested individual conductance pairs for linear dependence to identify correlations. Contrary to experimental evidence, in which all conductance correlations are positive, 32% of correlations seen in this database were negative relationships. In addition, 80% of correlations seen here involved at least one calcium conductance, which have been difficult to measure experimentally. Similar to experimental results, each activity type investigated had a unique combination of correlated conductances. Finally, we found that populations of models that conform to a specific conductance correlation have a higher likelihood of exhibiting a particular feature of electrical activity. We conclude that regulating conductance ratios can support proper electrical activity of a wide range of cell types, particularly when the identity of the cell is well-defined by one or two features of its activity. Furthermore, we predict that previously unseen negative correlations and correlations involving calcium conductances are biologically plausible. In well studied neuronal networks, it is often observed that each neuron has a specific role in the function of the circuit. In some cases, this role is unique and vital, and the health of the animal depends on a robust cellular identity. One example of this occurs in the pyloric circuit of the crustacean stomatogastric ganglion (STG). This well-studied system must produce robust rhythmic activity for successful digestion [1], and does so through the dependable cellular properties of its component neurons. These neurons are identified by their reliable network activity, morphology, and connectivity [1]. This reliability is surprising, because identified cells can have different sets of ion channel maximal conductances in different animals of a population, despite generating the same predictable electrical activity [2], [3]. Furthermore, these ion channels are constantly replaced or modified due to synaptic input, sensory feedback, and neuromodulatory action [4], [5]. In light of these sources of variability, how stomatogastric cells produce consistent, stable output is an open question. Recent experimental evidence suggests that coordinated expression and regulation of ion channels may play a role in constraining cellular electrical properties. The first such relationship found in the lobster STG was a positive co-regulation between the transient K+ current (IA) and the hyperpolarization-activated mixed ion current (Ih) [6], [7]. The investigators discovered that cellular injection of mRNA coding for the ion channel underlying IA caused an endogenous increase in the opposing Ih current, without changing the electrical activity of the cell. The same conductance pair was shown to be correlated in a population of identified neurons in the crab STG, and this correlation was found to be independent of the effects of neuromodulators [8]. The latter study compared correlations of current density in the pyloric dilator (PD) neuron before and after removal of top-down neuromodulatory input, and found two additional K+ conductance correlations that were neuromodulator-dependent. A variety of positive linear correlations in ion channel mRNA copy numbers have been found in other identified STG cell types, as well [9]. Each cell type appears to express a unique set of ion-channel correlation types and linear relationship slopes. In some cell types, correlations have been seen to involve three or four channel types. Similar relationships, involving conductances [10], [11], [12], or ion-channel kinetics [13], have been found in other model systems at the single cell (co-regulation) and population (correlation) levels. Combined, the data suggest a means for constraining cellular activity despite the often several-fold conductance variability seen in these cell types. To date, all experimentally demonstrated conductance correlations within a wild type population of identified neurons have had a positive slope; no negative correlations have been found. This finding raises several questions about the mechanisms underlying the conductance correlations and their relation to maintenance or definition of activity type. At this time, the mechanisms are unknown, though there are several possibilities. Two membrane channel genes may be concurrently regulated by a single transcription factor in a cell-type specific manner [14]. Alternatively, a cleaved section of one ion channel can act as the transcription factor (or repressor) for another [15]. A unidirectional mechanism of this type may explain why the IA–IH co-regulation is not reciprocal in STG neurons [16]. In addition, correlations may be controlled by coordinated post-transcriptional or -translational modification of one or more channel gene products [17], [18]. Though there is evidence for activity-independent co-regulation [6], [8], changes in activity are needed for co-regulation to be expressed during Xenopus laevis development [11]. This raises the possibility that activity-dependent regulatory cascades may also be involved in conductance co-regulation in some cases [19], [20]. A number of conductance correlations are also neuromodulator dependent [8]. There is a possibility that any of the above mechanisms are influenced by the presence of neuromodulator, and that correlation differences between cell types are due to differences in neuromodulator sensitivity [21], [22]. In addition to questions about the underlying mechanisms, questions remain regarding the cellular effects of linear conductance correlations. The effects on activity are likely different for each conductance pair; however, recent modeling studies have shed light on general trends in the structure of the conductance space with regard to activity type. Goldman et al. undertook a study of the pair-wise conductance space of stomatogastric neurons and neuron models, and demonstrated quasi-linear grouping of broadly categorized activity types [23]. Similar results have been obtained with a high-dimensional visualization technique of a model neuron database, wherein all conductance parameters are considered [24]. Hand-tuned models have also been a useful tool for demonstration of conductance correlation effects on activity. In one stomatogastric neuron model, a linear relationship between IA and IH maintains a specific number of spikes per burst [6]. Further theoretical studies demonstrated that correlated changes in these conductances can adjust neuronal input-output gain, while maintaining other features of activity [25]. Parameter changes in a leech heartbeat neuron model with narrowly constrained burst period and spike frequency demonstrated a correlation in the spike-mediated synaptic conductance and the inactivation time constant of the transient calcium current ICaS [26]. A tempting conclusion to draw from these results would be that as more features of activity are constrained, more correlations may be required to maintain proper activity. This may not be true, as has been suggested previously [26]. Taylor et al. recently published a detailed model of the lateral pyloric (LP) neuron constrained to reproduce 9 experimentally measured activity characteristics, but found no strong linear correlations between conductance parameters [27]. Here, we expand on previous work by investigating the general relationship between the presence of conductance correlations and the manifest features of activity type. We hypothesize that conductance correlations do contribute to the robustness of critical features of electrical activity. To test this, we use a database of generic STG conductance-based model neurons [28]. These model neurons were first partitioned into groups based on ranges of a single characteristic of the electrical activity, or combinations of biologically-inspired activity characteristics. The conductance space for each group was examined and individual conductance pairs were tested for linear dependence. The conductance correlations found were further evaluated for their effect on activity type by building correlation-based populations of models. Our results show that while regulating conductance ratios can support the maintenance of a single cellular activity characteristic, the effects of correlations are less clear when multiple characteristics are required to specify activity type. A model neuron database was used to investigate the relationship between conductance correlations and intrinsic activity type. This database has been previously described [28]. Briefly, a single-compartment conductance-based model was used. Seven of the eight conductance types used in this model were derived from experiments on unidentified stomatogastric cells in culture [29]. They include: a fast Na+ (gNa), fast transient Ca2+ (gCaT), slow transient Ca2+ (gCaS), fast transient K+ (gA), Ca2+-dependent K+ (gKCa), delayed-rectifier K+ (gKd), and a voltage-independent leak conductance (gleak). The hyperpolarization-activated mixed-ion inward conductance (gH) was modeled after that found in guinea pig lateral geniculate relay neurons [30]. Each maximal conductance parameter was independently varied over 6 equidistant values, ranging from zero to a physiologically relevant maximum. By simulating the model neurons corresponding to all 68 = 1,679,616 possible combinations of these parameters, a large variety of intrinsic electrical activity patterns were created. This systematic variation of parameters created an eight-dimensional grid of simulated parameter sets within the conductance space of the model. A single model neuron, with its particular intrinsic activity type, inhabits one grid point in this eight-dimensional space. The currents used to generate this model were not based on any one STG cell type. To approximate distinctions between cell types, which are identified in part by activity, the database was partitioned based on intrinsic activity type of each model neuron. The first level of categorization divided the model neurons into five subsections: silent, periodic spiking, periodic bursting, irregular (non-periodic) spiking or irregular bursting (Figure 1). Silent models exhibited no membrane voltage maxima. Periodic spiking models had inter-maximum intervals that were consistent within 1% of their mean. Periodic bursting activity was detected and defined as follows. First, the peaks and troughs of the membrane potential were subjected to a spike detection threshold. Any peak greater than −30 mV was defined as a spike, and all others were ignored as sub-threshold activity. Next, the inter-burst interval was defined as any inter-spike interval that deviated from the length of the greatest inter-spike interval by less than 30%. These definitions are different from previous burst classification metrics [28] to avoid minor classification errors associated with alternating burst features. The second level of categorization further partitioned the periodic spiking and bursting models. The large group of periodic bursting neurons was either partitioned based on the duty cycle, defined as the fraction of the burst period taken up by the burst duration, or by the average slope between the inter-burst minimum and the start of the next burst, hereafter referred to as the rising phase of the slow wave (see Figure 2C). Briefly, the slope was calculated by first locating the inter-burst minimum (point 1 on the inset of Figure 2C). Then, the next crossing of the spike detection threshold (−30 mV) was recorded (point 5). The distance between the two points was divided into four equal sections (demarcated by points 2–4). Average slope was calculated between points 1 and 4, omitting the portion between points 4 and 5 to avoid any artifact generated by the sharp incline of the first spike in the burst. Initial slope (average slope between points 1 and 2) and central slope (average slope between points 2 and 4) were also investigated. These characteristics were chosen because both duty cycle and the average slope of the rise phase are thought to be regulated within a narrow range in biological cells [6], [31]. Periodic spiking neurons were similarly partitioned by spike frequency. Group size and boundaries were chosen based on population distributions shown in Figure 2. Clustering of models was seen in the spiking models (Figure 2A) and the bursting models segmented by the rising phase of the slow wave (Figure 2C, D). In the absence of clear subpopulations arising from model clustering, an effort was made to achieve optimal resolution within the conductance space by avoiding large differences in group size. For activity metrics without clear subpopulations, shifting the arbitrarily chosen population boundaries shown in Figure 2 did not drastically change the correlations found. Additional metrics were used to partition the periodic spiking and bursting models, such as spike height and the number of spikes per burst, respectively. However, these metrics resulted in a lower overall success rate for finding correlations and were therefore not reported here. The segmentation scheme described above investigates the relationship between correlations and single activity characteristics. We also looked for correlations in model populations based on multiple activity characteristics. To do this, we constructed model populations based on subsets of the pacemaker activity criteria previously described, including characteristics of the “slow wave” voltage oscillation underlying bursts in STG pacemaker neurons [28]. Briefly, the slow wave is the membrane potential of a bursting model after spikes have been subtracted. The peak of the slow wave was approximated by the last maximum in a burst, and the slow wave amplitude is then the difference between this maximum and the between-burst minimum. The ranges of activity characteristics used are based on experimental data from the spontaneously bursting pacemaker kernel of the STG, which consists of one anterior burster (AB) neuron electrically coupled to two PD neurons. The complete dataset, including the descriptive statistics used to classify models, is available online as a supplemental file (datasets S1 and S2). After model neurons were sorted based on intrinsic activity, conductance plots were generated for each activity subsection (Figure 1). As shown in Figure 3, these plots graph the value of one conductance parameter versus another, for a particular activity subsection. Each model in the activity subsection falls on one grid point on this plot, positioned by the maximal conductance parameters it was assigned for the pair in question. The other 6 conductance parameters were not constrained when plotting. The grid structure used to generate simulation points for this database creates a conductance space wherein all conductance values are initially equally represented. After segmentation of the database, this is no longer the case and each activity type has a unique distribution of each conductance parameter. Bursting models, for example, have a higher average value of gKCa than spiking models. These non-standard one-dimensional (1D) conductance distributions violate the assumptions of parametric tests and significance testing. Instead, we used two non-parametric tests with simple cutoff values to define correlations. For each activity subsection: if the value of one conductance was determined to be dependent on the value of another (by a chi-squared test of independence χ2>500), and this dependence was confirmed to have a linear trend (by a Spearman' s rank correlation |ρ|>0. 2), the relationship was considered a correlation. Note that, in the absence of significance testing, these definitions are not dependent on the number of observations (models in each population). This was chosen purposefully because, due to the sparse sampling of the conductance space, populations with very few models may not contain enough information to reliably identify correlations. Cutoff values independent of the number of observations ensures that those correlations reported are strong in all cases. In addition to the described statistics, a simple visual check for a linear dependence was performed as follows. Assuming independence between conductances, it is possible to generate the expected two-dimensional (2D) conductance distribution for a particular activity type by multiplying normalized single-conductance histograms for that activity type (hereafter referred to as an independence matrix). By looking at the deviations in the actual data from this independent assumption, a linear dependence or lack thereof is more clearly visible than when looking at the correlation plots alone (Figure 3E, F). We chose to use this simple, graphical representation of dependence as a visual check to distinguish correlations from conductance relationships that can be explained by independently varying parameters. Specifically, the independence matrix was created by multiplying two conductance histograms for a particular activity type (units in % of models of that activity type). Then, this independence plot was subtracted from the actual data (units in % of models of that activity type). Note that the resulting plot of percent difference (hereafter referred to as a difference matrix, shown in Figure 3E, F) is distinct from, but complementary to, the chi-squared test of independence. Defined correlations were used to generate populations of model neurons that fit within those correlation boundaries. The shape of each correlation was defined by setting a threshold of 3% (models/all models in the activity type, the latter hereafter referred to as typei) per grid point of the correlation plot raw data (see the white outline in Figure 3B for an example). The entire database, regardless of activity type, was scanned for models that fell within the boundaries of one defined correlation or a combination of correlations. We call the resulting “correlation-based” population cbi. If a correlation is successful in restricting activity to a particular type, it is expected that a large number of models in the correlation-based population would be of that type. Specifically, we would expect that there would be a greater percentage of typei models in cbi when compared to the original database. We therefore calculated a statistic we termed success percentage (%Success), by dividing the number of models in cbi that are of typei by the total number of models in cbi (%SuccessCorrelation-based). The percent of typei models in the original database (%SuccessOriginal) was also calculated (see percentages shown in Figure 1). The two were compared as follows: where O represents the original database, N denotes a function returning the number of models in the parameter population, and O∩typei should be read as “the intersection of the original database and the set of models of typei”. The success factor (fSuccess) is therefore a multiplicative factor by which implementing a correlation increases or decreases the likelihood of a particular activity type. An increase in % success for the correlation-based population versus the original database (fSuccess>1) indicates a correlation that is useful in supporting a desired activity type. As a control, fSuccess was also calculated for a randomly distributed conductance plot applied to a random conductance pair. For the case of multiple correlations, the control case employed the same number of randomly generated conductance plots as there are defined correlations for that activity type. As further verification of our methods, we also applied an ideal linear correlation shape to randomly selected conductance pairs and activity types, and found no unexpected increases in fSuccess (data not shown). A model neuron database partitioned by activity type was screened for pair-wise conductance relationships, to elucidate the possible functional role of linear conductance relationships seen in experiments [6], [8], [9]. However, linear dependence was not the only relationship type seen in this database. A wide variety of nonlinear relationship types were also seen (for examples, the correlation plots of activity type “spiking models 50–75 Hz” are used, Figure 4). Many conductance pairs were clearly independent of one another; examples include completely flat distributions (gH and gleak, Figure 4) or correlation plots with a striped appearance (gNa and gH, Figure 4). In these cases, one or both of the conductances involved has a flat 1D distribution, and therefore the activity type in question does not require a particular value or range of values of that conductance. Another type of relationship commonly seen was the “ramp” type. These conductance plots had a high concentration of models in one corner, few models in the opposite corner, and a gradient in between. Intuitively, this would suggest dependence. On the contrary, many of these apparent relationships could be explained by independent variation of conductances. For example, the gCaS and gA conductance plot shown in Figure 4 is one example of a ramp type relationship where the corresponding chi-squared statistic was low, and the percent-difference plot between actual data and the independence assumption (difference matrix) shows no interesting trend. Indeed, this relationship appears to be fully explained by independent variation of parameters, as many of the ramp-type relationships we saw were (Figure 3A, C). Finally, both positive (gNa and gCaT, Figure 4) and negative (gCaT and gCaS, Figure 4) linear correlations were seen and will be described in detail. The statistical criteria used to define correlations were chosen with the goal of distinguishing those conductance pairs linearly dependent on one another. To verify the statistics, each plot and its corresponding difference matrix were checked for visual confirmation of a linear trend (Figure 4). In a small number of cases (16/174) we found that a plot would fit the statistical criteria for linear dependence but linearity was not evident. This was most often an edge effect; for instance, a relationship wherein one or both conductances were zero for a majority of the models (Figure 5). Notably, most false positives were due to low average values of gKCa or gCaT or both, and were found in activity types such as silent and fast spiking models. False-positives, though they met the statistical criteria, were not considered correlations (see Supplemental files, Table S2). Statistical criteria were chosen based on minimization of false-positive results. Excluding those cases discussed above, our statistical criteria identified 174 pair-wise linear correlations out of a possible 1316 conductance pair combinations in 47 model sub-populations (see Figure 6B and Supplemental files Table S1). We did not tally the relationships that appeared linear but did not meet the cutoff criteria (apparent false-negatives) because they tended to occur only in activity subsections with relatively low numbers of models. A property of the chi-squared test of independence, that relationships with more data points will more easily reach a cutoff value, was intentionally used to compensate for the sparse sampling of the conductance space. One example of an apparent false negative is shown in Figure 7. The activity type “bursting models with a duty cycle greater than 0. 6” appears to have a linear dependence between gA and gKCa; however, the chi-squared statistic is less than 500 for this plot. A possible solution to avoid excluding this apparent correlation would be to use a chi-squared statistic that is scaled by the total number of models in the population, because this group contains only 1776 models compared to an average of 100,000 (Table S1). However, that approach would exclude other, possibly more reliable, relationships. For example, a scaled cutoff would exclude the gA and gKCa correlation from the activity type “bursting models with a duty cycle between 0. 2 and 0. 4” which appears very similar to the ‘false negative’ discussed above (Figure 7). We chose to use the raw statistic (unscaled) because we found this result counter-intuitive. A relationship found in a population with a large number of models should be more reliable than an equivalent relationship found in a population with very few models, especially on a sparse grid. This is not to say that relationships fitting our statistical criteria were not apparent in populations with low numbers of models. On the contrary, we found several correlations in this and other populations with relatively few models (Table S1). When model populations were defined by a single activity characteristic, a correlation was more likely to appear in a group with a narrow range of that characteristic (Figure 1). For example, the group “all periodic spikers” had no correlations; however, when this group was segmented by spike frequency every subsection had at least one correlation. Similarly, the group “all periodic bursters” demonstrated no correlations, whereas all of its subsections partitioned by duty cycle did. Although the categories without correlations each included over 200,000 model neurons (see Figure 1 and Supplemental files, Table S1), this phenomenon was not linked to the size of the activity subsection. For example, the group “bursters with duty cycle <0. 05” contains a large number of models (392,398) and has three correlations. It is reasonable then to assume that correlations arise by virtue of the narrowly defined activity type of a group rather than simply the number of models in a group. Very few conductance pairs were seen to demonstrate a positive correlation in one activity type and a negative correlation in others (Figure 6A). Thirteen conductance pairs exhibited only positive correlations, eight conductance pairs exhibited only negative correlations, and two conductance pairs had both relationship types. We found no correlations for the remaining five possible conductance pairs. As shown in Figure 7, the relationship between gA and gKCa is one example of a negative correlation. This conductance pair was negatively correlated for spiking models with a frequency of 25–75 Hz (subsections 25–50 Hz and 50–75 Hz) and bursting models with a duty cycle between 0. 2 and 0. 6 (subsections 0. 2–0. 4, and 0. 4–0. 6) (Figure 7). Interestingly, though all of the correlations for the gA/gKCa pair are negative, they appear to have a slightly different slope in each case. This is only one example of slope differences seen between activity types for a single conductance pair, though there are also cases of correlations with the same slope in several activity groups. In the latter case, correlations in different activity groups generally inhabit different areas of the conductance space (Figure 8). There was a strong presence of calcium conductance correlations in this database (Figure 6A). In the model sub-populations we investigated, there were 140 correlations involving one or both calcium conductances, compared to only 34 that did not involve either calcium conductance. All conductances showed correlations with at least one of the calcium conductances. The gKCa and gCaS conductance pair had the highest number of correlations and was strongly correlated in several activity types. In contrast with populations based on a single criterion, there was no straightforward trend in the appearance or disappearance of correlations when multiple activity criteria were used to generate a population. Individually, each population based on a single pacemaker criterion has a unique set of correlations (Figure 6B, bottom row). When criteria are combined, however, correlations appear or disappear with a seeming disregard to those seen in the “parent” populations. For example, starting with the population based on slow wave amplitude (SWA) between 10 and 30 mV, adding the slow wave peak (SWP) range as a criterion leads to a loss of the gCaT vs. gleak correlation. Adding duty cycle as a criterion brings back the gCaT vs. gleak correlation, even though the duty cycle parent population does not use it. Adding burst period again leads to loss of that correlation. There is no path from bottom to top in Figure 6 along which the number of correlations increases steadily with an increase in criteria. When all 5 criteria were used, no correlations were found, though this may be influenced by the small number of models that fit all 5 criteria (56). Finally, defined correlations were used to create correlation-based sub-populations of models as described in the Methods section. A correlation-based population was, in all cases, found to have a larger percentage of models of the desired activity type than the original database. We found a 2. 3 fold increase for individual correlations, on average (σ = 0. 9). Individual correlations ranged from having a small positive effect on success rate to increasing it 5 fold (Figure 9). This is in contrast to the control condition of random conductance relationships, in which decreases in success were as likely as increases. On average, there was no difference between the control condition and the original database (average fSuccess = 0. 98, σ = 0. 2). The difference between control and correlation-based populations was even larger when multiple correlations were used to generate each population. When the set of all correlations for an activity type was used to create a population of models, the percent success increased 10 fold on average (Table S3). In contrast, the average fSuccess for the control case with multiple random conductance distributions was below 1 (μ = 0. 89, σ = 0. 17). Our results illuminate the functional benefit of linear conductance correlations for maintaining activity type. When a population of models was gathered based on adherence to a correlation rule, there was always an increase in a particular feature of activity. In some cases, this increase was admittedly modest, particularly when the population was based on a single correlation. However, there were several cases in which the constraint on activity type was impressive (Figure 9). In one case, imposing a single correlation caused an increase in the desired activity type from 18% (in the original database) to 53% (in the new, correlation-based population). Another population increased its percentage of a single activity type by 72% when multiple correlations were used. Put another way, the combined correlation-based populations demonstrated up to 81% of a single activity type. We find this to be a notable contribution to robustness of activity. This model implicates two types of correlations not previously seen in experiments. As of this writing there have been no published calcium conductance correlations. We have shown that calcium correlations are plausible, and if implemented by a cell, would assist maintenance of a wide range of activity types. This is also true of correlations with a negative slope, which have not yet been seen experimentally. Thirty percent of all correlations seen (56/174) were negative, spanning 10 out of the 23 conductance pairs with correlations in this study (see Supplemental files, Table S1). Though the mechanisms underlying biological correlations are uncertain, and not represented in the details of our model, our results suggest that negative correlations can be just as useful as positive ones for maintaining cellular activity. Negative correlations have been hypothesized for conductances that may compensate for one another. For example, a study of the solution space of a multi-compartment cerebellar Purkinje cell model reports negative correlations between several pairs of conductances, including two calcium conductances that appear to be anti-correlated so as to preserve the total calcium influx into the cell [32]. When fitting model neuron parameters to reproduce specific experimentally recorded neuronal voltage traces, such functionally compensatory conductances can lead to irreducible model parameter uncertainty [33]. An analysis of the correlation between the two potassium conductances gKCa and gA shows this type of relationship in our model. The Ca2+-dependent K+ current is often associated with bursting cells, and contributes in our model to determining burst length. Keeping all other conductances constant in a bursting model neuron, a higher value of gKCa will correspond to a shorter burst length [34]. The A current is also a K+ current, though it is not calcium dependent and it acts on a much faster time scale. Keeping all other conductances constant in a bursting cell, a higher value of gA will result in a slower spike rate, or fewer spikes per burst [35]. The effects of these conductances appear to sum for bursting neurons with a duty cycle between 0. 2–0. 6 (Figure 7). To maintain duty cycle in this range, a model needs a minimal amount of one or both conductances to counter-balance depolarizing (inward) currents. The exception to this rule is a small population (∼300 models) of elliptic bursters in the 0. 4–0. 6 duty cycle group. These models all have the same low value of gNa (100 mS/cm2), which may allow the absence of both gKCa and gA. Interestingly, bursting models with a low duty cycle show no dependence between gA and gKCa. A second type of negative correlation was seen between gNa and gKd. Initially, this negative correlation was surprising. Both conductances are involved in generation of the action potential and would therefore be expected to require balance via a positive correlation. Instead, only negative correlations were seen, and they appeared in two activity subsections: spiking models with frequency greater than 75 Hz, and bursting neurons with a duty cycle less than 0. 05. We found, based on where these correlations appeared, that a negative relationship between these two conductances was useful for maintaining very fast spiking. If a model in either of these activity groups was adjusted to make both conductances very low, then the model became silent. If both conductances were very high, the spike would broaden and the spiking frequency would decrease. It is interesting to note that both the conductance pairs discussed so far have been found to be positively correlated experimentally in stomatogastric neurons [9]. However, the activity types found to have these correlations in the database are not typical of stomatogastric intrinsic activity types [1], [36], [37] and thus should not be compared directly to experiment. There are several reasons why directly relating our results to the published experimental data is a challenge. First, conductance is not directly measured in experiments. Mapping conductance (used in the database) to mRNA copy number or current density involves several tenuous assumptions. For example, conversion between mRNA copy number and conductance value has been studied for three conductance types in the STG, of which only two were found to relate linearly [3], [38]. Therefore, it cannot be assumed that the other 5 conductances types used in this model have a linear relationship between mRNA copy number and conductance value. A larger problem is the choice of comparison: which activity characteristics should be assigned to a biological cell in a particular experiment? Our analysis of activity type of the model did not consider the effects of current injection or neuromodulation, and therefore should be compared to intrinsic activity of the biological cells. Unfortunately, there are conflicting accounts of the intrinsic activity of cells in the stomatogastric ganglion. While they have reliable and identifiable activity types in the intact circuit, isolating a cell to measure intrinsic activity often involves direct harm to its own processes or neighboring cells. The method of isolation may influence activity in unknown ways. For example, when the PD neuron is cut off from all synaptic input and neuromodulation it can be spontaneously silent [39], spike tonically [39], [40], burst with a periodic rhythm [39] or burst irregularly [40]. The one exception in the STG is the AB neuron, which is an intrinsic burster and has the same intrinsic and network behavior. Unfortunately, no experiments have been done examining conductance correlations in the AB neuron. For these reasons, we chose to avoid comparing our results for a particular activity type with the experimental data for a single cell type. Even so, there are general comparisons that can be made between our results and experiment. For instance, we found differences in slope for the same correlation in different activity types (Figure 7). This is reminiscent of the cell-type specific correlations found by Schulz et al. ; the correlation slope for a single conductance pair was different in each cell type [9]. In addition, we saw correlations which maintained slope but differed between activity types with regard to intercept (Figure 8). We can predict from this result that the general location within the conductance space is also important for determining activity, which has been suggested before [23]. Furthermore, each identified cell type studied by Schulz et al. had a unique combination of correlated conductance pairs, similar to our results (see Supplemental files, Table S1). This suggests that not only the slope and location of correlations, but the combinations in which they are used, are important for definition of cellular identity. This view is bolstered by our results with correlation-based populations. We showed that combinations of correlations, drawn from a particular activity type, generated much larger increases in percent success compared to individual correlations or controls (Figure 9). Another important comparison to experiment involves the hyperpolarization-activated inward conductance, gH. Correlations involving this conductance were only found when the slope of the slow wave was used to identify activity type (Figure 6C). We found this result to be particularly interesting because correlations involving gH have been found repeatedly in experiments in the stomatogastric ganglion [6], [7], [8], [9]. In experiments on STG PD and LP cells, both the slope of the slow wave and the ratio of IA to IH were shown to be conserved after injection of shal mRNA [6]. This implies that the correlation between the values of IA and IH may have something to do with maintaining the shape of the slow wave. In these experiments, the average slope of the rising phase of the slow wave was found to range, roughly, from 0. 02–0. 08 mV/ms. We found this correlation in a slightly higher range when looking at the central 50% of the rise phase (>0. 1, see supplemental Table S1). Though these results are slightly different, both correlations were positive and both suggest that IH is involved in maintaining a specific activity characteristic: inter-burst dynamics. Interestingly, IH was not correlated in any other activity types. We hypothesize that this is because the other activity types are defined more by conductances which are active during the depolarized phase of the spike or burst. If this is the case, it would follow that correlations involving IH would be more prevalent if response to hyperpolarizing current injection were used as a metric for defining activity type. For cells dependent on inhibitory network connections for a bursting phenotype, such as LP and pyloric (PY) neurons in the STG, this may be the case and should be an interesting area of further research. Recently, Taylor et al published a study of a population of LP neuron models, constrained by 9 activity characteristics, in which no strong linear correlations were found. We report a similar result in that the usefulness of correlations for supporting activity characteristics did not scale up as we expected. When multiple biologically-realistic activity criteria were used to generate a population of models, the appearance and disappearance of correlations was not easily explained by the correlations found in the “parent” single-criteria populations. This suggests that the correlations which would be helpful for each feature individually might interact in complex ways, inhibiting or enhancing the influence of the other, when multiple activity characteristics are imposed on a group. In our case, a likely explanation for these results lies in the complexity of the conductance space. The conductance plots we analyzed were 2D approximations of an 8-dimensional space. This means that a perfect linear correlation between two parameters will appear as such. However, dependencies between multiple parameters will form subspaces within the overall conductance space that may, or may not, be detectable in a flattened 2D analysis. Furthermore, the perfect population definition for one conductance pair may not be ideal for another conductance pair, resulting in the occasional conductance plot that contains two local dependency rules (see Figure 5A for a possible example). Finally, the scale of analysis is important. We had the opportunity to examine raw experimental data from correlations published by Schulz et al. [9]. It has been previously shown that every STG cell type has a unique range of conductance values [3], [38]. By binning the experimental data based on the conductance ranges of each cell type, it was apparent that bin size could contribute to the appearance of correlations (analysis not shown). For example, when binning conductance measurements from the gastric mill (GM) cell type based on the range of conductance values present in the PD cell type, otherwise apparent correlations are lost. Our model database is limited in this way, due to the grid structure of the conductance space, which can be interpreted as a type of binning. Combined, this complex behavior can easily give rise to situations in which correlations appear, or disappear, as the population of models is further reduced. Though this result highlights the inability of our analysis to capture all possible linear dependencies between conductances, it is important to note that it does not shed doubt on the correlations we did find. Though our set of apparent correlations should not be considered an inclusive list for these activity types, the correlations found in our database are useful for considering the possible effects of conductance relationships on activity. We argue that the presence of a large number of defined conductance relationships lends to the validity of the database as a tool for investigating correlation utility. With this tool, we have shown that linear conductance correlations can shape neuronal activity. Furthermore, we made specific predictions about the presence of negative or gCa correlations and situations in which they might be useful. | Most motor neurons receive input from the brain before transmitting to the muscle, resulting in a muscle contraction. In some cases, a small group of motor neurons can act independently to control rhythmic muscle contractions. Locomotion in mammals is thought to arise, in a large part, due to neuronal networks of this type residing in the spinal cord. However, the cellular machinery that guarantees the needed rhythmic pattern of electrical activity in these neurons is not fully understood. Here, we use a small circuit that controls stomach contractions in crustaceans like crabs and lobsters, called the pyloric circuit, to investigate potential mechanisms for regulation of neuronal activity. Ion channel proteins are integral to determination of electrical activity type. Recently, experimental studies using cells of the pyloric circuit have shown correlations in the expression of these proteins. Our study uses a mathematical model of neuronal electrical activity to detail how these correlations may be influencing activity type. We found that correlations imposed on model parameters increase the likelihood of a desired behavior, and we therefore conclude that a biological cell utilizing ion-channel correlations will have the advantage of increased robustness of activity type. | lay_plos |
The pupil is primarily regulated by prevailing light levels but is also modulated by perceptual and attentional factors. We measured pupil-size in typical adult humans viewing a bistable-rotating cylinder, constructed so the luminance of the front surface changes with perceived direction of rotation. In some participants, pupil diameter oscillated in phase with the ambiguous perception, more dilated when the black surface was in front. Importantly, the magnitude of oscillation predicts autistic traits of participants, assessed by the Autism-Spectrum Quotient AQ. Further experiments suggest that these results are driven by differences in perceptual styles: high AQ participants focus on the front surface of the rotating cylinder, while those with low AQ distribute attention to both surfaces in a more global, holistic style. This is the first evidence that pupillometry reliably tracks inter-individual differences in perceptual styles; it does so quickly and objectively, without interfering with spontaneous perceptual strategies. A visual scene can be perceived at various hierarchical levels of structure, from the most local elements to the global organization. A dense patch of trees is perceived as a forest at a global level, while a progressively more local analysis reveals the individual trees, then their leaves, bark, etc. The basis of global perception is the structuring into larger units the ‘bits and pieces’ of visual information in order to perceive objects and their relations (de-Wit and Wagemans, 2015). The ability to form a whole from parts, ignoring details to form meaningful classes, is a major developmental step in childhood and a component of how we define intelligence – one of the tests for IQ in the standard Wechsler Scale. Different authors have developed relatively simple perceptual tasks that have become established indexes of the preference for local or global: the block design task (Kohs, 1920; Wechsler, 1955), the Rod-and-Frame Test (Witkin and Asch, 1948); the Embedded Figures Test (EFT: 5); and Navon’s hierarchical letters (Navon, 1977). The preference for local or global is also part of a general way of feeling and behaving, as described by personality traits. Perhaps, the clearest example is the tendency for local or detail-oriented perception in Autistic Spectrum Disorders (Happé and Frith, 2006; Mottron et al., 2006; Plaisted et al., 1999), compared with the more global or holistic perception in typical adults (Navon, 1977): autistic observers show slower responses to global structure (Van der Hallen et al., 2015) and enhanced processing of local features (Mottron et al., 2006; Muth et al., 2014). There is increasing interest in determining whether differences in the preference for local or global styles are also associated with autistic traits in the typical population, supporting the dimensional view of autistic disorders, where people with and without ASD diagnosis lay along a continuum and differ only quantitatively, not qualitatively (Baron-Cohen et al., 2001; Constantino and Todd, 2003; Ruzich et al., 2015; Skuse et al., 2009; Wheelwright et al., 2010). However, not all attempts have been successful at showing a local-global difference in neurotypical individuals (Cribb et al., 2016), which may a result from the limited reliability and validity of the tests, which correlate little with each other and probably measure very different combinations of abilities and constructs (Milne and Szczerbinski, 2009). Here, we report on a pupillometry-based paradigm that yields an objective index of local-global processing. We tested 50 randomly selected neuro-typical adults with variable degrees of autistic traits, as measured by the Autism-Spectrum Quotient (AQ), a tool developed to characterize the normal spectrum of subclinical autistic behaviours in the general population (Baron-Cohen et al., 2001). Our psychophysical task – simply reporting the apparent direction of rotation of a bistable stimulus – can be performed equally well with both local and global perceptual styles: either by attending to only the front surface, or by attending to the whole global rotation. By tagging the front and back surfaces with different luminance levels, the two styles should have different modulatory effects on pupil-size, which is has shown to be modulated by cognitive and top-down factors, such as perceived rather than physical luminance, and shifts in attention from dark to bright surfaces (Binda and Murray, 2015; Binda et al., 2014). Specifically, attending to a bright or dark surface is sufficient to evoke pupil constrictions or dilations respectively, tracking the focus of ‘feature-based’ attention (Binda et al., 2014). We therefore hypothesized that pupil-modulations would depend on whether participants attend locally to the front surface, or globally to both – implying that pupil-modulations in this task effectively index differences in the local-global preference across our participants. We tracked pupil-size while participants viewed an ambiguous dynamic stimulus comprising 150 leftward-moving white dots and 150 rightward-moving black dots, giving the immediate impression of a cylinder rotating in depth at 10 revolutions per minute (see Figure 1A and Video 1). As the depth of the dots was ambiguous, either dot-group (tagged by luminance and direction) could be seen in front, resulting in either clockwise or counter-clockwise rotation. Subjects continuously reported the perceived direction of rotation by keypress or joystick for three sessions, each lasting 10 min. The 50 participants were recruited in two groups of 25, and the two sessions were intended as self-replications of the experiment (see below, Figure 3). On average, perceived direction alternated every 5. 5 s (0. 21 ± 0. 01 perceptual switches per second); the between-subject variability of switch rate did not correlate with autistic traits (Pearson’s r = 0. 02 [-0. 26 0. 29], p=0. 901, Bayes Factor [BF]=0. 1). Figure 1B shows the timecourse of pupil-size, synchronized to the perceptual switch and averaged over all subjects, separately for perceptual phases with black or white foreground (blue and red, respectively). Two distinct kinds of pupil modulations are apparent. First, as reported previously for binocular rivalry (Einhäuser et al., 2008), the pupil dilated transiently at or just after each perceptual switch; this effect is seen on both blue and red traces, meaning that the dilation occurs irrespective of whether perception switches toward a black or a white foreground. However, there is also a second kind of pupil modulation that depends on the direction of the shift: the pupil was more dilated when the foreground was black and more constricted when it was white. This is similar to the pupillary modulation that occurs during binocular rivalry between stimuli of different luminances, leading to pupillary constriction when the brighter stimulus dominates (Naber et al., 2011). This luminance-dependent modulation is a purely perceptual effect, as the stimulus luminance is constant throughout the experiment. The constriction when the foreground was white probably results from participants attending more to the foreground than the background, as it is known that attending to white constricts the pupil (Binda et al., 2013). On the other hand, if participants were to distribute attention evenly between the back and the front surfaces (or rapidly switch attention between them), no pupil difference would be expected. As these two strategies correspond well to the local and global perceptual styles associated with high and low autistic traits (Happé and Frith, 2006; Grinter et al., 2009), pupil modulation should be more prominent in participants with higher AQ scores. Figure 1D shows that, in our sample of typical young adults, there was considerable variation in luminance-dependent pupil change pupil modulation. The signed amplitude of the modulation was significantly positive in 20 out of the 50 participants. Crucially, the amplitude of modulation, which we suggest is an index of perceptual style, was highly correlated with AQ scores (r = 0. 70, [0. 52: 0. 82] p<10−5, BF >105). Importantly, the correlation was specific to this particular pupillometric index: AQ scores did not correlate with the general dilation that followed all perceptual switches (r = 0. 06, [-0. 22: 0. 33], p=0. 678, BF = 0. 1: Figure 1C). To test the idea that the variability of pupil responses emerges from different (local-global) perceptual styles, we performed two further experiments. Firstly, we attempted to induce changes in viewing strategy by changing the instructions given to the participants, and looked for concomitant changes of pupil modulation. We retested twice more a small subset of 10 participants, under two different instruction sets: ‘try to attend to both surfaces and see the cylinder rotate as a single unit’; or ‘focus attention on the front surface alone’. Most subjects reported that they found the instructions difficult to follow, and that they tended to lapse into their more natural viewing style. Nevertheless, the instructions significantly affected the luminance-dependent pupil modulation (0. 005 ± 0. 007 mm when instruction favoured the global strategy; 0. 029 ± 0. 008 mm when they favoured the local strategy), which was greater when the local strategy was encouraged (paired t-test: t: (9) = −2. 72, p=0. 024). This supports the suggestion that pupil modulation is driven by perceptual style, and further also shows that the pupil can be a more sensitive (as well as more objective) probe of perceptual style than are subjective reports. In a second experiment, we modified the procedure to encourage all participants to view the stimulus in the same local manner, attending to only one surface. Instead of the continuous presentation used before, we presented brief (6 s) bursts of the stimulus and instructed subjects to which surface they should attend (Figure 2A). To monitor compliance, participants reported the number of speed changes in the dots of the cued surface (ignoring changes in the other surface), which they did with an average of 62 ± 2% correct responses (uncorrelated with AQ, r = 0. 24 [-0. 04 0. 49], p=0. 090, BF = 0. 5). Under these conditions, there was a strong modulation of pupil size in the averaged data, depending on whether the white or the dark surface was attended (Figure 2B), consistent with the reported effects of feature-based attention on pupil size (Binda et al., 2014). However, in this experiment, where viewing style was constrained by the task, all subjects tended to show front-color dependent pupil modulation, and it was not correlated with AQ (r = 0. 20 [-0. 08: 0. 46], p=0. 156, BF = 0. 3, Figure 2C). Having established that the correlation between pupil difference and AQ scores is specific to the free-viewing of our bistable stimulus, we went on to check the reliability of the effect. We measured the correlation separately in the two groups of 25 participants who participated in the two sessions of the experiment (and are combined in the plots of Figure 1B–D). The correlation between AQ and pupil difference was strong and significant in both groups (Figure 3A, first subset: r = 0. 75, p<10−4, BF >103; Figure 3B, second subset: r = 0. 64, p<0. 001, BF = 54. 3). We further replicated the effect in a slightly different condition, tested in 26 participants (swapping the motion directions of white and black dots, Figure 3C, r = 0. 66 p<0. 001, BF = 85. 3); the three correlation coefficients are statistically indistinguishable (Fisher' s Z test; all p-values>0. 23), implying that the results are robust. As a further check on the reliability of the results, we also analyzed the correlations between pupil difference in the 50-participant group (Figure 1D), and each of the five subscales of the AQ questionnaire (normally distributed Figure 3D). All but one correlation coefficients were positive and significant (Figure 3E). The strongest correlations were with the Communication and Social Skills subscales, and weakest with ‘Attention to Detail’ scores (discussed later). The bottom row of Figure 3E shows the correlation of each subscale with the rest of the questionnaire (the sum of the other four subscales), and also the pupil-difference index with the overall AQ score. Note that the correlation of the pupil-difference index with the total AQ score was higher than those between any subscale and the other four subscales. The results reported in Figures 1–3 show that pupil modulation reliably indexes attention to the front or both surfaces, depending on the participant’s AQ score. In principle, perceptual style should also be measurable by psychophysical techniques, with enhanced performance on the attended surface (s) (Carrasco, 2011). To compare our novel pupillometry approach with more standard psychophysical methods, we measured correlations between AQ and ability to detect subtle changes on the front or rear surface. As in the main experiment, participants continuously tracked the rotation of the cylinder over 10 min long sessions; but here they also reported brief changes in speed that occurred randomly on the surface formed either by white or black dots, at the front or the rear depending to the perceived rotation of the cylinder. As in the main experiment, pupils dilate more when the foreground is black compared with when it is white (Figure 4A). Speed-change detection performance was generally better when targets occurred on the front than on the rear surface (Figure 4C paired t-test on d-prime values: t (24) =4. 43, p<0. 001). However, AQ correlated neither with the difference in performance (Figure 4D, r = −0. 03 [-0. 42 0. 37], p=0. 869, BF = 0. 2) nor with the pupil difference (Figure 4B r = −0. 27 [-0. 60 0. 14], p=0. 187, BF = 0. 4). This shows that the double-task setting (necessary to obtain a psychophysical index of attention distribution) interfered with participants’ natural viewing style, biasing all towards a ‘local’ processing style, focusing on one surface at a time to detect the speed changes. In most cases, the surface was the front one. This clearly undermined the possibility of estimating the inter-individual differences that spontaneously emerge in the free-viewing conditions of the main experiment. We measured pupil size while subjects viewed a structure-from-motion stimulus perceived as a cylinder rotating in 3D with bistable direction. In some but not all participants, pupil-size modulated in synchrony with their bistable perception, more constricted when the white surface was perceived in front. The magnitude of the modulation was tightly correlated with the Autistic Quotient scores, consistent with the view that stronger autistic traits accompany a preference for focusing on local detail, as opposed to globally attending the whole stimulus configuration. Some participants spontaneously reported their perceptual experience: of these some reported that they attending locally to only the front surface, while others reported that they had to attend globally to the whole configuration, both front and rear, in order to perceive the rotation. These spontaneous reports (respectively from participants with high and low AQ) provide qualitative support for our interpretation. Although preference for local versus global has been extensively investigated and has come to be a defining feature of Autistic Spectrum Disorders (Van der Hallen et al., 2015; Muth et al., 2014), its underlying mechanisms are essentially unknown. It could be a perceptual style, depending on differences in the bottom-up transmission of sensory information (Mottron et al., 2006; Mottron et al., 2003; Plaisted et al., 2003), but it could also have a cognitive or attentional basis, depending on a preference to maintain a narrow focus of attention (Happé and Frith, 2006; Plaisted et al., 1999) or a relative inability to divide or switch attention between multiple targets (Behrmann et al., 2006; Frith and Happé, 1994; Happé and Frith, 1996; Happé and Booth, 2008). Our results are equally supportive of all three possibilities, as are the classic tests for global-local preference, such as Navon letters or embedded figures. While our results do not distinguish between the different possible explanations of perceptual styles, they clearly show that autistic traits are associated with a more local perceptual style. There is an ongoing dispute about whether autistic traits are distributed continuously amongst the population with ASD diagnosis at an extreme, or whether the distinction is more categorical (Baron-Cohen et al., 2001; Constantino and Todd, 2003; Ruzich et al., 2015; Skuse et al., 2009; Wheelwright et al., 2010). The hypothesis of a ‘Broader Autistic Phenotype’ strongly predicts that local-global preference should be continuously distributed and correlated with autistic traits in the neurotypical population (Cribb et al., 2016). However, many of the classic tests show very low or insignificant correlations with autistic traits (Cribb et al., 2016). Our experiment describes a pupillometry index with the strongest correlation with autistic traits reported so far in a large group of participants, explaining about 50% of the variance in AQ scores. This correlation may appear surprisingly high, given that it is measured between two different tasks. However, each of these separate tasks has high reliability. The test-retest reliability of the autistic questionnaire has been reported to be around 0. 75 (Baron-Cohen et al., 2001; Hoekstra et al., 2008; Kurita et al., 2005). In the 22 participants who performed both the main experiment and the swapped-direction, the correlation of pupil modulation in the two tasks was 0. 68 ([0. 36: 0. 86], p<0. 001, BF = 62. 7). Considering the 95% confidence interval range (r = 0. 70, [0. 52: 0. 82]), the obtained correlations are not unrealistic, and certainly highly significant. Although the autistic questionnaire is well validated (Baron-Cohen et al., 2001), not all items have the same validity. We also observed variability in the strength of correlation of the pupil index and the AQ subscales, which was strongest for the Communication and Social Skills subscales and, somewhat surprisingly, weakest for the ‘Attention to Detail’ scores. However, the ‘Attention to Detail’ subscale seems to have the lowest construct validity of the five subscales: it had the lowest performance in classifying individuals with and without ASD diagnosis in a sample of 350 adults, with 130 ASD diagnoses (Lundqvist and Lindner, 2017), it had the lowest correlation with the general AQ score in a sample of 2343 typical individuals (Palmer et al., 2015), and, in our sample, scores on the ‘Attention to Detail’ subscale did not correlate significantly with the overall AQ score. It is also worth noting that among the items contributing to the ‘Attention to Detail’ subscale, only four actually relate to perceptual phenomena (e. g. ‘I usually concentrate more on the whole picture, rather than the small details. ”), the others being focused on memory and cognition (e. g. ‘I am fascinated by numbers. ”). All this highlights a general difficulty in measuring local-global preference, either with self-report or with laboratory tests. However, by combining the objectivity of pupillometry and the flexibility of a perceptual task involving bistable perception, we can achieve a very reliable and precise measure of inter-individual differences. There is growing interest in relating differences among individuals in their performance in perceptual tasks, with states or traits of their personality (Tadin, 2015; Eldar et al., 2013; Antinori et al., 2016; Wilson et al., 2016; Maclean and Arnell, 2010). However, the amount of explained variance is usually lower than in our experiment, which is a rare case where a physiological measure obtained from a laboratory experiment explains a large portion of inter-individual differences in the way we feel and behave. To obtain the clear correlation with AQ, free-viewing of the bistable stimulus was essential: the correlation between pupillometry and AQ scores was eliminated when cueing attention, either explicitly, by instructing participants to attend globally/locally or by indicating that the surface formed by white or black dots alone is task-relevant, or implicitly, by introducing a task that is best performed by attending a single surface at a time. Although previous work has shown differences in bistable viewing that are related to autism [ (Robertson et al., 2013); but see also (Said et al., 2013) ], we found that the dynamics of perceptual oscillations between the two directions of motion were uncorrelated with autistic traits. The correlation with AQ was only revealed only when taking pupillometry measures to index how attention was distributed during the bistable perception. The current study strengthens previous claims that pupil size is modulated not only by light, but also by perceptual ‘top-down’ processes (Binda and Murray, 2015; Mathôt and Van der Stigchel, 2015; Laeng and Endestad, 2012). The neural substrates of these modulatory mechanisms are beginning to be unveiled, involving pre-frontal input into the circuit of the pupillary response to light (Ebitz and Moore, 2017), which in turn may include the visual cortex (Binda and Gamlin, 2017). These multiple influences converge into a simple subcortical system that controls a unidimensional variable: pupil size. As we show here, under appropriate conditions (free viewing a bistable stimulus with brighter and darker components), this variable can provide an objective physiological correlate of conscious perception, so reliable that it can successfully reveal subtle inter-individual differences that are hard to study psychophysically. The method is quick, easy, and objective, and requires only relatively simple, portable apparatus. We hope it can form the basis for tests suitable for clinical populations, possibly providing a new powerful tool for identifying anomalies in perception that are predictive of ASD. We recruited a total of 62 subjects (42 women; age (mean ± SD): 25. 53 ± 4. 04), in three groups (25 in the first, 26 in the second and 11 in the last). All were students from the University of Pisa or Florence, in at least their third year. All reported normal or corrected-to-normal vision, and had no diagnosed neurological condition. The number of participants recruited for the study was selected to provide a large effect size as indicated by a priori power analysis (effect size: 0. 50, α = 0. 05, two-tail) that reveals that in order to reach a power (1−β) of 0. 8 a sample size of 26 subjects was needed. Experimental procedures were approved by the regional ethics committee [Comitato Etico Pediatrico Regionale—Azienda Ospedaliero-Universitaria Meyer—Firenze (FI) ] and are in line with the declaration of Helsinki; participants gave their written informed consent. All participants completed the Autistic-traits Quotient questionnaire, self-administered with the validated Italian version (Baron-Cohen et al., 2001; Ruta et al., 2012). This contains 50 items, grouped in five subscales: Attention Switching, Attention to Detail, Imagination, Communication and Social Skills. For each question, participants read a statement and selected the degree to which the statement best described them: ‘‘strongly agree’’, ‘‘slightly agree’’, ‘‘slightly disagree’’, and ‘‘strongly disagree’’ (in Italian). Items were scored in the standard manner as described in the original paper (Baron-Cohen et al., 2001): 1 when the participant’s response was characteristic of ASD (slightly or strongly), 0 otherwise. Total scores ranged between 0 and 50, with higher scores indicating higher degrees of autistic traits. All tested subjects scored below 32, which is the threshold above which a clinical assessment is recommended (Baron-Cohen et al., 2001). The mean (SD) of the scores was 14. 85 (6. 73); scores were normally distributed (see Figure 3D), as measured by the Jarque-Bera goodness-of-fit test of composite normality (JB = 1. 42 p=0. 37). The experiment was performed in a quiet room with artificial illumination of 100 lux. Subjects sat in front of a monitor screen, subtending 41 × 30° at 57 cm distance, with their heads stabilized by chin rest. Viewing was binocular. Stimuli were generated with the PsychoPhysics Toolbox routines (Brainard, 1997; Pelli, 1997) for MATLAB (MATLAB r2010a, The MathWorks) and presented on a 22-inch CRT colour monitor (120 Hz, 800 × 600 pixels; Barco Calibrator), driven by a Macbook Pro Retina (OS X Yosemite, 10. 10. 5). Two-dimensional eye position and pupil diameter were monitored either with a CRS LiveTrack system (Cambridge Research Systems) at 60 Hz, or with an Eyelink1000 Plus (SR Research) at 1000 Hz. We verified that although the two systems have different precision and accuracy, they yielded comparable results in our experiments. Both systems use an infrared camera mounted below the screen. Pupil diameter measures were transformed from pixels to millimeters after calibrating the tracker with an artificial 4 mm pupil, positioned at the approximate location of the subjects’ left eye. Eye position recordings were linearized by means of a standard 9-point calibration routine performed at the beginning of each session. Different subsets of participants took part in the four experiments (main, swapped motion directions, feature-based attention, double-task). For the ‘main’ experiment, we recruited a total of 51 participants of which one was excluded (see below). We recruited and tested them in two groups (subjects 1–25 and 26–51), intended as self-replications each with 25 participants (after the exclusion of one participant based on criteria detailed below). Trials began with subjects fixating a red dot (0. 15° diameter) shown at the centre of a grey background (12. 4 cd/m2). The stimulus comprised a centrally positioned 8 × 14° rectangle which appeared to be a cylinder rotating about its vertical axis (Figure 1A). The 3D illusion was generated by presenting a total of 300 randomly positioned dots (each 0. 30° diameter) moving around a virtual vertical axis with an angular velocity of 60 deg/s (10 rotations per minute): the linear velocity followed a cosine function, 3. 9°/s at screen centre. Dots were black (0. 05 cd/m2) when they moved rightwards (half at any one time) and white (55 cd/m2) when they moved leftwards. The resulting stimulus was compatible with two perceptual interpretations: a cylinder rotating anticlockwise (when viewed from above) with black surface in the front and white surface at rear; or clockwise, with white surface in the front, black surface at rear. The two perceptual interpretations alternated spontaneously in all participants, who continuously reported their percept (clockwise or anticlockwise rotation of the cylinder), either by holding down one of two keyboard arrow keys or by joystick. There was no response button for uncertain or mixed percepts: subjects were instructed to report which of the two percepts was dominant if in doubt. The stimulus was played for 10 trials of 59 s each, during which participants continuously reported whether the rotation was clockwise or anticlockwise. Participants were instructed to minimize blinks and maintain their gaze on the fixation spot at all times, except during a 1 s inter-trial pause, marked by a change of colour of the fixation spot (which turned from red to black). Each participant completed a minimum of three runs, in a single session. A subsample of 27 participants (19 of the first group of participants, 4 of the second group, 4 of the last group, one excluded as explained below) were also tested in the ‘swapped motion direction’ experiment – same as in the ‘main’ experiment, except that black dots moved leftward, and white dots moved rightward. A small subset of participants (N = 10) were re-tested with the same stimuli and procedures as in the ‘main experiment’, but different instructions. These were meant to explicitly encourage a global or a local distribution of attention. In two separate sessions (randomized order), participants were either told to ‘try to attend to both surfaces and see the cylinder rotate as a single unit’ (encouraging global viewing), or to ‘focus attention on the front surface alone’ (encouraging local viewing). Each session lasted about 20 min and included two runs of ten trials each. Another subsample of 25 participants (6 from the first group, 9 from the second group, 10 from the last, chosen for the disposability for a second testing session) took part in the ‘double-task’ experiment, with the same trial structure as the ‘main’ experiment. The primary task was unaltered, with the participant reporting whether they perceived clockwise or anticlockwise rotation of the cylinder (using a joystick rather than the keyboard to minimize interference with the secondary task). Meanwhile, subtle speed increments (1 frame duration, 600 deg/s angular velocity increment) occurred for either the black or the white dots (forming the front or the rear surface depending on the participant’s perception), every 3 s on average (with 2 s minimum separation between speed increments). Participants were asked to press the space bar as soon as they detected a speed increment (on either surface). Any bar press within 2 s from a speed increment was counted as a hit; any bar press that happened more than 2 s away from any speed change was counted as a false alarm. D-prime values were computed from z-transformed hits and false alarms, separately for speed increments occurring on the front and rear surface. For each of these conditions, a minimum of two runs of 10 trials each were acquired (approximately 20 min). Finally, 50 participants (18 from the first group, 22 from the second group, 10 from the last one) were tested in the ‘feature-based attention’ experiment. Trials were only 10 s long. During a pre-stimulus 2 s interval, no dots were shown and a letter (0. 5° wide, either ‘B’ or ‘N’, for bianco or nero, Italian for white or black) was shown at fixation and cued subjects to attend selectively to only white or black dots. Next came the two groups of dots, moving with the same direction and speed as in the main experiment, but lasting only 6 s. During this time, between 0 and 3 speed increments could occur on the cued and uncued surface. Upon extinction of the dots, the participant had 2 s to report by keypress how many speed increments occurred on the cued surface, ignoring speed increments on the uncued surface. In this case the participants did not report the perceived direction of rotation of the cylinder, which may or may not have perceived as a 3D object rather than two independent clouds of dots. Participants performed well above chance, with an average d-prime of 2. 36 ± 0. 09. We tracked pupil diameter during the 6 s stimulus interval, separating trials where the white and the black dots were cued. This experiment was performed in two runs of 50 trials each (approximately 15 min). An off-line analysis examined the eye-tracking output to exclude time-points with unrealistic pupil-size recordings (smaller than 1 mm, likely due to blinks, or larger than 7 mm, likely due to eyelash interference). We further excluded perceptual phases lasting less than 1 s (often finger errors) and longer than 15 s (marking trials with too few oscillations to measure bistability). These criteria led to the exclusion of one participant for the ‘main’ experiment, and one for the ‘swapped motion direction’ experiment (who had less than 10 usable phases), leaving 50 participants for the ‘main’ experiment, for which the percentage of excluded phases is 27. 39 ± 2. 18%, and 26 participants for the ‘swapped motion direction’ and ‘double-task’ experiments, for which the percentages of excluded phases were similar (23. 32 ± 2. 98% and 22. 58 ± 3. 21% respectively). No trials and no participants were excluded for the ‘feature-based attention’ experiment. In all three bistable experiments (the ‘main’ experiment, the ‘swapped motion direction’ and ‘double-task’ experiments), dark and white dots were equally likely seen as foreground (percentage of time of the dark foreground percept, respectively: 51. 85 ± 0. 99%, 51. 13 ± 1. 43%, 51. 34 ± 0. 99%, never significantly different from 50%). Pupil traces were parsed into epochs locked to each perceptual switch (when the subject changed reported perception). We aligned traces to the switch (zero in Figure 1B), and labeled the phases according to the perceived direction of rotation. For the ‘feature-based attention’ experiment, pupil traces were time-locked to stimulus onset and separated based on whether the black or white surface was cued (Figure 2B). For all experiments, we subtracted from each trace a baseline measure of pupil size, defined as the mean pupil size in the 150 ms immediately preceding or following the switch (for negative and positive traces, respectively), or stimulus onset for the ‘feature based attention’ experiment. The resulting traces were averaged across trials and participants, separately for the two perceptual phases (or attention cues for the feature-based attention experiment), to give, Figure 1B and Figure 2B. From these, and also for the individual traces, we defined two summary statistics: the difference of pupil traces between the two types of epochs, and overall mean pupil trace across all epochs. For the ‘main’, the ‘swapped motion direction’ and the ‘double-task’ experiments, these indices were computed after averaging pupil measurements over the first (or first and last) 1 s of each epoch, the minimum phase duration, ensuring that all phases contribute equally to the mean. However, we also verified our main result (correlations with AQ scores) with different epoch definitions. For the ‘feature-based attention’ experiment, the difference of pupil traces between trials where the white and black dots were cued was computed in the interval between 1 and 3 s from stimulus onset (where the effect of attention is expected to peak [Binda et al., 2014]). The datasets generated during the current study and scripts used for analyses are available on Dryad, at the following address: http: //dx. doi. org/10. 5061/dryad. b3ng3 | The pupils control how much light reaches the eye. They become smaller in bright light and larger in darkness to let more light in. Other factors can also affect pupil size. For example, the pupils slightly constrict when a person focuses on brighter objects and they enlarge when focusing on a darker object. Tracking changes in pupil size can tell scientists what someone is focusing on. This can be helpful because different people distribute their attention differently. Some tend to focus on the big picture, others tune into individual details. People with autism spectrum disorders (ASD) tend to focus on the details. Now, Turi et al. showed that measuring pupil size changes during a simple visual task could identify typical people who have milder versions of the characteristics seen in people with ASD. In the experiments, 50 young adults without a diagnosis of an ASD filled out a questionnaire designed to assess how many ASD-like traits they have. Next, the participants watched an illusion of a cylinder with a light and dark side rotating. As they watched, the pupil size of people with more ASD-linked behaviors fluctuated more than the pupil size of those with few such characteristics. The pupils of people with ASD-type traits became larger when they perceived the dark side of the cylinder to be forward, and smaller when the light side appeared. This suggests they are focusing on the front of the cylinder. Future studies are needed to see if similar pupil fluctuations occur in people diagnosed with ASD. Turi et al. predict that pupil changes will be even more dramatic in people with ASD. If this is the case, these pupil measurements could be used to help diagnose ASD or determine the severity of symptoms. | lay_elife |
This is a continuation of application Ser. No. 343,779, filed Mar. 22, 1973, now abandoned. BACKGROUND OF THE INVENTION 1. Field of the Invention The invention concerns a respirator and more particularly a respirator in which flow and pressure of the respiration gas are automatically regulated during inspiration and expiration. 2. Description of the Prior Art Respirators serve to replace or assist the respiratory funciton in patients having deficient or insufficient spontaneous respiration. Further, respirators can be employed for anaesthesia. The usual respirators are controlled systems, that is their functional operating sequence is governed by predefined input variables. A group of known respirators, encompasses the so-called pressure-controlled systems, wherein respiration gas under pressure is supplied to the patient, with a control valve interrupting the respiration gas supply if a certain predetermined pressure is built up in the line leading to the patient. A further group encompasses the so-called volume-controlled systems, by which a quantity of the respiration gas measured according to volume is supplied to the patient. Both types of respirators have a disadvantage which is fundamentally inherent to all controlled systems; specifically, they can not respond in a compensating manner to variations of the lung mechanics which cannot be predicted. There have also already been proposed automatically regulated respirators, which are not suitable "however" for various reasons for general, routine-type use in hospitals. One reason is that the measurement of the controlled condition calls for a complicated mechanism which on the one hand is expensive and on the other hand is trouble-prone. SUMMARY The present invention is grounded in solving the problem of providing an automatically regulated respirator which is free from the disadvantages of known systems and which is suitable for a wide range of applications because of its simple and inexpensive manufacture. In accordance with the invention, this is achieved by a respirator with a flow and pressure measuring device disposed directly adjacent the patient connection, to measure the flow and pressure of the respiration gas and convert these parameters into electrical signals, a valve system disposed between the respiration gas source and the flow and pressure measuring device for the control of flow and pressure of the respiration gas during inspiration and expiration, and an electronic regulating unit which forms a regulating circuit together with the measuring device and the valve system to compare the electrical signals with standard values and to generate a correction signal which regulates the valve operation. BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 shows a block diagram of an automatically regulated respirator according to the invention. Fig. 2 shows a perspective view of a preferred embodiment of the regulating valve, of the inspiration/expiration valve, and of the measuring head in the assembled state. FIG. 3 shows the regulating valve, partially sectioned in the plane A--A of FIG. 2. FIG. 4 shows a longitudinal section along the plane B--B of FIG. 2. FIG. 5 shows a schematic plan view of the measuring head. FIG. 6 shows a longitudinal section of a portion of the measuring head in FIG. 4 in enlarged scale. FIG. 7 shows an electrical block diagram of the regulating unit 13. FIG. 8 shows a block diagram of the electrical circuit for the automatic zero adjustment of the flow signal. FIG. 9 shows the voltage variation curves at designated points of the circuit shown in FIG. 8. DESCRIPTION OF THE PREFERRED EMBODIMENT The respirator shown in FIG. 1 in block diagram form and partially in FIG. 2 perspectively, serves to supply respiratory gas to a patient designated as 11 from a source (not shown) and to remove such gas from the patient, in a definite rhythm similar to the natural respiration. Either a pressure generator or a central supply system, as is usually installed in larger hospitals, can serve as the respiration gas source. Any desired gas or gas mixture may be employed, for example pure respiration gases such as air, oxygen, or mixtures with narcotic gas such as ether, laughing gas etc. The respiration gas delivered should preferably have a pressure of 1.2 atm. Further, the respiration gas should be adjusted optimally for the requirements of respiration with respect to moisture content and temperature, that is have a temperature of 37°C and be saturated to more than 90% with water. The devices necessary for this purpose are known per se and do not have to be described here. A line 1 serves for the connection of the respirator to the respiration gas source and leads to a regulating valve 2. The regulating valve 2 serves for the switching between inspiration phase and expiration phase and simultaneously for the control of flow and pressure in the two switching positions. In the inspiration phase, the valve 2 connects the line 1 with a line 3, and, in the expiration phase the valve 2 connects the line 1 with a line 4. In each phase, the magnitude of pressure and flow is controlled by a larger or smaller opening of the valve. The two lines 3 and 4, each lead to an inspiration/expiration valve 5 for convenience hereinafter called I/E valve. Apart from the two lines 3 and 4, also connected to the I/E valve are a patient line 6 and an expiration line 7. Line 7 may either be opened to the atmosphere or for the purpose of recovering the expiration gas may be connected via a line 8 returning to the respiration gas source. The patient line 6 leads via a measuring head 9 to a tracheal tube 10 for intubation of a patient 11, the measuring head 9 being provided for the measurement of the flow and pressure of the respiration gas signals are generated which are representative of the flow and pressure variation of the respiration gas. An electrical lead 12 serves for the transmission of these signals to an electronic regulating unit 13. Standard signals for the respiration function, are supplied via a line 14 to the regulating unit 13. In the regulating unit 13 the signals from measuring head 9 are processed, compared with the standard signals, and a correction signal is obtained for adjustment of the regulating valve 2. An electrical output lead 15 serves for the transmission of the correction signals to the drive mechanism of the regulating valve 2 for adjustment purposes. As is especially visible from FIG. 2, the individual elements of the control section, i.e. regulating valve 2, I/E valve 5 and measuring head 9, are assembled into a single unit. This unit is exceptionally compact its dimensions being approximately 15 × 10 × 5 cm. The regulating valve 2, the I/E valve 5, the measuring head 9, and the regulating unit 13, are individually described below. The regulating valve 2 as illustrated in cross-section in FIG. 3, includes a substantially hollow-cylindrical housing 16 with an inner bulkhead 17 (running perpendicular to its axis) at one side of which there is attached a commercially available servo-motor 18. The housing side within which the motor 18 is located, is expediently provided with cooling ribs 19. At the side of motor 18, the housing is sealed off with a cover (now shown). The motor-shaft is extended on the one side beyond bulkhead 17 and outwardly on the other side beyond the cover. The other side of the housing 16 contains the actual valve part. The shaft section is projected within this part of the housing and terminates in the form of a cup-shaped rotor 21. The external diameter of the cylindrical part of the rotor is slightly smaller, for example 0.1 mm, than the internal diameter of the housing. At one position of the cylindrical wall of the rotor 21, an annular boring 22 is provided. The wall of the housing 16 has two borings 23 and 24. The axes of the rotor boring 22 and the two housing borings 23 and 24, lie in a plane perpendicular to the axis of the housing. The distance between the centers of the two housing borings 23 and 24, is greater than the diameter of the boring 22. By appropriate angular position of the rotor, either the rotor boring 22 is brought completely or partially to coincidence with one of the two housing borings 23 or 24, or the housing borings 23 and 24 are closed by the rotor wall. A part of the housing 16 in which the rotor is seated i.e. the actual valve part, is enclosed by a plastic block 25, which has an inlet line 1 leading to the regulating valve 2 and outlet lines 3 and 4 leading away from the regulating valve. Block 25 is also part of a housing containing the I/E valve 5 and the measuring head 9. The input line 1 extends into the interior of the housing and of the rotor, while the output lines 3 and 4 connect the housing borings 23 and 24 with the corresponding inputs of the I/E valve. The outer plastic block 25 is readily detachable from the housing 16, i.e. connected for example with clamps or a bayonet connection. For the venting of the space between the rear of the rotor 21 and the bulkhead 17, there is provided a boring 26 in the housing wall 16. About the other side of the motor 18, where the shaft 20 is likewise led out, is located a control device 27 for the determination of the angular position of the motor 18 or of the rotor 21. The control device 27 consists essentially of a rotary capacitor with rotatable plates mounted on the shaft 20 and fixed plates which are mounted in insulated manner on the housing and project into the space between the rotatable plates. With application of a voltage between the rotatable and the fixed plates, the angular position of the motor 18 or of the rotor 21 can clearly be determined by the variable capacity. The control device 27 for the determination of the angular position shall not be described in detail as the manufacture of such a device is obvious to one skilled in the art. The operation of the regulating valve 2 is based on the rotation of the rotor 21 by the servo-motor 18 according to the control signals, so that the rotor boring 22 is selectively brought over one of the housing borings 23 and 24. Respiration gas is delivered under pressure through the inlet line 1. At the beginning of the inspiration phase, the rotor 21 is so rotated that the rotor boring 22 is positioned over the housing boring 23. The respiration gas can consequently flow via the line 3 to the input of the I/E valve. Flow and pressure of the respiration gas can be infinitely controlled between the value zero and a maximum value, through a number of relative displacement positions between the rotor boring 22 and the housing boring 23, thus varying the cross-section of the common aperture. At the commencement of the expiratory phase the rotor 21 is so rotated that the rotor boring 22 is aligned with the housing boring 24. The gas flows now via the line 4 to the second inlet of the I/E valve, where it is used, as is shown in the following, to generate a negative pressure to assist expiratory respiration. Also in this position flow and pressure of the gas flowing in the line 4 are similarly infinitely controllable between the value zero and a maximum value, by relative displacement of the borings. It may be desirable to interpose pause intervals between the two phases of a respiration cycle whereby neither respiration gas is supplied to the patient nor is the expiration assisted by production of a negative pressure. In this case, the rotor 21 is so rotated that the rotor boring 22 has no common aperture with either of the two housing borings 23 and 24. To minimize the rotation angle of the rotor, for this case the position between the two housing borings 23 and 24 is expediently chosen. In the construction of the valve 2, no friction washers are provided because these would considerably impair the operating speed. The very small separation between the external surface of the rotor 21 and the internal wall of the housing 16 achieves sufficient sealing tightness to make any resulting loss of respiration gas insignificant. Moreover, the vent boring 26 prevents respiratory gas passing via the motor chamber into the monitoring control device which might otherwise cause disturbances there. As already mentioned, the plastic block 25 which contains the lines 1, 3 and 4 is part of a housing in which the I/E valve is also housed. FIG. 4 shows a section through regulating valve 2, I/E valve 5 and measuring head 9, directed along the longitudinal axis of the I/E valve. The I/E valve has a first or main channel 28 and a second or auxiliary channel 29 running perpendicular thereto and opening approximately in the center thereof. One end 30 of the main channel 28 serves as inlet and communicates directly with the line 3 coming from the regulating valve. The other end 31 of the main channel 28 provides communication between the I/E valve and the line 6 of the measuring head 9. The auxiliary channel 29, in the internal region neighboring the main channel, has firstly a zone of cylindrical form and then widens out conically in an adjacent zone 32. Its end 33 represents the outlet of the I/E valve and serves for the connection to the line 7. In the main channel 28 there is placed co-axially thereto and in the region of the outlet of the side channel, an elongated bullet-shaped body 34, which is denoted in the following as circumfluous body. With respect to its cross-section the circumfluous body 34 has essentially three zones. In the first zone 35 facing the inlet 30, the body 34 has a conical form with a spherically-rounded end surface, in the central zone 36 a cylindrical form and in the adjacent, third zone 37 again a conical form with spherical rounding at the end. The transition of the cylindrical zone 36 into the conical zone 37 lies in a plane set through the axis of the auxiliary channel, perpendicular to the axis of the main channel. From the spherically-rounded front end of the circumfluous body 34 facing the measuring head 9, a boring 38 runs first co-axially to the main channel 28 and then, bent at 90°, co-axially to the auxiliary channel 29 up to the part of the exterior surface of the circumfluous body 34 opposite the region of intersection with the auxiliary channel. A thin tube 39 is installed in corresponding borings of the housing of the I/E valve co-axial to the auxiliary channel and through the circumfluous body. The tube 39 opens into the cylindrical zone of the auxiliary channel 29. In the zone of the conical part 35 facing the inlet 30, the circumfluous body 34 has a number of radially-arranged vanes or guide vanes 40, which are inclined with respect to planes cutting them through the axis of the main channel at a small angle, for example 6°. The vanes 40 serve on the one hand for the mounting and centering of the circumfluous body 34 and on the other hand, by their inclination thereto, serve for imparting a swirling to the respiration gas flowing through the tube. The space 41 between the circumfluous body 34 and the line 6 serves as diffuser. Likewise the widened-out part 32 of the side channel 29 serves as diffuser. In operation, as already described, gas (fixed at an average pressure of ca. 1.2 atm. by the regulating valve 2) is alternately supplied through the lines 3 and 4. In the inspiration phase the gas comes through the inlet 30 into the main channel and flows past the circumfluous body 34. By the reduction of the circumfluous body cross-section in the conical region 35, the flow-rate of the gas increases considerably, that is its pressure energy is converted into kinetic energy. In the zone of the outlet of the auxiliary channel 29, the respiration gas has atmospheric pressure. There does not exist therefore a pressure gradient between the main channel and the auxiliary channel and consequently practically no respiration gas flows through the auxiliary channel during the inspiration phase. The cylindrical part 36 of the circumflous body was selected relatively long so that a laminar flow can develop. In the diffusion region 41 the pressure energy is recovered from the kinetic energy of the gas and a pressure of ca. 1.1 atm. is attained at the end 31 of the main channel. By means of the vanes 40 a swirling effect is conveyed to the gas coming from the inlet 30 so that a smooth separation of flow is given at the end of the circumfluous body and a separation of flow from the wall of the main channel (which would give an undesired pressure distribution) is avoided. In the expiration phase the gas expired from the patient, proceeds through the measuring head 9 and the line 6 into the region of the diffuser 41 and through the boring 38 into the auxiliary channel 29. Since the free cross-section remaining between the cylindrical zone 36 of the circumfluous body and the wall of the main channel is smaller than the boring 38 cross-section, practically no expiration gas from end 31 proceeds past the circumfluous body 34 to the input side of the main channel. The boring 38 is of such size that the expiration phase functions well without external assistance. During the expiration phase the gas, held under pressure by the regulating valve 2, is led in the line 4 to the tube 39 and through this into the auxiliary channel 29. Because of the small cross-section of the tube 39, the gas has a high velocity therein, with which it also discharges into the auxiliary channel 29. This provides an aspirator action with which a negative pressure can be produced on the patient side to assist the expiration phase. Further the suction caused by the respirator action assists to virtually eliminate the passage of any expiration gas past the circumfluous body to the inlet side of the main channel. In the diffusion region 32 of the auxiliary channel the kinetic energy of the gas is again converted into pressure energy so that the gas again has atmospheric pressure at the outlet 33. The I/E valve has two essential advantages with respect to previously known I/E valves. Since it has no moving parts, no functional disturbances occur due to depositions of liquid drops and solid materials contained in the expiration gas. Practically no obstruction of the channels can occur since in each case they are blown free in the succeeding inspiration phase. The second advantage exists in that the I/E valve can be manufactured completely from plastic because of its simplicity of construction and can be integrated with the manifold block 25 of the regulating valve and the channel of the measuring head 9. From FIG. 4 the construction of the measuring head 9 is also visible. This consists essentially of two parts, of which the first 42 contains the patient line 6, through which the respiration gas flows during inspiration phase and expiration phase, while the second part 43 contains the actual measuring device. The part 42 of the measuring head containing the patient line 6 consists of a plastic block and is a component of the aforementioned housing also containing the I/E valve and the plastic block 25. The second part 43 containing the measuring device is, on the other hand, mechanically connected with the regulating valve 2, or with the motor 18. The housing, consisting of the plastic block 25, the I/E valve 5 and the manifold part 42 of the measuring head 9, represents an interchangeable element, which is connected by an easily-detachable mechanism with the fixed parts of the respirator, to which belong the actual valve part of the regulating valve 2, the motor 18 and the control device 27 for the determination of the angular position of the motor 18 as well as the actual measuring part 43 of the measuring head 9. This interchangeable housing is designed as a throw-away part, so that it can be replaced after a single use (i.e. for one patient) and therefore a purification and a sterilization are obviated. For its manufacture a synthetic material is chosen however, which also makes possible a sterilization, so that in a case of emergency in which no new parts are available, the readiness for use of the respirator is ensured. For the connection of the channel representing the patient line 6 with the actual measuring device 43, channels 44 are provided. These channels 44 open with one end at specific measuring points of the patient line, while their other end opens in each case at the outer limitation of the part 42. If the interchangeable housing is assembled with the fixed parts, the external outlets of the channels 44 lie opposite corresponding openings of the actual measuring device 43 and are there, as is shown in the following, sealed from the surroundings. Firstly, the principal of the measuring head is described by way of the FIG. 5 in which there is shown a plan of the interchangeable part 42, and in schematic representation the actual measuring part 43 of the measuring head 9. Part 42 consists of a substantially L-shaped plastic block with rectangular cross-section. In its interior the patient line 6 runs as a channel inclined substantially at 90°. In its first part 45 connecting to the I/E valve, the channel 6 has an annular cross-section and after a transition region 46 a rectangular cross-section. The outside-bend or outside-curve wall 47 (with respect to the bend of the channel) runs firstly parallel to the axis and breaks away, from a specific plane perpendicular to the axis, into a bend 48 with a radius of curvature equal to the longer side of the rectangular cross-section. In the other leg whose axis runs perpendicular to the aforesaid axis, the outside wall 49 also runs firstly parallel to the axis, to subsequently continue into a bend 50 exactly symmetrical to the bend 48. The inside-bend or inside-curve wall also has two sections 51, 52 parallel to the axes, which lie opposite the axes-parallel sections 47, 49 of the outside wall, while its section opposite the bends 48, 50 runs at an angle of 45°, without curvature however. For the flow and pressure measurement four measuring points are provided, to which the connection channels 44a, 44b, 44c, 44d lead. As already mentioned, these channels 44 represent the connection between the patient line 6 and the actual measuring device. The two connection channels 44a and 44b open at the points in the patient line, to which the axes-parallel sections 51, 52 of the inside wall bend into the section running at an angle of 45°. The two other connection channels 44c and 44d open at the outside wall of the channel 6 at a position in each case at which the axes of the two side-arms of the channel 6 strike the curved wall-sections 48 and 50. In the actual measuring part 43 two channels 53, 54 are provided, which on the one hand (if the interchangeable part 42 is assembled) are in connection with the connection channels 44a, 44b and on the other hand are connected with one another via a central cavity 55. From the cavity 55 two further channels 56 and 57 lead to the connection channels 44c, 44d opening at the outside wall of the patient line 6. The two channels 56, 57 have in each case at both of their ends (i.e. thus in the vicinity of the cavity 55 and in the vicinity of their connections to the connection channels 44c, 44d) pneumatic orifice plates 58, 59 or 60, 61. Between the two orifice plates 59, 61 (arranged on the side of the connection channels 44c, 44d) and the two connection channels there is mounted in each case, in the immediate vicinity of the orifice plate opening and as concentrically as possible thereto, a temperature-dependent resistor (abbreviated in the following to thermistor) 62, 63. The connecting leads of the thermistors 62, 63 are led out at the side of the channels and connected with the electronic part of the measuring device (to be described later). The two channels 56, 57 are connected "moreover" with a channel 64 and 65 each, which in their turn are in connection via pneumatic resistors 66, 67 with a combined channel 68. The channel 68 is connected to a pressure gas source (not shown). From the central cavity 55 a further channel 69 leads to the outside air. This channel contains pneumatic orifice plates 70, 71 (one mounted in the vicinity of the cavity 55 and one mounted in the vicinity of its outlet to the outside air) and two pneumatic, resistors 72, 73 between these two orifice plates. Between the pneumatic orifice plate 70 and the cavity 55 a thermistor 74 is mounted in the immediate vicinity of the orifice plate opening and between the pneumatic orifice plate 71 and the outside air a thermistor 75 is mounted in the immediate vicinity of the orifice plate opening. The part of the channel 69 lying between the two pneumatic resistors 72, 73 is connected via a connection channel 76 and a further pneumatic resistor 77 with the channel 68. Finally, another channel 78 leads from the cavity 55 to the outside air. In the channel 78 there opens a narrow connection line 79 (to the connection channel 76) and a likewise narrow passageway 80 (opposite the line 79) in which a thermistor 82 is mounted behind an orifice plate 81 and which opens to the outside air. A pneumatic resistor 78' is mounted in the channel 78. The connecting leads of the thermistors 74, 75 and 82 are likewise led out at the side of the channels and lead to the electronic part of the measuring device. The function of the measuring device is as follows: from a pressure gas source a gas stream is supplied via the channel 68, which distributes itself to the individual branches of the device. A part of the gas stream proceeds through the pneumatic resistors 66 and 67 into the channels 64, 65 and further into the channels 56 and 57. Since in the channels 56 and 57 similar processes occur, the description can be limited in the following to one 56 of the two channels. In the channel 56 the gas stream arriving through the channel 64 again splits up into two branches of which one flows through the opening of the orifice plate 58 into the cavity 55, while the other proceeds through the opening of the orifice plate 59 past the thermistor 62 into the connection channel 44c and from there into the patient line 6. These two branch streams serve for the measurement of the inspiratory flow in the patient line 6. If no gas flows in the patient line 6, the same pressure exists at all measuring points. The two branch streams flowing through the openings of the orifice plates 58 and 59 occur accordingly at the same pressure and remain unchanged for the time being. During the inspiration however the respiration gas coming from the I/E valve flows through the patient line and "bounds" onto the curved part 48 of the outside wall. In this stream a higher pressure exists at the outlet of the connection channel 44c than in the region of the inside wall of the channel (i.e. at the outlets of the connection channels 44a and 44b) and therewith a higher pressure also exists than in the cavity 55. By this pressure difference the two branch streams vary in the channel 56 in a manner such that the stream directed through the opening of the orifice plate 59 becomes smaller and the stream directed through the opening of the orifice plate 58 becomes larger. This variation of the branch stream proceeding through the opening of the orifice plate 59 is measured with the air of the thermistor 62. For the measurement the impedance of the thermistor is regulated to a constant value. With an equivalent gas stream coming through the orifice plate 59 the thermistor is equivalently cooled, by which means an equilibrium appears in the stationary state. In the non-stationary state, that is if an inspiratory flow exists and therewith the branch stream coming through the opening of the orifice plate 59 varies, the heat conducted by the gas also varies and therewith in turn the power loss of the thermistor 62. The electric current supplied to the thermistor 62 is measured and a variation of the power loss is accordingly determined. It has been found that the variation of the rates of the two branch streams in the channel 56, and therewith also the heat conduction brought about at the thermistor 62 by one of the two branch streams as well as ultimately also the power loss variation of the thermistor or the signal recording this power loss variation, shows a clear dependence of the variation of the inspiratory flow existing in the patient line 6. Therewith an electric signal recording the flow can be obtained. The flow signal is, for the purpose of regulation, supplied to the electronic circuit (to be described later). The measurement of the expiratory flow proceeds in the same manner with the aid of the thermistor 63 arranged in the channel 57. For simplification it has been assumed here that during the inspiration no signal is obtained at the thermistor 63 and during the expiration no signal is obtained at the thermistor 62. Strictly speaking this is not correct, however these undesired signals are considerably smaller than the signals used for the measurement and can be readily eliminated in the subsequent processing. One part of the gas stream (brought up through the line 68) proceeds via the pneumatic resistor into the channel 76 and is used for the pressure measurement. The stream proceeds into the channel 69 and proceeds here via the two pneumatic resistors 72 and 73 to the orifice plates 70 and 71. If barometric pressure exists in the cavity 55, the gas streams proceeding through the openings of the orifice plates 70 and 71 and therewith the cooling capacity of the thermistors 74 and 75 are of the same magnitude. With a pressure difference between the pressure existing in the cavity 55 and the atmospheric pressure, a difference of the gas streams cooling the thermistors 74 and 75 is also produced, by which means a difference signal can be obtained. It has been found that this difference signal is proportional to the desired pressure difference. One part of the gas stream proceeding into the channel 76 is led via the line 79 into the channel 78 and serves for the zero adjustment of the pressure signal. Since the channel 78 represents a connection between the cavity 55 and the outside air, a flow is formed in one or the other direction depending on the sign of the pressure difference between the pressure existing in the cavity (denoted as buccal pressure P bucc ) and the atmospheric pressure. If no pressure difference exists between the cavity 55 and the outside air and therewith no gas flows into the channel 78, this condition corresponding to the desired zero point, the gas arriving through the line 79 proceeds into the passageway 80 and through the opening of the orifice plate 81 to the thermistor 82. Since during the respiration the buccal pressure alternates from time to time between a value lying below the barometric pressure and one lying above the barometric pressure, the desired zero state exists briefly from time to time in the channel 78. In this short time period, a signal is given from the thermistor 82. This signal can moreover be used to adjust the measured pressure signal to zero in every respiration cycle. This device for the zero adjustment saves a frequent manual zero adjustment. Just as for the obtained pressure signals, a zero adjustment is also necessary for the flow signals, since the zero lines for both signals have a tendency to shift because of various external effects. The zero adjustment of the flow signals is however, as is shown later, electronically undertaken. While in FIG. 5 the actual measuring device for the flow and pressure measurement is shown schematically, from FIG. 4 the actual construction can be seen. All channels as well as the cavity 55 are housed on the inside of one of two parts of a metal block 83 lying flat on one another. They are partially recessed in the separating surfaces between the two parts of the metal block 83, partially composed of borings perpendicular to these separating surfaces. The section resolved in FIG. 4 runs through a section of the channel 68, the pneumatic resistor 66, the channels 64 and 56, the pneumatic orifice plate 59, the thermistor 62 and the channel 44c. For the illustration of the construction a description of the measuring point shown in this section is sufficient, since the other measuring points correspond in principle to the one in question. This measuring point is shown in FIG. 6 in enlarged scale. The pneumatic resistors are composed for example from metal capillaries which are brought to the desired resistance value by compression at a specific point. They can be pressed into the provided connection borings between the individual channels in a manner known per se. The boring representing a part of the channel 56, opens into the front end of the metal block 83 opposite the interchangeable part of the measuring head 9, that is opposite the connection channel 44c. Concentrically to the outlet of this boring an annular groove or channel 84 is recessed into the front end of the metal block. The internal diameter of this groove is somewhat larger than the diameter of the boring, so that a rim remains. Over this rim is inverted a cylinder-like sleeve 59 closed at one end, which has a concentric aperture in its flat section. This sleeve represents the pneumatic orifice plate. Moreover a further cylindrical sleeve 85 opened at both ends of an electrically insulated material is inserted into the groove. A further sleeve 86 of the same diameter is mounted on the sleeve 85. The thermistor 62 is clamped between the two sleeves 85 and 86. Its connecting wires pass outwards through both sides and are soldered onto a printed circuit board 87. The sleeve 86 passes through the circuit board 87 and is mounted on a rubber plate 88. The rubber plate 88 has an aperture 89 and this aperture has annular encircling sealing washers 90. The whole arrangement is housed in a housing 91, which has a corresponding opening on the side of the interchangeable part 42 in which the rubber plate 88 is installed. In the assembled state the rubber plate is pressed with the sealing washers 90 onto the surface of the interchangeable part 42 such that a sealing from the surroundings results. For the thermostatical stabilizing of the metal block 83, a power transistor is mounted on its other side. The interspace between the metal block 83 and the wall of the housing 91 is sealed from the surroundings and is in contact with the supply of the gas used for the measurement. Since nitrogen is usually used for the measurement, there is provided therewith a protection from explosion in this space which contains the power transistor 92. As already mentioned, the regulation of the respirator is effected by the electronic regulating unit 13, which controls the regulating valve 2, by means of both, measured actual values of the flow and pressure and of given standard values. In the present embodiment a flow regulation normally takes place during the inspiration while a pressure regulation takes place during the exspiration. This type of regulation has proved especially expedient for a number of reasons as follows. During the inspiration a specific standard volume of the respiration gas is supplied to the patient. At the commencement of the expiration the final inspiratory pressure is taken as starting value for the pressure regulation and the buccal pressure is brought according to a definite curve to the value zero or barometric pressure. The curves of the flow and pressure variation are presented as standard curves; simultaneously the corresponding actual values are measured and serve for the correction of the standard curves. There has been fixed as an overriding condition that the standard minute volume is adhered to. For this purpose the volume is calculated with the aid of the flow measurement and used for the correction of one or more of the other standard values. As a further condition it has been intended that during the inspiration the buccal pressure and its increase per unit time must not exceed specific maximum values. If during the flow regulation one of these maximum values is attained in the inspiration, the regulating unit switches to pressure regulation until the flow curve brings about a pressure or pressure increase lying below the maximum values. Construction and function of the regulating unit 13 are visible from FIG. 7 in which a block diagram of this device is shown. In FIG. 7 and in the following description, details of the circuit are not dealt with since the functions represented by blocks can usually be realized as several different types which are familiar to one skilled in the art. Fundamental of the invention is, above all, the combination of the individual functions, especially those of several underlying control loops. Firstly, there is again visible in the FIG. 7, the control section consisting of regulating valve 2, I/E valve 5 and measuring head 9. The first of the underlying control loops serves for the position regulation of the servo-motor 18 of the regulating valve 2. For this purpose the motor 18 is connected with a circuit 100 for the position regulation, abbreviated in the following to position regulator, into which the rotating plate condenser (mounted on the shaft of the motor) of the control device 27 is connected, by which means the loop is closed. The position regulator 100 is connected to a circuit 101 for the switching between flow and pressure regulation, abbreviated in the following to switching circuit. The switching circuit 101 has three inputs, of which the first comes from a time generator 102, which produces by means of given values a chronological operating sequence. The two other inputs of the switching circuit 101 are connected at two adder points 103', 104' with the outputs of two curve generators 103, 104 and with the feed-backs of the flow and pressure control loops. The curve generators produce by means of given values and correction values obtained from within the regulating device, the flow and pressure curves according to which the inspiration and the expiration elapse. The adder points 103', 104' are formed essentially from subtraction circuits in which the measured or actual-signals are subtracted from the standard signals and an error signal is obtained. The loop, consisting of switching circuit 101, position regulator 100, control valve 2, I/E valve 5, flow measuring device 105 and adder point 103', represents the flow control loop. Appropriately the adder point 104' is connected with the output of the pressure measuring device 105', by which means the pressure control loop is formed. The standard curve generators 103, 104 have several inputs, of which one each is connected with an external operating element 106, which serves for the adjustment of the desired standard curve. The desired standard curve defines that flow or pressure variation which was obtained without disturbances. If disturbances appear the chosen standard curve is modified accordingly in each case. Each second input of the curve generators 103 and 104 is connected with the time generator 102, from which the conditions for the length in time of a respiration cycle and the ratio of the inspiration to the expiration phase are furnished. A third input of the flow curve generator 103 is connected with an external operating element 107 for the adjustment of the standard minute volume. The amplitude of the flow curve depends on the choice of the standard minute volume. A third input of the pressure curve generator 104 is connected with a logic circuit 108 in which disturbances such as spontaneous respiration, coughing, exceeding the maximum pressure or maximum pressure increase, are processed and used for the instigation of certain variations of the course of the respiration cycle. Thus, for example, as already mentioned, the system is switched over to pressure control with an exceeding of the maximum pressure or of the maximum pressure increase during the inspiration. On the other hand the respiration cycle is discontinued in the case of coughing or spontaneous respiration and recommenced at the end of this disturbance. For this purpose a second output of the logic 108 is connected with the time generator 102. The pressure curve generator 104 has a fourth input, which is connected with a measuring device 109 for the measurement of the final inspiratory pressure. The final inspiratory pressure is delivered therefore to the pressure curve generator 104, because it is used as starting value for the pressure curve to follow during the expiration. In this manner a kink is avoided in the pressure curve, which is extremely undesired. The input of the measuring device 109 is connected to the output of the pressure measuring device 105'. The time generator 102 has three inputs, of which one, as already mentioned, is connected with the logic 108. A further input is connected with an external operating element 110 for the selection or adjustment of the respiration frequency. The third input of the time generator 102 is connected via a correction circuit 111 with an external operating element 112 for the selection or adjustment of the time ratio between inspiration and expiration. This adjusted ratio is modified accordingly by the correction circuit 111. For this purpose the correction circuit 111 is connected with a comparator circuit 113 for the comparison of the actual gas volume per minute (actual minute volume) supplied to the patient with the adjusted standard minute volume. The actual volume is calculated in an integration circuit 114 from the measured values of the expiratory flow. The expiratory flow was chosen for the calculation because with its employment the effect of a possible leak is readily eliminated. The calculated volume can be supplied via an output 115 to an indicator or recording unit. The logic circuit 108 is connected with a comparator circuit 116 which carries out a comparison between the actual pressure and the maximum permissible pressure as well as between the rise gradient of the pressure and a corresponding maximum value. The two maximum values for pressure and rise gradient of the pressure are given by the aid of external operating elements 117 and 118. For the determination of the actual values the comparator circuit 116 is connected with the pressure measuring device 105'. The limitation of the buccal pressure and the rise gradient of rise gradient velocity of the buccal pressure has been provided because too high a pressure or too rapid a pressure rise can give the patient pains. Thus, for example, with rib injuries or similar injuries the maximum values are set as low as possible. The pressure signal coming from the pressure measuring device 105' is supplied to an indicator or recording unit 119. As already mentioned, an automatic zero adjustment is provided for the flow and the pressure measurement. The automatic zero adjustment is contained in the flow and pressure measuring devices 105, 105'. For the pressure measurement there has already been described the automatic zero adjustment whose zero adjustment signal is pneumatically obtained. For the flow measurement the zero adjustment is carried out completely electronically. The circuit shown in FIG. 8 which is contained in the flow measuring device 105 serves for this purpose. For the description of the function of this circuit FIG. 9, which reproduces the signals at various points (denoted by a-h) of the circuit, is also taken into consideration. At the input 120 the signal (a) indicating the inspiratory flow, which comes essentially from the thermistor 62, is obtained. This signal is idealized insofar as during the expiration no pressure variation has been assumed in the measuring point. In reality an amplitude is also produced during the expiration which is however essentially smaller than that during the inspiration. Since the signals, as will be shown subsequently, are subtracted or added, the small amplitudes in the phase not measured in each case are negligible. There is obtained at the input 121 the signal b indicating the flow variation during expiration, which comes essentially from the thermistor 63 and for which the same is valid as has been stated for the signal a. The two signals a and b have no defined zero lines. From the two inputs 120 and 121 each connection leads to a subtraction circuit 122 and to an addition circuit 123. At the output of the subtraction circuit 122 the signal g is obtained which already substantially represents the flow signal, however has no defined zero line. At the output of the addition circuit 123 the signal c is produced. The output of the addition circuit 123 is connected with an audio link 124. The input of the audio link is connected via a capacitor 125 with the base of a transistor 126 whose emitter is earthed. A resistor 127 lies between the base of the transistor 126 and earth. In this circuit the signal d is produced at the base of the transistor 126 and the signal e is produced at its collector which represents the output of the audio link. For the improvement of the precision in the present case an operational amplifier with similar functioning is provided. This signal is supplied to a monostable multivibrator 128 at whose output the signal f is obtained. A switch 129, which momentarily short circuits to earth the signal coming from the subtraction circuit 122 with each change of inspiration phase to expiration phase and vice versa, is actuated essentially by the signal f. At the output of the switch 129 there is obtained therewith the signal h which has by this manner a clearly defined zero line. The output signal h is the signal which is fed back to the input of the control circuit 101 for the purpose of the flow regulation and which is supplied to the integration circuit 114 for the purpose of the volume calculation. A very large band width is produced by the system of the underlying control loop which is clearly not fully utilized but ensures an increase of the precision in the working range. | A respirator used to replace or assist the respiratory function and automatically regulate the respiration gas flow and pressure during the inspiration phase and expiration phase comprising means for measuring the flow and pressure of respiration gas and providing electrical output signals, valve means including an adjustable valve for controlling the flow and pressure of respiration gas past the measuring means, and electronic regulating means connected with the measuring means and valve means for comparing the electrical signals with predetermined values including standard values and for generating a regulating signal for adjusting the valve means. | big_patent |
SECTION 1. SHORT TITLE. This Act may be cited as the ``Subsidized Stafford Loan Reduced Interest Rate Extension Act of 2012''. TITLE I--EXTENSION OF REDUCED INTEREST RATE SEC. 101. INTEREST RATE EXTENSION. Section 455(b)(7)(D) of the Higher Education Act of 1965 (20 U.S.C. 1087e(b)(7)(D)) is amended-- (1) in the matter preceding clause (i), by striking ``and before July 1, 2012,'' and inserting ``and before July 1, 2013,''; and (2) in clause (v), by striking ``and before July 1, 2012,'' and inserting ``and before July 1, 2013,''. TITLE II--IMPROPER PAYMENTS ELIMINATION AND RECOVERY IMPROVEMENT SEC. 201. SHORT TITLE. This title may be cited as the ``Improper Payments Elimination and Recovery Improvement Act of 2012''. SEC. 202. DEFINITION. In this title, the term ``agency'' means an executive agency defined under section 105 of title 5, United States Code. SEC. 203. IMPROVING THE DETERMINATION OF IMPROPER PAYMENTS BY FEDERAL AGENCIES. (a) In General.--The Director of the Office of Management and Budget shall on an annual basis-- (1) identify a list of high-priority Federal programs for greater levels of oversight and review-- (A) in which the highest dollar value or majority of governmentwide improper payments occur; or (B) for which there is a higher risk of improper payments; (2) in coordination with the agency responsible for administering the high-priority program-- (A) establish semi-annual or quarterly targets and actions for reducing improper payments associated with each high-priority program; or (B) if such targets are in effect on the date of enactment of this Act, establish supplemental targets; and (3) determine the entities that have received the greatest amount of improper payments (or, if improper payments are identified solely on the basis of a sample, the entities that have received the greatest amount of improper payments in the applicable sample). (b) Report on High-Dollar Improper Payments.-- (1) In general.--Subject to Federal privacy policies and to the extent permitted by law, each agency on a quarterly basis shall submit to the Inspector General of that agency, and make available to the public (including availability through the Internet), a report on any high-dollar improper payments identified by the agency. (2) Contents.--Each report under this subsection-- (A) shall describe-- (i) any action the agency-- (I) has taken or plans to take to recover improper payments; and (II) intends to take to prevent future improper payments; and (B) shall not include any referrals the agency made or anticipates making to the Department of Justice, or any information provided in connection with such referrals. (3) Availability of information to inspector general.-- Paragraph (2)(B) shall not prohibit any referral or information being made available to an Inspector General as otherwise provided by law. (4) Assessment.--After the review of each report under this subsection, the Inspector General shall-- (A) assess the level of risk associated with the applicable program and the quality of the improper payment estimates and methodology of the agency; (B) determine the extent of additional oversight or financial controls warranted to identify and prevent improper payments; and (C) provide the head of the agency with any recommendations, for modifying any plans of the agency, including improvements for improper payments determination and estimation methodology. (c) Improved Estimates.-- (1) In general.--Not later than 180 days after the date of enactment of this Act, the Director of the Office of Management and Budget shall provide guidance to agencies for improving the estimates of improper payments under the Improper Payments Information Act of 2002 (31 U.S.C. 3321 note). (2) Guidance.--Guidance under this subsection shall-- (A) strengthen the estimation process of agencies by reviewing the underlying validity of payments to ensure amounts being billed are proper; and (B) include-- (i) access to more complete data as part of reviews; (ii) ending reliance on self-reporting of improper payments as a replacement for estimates, and relying on the development of a robust process to estimate and identify improper payments across the agency; (iii) all overpayments in the improper payments estimate, regardless of whether improperly paid funds have been or are being recovered; (iv) ensuring that-- (I) the review of payments to employees shall include analysis of employee data, including pay grade data, locality pay, and other factors that affect pay; and (II) reviews address high-risk or high-dollar personnel payments, including travel, pay, and purchase cards; (v) reassessing high-risk programs to better reflect the unique processes, procedures, and risks of improper payments, including assessments for each program to reflect different risk components and better direct corrective actions; and (vi) confirming that inter-agency transfers are proper using a methodology comparable to that used to assess program level improper payments. SEC. 204. IMPROPER PAYMENTS INFORMATION. Section 2(a)(3)(A)(ii) of the Improper Payments Information Act of 2002 (31 U.S.C. 3321 note) is amended by striking ``with respect to fiscal years following September 30th of a fiscal year beginning before fiscal year 2013 as determined by the Office of Management and Budget'' and inserting ``with respect to fiscal year 2014 and each fiscal year thereafter''. SEC. 205. DO NOT PAY INITIATIVE. (a) Prepayment and Preaward Procedures.-- (1) In general.--Each agency shall review prepayment and preaward procedures and ensure that a thorough review of available databases with relevant information on eligibility occurs to determine program or award eligibility and prevent improper payments before the release of any Federal funds, to the extent permitted by law. (2) Databases.--At a minimum, each agency shall, before payment and award, check the following databases (if applicable and permitted by law) to verify eligibility: (A) The Death Master File of the Social Security Administration. (B) The General Services Administration's Excluded Parties List System. (C) The Debt Check Database of the Department of the Treasury. (D) The Credit Alert System or Credit Alert Interactive Voice Response System of the Department of Housing and Urban Development. (E) The List of Excluded Individuals/Entities of the Office of Inspector General of the Department of Health and Human Services. (b) Do Not Pay List.-- (1) Establishment.--There is established the Do Not Pay List which shall consist of-- (A) the databases described under subsection (a)(2); and (B) any other database designated by the Director of the Office of Management and Budget in consultation with agencies. (2) Other databases.--In making designations of other databases under paragraph (1)(B), the Director of the Office of Management and Budget shall consider-- (A) any database that assists in preventing improper payments; and (B) the database of incarcerated individuals established under subsection (f). (3) Access and review by agencies.--For purposes of identifying and preventing improper payment, each agency shall have access to, and use of, the Do Not Pay List to determine payment or award eligibility when the Director of the Office of Management and Budget determines the Do Not Pay List is appropriately established for the agency. (4) Payment otherwise required.--When using the Do Not Pay List, an agency shall recognize that there may be circumstances under which the law requires a payment or award to be made to a recipient, regardless of whether that recipient is on the Do Not Pay List. (c) Database Integration Plan.--Not later than 60 days after the date of enactment of this Act, the Director of the Office of Management and Budget shall provide to the Congress a plan for-- (1) inclusion of other databases on the Do Not Pay List; (2) to the extent permitted by law, agency access to the Do Not Pay List; and (3) the multilateral data use agreements described under subsection (e). (d) Initial Working System.-- (1) Establishment.--Not later than 90 days after the date of enactment of this Act, the Director of the Office of Management and Budget shall establish a working system for prepayment and preaward review that includes the Do Not Pay List as described under this section. (2) Initial system.--The working system established under paragraph (1)-- (A) may be located within an appropriate agency; (B) shall include not less than 3 agencies; (C) shall include fraud and improper payments detection through predictive modeling and other analytic technologies and other techniques; and (D) may provide for the use of commercial database sources, commercial analysis, and other functionality for payment or award reviews, as determined appropriate by the Director of the Office of Management and Budget for verifying Federal data. (3) Application to all agencies.--Not later than January 1, 2013, each agency shall review all payments and awards for all programs of that agency through the system established under this subsection. (e) Multilateral Data Use Agreements.-- (1) In general.--Not later than 60 days after the date of enactment of this Act, the Director of the Office of Management and Budget shall develop a plan to establish a multilateral data use agreement authority to carry out this section, including access to databases such as the New Hire Database under section 453(j) of the Social Security Act (42 U.S.C. 653(j)). (2) General protocols and security.-- (A) In general.--The multilateral data use agreements shall be consistent with protocols to ensure the secure transfer and storage of any data provided to another entity or individual-- (i) under the provisions of, or amendments made by, this section; and (ii) consistent with applicable information, privacy, security, and disclosure laws, including-- (I) the regulations promulgated under the Health Insurance Portability and Accountability Act of 1996 and section 552a of title 5, United States Code; and (II) subject to any information systems security requirements under such laws or otherwise required by the Director of the Office of Management and Budget. (B) Consultation.--The Director of the Office of Management and Budget shall consult with-- (i) the Council of Inspectors General on Integrity and Efficiency before implementing this paragraph; and (ii) the Secretary of Health and Human Services, the Social Security Administrator, and the head of any other agency, as appropriate. (f) Development and Access to a Database of Incarcerated Individuals.-- (1) In general.--The Attorney General shall develop and maintain a database of individuals incarcerated at Federal and State facilities. (2) Availability and update.--The database developed under this subsection shall be-- (A) available to agencies to carry out this section and prevent waste, fraud, and abuse; and (B) updated no less frequently than on a weekly basis. (g) Plan To Improve the Social Security Administration Death Master File.-- (1) Establishment.--In conjunction with the Commissioner of Social Security and in consultation with stakeholders and the States, the Director of the Office of Management and Budget, shall establish a plan for improving the quality and timeliness of death data maintained by the Social Security Administration, including death information reported to the Commissioner under section 205(r) of the Social Security Act (42 U.5.C. 405(r)). (2) Actions under plan.--The plan established under this subsection shall include actions agencies are required to take to-- (A) increase the quality and frequency of access; (B) achieve a goal of at least daily access as appropriate; and (C) provide for all States to use modern, electronic means for providing data. (3) Report.--Not later than 120 days after the date of enactment of this Act, the Director of the Office of Management and Budget shall submit a report to Congress on the plan established under this subsection, including recommended legislation. SEC. 206. IMPROVING RECOVERY OF IMPROPER PAYMENTS. (a) In General.--The Director of the Office of Management and Budget shall determine-- (1) current and historical rates and amounts of recovery of improper payments (or, in cases in which improper payments are identified solely on the basis of a sample, recovery rates and amounts estimated on the basis of the applicable sample), including specific information of amounts and payments recovered by recovery audit contractors; and (2) targets for recovering improper payments, including specific information on amounts and payments recovered by recovery audit contractors. (b) Recovery Audit Contractor Programs.-- (1) Establishment.--Not later than 90 days after the date of enactment of this Act, the Director of the Office of Management and Budget shall establish a plan for no less than 10 Recovery Audit Contracting programs for the purpose of identifying and recovering overpayments and underpayments in 10 agencies. (2) Review of commercial payments.--Of the programs established under this subsection, 5 programs shall review commercial payments by an agency. (3) Duration.--Any program established under this subsection shall terminate not more than 3 years after the date on which the program is established. (4) Reports.-- (A) In general.--Not later than 3 months after the completion of a program, the head of the agency conducting the program shall submit a report on the program to Congress. (B) Contents.--Each report under this paragraph shall include-- (i) a description of the impact of the program on savings and recoveries; and (ii) such recommendations as the head of the agency considers appropriate on extending or expanding the program. | Subsidized Stafford Loan Reduced Interest Rate Extension Act of 2012 - Amends title IV (Student Assistance) of the Higher Education Act of 1965 to make the 3.4% interest rate on Direct Stafford loans first disbursed to undergraduate students between July 1, 2011, and July 1, 2012, applicable to Direct Stafford loans first disbursed to undergraduate students between July 1, 2011, and July 1, 2013. Improper Payments Elimination and Recovery Improvement Act of 2012 - Requires the Director of the Office of Management and Budget (OMB) to: (1) identify, on an annual basis, a list of high-priority federal programs for greater levels of oversight and review of improper payments; (2) coordinate with agencies responsible for administering high-priority programs to establish semi-annual or quarterly targets and actions for reducing improper payments; and (3) provide guidance to agencies for improving estimates of improper payments. Requires federal agencies to: (1) make quarterly reports to their Inspectors General on any high-dollar improper payments identified by such agencies, and (2) review prepayment and preaward procedures and available databases to determine program or award eligibility and prevent improper payments before releasing any federal funds. Establishes a Do Not Pay List based on information from databases maintained by the federal government, including the database of the Social Security Administration (SSA) reporting deaths of Social Security recipients. Requires the Director to: (1) determine the current and historical rates and amounts of recovery of improper payments and targets for recovering improper payments, and (2) establish a plan for at least 10 Recovery Audit Contracting programs to identify and recover overpayments and underpayments in 10 agencies. | billsum |
Depending on the epidemiological setting, a variable proportion of leprosy patients will suffer from excessive pro-inflammatory responses, termed type-1 reactions (T1R). The LRRK2 gene encodes a multi-functional protein that has been shown to modulate pro-inflammatory responses. Variants near the LRRK2 gene have been associated with leprosy in some but not in other studies. We hypothesized that LRRK2 was a T1R susceptibility gene and that inconsistent association results might reflect different proportions of patients with T1R in the different sample settings. Hence, we evaluated the association of LRRK2 variants with T1R susceptibility. An association scan of the LRRK2 locus was performed using 156 single-nucleotide polymorphisms (SNPs). Evidence of association was evaluated in two family-based samples: A set of T1R-affected and a second set of T1R-free families. Only SNPs significant for T1R-affected families with significant evidence of heterogeneity relative to T1R-free families were considered T1R-specific. An expression quantitative trait locus (eQTL) analysis was applied to evaluate the impact of T1R-specific SNPs on LRRK2 gene transcriptional levels. A total of 18 T1R-specific variants organized in four bins were detected. The core SNP capturing the T1R association was the LRRK2 missense variant M2397T (rs3761863) that affects LRRK2 protein turnover. Additionally, a bin of nine SNPs associated with T1R were eQTLs for LRRK2 in unstimulated whole blood cells but not after exposure to Mycobacterium leprae antigen. The results support a preferential association of LRRK2 variants with T1R. LRRK2 involvement in T1R is likely due to a pathological pro-inflammatory loop modulated by LRRK2 availability. Interestingly, the M2397T variant was reported in association with Crohn’s disease with the same risk allele as in T1R suggesting common inflammatory mechanism in these two distinct diseases. Leprosy is a chronic dermato-neurological infectious disease caused by M. leprae. Leprosy irrespective of its clinical presentation is curable by multi-drug therapy. The current global effort in leprosy control is focused on early recognition of leprosy cases and prevention of permanent disabilities [1]. A common complication of leprosy are excessive pro-inflammatory episodes termed type-1 reactions (T1R) [2]. If untreated, T1R can lead to irreversible nerve function impairment due to a pathological cellular immune response directed against host peripheral nerve cells [3]. Up to 50% of all leprosy cases can undergo T1R with the incidence varying according to endemic settings and criteria for case definition [4]. Why only a proportion of leprosy patients undergo T1R is not known. However, clinical and environmental factors have been associated with T1R outcome [5,6]. Individuals categorized as borderline in the Ridley and Jopling clinical spectrum of leprosy are at increased risk to develop T1R while patients with tuberculoid or lepromatous polar leprosy forms rarely develop T1R [6]. Positive bacillary index, PCR detection of M. leprae, and increased age at leprosy diagnosis are other factors associated with T1R-risk [6–8]. A number of studies have shown a consistent upregulation of pro-inflammatory cytokines, i. e. TNF, IFNγ and the chemokine IP10, in the blood of T1R patients [4,9–12]. In a prospective study, the transcriptional profile of leprosy cases destined for T1R displayed a distinct signature from leprosy patients that remained T1R-free [11]. A dysregulated balance between innate pro- and anti-inflammatory responses emerged from this study as a key factor in T1R outcome [11]. A number of genes have been shown to be associated with T1Rincluding TNFSF15 and the pathogen recognition genes TLR1, TLR2 and NOD2 [3,13]. All of these genes had also been found to be associated with leprosy per se in other studies [14–16]. Since leprosy patients are usually not stratified by their T1R status, it is possible that some of the leprosy per se associations were caused by T1R patient subgroups. For example, the TNFSF15 gene had initially been shown to be associated with leprosy per se by a genome wide association study (GWAS) in a Chinese population [14]. However, in a Vietnamese sample stratified by T1R status the association signal could be unambiguously assigned to the T1R group [13]. Of the genes reported by the GWAS, the TNFSF15 and LRRK2 were the only leprosy susceptibility genes not validated for association with leprosy per se in a Vietnamese population [17]. Like TNFSF15, LRRK2 is a gene with an uncertain role in leprosy per se susceptibility. Several groups have evaluated the association of the LRRK2 gene with leprosy susceptibility but results were inconsistent [17–20]. Given that T1R affects different proportions of leprosy cases according to the studied population, we wondered if inconsistencies in LRRK2 association with leprosy per se were due to different proportions of T1R in each setting. Here, we evaluated a possible role for LRRK2 in T1R-affected families and contrasted the results with T1R-free families. We identified a set of 18 SNPs in LRRK2 preferentially associated with T1R. These variants overlapped with previous associations reported for Crohn’s Disease (CD), Ulcerative Colitis (UC) and Inflammatory Bowel Disease (IBD) [21,22]. For the LRRK2 study, a total of 1372 individuals were selected [13]. These individuals were divided in two family-based samples. The first set of families contained 229 leprosy affected offspring that underwent T1R (T1R-affected) and their respective parents. The T1R-free subset was matched to the T1R-affected subset by leprosy clinical subtype of the offspring (Fig 1). Consequently, the second set of families included 229 leprosy affected offspring and their parents in which the offspring had no signs of leprosy reaction (T1R-free). There was no difference in gender and age at leprosy onset regarding T1R outcome between both subsets (S1 Table). The subjects included in the eQTL analyses were part of a study evaluating the transcriptional profile of leprosy patients prior to T1R onset [23]. Briefly, 53 newly diagnosed leprosy cases in the borderline spectrum (19 BT, 30 BB and 4 BL) were enrolled. A blood sample was collected from each subject within 3 months of leprosy diagnosis and none of the subjects suffered T1R at enrolment. The samples used in the current study were selected from our records at the Dermato Venerology Hospital, Ho Chi Ming City, Vietnam, as described previously [13]. Written informed consent was obtained from all subjects enrolled in the study and all subjects were anonymized. This study was approved by the regulatory authorities in Ho Chi Minh City, Vietnam, and the Research Ethics Board at the Research Institute of the McGill University Health Centre, Montreal, Canada. The investigation have been conducted according to the principles expressed in the Declaration of Helsinki. Genotypes for156 SNPs mapping to a 500 kb window overlapping the LRRK2 and MUC19 genes were obtained via the 660W-quad v1 Illumina array [24]. The variants selected covered 89% of the SNPs with a MAF > 5% at a r2 > 0. 5 for the Vietnamese (KHV) and Chinese (CHB) and 84% for Caucasians (CEU) populations from the 1000 genomes project [25]. All genotypes passed standard quality-control presenting call rates greater than 98%, less than 2 Mendelian Errors (ME) and were in Hardy-Weinberg Equilibrium (HWE) with P > 0. 05 in 763 leprosy unaffected parents from both T1R-affecte and T1R-free subsets. LRRK2 expression levels were obtained using Illumina HumanHT12 v4 BeadChips as previously described [23]. Briefly, whole blood from the 53 leprosy patients was divided in two aliquots. One aliquot was stimulated with M. leprae sonicate for 24hrs to 30 hrs while the second aliquot was incubated for the same time interval in the absence of M. leprae sonicate (non-stimulated). Total RNA was extracted from all aliquots and used for LRRK2 quantification. Family based SNP and haplotype association tests were performed using Transmission Disequilibrium Test (TDT) as implemented in FBAT 2. 0. 4 [26]. Association testing was carried out under the same genetic model in T1R-affected and T1R-free families and the P values for the best genetic model (additive or dominant) were displayed (Fig 1). Due to the highly correlated nature of the genotyped SNPs, we did not perform a correction for multiple testing. Subsequently, a formal heterogeneity test was performed to evaluate preferential association of genetic variants with T1R (Fig 1) by using a modified version of the FBAT statistics (FBATHet) as described by Gaschignard, J. et al [27]. A multivariate analysis was performed to test for independence of T1R-specific associations. For each SNP bin (r2 > 0. 5), SNP with the most significant evidence for association was included in the multivariate model. Multivariate analyses were done by stepwise conditional logistic regression (SAS v. 9. 3). Logistic regression was also used to estimate the odds ratio for each individual SNP in the T1R-affected subset. Briefly, the TDT evaluates the non-random transmission of alleles from heterozygote parents to affected offspring. We used the non-transmitted alleles from the TDT to create up to three unaffected pseudo-sibs per family, one for each possible genotype. We compared the original T1R-affected offspring with T1R-unaffected pseudo sibs in a matched case-control design as described in [28]. Under the additive model, the TDT and the conditional logistic regression statistics result in the same P values. The recombination rate in centimorgan by mega base (cM/Mb) according to the 1000 genomes and the LD structure of the LRRK2/MUC19 locus were obtained with the R packages Locuszoom v. 1. 3 [29] and snp. ploter v. 0. 5. 1 [30], respectively. The minor allele frequencies (MAF) and HWE for each SNP were estimated using Haploview 4. 2 [31]. For the eQTL analyses, the correlation between genotypes and gene expression levels was performed with a simple linear regression under both stimulated and non-stimulated conditions using R 3. 2. 0. Genotype associations of LRRK2 and PD were obtained from the PDgene database (www. pdgene. org). LRRK2 variants associated with PD were obtained from a GWAS meta-analysis of 14 studies with a total population sample of 12,771 PD cases and 93,386 controls [32]. Genotype associations of LRRK2 variants with IBD and CD were obtained from the IBDgenetics database (www. ibdgenetics. org). The IBDgenetics population sample consisted of 42,950 IBD cases (22,575 CD and 20,417 UC patients) and 53,536 controls [21,22]. A genome-wide association (GWAS) in a Chinese sample provided the most comprehensive study of LRRK2 in leprosy. Hence, we first analyzed six LRRK2 SNPs (rs1873613, rs10878220, rs1491938, rs12820920, rs11174812 and rs11173979) described in association with leprosy per se by a GWAS in a Chinese population. None of the six variants presented significant evidence of association with leprosy in the T1R-free subset (Table 1). In contrast, SNPs rs10878220, rs1491938 and rs12820920 that belonged to the same r2 < 0. 5 SNP bin were nominally associated with disease in the T1R-affected subset (Table 1). These SNPs were preferentially associated with T1R when a formal heterogeneity test was performed (Table 1). Moreover, the direction of association was the same as previously reported for leprosy per se. While SNP rs10878220 is located in the core promoter region of the LRRK2 gene, SNPs rs1491938 and rs12820920 are LRRK2 intronic variants (Table 1). The remaining three SNPs that did not show association with T1R are located 66kb to 367kp upstream of LRRK2 transcription start site (Table 1). To further evaluate the association of LRRK2 with T1R, an additional 150 SNP were selected from the LRRK2 gene region. Of all 156 SNPs evaluated for association in the T1R-affected subset, 34 showed P < 0. 05 including the initial three SNPs associated with T1R (Fig 2). In the T1R-free family subset, the SNP rs7972711 situated near the 3’ end of the MUC19 gene and rs10878434, rs7303525 and rs11564172 located near 3`end of LRRK2 provided evidence of association (P < 0. 05; Fig 2). None of the 34 SNPs associated with T1R showed evidence of association with leprosy in the T1R-free families (Fig 2). When formally tested for heterogeneity of association, 18 out of the 34 SNPs were preferentially associated with T1R (Fig 2 and Table 1). These 18 SNPs preferentially associated with T1R belong to two extended SNP bins (r2 > 0. 5) and two single SNP bins (Table 1). Notably, although not nominally significant these 18 SNPs showed the opposite allelic enriched in T1R-free subset relative to the T1R-affected subset (Table 1). The bin tagged by the missense M2397T variant (rs3761863) situated in the WD40 domain of LRRK2 displayed the strongest preferential association with T1R (P = 0. 003; odds ratio (OR) = 1. 49; 95% confidence interval (CI) = 1. 12–1. 97 and P Het = 0. 008 for M2397 allele under an additive model) in the same direction as previously reported for CD (Table 2). To test if the r2 > 0. 5 SNP bins were independently associated with T1R we performed a multivariate analysis including the most significantly associated SNP for each bin (Table 2). The multivariate analysis identified the missense M2397T variant as the main association with T1R (P multi = 0. 01; Table 2). However, a trend for independent association was observed for the SNP bin tagged by rs1031996 (P multi = 0. 05) and the single SNP bin rs1463739 (P multi = 0. 13; Table 2) while the association of T1R and rs1427271 disappeared. We further investigated the combined effect of M2397T and rs1031996 by conducting a haplotype analysis. We found that the M2397 allele was mostly observed in the presence of the rs1031996 C allele (48% of the T1R-affected offspring) and this haplotype displayed a strong risk effect (Table 3). Importantly, the trend towards protection observed on the T2397 background was not modulated by rs1031996 alleles. Hence, the haplotype analysis confirmed that the main effect of the LRRK2 gene on T1R susceptibility was driven by the M2397T variant. No haplotype association was observed when leprosy per se was considered as phenotype. To investigate if SNP alleles associated with T1R were correlated with LRRK2 transcriptional levels we performed an eQTL analysis. Of the 18 SNPs preferentially associated with T1R, nine variants belonging to the same SNP bin as the missense M2397T variant (r2 = 0. 5) were eQTLs for LRRK2 in non-stimulated whole blood of 53 individuals (Table 1, S1 Fig). When a tighter LD threshold (r2 = 0. 8) was considered, the eQTL variants were separated from the bin tagged by M2397T. This observation suggested that the modest eQTL effect of M2397T was due to the linkage disequilibrium with the causal eQTL. The strongest eQTL effect was observed for SNP rs2404580 with the T1R-risk “T” allele being associated with higher LRRK2 expression in unstimulated cells (P = 5. 1E-05; Fig 3). Following stimulation with M. leprae sonicate, an abrogation of the eQTL effect was observed for all nine SNPs (Figs 3 and S1). Clinical subtypes of leprosy had no detectable impact on the eQTL effect of LRRK2 genotypes. We identified an amino acid change M2397T in the WD40 domain of LRRK2 and a bin tagged by the variant the variant rs1031996 as being associated specifically with T1R. LRRK2 is a protein that exerts a diverse set of functions. LRRK2 mediates catalytic processes through its enzymatic ROC/COR domain; facilitates signal transduction through a MAPK domain and interacts with other proteins through three scaffold domains, an ankylin repeat (ANK), a leucine rich repeat (LRR) and a WD40 repeat domain [33]. Scaffold domains are also responsible for protein conformation and stability [33]. Although, LRRK2 displays multiple functions, the association of T1R with a variant in the WD40 domain of LRRK2 suggests that protein conformation and/or stability are key factors in T1R. Indeed, the M2397T variant in LRRK2 has previously been shown to impact on LRRK2 protein turnover [34]. The half-life of LRRK2 with the T1R-risk allele M2397 had been estimated at approximately 8 hrs which is substantially shorter than the estimated 18 hrs half-life of the T2397 allele of LRRK2 (Fig 4) [34]. Cytoplasmic LRRK2 forms a complex that arrests nuclear factor of activated T-cells (NFAT) in the cytoplasm [34]. A consequence of LRRK2 deficit in the cytoplasm is the translocation of NFAT to the nucleus, which strongly induces the transcription of pro-inflammatory cytokines (Fig 4) [35,36]. Thus, the association of the M2397 allele with T1R-risk is in agreement with an exacerbated pro-inflammatory response in T1R cases (Fig 4). While the M2397T variant is a strong candidate for functional impact in T1R, the association of the bin tagged by rs1031996 requires further investigations. A set of eQTLs for LRRK2 was observed in non-stimulated cell from leprosy patients. Due to LD the M2397 allele and the eQTL alleles that correlated with increased LRRK2 expression were preferentially observed on the same haplotype. This suggests that the effect of M2397 on faster LRRK2 turn-over is mitigated by higher levels of LRRK2 message. Interestingly, in the presence of mycobacterial antigen the compensatory effect of higher LRRK2 message for the more rapid turnover of the M2397 protein is strongly abrogated. This implies that the genetic effect of the M2397 allele will be more pronounced in the presence of mycobacterial antigen while it may be largely abolished without such antigen exposure. While these in-vitro findings in whole blood will need to be validated employing purified cell types and in-vivo studies in leprosy lesion, the results obtained provide an example of how environmental stimuli can modulate germline encoded genetic risk factors (Figs 3 and 4). LRRK2 variants associated with T1R overlapped previous reported LRRK2 associations with CD and PD [21,22,32]. In PD, rare coding variants in the enzymatic and kinase domains of LRRK2 were shown to be causally linked to PD [33]. In addition, common LRRK2 variants were shown in association with PD [32]. Two of these variants, rs1491932 and rs7970326, were observed in association with T1R and borderline evidence T1R specificity for the alleles opposite to PD. Common variants may tag rare variants with stronger effects. However, given our sample size we were unable to evaluate the role of rare variants in T1R. Of 18 SNPs preferentially associated with T1R, 17 were nominally associated with IBD with the same risk allele observed for T1R [22]. The only exception was rs1463739 a SNP located outside of LRRK2 in the 3’ region of MUC19 (Table 2 and Fig 2). When considering IBD subtypes, 14 T1R-risk variants were associated with risk of CD and 11 with risk of UC [22]. The M2397 allele is a risk factor for T1R, CD and UC, suggesting that a faster LRRK2 turnover leads to an increased pro-inflammatory response that is common to these diseases. T1R and CD may share susceptibility to mycobacterial species as common etiology [37,38]. In PD, pathogen involvement is controversial although some studies suggested that Helicobacter pylori and prions might play a role in disease susceptibility [39–41]. Further studies will be needed to understand the precise role of LRRK2 in the pathogenesis of these three diseases. | A major challenge of current leprosy control is the management of host pathological immune responses coined Type-1 Reactions (T1R). T1R are characterized by acute inflammatory episodes whereby cellular immune responses are directed against host peripheral nerve cells. T1R affects up half of all leprosy patients and are a major cause of leprosy-associated disabilities. Since there is evidence that host genetic factors predispose leprosy patients to T1R, we have conducted a candidate gene study to test if LRRK2 gene variants are T1R risk factors. The choice of LRRK2 was motivated by the fact that LRRK2 was associated with leprosy per se in some but not in other studies. We reasoned that this may reflect different proportions of leprosy patients with T1R in the different samples and that LRRK2 may in truth be a T1R susceptibility gene. Here, we show that variants overlapping the LRRK2 gene, reported as suggestive leprosy per se susceptibility factors in a previous genome-wide association study, are preferentially associated with T1R. The main SNP carrying most of the association signal is the amino-acid change M2397T (rs3761863) which is known to impact LRRK2 turnover. Interestingly, eQTL SNPs counterbalanced the effect of the M2397T variant but this compensatory mechanism was abrogated by Mycobacterium leprae antigen stimulation. | lay_plos |
SECTION 1. SHORT TITLE; TABLE OF CONTENTS. (a) Short Title.--This Act may be cited as the ``Paul Revere Freedom to Warn Act''. (b) Table of Contents.--The table of contents for this Act is as follows: Sec. 1. Short title; table of contents. Sec. 2. Discrimination against whistleblowers prohibited. Sec. 3. Enforcement action. Sec. 4. Remedies. Sec. 5. State secrets privilege. Sec. 6. Criminal penalties. Sec. 7. Rights retained by covered individual. Sec. 8. Notification. Sec. 9. Definitions. Sec. 10. Effective date; applicability. SEC. 2. DISCRIMINATION AGAINST WHISTLEBLOWERS PROHIBITED. It shall be unlawful for any person to discharge, demote, suspend, reprimand, investigate, or take or fail to take any other personnel action that in any manner discriminates against any covered individual, or in any other manner discriminate against any covered individual (including by a denial, suspension, or revocation of a security clearance or by any other security access determination, or by denial of award of a Federal contract or subcontract), or to threaten or recommend the discharge, demotion, suspension, reprimand, investigation, other personnel action (or rejection of such action) that in any manner discriminates against any covered individual, or other manner of discrimination if such action, discrimination, or recommendation is due, in whole or in part, to any lawful act done, perceived to have been done, or intended to be done by the covered individual-- (1) to provide information, cause information to be provided, or otherwise assist in an investigation or proceeding regarding any conduct which the covered individual reasonably believes constitutes evidence of a violation of any law, rule, or regulation, a threat to national or homeland security, a substantial and specific threat to public health or safety, or fraud, abuse of authority, waste, or mismanagement of public funds, if the information or assistance is provided to or the investigation is conducted by-- (A) a Federal, State, or local regulatory or law enforcement agency (including an office of Inspector General under the Inspector General Act of 1978); (B) any Member of Congress, any committee of Congress, or the Government Accountability Office; (C) any person with supervisory or managerial authority over the covered individual (or any other person who has the authority to investigate, discover, or terminate misconduct); or (D) a potential witness to or other person affected by or aware of the conduct described in this section; (2) to file, cause to be filed, testify, participate in, or otherwise assist in a proceeding or action filed or about to be filed relating to an alleged violation of any law, rule, or regulation; or (3) to refuse to violate or assist in the violation of any law, rule, or regulation. SEC. 3. ENFORCEMENT ACTION. (a) In General.--A covered individual who alleges discharge or other discrimination by any person in violation of section 2 may seek relief under section 4 by-- (1) filing a complaint with the Secretary of Labor; or (2) if the Secretary has not issued a final decision within 180 days after the filing of the complaint and there is no showing that such delay is due to the bad faith of the claimant, bringing an action at law or equity for de novo review by a jury in the appropriate district court of the United States, which shall have jurisdiction over such an action without regard to the amount in controversy. (b) Procedure.-- (1) In general.--An action under subsection (a)(1) shall be governed under the rules and procedures set forth in section 42121(b) of title 49, United States Code. (2) Exception.--Notification made under section 42121(b)(1) of title 49, United States Code, shall be made-- (A) to the person named in the complaint; and (B) to the person's employer or, in the case of a Federal contractor or subcontractor, to the instrumentality of the Government with which such contractor or subcontractor has entered into, or submitted an offer to enter into, a contract. (3) Burdens of proof.--An action brought under subsection (a)(2) shall be governed by the legal burdens of proof set forth in section 42121(b) of title 49, United States Code. (4) Statute of limitations.--An action under subsection (a) shall be commenced not later than 6 years after the date on which the alleged violation occurred. SEC. 4. REMEDIES. (a) In General.--A covered individual prevailing in any action under section 3(a) shall be entitled to all relief appropriate to make the covered individual whole. (b) Damages.--Relief for any action under subsection (a) may include-- (1) reinstatement with the same seniority status and employment grade or pay level (or the equivalent) that the covered individual would have had, but for the discrimination; (2) compensatory damages, including the amount of any back pay, with interest; (3) compensation for any special damages sustained as a result of the discrimination, including litigation costs, expert witness fees, and reasonable attorneys fees; and (4) punitive damages in an amount not to exceed the greater of 3 times the amount of any monetary damages awarded under this Act (apart from this paragraph) or $5,000,000. SEC. 5. STATE SECRETS PRIVILEGE. If, in any action brought under section 3(a)(2), the Government asserts as a defense the privilege commonly referred to as the ``state secrets privilege'' and the assertion of such privilege prevents the plaintiff from establishing a prima facie case in support of the plaintiff's claim, the court shall enter judgment for the plaintiff and shall determine the relief to be granted. SEC. 6. CRIMINAL PENALTIES. (a) In General.--Any person violating section 2 may be fined under title 18 of the United States Code, imprisoned not more than 10 years, or both. (b) Reporting Requirements.--The Department of Justice shall (based on such periodic reports and other information from the Department of Labor as the Department of Justice may require) submit to Congress an annual report on the enforcement of subsection (a). Each such report shall-- (1) identify each case in which formal charges under subsection (a) were brought; (2) describe the status or disposition of each such case; and (3) in any actions under section 3(a)(2) in which the covered individual was the prevailing party or the substantially prevailing party, indicate whether or not any formal charges under subsection (a) have been brought and, if not, the reasons therefor. SEC. 7. RIGHTS RETAINED BY COVERED INDIVIDUAL. Nothing in this Act shall be deemed to diminish the rights, privileges, or remedies of any covered individual under any Federal or State law, or under any collective bargaining agreement. The rights and remedies in this Act may not be waived by any agreement, policy, form, or condition of employment. SEC. 8. NOTIFICATION. The provisions of this Act shall be prominently posted in any place of employment to which this Act applies. SEC. 9. DEFINITIONS. For purposes of this Act-- (1) the term ``covered individual'' means an employee or a member of the uniformed services (as defined by section 2101(3) of title 5, United States Code)-- (A) serving in or under-- (i) an Executive agency (as defined by section 105 of such title 5), a military department (as defined by section 103 of such title 5), or any other instrumentality of the Government (which, for purposes of this Act, includes the Department of Homeland Security, the Transportation Security Administration, and any other instrumentality of the Government, notwithstanding any special personnel authorities which might be available to such instrumentality under law); (ii) a Federal contractor or subcontractor; or (iii) the Federal National Mortgage Association, the Federal Home Loan Mortgage Corporation, and any other federally chartered entity; or (B) employed by an employer within the meaning of section 701(b) of the Civil Rights Act of 1964 (42 U.S.C. 2000e(b)); (2) the term ``employee'' means-- (A) with respect to an employer referred to in paragraph (1)(A)(i), an employee as defined by section 2105 of title 5, United States Code; and (B) with respect to an employer referred to in paragraph (1)(A)(ii) or (1)(B), any officer, partner, employee, or agent; such term, as defined by subparagraph (A), includes an individual holding a position in an instrumentality of the Government identified in the parenthetical matter under paragraph (1)(A); (3) the term ``evidence'' means information that meets the standard for admissibility under the Federal Rules of Evidence, or is used as part of the record in support of a finding in an investigative report or decision by a government office with jurisdiction; (4) the term ``Federal contractor'' means a person who has entered into, or responded to a request for proposals or solicitation for bids to enter into, a contract with an instrumentality of the Government; (5) the term ``lawful'' means not specifically prohibited by law, except that, in the case of any information the disclosure of which is specifically prohibited by Federal statute or specifically required by Executive order to be kept secret in the interest of national defense or the conduct of foreign affairs, any disclosure of such information to any Member of Congress, committee of Congress, or other recipient authorized to receive such information, shall be deemed lawful; (6) the term ``law, rule, or regulation'' refers to a law of the United States and any rule or regulation prescribed under any such law; (7) the term ``person'' means a corporation, partnership, State entity, business association of any kind, trust, joint- stock company, or individual; (8) the term ``reasonably believes'', with respect to information provided by a covered individual, means only that a disinterested observer with knowledge of the essential facts known to and readily ascertained by the covered individual could conclude that the information constitutes evidence of conduct described under section 2(1); and (9) the term ``subcontractor'', with respect to a Federal contractor, means any person, other than the Federal contractor, who offers to furnish or furnishes any supplies, materials, equipment, or services of any kind under a contract with an instrumentality of the Government or a subcontract (at any tier) entered into under such a contract. SEC. 10. EFFECTIVE DATE; APPLICABILITY. (a) Effective Date.--This Act shall take effect 90 days after the date of the enactment of this Act. (b) Applicability.--This Act shall apply to-- (1) any administrative or judicial proceeding pending on the effective date of this Act; and (2) any administrative or judicial proceeding brought on or after the effective date of this Act. | Paul Revere Freedom to Warn Act - Makes it unlawful to take any adverse personnel action against any covered individual if such individual has acted lawfully to: (1) provide information or assistance in an investigation or proceeding regarding any conduct which the covered individual reasonably believes constitutes evidence of a violation of any law, rule, or regulation, a threat to national homeland security, a substantial and specific threat to public health or safety, or fraud, abuse of authority, waste, or mismanagement of public funds, if the information or assistance is provided to, or the investigation is conducted by, specified individuals, including law enforcement authorities, Members of Congress, or supervisors of such individual; (2) file, testify, participate in, or assist in a proceeding or action relating to an alleged violation of any law, rule, or regulation; or (3) refuse to violate or assist in the violation of any law, rule or regulation. Specifies the enforcement actions under which a covered individual who alleges discharge or other discrimination by any person in violation of such requirement may seek relief. Entitles a covered individual prevailing in any such action to all relief appropriate to make such individual whole. Sets forth criminal penalties for violations of this Act. Requires the Department of Justice to submit annual reports on the enforcement of such violations. Requires this Act's provisions to be prominently posted in places of employment to which it applies. | billsum |
A man has been convicted of rape after the victim spotted him six years after the incident, according to Fulton County prosecutors. Antonio White, 54, was sentenced to life without parole after he was convicted Friday night, Fulton County District Attorney Paul Howard said in a statement. The victim recognized White while she waited at the Five Points MARTA station in October 2013, six years after the incident, Howard said. “She was overcome by the moment and began to yell out that he was the man who had raped her six years earlier,” Howard said. “Hearing her cries for help, MARTA police arrested White and turned him over to Atlanta police.” In August 2007, the victim was walking to the Hamilton E. Holmes station when White drove up in his gray Chevrolet Lumina and asked if she wanted a ride, according to prosecutors. “The victim knew White from the former Carver Homes neighborhood in Southeast Atlanta where they both formerly lived, so she said OK,” Howard said. “Instead of driving her home, the defendant took the victim to an abandoned house on Bowen Avenue. “As White opened his door, the victim opened her door and ran, but he chased her with a gun,” Howard said. “The defendant grabbed the victim’s hair from behind and pulled her back inside the car at gunpoint. White then raped the victim inside his vehicle.” For years, the victim told family members and other people she had been raped, but she never officially reported the incident to police. Prosecutors presented evidence at the trial of four previous sexual assaults by White, who has one previous rape conviction, a robbery conviction and a guilty plea to sexual battery. In other news: An Atlanta man was sentenced to life in prison Friday after he was convicted of raping a woman – who reportedly recognized him during a chance sighting at a train station six years after the assault. Antonio White, 54, was waiting for a train at a MARTA subway station in Atlanta in October 2013 when he was spotted by his victim. “She was overcome by the moment and began to yell out that he was the man who had raped her six years earlier,” the Fulton County district attorney’s office said in a statement, adding it was the first time the woman had seen White since the attack. In August 2007, White spotted the unidentified victim walking to a MARTA subway station in Atlanta and offered to give her a ride. The woman accepted because she recognized him from the neighborhood where she previously lived, the district attorney’s office said. But instead of driving her to her destination, White took her to an abandoned house, prosecutors said. She attempted to run away, but White chased her with a gun and managed to pull her back inside the car, where he raped her, the district attorney’s office said. BALTIMORE TEEN CHARGED IN RAPE, MURDER OF 83-YEAR-OLD NEIGHBOR During the assault, prosecutors said White threatened the victim: “Shut up that screaming [expletive] or I’ll kill you.” The victim did not report the crime to the police and did not know the name of the man who raped her, the district attorney’s office said. She did, however, tell her friends and family about the assault. After the victim identified White at the subway station, MARTA police arrested him and turned him over to the Atlanta Police Department, WAGA-TV reported. PENNSYLVANIA MAN ACCUSED OF 150 RAPES MAKES FIRST KANSAS COURT APPEARANCE During the investigation, police learned White had sexually assaulted multiple other women between 1983 and 2008, prosecutors said. He also had prior convictions for rape and robbery and had pleaded guilty to sexual battery in the past. | A woman raped in 2007 had the jarring experience of spotting her rapist six years later in an Atlanta subway station, reports the Atlanta Journal Constitution. At that point, she didn't think much about what to do next. "She was overcome by the moment and began to yell out that he was the man who had raped her six years earlier," the Fulton County district attorney's office said in a statement, per Fox News. Police then arrested Antonio White, 54, who was sentenced to life in prison without parole following his rape conviction on Friday. The woman, who had only described the crime to friends and family at the time, told authorities how she had accepted a ride from White, whom she recognized as having lived in her former neighborhood. Rather than take her home, White drove the woman to an abandoned house and chased her with a gun as she tried to escape, ultimately pulling her back into the vehicle by her hair. Prosecutors say he raped her in the vehicle and threatened to kill her. At trial, they described four previous sexual assaults by White between 1983 and 2008. | multi_news |
TECHNICAL FIELD OF INVENTION [0001] This invention relates to control methods to use in Plant health management programs. Provides an active ingredient and a method of biological control against diseases caused by the root-knot-nematodes that belong to the genera Meloidogyne spp. In this particular form provides a strain of the fungus Pochonia chlamydosporia and a method of using it as a way of decrease or inhibit Meloidogyne spp. populations developing in soil and rhizosphere of susceptive plants. BACKGROUND ART [0002] The root-knot nematodes, like other plant pathogenic organisms can be controlled using several control methods, namely phisic, cultural, genetic, chemical and biological, and/or a combination of these, following different strategies, namely the Integrated Pest Management which arouse as the main strategy over the latest years. This strategy aims to decrease the undesirable side-effects of Pesticide use, and the optimisation of these and other control methods, ensuring at the same time the objectives of the plant growers and the minimization to the lowest possible level of the secondary effects on the environment and consumers. [0003] Although the cultural and physical control methods currently available, may achieve in particular conditions a certain degree of success, they are not suited for the most practical situations where the most important economic damages arouse from the presence of significant populations of Meloidogyne spp., because they require high levels of resources, namely economic and time. [0004] With genetic methods, a partial success has been achieved by plant breeding, but the strains currently available and economically more interesting for plant growers, can't resist or have low tolerance to Meloidogyne spp. attack. Nevertheless, can be observed great tolerance variability among plants and strains, and is expected that those with higher levels of tolerance are in better position to succeed in commercial growers. [0005] Chemical control methods are the main process used to limit the Meloidogyne spp. populations, especially in high value crops that use high amounts of input factors. [0006] There are 3 main pesticide groups that are in use to control nematodes: general fumigant biocides; fumigant nematicides; non-fumigant nematicides. [0007] The general biocides are the most effective, probably due to its physic properties that allow its even distribution in the soil, which is mainly due to the high vapour pressures at room temperature. One example of these compounds is the most widespread named metal-bromide. These compounds have several drawbacks: they have to be applied before crop settlement, because they are phytotoxic and demand a waiting period variable with the substance, usually between 1-4 weeks, and cause noxious side-effects on the environment. [0008] The fumigant nematicides, although effective under the appropriate conditions, present more variability in the efficacy, because they experience a higher degree of difficulty to spread in soil. They present also similar drawbacks to the previous group. [0009] The non-fumigant nematicides, although effective in favourable conditions, are usually less effective compared to the compounds of the previous groups, specially due the difficulty in spreading them evenly through the soil, although they can be used after crop settlement, which is an advantage, because they are less phytotoxic. [0010] All the referred pesticides have several undesirable side-effects, and although these vary with the substance, all have side-effects that can be considered a liability for public human health, and for the environment, if their use becomes widespread. [0011] In summary, for the major substances currently in use: [0012] General biocides: metal bromide goes to atmosphere and is a strong depletor of ozone layer, and its use is currently prohibited by the international environment agreements. The chlorpicrin and metyl-isothiocianate precursors (dazomet and metham-sodium) left dangerous residues in crops for several months and can contaminate underground water. [0013] Fumigant nematicides: The 1.3 D, the only allowed currently in most countries, left residues in soil for several months, can contaminate underground water, left residues in plants and is highly phytotoxic. [0014] Non-fumigant nematicides: All the 4 more widespread, left high levels of residues in crops, during large periods of time, which limit its homologation to long cycle crops and with long safety intervals which limits its efficacy. Because many of them act by inhibiting nematode actions and not by killing them, they can recover after the soil concentration lower to adequate levels, as happen with fenamifos and oxamil. Besides those, the substances carbofuran and aldicarb are highly dangerous for underground water; and their use is limited to situations where the risk of underground contamination is low. [0015] Biological control methods are an alternative to the methods presented above, but in spite of the strong pressure coming from the side effects of pesticides and lack of other viable alternatives, currently there are very few effective alternatives that can be used to control Meloidogyne spp. populations. [0016] These nematodes possess many natural enemies that can be searched for new methods of biological control. They belong to many different groups of organisms, like bacteria, insects, acariens, predator nematodes and fungi. Although many of these organisms have been studied, there are 4 that received the major efforts from the researchers over the years, although its effective use as part of a control method has proved to be difficult. These organisms are: the bacteria Pasteuria penetrans, and 3 types of fungus: species of the genus Artrobotrys spp. namely A. Dactyloides and A. Oligospora, the fungus Paecilomyces lilacinus and the fungus Pochonia chlamydosporia, named previously Diheterospora chlamydosporia or Verticillium chlamydosporium. [0017] Currently to any of these organisms, its possible identify two different strategies of control against Meloidogyne spp. populations. [0018] a) Promote the development of indigenous populations [0019] b) Introduce relatively small inoculations, or mass inoculations, with strains exogenous to the ecosystem, in order to enable the settlement of an aggressive population of nematode antagonists, capable of control the nematode population. [0020] To the present date it was not possible, with pontual exceptions in particular environments, not fully understanded and concerning both strategies, to produce a reliable and reproducible biological method of control against Meloidogyne spp. [0021] Concerning the fungus Pochonia chlamydosporia there are extensive published data (Kerry, 1995; Kerry & Bourne 1996; Kerry & Bourne, 2002; Bourne, et al., 1996; Leij et al., 1992; Leij et al. 1993), suggesting the possibility of using strains of this fungus as part of control strategies against Meloidogyne spp. mainly by the use of great amounts of spores, by mass inoculate them in the treated soils. [0022] To use the referred strategy its necessary that the selected strain meet the following requirements: i) possess a high agressivity against Meloidogyne spp. eggs, which is measured by the % parasitism, measured in Petri dishes, and should be higher than 30%; ii) high ability to colonize the roots, which is measure in Petri dishes and should be bigger than 80%; iii) high capacity to produce chlamydospores in solid media, using milled barley, and should be bigger than 10 6 chlamydospores/g media in order to permit enough spore production to built the inoculum for field inoculation. [0023] In addition, in pot tests, it was possible to determine the main variables related to parasitism, namely: i) the population recovered from soil, should be higher than 2×10 4 cfu/g soil (colony forming units); ii) the rhizosphere population should be bigger than 8×10 3 cfu/g root; and iii) the egg parasitism, extracted from infected roots with Meloidogyne spp. should be higher than 30% (parasitised eggs/total number of eggs). [0024] In spite of the results obtained in controlled conditions in laboratory tests, in pot tests and in microplots, it was not possible to confirm them in commercial crops, growing in the conditions similar to those of commercial growers, either using indigenous populations or with mass introduction of formulated spores of exogenous strains, grown specially to that purpose. [0025] In spite of that, and based only in laboratory tests and in pots there is a strain of Pochonia chlamydosporia named at the time, Verticillium chlamydosporia strain ac, whose use against Meloidogyne spp. is patented (WO91/01642). SOLUTIONS PROVIDED BY THIS INVENTION [0026] The majority of the strains discovered to date, are not good candidates to use in biological control, because they have a relative low virulence against Meloidogyne spp. eggs. This is shown by in-vitro tests, yielding a parasitism rate average that seldom surpass 40%. Besides that, in field conditions similar to commercial crops, and using as inoculum chlamydospores formulated with inert materials, it was not possible to date obtain significant population reductions with any of the tested strains. [0027] The strain PcMR has a high virulence against Meloidogyne spp. eggs, and shows a parasitism rate of 66% in in-vitro Petri dish selection tests. [0028] The strain PcMR, in conditions similar to those utilised by commercial growers, can control Meloidogyne spp. populations in root-knot-nematode infested soils. This control can be achieved at least in two consecutive years, in soils inoculated with chlamydospores formulated with sterilized fine sand (Ø 90-700 μm), with a rate of 5000 chlam./g soil, in the most superficial 20 cm of soil and can be shown by the following parameters: [0029] a) Meloidogyne population reductions to control of 74% in the total number of viable juveniles plus eggs (reduction between 56 and 91%, t-student 95% interval) in 1 st year, and 65% (reduction between 60 and 71%, t-student 95% interval) in the 2 nd year. [0030] b) Meloidogyne population reductions to control of 64% in the total number of viable juveniles+eggs (reduction between 49 and 79%, t-student 95% interval) in 1 st year, and 70% (reduction between 63 and 77%, t-student 95% interval) in the 2 nd year. [0031] c) An average PcMR egg parasitism (eggs collected from egg masses present in the outside of roots) of 45% (between 39 and 51%, t-student 95% interval) in 1 st year, and of 39% (between 34 and 43%, t-student 95% interval) in the 2 nd year. [0032] The strain PcMR is characterized in that it has all the other characteristics, consider essential to be utilized as biologic control agent, referred in background art, and characterized in the detailed description. DETAILED DESCRIPTION [0033] Morphologic Characterization of PcMR Strain: [0034] This strain of the fungus Pochonia chlamydosporia has all the morphologic typical characteristics of the fungus Pochonia chlamydosporia var. chlamydosporia, described by Gams, 1988. [0035] Selection tests outcome, using the methods stated in IOBC manual (Kerry & Bourne, 2002): [0036] Were utilized the 3 standard selection tests, the Barley root colonization test in semi-sterile conditions, the chlamydospore production test in vitro, the egg parasitism test in water-agar Petri dishes. [0037] PcMR yielded the following results, in these in-vitro selection tests: [0038] a) Barley roots colonization test: 81.4%. [0039] b) Chlamydospore production test: 2.2×10 7 chlamydospores/g of barley used in the chlamydospore production medium. [0040] c) Egg parasitism test: 66%. [0041] Green pepper pot tests according to IOBC methods (Kerry & Bourne, 2002): [0042] Experiment Conditions: [0043] The experiment was carry way in a growth chamber, at 25° C., with appropriate light regime to green pepper growth, and with manual irrigation. [0044] Plant utilized: Green Pepper, Capsicum annum var. Corteso. [0045] Soils used: 2 types: type 1: non-autoclaved sandy soil; type 2: autoclaved sandy soil. [0046] Fungus treatment: 3 strains tested and controlled (non-inoculated soil). All inoculations were performed with 5000 chlamydospores/g of soil. [0047] Nematode treatment with Meloidogyne incognita : were used 3 levels: level 1: 6000 juveniles, level 2: 2500 juveniles, level 3: control (non-inoculated). [0048] Each statistical unit, obtained by the combination of the different treatments stated was repeated 5 times. [0049] In pot tests the strain PcMR decreased significantly (S<0.05) the number of eggs/egg mass, in soil inoculated with 5000 chlamydospores/g of soil, compared to non-inoculated soil, in eggs collected from egg masses obtained from the green peppers grown in the pots with 2500 and 6000 juveniles of Meloidogyne incognita. [0050] Field Trials, in 2 consecutive years, in conditions matching those utilized by commercial growers (assay design and evaluation methods of assay parameters according to Kerry & Bourne, 2002) [0051] Assay Description: [0052] Site: Agricultural Experimental station of Patação, in Direcção Regional de Agricultura do Algarve, Algarve, Portugal. [0053] Plastic house structure: Plastic house length-40 m, width-7.2 m. [0054] The plastic house soil was a Meloidogyne javanica infested sandy soil, and had no indigenous population of P. chlamydosporia. [0055] Cultivated plant: Tomato, Lycopersicum esculentum var. sinatra. [0056] Plant density: 6 rows with 97 plants each, spaced 1.1 m between rows×0.4 m in the row. [0057] Statistical Design: [0058] 16 plots, each with 3 plant rows, 11 plants per row, in a total of 33 plants per plot. The plots were randomly distributed in the plastic house. [0059] Fungus treatment: only one strain tested: strain PcMR [0060] Were chosen 3 treatments and 1 control (without treatment). [0061] Treatment 1: nematicide application: methyl-bromide in pre-plantation. [0062] Treatment 2: nematicide application+PcMR: methyl-bromide in pre-plantation+PcMR treatment (same as treatment 3). [0063] Treatment 3: PcMR treatment: 2-3 inoculations, utilizing 5000 chlamydospores/g soil, inoculating 8 plants chosen in central row of the plot, and calculating the soil volume by multiplying soil area available for each plant×top 20 cm of soil. The inoculations started 2-3 weeks after planting. [0064] Treatment 4: control: no treatment applied. [0065] Each treatment was repeated 4 times. All the evaluated parameters were determined 3 times in each sample. [0066] The Results: [0067] 1 st year (Treatment 3 compared with control): Soil colonization, final population recovered at the end of assay: 2.7×10 4 cfu/g of soil (conf. int. t-student 95%, between 1.5 and 3.8×10 4 cfu/g of soil). [0068] Root colonization: 1.3×10 4 cfu/g of root (conf. int. t-student 95%, between 0.7 e 1.9×10 4 cfu/g of root) [0069] Egg parasitism in Meloidogyne eggs, tested in semi-selective media, from eggs extracted by mechanic methods from egg masses obtained from the field infected plants: average 45% (conf. int. t-student 95%, between 39 and 51%). [0070] Meloidogyne population reduction: Determination of the parameter: total viable juveniles plus eggs: average of 74% (conf. int. t-student 95%, between 56 e 91%). Determination of parameter: number of viable eggs/egg mass: average of 64% (conf. int. t-student 95%, between 49 e 79%). [0071] 2 nd year (Treatment 3 compared with control): Soil colonization, final population recovered at the end of assay: 2.8×10 4 cfu/g of soil (conf. int. t-student 95%, between 1.7 and 3.8×10 4 cfu/g of soil). [0072] Root colonization: 1.3×10 4 cfu/g of root (conf. int. t-student 95%, between 1.2 e 1.5×10 4 cfu/g of root). [0073] Egg parasitism in Meloidogyne eggs, tested in semi-selective media, from eggs extracted by mechanic methods from egg masses obtained from the field infected plants: average 39% (conf. int. t-student 95%, between 34 and 43%). [0074] Meloidogyne population reduction: Determination of the parameter: total viable juveniles plus eggs: average of 65% (conf. int. t-student 95%, between 60 e 71%). Determination of parameter: number of viable eggs/egg mass: average of 70% (conf. int. t-student 95%, between 63 e 77%). INDUSTRIAL APPLICATIONS [0075] A—This strain can be utilized in mass production of chlamydospores, using fermentors of variable size, using the appropriate technology to produce large amounts of chlamydospores, namely solid state fermentors using an appropriate substratum. Currently, several media can be used, namely media based in barley, rice, wheat or corn. These spores can then be extracted from liquid suspensions (international patent request PTI 04/000009), or through dry methods. [0076] B—Its possible to obtain formulated products from extracted chlamydospores referred in A by adding to them inert substances. These products can be applied in soils, in plants, in seeds or other growing media for plants. These products can be utilized as part of a biological control method against root-knot-nematode ( Meloidogyne spp.) which can be utilized in the majority of susceptible cultivated plants. EXAMPLE [0077] Control of a Meloidogyne spp. population, composed mainly by Meloidogyne javanica, in plastic house tomato crop, in Algarve, Portugal. [0078] Mass Production of Chlamydospores. [0079] 1—Production medium preparation (barley+sand medium) (Kerry & Bourne 2002): [0080] The barley is milled and sieved through a 2 mm sieve and then washed through a 44 μm, collecting all the residue collected in 44 μm. [0081] Sieve fine sand, through a 2 mm sieve, then wash it through a 44 μm, and then collect the particles between 2 mm and 44 μm. [0082] The production medium is obtained by mixing the collected fractions of milled barley and fine sand in 1:1 proportion, and let it dry to the appropriate moisture level. [0083] Place 50 ml of this mixture in small 250 ml Erlenmyer flasks (fermentors), close with cotton, seal with aluminium foil and then autoclave. [0084] 2—Inoculate each fermentor of 250 ml, in sterile conditions, with 4 agar plugs previously colonized with PcMR, wait 3 days at 25° C., and then shake gently to spread inoculum and then let incubate for 1 month. [0085] Chlamydospore Extraction. [0086] From a sufficient number of 250 ml fermentors, extract all the colonized medium with chlamydospores, homogenise and wash it to the feeding tank of the extraction and separation apparatus. Then operate the apparatus in order to correctly extract the chlamydospores (international patent request PTI 04/000009) and then collect the chlamydospores in appropriate vials, and keep them in the fridge at 4° C. [0087] Product Formulation: [0088] Prepare fine sand (Ø 90 to 700 mm), sterilize it in the autoclave, dry it overnight at 90° C. in the oven. [0089] Build up the inoculum by mixing the sand and chlamydospores, evenly at the 10:1 proportion, and then keep it at 4° C. until necessary. [0090] Plant Inoculation: [0091] With soil moisture at field capacity, apply an aqueous mixture of inoculum to the plant by spreading it on the soil around the plant, at the rate of 5000 chlamydospores/g soil, on the top 20 cm of the soil. Then slightly incorporate inoculum in soil surface and cover it with soil. [0092] Apply the inoculum as much times as needed to achieve a recovered population from soil of 2×10 4 cfu/g soil. [0093] Soil Monitorization: [0094] Monitoring Pochonia chlamydosporia var. chlamydosporia strain PcMR. Perform soil analysis, with the available methods, to determine the population of PcMR in cfu/g soil. (IOBC manual, Kerry & Bourne, 2002). BIBLIOGRAPHY [0000] Gams, W. (1988) A contribution to the knowledge of nematophagous species of Verticillium. Netherlands Journal of Plant Pathology, 94: 123-148. Kerry, B. R. and Bourne, J. M. (ed.) (2002) A Manual for Research on Ver - ticillium chlamydosporium, a Potential Biological Control Agent for Root-Knot Nematodes. IOBC/WPRS, Gent, Belgium, 84 pp. Kerry, B. R. (1995) Ecological considerations for the use of the nematophagous fungus, Verticillium chlamydosporium, to control plant parasitic nematodes. Canadian Journal of Botany 73(suppl. 1): S65-S70. Kerry, B. R. and Bourne, J. M. (1996) The importance of rhizosphere interactions in the biological control of plant parasitic nematodes-a case study using Verticillium chlamydosporium. Pesticide Science 47: 69-75. Bourne J. M., Kerry, B. R., and Leij, F. A. A. M. de (1996) The importance of the host plant on the interaction between root-knot nematodes ( Meloidogyne spp.) and the nematophagous fungus, Verticillium chlamydosporium Goddard. Biocontrol Science and Technology 6 (4), 539-548. Leij, F. A. A. M. de, Davies, K. G. and Kerry, B. R. (1992) The use of Ver - ticillium chlamydosporium Goddard and Pasteuria penetrans (Thorne) Sayre & Starr alone and in combination to control Meloidogyne incognita on tomato plants. Fundamental and Applied Nematology 15(3), 235-242. Leij, F. A. A. M. de, Kerry, B. R. and Dennehy, J. A. (1993) Verticillium chlamy - dosporium as a biological control agent for Meloidogyne incognita and M. hapla in pot and micro-plot tests. Nematologica, 39: 115-126. | This invention refers to isolation and utilization of the fungus Pochonia chlamydosporia var. chlamydosporia strain PcMR in biological control of nematodes, characterized in that it has a high virulence against Meloidogyne spp. in in-vitro microbiologic tests, pot tests and field tests. In these tests, the strain PcMR, isolated in Portugal, have a good performance in the ability to control Meloidogyne spp. populations and shows a good capacity to produce high amounts of chlamydospores used to produce inoculum and to colonize plant roots that might be infected by Meloidogyne spp. This strain can be utilized as part of biological control methods that effectively control root-knot-nematode populations belonging to genus Meloidogyne and this invention refers also to production and utilization of nematicides based on Pochonia chlamydosporia var. chlamydosporia strain PcMR and any organisms derived from this strain. | big_patent |
Exercise-induced cognitive improvements have traditionally been observed following aerobic exercise interventions; that is, sustained sessions of moderate intensity. Here, we tested the effect of a 6 week high-intensity training (HIT) regimen on measures of cognitive control and working memory in a multicenter, randomized (1: 1 allocation), placebo-controlled trial. 318 children aged 7-13 years were randomly assigned to a HIT or an active control group matched for enjoyment and motivation. In the primary analysis, we compared improvements on six cognitive tasks representing two cognitive constructs (N = 305). Secondary outcomes included genetic data and physiological measurements. The 6-week HIT regimen resulted in improvements on measures of cognitive control [BFM = 3. 38, g = 0. 31 (0. 09,0. 54) ] and working memory [BFM = 5233. 68, g = 0. 54 (0. 31,0. 77) ], moderated by BDNF genotype, with met66 carriers showing larger gains post-exercise than val66 homozygotes. This study suggests a promising alternative to enhance cognition, via short and potent exercise regimens. Funded by Centre for Brain Research. NCT03255499. Possibly the most reliable means to induce cognitive improvements behaviorally, physical exercise has become known for its effects on the brain in addition to its well-documented impact on the body (see for a review Moreau and Conway, 2013). Individuals with higher cardiovascular fitness typically show higher performance on a wide range of cognitive measures, from cognitive control (Pontifex et al., 2011) to working memory (Erickson et al., 2013) and executive functioning (Colcombe and Kramer, 2003; Hillman et al., 2008). In addition, long-term sport practice is associated with higher working memory capacity (Moreau, 2013), spatial ability (Moreau, 2012), and more efficient visual processing of movements (Güldenpenning et al., 2011). In neuroimaging studies, greater fitness indices have also been linked to differences in white matter integrity (Chaddock-Heyman et al., 2014; Voss et al., 2013), as well as with larger hippocampal (Weinstein et al., 2012) and cortical areas (Erickson et al., 2009; Makizako et al., 2015). These results are corroborated by increased long-term potentiation (LTP) in the visual system of physically fit individuals compared to the non-fit (Smallwood et al., 2015), a key finding given the primary role of LTP in major cognitive processes such as learning and memory (Bliss and Collingridge, 1993, Bliss and Lomo, 1973). Importantly, these correlational findings are further supported by longitudinal designs. Exercise interventions can elicit cognitive improvements in diverse populations ranging from children (Davis et al., 2011) to the elderly (Fabre et al., 2002), as well as in individuals with various clinical conditions such as developmental coordination disorders (Tsai et al., 2012) and schizophrenia (Pajonk et al., 2010). Furthermore, improvements appear to be dose-dependent (Davis et al., 2007; Vidoni et al., 2015), and are not restricted to atypical or clinical populations—adults at their cognitive peak show similar benefits (Gomez-Pinilla and Hillman, 2013; Moreau and Conway, 2013; Voss et al., 2011). In school settings, exercise interventions have shown to be associated with higher levels of academic achievement (Coe et al., 2006), and exercise regimens in children typically lead to improvements in various aspects of cognition, including executive function, cognitive control and memory (see for a review Tomporowski et al., 2015a). Interventions implemented in early stages of life allow capitalizing on higher cortical plasticity, potentially maximizing their impact (Cotman and Berchtold, 2002). The appeal of early interventions has motivated a whole line of research exploring the effect of physical exercise regimens on behavior, cognitive function, and scholastic performance (Castelli et al., 2007; Davis et al., 2007, Davis et al., 2011; Donnelly et al., 2016; Jackson et al., 2016; Pontifex et al., 2013). Consistent with these findings, Sibley et al. found a robust effect of physical exercise on cognitive function in children, in a meta-analytic assessment of the literature at the time (Sibley and Etnier, 2003). Most studies in this line of research have evaluated the impact of a rather specific type of regimen, based on aerobic exercise. Usually defined as a sustained regimen performed at moderate intensity (e. g., McArdle et al., 2006), aerobic exercise has come to be accepted as the form of exercise typically associated with neural changes and cognitive enhancement (Hillman et al., 2008; Thomas et al., 2012), for at least two reasons. First, current interventions are rooted in early findings in the animal literature, which typically investigated the effects of physical exercise in rodents—animals who naturally favor aerobic forms of exercise (Gould et al., 1999; Shors et al., 2001). Second, the most dramatic gains in cognition have been observed in the elderly (Erickson et al., 2015; but see also Etnier et al., 2006; Young et al., 2015, for a more nuanced view), a population for which moderate-intensity exercise is seemingly most adequate. Subsequent studies have stemmed from this line of research, therefore expanding the initial paradigm to a wider range of populations. Yet current trends of research suggest other promising directions. For example, regimens based on resistance training have shown sizeable effects on cognition (Best et al., 2015; Liu-Ambrose et al., 2012; van Uffelen et al., 2007), despite underlying mechanisms of improvements being potentially different from those elicited by aerobic exercise (Goekint et al., 2010). More complex forms of motor training that combine high physical and cognitive demands also appear encouraging (Moreau et al., 2015; Tomporowski et al., 2015b). Moreover, a compelling body of research in the field of exercise physiology indicates that interventions based on short, intense bursts of exercise can induce physiological changes that mirror those following aerobic exercise on a wide variety of outcomes. These include measures of cardiovascular function (Chrysohoou et al., 2015; Gayda et al., 2016), overall fitness (Benda et al., 2015), and general health (Milanović et al., 2015). In some cases, physiological improvements following high-intensity training (HIT) can even go beyond those typically following aerobic regimens (Rognmo et al., 2004). For example, HIT appears to be particularly effective to increase the release of neurotrophic factors essential to neuronal transmission, modulation and plasticity—potentially surpassing aerobic exercise regimens (Ferris et al., 2007). This body of research is promising, as it suggests a plausible mechanism by which intense bursts of exercise could meaningfully influence cognitive function and behavior. A few studies have partially tested this idea. Short bouts of exercise have been shown to alleviate some of the difficulties typically associated with Attention-deficit/hyperactivity disorder (ADHD) in children (Piepmeier et al., 2015), demonstrating the potential of this type of intervention to enhance cognitive abilities. The benefits reported in this study were not limited to children diagnosed with ADHD, however—typical children also exhibited cognitive improvements. More strikingly perhaps, Pontifex and colleagues found that a single 20 min bout of exercise was sufficient to improve cognitive function and scholastic performance in children (Pontifex et al., 2013). This is an impressive finding, given that exercise-induced cognitive improvements typically occur after longer training periods (Etnier et al., 2006; Sibley and Etnier, 2003). Importantly, these effects should be distinguished from short-term improvements immediately following acute bouts of exercise (Tomporowski, 2003), which typically dissipate after a few hours. The two types of outcomes (short-term consequences of single acute sessions vs. more durable benefits) are sometimes conflated, resulting in misleading conclusions (Jackson et al., 2016). The hypothesized mechanisms are, however, different—heightened state of alertness induced by neurotransmitter increases for the former (Tomporowski, 2003), and slower but more durable neurophysiological adaptations in the case of the latter (Erickson et al., 2011, Erickson et al., 2015; Moreau and Conway, 2013; Voss et al., 2013). One aspect that remains to be formerly investigated relates to the specific influence of exercise intensity. Although focused on short sessions, the aforementioned studies utilized regimens of moderate intensity—a reported 62–72% (Piepmeier et al., 2015) and 65–75% (Pontifex et al., 2013) of individual maximum heart rate, respectively. Yet based on findings from the physiological literature (e. g., Rognmo et al., 2004), there are clear mechanisms via which exercising at a high intensity could influence cognition in a meaningful manner. Arguably, HIT could elicit improvements above and beyond those typically following short sessions of moderate intensity, and provide a legitimate, time-efficient alternative to longer aerobic exercise regimens (Costigan et al., 2015). Together, the conjunction of promising early findings and clear mechanisms of action has prompted discussions to implement HIT interventions more systematically within the community (Gray et al., 2016). In an effort to better understand and predict individual responses to physical exercise interventions, several studies have investigated the role of specific genetic polymorphisms on the magnitude of exercise-induced improvements (Erickson et al., 2008, Erickson et al., 2013). Among these, many have focused on the brain-derived neurotrophic factor (BDNF) val66met polymorphism, given its direct influence on serum BDNF concentration (Lang et al., 2009). BDNF is known to support neuronal growth and has been shown to facilitate learning, a process that in turn induces BDNF production (Berchtold et al., 2001; Cotman and Berchtold, 2002; Kesslak et al., 1998). This dynamic coupling makes BDNF an important underlying factor of exercise-induced cognitive improvements. Consistent with this idea, it has been proposed that individuals whose particular BDNF polymorphism is associated with lower activity-dependent BDNF levels (met66 carriers) might benefit from exercise interventions to a greater extent than individuals whose activity-dependent BDNF levels are higher (val66 homozygotes). Similarly, a few studies have shown that individuals with lower cardiovascular function might maximize benefits from physical exercise interventions designed to improve cognitive function (Sofi et al., 2011; Strong et al., 2005). The implicit assumption is that although lack of physical exercise might be a limiting factor for individuals whose fitness level is low, more active individuals might be less impacted by an exercise intervention program (Heyn et al., 2004; Lautenschlager et al., 2008; Sniehotta et al., 2006). Despite the intuitive appeal of this assumption, several studies, including one from our group, have failed to find a positive correlation between exercise-induced cognitive improvements and the associated physiological changes (Moreau et al., 2015; Tsai et al., 2014). Arguably, the absence of a clear link between the hypothesized physiological mechanisms of improvements and tangible cognitive gains might stem from the plurality and complexity of variables underlying these changes, rendering coherent associations elusive. Despite similarities in the physiological mechanisms linking aerobic exercise and HIT on cognition, the precise impact of the latter on cognitive performance remains to be confirmed experimentally. In the present study, we tested the viability of HIT as a substitute for aerobic exercise to induce cognitive improvements in school populations. In particular, we postulated that HIT would result in improvements in measures of cognitive control and working memory, as both constructs have been linked to fitness levels (Pontifex et al., 2011) and appear to be malleable via aerobic regimens (Erickson et al., 2013). The choice of these constructs was also motivated by previous research showing the malleability of both cognitive control and working memory in training studies, thus providing theoretical and empirical support for the plausibility of expected improvements (Hampshire et al., 2012; Mishra et al., 2014). Consistent with recent efforts to better model and understand the mechanisms of cognitive improvement (Young et al., 2015; Moreau and Waldie, 2015), the present study also intended to address interindividual variability so as to isolate the underlying factors of improvement. Based on previous literature (Erickson et al., 2013; Moreau et al., 2015), we hypothesized that exercise training would elicit substantially larger cognitive benefits in individuals whose cardiovascular fitness is low, and in BDNF met66 carriers, whose activity-dependent BDNF levels are naturally limited. Finally, we expected physiological improvements with exercise, as typically induced from aerobic interventions (see for a review Gomez-Pinilla and Hillman, 2013). Participants in the exercise group saw a greater decrease in resting heart rate than controls, as demonstrated by a Bayesian ANCOVA with Condition (HIT vs. Control) as a fixed factor and baseline heart rate as a covariate. The full model was preferred to the model with baseline resting heart rate only: BFM = 40. 45, and was the most likely given our data: P (M | Data) =0. 93, assessed from equal prior probabilities (Figure 1A). In-depth analyses focused on individuals with elevated resting heart rate at baseline allowed further insights into the potency of our exercise intervention. Specifically, a Bayesian t-test on resting heart rate change showed a sizeable difference between the two groups, with larger gains for the HIT group (BF10 = 3. 47, with Mgain = 6. 11, SDgain = 11. 64 and Mgain = 1. 89, SDgain = 8. 63, for HIT and Control, respectively; Hedges’ g = 0. 41 (0. 09,0. 73). Test-retest reliability—assessed via a comparison between pretest and posttest resting heart rate for controls only—was acceptable (r = 0. 77, BF10 = 1. 75 e+51). Physiological data also provided important indications about workout intensity idiosyncrasies. We used resting heart rate at pretest to determine target intensities for each individual, such that: (1) HRTarget=HRReset+δ (HRMax−HRRest) where HRMax = 220 Age, and δ is set to. 80. This yielded an individual target range (HRTarget or above) while exercising. We then compared this range with the maximum intensity measured during each workout, to obtain an index of accuracy, or agreement, between target zone and actual effort. Results showed that participants did exercise at a suitable intensity overall, as expressed by the deviation from individual target heart rate values (MDev = 1. 65, SDDev = 5. 87; Figure 1B). Importantly, effort intensity was maintained stable across time, as demonstrated by moderate evidence favoring the null model over an alternate model that included time as a predictor of maximum heart rate in a Bayesian linear regression analysis [BFM = 2. 87, P (M | Data) =0. 74, Figure 1C]. Because individual resting heart rates tended to decrease throughout the intervention, sustained effort indicates that individuals incrementally increased workout volume, which was confirmed by additional measures such as step count [BFM = 2. 979e + 10, P (M | Data) ≈ 1, for the model that included Session as a covariate, Figure 1D]. Together, these results support the notion that the intervention was adaptive, allowing workout intensities tailored to each individual. Physiological improvements are informative in two key aspects: they provide corroborating evidence for the hypothesized changes associated with exercise, and they allow identifying idiosyncratic parameters often characteristic of training interventions. However, the main goal of a cognitive intervention is to elicit cognitive gains, which were the primary outcomes of the present intervention. In the following sections, we first identify latent constructs from cognitive assessments, before discussing the impact of the intervention on these two constructs. An exploratory factor analysis using principal component extraction and promax rotation was performed on all six cognitive measures at pretest. Although less common than orthogonal rotations, oblique rotations such as promax allow factors to correlate; this property is especially appropriate when the factors extracted are assumed to be correlated to some degree−a reasonable assumption given our design. The corresponding scree plot and eigenvalues (i. e. the variance in all variables accounted for by each factor) suggested a two-component solution (see factor loadings in Table 1 and Table 1—source data 1 and 2). Subsequent test of the two-factor model confirmed that the number of factors was sufficient (χ2 (4) =0. 59, p=0. 96; Bayesian Information Criterion, BIC = −22. 05). We refer to these two components hereafter as Cognitive Control and Working Memory. The correlation between the two factors was r = 0. 32. Uniqueness values indicated that the tasks spanned an adequate range within the sample space of each construct (Table 1). Here, we report cognitive improvements broken down by constructs, defined based on the factors extracted from the exploratory factor analysis. A Bayesian repeated measures ANOVA on Cognitive Control scores, with Session (pretest vs. posttest) as a within factor and Condition (HIT vs. Control) as a between factor, showed moderate evidence for the interaction model over the main effect model [BFM = 3. 38, p (M | Data) =0. 46; Table 2]. Participants in the HIT group showed larger improvements than controls from pretest to posttest (Mgain = 0. 25, SDgain = 0. 6 and Mgain = 0. 08, SDgain = 0. 47, respectively, Hedges’ g = 0. 31 [0. 09,0. 54]; Figure 2A, see also Figures 4–6). A Bayesian repeated measures ANOVA on Working Memory scores, with Session (pretest vs. posttest) as a within factor and Condition (HIT vs. Control) as a between factor showed strong evidence for the interaction model over the main effect model [BFM = 5233. 68, p (M | Data) ≈1; Table 3]. Participants in the HIT group showed larger improvements than controls from pretest to posttest (Mgain = 0. 48, SDgain = 0. 83 and Mgain = 0. 12, SDgain = 0. 44, respectively, Hedges’ g = 0. 54 [0. 31,0. 77]; Figure 2B). Because the cognitive improvements we reported are presumably based on physiological changes, we directly tested the relationship between the two types of variables. A Bayesian regression analysis showed that change in resting heart rate was a reliable predictor of cognitive gains in the HIT group, with respect to Cognitive Control (BF10 = 6. 34, p (M | Data) =0. 86). This was not the case in the Control group (BF10 = 0. 20, p (M | Data) =0. 16). The contrast was weaker when comparing Working Memory gains in the HIT group (BF10 = 0. 56, p (M | Data) =0. 36) with those of the Control group (BF10 = 0. 18, p (M | Data) =0. 15). Additional Bayesian regression analyses showed that lower resting heart rate at pretest did not predict improvements in either Cognitive Control or Working Memory in the HIT group (BF10 = 0. 41, p (M | Data) =0. 29. and BF10 = 0. 18, p (M | Data) =0. 15, respectively). This was also the case when the analyses were restricted to the. 75 quantile of individuals with the lowest resting heart rate at baseline (BF10 = 0. 34, p (M | Data) =0. 25. and BF10 = 0. 62, p (M | Data) =0. 38, respectively). Overall, baseline resting heart rate was a fairly noisy measure (M = 85. 2, SD = 14. 77, over the entire sample) and this might have contributed to the lack of clear impact of resting heart rate change on cognitive function. A subsample of our data allowed for a better understanding of individual differences in exercise-induced cognitive improvements. Specifically, we looked at the effect of variations in the BDNF polymorphism on cognitive gains in the HIT group, via a comparison between met66 carriers (i. e. met66/ met66 or val66/ met66) and non-carriers (val66 homozygotes). Separate Bayesian repeated measures ANOVAs on Cognitive Control and Working Memory scores, with Session (pretest vs. posttest) as a within factor and BDNF polymorphism (val66 homozygotes vs. met66 carriers) as a between factor showed strong evidence for the interaction model in both cases [BFM = 31. 17, p (M | Data) =0. 89, and BFM = 675. 92, p (M | Data) =0. 99, for Cognitive Control and Working Memory, respectively, see Table 4 and Table 5]. These findings suggest that met66 carriers benefited to a greater extent than non-carriers from the exercise intervention (Cognitive Control: Mgain = 0. 93, SDgain = 1. 20 and Mgain = 0. 05, SDgain = 0. 13, Hedges’ g = 1. 36 [0. 52,2. 2]; Working Memory, Mgain = 0. 87, SDgain = 0. 64 and Mgain = 0. 14, SDgain = 0. 24, Hedges’ g = 1. 83 [0. 94,2. 72]; Figure 3). Unequal baseline scores cannot fully account for this effect since evidence for differences in Cognitive Control was limited at pretest (BF10 = 4. 03, Error (%) =1. 55 e −6 from a Bayesian independent samples t-test) and more substantial, but unable to account for the full effect, for Working Memory (BF10 = 17. 47, Error (%) =1. 22 e −6 from a Bayesian independent samples t-test). Together, these findings indicate that although genetic variations in the BDNF polymorphism are associated with cognitive differences, the latter are malleable and can be reduced with physical exercise. All priors used in the reported analyses are default prior scales (Morey and Rouder, 2015). For Bayesian repeated measures ANOVA and ANCOVA, the prior scale on fixed effects is set to 0. 5, the prior scale on random effects to 1, and the prior scale on the covariate to 0. 354. The latter is also used in Bayesian Linear Regression. The Bayesian t-test uses a Cauchy prior with a width of √2/2 (~0. 707), that is half of parameter values lies within the interquartile range [−0. 707; 0. 707]. It is worth pointing out that the Bayesian repeated measures ANOVA that showed only moderate evidence for the effect of our HIT intervention on Cognitive Control shows stronger evidence with a slight variation on the prior scale. Although this variation in priors is consistent with our data and provides stronger evidence for our claim, we chose to report analyses with default prior scales, as these were the intended parameters a priori. For transparency, we plotted below the prior and posterior distribution for the comparison between Conditions (HIT vs. Control) for Cognitive Control (Figure 4), as well as the Bayes Factor robustness check (Figure 5). Both indicate that our findings are robust and supported by a wide range of priors, as corroborated by a sequential analysis (Figure 6). Broadly speaking, MCMC methods approximate the true posterior density p (θ | y) by constructing a Markov chain on the state space θ ∈ Θ. The probability of the subsequent state in a given chain can be defined as: (2) P (Xn+1=in+1|Xn=In), I ∈ θwhere {X0, X1,.. } is a sequence of random variables and θ is the state space. Accordingly, the state at time step n + 1 is dependent only on the state at time n. This process is best represented with a random walk where each vertex is defined by θ, and weighted by the transition probabilities: (3) pij=P (Xn+1=j|Xn=i), i, j∈θ In the analyses reported in the paper, MCMC was used to generate posterior samples via the Metropolis-Hastings algorithm (see for details Rubinstein and Kroese, 2011). All analyses were set at 10,000 iterations, with diagnostic checks for convergence. One chain per analysis was used for all analyses reported in the paper, with a thinning interval of 1 (i. e., no iteration was discarded). We reported Bayesian analyses throughout the paper. Because we understand that some readers may wish to compare these results with the equivalent frequentist analyses, we are providing all of these herein, in the order of presentation in the paper. Note that an a priori power analysis based on previous studies (Erickson et al., 2013; Moreau et al., 2015) indicated the need for a minimum N of 129 participants per group to detect an effect of d = 0. 35 on the primary outcome measures, with 1 – β = 0. 80 and α = 0. 05. The actual sample size of the present study (N = 152 and N = 153, for HIT and control groups, respectively) allowed an a priori power of. 86, given d and α constant. We present below analyses for each cognitive tasks included in this study. Descriptive statistics are reported in Table 6. Here, we report analyses for which our a priori hypotheses were null effects. These variables were collected either to control for potential confounds, or for exploratory purposes. There was no difference between groups regarding self-reported enjoyment or motivation (W = 12058, p=0. 54 and W = 11497, p=0. 86, respectively). This finding allows controlling for expectation effects, and thus stronger causal claims (Boot et al., 2013; Stothart et al., 2014). In addition, participants’ self-reported belief about cognitive malleability (i. e., mindset) indicated a statistically significant difference (p<0. 03) in favor of the control group (M = 7. 11, SD = 2. 65 and M = 6. 42, SD = 2. 74, respectively, Hedges’ g = 0. 26 [0. 03,0. 48]). There was no statistically significant difference between groups at either time points (pretest or posttest) in terms of ethnic background, age, gender, handedness, height, weight, diagnosis of learning disorder, brain trauma or epileptic seizures, current or past enrolment in a remediation or a cognitive training program, English as first language, videogaming experience, physical exercise, self-reported happiness, sleep quality, or general health. The present study reported the first experimental evidence that HIT can elicit robust cognitive improvements in children. We confirmed the main hypothesis that exercise could induce gains in both cognitive control and working memory, as assessed from multiple measures. This finding is particularly promising given that the two constructs are reliable predictors of success in many domains, including professional and academic (Deary et al., 2007); in the classroom, cognitive control and working memory have been associated with effective learning and overall achievement (Rohde and Thompson, 2007). Importantly, these effects are also meaningful at the level of single tasks. Our main findings thus emphasize the potency of short but intense exercise interventions to enhance cognition, and suggests that aerobic exercise is not the sole means to elicit cognitive gains, in line with a growing body of research (Liu-Ambrose et al., 2012; Moreau et al., 2015; Pesce et al., 2016; Tomporowski et al., 2015b). HIT appears to be a viable and promising alternative to longer workouts to enhance cognition. In addition to the main effect of training, we also postulated that specific genetic profiles would be correlated with different responses to training, with BDNF met66 carriers (i. e. met/met or val/met) benefiting from exercise to a greater extent. This hypothesis, confirmed by the present findings, was based on previous literature showing a relationship between BDNF polymorphism and serum BDNF (Lang et al., 2009), and the influence of exercise interventions on the latter (Leckie et al., 2014). BDNF met66 carriers showed greater gains from pretest to posttest on both cognitive constructs. As the substitution from valine to methionine at codon 66 typically results in decreases of activity-dependent secretion of BDNF at the synapse (Egan et al., 2003), BDNF met66 carriers are thought to be particularly impacted by post-exercise BDNF increases (Nascimento et al., 2015). Conversely, val66 homozygotes might benefit less from BDNF increases given above-average baseline levels. This finding is interesting because of its predictive power−controlling for BDNF polymorphism can allow more accurate forecasting of individual training responses, and better estimates of effect size. Given the current trend toward more personalized interventions (Medalia and Richardson, 2005; Moreau and Waldie, 2015), factoring in genetic information has the potential to refine and improve regimens, for each individual. Importantly, the main effect of exercise on cognitive function is unlikely to be explained by the placebo effect. Self-reported measures of enjoyment and motivation did not differ between groups, supporting the notion that both types of intervention were equally appealing to children, or at least not fundamentally different with respect to intrinsic motivating factors. In addition, we also controlled for mindset—the degree to which individuals believe their cognitive abilities can change over time (Dweck et al., 1995). Previous research suggests that individual mindsets may be of influence in cognitive growths: individuals with a more ‘malleable’ mindset are thought to be more likely to improve in cognitive and academic domains than individuals with a ‘fixed’ mindset (Paunesku et al., 2015). Although we did find a difference between conditions, it was to the advantage of children in the control group, who held a more malleable view about the dynamic properties of cognitive function than did the HIT group. This finding was reported as an additional analysis in the Results section, rather than in the main section, because the effect was not hypothesized a priori. In any case, and with warranted caution regarding post hoc interpretation, this effect would suggest that controls were more likely to improve over time, an assumption that was not corroborated by our main finding. If anything, this strengthens our main claim—greater improvements in the HIT condition are inconsistent with a differential placebo effect, a critical point in light of recent findings in the field of cognitive training (Foroughi et al., 2016). Effort-dependent variables provided additional insight about the mechanism underlying improvements. Consistent with previous interventional studies that have investigated the influence of exercise on cognition, we actively monitored several variables throughout the intervention. We could thus ensure that training was adequate, performed at a suitable intensity, and could test directly the dynamic coupling of these variables and their effects on cognitive outcomes. Indeed, we found that almost all participants stayed within the overall targeted range of effort required by the design of the workout and by initial individual measurements. This indicates a high degree of agreement, or fidelity, to the intended protocol—an essential component of the intervention given the underlying assumption that participants would exercise at a high intensity. Performed at a more moderate intensity, the same regimen becomes an aerobic training program, for which substantially longer time commitment might be required to elicit improvements. In addition, it is important to note that participants incrementally increased workout load, thus maintaining appropriate intensity throughout the intervention. Arguably, this adaptive property emerged from the design of the intervention, whereby participants were encouraged to exercise at maximum intensity at the time of the workout—an intrinsically individual and dynamic variable by definition. In the present study, the effect of exercise could also be appreciated on changes in resting heart rate from pretest to posttest, a finding that confirms previous research in the field (e. g., Moreau et al., 2015). More specifically, exercise appears to be a valuable regulating mechanism, with elevated resting heart rate values likely to normalize as a result of HIT. This effect could not be attributed to regression toward the mean, as it was not found in controls, thus suggesting that the intervention is especially beneficial to individuals who need it most. Together with the finding that HIT benefited more individuals with a genetic polymorphism (BDNF met66 carriers) that was associated with lower cognitive performance, this idea emphasizes the relevance of exercise interventions to individuals with specific genetic or physiological attributes to reduce interindividual differences (Gómez-Pinilla et al., 2001; Leckie et al., 2014). Disparities are genuine, yet targeted interventions allow low-performing individuals to improve dramatically. Our design allowed delving deeper into the relationship between exercise and cognitive improvements. In particular, we found that change in resting heart rate was predictive of cognitive control, but not working memory, gains. This finding is of interest because we postulated that the mechanisms of improvement were physiological, given that HIT has been shown to elicit neurophysiological changes similar to those following aerobic exercise regimens (Ferris et al., 2007). However, we should point out that baseline resting heart rate did not predict gains in cognitive control or working memory in the HIT group, emphasizing the inherent noise associated with the relationship between physiological and cognitive measures. This lack of clear association between both types of variables is fairly common in the literature, suggesting that the hypothesized mechanisms of improvements are difficult to elucidate (Moreau et al., 2015; Tsai et al., 2014). Potentially, this also suggests that other variables may be of importance, a question we attempted to address with additional measurements, in an exploratory manner (see Additional Analyses in the Results section). Regardless of the strength of the evidence reported in the present study, one might question the genuine impact of such a short training regimen. Although perhaps counterintuitive, the extreme potency of short, intense bursts of exercise has come to light in recent years (Lucas et al., 2015; Rognmo et al., 2004). In a review of the impact of HIT on public health, Biddle and Batterham, 2015 go as far as to premise their entire argument on the idea that the cardio-metabolic health outcomes are not to be questioned—rather, their only concern was about whether or not such regimens can be widely implemented and sustained over time (Biddle and Batterham, 2015). In a similar vein, Costigan and colleagues concluded that HIT is a time-efficient approach for improving cardiorespiratory fitness in adolescent populations (Costigan et al., 2015). These are strong, unequivocal statements, which reflect current views in exercise physiology—HIT has tremendous health benefits, with little, if any, disadvantages. More direct evidence for the impact of HIT on brain function comes from neurophysiological studies. In an experiment that directly assessed the impact of short bursts of exercise on BDNF levels, Ferris et al. found that exercise leads to BDNF increases, and that the magnitude of the increase is intensity-dependent (Ferris et al., 2007). This result emphasizes the importance of controlling exercise intensity in HIT studies, given that the main determinant of improvement appears to be the intensity of the workout. Indeed, previous studies have looked at the effect of short, but not intense, bursts of exercise on cognition, and found no clear evidence of improvement (Craft, 1983). The high-intensity component of this type of exercise regimen is intended to allow for higher workout intensity than traditional workouts, despite shorter overall volume. The brevity of exercise, on the other hand, is simply a byproduct of intensity—one cannot maintain a near-maximal exercise intensity for long periods of time, given that this type of regimen depletes energetic resources rapidly (Parolin et al., 1999). In terms of practical implications, this aspect is critical, as it allows designing shorter, more potent workouts. Despite our findings being in line with previous literature showing that short bursts of exercise can elicit potent cognitive improvements in children (Piepmeier et al., 2015; Pontifex et al., 2013), and, more generally, with a wider, more general literature linking physical exercise and cognition in children (Jackson et al., 2016; Sibley and Etnier, 2003), we should also point out a few limitations of the present study. These open up interesting avenues and directions for future research. First, the duration of training was not experimentally manipulated, and therefore the specific question of dose-dependence was not directly assessed. Therefore, we cannot claim that a 6 week regimen is optimal—larger cognitive improvements could possibly be elicited with longer durations, or, alternatively, similar improvements could be induced with a shorter intervention. Similarly, no follow-up tests were performed to assess durability of improvements post-training; although it should be noted that maintaining benefits is arguably less important with short, potent interventions such as the HIT regimen we proposed. In addition, both the duration of the intervention and the specific experimental protocol were constrained by external factors (e. g. feasibility, academic schedule). With respect to the potency of the intervention, however, this is also promising—as little as six weeks of training can induce noticeable improvements, with possible larger effects if physical exercise is sustained. Related to this idea, we had to work around constraints typically imposed by interventional studies; namely, the necessity to keep testing sessions time-efficient. Beyond logistic considerations, this was also intended to minimize the influence of cognitive fatigue on our results. Despite time constraints, we aimed to preserve testing diversity (i. e., number of tasks per construct) for a few reasons. First, we strived to provide estimates of constructs that minimize task-specific components and extract meaningful domain-general scores. In psychometric testing of working memory capacity, Foster and colleagues demonstrated that the majority of the variance explained by a single WM task is accounted for in the first few blocks, and that the predictive nature of the task remains largely unchanged for practical purposes when tasks are shortened (Foster et al., 2015). Second, simulation studies have shown that incorporating more tasks within constructs leads to a better signal-to-noise ratio, resulting in more meaningful measures of an underlying ability (Moreau et al., 2016). To further validate this approach, we piloted different versions of our testing tasks and determined that the validity of the versions we retained for the present study was acceptable, given appropriate reliability across task lengths. Second, because training occurred in schools, the environment was possibly less standardized and controlled than laboratory settings, highlighting typical tradeoffs between impoverished but highly reliable environments and ecological but less controlled settings. Our view remains that ecological validity is of primary importance when training cognitive abilities, because of the wide range of applications stemming from this line of research (Moreau and Conway, 2014). Accordingly, fixed, predictable training regimens are unlikely to favor durable improvements of cognitive function (Moreau and Conway, 2014; Moreau et al., 2015; Posner et al., 2015). We have previously stressed the importance of novelty and variety in cognitive training interventions (Moreau and Conway, 2014), and this limitation applies to exercise regimens as well. Importantly, this echoes similar views across research groups worldwide, which suggest that training-induced cognitive improvements are often restricted to specific activities (Harrison et al., 2013; Tomporowski et al., 2012), and are best nurtured within complex, dynamic environments (Diamond and Lee, 2011). Therefore, the regimen we have presented in this paper constitutes a potent short-term intervention, but more variety might be required to elicit long-lasting improvements. In a field that has suffered from setbacks such as lack of replication (e. g. Redick et al., 2013; Thompson et al., 2013) or common methodological flaws (Moreau et al., 2016), it is wise to remain cautious about preliminary studies and emphasize the need for replication. Despite these limitations, the present findings represent a promising first step toward reliable and affordable exercise-based cognitive interventions, highlighting effective alternatives to aerobic exercise. Together with complementary findings (e. g. Moreau et al., 2015), the type of physical exercise regimen we described in this paper could pave the way for novel exercise interventions particularly suited to school environments, which are often constrained by time and equipment. The high-intensity workout we designed did not require any special equipment or any instructors training, and each 10 min session was all-inclusive with warm-up and stretching. Therefore, this type of regimen could also be generalized to other populations; for instance, individuals whose schedule allows little time for exercise, or those who do not intrinsically enjoy exercising, could appreciate opportunities to shorten workouts while preserving the typical benefits of exercise. Adaptations to older populations also represent interesting opportunities considering the benefits typically associated with exercise regimens in these communities (Colcombe and Kramer, 2003). The present regimen might require adjustments, given that practicality in a specific context (e. g. managing time constraints in school or professional schedules) potentially differs from practical considerations in another (e. g. mitigating risks of injury in older populations). In any case, generalization is of the rationale, not necessarily of the specific workout that was designed for this intervention. Finally, it is important to acknowledge that physical exercise, regardless of the specific training regimens considered, is not a panacea when it comes to addressing cognitive deficits−in some cases, especially in the presence of specific conditions or disorders, more targeted or individualized interventions might be required (e. g., Moreau and Waldie, 2015), and the ability for exercise regimens to remediate core cognitive deficits might appear inherently limited. However, it remains that physical exercise is one of the most potent and wide-ranging means currently available to enhance cognition non-invasively, with a myriad of positive side effects. A total of 318 children participated in this study. Thirteen participants were not included in the analyses because of dropouts (N = 7), extensive missing data (N = 4) or problems in data collection (N = 2, see CONSORT flow diagram for details). Our final sample consisted of 305 children (Mage = 9. 9 (7–13), SDage = 1. 74,187 female, MBMI = 18. 3, SDBMI = 6. 26). They were recruited from six schools across New Zealand, providing a sample of various socioeconomic deciles (three public institutions, three private), locations (three urban institutions, three rural), and ethnic backgrounds representative for the country (70% New Zealand European, 20% Pacific, 7% Asian, 3% Other). The number of students involved per school ranged from 5 to 83 (M = 50. 8, SD = 31). All participants reported no history of brain trauma or epilepsy, and all had self-reported normal or corrected-to-normal vision. A subset of 22 children reported a learning disability diagnosis (dyslexia: 14, ADHD: 3, Autism spectrum disorder: 3, mild developmental delay: 2, Irlen syndrome: 2, dyscalculia: 1, dyspraxia: 1). Respective subsets of 284,99 and 32 participants underwent all assessments, measurements and genotyping described below. All the variables measured in the experiment are reported hereafter. Testing was conducted on school premises. All cognitive assessments were computer-based, administered in groups of a maximum of 15 students. This limit on the number of participants tested at a given time was implemented to minimize potential averse effects of group testing. These assessments have shown to be adequate measures of both cognitive control and working memory (Anderson-Hanley et al., 2012; Aron and Poldrack, 2005; Kane et al., 2004; Nee et al., 2007; Pajonk et al., 2010; Rudebeck et al., 2012; Unsworth and Engle, 2007). For each task, we measured accuracy and response time. Different stochastic variations of all tasks were used at pretest and posttest. Unless specified otherwise, the number of trials varied based on individual performance to allow reaching asymptotes, with a minimum and a maximum specified for each task. The reliability of this method for each task was assessed from a separate sample (N = 34, Mage = 10. 3 (8–12), 15 females), and deemed acceptable (all ρs > 0. 65) based on Spearman-Brown prophecy formula (Brown, 1910; Spearman, 1910). Specifically, reliability was calculated by comparing test scores on the asymptotic version vs, the maximal-length version, for each task (see trial length details below and online repository for source code data). The order below was the order of presentation for every participant at both pretest and posttest (i. e., Flanker – Go/no-go – Stroop – Backward digit span – Backward Corsi blocks – Visual 2-back). Both testing sessions were scheduled at the same time of the day, and lasted approximately one hour. Physiological measures were collected using FitbitChargeHRTM, powered by the MEMS tri-axial accelerometer. This multisensory wristband has shown adequate accuracy and reliability in previous studies for the measures of interest in the present study (e. g., de Zambotti et al., 2016). Measures included minutes of activity, calories burned, intensity, intensity range (sedentary, lightly active, fairly active, very active), steps and heart rate (measured by changes in blood volume using PurePulseTM LED lights). Participants provided information about the following: ethnic background, age, gender, handedness, height, weight, diagnosis of learning disorder, brain trauma or epileptic seizures, current or past enrolment in a remediation or a cognitive training program, and whether English was their first language. In addition, self-reported information was gathered to quantify videogaming and physical exercise habits (4-point Likert scale in both cases), as well as to evaluate overall health, happiness, sleep quality, and mindset (6-point Likert scale for each item). The latter was intended to capture beliefs about the malleability of cognitive ability in the context of schoolwork, that is, the extent to which students perceive academic achievement in a predominantly fixed or malleable manner (see for example Paunesku et al., 2015). All measures were collected prior to the intervention, but variables susceptible to change over time were reassessed post-intervention. DNA collection was performed using Oragene-DNA Self-Collection kits, in a manner consistent with the manufacturer' s instructions. DNA was subsequently extracted from all saliva samples according to a standardized procedure (Nishita et al., 2009). All resultant DNA samples were resuspended in Tris-EDTA buffer and were quantified used Nanodrop ND-1000 1-position spectrophotometer (Thermo Scientific, Waltham, MA, USA). DNA samples were diluted to 50 ng/μL. A modified version of the method described by Erickson et al., 2008 was used for DNA amplification. Amplification was carried out on the 113 bp polymorphic BDNF fragment, using the primers BDNF-F 5-GAG GCT TGC CAT CAT TGG CT-3 and BDNF-R 5-CGT GTA CAA GTC TGC GTC CT-3. Polymerase chain reaction (PCR) was conducted using 10X Taq buffer (2. 5 L μL), Taq polymerase (0. 125 μL), dNTPs (5 nmol), primers (10 pmol each), Q solution (5 μL), and DNA (100 ng) made up to 25 μL with dH2O. The PCR conditions consisted of denaturation at 95°C for 15 min, 30 cycles on a ThermoCycler (involving denaturation at 94°C for 30 s, annealing at 60°C for 30 s, and extension at 72°C for 30 s) and a final extension at 72°C. PCR product (6. 5 μL) was incubated with Pm1l at 37°C overnight. The digestion products were analyzed using a high-resolution agarose gel (4%) with a Quick Load 100 bp ladder (BioLabs) and a GelPilot Loading Dye (QIAGEN). After immersion in an ethidium bromide solution for 10 min, DNA was visualized under ultraviolet light. Enzyme digestion resulted in a 113 bp fragment for the BDNF met66 allele, and 78 and 35 bp fragments for the val66 allele. This procedure is consistent with the one described by Erickson et al. (2008). Participants were randomly assigned to either an exercise group (N = 152) or a control (N = 153) group (see Table 7). Randomization was computer-based, generated in R (Core Team R, 2016) by one of the authors (D. M.). Group allocation was performed at the individual level. Testers were blind to group allocation. The exercise intervention consisted of a high-intensity workout including the following: warm-up (2 min), short bursts (5 × 20 s, interleaved with incremental breaks (30 s, 40 s, 50 s, 60 s, and a shorter 20 s break after the last workout period), and stretching (2 min). The video-based workout did not require previous experience or knowledge, as it included basic fitness movements. All movements were designed so that participants could maintain their gaze fixed on the screen at all times. All instructions were provided both verbally (audio recording) and visually (on-screen captions). Complete details and script can be found in the online repository. A complete session lasted 10 min, and was scheduled every morning on weekdays. The control condition consisted of a blend of board games, computer games, and trivia quizzes, consistent with current recommendations regarding active control groups (Boot et al., 2013) and findings showing that aerobic exercise interventions typically do not differ from other regimens with respect to participants’ expectations (Stothart et al., 2014). Consistent with this assumption, self-reported feedback indicated no difference in enjoyment or motivation between conditions, and no difference in mindsets regarding cognitive malleability (Paunesku et al., 2015). Frequency and duration were matched between conditions. The intervention was 6 weeks long, with five sessions per week, for a total of 30 sessions. This translates to 300 min of actual exercise. There was no difference between groups regarding the number of sessions completed (M = 29. 05, SD = 1. 63, overall). Due to the nature of the intervention, class size was limited to 20 participants in both conditions. Participants were supervised at all times, to ensure a high degree of fidelity to the intended protocol. Participants did not differ between groups in any of the self-reported measures described previously, which include physical exercise habits. Note that participants did not exercise on the days of pretest and posttest, to prevent acute effects of physical exercise on cognitive performance (see Tomporowski, 2003). | Exercise has beneficial effects on the body and brain. People who perform well on tests of cardiovascular fitness also do well on tests of learning, memory and other cognitive skills. So far, studies have suggested that moderate intensity aerobic exercise that lasts for 30 to 40 minutes produces the greatest improvements in these brain abilities. Recently, short high-intensity workouts that combine cardiovascular exercise and strength training have become popular. Studies have shown that these brief bouts of strenuous exercise improve physical health, but do these benefits extend to the brain? It would also be helpful to know if the effect that exercise has on the brain depends on an individual's genetic makeup or physical health. This might help to match people to the type of exercise that will work best for them. Now, Moreau et al. show that just 10 minutes of high-intensity exercise a day over six weeks can boost the cognitive abilities of children. In the experiments, over 300 children between 7 and 13 years of age were randomly assigned to one of two groups: one that performed the high-intensity exercises, or a 'control' group that took part in less active activities - such as quizzes and playing computer games - over the same time period. The children who took part in the high-intensity training showed greater improvements in cognitive skills than the children in the control group. Specifically, the high-intensity exercise boosted working memory and left the children better able to focus on specific tasks, two skills that are important for academic success. Moreau et al. further found that the high-intensity exercises had the most benefit for the children who needed it most - those with poor cardiovascular health and those with gene variants that are linked to poorer cognitive skills. This suggests that genetic differences do alter the effects of exercise on the brain, but also shows that targeted exercise programs can offer everyone a chance to thrive. Moreau et al. suggest that exercise need not be time consuming to boost brain health; the key is to pack more intense exercise in shorter time periods. Further work could build on these findings to produce effective exercise routines that could ultimately form part of school curriculums, as well as proving useful to anyone who wishes to improve their cognitive skills. | lay_elife |
For the Record: For Trump, everything’s going to be alt-right Donald Trump gives a thumbs-up following his speech during a campaign rally in Austin, Texas, on Aug. 23, 2016. (Photo: Suzanne Cordeiro, AFP/Getty Images) On Thursday, the term “alt-right” entered the 2016 spotlight like never before. First, Hillary Clinton put out an ad tying Donald Trump to the alt-right. Then Trump denied he was alt-right. Then Clinton, that afternoon, called Trump alt-right for an entire speech. So, what does “alt-right” mean, anyway? It’s an online movement of white people—young white guys, mostly—including white supremacists, nationalists, nativists and plain ol’ racists. They’re anxious. They’re afraid. They worry whites are losing power and influence in America, and so they generally oppose immigration and multiculturalism. They post zany memes on Twitter and Reddit, hate “political correctness” and love the word “cuck” (short for “cuckold”), their sick burn for establishment Republicans. They also love Breitbart News, declared “the platform for the alt-right” last month by then-chairman, Steve Bannon. Bannon, of course, is now Trump’s campaign CEO. And that’s where our story begins... It’s For the Record: the politics newsletter from USA TODAY. Clinton: Trump’s embracing racists. Trump: You mean these decent people? CLOSE Hillary Clinton criticized Donald Trump today for embracing radical elements of the right. USA TODAY Clinton’s Thursday speech marked her harshest attack on Trump since July’s Democratic convention. She called him a bigot, one who “buys so easily into racially-tinged rumors” and let “a radical fringe take over the Republican party.” Trump “built his campaign on prejudice and paranoia,” Clinton said. As a prelude to her speech, Clinton’s camp dropped a new ad that morning highlighting KKK members and white nationalists who support Trump. “The reason a lot of Klan members like Donald Trump is because a lot of what he believes, we believe in,” a man in a KKK robe says in the ad. Trump called the ad shameful. He accused Clinton of hurling insults to deflect from a recent batch of embarrassing emails that sparked new questions about her ethics while secretary of state. "She lies, and she smears, and she paints decent Americans — you — as racists,” he told supporters. Mark Burns, an African-American pastor and Trump surrogate, blasted Clinton for “tying the Trump Campaign with horrific racial images.” Except Trump ties himself to racial controversy, too: He has regularly retweeted white supremacists throughout his campaign, giving their voices a platform to his 11 million followers. During a week in January, more than half of Trump’s retweets were white supremacists praising him, as New York magazine noted. The spotlight on Trump’s ties to white supremacists and/or the alt-right comes at a less than ideal time: He’s trying to win over African-American voters at present, roughly 1% of whom support him. Trump on immigration: between a border wall and a hard place Donald Trump holds a sign a supporter brought showing his support for Hispanic voters during a campaign stop in Louisville in March. (Photo: Pat McDonogh, The (Louisville) Courier-Journal) Now that we’re in the general, Trump needs Hispanic voters, too. Yet analysts have suggested that Trump’s description of undocumented Mexicans as “rapists,” paired with calls to deport 11 million immigrants and build a giant wall at the U.S.-Mexico border might complicate things. So Trump has recently softened his stance on immigration. Or is he just softening how he talks about it? Our own Eliza Collins compared Trump’s past and present immigration statements and … it’s not clear. In the past, Trump promised a full deportation, saying undocumented immigrants “will leave, they’re going to go back to where they came from.” This week, he said the U.S. should “work with them,” suggesting some undocumented immigrants could stay if they paid taxes. When asked on CNN whether Trump was shifting his immigration stance, campaign manager Kellyanne Conway said it remained “what it’s always been.” Either way, Trump’s put himself in a pickle: If he’s not actually shifting his immigration stance, he could be seen as blatantly pandering among Hispanic voters. If he is actually shifting his immigration, then he could be considered what his alt-right supporters call “a cuck.” Related: 76% of Americans believe undocumented immigrants are “as hard-working and honest as are U.S. citizens,” a new Pew poll found, and 67% said they were “no more likely than citizens to commit serious crimes.” More from the campaign trail Pro-immigration groups to Trump: You’re too late, buddy (USA TODAY) The Republicans are better off if Trump loses, but not by too much.’ (USA TODAY) A whole lotta conflicts of interest with the Clinton Foundation if Clinton wins (USA TODAY) ‘We haven’t seen a modern presidential campaign that is so lopsided in terms of advertising.’ (USA TODAY) New Quinnipiac poll: Clinton tops Trump by 10 points nationally (USA TODAY) CUCKS DIDN’T START THE FIRE! Of course they didn’t! It was the alt-right! You must see this beautifully cringe-inducing music video courtesy of American Renaissance. Read or Share this story: http://usat.ly/2btRpkT Looking for news you can trust? Subscribe to our free newsletters. Last week, when Donald Trump tapped the chairman of Breitbart Media to lead his campaign, he wasn’t simply turning to a trusted ally and veteran propagandist. By bringing on Stephen Bannon, Trump was signaling a wholehearted embrace of the “alt-right,” a once-motley assemblage of anti-immigrant, anti-Muslim, ethno-nationalistic provocateurs who have coalesced behind Trump and curried the GOP nominee’s favor on social media. In short, Trump has embraced the core readership of Breitbart News. “We’re the platform for the alt-right,” Bannon told me proudly when I interviewed him at the Republican National Convention (RNC) in July. Though disavowed by every other major conservative news outlet, the alt-right has been Bannon’s target audience ever since he took over Breitbart News from its late founder, Andrew Breitbart, four years ago. Under Bannon’s leadership, the site has plunged into the fever swamps of conservatism, cheering white nationalist groups as an “eclectic mix of renegades,” accusing President Barack Obama of importing “more hating Muslims,” and waging an incessant war against the purveyors of “political correctness.” “Andrew Breitbart despised racism. Truly despised it,” writes a former Breitbart News editor. “With Bannon embracing Trump, all that changed.” “Andrew Breitbart despised racism. Truly despised it,” former Breitbart editor-at-large Ben Shapiro wrote last week on the Daily Wire, a conservative website. “With Bannon embracing Trump, all that changed. Now Breitbart has become the alt-right go-to website, with [technology editor Milo] Yiannopoulos pushing white ethno-nationalism as a legitimate response to political correctness, and the comment section turning into a cesspool for white supremacist mememakers.” Exactly who and what defines the alt-right is hotly debated in conservative circles, but its most visible proponents—who tend to be young, white, and male—are united in a belief that traditional movement conservatism has failed. They often criticize immigration policies and a “globalist” agenda as examples of how the deck is stacked in favor of outsiders instead of “real Americans.” They bash social conservatives as ineffective sellouts to the GOP establishment, and rail against neo-conservative hawks for their embrace of Israel. They see themselves as a threat to the establishment, far bolder and edgier than Fox News. While often tapping into legitimate economic grievances, their social-media hashtags (such as #altright on Twitter) dredge up torrents of racist, sexist, and xenophobic memes. Trump’s new campaign chief denies that the alt-right is inherently racist. He describes its ideology as “nationalist,” though not necessarily white nationalist. Likening its approach to that of European nationalist parties such as France’s National Front, he says, “If you look at the identity movements over there in Europe, I think a lot of [them] are really ‘Polish identity’ or ‘German identity,’ not racial identity. It’s more identity toward a nation-state or their people as a nation.” (Never mind that National Front founder Jean Marie Le Pen has been fined in France for “inciting racial hatred.”) Bannon dismisses the alt-right’s appeal to racists as happenstance. “Look, are there some people that are white nationalists that are attracted to some of the philosophies of the alt-right? Maybe,” he says. “Are there some people that are anti-Semitic that are attracted? Maybe. Right? Maybe some people are attracted to the alt-right that are homophobes, right? But that’s just like, there are certain elements of the progressive left and the hard left that attract certain elements.” A Twitter analysis conducted by The Investigative Fund using Little Bird software found that these “elements” are more deeply connected to Breitbart News than more traditional conservative outlets. While only 5 percent of key influencers using the supremacist hashtag #whitegenocide follow the National Review, and 10 percent follow the Daily Caller, 31 percent follow Breitbart. The disparities are even starker for the anti-Muslim hashtag #counterjihad: National Review, 26 percent; the Daily Caller, 37 percent; Breitbart News, 62 percent. Bannon’s views often echo those of his devoted followers. He describes Islam as “a political ideology” and Sharia law as “like Nazism, fascism, and communism.” On his Sirius XM radio show, he heaped praise on Pamela Geller, whose American Freedom Defense Initiative has been labeled an anti-Muslim hate group by the Southern Poverty Law Center. Bannon called her “one of the leading experts in the country, if not the world,” on Islam. And he basically endorsed House Speaker Paul Ryan’s primary challenger, businessman Paul Nehlen, who floated the idea of deporting all Muslims from the United States. During our interview, Bannon took credit for fomenting “this populist nationalist movement” long before Trump came on the scene. During our interview, Bannon took credit for fomenting “this populist nationalist movement” long before Trump came on the scene. He credited Sen. Jeff Sessions (R-Ala.)—a Trump endorser and confidant who has suggested that civil rights advocacy groups were “un-American” and “Communist-inspired”—with laying the movement’s groundwork. Bannon also pointed to his own films, which include a Sarah Palin biopic and an “exposé” of the Occupy movement, as “very nationalistic films.” Trump, he said, “is very late to this party.” At Breitbart News, one of the most strident voices for the alt-right has been Yiannapolous, who was banned by Twitter during the RNC for inciting a racist pile-on of Ghostbusters actress Leslie Jones. Published back in March, his “Establishment Conservative’s Guide to the Alt Right” featured an illustration of a frog taunting an elephant—the frog image being a meme white supremacists had popularized on social media. The piece praised the anti-immigrant site VDare, the white nationalist site American Renaissance, and white nationalist leader Richard Spencer, as the alt-right’s “dangerously bright” intellectual core. On the RNC’s opening day, Yiannapolous spoke at a “Citizens for Trump” rally. He also co-hosted a party featuring anti-Muslim activist Geller and the Dutch far-right nationalist politician Geert Wilders. Yiannopolous has proved to be Breitbart‘s most vitriolic anti-Muslim presence, erasing the distinction many conservatives draw between Islam and “radical Islam.” After the Orlando shootings, Yiannopolous told Bannon on his weekly radio show that “there is a structural problem with this religion that is preventing its followers from assimilating properly into Western culture. There is something profoundly antithetical to our values about this particular religion.” Bannon has stoked racist themes himself, notably in a lengthy July post accusing the “Left” of a “plot to take down America” by fixating on police shootings of black citizens. He argued that the five police officers slain in Dallas were murdered “by a #BlackLivesMatter-type activist-turned-sniper.” And he accused the mainstream media of an Orwellian “bait-and-switch as reporters and their Democratic allies and mentors seek to twist the subject from topics they don’t like to discuss—murderers with evil motives—to topics they do like to discuss, such as gun control.” Bannon added, “[H]ere’s a thought: What if the people getting shot by the cops did things to deserve it? There are, after all, in this world, some people who are naturally aggressive and violent.” On Twitter, conservative Breitbart critic Bethany Mandel says she has been “called a ‘slimy Jewess’ and told that I ‘deserve the oven.'” Some Breitbart staffers who resisted the site’s transformation into a pro-Trump alt-right hub eventually resigned in protest. Several jumped ship after Corey Lewandowski, then Trump’s campaign manager, manhandled Breitbart News reporter Michelle Fields at a rally. (The site appeared to side with Lewandowski, and staffers were reportedly told not to question his account.) Among the departing staffers were Fields, who now writes for the Huffington Post, and Shapiro, who has emerged as one of Breitbart‘s most vociferous conservative critics. On Thursday, in the Washington Post, Shapiro upped the ante, describing the alt-right as a “movement shot through with racism and anti-Semitism,” and Breitbart News as “a party organ, a pathetic cog in the Trump-Media Complex and a gathering place for white nationalists.” The reception he and another conservative Jewish Breitbart critic, Bethany Mandel, have experienced in the Bannonosphere is revealing: In May, when Shapiro, who became editor-in-chief of the Daily Wire after leaving Breitbart, tweeted about the birth of his second child, he received a torrent of anti-Semitic tweets. “Into the gas chamber with all 4 of you,” one read. Another tweet depicted his family as lampshades. Mandel says she has been harassed on Twitter for months, “called a ‘slimy Jewess’ and told that I ‘deserve the oven.'” After Shapiro called out the anti-Semitism, Breitbart News published (under the byline of Pizza Party Ben) a post ridiculing Shapiro for “playing the victim on Twitter and throwing around allegations of anti-Semitism and racism, just like the people he used to mock.” Back at the RNC, Bannon dismissed Shapiro as a “whiner…I don’t think that the alt-right is anti-Semitic at all,” he told me. “Are there anti-Semitic people involved in the alt-right? Absolutely. Are there racist people involved in the alt-right? Absolutely. But I don’t believe that the movement overall is anti-Semitic.” In any case, Breitbart‘s conservative dissenters are fearful of what the Trump-Bannon alliance might bring. As Mandel puts it, “There’s no gray area here: Bannon is a bad guy. And he now has control of a major campaign for president.” This article was reported in partnership with The Investigative Fund at The Nation Institute. Additional reporting was done by Kalen Goodluck, Josh Harkinson, and Jaime Longoria. Hillary Clinton today used every tactic in the playbook of the identity-obsessed progressive Left: smears, name-calling and finger-wagging. This is precisely what the alt-right is responding to. They post offensive memes because they know it’ll wind up boring, grouchy grannies like Hillary. The speech codes and political correctness of the Left are what has given rise to this vibrant new movement, what has given rise to Donald Trump’s extraordinary popularity, and what gives rise to me — and my fabulous headlines! They were, by the way: “Birth Control Makes Women Unattractive And Crazy” (sorry, no offense, but it’s true!) and “Would You Rather Your Child Had Feminism Or Cancer?” (no prizes for guessing which readers chose). Little did I realise when I coined the #FeminismIsCancer hashtag that it would end up in the mouth of the Democratic nominee for President. But there’s something else that Clinton represents. While she obsesses over the ironic memes of dissident youths on the internet, there are Black Lives Matter activists calling for the deaths of white police officers, and there are teaching assistants in American universities hosting courses on “how to stop white males.” If there’s genuine, noxious and damaging racism in the United States, it isn’t coming from the alt-right, who are responding to it. Much of the alt-right’s rhetoric is designed to illuminate the absurdity of identitarian politics and the blatant hypocrisy and cruelty of the progressive Left. It’s working – and that’s why the Left is so scared. Hillary is using the same old failed leftist tactics of name-calling, vague allegations and guilt-by-association, but hoping for a different result this time. In other words, she is complaining about a phenomenon she helped to create, thinking she can beat it with the bad habits and lazy smears that fostered it in the first place. Good luck with that! A specter is haunting the dinner parties, fundraisers and think-tanks of the Establishment: the specter of the “alternative right.” Young, creative and eager to commit secular heresies, they have become public enemy number one to beltway conservatives — more hated, even, than Democrats or loopy progressives. The alternative right, more commonly known as the alt-right, is an amorphous movement. Some — mostly Establishment types — insist it’s little more than a vehicle for the worst dregs of human society: anti-Semites, white supremacists, and other members of the Stormfront set. They’re wrong. Previously an obscure subculture, the alt-right burst onto the national political scene in 2015. Although initially small in number, the alt-right has a youthful energy and jarring, taboo-defying rhetoric that have boosted its membership and made it impossible to ignore. It has already triggered a string of fearful op-eds and hit pieces from both Left and Right: Lefties dismiss it as racist, while the conservative press, always desperate to avoid charges of bigotry from the Left, has thrown these young readers and voters to the wolves as well. National Review attacked them as bitter members of the white working-class who worship “father-Führer” Donald Trump. Betsy Woodruff of The Daily Beast attacked Rush Limbaugh for sympathising with the “white supremacist alt-right.” BuzzFeed begrudgingly acknowledged that the movement has a “great feel for how the internet works,” while simultaneously accusing them of targeting “blacks, Jews, women, Latinos and Muslims.” The amount of column inches generated by the alt-right is a testament to their cultural punch. But so far, no one has really been able to explain the movement’s appeal and reach without desperate caveats and virtue-signalling to readers. Part of this is down to the alt-right’s addiction to provocation. The alt-right is a movement born out of the youthful, subversive, underground edges of the internet. 4chan and 8chan are hubs of alt-right activity. For years, members of these forums – political and non-political – have delighted in attention-grabbing, juvenile pranks. Long before the alt-right, 4channers turned trolling the national media into an in-house sport. Having once defended gamers, another group accused of harbouring the worst dregs of human society, we feel compelled to take a closer look at the force that’s alarming so many. Are they really just the second coming of 1980s skinheads, or something more subtle? We’ve spent the past month tracking down the elusive, often anonymous members of the alt-right, and working out exactly what they stand for. THE INTELLECTUALS There are many things that separate the alternative right from old-school racist skinheads (to whom they are often idiotically compared), but one thing stands out above all else: intelligence. Skinheads, by and large, are low-information, low-IQ thugs driven by the thrill of violence and tribal hatred. The alternative right are a much smarter group of people — which perhaps suggests why the Left hates them so much. They’re dangerously bright. The origins of the alternative right can be found in thinkers as diverse as Oswald Spengler, H.L Mencken, Julius Evola, Sam Francis, and the paleoconservative movement that rallied around the presidential campaigns of Pat Buchanan. The French New Right also serve as a source of inspiration for many leaders of the alt-right. The media empire of the modern-day alternative right coalesced around Richard Spencer during his editorship of Taki’s Magazine. In 2010, Spencer founded AlternativeRight.com, which would become a center of alt-right thought. Alongside other nodes like Steve Sailer’s blog, VDARE and American Renaissance, AlternativeRight.com became a gathering point for an eclectic mix of renegades who objected to the established political consensus in some form or another. All of these websites have been accused of racism. The so-called online “manosphere,” the nemeses of left-wing feminism, quickly became one of the alt-right’s most distinctive constituencies. Gay masculinist author Jack Donovan, who edited AlternativeRight’s gender articles, was an early advocate for incorporating masculinist principles in the alt-right. His book, The Way Of Men, contains many a wistful quote about the loss of manliness that accompanies modern, globalized societies. It’s tragic to think that heroic man’s great destiny is to become economic man, that men will be reduced to craven creatures who crawl across the globe competing for money, who spend their nights dreaming up new ways to swindle each other. That’s the path we’re on now. Steve Sailer, meanwhile, helped spark the “human biodiversity” movement, a group of bloggers and researchers who strode eagerly into the minefield of scientific race differences — in a much less measured tone than former New York Times science editor Nicholas Wade. Isolationists, pro-Russians and ex-Ron Paul supporters frustrated with continued neoconservative domination of the Republican party were also drawn to the alt-right, who are almost as likely as the anti-war left to object to overseas entanglements. Elsewhere on the internet, another fearsomely intelligent group of thinkers prepared to assault the secular religions of the establishment: the neoreactionaries, also known as #NRx. Neoreactionaries appeared quite by accident, growing from debates on LessWrong.com, a community blog set up by Silicon Valley machine intelligence researcher Eliezer Yudkowsky. The purpose of the blog was to explore ways to apply the latest research on cognitive science to overcome human bias, including bias in political thought and philosophy. LessWrong urged its community members to think like machines rather than humans. Contributors were encouraged to strip away self-censorship, concern for one’s social standing, concern for other people’s feelings, and any other inhibitors to rational thought. It’s not hard to see how a group of heretical, piety-destroying thinkers emerged from this environment — nor how their rational approach might clash with the feelings-first mentality of much contemporary journalism and even academic writing. Led by philosopher Nick Land and computer scientist Curtis Yarvin, this group began a gleeful demolition of the age-old biases of western political discourse. Liberalism, democracy and egalitarianism were all put under the microscope of the neoreactionaries, who found them wanting. Liberal democracy, they argued, had no better a historical track record than monarchy, while egalitarianism flew in the face of every piece of research on hereditary intelligence. Asking people to see each other as human beings rather than members of a demographic in-group, meanwhile, ignored every piece of research on tribal psychology. While they can certainly be accused of being overly-eager to bridge the gap between fact and value (the truth of tribal psychology doesn’t necessarily mean we should embrace or encourage it), these were the first shoots of a new conservative ideology — one that many were waiting for. NATURAL CONSERVATIVES Natural conservatives can broadly be described as the group that the intellectuals above were writing for. They are mostly white, mostly male middle-American radicals, who are unapologetically embracing a new identity politics that prioritises the interests of their own demographic. In their politics, these new conservatives are only following their natural instincts — the same instincts that motivate conservatives across the globe. These motivations have been painstakingly researched by social psychologist Jonathan Haidt, and an instinct keenly felt by a huge swathe of the political population: the conservative instinct. The conservative instinct, as described by Haidt, includes a preference for homogeneity over diversity, for stability over change, and for hierarchy and order over radical egalitarianism. Their instinctive wariness of the foreign and the unfamiliar is an instinct that we all share – an evolutionary safeguard against excessive, potentially perilous curiosity – but natural conservatives feel it with more intensity. They instinctively prefer familiar societies, familiar norms, and familiar institutions. An establishment Republican, with their overriding belief in the glory of the free market, might be moved to tear down a cathedral and replace it with a strip mall if it made economic sense. Such an act would horrify a natural conservative. Immigration policy follows a similar pattern: by the numbers, cheap foreign workers on H1B visas make perfect economic sense. But natural conservatives have other concerns: chiefly, the preservation of their own tribe and its culture. For natural conservatives, culture, not economic efficiency, is the paramount value. More specifically, they value the greatest cultural expressions of their tribe. Their perfect society does not necessarily produce a soaring GDP, but it does produce symphonies, basilicas and Old Masters. The natural conservative tendency within the alt-right points to these apotheoses of western European culture and declares them valuable and worth preserving and protecting. Needless to say, natural conservatives’ concern with the flourishing of their own culture comes up against an intractable nemesis in the regressive left, which is currently intent on tearing down statues of Cecil Rhodes and Queen Victoria in the UK, and erasing the name of Woodrow Wilson from Princeton in the U.S. These attempts to scrub western history of its great figures are particularly galling to the alt-right, who in addition to the preservation of western culture, care deeply about heroes and heroic virtues. This follows decades in which left-wingers on campus sought to remove the study of “dead white males” from the focus of western history and literature curricula. An establishment conservative might be mildly irked by such behaviour as they switch between the State of the Union and the business channels, but to a natural conservative, such cultural vandalism may just be their highest priority. In fairness, many establishment conservatives aren’t keen on this stuff either — but the alt-right would argue that they’re too afraid of being called “racist” to seriously fight against it. Which is why they haven’t. Certainly, the rise of Donald Trump, perhaps the first truly cultural candidate for President since Buchanan, suggests grassroots appetite for more robust protection of the western European and American way of life. Alt-righters describe establishment conservatives who care more about the free market than preserving western culture, and who are happy to endanger the latter with mass immigration where it serves the purposes of big business, as “cuckservatives.” Halting, or drastically slowing, immigration is a major priority for the alt-right. While eschewing bigotry on a personal level, the movement is frightened by the prospect of demographic displacement represented by immigration. The alt-right do not hold a utopian view of the human condition: just as they are inclined to prioritise the interests of their tribe, they recognise that other groups – Mexicans, African-Americans or Muslims – are likely to do the same. As communities become comprised of different peoples, the culture and politics of those communities become an expression of their constituent peoples. You’ll often encounter doomsday rhetoric in alt-right online communities: that’s because many of them instinctively feel that once large enough and ethnically distinct enough groups are brought together, they will inevitably come to blows. In short, they doubt that full “integration” is ever possible. If it is, it won’t be successful in the “kumbaya” sense. Border walls are a much safer option. The alt-right’s intellectuals would also argue that culture is inseparable from race. The alt-right believe that some degree of separation between peoples is necessary for a culture to be preserved. A Mosque next to an English street full of houses bearing the flag of St. George, according to alt-righters, is neither an English street nor a Muslim street — separation is necessary for distinctiveness. Some alt-righters make a more subtle argument. They say that when different groups are brought together, the common culture starts to appeal to the lowest common denominator. Instead of mosques or English houses, you get atheism and stucco. Ironically, it’s a position that has much in common with leftist opposition to so-called “cultural appropriation,” a similarity openly acknowledged by the alt-right. It’s arguable that natural conservatives haven’t had real political representation for decades. Since the 1980s, establishment Republicans have obsessed over economics and foreign policy, fiercely defending the Reagan-Thatcher economic consensus at home and neoconservative interventionism abroad. In matters of culture and morality, the issues that natural conservatives really care about, all territory has been ceded to the Left, which now controls the academy, the entertainment industry and the press. For those who believe in the late Andrew Breitbart’s dictum that politics is downstream from culture, the number of writers, political candidates and media personalities who actually believe that culture is the most important battleground can be dispiriting. (Though Milo is trying his best.) Natural liberals, who instinctively enjoy diversity and are happy with radical social change – so long as it’s in an egalitarian direction – are now represented by both sides of the political establishment. Natural conservatives, meanwhile, have been slowly abandoned by Republicans — and other conservative parties in other countries. Having lost faith in their former representatives, they now turn to new ones — Donald Trump and the alternative right. There are principled objections to the tribal concerns of the alt-right, but Establishment conservatives have tended not to express them, instead turning nasty in the course of their panicked backlash. National Review writer Kevin Williamson, in a recent article attacking the sort of voters who back Trump, said that white working-class communities “deserve to die.” Although the alt-right consists mostly of college-educated men, it sympathises with the white working classes and, based on our interviews, feels a sense of noblesse oblige. National Review has been just as directly unpleasant about the alt-right as it has, on occasion, been about white Americans in general. In response to concerns from white voters that they’re going to go extinct, the response of the Establishment — the conservative Establishment — has been to openly welcome that extinction. It’s true that Donald Trump would not be possible without the oppressive hectoring of the progressive Left, but the entire media is to blame for the environment in which this new movement has emerged. For decades, the concerns of those who cherish western culture have been openly ridiculed and dismissed as racist. The alt-right is the inevitable result. No matter how silly, irrational, tribal or even hateful the Establishment may think the alt-right’s concerns are, they can’t be ignored, because they aren’t going anywhere. As Haidt reminds us, their politics is a reflection of their natural inclinations. In other words, the Left can’t language-police and name-call them away, which have for the last twenty years been the only progressive responses to dissent, and the Right can’t snobbishly dissociate itself from them and hope they go away either. THE MEME TEAM Earlier, we mentioned the pressure to self-censor. But whenever such pressure arises in a society, there will always be a young, rebellious contingent who feel a mischievous urge to blaspheme, break all the rules, and say the unsayable. Why? Because it’s funny! As Curtis Yarvin explains via email: “If you spend 75 years building a pseudo-religion around anything – an ethnic group, a plaster saint, sexual chastity or the Flying Spaghetti Monster – don’t be surprised when clever 19-year-olds discover that insulting it is now the funniest fucking thing in the world. Because it is.” These young rebels, a subset of the alt-right, aren’t drawn to it because of an intellectual awakening, or because they’re instinctively conservative. Ironically, they’re drawn to the alt-right for the same reason that young Baby Boomers were drawn to the New Left in the 1960s: because it promises fun, transgression, and a challenge to social norms they just don’t understand. Just as the kids of the 60s shocked their parents with promiscuity, long hair and rock’n’roll, so too do the alt-right’s young meme brigades shock older generations with outrageous caricatures, from the Jewish “Shlomo Shekelburg” to “Remove Kebab,” an internet in-joke about the Bosnian genocide. These caricatures are often spliced together with Millennial pop culture references, from old 4chan memes like pepe the frog, to anime and My Little Pony references. Are they actually bigots? No more than death metal devotees in the 80s were actually Satanists. For them, it’s simply a means to fluster their grandparents. Currently, the Grandfather-in-Chief is Republican consultant Rick Wilson, who attracted the attention of this group on Twitter after attacking them as “childless single men who jerk off to anime.” Responding in kind, they proceeded to unleash all the weapons of mass trolling that anonymous subcultures are notorious for — and brilliant at. From digging up the most embarrassing parts of his family’s internet history to ordering unwanted pizzas to his house and bombarding his feed with anime and Nazi propaganda, the alt-right’s meme team, in typically juvenile but undeniably hysterical fashion, revealed their true motivations: not racism, the restoration of monarchy or traditional gender roles, but lulz. It’s hard to know for certain, but we suspect that unlike the core of the alt-right, these young renegades aren’t necessarily instinctive conservatives. Indeed, their irreverence, lack of respect of social norms, and willingness to stomp on other people’s feelings suggest they may actually be instinctive libertarians. Certainly that’s the case for a joyful contingent of Trump supporters who spend hours creating memes celebrating the “God Emperor” and tormenting his adversaries, such as Yiannopoulos ally @PizzaPartyBen, who has amassed 40,000 followers on Twitter with his raucous antics. Were this the 1960s, the meme team would probably be the most hellraising members of the New Left: swearing on TV, mocking Christianity, and preaching the virtues of drugs and free love. It’s hard to imagine them reading Evola, musing at St. Peter’s Basilica or settling down in a traditional family unit. They may be be inclined to sympathise to those causes, but mainly because it annoys the right people. Young people perhaps aren’t primarily attracted to the alt-right because they’re instinctively drawn to its ideology: they’re drawn to it because it seems fresh, daring and funny, while the doctrines of their parents and grandparents seem unexciting, overly-controlling and overly-serious. Of course, there is plenty of overlap. Some true believers like to meme too. If you’re a Buzzfeed writer or a Commentary editor reading this and thinking… how childish, well. You only have yourself to blame for pompously stomping on free expression and giving in to the worst and most authoritarian instincts of the progressive left. This new outburst of creativity and taboo-shattering is the result. Of course, just as was the case in history, the parents and grandparents just won’t understand, man. That’s down to the age difference. Millennials aren’t old enough to remember the Second World War or the horrors of the Holocaust. They are barely old enough to remember Rwanda or 9/11. Racism, for them, is a monster under the bed, a story told by their parents to frighten them into being good little children. As with Father Christmas, Millennials have trouble believing it’s actually real. They’ve never actually seen it for themselves — and they don’t believe that the memes they post on /pol/ are actually racist. In fact, they know they’re not — they do it because it gets a reaction. Barely a month passes without a long feature in a new media outlet about the rampant sexism, racism or homophobia of online image boards. For regular posters at these boards, that’s mission accomplished. Another, more palatable, interpretation of these memes is that they are clearly racist, but that there is very little sincerity behind them. The funny thing is, being Millennials, they’re often quite diverse. Just visit a /pol/ thread, where posters’ nationalities are identified with small flags next to their posting IDs. You’ll see flags from the west, the Balkans, Turkey, the Middle East, South America, and even, sometimes, Africa. Everyone on the anonymous board hurls the most vicious slurs and stereotypes each other, but like jocks busting each other’s balls at the college bar, it’s obvious that there’s little real hatred present. That is, until the 1488ers show up. THE ‘1488rs’ Anything associated as closely with racism and bigotry as the alternative right will inevitably attract real racists and bigots. Calmer members of the alternative right refer darkly to these people as the “1488ers,” and for all their talk of there being “no enemies to the right,” it’s clear from the many conversations we’ve had with alt-righters that many would rather the 1488ers didn’t exist. These are the people that the alt-right’s opponents wish constituted the entire movement. They’re less concerned with the welfare of their own tribe than their fantasies of destroying others. 1488ers would likely denounce this article as the product of a degenerate homosexual and an ethnic mongrel. Why “1488”? It’s a reference to two well-known Neo Nazi slogans, the first being the so-called 14 Words: “We Must Secure The Existence Of Our People And A Future For White Children.” The second part of the number, 88, is a reference to the 8th letter of the alphabet – H. Thus, “88” becomes “HH” which becomes “Heil Hitler.” Not very edifying stuff. But if you want to use the 1488ers to tarnish the entire alt-right, you need to do the same with Islamist killers and Islam and third-wave feminist wackos with the entire history and purpose of feminism. Which you might well be fine with — but let’s be consistent. Alt-right vlogger Paul “RamZPaul” Ramsey describes them as “LARPers” or Live-Action Role Players: a disparaging comparison to nerdy nostalgists who dress up as medieval warriors. Paul even goes as far as to suggest some in this “toxic mix of kooks and ex-cons” may be there solely to discredit the more reasonable white identitarians. Hitler is dead. Quit larping that it is perpetually 1933 Germany. https://t.co/Qg6vXRZkdX — RAMZPAUL (@ramzpaul) December 29, 2015 1488 is primarily about: 1) LARPING 2) discrediting White identity via guilt by association https://t.co/vuO8ZvJ1mg — RAMZPAUL (@ramzpaul) February 24, 2016 I have no interest in the 1488 crowd. A toxic mix of kooks and ex-cons turned informants. LARPing the Third Reich. https://t.co/xEwPdONnlF — RAMZPAUL (@ramzpaul) February 24, 2016 Every ideology has them. Humourless ideologues who have no lives beyond their political crusade, and live for the destruction of the great. They can be found on Stormfront and other sites, not just joking about the race war, but eagerly planning it. They are known as “Stormfags” by the rest of the internet. Based on our research we believe this stands in stark contrast with the rest of the alt-right, who focus more on building communities and lifestyles based around their values than plotting violent revolution. 1488ers are the equivalent of the Black Lives Matter supporters who call for the deaths of policemen, or feminists who unironically want to #KillAllMen. Of course, the difference is that while the media pretend the latter are either non-existent, or a tiny extremist minority, they consider 1488ers to constitute the whole of the alt-right. Those looking for Nazis under the bed can rest assured that they do exist. On the other hand, there’s just not very many of them, no-one really likes them, and they’re unlikely to achieve anything significant in the alt-right. What little remains of old-school white supremacy and the KKK in America constitutes a tiny, irrelevant contingent with no purchase on public life and no support even from what the media would call the “far-Right.” (Admittedly, these days that includes anyone who votes Republican.) THE ESTABLISHMENT’S FRANKENSTEIN Not all alt-righters will agree with our taxonomy of the movement. Hacker and white nationalist Andrew Auernheimer, better known as weev, responded in typically jaw-dropping fashion to our enquiries: “The tireless attempts of you Jews to smear us decent Nazis is shameful.” Delving into the depths of the alternative right, it quickly becomes apparent that the movement is best defined by what it stands against rather than what it stands for. There are a myriad of disagreements between its supporters over what they should build, but virtual unity over what they should destroy. For decades – since the 1960s, in fact – the media and political establishment have held a consensus over what’s acceptable and unacceptable to discuss in polite society. The politics of identity, when it comes from women, LGBT people, blacks and other non-white, non-straight, non-male demographics is seen as acceptable — even when it descends into outright hatred. Any discussion of white identity, or white interests, is seen as a heretical offence. It’s a fact observed as early as 2008 by Yarvin: Ethnic pride is one thing. Hostility is another. But – as progressives often observe – they tend to travel together. It strikes me as quite incontrovertible that if an alien anthropologist were to visit Earth and collate expressions of hostility toward human subpopulations in Western culture today, the overwhelming majority would be anti-European. Anti-Europeanism is widely taught in schools and universities today. Its converse most certainly is not. So here is my challenge for progressives, multiculturalists, “dynamists,” and the like: if your antiracism is what it claims to be, if it is no more than Voltaire 3.0, why do non-European ethnocentrism and anti-European hostility not seem to bother you in the slightest? Do they maybe even strike you as, um, slightly cool? The current consensus offers, at best, mild condemnation of identity politics on the Left, and zero tolerance for identity politics on the right. Even for us – a gay man of Jewish descent and a mixed-ethnic half-Pakistani – the dangers of writing on this topic loom large. Though we do not identify with the alt-right, even writing an article about them is akin to prancing through a minefield. The pressure to self-censor must be almost overwhelming for straight white men — and, for most of them, it appears to be, which explains why so much of the alt-right operates anonymously. While movements like third-wave feminism and Black Lives Matter often draw criticism from conservatives and libertarians, advocacy on behalf of those causes is not a career-ending offence. Quite the reverse. It’s possible to build successful and lucrative careers off the back of those movements. Just look at Al Sharpton, Anita Sarkeesian and Deray Mckesson. In the past five years, left-wing identity politics underwent a renaissance just as the crisis of white males – especially young white males – in the west became obvious. As feminism entered its “fourth wave,” obsessed with trivialities like online trolling, “sexist t-shirts” and “microaggressions,” male suicide rates were reaching crisis levels. As minority advocates on college campuses raised Hell about offensive Halloween costumes and demanded safe spaces in which they could be insulated from differing points of view, working-class white males became the least likely group to attend university in the U.K. To politically alert Millennials, the contrast between the truly marginalized and those merely claiming victim status has become stark. The Establishment bears much of the blame. Had they been serious about defending humanism, liberalism and universalism, the rise of the alternative right might have been arrested. All they had to do was argue for common humanity in the face of black and feminist identity politics, for free speech in the face of the regressive Left’s censorship sprees, and for universal values in the face of left-wing moral relativism. Instead, they turned a blind eye to the rise of tribal, identitarian movements on the Left while mercilessly suppressing any hint of them on the Right. It was this double standard, more than anything else, that gave rise to the alternative right. It’s also responsible, at least in part, for the rise of Donald Trump. While the alt-right is too sophisticated to be mistaken for a mindless knee-jerk reaction, opposition to this prevailing consensus is the glue that holds it together. Some enjoy violating social norms for shock value, while others take a more intellectual approach, but all oppose the pieties and hypocrisies of the current consensus — from both Left and Right — in some form or another. In that, the alt-right has much in common with the cultural libertarian movement first identified in these pages. And there are many people who would identify with both labels. THE MASK OF RACISM To young people and the politically disengaged, debate in the public square today appears topsy-turvy. The regressive Left loudly insists that it stands for equality and racial justice while praising acts of racial violence and forcing white people to sit at the back of the bus (or, more accurately, the back of the campus — or in another campus altogether). It defends absurd feminist positions with no basis in fact and ridicules and demeans people on the basis of their skin colour, sexual orientation and gender. Meanwhile, the alt-right openly crack jokes about the Holocaust, loudly — albeit almost entirely satirically — expresses its horror at “race-mixing,” and denounces the “degeneracy” of homosexuals… while inviting Jewish gays and mixed-race Breitbart reporters to their secret dinner parties. What gives? If you’re this far down the article, you’ll know some of the answers already. For the meme brigade, it’s just about having fun. They have no real problem with race-mixing, homosexuality, or even diverse societies: it’s just fun to watch the mayhem and outrage that erupts when those secular shibboleths are openly mocked. These younger mischief-makers instinctively understand who the authoritarians are and why and how to poke fun at them. The intellectuals are animated by a similar thrill: after being taken for granted for centuries, they’re the ones who get to pick apart some of the Enlightenment’s dead dogmas. The 1488ers just hate everyone; fortunately they keep mostly to themselves. The really interesting members of the alt-right though, and the most numerous, are the natural conservatives. They are perhaps psychologically inclined to be unsettled by threats to western culture from mass immigration and maybe by non-straight relationships. Yet, unlike the 1488ers, the presence of such doesn’t send them into fits of rage. They want to build their homogeneous communities, sure — but they don’t want to commit any pogroms along the way. Indeed, they would prefer non-violent solutions. They’re also aware that there are millions of people who don’t share their inclinations. These are the instinctive liberals, the second half of Haidt’s psychological map of western polities — the people who are comfortable with diversity, promiscuity, homosexuality, and all other features of the cultural consensus. Natural conservatives know that a zero-sum battle with this group would end in stalemate or defeat. Their goal is a new consensus, where liberals compromise or at least allow conservative areas of their countries to reject the status quo on race, immigration and gender. Others, especially neoreactionaries, seek exit: a peaceful separation from liberal cultures. Should the liberal tribe (and let’s not deny it any longer – that’s both the Democratic and GOP Establishments these days) do business with them? Well, the risk otherwise is that the 1488ers start persuading people that their solution to natural conservatives’ problems is the only viable one. The bulk of their demands, after all, are not so audacious: they want their own communities, populated by their own people, and governed by their own values. In short, they want what every people fighting for self-determination in history have ever wanted, and what progressives are always telling us people should be allowed — unless those people are white. This hypocrisy is what has led so many Trump voters — groups who have in many cases not voted since the 1970s or 80s — to come out of the woodwork and stand up for their values and culture. The Establishment need to read their Haidt and realise that this group isn’t going away. There will be no “progress” that erases the natural affinities of conservatives. We can no longer pretend that divides over free trade and the minutiae of healthcare reform really represent both sides of the political spectrum in America. The alt-right is here, and here to stay. Allum Bokhari is a reporter for Breitbart. He can be followed on Twitter at @LibertarianBlue. Milo Yiannopoulos is a senior editor for Breitbart. He can be followed at @Nero. You can follow their work by downloading the Milo Alert! app and hear them on The Milo Yiannopoulos Show. The alternative right has come under fire from Hillary Clinton and establishment Republicans, but it has been seeping into American politics for years as a far-right option for conservatives. Here's what you need to know about the alt-right movement. (Jenny Starrs/The Washington Post) On Thursday, with an unusual amount of fanfare, Hillary Clinton will give a speech denouncing the "alt-right" and delineating ways in which Donald Trump has inflamed racist sentiment. On the alt-right itself, the speech is being welcomed as a sort of coming-out party; alt-right figures are finding their phones and email boxes glowing with new messages, asking to explain who they are and what they think. [Clinton plans Thursday address in Nevada on Trump and the ‘alt-right’] While reporters like Rosie Gray, Olivia Nuzzi, and Benjy Sarlin have reported on the alt-right's success for a year, and while the Southern Poverty Law Center has closely monitored its success, the movement remains elastically defined, harboring some terms and personalities that remain obscure or impenetrable. This is a guide — which can and will be updated — to the basics. 'The Camp of the Saints' A 1973 French novel by Jean Raspail, published as "Le Camp des Saints," which envisions an immigrant invasion of France, and which many on the alt-right view as prophetic. In a 2005 essay for the American Conservative, after riots in France, commentator (and future Michelle Bachmann collaborator) Jim Pinkerton cited Raspail's novel at length to ask why Europe had not realized it was committing "national suicide." As Raspail describes the scene aboard the immigrant convoy, “Everywhere, rivers of sperm. Streaming over bodies, oozing between breasts, and buttocks, and thighs, and lips, and fingers … a welter of dung and debauch.” But France is persuaded that these people are a “million Christs,” whose arrival will “signal the dawn of a just, new day.” In other words, Raspail writes, what the French are lacking is a proper sense of national-racial consciousness, “the knowledge that one’s own is best, the triumphant joy at feeling oneself to be part of humanity’s finest.” Instead, he concludes, after having been beaten down by decades of multicultural propaganda, “the white race” has become “nothing more than a million sheep.” Raspail's vision has been cited frequently at Breitbart News, especially when a major Western leader criticizes anti-immigrant sentiment. "Now, as in the novel, prominent political officials are urging on ever larger waves," wrote Breitbart's Julia Hahn in 2015. "Secular and religious leaders hold hands to pressure blue collar citizens to drop their resistance; media elites and celebrities zealously cheer the opportunity that the migrants provide to atone for the alleged sins of the West — for the chance to rebalance the wealth and power of the world by allowing poor migrants from failed states to rush in to claim its treasures." Trump campaign CEO Stephen Bannon, a former Breitbart executive. (Carlo Allegri/Reuters) 'Cuckservative' A portmanteau, from "cuckold" and "conservative," used to troll people who call themselves conservative but support immigration reform and multiculturalism. The implication: A white American who allows mass immigration into his country is no different than a man allowing other men to sleep with his wife. 'It's the Current Year!' A logical fallacy, popularized on 4chan and Reddit, in which an idea can be dismissed because "it's 2016" (i.e., the world and history have moved on, and there is nothing left to discuss). It's frequently identified with HBO's John Oliver, whose commentaries (circulated widely on progressive news sites) often label ideas as ridiculous because, well, it's 2016. Jared Taylor The founder of American Renaissance, a magazine, then conference, then website about white identity. Ever game to talk to media — though critical of the term "alt-right" — he's used the publication and conference to encourage white nationalists to expand on their ideas. Pepe the Frog A cartoon that originated on MySpace but was adopted by Trump supporters and alt-right trolls, as reporter Olivia Nuzzi explained at length this year. Peter Brimelow The founder of VDare, a clearinghouse of news and opinion about immigration, which he founded after his immigration book "Alien Nation" became a taboo bestseller. 'The Political Cesspool' A white nationalist podcast and radio show that began in 2004 and grew its following during Barack Obama's presidency, and became notorious after Donald Trump Jr. appeared to promote his father's presidential campaign. The Robert A. Taft Club A defunct political group co-founded by the future alt-right leaders Richard Spencer and Kevin DeAnna in northern Virginia in 2006. A third co-founder, Marcus Epstein, described its mission in a message inviting students and journalists to one of its first events: It focuses to foment a lively debate about important issues that divide the Right, but must be addressed such as foreign policy, immigration, and multiculturalism. While not shying away from the tough issues, the Robert Taft Club hopes to deal with these issues without resorting to sectarian squabbling. The typical Taft Club forum featured free beer and a debate between mainstream conservative figures and those more associated with nationalism. Over several years, the Club's guests included Jared Taylor, John Derbyshire, leaders of the right-wing Belgian party Vlaams Belang, and then-Texas congressman Ron Paul during his first Republican presidential bid. Joe Sobran A columnist purged from National Review in 1993, a decision that that magazine's founder William F. Buckley explained, in a very lengthy essay, as a strike against anti-Semitism. "Individualists have been replaced by apparatchiks," Sobran said bitterly in 2002. "Zionism has infiltrated conservatism in much the same way Communism once infiltrated liberalism. " Marine Le Pen The leader of France's National Front, a far-right party long led by her father, but thriving amid the backlash to mass Muslim immigration and the unpopular Socialist government of President François Hollande. Nigel Farage The longtime leader of the United Kingdom Independence Party (UKIP), and member of the European Parliament, whose populist campaigning helped produce an upset win for the "Brexit" campaign. Like Le Pen, he's seen as proof that alt-right politics can win, even in a hostile media environment. Richard Spencer The president of the Virginia-based National Policy Institute and founder of the defunct website Alternative Right, which was "dedicated to heretical perspectives on society and culture — popular, high, and otherwise — particularly those informed by radical, traditionalist, and nationalist outlooks." One of the most media-savvy thinkers in the movement, Spencer was an early supporter of what Trump's campaign represented; before that, he helped find and promote young alt-right thinkers. In addition to shaping what "alt-right" meant, Spencer coined the term "identitarian" to distinguish white people who wanted to defend their culture but rejected the label of "racism." Sam Francis An influential conservative thinker cast out of the movement's mainstream — and fired from his Washington Times column — for speaking at the 1994 American Renaissance conference. Subsequently, he became a sort of martyr for nationalist writers and thinkers. Throughout his career, he argued that cultural liberalism was not as popular or inevitable as its promoters claimed. "Whites need to form their racial consciousness in conformity not only with what we now know about the scientific reality of race but also with the moral and political traditions of Western Man-White Man," Francis wrote in 2005. "The purpose of white racial consciousness and identity is not simply to serve as a balance against the aggression and domination of other races but also to preserve, protect, and help revitalize the legacy of the civilization that our own ancestors created and handed down to us, for its own sake, because it is ours, and because, by the standard of the values and ideals we as a race and a civilization have articulated, it is better." Walt Bismarck A musician and video editor who grew a following (under the sobriquet "Uncuck the Right") with pop song parodies rewritten around alt-right themes. "The alt-right does not comprise obese low church Protestant Baby Boomers with 103 IQs," he explained to Fusion in 2015. "We’re a bunch of eccentric hipsters and neckbeards who understand how the Left works, and how to create legitimately subversive and effective propaganda." Youth for Western Civilization A now-defunct alliance of student groups, founded in 2008 by the alt-right activist Kevin DeAnna, with the hopes of capitalizing on young people's support for Ron Paul by creating a revolutionary, anti-state, anti-multicultural movement. "We are essentially just echoing standard conservative rhetoric on immigration, multiculturalism, and American identity," DeAnna wrote in 2009. "But even this moderate approach is too much for leftists. Calls to completely transform the structure of the American economy meet far less opposition than suggesting the enforcement of existing immigration laws. I submit this tells us what the real forbidden issues are in America today and where the Left really sees the battle lines falling." In just three years, YWC became a controversial campus force, drawing negative attention from the Southern Poverty Law Center. It petered out as DeAnna ran out of time and interest, but some YWC veterans went on to become alt-right or white nationalist activists, such as Towson University's Matthew Heimbach. When YWC folded, he attempted to form a white student union; four years later, cameras captured him roughly ejecting a Black Lives Matter activist from a Donald Trump rally. Published on Aug 24, 2016 What is the Alt Right? What do we desire? Hillary Clinton is schedule to speak on August 25, 2016 to damn the Alt Right for our desire for self-determination - a home. http://www.breitbart.com/2016-preside... | Suddenly, "alt-right" is the political phrase of the moment. Hillary Clinton on Thursday called out Donald Trump for being linked to what she calls a racist, "paranoid fringe" movement, while Trump denied even knowing what the phrase means. Coverage: What Clinton said, per Gothamist: "These are race-baiting ideas, anti-Muslim and anti-immigrant ideas, anti-woman-all key tenets making up an emerging racist ideology known as the 'alt-right.'... The names may have changed. Racists now call themselves 'racialists.' White supremacists now call themselves 'white nationalists.' The paranoid fringe now calls itself 'alt-right.' But the hate burns just as bright." What Trump said, per the Hill, on CNN: "Nobody even knows what it is, and she didn't know what it was. There's no alt-right or alt-left. All that I'm embracing is common sense." Trump campaign CEO Steve Bannon, formerly of Breitbart Media, told Mother Jones last week that Breitbart is the "platform for the alt-right." Read the interview here. (Asked about it on CNN, Trump responded, "I don't know what Steve said. I can only speak for myself.") CNN's Brian Stelter has an explainer video on the movement here. The Washington Post's Dave Weigel has a primer here. At Breitbart, editor Milo Yiannopoulos says Clinton herself is responsible for the rise of the alt-right. Earlier this year, he wrote "An Establishment Conservative's Guide to the Alt-Right." Read it here. A USA Today columnist offers a definition: "It's an online movement of white people-young white guys, mostly-including white supremacists, nationalists, nativists and plain ol' racists. They're anxious. They're afraid. They worry whites are losing power and influence in America, and so they generally oppose immigration and multiculturalism." This video blogger and alt-right supporter says the movement is "about us having a home, a place where we can be with people like ourselves." When Clinton called out the alt-right movement, the alt-right loved it. See tweets rounded up by Slate. | multi_news |
SECTION 1. SHORT TITLE. This Act may be cited as ``The Separate Enrollment and Line Item Veto Act of 1999''. SEC. 2. STRUCTURE OF LEGISLATION. (a) Appropriations Legislation.-- (1) In general.--The Committee on Appropriations of either the House or the Senate shall not report an appropriation measure that fails to contain such level of detail on the allocation of an item of appropriation proposed by that House as is set forth in the committee report accompanying such bill. (2) Point of order.--If an appropriation measure is reported to the House or Senate that fails to contain the level of detail on the allocation of an item of appropriation as required in paragraph (1), it shall not be in order in that House to consider such measure. If a point of order under this paragraph is sustained, the measure shall be recommitted to the Committee on Appropriations of that House. (b) Authorization Legislation.-- (1) In general.--A committee of either the House or the Senate shall not report an authorization measure that contains new direct spending or new targeted tax benefits unless such measure presents each new direct spending or new targeted tax benefit as a separate item and the accompanying committee report for that measure shall contain such level of detail as is necessary to clearly identify the allocation of new direct spending or new targeted tax benefits. (2) Point of order.--If an authorization measure is reported to the House or Senate that fails to comply with paragraph (1), it shall not be in order in that House to consider such measure. If a point of order under this paragraph is sustained, the measure shall be recommitted to the committee of jurisdiction of that House. (c) Conference Reports.-- (1) Appropriations.--A committee of conference to which is committed an appropriations measure shall not file a conference report in either House that fails to contain the level of detail on the allocation of an item of appropriation as is set forth in the statement of managers accompanying that report. (2) Authorizations.--A committee of conference to which is committed an authorization measure shall not file a conference report in either House unless such measure presents each direct spending or targeted tax benefit as a separate item and the statement of managers accompanying that report clearly identifies each such item. (3) Point of order.--If a conference report is presented to the House or Senate that fails to comply with either paragraph (1) or (2), it shall not be in order in that House to consider such conference report. If a point of order under this paragraph is sustained in the House to first consider the conference report, the measure shall be deemed recommitted to the committee of conference. SEC. 3. WAIVERS AND APPEALS. Any provision of section 2 may be waived or suspended in the House or Senate only by an affirmative vote of three-fifths of the Members of that House duly chosen and sworn. An affirmative vote of three-fifths of the Members duly chosen and sworn shall be required to sustain an appeal of the ruling of the Chair on a point of order raised under that section. SEC. 4. SEPARATE ENROLLMENT. (a) In General.-- (1) Enrollment.--Notwithstanding any other provision of law, when any appropriation or authorization measure first passes both Houses of Congress in the same form, the Secretary of the Senate (in the case of a measure originating in the Senate) or the Clerk of the House of Representatives (in the case of a measure originating in the House of Representatives) shall disaggregate the items as referenced in section 5(4) and assign each item a new bill number. After disaggregation each item shall be treated as a separate bill to be considered under the following subsections. The remainder of the bill not so disaggregated shall constitute a separate bill and shall be considered with the other disaggregated bills pursuant to subsection (b). (2) Form.--A bill that is required to be disaggregated into separate bills pursuant to paragraph (1)-- (A) shall be disaggregated without substantive revision; and (B) shall bear the designation of the measure of which it was an item prior to such disaggregation, together with such other designation as may be necessary to distinguish such measure from other measures disaggregated pursuant to paragraph (1) with respect to the same measure. (b) Procedure.--The new bills resulting from the disaggregation described in subsection (a)(1) shall be immediately placed on the appropriate calendar in the House of origination, and upon passage, placed on the appropriate calendar in the other House. They shall be the next order of business in each House and they shall be considered and voted on en bloc and shall not be subject to amendment. A motion to proceed to the bills shall be nondebatable. Debate in the House of Representatives or the Senate on the bill shall be limited to not more than 1 hour, which shall be divided equally between the majority leader and the minority leader. A motion further to limit debate is not debatable. A motion to recommit the bills is not in order, and it is not in order to move to reconsider the vote by which the bills are agreed to or disagreed to. SEC. 5. DEFINITIONS. In this Act: (1) Appropriation measure.--The term ``appropriation measure'' means any general or special appropriation bill or any bill or joint resolution making supplemental, deficiency, or continuing appropriations. (2) Authorization measure.--The term ``authorization measure'' means any measure other than an appropriations measure that contains a provision providing direct spending or targeted tax benefits. (3) Direct spending.--The term ``direct spending'' shall have the same meaning given to such term in section 250(c)(8) of the Balanced Budget and Emergency Deficit Control Act of 1985. (4) Item.--The term ``item'' means-- (A) with respect to an appropriations measure-- (i) any numbered section, (ii) any unnumbered paragraph, or (iii) any allocation or suballocation of an appropriation, made in compliance with section 2(a), contained in a numbered section or an unnumbered paragraph but shall not include a provision which does not appropriate funds, direct the President to expend funds for any specific project, or create an express or implied obligation to expend funds and-- (I) rescinds or cancels existing budget authority; (II) only limits, conditions, or otherwise restricts the President's authority to spend otherwise appropriated funds; or (III) conditions on an item of appropriation not involving a positive allocation of funds by explicitly prohibiting the use of any funds; and (B) with respect to an authorization measure-- (i) any numbered section, or (ii) any unnumbered paragraph, that contains new direct spending or a new targeted tax benefit presented and identified in conformance with section 2(b). (5) The term ``targeted tax benefit'' means any provision-- (A) estimated by the Joint Committee on Taxation as losing revenue for any one of the three following periods-- (i) the first fiscal year covered by the most recently adopted concurrent resolution on the budget; (ii) the period of the 5 fiscal years covered by the most recently adopted concurrent resolution on the budget; or (iii) the period of the 5 fiscal years following the first 5 years covered by the most recently adopted concurrent resolution on the budget; and (B) having the practical effect of providing more favorable tax treatment to a particular taxpayer or limited group of taxpayers when compared with other similarly situated taxpayers. SEC. 6. JUDICIAL REVIEW. (a) Expedited Review.-- (1) Member of congress.--Any Member of Congress may bring an action, in the United States District Court for the District of Columbia, for declaratory judgment and injunctive relief on the ground that a provision of this Act violates the Constitution. (2) Intervention by houses.--A copy of any complaint in an action brought under paragraph (1) shall be promptly delivered to the Secretary of the Senate and the Clerk of the House of Representatives, and each House of Congress shall have the right to intervene in such action. (3) Panel.--Any action brought under paragraph (1) shall be heard and determined by a three-judge court in accordance with section 2284 of title 28, United States Code. (4) Authority of houses.--Nothing in this section or in any other law shall infringe upon the right of the House of Representatives or the Senate to intervene in an action brought under paragraph (1) without the necessity of adopting a resolution to authorize such intervention. (b) Appeal to Supreme Court.--Notwithstanding any other provisions of law, any order of the United States District Court for the District of Columbia which is issued pursuant to an action brought under paragraph (1) of subsection (a) shall be reviewable by appeal directly to the Supreme Court of the United States. Any such appeal shall be taken by a notice of appeal filed within 10 days after such order is entered; and the jurisdictional statement shall be filed within 30 days after such order is entered. No stay of an order issued pursuant to an action brought under paragraph (1) of subsection (a) shall be issued by a single Justice of the Supreme Court. (c) Expedited Consideration.--It shall be the duty of the District Court for the District of Columbia and the Supreme Court of the United States to advance on the docket and to expedite to the greatest possible extent the disposition of any matter brought under subsection (a). (d) Severability.--If any provision of this Act, or the application of such provision to any person or circumstance is held unconstitutional, the remainder of this Act and the application of the provisions of such Act to any person or circumstance shall not be affected thereby. SEC. 7. TREATMENT OF EMERGENCY SPENDING. (a) Emergency Appropriations.--Section 251(b)(2)(D)(i) of the Balanced Budget and Emergency Deficit Control Act of 1985 is amended by adding at the end the following new sentence: ``However, OMB shall not adjust any discretionary spending limit under this clause for any statute that designates appropriations as emergency requirements if that statute contains an appropriation for any other matter, event, or occurrence, but that statute may contain rescissions of budget authority.''. (b) Emergency Legislation.--Section 252(e) of the Balanced Budget and Emergency Deficit Control Act of 1985 is amended by adding at the end the following new sentence: ``However, OMB shall not designate any such amounts of new budget authority, outlays, or receipts as emergency requirements in the report required under subsection (d) if that statute contains any other provisions that are not so designated, but that statute may contain provisions that reduce direct spending.''. (c) New Point of Order.--Part A of title IV of the Congressional Budget Act of 1974 is amended by adding at the end the following new section: ``point of order regarding emergencies ``Sec. 407. It shall not be in order in the House of Representatives or the Senate to consider any bill or joint resolution, or amendment thereto or conference report thereon, containing an emergency designation for purposes of section 251(b)(2)(D) or 252(e) of the Balanced Budget and Emergency Deficit Control Act of 1985 if it also provides an appropriation or direct spending for any other item or contains any other matter, but that bill or joint resolution, amendment, or conference report may contain rescissions of budget authority or reductions of direct spending, or that amendment may reduce for that emergency.''. (d) Conforming Amendment.--The table of contents set forth in section 1(b) of the Congressional Budget and Impoundment Control Act of 1974 is amended by inserting after the item relating to section 406 the following new item: ``Sec. 407. Point of order regarding emergencies.''. SEC. 8. SAVINGS FROM RESCISSION BILLS USED FOR DEFICIT REDUCTION. (a) In General.--Not later than 45 days of continuous session after the President vetoes an appropriations measure or an authorization measure, the President shall-- (1) with respect to appropriations measures, reduce the discretionary spending limits under section 601 of the Congressional Budget Act of 1974 for the budget year and each outyear by the amount by which the measure would have increased the deficit in each respective year; and (2) with respect to a repeal of direct spending, or a targeted tax benefit, reduce the balances for the budget year and each outyear under section 252(b) of the Balanced Budget and Emergency Deficit Control Act of 1985 by the amount by which the measure would have increased the deficit in each respective year. (b) Exceptions.-- (1) In general.--This section shall not apply if the vetoed appropriations measure or authorization measure becomes law, over the objections of the President, before the President orders the reduction required by subsections (a)(1) or (a)(2). (2) Restoration of limits.--If the vetoed appropriations measure or authorization measure becomes law, over the objections of the President, after the President has ordered the reductions required by subsections (a)(1) or (a)(2), then the President shall restore the discretionary spending limits under section 601 of the Congressional Budget Act of 1974 or the balances under section 252(b) of the Balanced Budget and Emergency Deficit Control Act of 1985 to reflect the positions existing before the reduction ordered by the President in compliance with subsection (a). SEC. 9. EVALUATION AND SUNSET OF TAX EXPENDITURES. (a) Legislation for Sunsetting Tax Expenditures.--The President shall submit legislation for the periodic review, reauthorization, and sunset of tax expenditures with his fiscal year 2000 budget. (b) Budget Contents and Submission to Congress.--Section 1105(a) of title 31, United States Code, is amended by adding after paragraph (30) the following: ``(31) beginning with fiscal year 2002, a Federal Government performance plan for measuring the overall effectiveness of tax expenditures, including a schedule for periodically assessing the effects of specific tax expenditures in achieving performance goals.''. (c) Pilot Projects.--Section 1118(c) of title 31, United States Code, is amended by-- (1) striking ``and'' after the semicolon in paragraph (2); (2) redesignating paragraph (3) as paragraph (4); and (3) adding after paragraph (2) the following: ``(3) describe the framework to be utilized by the Director of the Office of Management and Budget, after consultation with the Secretary of the Treasury, the Comptroller General of the United States, and the Joint Committee on Taxation, for undertaking periodic analyses of the effects of tax expenditures in achieving performance goals and the relationship between tax expenditures and spending programs; and''. (d) Congressional Budget Act.--Part A of title IV of the Congressional Budget Act of 1974 is amended by adding at the end thereof the following: ``tax expenditures ``Sec. 408. It shall not be in order in the House of Representatives or the Senate to consider any bill, joint resolution, amendment, motion, or conference report that contains a tax expenditure unless the bill, joint resolution, amendment, motion, or conference report provides that the tax expenditure will terminate not later than 10 years after the date of enactment of the tax expenditure.''. SEC. 10. SEVERABILITY. If any provision of this Act, or the application of such provision to any person or circumstance is held unconstitutional, the remainder of this Act and the application of the provisions of such Act to any person or circumstance shall not be affected thereby. SEC. 11. EFFECTIVE DATE. The provisions of this Act and the amendments made by this Act shall apply to measures passed by the Congress beginning with the date of the enactment of this Act and ending on September 30, 2004. | Separate Enrollment and Line Item Veto Act of 1999 - Prohibits the Committee on Appropriations of either the House of Representatives or the Senate from reporting an appropriation measure that fails to contain such level of detail on the allocation of an item of appropriation proposed by that House as is set forth in the accompanying committee report. Prohibits a congressional committee from reporting an authorization measure containing new direct spending or new targeted tax benefits unless such measure presents such items separately and the accompanying committee report contains the necessary level of detail. Prohibits the filing of conference reports on appropriations measures that fail to contain such level of detail on the allocation of an item as is set forth in the accompanying statement of managers. (Sec. 3) Allows the waiver or appeal of such prohibitions by a three-fifths vote of the appropriate House. (Sec. 4) Requires separate enrollment of each item of appropriation or authorization in measures passed by both Houses in identical form. Provides for congressional consideration of such bills. (Sec. 6) Provides for expedited judicial review of provisions of this Act in the U.S. District Court for the District of Columbia and direct appeals to the Supreme Court. (Sec. 7) Amends the Balanced Budget and Emergency Deficit Control Act of 1985 (Gramm-Rudman-Hollings Act) and the Congressional Budget Act of 1974 to prohibit the inclusion of nonemergency spending proposals in emergency spending legislation. Allows such proposals to contain rescissions of budget authority or provisions that reduce direct spending. (Sec. 8) Requires savings from rescissions bills to be used for deficit reduction. (Sec. 9) Requires the President to submit legislation for the periodic review, reauthorization, and sunset of tax expenditures with the FY 2000 budget. Requires the inclusion in the budget beginning with FY 2002 of a performance plan for measuring the overall effectiveness of tax expenditures, including a schedule for periodically assessing the effects of specific tax expenditures in achieving performance goals. Directs the Director of the Office of Management and Budget to include as a pilot project the periodic analyses of such goals and the relationship between tax expenditures and spending programs. Amends the Congressional Budget Act of 1974 to prohibit consideration in the House and the Senate of legislation that contains a tax expenditure unless the expenditure terminates not later than ten years after the date of its enactment. (Sec. 11) Makes this Act effective until the end of FY 2004. | billsum |
(CNN) -- A Delaware pediatrician was indicted Wednesday on 58 additional counts of rape and other sexual offenses in the alleged child abuse of his patients, state Attorney General Beau Biden announced Monday. Dr. Earl Bradley, 56, previously pleaded not guilty to 471 felony counts involving 102 girls and one boy. Wednesday's indictment -- which includes charges of rape, unlawful sexual contact, continuous sexual abuse of a child and sexual exploitation -- involves 24 girls he saw as patients from 1999 to 2009. Biden has said the charges are based on "video and digital evidence" seized from Bradley's home and medical practice in December, when the doctor was arrested. Bradley, whose practice was in Lewes, Delaware, also has medical licenses in Pennsylvania, New Jersey and Florida. Authorities have said they have contacted officials in those states. In a February 24 interview with CNN, Bradley's attorney said he would base his client's defense on mental health. "Most of the evidence in this case comes from videotapes -- it's kind of hard to argue with videotapes," Eugene Maurer Jr. said. He added, "The issue in this case is going to come down to his mental health at the time." The Delaware pediatrician accused in a horrific patient sexual abuse case was cleared in a similar complaint by a hospital just two years before he allegedly began a molestation spree involving hundreds of children at his private practice, authorities said. The Beebe Medical Center in Delaware released a statement on its website revealing that a nurse had complained that Dr. Earl Bradley may have been inappropriately touching young female patients, particularly when taking urine samples during examination, in 1996. The 471 criminal counts Bradley now faces are alleged to have begun in 1998, according to court documents obtained by ABCNews.com. Bradley is accused of videotaping the sex acts with more than 100 patients at his Lewes, Del., practice called BayBees Pediatrics while the children's parents waited in another room. Play A lawyer for one of Bradley's alleged victims and one of 18 families suing Beebe Medical Center for negligence told ABCNews.com that he wonders if the victims could have been spared had the hospital done more at the time of the initial complaints. "What the 1996 revelation signifies to me, is an incredible red flag that should have been dealt with properly and if it had, we think that folks could have been saved," said attorney Chase T. Brockstedt. An internal investigation of the allegations was conducted at the time, according to Beebe, but was done through "peer review," rather than by reporting it to the state medical board. This way, the hospital could investigate "without fear that the results of the investigation will become available for malpractice litigation," Brockstedt said. It was determined during the internal review that Bradley's techniques involving female patients were "within mainstream of current pediatric practices," and Bradley continued to work at the hospital in various capacities until 2009, the hospital said. Beebe released the information to authorities only after they were subpoenaed following Bradley's December 2009 arrest. Beebe CEO Jeffrey M. Fried was not made available to ABCNews.com for an interview, but told The News Journal that, "Once you know what happened, you see what a diabolical person he was. It makes your stomach turn." Beebe said in the statement, "People's lives have been forever altered by this diabolical monster." Bradley, 56, is being held at the Vaughn Correctional Center on $4.7 million bail and pleaded not guilty to the charges in February. His public defender did not immediately return messages left by ABCNews.com. If convicted, Bradley faces a mandatory sentence of life in prison without the possibility of parole. "I cannot say certain things that I am feeling, and I am feeling a great deal. I am determined to see that this defendant will never, ever be in a position again to hurt another child," Delaware Attorney General Beau Biden said in a statement following the February indictment. The 160-page indictment describes in detail Bradley's alleged behavior between 1998 and 2009. Of the 103 children mentioned in the indictment, only one of them was male. Among the 471-count indictment includes allegations that Bradley forced children to perform sex acts on him as recently as Dec. 13. Some of the children were allegedly molested continuously over a series of days or months. One victim was allegedly raped by Bradley from June 2007 until February 2009, and another was molested "continuously" between November 2008 and November 2009. A search of Bradley's medical practice late last year led to the analysis of more than 13 hours of video files, computers and hard drives and more than 7,000 patient files. Biden told ABCNews.com at the time of Bradley's arrest that "this is a very very troubling case, and we here in the state of Delaware are pursuing it aggressively to the fullest extent of the law." "The thing that makes this case so troubling and horrific is the alleged abuse of trust in this matter," said Biden. "These are highly specialized cases where no one in the community could believe someone could do this to a child, to a child who is voiceless." Biden said that some of the alleged victims were just months old. Pediatrician Earl Bradley Accused of Sexually Abusing More Than 100 Patients According to court documents obtained by ABCNews.com, the investigation was spurred after a 2-year-old girl told her mother that Bradley had touched her genitals and "hurt her" during a Dec. 7 appointment. According to the child's mother, who is not named in the court documents, Bradley first examined the toddler in her presence, but then "removed the victim to the basement of the office, where a toy room is located." The mother told authorities that she permitted her daughter to be alone with Bradley "because of his position as a doctor" and because she "trusted him." But on the way home from the doctor the mother alleges that her daughter said Bradley had sexually abused her while they were alone in the basement. The girl's father, who had taken to her to a previous appointment with Bradley, also told authorities his daughter had made a similar complaint to him in November. In 160 pages of disturbing court documents, Bradley is accused of videotaping sexual acts with his young patients and molesting them repeatedly while their parents waited in nearby examination rooms. Bradley, who had been in the 1,300-person town of Lewes since 1994, is accused of abusing his patients for the past 11 years. According to the arrest documents, the examination room known as the Pinocchio Room because of its decorations was seen frequently on the video tapes obtained by authorities that reportedly show Bradley undressing his patients and performing sex acts on them. In another room decked out with "The Little Mermaid" paraphernalia, equally horrific abuse allegedly occurred at the hands of Bradley, who investigators said was seen on the tapes muzzling screaming children as they tried to flee the abuse. Described as a 6-foot, 225-pound man, Bradley allegedly had a "violently enraged expression on his face" as he yelled to a 2-year-old patient to perform sexual acts on him. That particular video was described by the investigating officer in court documents as "one of the most violent and brutal attacks on a child of any age" that he had ever seen. | A Delaware pediatrician charged with sexually assaulting more than 100 of his small patients has been hit with another 58 counts of sex offenses, reports CNN, boosting the total against him to a record 529 charges. Dr. Earl Bradley, 56, has pleaded not guilty to 471 felony counts involving sex assaults on 102 girls and a boy. The new charges, including rape and continuous sexual abuse, involve 24 other girls. Some of his victims were just months old, according to authorities. One investigator called the assaults "the most brutal on a child I have ever seen." Much of the evidence against the 6-foot, 224-pound pediatrician comes from videotapes of the assaults created by Bradley himself, say police. "It's kind of hard to argue with videotapes," said his attorney, who plans a mental instability defense. Bradley is charged with attacking his patients in special "play rooms," where parents were not allowed. He was cleared of wrongdoing by a Delaware hospital just 2 years before his arrest late last year when a nurse reported inappropriate touching of his young female patients, reports ABC News. | multi_news |
The dominance of the major transcript isoform relative to other isoforms from the same gene generated by alternative splicing (AS) is essential to the maintenance of normal cellular physiology. However, the underlying principles that determine such dominance remain unknown. Here, we analyzed the physical AS process and found that it can be modeled by a stochastic minimization process, which causes the scaled expression levels of all transcript isoforms to follow the same Weibull extreme value distribution. Surprisingly, we also found a simple equation to describe the median frequency of transcript isoforms of different dominance. This two-parameter Weibull model provides the statistical distribution of all isoforms of all transcribed genes, and reveals that previously unexplained observations concerning relative isoform expression derive from these principles. Most genes of eukaryotic organisms, unlike those of prokaryotes, may each generate multiple different mature transcript isoforms which can encode proteins with distinct or even opposing functions [1–6]. It has also been shown that the dominance of the major transcript isoform from a single gene may radically affect cell function, identity and fate, and that disruption of this dominance may result in human disease, including abnormal osteoclast genesis, tumorigenesis and Parkinson’s disease [3–11]. In addition, three intriguing observations have been reported regarding the frequency distribution of transcript isoforms that point to universal principles governing gene transcript isoform expression: (1) genes tend to express all their isoforms simultaneously but at different levels; (2) the major and minor dominant isoform of a gene usually accounts for over 30% and 15% of total transcript expression, respectively; and (3) for any two distinct transcript isoforms from the same gene, one of them is always significantly dominant [3–14]. However, the mechanisms underlying these fundamental observations remain unclear. Indeed, the overall expression and frequency distribution of all isoforms of entire transcriptomes has rarely been subjected to systematic analysis. Many studies have been performed to identify cis-acting elements, trans-acting factors and the specific biological processes involved in AS [1,15–18]. Essentially, the AS process contains two major steps: (1) intron identification by the binding of U1 and U2AF proteins to the 5’ and 3’ splice sites, respectively; and (2) intron splicing by the release of U1 and the additional binding of U4-6 snRNP [1,15–18]. We focused on intron identification as this decides the fate of the pre-mRNA by determining which transcript isoforms will be produced. U1 and U2AF engage in random three-dimensional (association/dissociation) and one-dimensional (sliding) Brownian search (Fig 1A) [19,20]. The binding of U1 and U2AF to the splice sites is ATP-independent, weak and reversible, and becomes stable only after the ATP-dependent binding of U2 snRNP (Fig 1B) [21]. Usually, in a segment of pre-mRNA presented for AS, many candidate splice sites exist and compete for the binding of U1 and U2AF. The lower the potential energy of the splice sites, the stronger the binding and the more time allowed for the formation of a stable A-complex, the more corresponding mature mRNA will be produced. This indicates that the process of AS is stochastic: the product of a transcript isoform from a pre-mRNA is probabilistically determined by the binding energy of splicing factors at splice sites. Mathematically, this process represents a stochastic minimization process in which U1 and U2AF dynamically search their global or local minimal potential energy sites on the pre-mRNA segment since non-minimal potential energy sites are not stable thus don’t have enough time to allow the formation of stable A-complex. A mature mRNA will undergo multiple rounds of the minimization process if the corresponding pre-mRNA has multiple introns to remove (Fig 1B). This suggests that the expression levels of transcript isoforms may follow an extreme value distribution, of which there are only three types whatever the original distribution of the random variables; namely, Gumbel distribution (Type I), Frechet distribution (Type II) and Weibull distribution (Type III). These three distributions can be transformed to each other by a simple mathematical transformation of the original random variable [22–24]. We tested the three candidate distributions by performing whole transcriptome deep sequencing (RNA-seq) on highly purified resting and activated peripheral blood human CD4 T cell subsets (naïve, central memory, transitional memory and effector memory) from 9 healthy donors (S1 Table). Because the AS mechanism is the same for all multiple-exon genes, the distribution function that their transcript isoforms follow should be similar. However, certain parameters of the distribution function may differ according to the level of expression of a gene as determined by the activation state of the cell. Thus, a scaling parameter must be applied to the raw expression levels of transcript isoforms from different cell conditions and genes. A simple scaling factor is the average expression level of all transcript isoforms from the same gene, as it positively correlates with the gene’s expression level but is independent of isoform number. The smaller the isoform number M, the greater the inaccuracy in the estimation of the scaling factor, and, for accuracy, only genes with M≥5 are used here. Our analysis reveals that the expression levels of transcript isoforms of a gene follows a type III Weibull extreme value distribution—W (x, a, b) (Fig 1C). W (x) is the probability of a transcript isoform with expression level x; b is the scale parameter, which will change with the expression level of gene; and a is the shape parameter, which is specific to the AS mechanism and should be constant for all genes. The approximate values of parameters a (0. 44) and b (0. 6) in Fig 1C are estimated by simple curve fitting, and thus are somewhat inaccurate. For the Weibull distribution, a simple formula links a, b and the population mean of μ [24,25], μ=bΓ (1+1a) (2) Γ represents Gamma function. For a gene with M transcript isoforms and expression level of E, the sample mean x¯=E/M. When the isoform number M is sufficiently large, the sample mean approaches the population mean, giving an approximate formula that connects transcript isoform number M, gene expression level E and two parameters a and b, E≈bMΓ (1+1a) (3) Of note, the analysis above shows that the correct scale factor b should be x¯/Γ (1+1a), not x¯. Although the experimental data fit very well with the Weibull distribution empirically, the statistical test of the fitness-of-fit is not significant for four reasons. First, our model is a very simple one, which considers only the most important factor influencing AS, the strength of splice site binding, and disregards many other factors such as co-transcriptional splicing, histone modifications on chromatin, poison exons, non-sense mediated degradation (NMD) and so on; Second, there is bias in the estimation of the scale factor, and furthermore this bias changes with isoform number M. Third, it is well known that current annotations for human transcript isoforms are incomplete; thus, the transcript isoform number M used for many genes is not accurate. Fourth, although significant improvement has been made in the accuracy of calculation of transcript isoform expression levels, current algorithms nevertheless remain imperfect. We defined the frequency of a gene’s transcript isoform as the ratio of its expression level relative to the expression level of the gene, which equals the sum of expression levels of all transcript isoforms from that gene (Fig 2A). Thus, for a gene with M different transcript isoforms where each isoform has the rank k in the hierarchy of expression levels, we use f (k, M) to represent the frequency of the kth dominant isoform. As 1≤k≤M, so f (1, M) ≥ f (2, M) ≥ … ≥ f (M, M). f (k, M) was entirely stochastic, differed among genes and changed with cell activation state, except for f (1,1) which was always 100% as long as the corresponding gene was expressed. For example, f (1,2) —the frequency of the most dominant isoform of a gene with two transcript isoforms—varied between 50% and 100%. f (2,2) —the frequency of the second most dominant isoform—varied between 0 and 50%. For a specific gene, both the frequencies and the ranks of its isoforms may change with cell condition, such that the most dominant isoform of a gene in one condition may become a less dominant isoform in other conditions. Thus, for the same gene (same M) and same k, f (k, M) may represent the frequency of a different isoform under different cell conditions. While notable, this property is inconsequential as the following analyses explore the relationship solely between the frequency and rank of transcript isoforms. To analyze the frequency distribution of transcript isoforms, we grouped genes according to the number of their isoforms from group 1, which contains genes with one isoform, through group M which contains genes with M isoforms. The variation in isoform frequency with k and M is illustrated straightforwardly in a boxplot (Fig 2B) which shows that the frequency of the most dominant isoform decreases with M. This trend becomes more apparent if we focus solely on the median value, mf (k, M). The median frequency of the most dominant isoform mf (1, M) decreases from 100% when M = 1, to approximately 50% when M = 10, and approximately 30% when M = 30. In contrast, the median frequency of the second most dominant isoform mf (2, M) initially increases, peaking when M = 6, and then decreases with M. The median frequencies of other isoforms (k>2) show a similar trend. These results confirm and extend a previous report where only the most dominant isoform (k = 1) was analyzed [13]. Notably, our model was not only able to explain and provide the overall distribution of the scaled expression levels of all transcript isoforms, it could also provide the frequency distribution of all transcript isoforms. We may show this using a Monte Carlo simulation. The scale parameter b was set as 1 as it has no influence on this simulation of transcript isoform frequency. First, we showed that all mf (k, M) can be explained by our model and, in the process, also showed how mf (k, M) could give a more accurate estimation of the shape parameter a—approximately 0. 44. We randomly selected a number in the range (0,1) as the value for a and performed the following computation: for genes with M transcript isoforms, we randomly extracted M numbers from the Weibull distribution W (a, 1) as the expression levels of the M simulated isoforms, which can then be transformed to their frequencies. We repeated this process 10,000 times for each M to obtain the simulated median frequency mf (k, M) and then compared it with the corresponding mf (k, M) from our experimental data. The Euclidian distance of all mf (k, M) from simulated data and experimental data in Fig 2B was calculated and reached the minimal value when a was 0. 39 (Fig 2C). Notably, 0. 39 is also the exact solution of equation 1+1/a = Γ (1+1/a). Fig 2B shows that when a = 0. 39, values for mf (k, M) calculated from the simulated data (red curves) are very close to those from the experimental data (box plot). The shape parameter a calculated by the Monte Carlo simulation is more accurate than that calculated by simple curve fitting for two reasons. First, the median value of a distribution is very stable, and sampling error and outliers has relatively less influence on its estimation. Second, the bias in the estimation of scale factor is same for both the experimental and simulated datasets and thus its influence is canceled out. To help understand how mf (k, M) changes with a, similar figures with a = 0. 2 and a = 0. 6 are also given in the supplemental material (S1 Fig). Fig 2B reveals that the median frequency of the most dominant isoform, mf (1, M), decreases with M and has no lower limit. This finding contradicts a previous observation that the frequency of the most dominant isoform is at least 30%, even for a gene with many isoforms [13]. Second, we showed that the frequency distribution of all transcript isoforms as well as each f (k, M) can be given by our model. Repeating the previous Monte Carlo simulation with a = 0. 39, we obtained the frequency distribution of all transcript isoforms for different gene groups (different M) (Fig 3) and each f (k, M) (Fig 4 and S2 Fig) from the simulated data. Here, we use Kullback-Leibler divergence (KLd) to evaluate the difference between the two distributions, which represents the amount of information lost when we used the simulation of our Weibull model to represent the frequency distribution of the experimental data. We found that for most frequency distributions analyzed, the amount of information lost is smaller than 0. 05 (mean = 0. 026, median = 0. 020). This shows that the frequency distribution from the simulated data (red curve) is highly consistent with that from the experimental data (black curve), although the shape and range of the distribution change with k and M. Thus, although the expression levels of transcript isoforms change with a particular gene, cell condition and rank, their overall frequency distributions do not change and can be described by our model. Third, these distributions enable statistical analysis of transcript isoform usage such as defining significantly dominant transcript isoforms. For genes with two, five, ten, 20 and 30 transcript isoforms, an isoform may be called significantly dominant if its frequency is above 0. 99,0. 852,0. 529,0. 269 and 0. 174, respectively, since the probability that an isoform randomly selected has frequencies above these thresholds is less than 5% (S2 Table). Thus, the ability to define such thresholds may be used as a statistical framework to discover functionally dominant transcript isoforms with relevance to disease states. The frequency distribution of transcript isoforms changes with their rank k and the isoform number M of the gene from which they are spliced. Nonetheless, our model was able to give the distribution of each f (k, M) as well as their median frequency mf (k, M). Remarkably, we also found that all mf (k, M) could be described by a simple formula: mf (k, M) =1/ (kM×e (1+kM) 2) ∑m=1M1/ (mM×e (1+mM) 2) =1k×e− (1+kM) 2∑m=1M1m×e− (1+mM) 2=e− (1+kM) 2k×HM (4) Here, HM is the Mth generalized harmonic number: HM=∑m=1M1m×e− (1+mM) 2 (5) Fig 2B shows that the median frequencies computed by the formula above (blue curves) are very close to the values from the experimental data and simulated data across all values of k and M, indicating that the median frequency of the kth dominant isoform of a gene with M isoforms is proportional to 1k×e− (1+kM) 2, which thus can be taken as its frequency index. Four important methodological points were addressed. First, to exclude the possibility that our results emerged from an intrinsic property of the analysis software, we reanalyzed the entire dataset with an independent software package, Salmon [26,27], which, in contrast to Cufflinks, requires no sequence alignment. The similarity of the results derived from these two approaches indicates that the isoform frequency distribution we observed is robust and software-independent (S3 Fig). Second, to exclude the possibility that our results emerged from the Expectation Maximization (EM) algorithm used by most software packages, we created two simulated RNA-seq datasets with transcript isoform expression level following a Normal distribution N (20,2) and a Weibull distribution W (0. 39,10), respectively (See Materials and methods for details). The results based on the simulated RNA-seq dataset from the Normal distribution were markedly different from those from our experimental RNA-seq data (S4 Fig). In contrast, the results based on the simulated RNA-seq dataset from the Weibull distribution showed a perfect match with our experimental RNA-seq data (S5 Fig). Third, to exclude the possibility that our results emerged purely as a function of the particular dataset we used, we analyzed 18 different pre-existing RNA-seq datasets derived from embryonic stem cells, cancers and human cell lines (S3 Table). We obtained similar results in every case (S6 and S7 Figs). Finally, the result based on a merged gene set (Euclidian distance 0. 158) showed a closer match with our formula than the result based solely on the Ensembl gene set (Euclidian distance 0. 160; S8 Fig), which reflects the incomplete nature of existing datasets. The correctness of our model is strongly supported by two points. First is the simplicity of the model in that it requires only one shape parameter. Second is its general applicability in explaining the scaled expression level of all transcript isoforms, the frequency distribution of transcript isoforms of genes with different isoform number (M), and furthermore the frequency distribution of individual transcript isoforms of different rank (k). Here, we did not perform a strict mathematical deduction of how stochastic searching of minimal-energy U1 and U2AF binding sites leads to the Weibull distribution as this would be extremely difficult if not impossible, as Weibull himself discussed in his original paper: “it is utterly hopeless to expect a theoretical basis for distribution functions of random variables such as strength properties of materials or of machine parts of particle size” [25]. Currently, our model takes the isoform annotation of all genes given by the user as input (we recommend the latest Ensembl transcript annotation), and it does not explain or predict the isoform number of genes. It should be noted that the binding potential energy landscape of U1 and U2AF on a specific pre-mRNA segment is not static but dynamic, and may change with the binding of other tissue-specific or non-specific auxiliary proteins on cis-acting AS elements induced by external signals; thus, genes may have different major transcript isoforms under different conditions. We analyzed the change of dominancy rank of transcript isoforms for every expressed gene in the four T cell subsets under the two cell conditions: resting and after in vitro activation. Using the Ensembl gene set, 540 genes underwent transformation of the most dominant transcript isoforms between resting and activated conditions across all four T cell subsets, another 891 genes underwent transformation of the most dominant isoform for three of the four T cell subsets (S4 Table). The biological processes enriched in the 540 genes are very diverse and include regulation of cellular response to stress, virus-host interaction, chromosome organization, transcription, translation and protein metabolism (S9 Fig). This suggests that a T cell may express not only different genes but also different transcript isoforms depending on its activation state. For example, BRD4 (bromodomain containing 4), an inhibitor gene of HIV-1 infection [28], has 11 known transcript isoforms. Of them, ENST00000371835 is the most dominant isoform in the activated condition of all four T cell subsets and the second most dominant in the resting condition, while ENST00000263377 is the most dominant isoform in the resting condition in all four T cell subsets and the second most dominant in the activated condition (Fig 5A). SRSF7 (serine/arginine-rich splicing factor 7), a splicing factor and inhibitor of HIV-1 Tat-mediated transactivation [29], has 12 distinct transcript isoforms. Of them, ENST00000409276 is the most dominant transcript isoform in the stimulated condition across all four T cell subsets, whereas ENST00000477635 is the most dominant transcript isoform in resting condition across all four T cell subsets (Fig 5B). Taken together, these and our previous results demonstrate that the dominancy rank of transcript isoforms of a gene can be regulated by external stimuli, but that the frequency distribution of transcript isoforms at each rank remains constant. It has been reported that the number of isoforms expressed increases with the number of isoforms annotated per gene [13]. We redid the analysis with our own RNA-seq data and confirmed these findings and, more importantly, can provide an explanation. To calculate the expected number of isoforms expressed, we still used the expression level of transcripts from the simulated Weibull distribution W (0. 39). Different genes have different expression levels; thus it is reasonable to select a cutoff of frequency rather the absolute expression level to define whether a transcript isoform would be theoretically detected. Here, we use 0. 001 as the frequency cutoff and thus define undetectable transcript isoforms as those whose frequency is below 0. 001. The boxplot is the observed result from our RNA-seq data (Fig 6). The red curve is the expected median calculated from our model guided by two assumptions: 1) genes express all their transcript isoforms simultaneously; 2) the scaled expression level of transcript isoform follows W (0. 39). There is excellent concordance between the two plots, and they both show that the number of isoforms expressed increases with the number of transcripts annotated per gene. Our model may be applied to a number of key observations that have been made in previous studies on the usage of AS transcript isoforms but for which mechanistic explanations have been lacking. The first observation is that genes tend to express all their transcript isoforms simultaneously but at different levels [13]. We can explain this mathematically because the Weibull distribution, when a<1 (here a = 0. 39), peaks at 0 and then decreases at a rate greater than an exponential distribution. Thus the expression level of most transcript isoforms will be slightly higher than 0, while the expression level of the remaining transcript isoforms will be considerably higher than 0 and will differ from each other. The second observation is that the major and minor isoforms of a gene usually account for over 30% and 15% of total transcript expression, respectively [12–14]. The theoretical percentage of human genes with f (1, M) ≥ 30% can be calculated from the weighted number of genes in each gene group. We first calculated the percent of simulated genes with f (1, M) ≥ 30% for each gene group in the simulated dataset and then used the result to weight the number of human genes in that group. The result revealed that 93% of genes that undergo AS will have f (1, M) >30%. Similar analysis revealed that 60% of genes that undergo AS will have f (2, M) ≥ 15%. The third observation is that, no matter how many different transcript isoforms a gene has, if we focus only on two of them, such as the two with opposing function, one will always be significantly dominant [3–11]. The frequency distribution of f (1,2) (Fig 4A) shows that for any two isoforms from the same gene, the possibility of the dominant isoform having frequency ≥80% is greater than 73%. This explains how cells can maintain the dominancy of one transcript isoform over others including those with opposing functions. Essentially, our results (Fig 4B) demonstrate that the frequency distribution of the second-most dominant transcript isoform changes with M; thus, a more rational way to define the minor transcript isoform may be to use an M-related dynamic threshold based on the distribution of f (2, M). In conclusion, we have derived a mathematical model that describes AS based on its physical process. Alternative splicing is a very complex biological process, and many factors contribute to the splicing of a pre-mRNA segment, such as strength of binding of AS complex on splice sites, co-transcriptional splicing, histone modifications on chromatin, poison exons, non-sense mediated degradation (NMD) and so on. Our model only considers the most important of these factors, the strength of splice site binding, and disregards all other factors. In this sense, it is a simple model which nevertheless succeeds very well in explaining many observation regarding AS. AS in our model and in this manuscript refers to the biological process that splices the same pre-mRNA into different transcript isoforms. It covers all five basic modes of alternative splicing: exon skipping or cassette exon, mutually exclusive exons, alternative donor site (alternative 5’ splice site), alternative acceptor site (alternative 3’ splice site) and intron retention. Our model suggests that: (1) AS is a stochastic process such that the relative expression level of different transcript isoforms from the same gene is probabilistically determined by the binding energy of splicing factors at their splice sites; (2) the expression levels of transcript isoforms of a gene follow the Weibull distribution W (0. 39, b), here b is a scale parameter dependent on the expression level of the gene, and the scaled expression levels of different transcript isoforms from all genes follow the same Weibull distribution W (0. 39); and (3) the frequency distributions of all transcript isoforms can be calculated from the Monte Carlo simulation of the Weibull distribution W (0. 39). This indicates that the expression of a transcript isoform is not a deterministic event but rather a stochastic event, and the detection of a transcript isoform in an RNA-seq dataset depends on both the expression level of its related gene and sequencing depth. We found a simple formula to describe the median frequency of each transcript isoform. Our analysis also provides transcriptome-wide evidence that the dominance rank of transcript isoforms is altered by distinct external signals and identifies 540 genes that switch their major transcript isoform usage in all four T cell subsets studied. Additionally, our analysis reveals that the AS process has an intrinsic tendency to maintain the dominancy of one transcript isoform over others including those with opposing function. Finally, by incorporating previously unexplained observations, the application of our model to describing the statistical distributions of scaled expression level and frequency of transcript isoforms provides a theoretical foundation for understanding the principles that govern relative transcript isoform generation, which in turn regulates cell identity, function and fate. Nine healthy study volunteers were recruited through the NIH Department of Transfusion Medicine and gave informed consent for leukapheresis. The study was approved by the NIH Institutional Review Board. Leukaphereses were performed at the NIH Blood Bank, followed by immediate isolation of PBMC by density gradient centrifugation. CD4 T cells were then isolated using the CD4+ T Cell Isolation Kit II (Miltenyi), counted, and viably cryopreserved in a freeze medium containing 10% DMSO and 90% sterile filtered, heat inactivated fetal calf serum. Viably cryopreserved peripheral blood CD4 T cells were thawed and stained with ViViD viability dye (Molecular Probes) and fluorescently-labeled monoclonal antibodies against cell surface markers. Staining antibodies included CD3-H7-Allophycocyanin (H7-APC; BD), CD27-Cyanin5-Phycoerythrin (Cy5-PE; Coulter), CD45RO-Texas Red-PE (Coulter), CCR7-Alexa680 (Pharmingen), CD8-Quantum dot-655 (QD655; Invitrogen), CD4-Quantum dot-605 (QD605; Invitrogen), CD19-Pacific Blue (Invitrogen), and CD14-Pacific Blue (Invitrogen). After excluding non-viable cells and those expressing CD19 and CD14, all viable CD3+CD4+CD8- events were gated to collect TN (CD27+CD45RO-), TCM (CD27+CD45RO+CCR7+), TTM (CD27+CD45RO+CCR7-) and TEM (CD27-CD45RO+) populations (S1 Table). Cells were sorted at 4°C and collected in sterile filtered, heat inactivated fetal calf serum. Sorted CD4 TN, TCM, TTM, and TEM subsets were divided into two equal portions to allow comparison between stimulated and unstimulated conditions. Unstimulated portions were immediately subjected to nucleic acid extraction. For stimulation cultures, cells were sedimented at 420g for 7 minutes at 4°C and resuspended in complete culture medium (RPMI 1640 + 10% heat inactivated, sterile filtered calf serum + Penicillin/Streptomycin/Glutamine). They were then combined with T cell activation/expansion beads (anti-CD3/anti-CD2/anti-CD28; Miltenyi) at a 1: 2 bead: cell ratio at a final concentration of 2 x 106 cells/mL and incubated at 37°C for 5–6 hours. Following this incubation, stimulated CD4 T cell subsets were subjected to nucleic acid extraction. Cell subsets were lysed in RNAzol RT reagent (Molecular Research Centers) and homogenized by pipetting. Total RNA was then extracted according to the manufacturer’s instructions. Extracted RNA in pellet form was dissolved in RNAse-free water and used for mRNA library construction. Sequencing libraries were prepared and sequenced as previously described [30]. In brief, total RNA was enriched for polyadenylated species by two sequential rounds of binding to oligo-dT dynabeads (Life Technologies), chemically fragmented in the presence of Mg2+, and reverse transcribed using Superscript III reverse transcriptase (Life Technologies). Second strand cDNA synthesis, end repair, A-tailing, and sequencing adaptor ligation were performed using NEBNext enzyme modules (New England Biolands). Libraries were amplified using universal and indexed primers from the NEBNext system with Kapa 2x Hot Start Readymix (Kapa Biosystems). Amplified libraries were size-selected using Beckman-Coulter Ampure XP beads, quantified by qPCR using the Kapa Library Quantification Kit for Illumina (Kapa Biosystems), and checked for sizing by electrophoresis on a BioAnalyzer (Agilent). Completed libraries were loaded on Illumina Truseq Paired-End v2 Cluster Kits and sequenced in 2 x 100 base paired-end runs on an Illumina HiSeq 2000 sequencer. The final dataset comprised 1. 27×109 reads pairs in total, with each cell condition corresponding to 1. 59×108 reads pairs and each sample corresponding to 1. 76×107 reads pairs on average. Trimmomatic (version 0. 22) was used to remove adapters and low quality bases [31]. The trimmed paired-end reads were mapped to the reference human genome (Hg19) using Tophat (version 2. 0. 8) and assembled with Cufflinks (version 2. 2. 1) [32–34]. Cuffmerge was used to merge all novel assemblies and the known human gene set (Ensembl “Homo_sapiens. GRCh37. 74. gtf”) to create a merged non-redundant transcript annotation. Finally, Cuffdiff was used to evaluate the expression of genes and their transcript isoforms. All genes with FPKM>1 are included in our analysis. To prove our results are software independent, another software, Salmon (version 0. 8. 2) was also used to evaluate the expression of genes and their transcript isoforms [26]. Supposing f1 and f2 are two 9×30 frequency matrices, where an element in row k and column M represents the median frequency of the kth most dominant transcript isoform of gene with M isoforms, k≤M. The frequency matrix data may be derived from experimental RNA-seq data, formula (4) or from a simulation of the Weibull distribution W (0. 39), such as in Fig 2B. The Euclidian distance of the two matrixes can be calculated from following formula, distance (f1, f2) =∑k=19∑M=k30 (f1 (k, M) −f2 (k, M) ) 2 Supposing P and Q are two probability distributions, P is from experimental data, Q is from simulated data of Weibull distribution, the Kullback-Leibler divergence (KLd) between P and Q is defined by following formula, KLd (P∥Q) =∫−∞∞p (x) logp (x) q (x) dx When P and Q are discrete probability distributions, KLd (P∥Q) =∑iP (i) logP (i) Q (i) KLd represents the amount of information lost when Q is used to approximate P. The information content or entropy of a distribution P is defined as, Entropy (P) =∫−∞∞p (x) log (p (x) ) dx When P is a discrete probability distribution, Entropy (P) =∑iP (i) log (P (i) ) The KLd and entropy in this study are calculated by the KL. plugin function in the R “entropy” package. First, we extracted transcript isoform sequences for all human genes from the reference genome (Hg19) according to the Ensembl annotation (Ensembl “Homo_sapiens. GRCh37. 74. gtf”). For a gene with M isoforms, we randomly extracted M values from N (20,2) or W (0. 39,10) as their expression levels E and proceeded thus: for a transcript isoform with length L and expression level E, we randomly extracted R = int (E*L/100/2+0. 5) read pairs to uniformly cover the transcript isoform. Each read pair has 100bp on each end and an average insert length of 100bp. This ensured that the transcript isoform had an expression level of E. We then added reads info and quality info for each read pair. We repeated this process for all transcript isoforms to create simulated FASTQ files. The whole process was repeated ten times to create ten different sequence data for the Normal distribution N (20,2) and the Weibull distribution W (0. 39,10), respectively. We estimated the value of parameters a (0. 44) and b (0. 6) by curve fitting, which is not accurate due to bias in the estimation of the scale factor for each gene as shown below. To illustrate why the shape parameter computed from curve fitting is inaccurate, we performed the same scale transformation on the simulated dataset. The expression values in the simulated dataset strictly follow the Weibull distribution W (0. 39,1) as they are produced from this distribution. However, the scaled expression values do not follow the Weibull distribution, and their range is from 0 to M (S10 Fig). The scaled expression is always 1 when M = 1. It has two peaks at 0 and 2 when M = 2. The larger the M, the closer the scaled expression (black histogram) and original expression (red curve). The difference becomes small when M = 5. The most distinct difference lies in the maximal value. For original expression, there is no upper bound for the maximal value although the higher the expression the less chance it appears. For scaled expression, the maximal value is bound by M. Traditionally, the Weibull plot is used to calculate the shape parameter of the Weibull distribution [24,25]. However, this method cannot be applied here for two reasons. First, the scale parameter and distribution is different for each gene and each condition. Second, the isoform number is limited for each gene, and there is bias in the estimation of the scale parameter for each gene. The comparison of the Weibull plot between the scaled and original values from same simulated data shows that the larger the M, the closer the values (S11 Fig). This indicates that we may use genes with a large M to calculate the shape parameter. However, the larger the M, the fewer the genes with that number of different transcript isoforms. Since the simulation from the Weibull distribution W (a = 0. 39) explains the frequency distribution of each f (k, M), it is reasonable to try to deduce mf (k, M) and the distribution of f (k, M) by pure mathematical theoretical deduction. A theoretical deduction requires the distribution of sums of random variables from the Weibull distribution. Unfortunately, there are currently no approximation formulae that describe the distribution of sums of Weibull random variables [35]. This renders it impossible to find a closed form formula to describe the distribution of each f (k, M). It is similarly impossible to obtain formula (4) from Weibull model by theoretical deduction. Supplemental Information includes 11 figures and four tables can be found with this article online. | Alternative RNA splicing within eukaryotic cells enables each gene to generate multiple different mature transcripts which further encode proteins with distinct or even opposing functions. The relative frequencies of the transcript isoforms generated by a particular gene are essential to the maintenance of normal cellular physiology; however, the underlying mechanisms and principles that govern these frequencies are unknown. We analyzed the frequency distribution of all transcript isoforms in highly purified human T cell subsets and built a simple mathematical model, based on the physical process of alternative splicing, which provides statistical principles that govern this process. This model matches very well with the observed distributions of expression levels and relative frequencies of all transcript isoforms from different tissues and cell lines. Notably, we used this model to elucidate many previously unexplained observations concerning transcript isoform expression. More importantly, this model reveals the existence of simple statistical principles that can be applied to understanding an essential and complex biological process such as alternative splicing. | lay_plos |
Abstract Background Previous studies have shown that young fatherhood is associated with higher later-life mortality. It is unclear whether the association is credible, in the sense that mortality and young fatherhood appear to be associated because both are determined by family-related environmental, socioeconomic and genetic characteristics. Methods We used a household-based 10% sample drawn from the 1950 Finnish census to estimate all-cause mortality of fathers born during 1940–1950. The fathers were followed from age 45 until death, or the end of age 54. We used a standard Cox model and a sibling fixed-effects Cox model to examine whether the effect of young fatherhood was independent of observed adulthood characteristics and unobserved early-life characteristics shared by brothers. Results Men who had their first child before the age of 22 or at ages 22–24 had higher mortality as compared with their brothers who had their first child at the median or mean age of 25–26. Men who had their first child later at ages 30–44 had lower mortality than their brothers who had a first child before the age of 25. The pattern of results from a standard model was similar to that obtained from a fixed-effects sibling model. Men who become fathers at a young age may have an increased risk of dying during middle age — in their late 40s or early 50s, new research finds. In the large Finnish study, researchers found that men who had their first child by age 22 were 26 percent more likely to die in middle age, compared with men who fathered their first child at age 25 or 26. For men who became fathers slightly later, between ages 22 and 24, the risk of dying in middle age was 14 percent higher than that of men who fathered their first child at age 25 or 26. [Beyond Vegetables and Exercise: 5 Surprising Ways to Be Heart Healthy] The findings suggest that young fathers have poorer health than men who become fathers at age 25 or older, but it's not clear why, the researchers said. Future research may tease apart the link between young fatherhood and how a man's family environment, early life circumstances and genetics may affect his risk of midlife death, the researchers wrote in the study, published online today (Aug. 3) in the Journal of Epidemiology & Community Health. It's possible that early fatherhood may interrupt career plans and push young dads into lower-paying jobs, which could impair their health, the researchers said. "Parenting at a young age can be challenging, and it is important that family members and health professionals recognize that it is not only the young mothers, but also the young fathers that may need support," said the study lead author, Elina Einiö, a postdoctoral social researcher at the University of Helsinki. Early fatherhood Previous research had found a link between early fatherhood and an increased risk of early death, but the new study is the first to use brothers as a control group, Einiö said. Other studies have compared young fathers to dads in the general public, but that made it difficult to control for genetic and environmental issues that may also affect men's risk of middle-aged death, Einiö said. "It was unclear whether the association [in earlier studies] was credible," she said. In the new study, Einiö and her colleagues used the men's brothers as a control group, as they were more likely to share genetic and environmental factors than other men in the population. However, the researchers also compared the fathers to each other, regardless of family ties. They collected data on 30,500 men born between 1940 and 1950 in Finland, who all became fathers sometime before they reached age 45. The researchers tracked these fathers until they reached age 54. They found that 15 percent of the men had their first child by age 22, whereas 29 percent had their firstborn between ages 22 and 24, and 18 percent became fathers at ages 25 or 26. The rest of the men (38 percent) became dads at age 27 or older. During the study, about 1 in 20 of the fathers died. The main causes of death were heart attacks (21 percent) and diseases related to alcohol poisoning (16 percent). It turned out that those who became fathers at the oldest ages had the lowest mortality risk. Men who fathered their firstborn between ages 30 and 44 were 25 percent less likely to die in middle age than those who became dads at age 25 or 26. [Sexy Swimmers: 7 Facts About Sperm] Sibling comparisons The researchers also looked at 1,124 of the men's brothers, and found the link held: The brothers who fathered their firstborn by age 22 were 73 percent more likely to die during middle age than the brothers who had their firstborn at age 25 or 26. The findings held true regardless of the men's year of birth, early life circumstances, educational levels, marital status, region of residence and number of children, the researchers said. "The findings of our study suggest that the association between young fatherhood and midlife mortality is likely to be causal," and not simply a correlation, the researchers wrote in the study. It's unclear whether the factors at work when these men became fathers will also affect today's young fathers, but different, new stressors may also impact their health, the researchers wrote. "The findings of our study provide evidence of a need to support young fathers struggling with the demands of family life in order to promote good health behaviors and future health," they wrote in the study. Follow Laura Geggel on Twitter @LauraGeggel. Follow Live Science @livescience, Facebook & Google+. Original article on Live Science. | The younger men are when they become fathers, the more likely they are to die by their 40s or 50s, according to a large new study out of Finland. Researchers say they've seen this link before, but wanted to test it using brothers who share similar DNA to better control for genetic and environmental differences, reports LiveScience. So they looked at 30,500 men born between 1940 and 1950 in Finland, all of whom became fathers before age 45, and then tracked these fathers until they reached 54. Among these dads, 15% had their first child by age 22, 29% between ages 22 and 24, 18% at ages 25 or 26, and 38% at age 27 or older. Reporting in the Journal of Epidemiology and Community Health, the researchers say that the older men are when they father their first child, the less likely they are to die in middle age. In fact, those who had their first child by age 22 were 26% more likely to die in middle age than those who had kids at 25 or 26, while men who fathered their first child between 30 and 45 were 25% less likely to die in middle age than those who were 25 or 26. The researchers also followed 1,124 of the men's brothers, and the link held. In fact, those who fathered their first kid by age 22 were 73% more likely to die during middle age than the brothers who had their firstborn at age 25 or 26-regardless of early life circumstances, education, marital status, region of residence, or number of kids. Thus this "association between young fatherhood and midlife mortality is likely to be causal," they write. The cause has yet to be nailed down, but they surmise that early fatherhood may result in lower education levels, and thus lower-paying jobs, and thus less access to health care. | multi_news |
Future HIV vaccines are expected to induce effective Th1 cell-mediated and Env-specific antibody responses that are necessary to offer protective immunity to HIV infection. However, HIV infections are highly prevalent in helminth endemic areas. Helminth infections induce polarised Th2 responses that may impair HIV vaccine-generated Th1 responses. In this study, we tested if Schistosoma mansoni (Sm) infection altered immune responses to SAAVI candidate HIV vaccines (DNA and MVA) and an HIV-1 gp140 Env protein vaccine (gp140) and whether parasite elimination by chemotherapy or the presence of Sm eggs (SmE) in the absence of active infection influenced the immunogenicity of these vaccines. In addition, we evaluated helminth-associated pathology in DNA and MVA vaccination groups. Mice were chronically infected with Sm and vaccinated with DNA+MVA in a prime+boost combination or MVA+gp140 in concurrent combination regimens. Some Sm-infected mice were treated with praziquantel (PZQ) prior to vaccinations. Other mice were inoculated with SmE before receiving vaccinations. Unvaccinated mice without Sm infection or SmE inoculation served as controls. HIV responses were evaluated in the blood and spleen while Sm-associated pathology was evaluated in the livers. Sm-infected mice had significantly lower magnitudes of HIV-specific cellular responses after vaccination with DNA+MVA or MVA+gp140 compared to uninfected control mice. Similarly, gp140 Env-specific antibody responses were significantly lower in vaccinated Sm-infected mice compared to controls. Treatment with PZQ partially restored cellular but not humoral immune responses in vaccinated Sm-infected mice. Gp140 Env-specific antibody responses were attenuated in mice that were inoculated with SmE compared to controls. Lastly, Sm-infected mice that were vaccinated with DNA+MVA displayed exacerbated liver pathology as indicated by larger granulomas and increased hepatosplenomegaly when compared with unvaccinated Sm-infected mice. This study shows that chronic schistosomiasis attenuates both HIV-specific T-cell and antibody responses and parasite elimination by chemotherapy may partially restore cellular but not antibody immunity, with additional data suggesting that the presence of SmE retained in the tissues after antihelminthic therapy contributes to lack of full immune restoration. Our data further suggest that helminthiasis may compromise HIV vaccine safety. Overall, these findings suggested a potential negative impact on future HIV vaccinations by helminthiasis in endemic areas. Human immunodeficiency virus (HIV) and parasitic helminthic worm infections are highly prevalent and geographically overlap each other in sub Saharan Africa (SSA) [1,2]. A majority of inhabitants harbor at least one or more species of parasitic helminth infection [3–6] and an estimated 50% of the chronically infected individuals living in high-risk rural communities are co-infected with HIV [7]. Furthermore, re-infections after successful treatments are also very common in endemic areas. Therefore, it is very likely that successful future HIV vaccines will be administered to people who already have ongoing helminthiasis or have been previously infected and treated. Current HIV-1 vaccine research suggests that a successful HIV vaccine will need to induce effective T cell and functional antibody responses, where a key component of immune protection would be conferred through a T helper 1 (Th1) immune pathway [8,9]. Induction of potent T cell mediated immune responses has previously been demonstrated using heterologous prime-boost vaccination strategies that utilise DNA and viral vaccine vectors such as modified Vaccinia Ankara (MVA) [10–14], while induction of durable antibody immune responses may require immunisation with HIV envelope protein-based vaccines [15–18]. It is widely accepted that an ideal HIV vaccine should induce both anti-HIV cellular responses and HIV Env-specific antibodies to destroy virus-infected cells and neutralize viruses at portals of entry respectively in order to clear the virus before dissemination into the tissues or block viral entry at the mucosal sites [8,9, 19,20]. During chronic schistosomiasis, parasite eggs are lodged in the liver and intestinal tissue [21,22] resulting in predominantly T-helper 2 (Th2) immune responses [23–27] and the induction of anti-inflammatory regulatory T-cells (Treg) which suppress the innate and adaptive T- and B-cell responses [24,28,29]. This has been shown to lead to general hyporesponsiveness which may adversely impact standard immunizations, by suppressing immune responses to Th1-type vaccine and impairing the expansion of pathogen-specific cytotoxic T lymphocyte (CTL) responses [30–37]. Parasitic helminth infections are currently treated with chemotherapeutic drugs such as praziquantel (PZQ) for schistosomiasis [38–40] and ivermectin or mebendazole for geohelminths [41–43], which are cost-effective interventions. However, re-infection after effective treatment is common and frequent in populations in endemic areas [44]. Several animal and clinical studies have reported that helminth infections impair the outcome of a variety of vaccines, including Salmonella [45]; BCG [30,46–48], tetanus [46,49–51], diphtheria toxoid [52], HBV [53], pneumococcal [54] and live attenuated oral cholera vaccines [55]. However, elimination of helminth infection has also been shown to at least partially restore this abrogation [56]. Furthermore, individuals treated with antihelminthics show higher frequencies of BCG-specific IFN-γ and IL-12 producing cells than untreated helminth infected individuals [30]. Previous HIV vaccines studies reported reduced vaccine-induced immunity in schistosome-infected mice [57] and partial restoration after elimination of helminths [58,59]. However, it is not clear if antibody responses are attenuated as these studies evaluated only cellular responses to Gag as they were monovalent candidate vaccines. Current vaccine candidates and future successful vaccines will likely include multiple immunogens, including Env, in order to broaden the vaccine targets and the capacity to cross-neutralise the majority of transmitted viruses. [60,61]. It is well-accepted that helminth-induced Th2 responses play an important role in host protection [62,63]. Since Th1 and Th2 display reciprocal antagonist, it would be anticipated that HIV vaccine-generated Th1 responses may reduce host immunity against helminth-associated pathology, thereby compromising the safety of an otherwise effective HIV T-cell vaccine. Poxvirus-vectored HIV vaccines are promising candidates for induction of T cell responses and therefore this is a relevant safety issue which remains under-investigated in the helminthic infection background. We have previously described the development of two multigene candidate vaccines, the SAAVI DNA-C2 and SAAVI MVA-C, which express matched HIV-1 subtype C proteins (Gag, RT, Tat, Nef and Env) [11,64–66]. These vaccine candidates have been evaluated further in nonhuman primates [18,67] and Phase 1 clinical trials [15,16]. Also, we have evaluated these vaccines in combination with an HIV-1C gp140ΔV2 Env protein [11,15,16,18,64,65,67]. In the current study, we investigated the impact of chronic schistosomiasis on the immunogenicity of these vaccines in a mouse model and whether the elimination of worms by antihelminthic chemotherapy prior to immunization benefits vaccination outcome. We further investigated whether the S. mansoni eggs (SmE) in the absence of active infection, which mimics the state whereby SmE remain trapped in the tissues shortly after antihelminthic treatment, has an adverse effect on vaccine immunogenicity. Lastly, we evaluated helminth-induced pathology to predict HIV vaccine safety in helminth endemic areas. Our findings show that mice infected with S. mansoni displayed reduced magnitudes of vaccine-specific cellular and humoral responses and anthelminthic treatment with PZQ failed to restore levels of anti-gp140 antibodies while partially reversing the adverse impact on cellular responses. Unexpectedly, vaccination with a T-cell based vaccine regimen was observed to worsen helminth-associated pathology suggesting potential safety concerns in future mass HIV vaccination in helminth endemic areas. To assess the impact of Sm-infection on systemic immune responses, we quantified systemic Th1 and Th2 immune responses in uninfected and Sm-infected mice following MVA+gp140 and DNA+MVA vaccination. Con A stimulation of splenocytes from Sm-infected mice resulted in a significantly reduced IFN-γ: IL-4 ratio in DNA+MVA (p<0. 05) and MVA+gp140 (p<0. 01) vaccine regimens compared to Sm-uninfected mice (Fig 1A). Similarly, Con A-stimulated splenocytes from Sm-infected mice produced significantly lower (p<0. 05) levels of IFN-γ and IL-2 ELISpot responses compared to splenocytes from uninfected mice (Fig 1B). Furthermore, Sm-infected mice had a reduced frequency (p<0. 05) of cytokine-producing CD4+ and CD8+ T cells after re-stimulation of splenocytes with PMA/Ionomycin compared to splenocytes from uninfected control mice (Fig 1C). Moreover, vaccinated Sm-infected mice displayed an impaired type 1 antibody response, indicated by reduced amount of type 1 total antibody isotypes [ (IgG2a (p<0. 001), IgG2b (p<0. 001) ] and increased type 2 antibody isotype [ (total IgG1 (p<0. 001) (Type 2-associated antibodies), IgM (p<0. 001) ] compared to uninfected but vaccinated mice and uninfected control mice (Fig 1D). Sm-infection was accompanied with increased levels of the regulatory cytokine IL-10 (S1C and S1I Fig). To determine the effect of chronic Sm infection on the HIV-1 vaccine-specific T cell immunity, Sm-infected and uninfected mice were vaccinated with either DNA+MVA or MVA+gp140 vaccine regimens and vaccine-specific T cell responses were determined using ELISpot, CBA and flow cytometry. To determine if elimination of schistosome infection prior to vaccination could reverse the effect on those responses, groups of mice were treated with PZQ before vaccinations. Vaccination with DNA+MVA induced significantly higher cumulative HIV-1 specific IFN-γ (2014 ± 177. 4 SFU/106 splenocytes) and IL-2 (174. 1 ± 71. 13 SFU/106 splenocytes) ELISpot responses in uninfected mice compared to Sm-infected mice (IFN-γ: 1420 ± 61. 54 SFU/106 splenocytes and IL-2: 0 SFU/106 splenocytes) (Fig 2A and 2B). Responses to the RT (CD8) peptide induced the highest number of IFN-γ secreting CD8+ and CD4+ T cells (1019 ± 217. 3 SFU/106 splenocytes) compared to other peptides in uninfected mice vaccinated with DNA+MVA. However, IL-2 SFU/106 cells were similar among different peptides stimulations. Similarly, vaccination with MVA+gp140 induced significantly higher cumulative HIV-1 specific IFN-γ (1838 ± 173. 3 SFU/106 splenocytes) and IL-2 (197. 7 ± 20. 12 SFU/106 splenocytes) ELISpot responses in uninfected mice compared to Sm-infected mice (IFN-γ: 1166 ± 132. 2 SFU/106 splenocytes) and IL-2: 11. 89 ± 5. 951 SFU/106 splenocytes) (Fig 2A and 2B). Responses to the Env (CD8) peptide induced the highest number of IFN-γ secreting CD8+ and CD4+ T cells (553. 3 ± 55. 86 SFU/106 splenocytes) compared to other peptides in uninfected mice vaccinated with MVA+gp140. However, IL-2 SFU/106 cells were similar among different peptides stimulations. Vaccination after PZQ treatment had varying effects on the magnitudes of ELISpot responses. For DNA+MVA vaccine regimen, the cumulative magnitude of IFN-γ but not IL-2 SFU/106 cells was still significantly lower in treated mice compared with uninfected mice indicating partial restoration of responses to normal magnitudes (Fig 2A and 2B). In contrast, the cumulative magnitudes of both IFN-γ and IL-2 SFU/106 cells between PZQ-treated and vaccinated mice and uninfected mice were similar for MVA+gp140 vaccine regimen, indicating restoration to near normal SFU/106 cells (Fig 2A and 2B). Th1 cytokine levels were significantly reduced in Sm-infected mice. As shown in Fig 2C–2E, significantly higher levels of net cumulative IFN-γ (6523 ± 282. 0 pg/ml); IL-2 (84. 86 ± 0. 3147 pg/ml) and TNF-α (251. 2 ± 30. 33 pg/ml) (Fig 2C–2E) were released by splenocytes from uninfected mice in the DNA+MVA vaccine regimen compared to lower levels of IFN-γ (1899 ± 244. 6 pg/ml); IL-2 (23. 55 ± 4. 094 pg/ml) and TNF-α (122. 9 ± 17. 45 pg/ml) released in Sm-infected vaccinated mice (Fig 2C–2E). Similarly, for the MVA+gp140 vaccine regimen, significantly higher levels of net cumulative Th1 cytokines: IFN-γ (2416 pg/ml); IL-2 (63. 79 pg/ml) and lower TNF-α (112. 48 pg/ml) were released from splenocytes of uninfected vaccinated mice compared to lower levels of IFN-γ (789 pg/ml); IL-2 (4. 0 pg/ml) and higher TNF-α (123. 87 pg/ml) released in Sm-infected vaccinated mice (Fig 2C–2E). After treatment with PZQ, the levels of IFN-γ and IL-2 were observed to be significantly higher compared to Sm-infected mice for both DNA+MVA and MVA+gp140 vaccine regimens but noticeably lower than those of uninfected vaccinated mice, indicating only partial restoration to normal magnitudes. Furthermore, the frequencies of vaccine-specific cytokine (IFN-γ, IL-2 and TNF-α) producing T cells as determined by flow cytometry showed a similar general trend whereby lower levels of HIV-specific T cells were detected in Sm-infected mice compared with uninfected animals (Fig 2F and 2G). For both vaccine regimens, Pol- and Env- specific CD4+ T cells were undetectable in Sm-infected vaccinated mice whilst they were readily detected at similar levels in both uninfected and PZQ-treated vaccinated mice indicating restoration of cytokine responses by PZQ treatment (Fig 2F). However, Pol- and Env- specific CD8+ T cells were detected in Sm-infected mice at similar levels as the uninfected and PZQ-treated vaccine group except in the MVA+gp140 vaccine regimen where a significantly higher percentage of cumulative cytokine-producing CD8+ T cells in response to the Pol and Env CD8 peptides stimulation was observed in uninfected vaccinated mice (1. 79 ± 0. 04%) compared to Sm-infected vaccinated (1. 49 ± 0. 06%) mice. (Fig 2G). Most of the cytokine producing CD8+ and CD4+ T cells belonged to the effector memory phenotype (Fig 2H) and the profiles of the memory phenotypes were similar in both uninfected and PZQ-treated vaccinated groups. To determine the effect of helminth infection of the development of Env-specific antibody responses, mice were infected with Sm and vaccinated with MVA+gp140 and humoral responses were determined by ELISA. Uninfected and vaccinated mice produced higher amounts of gp140-specific IgG antibodies compared to Sm-infected vaccinated mice across all IgG isotypes (IgG1 [1681 ± 373. 9 vs 140. 8 ± 29. 42 AUs]; IgG2a [4746 ± 1154 vs 71. 14 ± 15. 98 AUs]; IgG2b [2247 ± 553. 9 vs 45. 23 ± 12. 65 AUs]). Treatment of infected mice with PZQ did not restore vaccine-specific antibody responses in infected mice as indicate by significantly lower titers of gp140-specific IgG antibodies across all IgG isotypes (IgG1 [287. 0 ± 79. 96 AUs], IgG2a [866. 1 ± 514. 3 AUs], IgG2b [126. 3 ± 28. 41 AUs]) compared to uninfected vaccinated control mice (Fig 3). We next sought to investigate whether Sm eggs (SmE) alone are capable of attenuating HIV vaccine-specific responses in the absence of an active Sm infection. To achieve this, we sensitized mice with 2 500 SmE intraperitoneally, challenged them with 2 500 SmE intravenously 14 days later and vaccinated them with the MVA+gp140 vaccine regimen. Cumulative cellular responses to the HIV peptides were measured in the spleens using CBA and ELISpot (Fig 4A–4E) and gp140 Env-specific antibodies in the sera (Fig 4F). Cumulative HIV-1 IFN-γ SFU/106 (Fig 4A), and IL-2 SFU/106 (Fig 4B) in SmE-inoculated vaccinated mice were noticeably lower, but not significantly when compared to SmE-free vaccinated mice. Similarly, levels of cumulative IFN-γ; TNF-α and IL-2 secreted by splenocytes were noticeably lower in SmE-inoculated vaccinated mice compared to those secreted by splenocytes from SmE-free vaccinated mice (Fig 4C–4E respectively). However, SmE-inoculated mice had significantly lower amounts of gp140-specific IgG1 (656. 8 ± 177. 1 versus 1203 ± 152. 0 AUs), IgG2a (71. 14 ± 15. 98 versus 238. 1 ± 34. 33 AUs), and IgG2b antibodies (82. 73 ± 18. 20 versus 218. 3 ± 41. 86 AUs) compared to SmE-free mice (Fig 4F), indicating broad attenuation of gp140 Env-specific antibody responses. Furthermore, we confirmed that SmE alone, in the absence of active infection, is capable of skewing the Th1/Th2 profile towards a Th2 response. As shown in Fig 4G, at 9 weeks post inoculation, the IFN-γ: IL-4 ratio was significantly lower in vaccinated SmE-inoculated (367. 0 ± 38. 34 pg/ml) compared to SmE-free (597. 2 ± 72. 93 pg/ml) vaccinated mice after stimulation with Con A. Similarly, stimulation of splenocytes with SEA resulted in a trend towards reduced IFN-γ: IL-4 ratio for SmE-inoculated mice compared to uninfected but vaccinated mice (S2 Fig). We investigated whether vaccination with DNA+MVA exacerbates helminth associated pathology in chronically infected mice by determining granuloma sizes and hydroxyproline content in mouse livers and assessing hepatosplenomegaly. We also investigated whether treatment with PZQ prior to vaccination with the DNA+MVA regimen ameliorates tissue pathology in infected mice. Sm-infected and vaccinated mice developed significantly larger (60. 27 ± 2. 37 mm2) granulomas when compared to all the other groups (vaccinated Sm-infected-PZQ treated [41. 29 ± 1. 66 mm2]; Sm-infected alone [40. 79 ± 2. 38 mm2] and Sm-infected-PZQ treated vaccinated [42. 36 ± 2. 26 mm2]) (Fig 5A and 5B). Sm-infected mice that were either vaccinated or unvaccinated developed hepatosplenomegaly as indicated by larger spleens and livers compared to vaccinated infected mice, naïve mice and vaccinated Sm-infected-PZQ treated mice (Fig 5C and 5D). No difference in hydroxyproline content was observed between unvaccinated Sm-infected and vaccinated Sm-infected mice (Fig 5E). Surprisingly, high levels of hydroxyproline content were observed in unvaccinated Sm-infected-PZQ treated mice compared to Sm-infected (Fig 5E). However, Sm-infected mice had significantly higher number of eggs per gram of liver compared to unvaccinated and vaccinated Sm-infected mice that were treated with PZQ (Fig 5F). The present study investigated the impact of chronic schistosomiasis on the induction of T cell-mediated and antibody responses to candidate HIV vaccines in a mouse model, whether attenuation of vaccine responses can be reversed by pre-vaccination anti-helminthic treatment, and if vaccination with a T cell-based candidate vaccine has an adverse effect on helminth-associated pathology. Firstly, we sought to establish that the mouse model of chronic schistosomiasis worked well as to be expected on our hands. As we expected, our data confirmed that prior to vaccination, Sm-infected mice elicited predominantly Th2 responses and a decreased Th1 cytokine profile (Fig 1A and 1B; S1 Fig), had impaired Th1 cytokine-producing CD8+ and CD4+ T cells (Fig 1C) and an increase in Th2 total antibodies in serum (Fig 1D) as well as enlarged spleens and livers (Fig 5C and 5D) compared to uninfected mice. These findings are consistent with previous reports that demonstrate that Sm infection and a host of other helminths skews the host’s immune responses from a Th1 towards a Th2 type with egg deposition and increased production of IL-4 as key driving forces [23,26,27,63,68–72]. Our data also agrees with previous studies that have demonstrated that IL-10 is also responsible for down-regulation of Th1 responses that is observed in schistosome infections [73,74]. IL-10 has been shown to mediate this down-regulation via an activation-induced cell death process resulting in apoptosis of CD4+ and CD8+ T cells which is also linked to the onset of egg-laying by the helminth parasite and formation of granulomas [75–77]. Our data also suggested a correlation between the decrease of cytokine-producing CD8+ T cells and levels of IgG2a and IgG2b antibodies observed in unvaccinated Sm and vaccinated Sm-infected mice. Previous studies have reported that cytokines produced by CD4+ and CD8+ T cells play an important role in the regulation of the humoral immune response and isotype switching [78–80]. The immunological consequence of a predominant Th2 biasing demonstrated in Sm-infected mice agrees with the concept of reciprocal antagonism between Th1 and Th2 as previously suggested [62,81]. Secretion of IFN-γ and IL-2 by T cells has been associated with suppression of viral replication in HIV-infected individuals and better proliferation of HIV-1-specific CD4+ and CD8+ T cells, suggesting that production of these Th1 cytokines by candidate HIV vaccines is a good indicator of vaccine-mediated immune protection [82] and good indications of polyfunctional CD4+ T cell responses [83]. In this study, we observed that the presence of Sm infection prevented optimal generation of vaccine-specific T cell responses following immunization with SAAVI vaccine candidates (Fig 2). As shown in Fig 2C–2E, vaccine-specific Th1 cumulative cytokine levels (IFN-γ, IL-2 and TNF-α) in recall responses to HIV peptides were significantly reduced in vaccinated Sm-infected compared to Sm-free mice vaccinated with DNA+MVA and MVA+gp140, with exception to TNF-α in MVA+gp140 vaccinated groups which were similar in both vaccinated Sm-infected and Sm-uninfected but vaccinated mice. Similarly, the magnitudes of vaccine-specific cumulative IFN-γ and IL-2 T cell responses measured by ELISpot were significantly lower in Sm-infected vaccinated mice compared to Sm-uninfected mice vaccinated with DNA+MVA and MVA+gp140 (Fig 2A and 2B). A similar decline has been reported in Sm-infected mice compared to uninfected controls following immunization with a DNA-vectored HIV-1 vaccine [57]. Also, the frequencies of vaccine-specific cytokine-producing CD4+ and CD8+ T cells in Sm-infected mice were observed in the current study (Fig 2F and 2G), which may translate to a decrease in antibody population [84]. However, it was noted that the cytokine-producing T cells were predominantly of the effector memory phenotype in both Sm-infected and uninfected vaccinated mice (Fig 2H). Vaccine-induced effector memory T cells have been associated with protection against mucosal SIV challenge in vaccinated rhesus monkeys [85]. In this study, the downregulation of these vaccine-specific cellular responses demonstrates the ability of Sm infection to negatively affect protective potential of candidate HIV-1 vaccines as have suggested by others [57,59]. The antibody responses to the gp140-Env protein were significantly impaired in Sm-infected vaccinated mice (Fig 3). The mean concentration of anti-gp140 antibodies in Sm-infected mice vaccinated with MVA+gp140 was significantly lower than that in Sm-free vaccinated control for all IgG isotypes (IgG1; IgG2 and IgG2b). Antibody responses to specific HIV antigens have been proposed to correlate with protection [82,86]; thus, this was an interesting finding with far-reaching implications for future vaccine development. Elimination of helminth parasites with an antihelminth drug prior to immunization was expected to restore normal vaccine T cell responsiveness as previously demonstrated [59,87,88]. Our results show that treating mice with PZQ reversed tissue pathology as indicated by reduced spleen and liver weights sizes of granulomas and SmE deposition in the liver tissue in PZQ-treated mice compared with untreated mice (Fig 5B–5D and 5F) is consistent with earlier reports [89,90]. Treatment with PZQ has been shown to eliminate adult worms with no direct impact on the SmE already trapped in the tissues other than preventing continued egg deposition in treated subjects [38–40] and potentially restoring normal T cell immune responsiveness. However, our immunological data showed that treating mice prior to vaccination only partially restored the hosts’ vaccine-specific T cells responses (Fig 2). Surprisingly, the partial recovery of these responses did not translate in reduction of the magnitudes of Th2 cytokine responses (S1 Fig). Anti-inflammatory cytokines such as IL-10 remained elevated despite treatment with antihelminth (S1C and S1I Fig) whilst previous studies in which PZQ was used reported similar findings [58,59]. However, it was unclear if the antibody responses were affected. In our study, Th1-type gp140-Env-specific antibody responses in Sm-infected mice were significantly lower despite treatment with PZQ (Fig 3). To our knowledge, no study has evaluated the ability of antihelminthic treatment in the restoration of antibody responses to HIV vaccines. However, it is has been suggested that the duration of infection prior and post treatment is an important factor which determines subsequent restoration of normal responses to vaccination [53,89,90]. A study by Chen et al., showed a recovery of immune balance 16 weeks post-treatment [53]. Also, findings from the studies conducted by Da’dara’s group and Shollenberger’s groups, demonstrated that normal immune responses can be achieved 2–10 weeks post-treatment [58,59]. In contrast to our study, only a 1. 5-week post-treatment period was allowed prior to commencing the vaccinations. Future studies should investigate varying post-treatment periods including multiple vaccinations to establish the optimal recovery durations to start vaccinations after antihelminthic treatment. This is particularly relevant if there will arise a need to integrate future HIV vaccinations with helminthic worms control programmes to improve the vaccination outcomes in helminth-endemic areas. This study went further to demonstrate that Th1 cellular responses elicited by DNA- and MVA- vectored HIV-1 vaccines exacerbated helminth-induced pathology. Sm-infected mice vaccinated with a DNA+MVA regimen had significantly larger granulomas as well as enlarged spleens and livers compared to Sm-infected unvaccinated groups (Fig 5B and 5C). Treatment significantly reduced the pathology; however, a considerable number of eggs were still present in the liver tissues of treated mice (Fig 5F). Surprisingly, the amount of hydroxyproline content, which is a measure of collagen content, was significantly higher in PZQ-treated uninfected mice compared with unvaccinated Sm-infected groups (Fig 5E), suggesting that PZQ treatment may contribute to increased fibrosis of the liver (Fig 5E) as an adverse side effect. A recent study showed that a novel experimental drug (Paeoniflorin) used for treating schistosomiasis managed to control sclerosis better than PZQ [91], pointing to a possible future replacement of PZQ as the antihelminthic drug of choice. Nevertheless, PZQ treatment resulted in reduced number of eggs per gram of liver tissue when compared with Sm-infected untreated mice (Fig 5F). These findings highlighted the scientific challenges in the development of HIV vaccines for SSA, where parasitic helminthiasis is endemic. As discussed above, this study found lack of restoration of vaccine-specific responses upon PZQ-treatment prior to vaccinations while a substantial level of SmE burden was observed in PZQ-treated mice several weeks post-treatment. We, therefore investigated if Sm eggs in the absence of a live infection could result in downregulation of HIV-specific responses. Following an established Sm-egg model [92], mice were inoculated with S. mansoni eggs and then vaccinated with candidate HIV vaccines to evaluate how these eggs affect vaccination outcomes. The IFN-γ: IL-4 ratio for the SmE-sensitized vaccinated mice was significantly smaller than the unsensitized vaccinated mice (Fig 4G), indicating a considerable elevation of Th2 cytokines and down regulation of Th1 in SmE-inoculated mice comparable to SmE-unsensitized mice. However, this polarized Th2 immune responses appear to have had only partial effects on the vaccine-specific T cell responses (Fig 4A–4E). Although reduced, the decrease in vaccine-specific cellular responses observed in SmE-inoculated mice was not significant. Surprisingly as with Sm live infection, antibody responses to HIV Env-gp140 were significantly reduced in the presence of SmE (Fig 4F). As suggested previously [72], this finding confirms that SmE trapped in the tissues play a critical role in attenuating the host’s vaccine-specific responses in Sm-infected individual and may explain why both cellular and antibody responses are still suppressed despite treatment in PZQ-treated groups. Thus, the possible mechanism by which Sm infection suppresses these HIV-specific cellular and humoral responses appear to involve the deposition of SmE in the tissues, which stimulates increased production of IL-4 and IL-10 with concomitant polarization of Th2 immune responses. This in turn may promote activation-induced apoptosis of HIV-specific CD4+ and CD8+ T cells resulting in attenuated induction of Th1 immune responses which are key components of HIV vaccine-specific responses. This finding further highlights another challenge that even after antihelminth treatment with PZQ, generation of optimal vaccine responses may not be achieved as helminth eggs left trapped in the tissues could still attenuate HIV vaccine-induced immune responses. In light of these findings, this study suggests that, whilst elimination of worms can offer an affordable and a simple means of antihelminthic treatment, only partially restoration of immune responsiveness to T cell-based vaccines for HIV-1 and other infectious diseases in helminth endemic settings may be achieved. Thus, it would be important to evaluate vaccine delivery systems that can potentially overcome the negative impact of concurrent helminthiasis as previously suggested [93]. An alternative avenue would be the discovery of antihelminthic drugs which are effective in elimination of SmE from the host’s tissues in addition to the elimination of the parasitic worms. Although, this study gives further information on the impact of helminth infection on the immunogenicity of HIV vaccines, not all immunological aspects could be elucidated. Thus, this study justifies further investigations with use of a nonhuman primate model such as baboons (immune system is highly similar to humans) to obtain a better understanding of these immune responses. The present study demonstrated that chronic helminth infection is associated with Th2-driven attenuation of both T cell and antibody response to HIV vaccines, and elimination of worm by chemotherapy partially restored T cell responses but not necessarily antibody responses. This study further demonstrated that vaccinating helminth-infected individuals with HIV vaccines that induce strong cellular responses may increase the pathology induced by the parasites, rendering the vaccine unsafe in helminth endemic areas. Lastly, this study suggests that the often-suggested integration of antihelminthic treatment programme with a successful future HIV vaccine might not result in improved vaccination outcome unless alternative antihelminthic drugs with a capacity to eliminate schistosome eggs from tissues are developed. In addition, we recommend that HIV vaccine development programs should consider designing vaccines that can overcome the adverse effects of helminth-induced immunity. Biomphalaria glabrata snails (Strain NMRI, NR-21962), infected with Schistosoma mansoni (Strain NMRI) were provided by the Schistosome Research Reagent Resource Center (NIAID, NIH, USA) and maintained in our laboratory for generation of live S. mansoni (Sm) cercariae that were used in this study. S. mansoni eggs (SmE) were purchased from the Theodor Bilharz Research Institute (Schistosome Biological Supply Center, Egypt) and stored at -80°C until use. The integrity and viability of the eggs were evaluated using a light microscope prior to use. Both DNA- and MVA-vectored HIV-1 vaccines have been shown to elicit strong T cell responses in mice [11], nonhuman primates [18,67] and clinical trials [16]. Female BALB/c mice (6–8 weeks old) were purchased from South African Vaccine Producers (SAVP) (Johannesburg, South Africa), housed in an Animal Biosafety Level 2 facility at the University of Cape Town and maintained in accordance with the South African National Guidelines for Use of Animals for Scientific Purposes (SANS Code 10386: 2008) which is also in line with EU Directive 2010/63/EU. Experimental protocols performed in this study were reviewed and approved by the Animal Ethics Committee of the University of Cape Town (UCT AEC: protocol number: 014/026) and performed by qualified personnel in compliance with the South African Veterinary Council regulations. A mixture of ketamine hydrochloride and xylazine was used to anaesthesise mice for all procedures that involved intramuscular or intravenous injections, infection with live Schistosoma mansoni cercariae, collection of blood by cardiac puncture and preparation for euthanasia. Euthanasia was done by cervical dislocation while the animals were under anaesthesia (induced with a mixture of ketamine and xylazine as describe above). The study comprised of three experiments. In Experiment 1 and 2 (Table 1), mice were randomly allocated to six groups (5–8 mice per group) per experiment. Mice receiving exposure to Sm (4 groups) were infected percutaneously via the abdomen with 35 live S. mansoni cercariae at the beginning of the experimentation. Those receiving antihelminthic treatment were given two doses of PZQ (Sigma Aldrich, USA) by oral gavage (500 mg/kg; diluted in water containing 2% Kolliphor EL [Sigma Aldrich, USA]) three days apart, between 8 and 8. 5 weeks post infection. Animals were vaccinated twice, 4 weeks apart, starting at 10 weeks post infection (Table 1). In Experiment 3 (Table 2), mice were allocated to 4 groups (5 mice per group). Two groups were inoculated twice with SmE (2500 eggs per mouse), 14 days apart, initially by intraperitoneal route, and subsequently by intravenous route. As in Experiments 1 and 2, mice were vaccinated twice, 4 weeks apart, starting at 1 week after the second inoculation with SmE (Table 2). Vaccinations with DNA-vectored (100μg DNA per mouse) and MVA-vectored (106 plaque forming units per mouse) HIV-1 vaccines were given intramuscularly. Vaccination with HIV-1 gp140 Env (10μg protein per mouse formulated in Imject Alum adjuvant [Thermo Scientific, USA]) was administered subcutaneously. DNA+MVA vaccine regimens were given as DNA prime and MVA boost vaccine regimens whilst MVA+gp140 Env were given concurrently. Twelve days following the last vaccination, blood was collected by cardiac punctured, mice were euthanised and spleens and livers were harvested for evaluation of HIV immune responses and helminth-induced pathology. Splenocytes were prepared using a standard protocol [95] and stimulated with HIV peptides or mitogen stimulant at 2μg/ml (S1 Table). IFN-γ and IL-2 ELISpot assays were carried out as previously described [11]. Cytometric bead array (CBA) assays were carried out as previously described [96]. Intracellular cytokine staining (ICS) and flow cytometry analysis was performed as previously described [12,14] with minor modifications. Briefly, cells were stained with a viability dye, violet amine reactive dye (ViViD; Invitrogen, USA), at a pre-determined optimal concentration before staining for cell surface molecules with the following fluorochrome-conjugated antibodies: anti-CD3-Alexa 700, anti-CD4-PE-Cy7, anti-αCD8-APC-Cy7, anti-CD62L-APC, and anti-CD44-FITC diluted to 0. 2μg in staining buffer (BD Biosciences, USA). Further intracellular cytokine staining was done with pooled PE-conjugated anti-TNF (0. 2μg) anti-IL-2 (0. 06μg) and anti-IFN-γ (0. 06μg) antibodies diluted in Perm/Wash buffer (BD Biosciences, USA). To measure the level of HIV gp140 Env-specific antibodies in mouse sera, a standardised ELISA assay was established as previously described [97]. Briefly, ELISA plates were coated with 0. 5μg/ml of gp140 protein diluted in PBS and incubated overnight at 4°C. Test mouse sera (diluted 1: 1000) were tested in duplicates. Mouse sera obtained from unvaccinated mice was used as a negative control while a reference serum sample prepared from mice previously vaccinated with HIV gp140 protein was used in 12 two-fold dilutions, starting at 1: 100, to generate a standard curve. For detection, appropriate secondary anti-mouse antibodies conjugated with horseradish peroxidase were used including the three anti-mouse IgG isotypes (IgG1; IgG2a and IgG2b; Southern Biotechnology). After colour development using tetramethyl-benzidine substrate (TMB; KPL, USA), the optical density (OD) was measured at 450nm (with a reference filter set at 540 nm) using a microplate reader (Molecular Devices Corporation, USA). Based on the constants of the standard curve generated from the serially diluted reference sample, the reciprocal dilution giving an OD value of 1 (against gp140) was assigned a value of 1000 antibody units (AUs). The negative control (unvaccinated mouse serum) was assigned a reciprocal dilution of 0 and zero AUs. A reference sample was used on each ELISA plate to generate a standard curve from which the assigned AUs were used to extrapolate for test samples at a fixed dilution of 1: 1000. A cut-off value for positive antibody responses was set at 2 x the OD value of the negative control serum (unvaccinated) and those below the cut of value were assigned an antibody unit of zero. Spleens and livers were weighed prior to processing for immunological evaluation in the laboratory to determine if HIV vaccination worsens helminth-associated pathology. Livers were then fixed in 4% (v/v) buffered formalin solution. The fixed samples were then embedded in wax and processed. Sections (5–7μm) were stained with hematoxylin and eosin (H&E) (Sigma Aldrich, USA) to show aggregation of white blood cells around the Sm eggs and Chromotrop-aniline blue solution (CAB) (Sigma Aldrich, USA) and counterstained with Weigert' s hematoxylin (Sigma Aldrich, USA) to stain for collagen. Micrographs of liver granuloma were captured using a Nikon 90i wide-field microscope using a 5. 0 megapixel colour digital camera running Nikon’s NIS-Elements v. 4. 30 software (Nikon Instruments Inc., USA). The area of each granuloma containing a single egg was measured with the ImageJ 1. 34 software (National Institutes of Health, USA). A total of 25–30 granulomas per slide per mouse were included in the analyses. Data was presented as a mean area of each granuloma containing a single egg. The number of eggs per gram of liver was determined by counting individual eggs from hydrolysed liver under a microscope. Hydroxyproline content, which is a direct measure of collagen content in liver was determined using a modified hydroxyproline protocol by Bergman and Loxley [98]. Briefly, liver samples were weighed, hydrolyzed and added to a 40mg Dowex/Norit mixture. The supernatants were neutralised with 1% phenolphthalein and titrated against 10 M NaOH. An aliquot was mixed with isopropanol and added to chloramine-T/citrate buffer solution (pH 6. 5). Erlich’s reagent (95% ethanol containing dimethylaminobenzaldehyde (DMAB) and concentrated hydrochloric acid) was added and absorbance was read at 570 nm. Hydroxyproline levels were calculated using 4-hydroxy-L-proline (Sigma Aldrich, USA) as a standard, and results were expressed as μmoles hydroxyproline per weight of tissue that contained 104 eggs. Statistical analysis was performed using Prism version 5. 0 (GraphPad Software, USA). The t-test for independent unpaired non-parametric comparisons was applied to assess the level of significance between means ±SEM. Three independent experiments were conducted and all tests were two-tailed. p values <0. 05 were considered as significant. The false discovery rate (FDR) with Benjamini-Hochberg-adjusted p<0. 05 was performed as previously described [99]. | Chronic parasitic worm infections are thought to reduce the efficacy of vaccines. Given that HIV and worm infections are common in sub-Saharan Africa (SSA) and their geographical distribution vastly overlaps, it is likely that future HIV vaccines in SSA will be administered to a large proportion of people with chronic worm infections. This study examined the impact of S. mansoni worm infections on the immunogenicity of candidate HIV vaccines in a mouse model. S. mansoni worm-infected animals had lower magnitudes of HIV vaccine responses compared with uninfected animals and elimination of worms by praziquantel treatment prior to vaccination conferred only partial restoration of normal immune responses to vaccination. The presence of S. mansoni eggs trapped in the tissues in the absence of live infection was associated with poor vaccine responses. In addition, this study found that effective immunization with some HIV vaccine regimens could potentially worsen worm-associated pathology when given to infected individuals. These novel findings suggest further research in HIV vaccines and future vaccination policies regarding the current clinical vaccines and future HIV vaccination with respect to parasitic worm infections especially in SSA. | lay_plos |
[PREVIOUSLY_ON]
Rebekah: Hello, Nik.
Klaus: (gapes at her, clearly shocked, but eventually smiles) Rebekah.
Freya: You know who I am.
Rebekah: Freya.
Freya: Tell our brothers I'll be coming to see them soon.
Mary: Do you renounce your Alpha status?
Hayley: These attacks, they're only gonna get worse. We need to get married as soon as possible.
Finn: I curse you to this body, a meaningless, lonely death.
Elijah: Niklaus.
Klaus (on phone): Finn knows about Hope, about everything. He is on his way.
(Gas hissing)
Elijah: Gas has pervaded every single room in this home. Good-bye, brother.
[SCENE_BREAK]
[ BACK ROADS OF ARKANSAS ]
(It is night time now, and Cami and Hope are speeding down the road in the SUV. Hope is crying, and Cami turns back to comfort her. Cami looks panicked)
Cami: (anxious) It's okay, sweet girl. I just need to find a pay phone.
(Cami finally finds a run-down auto garage and pulls in. There is a car with its hood popped in the parking lot, but otherwise it is completely deserted. Once the car is parked, Cami gets out and rushes over to pick up the still-crying Hope from the back seat before they head toward the pay phone. After a moment, Cami trips and accidentally drops all of her change onto the ground)
Cami: (frustrated) Damn it! (Hope starts to cry harder, and Cami looks guilty) Oh, I'm sorry! I'm sorry. You're being so brave, the least I can do is watch my...
(Cami cuts herself off when she hears a metallic noise nearby and freezes in place. She gulps nervously before raising her voice)
Cami: Whoever's out there, if you try anything, I will gouge out your eyes!
(She pants nervously before hearing a voice behind her. It's Elijah, whose clothes are burnt in places and whose body is covered in ash, but is otherwise unscathed)
Elijah: Actually, Camille, that's probably not necessary.
(He reaches his hand out to her to communicate that she should come over to him quickly. As she does, she looks at him in panic)
Cami: What the hell happened back there?
Elijah: (ushers her toward the SUV) That's a discussion for the car. (He checks to make sure no one is watching them) Let's move!
[SCENE_BREAK]
[ MIKAELSON COMPOUND ]
(Hayley is in her bedroom, quickly packing up a bag of clothes, when Klaus enters the room)
Klaus: What do you think you're doing?
Hayley: (continues packing) Elijah said they're on the road. So, I'm going to go to them and get my daughter.
Klaus: Hayley...
Hayley: (cuts him off) Do not tell me that it's not safe! I'll tell you what's not safe: blowing up a house just to keep your evil brother from finding her!
Klaus: (sighs) We will deal with Finn.
Hayley: (raises her voice) And then what? Every time you kill him, he's just going to jump into another body.
Klaus: We tried running. We tried hiding. Neither will work.
Hayley: So what's your bright idea?
Klaus: As it happens, I am working on a plan as we speak! One which will be bolstered greatly if you just calm down and keep your eye on the prize!
Hayley: (talks over him) Do not manage me, I have every right to...
Klaus: (interrupts her) Hayley, you are getting married today. An act which will seal the loyalty of all the wolves that answer to Finn! (Hayley gives him a look) You will be queen to an army. (He takes the bag out of her hands and sets it on the bed) And a queen does not run.
[SCENE_BREAK]
[ ST. ANNE'S CHURCH ]
(In Davina's attic room, Josh is groaning in pain and pinching the bridge of his nose as though he has a headache. Davina returns to the room to check on him)
Davina: Well, you still don't look so good.
Josh: Ugh, this is worse than the Halloween Hangover of 2011. (Davina giggles, and Josh sighs) I went after Aiden, didn't I?
(Davina's smile falls slightly, and she kneels in front of him to look him in the eyes)
Davina: You know, he was up all day, all night with you until Marcel called and said that the spell was broken.
Josh: (nods) You guys totally saved my ass.
(Suddenly, Kol walks into the room, and Davina rolls her eyes playfully)
Kol: Yeah, well, don't mention it! (He tosses Josh a blood bag) There you go, here's a little go-juice. So, you should be, well... (He gestures toward the door) ...going.
(Davina giggles and gives Josh a hug before he leaves)
Davina: Bye.
(Once Josh has left, Kol walks into the bedroom toward Davina)
Davina: (crosses her arms) So, where were you yesterday?
Kol: Well, you know, being a hero, saving the day, the usual!
Davina: Ah. And you didn't think I could help?
Kol: Well, you were with your mate!
Davina: You couldn't have called me? It takes ten seconds.
Kol: (gasps mockingly) Oh-ho, this is a right proper spat we're having? It's almost as if we're, uh... oh, what is that phrase? "Going steady?"
Davina: (rolls her eyes) You wish.
Kol: Ah, maybe! Maybe I could make it up to you? By, uh, well... finishing a mystical dagger? What do you say?
[SCENE_BREAK]
[ THE BAYOU ]
(Aiden and Jackson are standing out on the docks of the lake, where they're having the traditional Viking funeral for the Alphas who were killed in The Devil is Damned. They both look torn)
Aiden: All those Alphas... dead. That's a lot of wolves that are gonna want revenge.
Jackson: We make it clear the witches were behind this. The vampires were manipulated by dark magic. This... wasn't their fault.
(Their conversation is interrupted by Klaus, who comes up behind them and greets them with Hayley's bag from earlier, only it is now covered in blood. He throws it at their feet)
Klaus: Gentlemen. I come bearing gifts.
Jackson: (wary) The hell did you do?
Klaus: I removed the heads of the wolf leaders who refused to relinquish Finn's moonlight rings!
Jackson: (unamused) You brought me a bag of werewolf heads?
Klaus: (shrugs) Well, I'd hoped you'd see it as an early wedding present! Besides, I can't have witch sympathizers in my army.
Jackson: (approaches him) The wolves are not now, nor ever will be your army.
Klaus: You know, you are a brave and selfless leader, Jackson. (Jackson nods in agreement) And I'm positive you'll remain so for the entire duration of your reign. (Confused, Aiden joins Jackson's side as Klaus continues) Festivities begin at eight at my compound. Spread the word. And, do arrive early enough to clean yourself up. It's your wedding day, for God's sake!
(Klaus smiles at them before turning and leaving. Aiden and Jackson look at each other, clearly confused and suspicious. Jackson kicks at the bag of werewolf heads)
Jackson: Am I crazy, or did he just do his version of a nice thing?
[SCENE_BREAK]
[ ARKANSAS MORGUE / LAFAYETTE CEMETERY ]
(Freya has just entered the morgue, where she finds two bodies in black body bags laying on the autopsy tables. She walks toward the nearest one and unzips the bag to find Finn's body, which is badly burnt on one side. She lays her hand on his chest)
(At the cemetery, Davina and Kol are in the Claire tomb, preparing to do the Kemiya spell to turn the silver dagger into gold so it can be used against Klaus. Kol lights a bunsen burner as Davina sets up the ingredients)
Kol: Are you ready?
Davina: I don't know. Never done this before, remember?
Kol: I've never actually completed the spell itself, but, then, I've never had an accomplice as powerful as you.
(He pulls the silver dagger out of his trunk and holds it up as he smiles at her. She giggles as he returns to her)
Davina: So, let's do it, then!
Kol: Let's do it.
(At the morgue, Freya looks more closely at Finn's burned body, revealing that he's wearing her blue pendant talisman. She smiles at him)
(At the Claire tomb, Davina and Kol are both holding onto the handle of the dagger as they hold it into the flame of the bunsen burner as they cast the spell)
Kol & Davina: A Loki gae la lidi. A Loki gae la lidi.
(At the morgue, Freya is pouring a handful of salt over Finn's body as she casts her own spell)
Freya: (chants) Helbred bransar, belaste herte, begin panet. Helbred bransar, belaste herte, begin panet.
(The spell continues at the Claire tomb)
Kol & Davina: A Loki gae la lidi. A Loki gae la lidi.
(The spell continues at the morgue)
Freya: (chants louder) Helbred bransar, belaste herte, begin panet. Helbred bransar, belaste herte, begin panet.
(At the Claire tomb, the spell continues)
Kol & Davina: A Loki gae la lidi. A Loki gae la lidi.
(Suddenly, the dagger becomes so hot that both Kol and Davina drop it onto the floor. They look down at it in shock before Davina kneels and picks up the dagger, which has turned from silver to gold. Davina smiles at him proudly)
Kol: (amazed) I think it worked!
(Davina can't help but chuckle in amazement. Kol looks at her in the eyes before leaning forward and kissing her gently. Davina pulls away briefly to set the dagger on the table before she wraps her arms around his neck and kisses him more passionately)
(Freya is finishing her spell at the morgue)
Freya: (chants louder) Helbreth renzar bilaste herte begimbi. Helbreth renzar bilaste herte begimbi.
(At the Claire tomb, Kol suddenly stops kissing Davina and gasps in shock. As he backs away from her, his nose starts to bleed pretty heavily. He leans against the nearby table and wipes the blood from his lip with one hand)
Davina: (worried) Kol, are you okay?
(At the morgue, Freya's blue pendant talisman starts to glow around Finn's neck as her spell takes effect)
Freya: Helbreth renzar bilaste herte behimbi. Helbreth renzar bilaste herte behimbi
(Suddenly, Finn awakens with a gasp, though his burns are not fully healed)
[SCENE_BREAK]
[ LAFAYETTE CEMETERY ]
(At the Claire tomb, Kol is recovering from his nosebleed, mopping up the blood with a tissue as Davina watches him, clearly concerned)
Davina: Kol, are you sure you're alright?
(Kol is turned away from her, and looks extremely scared and worried, but when he turns to face her, he puts on a fake smile)
Kol: I'm fine! Oh, a spell like that takes his toll, doesn't it?
(Davina looks as though she doesn't believe him, but humors him anyway and nods in agreement)
Kol: (points to the dagger) And you hide that for now, okay? Because, uh, well, today, we've got a reason to celebrate. (He walks toward her) Come to the wedding with me.
Davina: (smiles and hands him the dagger) Fine. But, if there's music? We're dancing.
(Davina turns and leaves the tomb to get ready. Once she's gone, Kol's smile falls, and he pulls out his phone and dials a number)
Kol: (on the phone) Beks? Beks, it's your brother. I need a favor.
[SCENE_BREAK]
[ MIKAELSON COMPOUND ]
(Hayley is in her bedroom, looking through a wardrobe of old dresses, hats, and jewelry, when Rebekah appears in the doorway, hiding something in her hand from view)
Rebekah: Alright, bad news first: I will have to miss your nuptials. (Hayley looks at her in disappointment) Kol has a bit of a life-and-death matter he needs help dealing with.
Hayley: Anything I can do, Rebekah?
Rebekah: It's witch business. Which now seems to be my thing. (Hayley laughs and rolls her eyes) Besides, you have enough to deal with! On that note... (She pulls out a white lace wedding dress that she was hiding behind the door frame, and Hayley's eyes widen in surprise) It's white, which won't fool anyone. But you can't get married in skinny jeans and combat boots.
(She hands Hayley the dress, and Hayley hums in gratitude)
Hayley: Oh, Rebekah... Thank you.
(Rebekah smiles at her and watches as Hayley stands in front of her full-length mirror and holds the dress against her body to see how it looks)
Hayley: It's beautiful.
Rebekah: Well, I happen to be the only woman alive who's commissioned wedding dresses in five different centuries. Never made it down the aisle, of course.
Hayley: Well, it's not too late! From what I understand, Marcel is still single... (Rebekah smiles shyly and licks her lips) Have you told him yet that you're... you?
Rebekah: I thought I'd hold off on that for the moment. Only complicates matters. And, I'm not exactly sure how long this... (She gestures at her body) .. is going to last.
Hayley: (chuckles) Right.
Rebekah: (smiles and walks toward her) Hayley... I wanted to say... Now, you might be marrying Jackson Kenner of the boozy backwater Kenners, but you're still one of us. A Mikaelson. Always will be.
Hayley: (smiles happily) Gosh, that would be such a nice compliment if it didn't link me to so many homicidal lunatics.
(Rebekah smiles and winks at Hayley before she turns and leaves the room. Hayley turns back to the mirror and admires her new dress)
[SCENE_BREAK]
[ FRENCH QUARTER ]
(Josh is waiting around in an alley, pacing nervously, when Aiden turns into the alley from the street and walks toward him)
Josh: (anxious) Thanks for coming. Look, I know I owe you a... (Aiden immediately opens his arms and lunges toward him to give him a big hug) .. massive apology...
Aiden: You scared the hell out of me.
Josh: I know, and I'm sorry. I just...
(Josh cups the side of Aiden's face gently with his hand, but Aiden nervously pulls it away)
Aiden: Look, um... Hayley and Jackson are getting married today. Quarter's gonna be packed with wolves. 'Kay, and after this wedding? Our whole pack will inherit Hayley's control of her wolf form! That means werewolves will be able to turn at will; we'll be that much more deadly to vampires. To you. (Josh looks at him with a hurt expression) I... think we need a time-out.
Josh: (confused) Whoa, whoa! I'm sorry, is this the world's worst breakup speech? Because if so, just say it like it is: you wanna call things off because it would look bad for the werewolf VP to be dating... me.
Aiden: No! Dating me puts a target on your back! I'm just trying to keep you safe!
Josh: (angry) Okay, you know what? I can't remember the last time I was safe.
(Josh, visibly hurt, sighs and walks away from him. Aiden looks upset)
Aiden: Josh...
(Josh keeps walking and doesn't look back, leaving Aiden alone in the alley)
[SCENE_BREAK]
[ MIKAELSON COMPOUND ]
(The ballroom of the compound is full of decorators and caterers who are setting up for the impending wedding. Jackson has just arrived, and he and Hayley walk around and take it all in)
Jackson: Ohhhh, I so do not belong in your world.
Hayley: (scoffs) My world? Give me some credit. I'm not exactly the girl who sat around fantasizing about her wedding day. And, if I had... (She gestures to a huge wedding cake being pushed on a cart by a caterer) .. it probably would have looked a little less... this.
Jackson: Yeah... (He turns and looks at Hayley) You getting cold feet?
Hayley: (shakes head) No. This is what's right. For Hope, for our pack.
(Jackson nods in agreement as Klaus enters the room)
Klaus: Hayley. If I might intrude, there's someone who wishes to say hello.
(Hayley and Jackson share a look before she joins Klaus and walks into the courtyard, with Jackson following behind her. Just as they make it into the courtyard, Cami and Elijah, who is now wearing fresh clothes, walk in from the opposite direction. Cami has Hope in her arms, and they smile at them)
Hayley: (to Klaus) You brought them here? Finn could be anywhere!
Klaus: I've taken precautions. There'll be no uninvited guests at your wedding, and after, your wolves will be the first line of defense to this home. No more running, Queen.
(Klaus smiles at her and gestures for her to go see Hope. Elijah nods at them encouragingly. Hayley goes and takes Hope out of Cami's arms, and Hope immediately begins cooing and babbling happily when she finally gets to see her mother. Hayley and Cami both giggle contently as Hayley walks toward Jackson)
Hayley: Hope, this is Jackson. Jackson, this is Hope.
(Jackson smiles when he sees her, and Hope stares at him in wonder. When Jackson holds out his hand, Hope clutches his fingers tightly and coos at him. Hayley and Jackson can't help but smile at her, while Elijah watches them from afar, looking troubled)
(In Klaus' study, Klaus pours himself a shot of bourbon and drinks it quickly as Elijah joins him. Klaus pours himself another shot and doesn't turn to face his brother right away)
Elijah: I thought your daughter's return would please you.
Klaus: I'm overjoyed. (He drinks the second shot) And I'll be even more so provided you do nothing to dissuade Hayley from going through with this wedding.
Elijah: (confused) Is there something you wish to discuss, Niklaus?
Klaus: (turns to face Elijah) Everyone knows you're in love with her. But, Hayley has a duty to this family, and so do you.
Elijah: I was under the impression this was Hayley's choice. Tell me you did nothing to bully this decision.
Klaus: We're mobilizing an army. She will do what is asked of her. And you will do nothing to prevent that.
Elijah: (strides over to Klaus to look him in the eye) Unless, of course, I learn that she was pressured into sacrificing her freedom in the name of some political alliance.
Klaus: Hayley is putting family first. I suggest you do the same.
Elijah: Family is always first.
Klaus: (claps Elijah on the shoulder) Well, then we won't have a problem, will we?
[SCENE_BREAK]
[ LAFAYETTE CEMETERY ]
(In the Claire tomb, Rebekah and Kol are holding both of each others hands as they do a spell to try to heal Kol, surrounded by lit candles. Kol channels Rebekah's power and mutters the incantation)
Kol: (chanting) Voltre mezino cadau. Voltre mezino cadau.
(Suddenly, Kol gets a piercing ringing sound in his ears as his nose starts to bleed again. He angrily lets go of Rebekah's hands and swipes many of the materials off of the table and onto the floor as he yells in frustration. Rebekah looks at him sympathetically and reaches out to rub his arm)
Kol: NO!
Rebekah: Calm down. You need to keep your wits if you're to beat this.
(Kol sits down on the bench by the table, looking exhausted and defeated)
Kol: There is no beating it. (Rebekah looks at him sadly) It's latched on like a vice.
Rebekah: So you're giving up? What happened to the brother I used to know? The one who laughed death in the face?
Kol: (quietly) It's a lot easier to do when you haven't died already once. And I hated it the first time. The ironic thing is that I actually preferred this go-round. Being a witch. No heightened emotion, no bloodlust. It was just me. For a while, anyway.
(He laughs bitterly. Rebekah is speechless)
[SCENE_BREAK]
[ MIKAELSON COMPOUND ]
(Hayley is getting dressed in her bedroom in preparation for the wedding. She's wearing the wedding dress Rebekah got her, and her hair is loose, with braids on the sides that are secured in the back with small white flowers. Hope is sitting on the floor, playing with her toys and cooing happily next to her)
Hayley: Ah-ha-ha! I still can't believe that you're here! (She kneels down in front of Hope) Now, Mommy has to go and do this big thing, but don't worry! Because Cami will watch you, and after that, I'm not letting you out of my sight!
(Hope makes a squeaky excited noise and chews on a little teddy bear as she leans into Hayley's face, and Hayley laughs, clearly thrilled to have her there. After a moment, Elijah appears in the doorway and watches them silently for a moment before knocking on the door to get her attention. When Hayley sees him, she smiles, and he watches her as she stands to her feet)
Elijah: You look perfect. (He hesitates for a moment) Hayley, I understand that this arrangement is important to your cause, and I will do nothing to dissuade you from it. I would be remiss if I didn't tell you, at least once...
Hayley: (quietly cuts him off) Don't. Don't say it. You know, ever since the first day that I met you, I have felt everything for you. And all this time, you were never able to say how you feel about me. And I get it... you can't just be the guy who says how he feels. (She looks down at her engagement ring and wrings her hands nervously) But Jackson is. And I think that I can be happy with him. (Elijah looks at her with tears in his eyes, and silently starts to cry) And I just wanna be happy, Elijah. So, whatever you're going to say to me... (She shakes her head) Please don't say it.
(Elijah looks devastated, but nods in understanding before leaving the room without another word. Hayley looks sad and guilty)
(In another room, Jackson is getting ready for the ceremony. He's trying to tie his tie, but his hands are shaking nervously, and he frustratedly unknots the tie and sighs. Aiden, standing behind him, chuckles in amusement as he pours them drinks)
Aiden: Need a hand with that?
Jackson: (tosses the tie aside in annoyance) No.
Aiden: Look, I'm sorry Oliver couldn't be here.
Jackson: Yeah, me too. (He takes the drink Aiden offers him) Although, I'm sure he'd have a lot to say about me in this get-up in the vampire house!
(They both laugh and drink)
Aiden: So, you really think the wolves will be cool with the vampires?
Jackson: (nods) And if not, screw 'em! I mean, we're done chasing old grudges! We got real enemies to worry about. (Aiden takes a huge gulp of his drink, looking nervous, and Jackson suddenly realizes what Aiden meant) This ain't about vamps in general, now, is it?
Aiden: (embarrassed) Look, I know you know about me and Josh. (Jackson nods) And I'm not asking you for permission, okay?
Jackson: Good! 'Cause I'm pretty sure we had the "love is love" talk when you were seventeen.
Aiden: (rolls his eyes) Yeah.
Jackson: (laughs) Hey, he's good enough for you? (He shrugs) Then it's good.
Aiden: I just don't want him caught in the crossfire, you know?
Jackson: (claps him on the shoulder) That won't be easy. But we got no chance of winning this fight without something to fight for.
(Aiden nods, and Jackson claps him affectionately on the shoulder again. Hayley appears in the doorway, and when Aiden notices her, Jackson follows his gaze and turn to find Hayley smiling at him. He's taken away by the sight of her in her wedding dress)
Jackson: (stunned) Wow.
(Hayley laughs nervously)
Aiden: (smiles) I think that's my cue.
(Aiden leaves Hayley and Jackson alone in the room)
Hayley: There's no rule about seeing each other before the ritual, is there?
(Jackson smiles widely and shakes his head. Hayley notices Jackson's tie laying on the chair and gestures toward it before walking to pick it up)
Jackson: (laughs nervously) Um, heh-heh... is everything okay?
Hayley: (walks over to start tying his tie around his neck) Yeah! I was just getting a little nervous. (She smiles and chuckles) Wanted to see you, make sure you weren't wearing a flannel tux.
(Jackson laughs nervously again and watches as she ties his tie and straightens his collar)
Jackson: (whispers) Thank you. Um, I'm actually glad you're here; I got something for ya. (He walks over to the table and picks up a small wooden box. He opens it to show her a silver chain with a crescent moon pendant with a green stone in it) Um, the, uh... the stone is moss agate. You were born under a planting moon, so it's your mineral totem. It symbolizes healing and courage, and after everything that you've done for our pack, I'd say that's just about right.
Hayley: I don't know what to say, Jackson.
(Jackson takes out the necklace and walks around Hayley to fasten it around her neck)
Jackson: You're wearing Rebekah's dress, getting married in Klaus' house. I figured you should have at least one thing that is just yours.
(Hayley smiles, clearly touched by his words)
[SCENE_BREAK]
[ MIKAELSON COMPOUND - UNIFICATION CEREMONY ]
(The guests, both vampires and werewolves, are all seated in the ballroom just before the ceremony is about to begin. Davina enters the room in a blue velvet dress and gets a program from a young girl. She looks around for a seat until Josh calls out to her)
Josh: Davina!
(Davina smiles at him and sits down next to him. A moment later, Aiden arrives. When he sees Josh and Davina sitting on his left, he walks over to them)
Aiden: (to Davina) May I?
(She kindly gets up so Aiden can sit next to Josh, and when he's seated, she sits next to Aiden. Aiden smiles at Josh and reaches for his hand, squeezing it tightly. Hayley slowly walks to the doorway to the ballroom and stands still for a moment as all the guests stand to greet her. She nods at various people when she sees them and waits for Jackson, who appears behind her a moment later. Hayley and Jackson clasp hands and smile at each other as they walk down the aisle. When they reach the two staircases, they split apart so they can each walk up the two staircases and meet on the balcony, where Mary is waiting for them to preside over the ceremony. An orchestra plays as they make their ascent, and they smile at each other from across the room until they make it to the altar)
Mary: Please, be seated. (The guests all sit back in their chairs) We gather together as a community seeking peace, inspired by this couple standing before you. (Across the balcony on the other side of the courtyard, Klaus stands and watches the ceremony. Hayley catches his eye and smiles at him, but his face remains blank. Elijah joins him a moment later and stands at his side) There was a time when werewolves saw themselves not as cursed, but blessed with connection to our most pure selves. And tonight, we honor that blessing with the long-awaited unification of the two Crescent bloodlines. (Mary performs a hand-fasting by binding Hayley and Jackson's wrists with baby's breath flowers) In doing so, we choose to embrace Hayley's vampire nature. With this union, Hayley will share her unique gifts with her pack. (Mary places a long lit match into Hayley and Jackson's linked hands to light their ceremonial candle) And now, your vows.
Jackson: (takes a deep breath) I pledge to honor you and defend you and yours above all others.
Hayley: To share in blessings and burdens. To be your advocate, your champion.
Jackson: To be your comfort, your sanctuary, and for as long as we both shall live.
Hayley: To be your family.
Jackson: To be your family.
(They smile at each other and light their candle together. Elijah looks as though he's about to cry. Mary smiles at them)
Mary: You two have endured all the traditional werewolf rituals and trials. There is only one remaining. Jackson, you may kiss your bride.
(Jackson looks almost scared, but Hayley smiles at him encouragingly. He stares at her for a long moment before he cups her face in his hand and kisses her. Elijah looks away, and Klaus looks at him sympathetically and sighs. When Hayley and Jackson finally pull away, both of their eyes glow gold, indicating that the Unification Ceremony worked. They smile at each other happily and turn to look at the crowd. The guests all look at each other curiously. Josh and Davina are smiling, and when Aiden looks back to smile at Josh, his eyes glow gold as well. Everyone begins to smile and chatter, all thrilled that the ceremony has empowered their pack. Marcel and Gia look at each other, slightly nervous at this new development, but Klaus looks relieved)
(Klaus walks out onto the balcony overlooking the courtyard, where the reception is being held. Elijah follows behind him and joins him, wagging his finger at him knowingly)
Elijah: I know that look. I see it all too often. What are you planning?
Klaus: I'm sure I don't know what you're talking about.
Elijah: Jackson marries the mother of your child, in our home, solidifying his rule over the wolves you yourself once sought to control. Talk to me.
Klaus: Let Jackson have Hayley. Although, he isn't exactly fit to lead an army tasked with protecting my daughter. His reign will be short-lived.
Elijah: Niklaus, you cannot honestly believe that I would allow you to harm Jackson on the day of Hayley's wedding...
Klaus: (cuts him off) He's not one of us, Elijah! He's mortal. Mortals perish.
(Klaus goes to walk away, but Elijah grabs him roughly by the arm and stops him)
Elijah: What are you doing?
Klaus: Confide in me, brother. How do you feel when you see Hayley look at him?
Elijah: Niklaus, I'm warning you...
Klaus: (interrupts him) Face the facts! You're even now forcing yourself to deny you want Jackson dead just as much as I do. In fact, I think you want it more.
(Klaus smiles at him and walks away, leaving Elijah alone to think)
[SCENE_BREAK]
[ FRENCH QUARTER / MIKAELSON COMPOUND ]
(A parade is being held in the streets in celebration of Hayley and Jackson's wedding. It is led by several police officers on motorcycles, followed by Hayley, who has a white lace parasol in her hand and is dancing with Jackson at her side. Behind them is a marching band playing a bright jazz tune, along with the rest of the wedding guests, who are dancing and having a great time in the streets. Klaus watches the parade march down the main street when Elijah once again joins him)
Klaus: (annoyed) Are you to be my chaperon the entire evening, brother?
Elijah: Chaperon, steward, babysitter... whichever term tickles your fancy most, brother. You're welcome to indulge whomever and whatever you please. I would only ask that you refrain from any homicidal behavior...
Klaus: Just one day back, and you're already more than fulfilled your quota for irritating brotherly conduct.
(He turns to leave, but Elijah follows him)
Elijah: Let me make myself quite clear, Niklaus. So long as Jackson brings even an inkling of joy into Hayley's life, you are not to go near him.
Klaus: It's amusing listening to you defend the man who married the woman you love. But then, you've always coveted that which is not yours to have.
Elijah: Just listen to yourself! Fueled by your delusions of persecution! Think, Niklaus... if you kill Jackson, the wolves will descend into chaos. You're acting out of fear, terrified that Jackson might be a better father to Hope.
Klaus: Do not bring the child into this.
Elijah: Your child arrived here today, her security strengthened by those wolves that would defend her, and you would jeopardize that alliance? (He shakes his head) Niklaus, you yourself have mentioned that had you been raised by Ansel, you might have been a better man. Now, perhaps, a better man has entered Hope's life, and having seen that, you are shaken to your core.
Klaus: (angry) You have the audacity to analyze me? That's ambitious, considering your psychological difficulties. How was your time with my therapist? (Elijah looks at him, clearly offended) Was it helpful? Because it was a great risk leaving you alone with her! These days, who knows what you might do?
Elijah: (unamused) I have stood by you, and I have defended you for as long as I can recall. But, in the name of your daughter and her mother, I will not watch you commit this evil.
(Klaus stares at him angrily, but says nothing)
(Outside, the parade is still going on, and Davina and Josh are walking down the street together as they talk)
Davina: You okay?
Josh: Oh yeah, totally! My boyfriend and his buddies are super-wolves now...
(They turn and look behind them as Aiden shows off his new powers by jumping onto a car parked on the street and then doing a back-flip off of it and back onto the street. Aiden laughs happily and wraps his arms around the necks of his werewolf friends)
Josh: (smiles) It's gonna be awesome.
Davina: (chuckles) Hey, at least your boyfriend showed up.
Josh: Aw, come on!
(He grabs her hand and spins her around. She laughs happily until she notices a familiar figure standing in the nearby alley. It's Kol, looking pale and strung-out as he leans against the wall of the building. When she realizes it's him, Davina looks worried)
Davina: Is that...?
(Josh shoves Davina toward him so she can check on him. When she approaches Kol, he laughs bitterly with tears in his eyes)
Davina: Kol? What's wrong?
Kol: (laughs again) Aren't you a sight?
(Davina grabs Kol's hands, which are shaking, and looks even more concerned)
Davina: Kol, you're ice cold.
Kol: (squeezes her hands) Now, you listen to me, okay? Finn... he got a bit perturbed when we went to rescue Josh. And I... (Davina shakes her head in disbelief) I thought it was going to be okay, but... I'm running out of time.
[SCENE_BREAK]
[ MIKAELSON COMPOUND ]
(Cami is in Hope's nursery, holding a slightly-fussy Hope in her arms as she looks out the window. She turns when she hears Klaus approaching behind her)
Klaus: I heard her crying.
Cami: Oh, she's probably just teething.
Klaus: (walks closer to Cami) Thank you for taking care of her.
Cami: (smiles) She's actually pretty low-maintenance. She's been smiling and looking all around. I think she likes it here. Feels like home. (She watches Klaus smile at Hope) You wanna hold her? (Klaus doesn't move, he just speechlessly stares at Hope. Cami licks her lips nervously) I know this all must be pretty overwhelming, but some advice I learned from developmental psych? Happy mom, happy dad... happy baby.
(Klaus looks at Hope, who coos at him, and he smiles)
(Downstairs, the reception is still going on in the courtyard. Some people are eating and drinking, others are dancing, but everyone is mingling together while caterers come around with hors d'oeuvres and trays of drinks. Hayley and Jackson are dancing together in the middle of the room when Aiden walks over to where Josh is standing off to the side)
Josh: Look, I know that this won't be easy...
(Once again, Aiden cuts him off before he can finish, but this time, he kisses him softly in front of everyone. They keep kissing for a long moment, and when they pull away, they smile at each other)
(While Hayley and Jackson dance together, Hayley looks up to see white flower petals raining down on them, and Jackson smiles widely as he spins her around. Up on the balcony, Elijah watches them dance silently, looking slightly jealous. After a moment, Klaus comes down to the courtyard with Hope in his arms and gestures for the band to stop playing so he can make a speech)
Klaus: Ladies and gentlemen, may I have your attention please? Hayley, if you would join me, I would like to propose a toast.
(Hayley looks surprised, but she reluctantly joins Klaus at the front of the room and takes Hope into her arms)
Klaus: I want to welcome you all. As you know, last spring, Hayley and I had a daughter. Due to tragic circumstance, she was lost. Now, she has returned home. Her name is Hope. (The guests gasp and begin to murmur amongst themselves about this revelation) She will live here among you, her pack. Her family. We implore you... protect our daughter. Teach her. Love her, as one of your own. (In the crowd, Jackson nods at Klaus in agreement. Klaus takes a glass of champagne from a nearby tray. Elijah continues to watch from the balcony, and Klaus smiles at him before continuing his toast) Jackson, I invite you and your bride to live here, uniting your proud and noble people in peace. Welcome to the family, mate. (Jackson nods at him again, and Klaus holds up his glass) To Jackson and Hayley!
Various guests: Cheers!
(Klaus holds his glass up to Elijah and smiles at him devilishly)
[SCENE_BREAK]
[ LAFAYETTE CEMETERY ]
(In the Claire tomb, Kol is sitting in a chair while Davina and Rebekah go through all of their magical notes and texts to find a solution. Davina finds a passage in a book and places it on the table)
Davina: Transubstantiation. We can combine it with a protection spell.
Rebekah: Finn's spell blocks that. The body calls it, and then he dies. We need to jump him into a new one.
Davina: (frustrated) We can't just pick another body at a farmer's market, we don't even have a spell for that!
Kol: (pale and weak) Beks? Can you give me a moment alone with Davina?
(Rebekah leaves, and Kol stands to his feet. Davina looks at him with tears in her eyes)
Kol: I believe I owe you a dance.
(He holds out his hand, and Davina takes it, trying not to cry)
[SCENE_BREAK]
[ MIKAELSON COMPOUND ]
(Elijah is walking down the hall upstairs when Hayley approaches him from behind. Elijah, sensing her presence, stops, but doesn't turn around)
Elijah: A lovely ceremony.
Hayley: It was. Thank you for being there.
Elijah: With the wolves unified, Hope is safe. Everything is exactly as you intended. (Hayley looks uncomfortable, and Elijah sighs) And, in the interest of maintaining the peace, I have decided to join Marcel in Algiers. I believe with the correct instruction and guidance, this community may well prosper.
Hayley: (surprised) You're moving out? Elijah, this is your home.
Elijah: (smiles sadly) In my long, long life, I've called many places home, Hayley.
Hayley: I didn't know that Klaus would ask Jack and me to live here, but that... that doesn't mean that you have to leave.
(Hayley and Elijah's conversation is interrupted by Rebekah, who rushes over to them)
Rebekah: Elijah.
(Elijah squints at her, not recognizing her new appearance right away)
(In the nursery, Klaus is leaning over Hope's crib, watching her as she fidgets around. When he hears Elijah and Rebekah come into the room, he sighs in annoyance)
Klaus: Must you intrude on every moment?
Elijah: I'm not here to quarrel, brother.
Rebekah: It's Kol. I couldn't help him. (Klaus turns to her with a shocked expression on his face) He's not gonna last the night.
(Downstairs, Marcel and Gia are pouring champagne glasses for the guests when they notice Klaus, Elijah, and Rebekah march down the steps and rush out the door. Marcel glances over at Rebekah, and though he's not yet seen her new vessel, he seems to recognize her spirit anyway. He sets down the bottle of wine and moves to get a better look)
Gia: (confused) Do you know that girl?
Marcel: I think I might.
[SCENE_BREAK]
[ LAFAYETTE CEMETERY ]
(Kol and Davina are slow-dancing outside of the Claire tomb to the sound of an old-fashioned song on the gramophone player. Both of their eyes are red and rimmed with tears as the cry into each others' shoulders. They pull away so Kol can look her in the eyes)
Kol: I know that we're in a cemetery, and I happen to be terminal... (He laughs through his tears) ...but you've got to admit, the stars are lovely.
Davina: (shakes her head tearfully) How can you joke right now?
Kol: I'm not. We're under the same stars. It's some guy, and he's with his girl, and he thinks he's got all the time in the world, and he's right. (He laughs again, and Davina starts to cry as he leans his forehead against hers) And I hate him.
(Suddenly, Kol doubles over in a coughing fit, and Davina looks panicked)
Davina: (lifts his head) Are you okay?
Kol: (smiles) Yeah. (Davina takes his hand in hers and kisses it) I think I want to be alone for this bit.
(He continues to cough as he walks away from Davina and toward one of the tombs. However, he stops when he hears a voice behind him. He turns and finds Elijah, Klaus, and Rebekah standing in the aisle)
Elijah: I'm afraid that's not an option.
Klaus: "Always and forever" is not something that you just weasel out of, brother.
(Kol stares at his siblings in surprise and manages a weak smile, which Klaus returns)
[SCENE_BREAK]
[ MIKAELSON COMPOUND ]
(Jackson is standing on the terrace overlooking the French Quarter from Hayley's bedroom when Hayley comes in and joins him)
Hayley: Everybody's headed home. (She smiles shyly) Now what?
Jackson: Ahhh, wedding night. Awkward. (They both laugh nervously) Well, knowing you, I'm thinking you just wanna stay up all night watching Hope sleep. That sounds good to me.
Hayley: (hesitates before walking toward him) Jack... when I told you that the wedding was the right thing to do for our pack, there's something else I should have told you. (Jackson looks nervous and braces himself) I should have told you that... when I first got to New Orleans, I was terrified, pregnant... for a long time, basically alone. And then, one night, a wolf appeared out of the woods, and I knew that I was safe. From the moment I saw you, I... I knew I could trust you. You never made me feel ashamed about being a hybrid, or questioned why I had to lie to you about Hope.
Jackson: Well, I told you... I love you. I meant it.
Hayley: (nods) Yeah... and that's the first time that anyone's said that to me. I want you to know that I didn't marry you for all those people. I married you for me. That's what I should have told you.
(Jackson looks at her, so surprised by this confession that he quickly pulls her toward him and kisses her, and they continue kissing passionately on the balcony)
[SCENE_BREAK]
[ LAFAYETTE CEMETERY ]
(Davina, Rebekah, Klaus and Elijah have moved Kol into the tomb where Esther once imprisoned Elijah, which is surrounded by candles. Davina is sitting a few feet away, whispering a spell over a necklace that she's gripping tightly in her hands. There are lit candles scattered all over the room where Kol is laying on the floor, surrounded by his brothers and sister. Kol's nose and mouth are both bleeding and he groans in pain)
Kol: (grips the lapel of Klaus' coat) All my life, all I ever wanted was for you lot to care about me.
(Klaus struggles to hold back his tears, and before Kol can laugh, he's overcome by another coughing fit that startles Rebekah. She sits down on the ground, and she and Elijah rub Kol's back as he coughs before leaning him backward so she can cradle Kol's head in her lap. Kol grabs onto Rebekah's hand and squeezes it as he groans)
Rebekah: (crying) Kol, listen to me. You don't have long. You're going to die. (Kol can no longer hold back his tears, and he grips her arm tighter) But you will die a witch, and we will consecrate your body. You will join the ancestors of the French Quarter, and those spirits can be brought back. And, I promise you, brother, I will not leave this body until I find a way to bring you home.
(Kol smiles at her, despite his pain, but quickly begins to cough even harder. Davina finally finishes her spell and crawls toward Kol to give him the necklace)
Davina: (frantic and in tears) I tried a different spell.
Kol: (takes her hand and squeezes it) It's okay. I'm not scared.
(Davina tries her best to smile at him through her tears, and Kol manages one last laugh before he dies in Rebekah's arms. Davina breaks down in sobs, and Elijah and Klaus begin crying freely as well over Kol's body)
[SCENE_BREAK]
[ ARKANSAS MORGUE / MIKAELSON COMPOUND ]
(Finn, finally healed completely, gingerly sits up on the autopsy table with Freya's help)
Finn: (sighs in relief) What did you do?
Freya: It took a while, but I healed you. Brought you back from death, using this.
(She points to her blue pendant talisman in Finn's hands, which he examines)
Finn: Your pendant. (He laughs in wonder) You said it would protect me.
Freya: It's a talisman used to focus my power. I knew it would be dangerous to face Elijah. I didn't want you to be hurt.
Finn: Just like when we were children. (He stands to his feet and groans slightly, visibly sore after his resurrection) I seem to recall you were always defending me.
Freya: (takes his hand and squeezes it) You're my brother, Finn. I love you. And we'll need to protect each other. If Dahlia has sensed Klaus' child, she's already on her way.
Finn: Niklaus used magic to cloak the baby from my mother. It's going to take a bit of time before Dahlia finds her...
Freya: (cuts him off) You don't understand. Esther was nothing compared to Dahlia. I was taken when I was only five years old. I had just started to display a potential for magic. It is the magic that will draw Dahlia. It will serve as a beacon, calling her.
(At the compound, Hayley holds Hope in her arms in the nursery and dotes on her while Jackson watches from the doorway)
Freya: (V.O.): Dahlia will come, and she will take what's hers.
(Hope burrows into Hayley's shoulder as Hayley looks over at Jackson and smiles)
[ END ] | Cami rushes away from the house and stops at a payphone where Elijah meets her and Hope. Klaus offers his house for the location of Jackson and Hayley's wedding. Just before the wedding Elijah and Cami arrive at the house and Hope is reunited with her parents. Hayley doesn't want Elijah to change her mind about the wedding and she goes on to marry Jackson. During the reception, Klaus makes a toast inviting the newly weds to live in the house. Elijah thinks that Klaus dislikes Jackson as he will end up being a better father figure than he is, and Klaus thinks Elijah wants Jackson dead because he is in love with Hayley. Elijah tells Hayley he may move out. Meanwhile, Aiden and Josh are comfortable together and Kol and Devina complete the dagger they have been working on and Freya raises Finn back from the dead. The episode ends with Elijah, Rebekah, Klaus and Davina surrounding Kol as he dies, and Rebekah promising to resurrect him. | summ_screen_fd |
Scene: Sheldon and Leonard's apartment.
Sheldon: I've been thinking about time travel again.
Leonard: Why, did you hit a roadblock with invisibility?
Sheldon: Put it on the back burner. Anyway, it occurs to me, if I ever did perfect a time machine, I'd just go into the past and give it to myself, thus eliminating the need for me to invent it in the first place.
Leonard: Interesting.
Sheldon: Yeah, it really takes the pressure off.
Leonard: Sounds like a breakthrough, should I call the science magazines and tell them to hold the front cover? (Exiting the apartment.)
Sheldon: It's time travel, Leonard, I will have already done that.
Leonard: Then I guess congratulations are in order.
Sheldon: No, congratulations will have been in order. You know, I'm not going to enjoy this party.
Leonard: I know, I'm familiar with you.
Sheldon: At the last department party, Dr Finkleday cornered me and talked about spelunking for 45 minutes.
Leonard: Yes, I was there.
Sheldon: You know what's interesting about caves, Leonard?
Leonard: What?
Sheldon: Nothing.
Leonard: Well then we'll avoid Finkleday, we'll meet the new department head, congratulate him, shake his hand and go.
Sheldon: How's this? Pleased to meet you, Dr Gablehouser. How fortunate for you that the University has chosen to hire you, despite the fact that you've done no original research in 25 years, and instead have written a series of popular books that reduce the great concepts of science to a series of anecdotes, each one dumbed down to accommodate the duration of an average bowel movement. Mahalo.
Leonard: Mahalo's a nice touch.
Sheldon: Do you know there are only eight consonants in the Hawaiian language.
Leonard: Interesting, you should lead with that.
Scene: The department party. Sheldon, Raj and Leonard are at the buffet table.
Raj: Oh, God, Look at this buffet. I love America.
Leonard: You don't have buffets in India?
Raj: Of course, but it's all Indian food. You can't find a bagel in Mumbai to save your life. Schmear me.
Sheldon: Well here's an interesting turn of events.
Leonard: What. (Sees Howard entering with a statuesque blonde) Howard brought a date?
Sheldon: A more plausible explanation is that his work in robotics has made an amazing leap forward.
Howard: Hey, what up, science bitches? May I introduce my special lady friend, Summer. (Puts arm around her.)
Summer: I already told you, touching's extra.
Howard: Right. Sorry.
Leonard (to Sheldon): Here comes our new boss, be polite.
Gablehouser: Hi fellas, Eric Gablehouser.
Howard: Howard Wolowitz.
Gablehouser: Howard, nice to meet you, and you are?
Sheldon: An actual real scientist. (To Leonard) How was that?
Scene: The stairwell of the apartment building. Sheldon is carrying a box of his things.
Sheldon: I can't believe he fired me.
Leonard: Well, you did call him a glorified high-school science teacher whose last successful experiment was lighting his own farts.
Sheldon: In my defence, I prefaced that by saying "with all due respect." Credit sequence.
Scene: The apartment, Sheldon is in the kitchen cooking, Leonard enters.
Leonard: Morning
Sheldon: Morning.
Leonard: You're making eggs for breakfast?
Sheldon: This isn't breakfast, it's an experiment.
Leonard: Huh? Cos it looks a lot like breakfast.
Sheldon: I finally have the time to test my hypothesis, about the separation of the water molecules from the egg proteins, and its impact vis-a-vis taste.
Leonard: Sounds yummy. I look forward to your work with bacon.
Sheldon: As do I.
Leonard: You know, I'm sure if you just apologised to Gablehauser he would give you your job back.
Sheldon: I don't want my job back. I've spent the last three and a half years staring at greaseboards full of equations. Before that I spent four years working on my thesis. Before that I was in college, and before that, I was in the fifth grade. This is my first day off in decades, and I'm going to savour it.
Leonard: Okay. I'll let you get back to fixing your eggs.
Sheldon: I'm not just fixing my eggs, I'm fixing everyone's eggs.
Leonard: And we all thank you.
(Sheldon takes his eggs and sits down. Takes a photograph of them. Writes in his notebook, then takes a forkful. Writes in notebook again.)
Sheldon: Use new eggs. (There is a knock on the door).
Penny (popping her head round): Hi, hey. I'm running out to the market, do you guys need anything?
Sheldon: Oh, well this would be one of those circumstances that people unfamiliar with the law of large numbers would call a coincidence.
Penny: I'm sorry?
Sheldon: I need eggs. Four dozen should suffice.
Penny: Four dozen?
Sheldon: Yes, and evenly distributed amongst brown, white, free range, large, extra-large and jumbo.
Penny: Okay, one more time?
Sheldon: Never mind, you won't get it right, I'd better come with you.
Penny: Oh, yay!
Scene: Penny's car
Penny: How come you didn't go into work today.
Sheldon: I'm taking a sabbatical, because I won't kow-tow to mediocre minds.
Penny: So you got canned, huh?
Sheldon: Theoretical physicists do not get canned. But yeah.
Penny: Well, maybe it's all for the best, you know I always say, when one door closes, another one opens.
Sheldon: No it doesn't. Not unless the two doors are connected by relays, or there are motion sensors involved.
Penny: No, no, I meant...
Sheldon: Or the first door closing causes a change of air pressure that acts upon the second door.
Penny: Never mind.
Sheldon: Slow down. Slow down, please slow down.
Penny: We're fine.
Sheldon: Look, you're not leaving yourself enough space between cars.
Penny: Oh, sure I am.
Sheldon: No, no. Let me do the math for you, this car weighs let's say 4,000lb, now add say 140 for me, 120 for you.
Penny: 120?
Sheldon: Oh, I'm sorry, did I insult you? Is your body mass somehow tied into your self worth?
Penny: Well, yeah.
Sheldon: Interesting. Anyway, that gives us a total weight of, let's say, 4,400lb.
Penny: Let's say 4,390.
Sheldon: Fine. We're travelling forward at, good Lord, 51 miles an hour. Now let's assume that your brakes are new and the callipers are aligned, still, by the time we come to a stop, we'll be occupying the same space as that Buick in front of us, an impossibility that nature will quickly resolve into death, mutilation and... oh look, they built a new put-put course.
Scene: The supermarket.
Sheldon: This is great. Look at me, out in the real world of ordinary people, just living their ordinary, colourless, workaday lives.
Penny: Thank you.
Sheldon: No, thank you. And thank you, ordinary person. Hey, you want to hear an interesting thing about tomatoes.
Penny: Uh, no, no not really. Listen, didn't you say you needed some eggs.
Sheldon: Uh, yes, but anyone who knows anything about the dynamics of bacterial growth knows to pick up their refrigerated foods on the way out of the supermarket.
Penny: Oh, okay, well maybe you should start heading on out then.
Sheldon: No, this is fun. Oh, the thing about tomatoes, and I think you'll really enjoy this, is, they're shelved with the vegetables, but they're technically a fruit.
Penny: Interesting.
Sheldon: Isn't it?
Penny: No, I mean what you find enjoyable.
Sheldon (as Penny selects vitamin supplements): Oh boy.
Penny: What now?
Sheldon: Well, there's some value to taking a multivitamin, but the human body can only absorb so much, what you're buying here are the ingredients for very expensive urine.
Penny: Well, maybe that's what I was going for.
Sheldon: Well then you'll want some manganese.
Scene: On the stairwell of the apartment building.
Sheldon: That was fun. Maybe tomorrow we can go to one of those big warehouse stores.
Penny: Oh, I don't know Sheldon, it's going to take me a while to recover from all the fun I had today.
Sheldon: Are you sure. There are a lot of advantages to buying in bulk. For example, I noticed that you purchase your tampons one month's supply at a time.
Penny: What?
Sheldon: Well think about it, it's a product that doesn't spoil, and you're going to be needing them for at least the next thirty years.
Penny: You want me to buy thirty years worth of tampons?
Sheldon: Well, thirty, thirty five, hey, when did your mother go into menopause?
Penny: Okay, I'm not talking about this with you.
Sheldon: Oh, Penny, this is a natural human process, and we're talking about statistically significant savings. Now, if you assume 15 tampons per cycle and a 28 day cycle, are you fairly regular? (Penny shuts door in his face.) Okay, no warehouse store, but we're still on for put-put golf, right?
[SCENE_BREAK]
Scene: The apartment, Sheldon has several bowls containing goldfish.
Leonard (entering): Hey, I just ran into Penny, she seemed upset about something.
Sheldon: I think it's her time of the month. I marked the calendar for future reference.
Leonard: What's with the fish?
Sheldon: It's an experiment.
Leonard: What happened to your scrambled egg research?
Sheldon: Oh, that was a dead end. Scrambled eggs are as good as they're ever going to be.
Leonard: So... fish.
Sheldon: I read an article about Japanese scientists, who inserted DNA from luminous jellyfish into other animals, and I thought hey, fish nightlights.
Leonard: Fish nightlights.
Sheldon: It's a billion dollar idea. Shhhhh!
Leonard: Mum's the word. Sheldon, are you sure you don't want to just apologise to Gablehauser and get your job back.
Sheldon: Oh, no, no, no. No, I've too much to do.
Leonard: Like luminous fish.
Sheldon: Shhhhh!
Leonard: Right... I didn't....
Sheldon: That's just the beginning. I also have an idea for a bulk mail-order feminine hygiene company. Oh, glow in the dark tampons! Leonard, we're going to be rich.
Scene: The stairwell of the apartment building.
Leonard: Thank you for coming on such short notice.
Mrs Cooper: You did the right thing calling.
Leonard: I didn't know what else to do, he's lost all focus, every day he's got a new obsession. (They enter the apartment. Sheldon is weaving on a loom. He is wrapped in a poncho.) This is a particularly disturbing one.
Sheldon (looking round): Mommy.
Mrs Cooper: Hi baby.
Sheldon (mouths): You called my mother?
Mrs Cooper: Oh, you got yourself a loom, how nice.
Sheldon: Thank you.
Mrs Cooper: Honey, why did you get a loom?
Sheldon: I was working with luminous fish, and I thought, hey, loom! Mom, what are you doing here?
Mrs Cooper: Leonard called me.
Sheldon: I know, but why?
Leonard: Because one of the great minds of the twenty-first century is raising glow-in-the-dark fish and weaving sarapes.
Sheldon: This is not a sarape. This is a poncho. A sarape is open at the sides, a poncho is closed, this is a poncho, and neither is a reason to call someone's mother.
Leonard: Really, when was the last time you left the house.
Sheldon: I went to the market with Penny.
Leonard: That was three weeks ago.
Sheldon: Well then buckle up, in the next four to eight days she's going to get very crabby.
Mrs Cooper: Sweetheart, your little friend is concerned about you.
Sheldon: Yes, well I'm not a child, I'm a grown man capable of living my life as I see fit. And I certainly don't need someone telling on me to my mother.
Leonard: Where are you going?
Sheldon: To my room, and no-one's allowed in.
Mrs Cooper: He gets his temper from his daddy.
Leonard: Oh.
Mrs Cooper: He's got my eyes.
Leonard: I see.
Mrs Cooper: All that science stuff, that comes from Jesus.
Scene: Everyone but Sheldon is in the kitchen of the apartment.
Leonard: Sheldon? Your mum made dinner.
Sheldon (off): I'm not hungry.
Mrs Cooper: Oh, Leonard, don't trouble yourself, he's stubborn. He may stay in there 'til the Rapture.
Penny: Are we so sure that's a bad thing?
Mrs Cooper: I'll tell ya, I love the boy to death, but he has been difficult since he fell out of me at the K-Mart.
Howard: Excuse me for being so bold, but I now see where Sheldon gets his smouldering good looks.
Mrs Cooper: Oh, honey that ain't going to work, but you keep trying. (To Raj) I made chicken, I hope that isn't one of the animals that you people think is magic? You know, we have an Indian gentleman at our church, a Dr Patel, it's a beautiful story, the lord spoke to him, and moved him to give us all 20% off on lasic, you know, those that needed it.
Leonard: That is a lovely story, um, are we going to do anything about Sheldon?
Mrs Cooper: Oh, we will, you have to take your time with Sheldon. His father, God rest his soul, used to say to me, Mary, you have to take your time with Sheldon.
Leonard: Sounds like a wise man.
Mrs Cooper: Oh, not so wise, he was trying to fight a bobcat for some licquorish. So, everybody grab a plate, and a pretty place mat that Shelly wove.
Penny: Has Shelly ever freaked out like this before.
Mrs Cooper: Oh, all the time, I remember one summer when he was thirteen, he built a small nuclear reactor in the shed and told everybody he was going to provide free electricity for the whole town, well the only problem was he had no, whatchacall, fissionable materials. Anyway, when he went on the internets to get some, a man from the government came by and sat him down real gentle and told him it's against the law to have yellow cake uranium in a shed.
Penny: What happened?
Mrs Cooper: Well, the poor boy had a fit, locked himself in his room and built a sonic death ray.
Leonard: A death ray?
Mrs Cooper: Well, that's what he called it, didn't even slow down the neighbour kids. It pissed our dog off to no end. You know, you two make a cute couple. Both Leonard and Penny laugh, a little too forced.
Leonard: No, we're not, we're not, not a couple, two singles, like those individually wrapped slices of cheese that.... are friends.
Mrs Cooper: Did I pluck a nerve there?
Howard: Oh yeah.
Mrs Cooper: Okay. Alright everybody, it's time to eat. (Everybody begins to do so) Oh Lord, we thank you for this meal, all your bounty, and we pray that you help Sheldon get back on his rocker. (To Raj and Howard) Now after a moment of silent meditation I'm going to end with "In Jesus' Name" but you two don't feel any obligation to join in. Unless, of course, the holy spirit moves you. Time shift
Penny: Oh my God, this is the best cobbler I've ever had.
Mrs Cooper: It was always Sheldon' s favourite. You know what the secret ingredient is?
Penny: Love?
Mrs Cooper: Lard. Sheldon emerges from the bedroom area.
Howard: Hey, look who's come out....
Mrs Cooper: Shhh! You'll spook him. He's like a baby deer, you gotta let him come to you. Sheldon crosses to the cobbler, takes some and puts it on a plate. Looks round at the group in the matter of a frightened animal. Everyone but Leonard looks down at their meal.
Leonard: This is ridiculous. Dammit, Sheldon, snap out of it. You're a physicist, you belong at the University doing research, not hiding in your room. (Sheldon scuttles away)
Mrs Cooper: You don't hunt, do you?
Scene: Sheldon's bedroom. He is building a model of some kind of double helix. There is a knock on the door.
Mrs Cooper (entering): Good morning, snicker-doodle.
Sheldon: Morning.
Mrs Cooper: Oh, well that looks awful fancy, what is that?
Sheldon: It's my idea of what DNA would look like in a silicon based life form.
Mrs Cooper: But intelligently designed by a creator, right?
Sheldon: What do you want, mom?
Mrs Cooper: You know how your daddy used to say that you can only fish for so long before you got to throw a stick of dynamite in the water?
Sheldon: Yeah.
Mrs Cooper: Well, I'm done fishing. (Throwing a pair of trousers on the bed) You put those on.
Sheldon: What for?
Mrs Cooper: Because you're going to go down to your office, you're going to apologise to your boss, and get your job back.
Sheldon: No.
Mrs Cooper: I'm sorry, did I start that sentence with the words "if it please your highness?"
Sheldon: I'm not going to apologise, I didn't say anything that wasn't true.
Mrs Cooper: Now you listen here, I have been telling you since you were four years old, it's okay to be smarter than everybody but you can't go around pointing it out.
Sheldon: Why not?
Mrs Cooper: Because people don't like it. Remember all the ass-kickings you got from the neighbour kids? Now let's get cracking. Shower, shirt, shoes, and let's shove off. (Exits)
Sheldon: Wouldn't have been any ass-kickings if that stupid death ray had worked.
Scene: The kitchen
Mrs Cooper: Problem solved.
Leonard: Really? That's impressive.
Mrs Cooper: Leonard, the Lord never gives us more than we can handle. Thankfully he blessed me with two other children who are dumb as soup.
Scene: Dr Gablehouser's office
Mrs Cooper: Excuse me, Dr Gablehouser, are you busy?
Gablehouser: Well, actually....
Mrs Cooper: Sheldon, he's just doodling, get in here.
Sheldon: Dr Gablehouser.
Gablehouser: Dr Cooper.
Mrs Cooper: Let's go, baby, we're losing daylight.
Sheldon: Um, as you know, several weeks ago in our first encounter we may have gotten off on the wrong foot, when I called you an idiot. And I just wanted to say that I was wrong. To point it out.
Gablehouser (to Mrs Cooper): I'm sorry, we haven't been introduced. Dr Eric Gablehouser.
Mrs Cooper: Mary Cooper, Sheldon's mom.
Gablehouser: Now that's impossible, you must have had him when you were a teenager.
Mrs Cooper: Oh, aren't you sweet, his father's dead.
Gablehouser: Recently?
Mrs Cooper: Long enough.
Gablehouser (indicating chair): Please. Sheldon, shouldn't you be working?
Sheldon (leaving): Okay.
Leonard: Hey, how did it go?
Sheldon: I got my job back.
Leonard: Really? What happened?
Sheldon: I'm not quite sure. It involves a part of the human experience that has always eluded me.
Leonard: That narrows it down.
Scene: Sheldon's bedroom. Mrs Cooper is tucking him in.
Mrs Cooper: I'm very proud of you honey, you showed a lot of courage today.
Sheldon: Thanks, mom. Mom?
Mrs Cooper: Mmm-hmm?
Sheldon: Is Dr Gablehouser going to be my new daddy?
Mrs Cooper: We'll see. Sleep tight. Sheldon turns over to sleep in the glow of a luminous goldfish. | At the university Sheldon is fired from his job as a physicist after insulting his new boss Dr. Eric Gablehauser. Sheldon's change of circumstance triggers a downward spiral of depression in which he fails to improve scrambled eggs, develops luminous fish for nightlights, and weaves on a hand loom. Worried, Leonard calls Sheldon's mother, Mary Cooper. When she visits, the men realize she is the complete opposite of their expectations: she is sweet, down-to-earth, a devout Christian, and a loving and caring mother. Mary finally forces Sheldon to apologize, who is given his job back after she flirts with Dr. Gablehauser. | summ_screen_fd |
Existing theories of movement planning suggest that it takes time to select and prepare the actions required to achieve a given goal. These theories often appeal to circumstances where planning apparently goes awry. For instance, if reaction times are forced to be very low, movement trajectories are often directed between two potential targets. These intermediate movements are generally interpreted as errors of movement planning, arising either from planning being incomplete or from parallel movement plans interfering with one another. Here we present an alternative view: that intermediate movements reflect uncertainty about movement goals. We show how intermediate movements are predicted by an optimal feedback control model that incorporates an ongoing decision about movement goals. According to this view, intermediate movements reflect an exploitation of compatibility between goals. Consequently, reducing the compatibility between goals should reduce the incidence of intermediate movements. In human subjects, we varied the compatibility between potential movement goals in two distinct ways: by varying the spatial separation between targets and by introducing a virtual barrier constraining trajectories to the target and penalizing intermediate movements. In both cases we found that decreasing goal compatibility led to a decreasing incidence of intermediate movements. Our results and theory suggest a more integrated view of decision-making and movement planning in which the primary bottleneck to generating a movement is deciding upon task goals. Determining how to move to achieve a given goal is rapid and automatic. In the reaction time before a movement is initiated, two distinct processes are thought to occur: first, the exact goals of the movement must be decided upon and, second, the actions that will achieve the chosen goal must be selected and/or prepared [1]. Decisions about high-level movement goals have been well-characterized in terms of an accumulation of sensory evidence over time [2,3]. The process of selecting and/or preparing the actions to achieve a chosen goal, which we refer to here as movement planning, is classically thought to require further time-consuming computations [4,5, 6]. The relative contribution of goal selection and movement planning to the reaction time remains a matter of considerable debate [7,8]. One way to study the process of movement planning is to interrupt it and examine the behavioral consequences. When a reaching movement is released at a lower-than-normal reaction time, movements appear to be biased away from the target stimulus towards a ‘default’ movement [4,9, 10]. As preparation time increases, movements gradually converge on the target. Similar intermediate movements are observed if a target jumps shortly before movement onset [11,12,13,14,15] or in tasks that either deliberately or inadvertently create ambiguity about task goals [16,17,18,19]. These intermediate movements have been variously interpreted as reflecting incomplete movement planning or interference between parallel plans to each potential goal. Either interpretation suggests that intermediate movements occur as an unintentional artifact of stressing an underlying planning mechanism. Although generally well accepted, these existing interpretations of intermediate movements are at odds with more contemporary theories of movement execution based on optimal control theory [20]. According to this theory, a single, flexible feedback control policy can be sufficient to generate a wide variety of movements and rapidly switch between them based on new sensory observations [21,22]. Such an organization dispenses with the need to re-plan a movement each time the movement goal changes and is incompatible with replanning-based explanations for intermediate movements. Here we show how intermediate movements can be understood within an optimal control framework if the control policy takes into account an evolving decision about the location of movement goals. Our theory, therefore, frames intermediate movements as reflecting a deliberate plan to deal with uncertainty about movement goals, rather than as resulting from erroneous movement planning. A critical prediction of this theory is that intermediate movements should only occur when potential movement goals are compatible, i. e. when they require kinematically similar movements. Given compatible goals, an intermediate movement can bring the hand closer to both goals simultaneously, pending the arrival of further information about which goal to ultimately commit to [23,24]. If the compatibility between goals is eliminated, either by separating the goals more widely in space [4,25] or by imposing an obstacle between them, intermediate movements no longer offer this advantage and all movements should instead be directed to one target or another. We verified these predictions in three experiments that varied the compatibility between movement goals in two distinct ways. Subjects were trained to initiate their movements at a precise time during each trial (Fig. 1A) and make center-out movements to “shoot” through a target. On a subset (30%) of trials, the target jumped by ±45°, ±90° or ±135° at an unpredictable time (between 150ms and 550ms) prior to movement initiation (Fig. 1B). We were interested in how subjects’ behavior (specifically, the initial direction of their movement) depended on the amount of re-preparation time (rPT) available. That is, the amount of time that elapsed between when the target jumped and when the subject initiated their movement. Fig. 2 shows data from a representative subject. On trials in which the target jumped by 45°, we observed a continuous relationship between rPT and initial reach angle (Fig. 2A, B). For movements initiated less than 200ms after the target jump, movements were directed towards the original target location. Between 200ms and 350ms, the initial reach angle changed gradually from the original to the new target direction as the rPT increased. For movements initiated more than 350ms after the target jump, this subject was consistently able to compensate for the change in target location. A similar pattern held for the behavior in response to 90° jumps, only that the subject adhered to the initial reach direction slightly longer and the transition between targets was steeper (Fig. 2C, D). For the largest target jumps (135° change in reach direction), there was no clear transitional period between targets. Instead, behavior switched abruptly at around 350ms from movements directed towards the initial target to movements directed towards the post-jump target (Fig 2E, F). All subjects showed the same qualitative pattern of gradual adjustment of reach direction for small target jumps, and more abrupt adjustment for larger target jumps. Interestingly, there were still a small number of intermediate movements generated following large target jumps (Fig. 2 E, F), suggesting a continuous underlying change in reaching direction, albeit so rapid that the transition appeared abrupt. To characterize this behavior quantitatively, we fit sigmoid functions to the relationship between rPT and initial reach direction (see Methods, Eq. (2) ). This yielded two parameters for each subject, for each jump amplitude. The first parameter, τ, reflects the timescale over which this transition occurred. The second parameter, t50 (s) reflects the latency of the change in initial movement direction. Comparing the fitted sigmoid parameters for all subjects, we found that the slope of the sigmoid, τ differed significantly across jump amplitudes (F (2,18) = 22. 6; p<0. 0001) (Fig. 3A). Post-hoc pairwise comparisons of τ across jump amplitudes were all significant (p<0. 05). Thus the transition in initial reach direction was consistently more abrupt for large amplitude target jumps than for smaller amplitude jumps, confirming our predictions. Next, we examined the latency of compensation for the target jump. We found that t50, the time at which subjects would make an exactly intermediate movement, also depended on jump amplitude (F (2,18) = 11. 96; p<0. 001) (Fig. 3B). Visual inspection of the data suggests that the re-preparation time required for complete compensation, i. e. the shortest delay at which movements directed to the post-jump target were reliably observed, seemed to be consistent across jump amplitudes. To test this hypothesis, we considered the time t95 at which each fitted sigmoid reached 95% compensation. This measure showed no significant difference across jump amplitudes (F (2,18) = 1. 3; p = 0. 30) (Fig. 3C). Conversely, differences in the times at which compensation began (t05; 5% of sigmoid height) were highly significant across subjects (F (2,18) = 32. 04; p<. 0001). Thus, target jumps of larger amplitude angles did not require a longer period of re-preparation, despite requiring a larger change in movement direction. The primary difference in behavior across jump amplitudes was in the time at which the change in target location began to be reflected in subjects’ movements. Despite extensive training, all subjects exhibited considerable variability in their movement initiation times. The standard deviation in movement initiation time, averaged across subjects, was 79±21ms. This value was quite large when compared with the timescales over which subjects’ behavior changed (∼100ms). We considered whether the delay between the target jump and the intended time of movement initiation (i. e. the delay between the target jump and the fourth tone) could serve as a better predictor of behavior than rPT. To test this possibility, we repeated our analysis using the absolute time of the target jump instead of the delay between target jump and movement onset (rPT). The total log-likelihood of the sigmoid fit (including data points classified as outliers) was significantly worse (F (1,6) = 48. 2; p<0. 001) ). Thus we can conclude that behavior depended specifically on the actual time of movement initiation and not on the intended time of movement initiation. An important feature of Experiment 1 is that although the disappearance of the initial target is unambiguous, the target could have jumped to a number of different locations. We performed a second experiment to determine to what degree, if any, this ambiguity affected the timecourse of compensation for the jump. In Experiment 2, the target only appeared in two possible locations within each block (Fig. 4), such that whenever the target jumped, the location it jumped to was always known unambiguously. Despite the difference in paradigms, subject behavior was identical to that seen in Experiment 1. The initial reach direction changed gradually as a function of rPT for a 45° separation between targets, but changed abruptly when the targets were separated by 135°. As in Experiment 1, the extent of intermediate movements (slope of the sigmoid) differed significantly across jump amplitudes (F (1,5) = 24. 66, p<0. 01), as did the latency, t50 (F (1,5) = 36. 2, p<. 01). The time at which compensation was complete, t95, was similar across target separations, although appeared to be slightly earlier for the 135° than 45° separation (F (1,5) = 5. 62, p = 0. 06). We can conclude from the results of Experiment 2 that ambiguity about the precise location of the post-jump target did not significantly influence behavior. In Experiments 1 and 2, we showed that decreasing compatibility between goals by increasing their angular separation led to a more rapid transition between movement directions, with a corresponding decrease in the incidence of intermediate movements. This result is consistent with our hypothesis that intermediate movements constitute an intelligent solution to the problem of moving amid goal uncertainty. However, varying the jump amplitude also affected the degree of similarity between the motor commands required before and after the jump. Intermediate movements may have emerged from interference between overlapping movement representations, rather than because of an exploitation of task-level compatibility between goals. In Experiment 3, we controlled for this possibility by using an alternative approach to reducing the compatibility between the pre-jump and post-jump targets that allowed us to vary the compatibility between goals while keeping the pre-jump and post-jump goals consistent across conditions. We created a series of virtual barriers between adjacent targets (Fig. 5A). Any intermediate movements were penalized by playing an unpleasant rasping tone and withholding points and other success cues from subjects on trials in which they collided with the barrier. We tested the behavior of subjects in response to target jumps of 45° amplitude both with and without these barriers present (Fig. 5B). As before, when the barriers were not present, subjects exhibited a gradual change in movement direction with a large number of intermediate movements. With the barriers present, however, instead of this gradual change in behavior, subjects switched more abruptly from one direction to another. Applying the same analysis as in Experiment 1, we found a significant difference in the timescales of the change in initial movement direction, τ, across conditions (F (1,7) = 30. 75; p<. 001) and also in the latency t50 (F (1,7) = 29. 98; p<. 0001). The time required to fully compensate for the target jump (t95) was not significantly different across conditions (F (1,7) = 2. 57; p = 0. 15). Importantly, differences in behavior between the barrier and no barrier conditions cannot be attributed to an increase in accuracy demands when barriers were present: variability in initial reach direction on non-jump trials was not strongly affected by the presence of a barrier (s. d. in initial reach direction without barrier = 4. 8±. 6°, with barrier = 4. 4±. 9°; F (1,7) = 3. 58; p = 0. 1). Overall, we found that the behavior following 45° jumps with barriers was qualitatively similar to that observed for 135° jumps in Experiment 1 when movement paths were unconstrained. This confirmed our hypothesis that goal compatibility, and not the magnitude of difference in required movement directions, was the key determinant of behavior following a target jump. Our experimental results showed a clear pattern whereby the timecourse of adjustment in behavior following a target jump, and, consequently, the incidence of intermediate movements, dependeds upon the compatibility between the pre-jump and post-jump goals. For nearby targets, which are highly compatible, initial movement direction was adjusted gradually as a function of available re-preparation time. When the potential movement goals were incompatible with one another, initial movement direction switched abruptly at a clear threshold rPT. We formalized our intuition about these results through a mathematical model (Fig. 6). We suppose that, in the immediate aftermath of the target jump, the subject must determine based on sensory information whether the target has jumped and, if so, where it has jumped to. Although information about the target was presented discretely and unambiguously, the detection of such unambiguous stimuli still may entail significant uncertainty. We modeled this simple decision about the location of the target as a process of noisy evidence accumulation. Critically, this implies that subjects were transiently uncertain as to the true location of the target, but became more confident as more time elapsed following the target jump. We combined this decision-making process with an optimal control model of movement generation. We assume that each potential target is associated with some accuracy cost Jx that rewards movements that pass through the goal region and penalizes movements that do not (see Methods). We augment the usual state of the motor apparatus, xt, with a dynamic stochastic variable rt reflecting accumulating evidence about the true identity of the target. Paralleling standard models of decision-making [3,26], rt represents the log odds ratio of the belief pt that the initial target is the true target location: rt=log (pt1−pt). rt is allowed to vary from −∞ (certain that the target has not jumped) to +∞ (certain that the target has jumped). Together with an effort cost Ju, the overall expected endpoint cost is then given by a sum of the accuracy costs associated with each target, weighted by their beliefs (Equation 9). Optimizing this cost yields a single, fixed control policy ut = π (xt, rt, t) that guides responses to fluctuations in both the state of the plant and beliefs about the target. In principle this model could be used to predict full movement trajectories. However, the critical prediction of interest is the decision about what direction to move in at the very start of the movement given the prospect of gaining further evidence later on during movement i. e. whether or not an intermediate movement should be generated. A general solution for this class of control problem is intractable for high-dimensional plants due to the non-quadratic form of the cost function. We therefore examined the behavior predicted by this theory in a simplified one-dimensional model of the center-out reaching task. In this model, the optimal initial reach direction is driven solely by the belief state at movement onset, r0, and the compatibility between potential goals. We cannot precisely know the timecourse of changes in belief in subjects following the target jump (i. e. how r0 varies as a function of time since the target jump). However, we assume that the change in belief should follow the same monotonic timecourse regardless of the size of the target jump. Thus the model explains differences in behavior across different jump amplitudes as being due to there being different optimal initial reach directions associated with similar belief states. Our aim in the model was to demonstrate that decreasing the compatibility between goals (by increasing the distance between them) leads to more abrupt changes in behavior as a function of preparation time. Fig. 6D illustrates the predicted initial reach direction in this simplified model as a function of the belief about the target location at movement onset. As can be clearly seen, the sensitivity of behavior to the belief at the time of movement onset depends significantly on the separation between targets. Our theory also naturally explains the absence of intermediate movements in the presence of a barrier, as seen in Experiment 3. The presence of the barrier reduces the decision about initial reach direction to a discrete choice, in which case the subject should select the most likely target direction at the time of movement onset, leading to step-like behavior. Although the simulations clearly demonstrate the relationship between goal compatibility and the timescale of the change in reach direction, our simulations also reveal an interesting discrepancy between the theory and the data. In the model, behavior across all target jump amplitudes is aligned for exactly halfway intermediate movements, i. e. where r0 = 0. Consequently, large-amplitude target jumps are predicted to be fully compensated earlier than small-amplitude jumps. In the data, however, we observed that full compensation for the target jump occurred at similar delays across all jump amplitudes. One potential explanation for this is that in the model, the pre-jump and post-jump targets are treated symmetrically. The fact that the target jumped on only a subset of trials may have biased subjects towards the initial target. Subjects may even possess an innate bias towards their original movement plan in the event that circumstances unexpectedly change. Asymmetry may also have arisen from differences in confidence in the exact target location between pre- and post-jump targets (although the results of Experiment 2 allow us to largely rule out this possibility). There are therefore a variety of asymmetries between the pre and post-jump targets that are not captured by the basic model. We attempted to accommodate these effects within the model through an asymmetric cost function which effectively penalized a miss more heavily when the true target turns out to be the original target. We found that imposing such an asymmetric cost was able to better reproduce the observed pattern of behavior (Fig. 6E). While admittedly post-hoc, these results demonstrate that the basic modeling framework can feasibly be extended to account for this aspect of the data. Our experiments extend and reinterpret a classic series of studies by Ghez and colleagues, who developed the timed-response paradigm to examine movement planning and preparation [4,9, 32,33]. In their experiments, only two movement directions were possible in each trial (as opposed to 8 in our experiments) and ambiguity was created by providing no target information at all until shortly before movement onset (as opposed to jumping an existing target as in our experiments). Their results were qualitatively similar to our own, with intermediate movements occurring at low preparation times when targets were narrowly separated but not when widely separated. An advantage of our target-jump approach is that we were able to control the initial belief state of the subject which, along with the low proportion of jump trials and multiple potential targets, made it unlikely that intermediate movements were the result of subjects adopting a deliberate aiming or guessing strategy. Our experimental results therefore reinforce the view that intermediate movements seen at low preparation times reflect an implicit property of the motor system rather than an explicit strategy. Ghez and colleagues suggested that intermediate movements at narrow separations were due to incomplete specification (i. e. preparation) of the motor commands required for movement. This theory cannot, however, explain why intermediate movements do not occur for more widely separated targets or in the presence of a barrier. Ghez and colleagues therefore suggested the existence of two distinct mechanisms of movement planning: a discrete mechanism responsible for the abrupt behavior seen at wider separations and a continuous re-specification mechanism operating at more narrow separations (see also [25]). Our theory offers a more satisfying and parsimonious explanation for these contrasting modes of behavior: that they reflect qualitatively different solution regimes to the same optimization problem. Some authors have suggested that intermediate movements following a target jump occur because the target is perceived to be at a location intermediate between goal locations [12,13]. Similarly, intermediate movements could potentially reflect an interpolation between movement plans, rather than between perceived goal locations. In either case, such interpolation mechanisms can plausibly account for the pattern of intermediate movements following small (45°) target jumps, and would also predict an abrupt switch in reach directions following 180° jumps. However, interpolation would predict only a relatively modest change in the pattern of intermediate movements as the target separation increased from 45° to 90° to 135°. In particular, this kind of model predicts a far smaller difference in behavior between the 45° case and the 135° case than suggested by our data. In particular, individual subjects tended to show an abrupt transition between movement direction following 135° jumps (e. g. Fig. 2E). This abrupt transition is consistent with the findings of Ghez et al. [4] who reported no intermediate movements when potential targets were separated by 120°. A model based on a direct interpolation between movement plans or goals does not predict such an abrupt transition until the jump amplitude becomes close to 180° and cannot therefore fully account for our findings. Intermediate movements are often interpreted as evidence for interference between parallel movement plans. Tasks that directly manipulate goal uncertainty, either by delaying disclosure of goal information [4,16,17], by presenting distractors [18], or by providing deliberately ambiguous cues [34], yield intermediate movements. More abstract cognitive decisions can have a similar effect [19]. In all cases, ambiguity about the goals of the movement is believed to lead to interference between associated movement plans, which ultimately leads to errant intermediate movements being generated. The existence of such intermediate movements is therefore thought to offer insights into the underlying mechanism of movement planning. One would expect that such interference, arising from low-level mechanisms, should be unavoidable. This is, however, inconsistent with our result in Experiment 3 in which subjects could easily eliminate intermediate movements in the presence of a virtual barrier. While it may be possible to augment mechanistic models with a means to over-rule the generation of intermediate movements where necessary, this raises serious questions about why intermediate movements should ever be permitted. Our alternative interpretation, analogous to previous proposals by Hudson, Landy and Maloney [24], is that intermediate movements reflect a single, deliberate movement plan chosen to maximize performance in the task amid ambiguity about the goal, explaining the presence or absence of intermediate movements without requiring any assumptions about the underlying planning mechanism. The basic theoretical framework presented here provides a promising unifying framework for describing perception and action. However, the intractability of obtaining the optimal policy for such models is a severe limitation. Recent advances in solution methods for optimal control problems [35] are inapplicable due to the structure of our control problem. Specifically, efficient solution methods require that noise and control act in the same dimensions. The structure of our control problem violates this requirement, since the decision variable has noisy dynamics but is not controllable. The development of efficient numerical methods applicable to the specific class of problems described here would be valuable in generating more precise predictions of the general theory. Optimal control theory has previously been invoked to account for intermediate movement strategies [36,37]. Such theories suggest intermediate movements as a strategy to exploit execution noise: when multiple, equally valid targets are present, it is better to aim for the middle and let execution noise dictate which target to ultimately hit. Although this theory potentially explains the presence of intermediate movements in the presence of multiple targets, we believe it is insufficient to account for our findings since the amount of execution noise required to predict an intermediate movement given a 90° separation between targets is infeasibly large. Although our model accurately accounted for the incidence of intermediate movements, one aspect of the data that was not predicted was the fact that the target jump was fully corrected for at about the same delay across all conditions. The model predicts that perfect compensation will be seen earlier under incompatible conditions compared to compatible ones. This discrepancy could potentially be attributable to our model assuming that the pre-jump and post-jump targets should be treated as equivalent, whereas in reality there are important differences between them. The results of Experiment 2 allow us to rule out the possibility that ambiguity about the location of the post-target jump was a major source of asymmetry. It is possible that deteriorating quality of peripheral vision at more eccentric target locations [38] could account for unexpected differences across conditions, although it would be quite a coincidence for this to lead to such close temporal alignment. Additional sources of asymmetry may arise from intrinsic biases in subjects’ decision-making processes; subjects may be inherently biased against changing their minds [34]. Indeed, given that the target jumped in only 30% of trials, it was more likely a priori that the target would remain in its original location. An alternative explanation for the consistent time at which compensation for the jump became complete is that an underlying mechanistic constraint on movement preparation limited the ability to generate an accurate response to the changed target location. It is unclear how intermediate movements might be reconciled with such a constraint. Nevertheless, our theory provides a rational account of why intermediate movements should ever be allowed to occur, instead of simply always switching abruptly between movement directions. Our normative model suggests an alternative interpretation of a number of well-established neural correlates of movement planning and preparation. High-level movement goals appear to be represented in dorsal [39] and ventral [40] premotor cortex and also posterior parietal cortex [41]. When multiple potential goals are presented, these goals are represented simultaneously [39,42]. Conventionally, activity associated with a single goal is construed as representing a specific movement plan; simultaneous responses when multiple targets are present is thought to reflect multiple such plans occurring in parallel [23]. Our theory provides an alternative view: that the overall pattern of activity across this population represents a global belief state (a multi-target analog of our binary decision variable rt) over all possible movement goals; details of how to achieve these goals will be determined by a downstream site, possibly primary motor cortex, which is responsible for implementing a single control policy associated with this global belief state. Computational models have suggested that lateral connectivity within a network representing task goals may provide a mechanism whereby intermediate movements are generated [27,43,44]. Excitatory connections between units tuned to similar movement directions can lead to two peaks of activity becoming merged and thus leading to intermediate movements. These theories therefore explain intermediate movements as a by-product of an underlying planning mechanism. Inhibitory connections between units representing dissimilar movements create winner-take-all dynamics when potential goals are more widely separated. Our results show, however, that intermediate movements are not obligatory; in the presence of a barrier they can be suppressed. This absence of intermediate movements could potentially be explained by inhibition of units representing movement directions that would hit the barrier. However, if it is so easy to eliminate intermediate movements in such a model, it is unclear why they should be permitted in the absence of a barrier. It is currently unclear exactly how control policies are represented in the brain. However, recent theories have suggested that the state of motor cortex at the time of movement onset is sufficient to encode the full sequence of feedforward motor commands required to execute a movement [45,46]. Typically, neural activity converges onto a movement-specific preparatory state over a period of around 100ms following stimulus presentation [47,48]. It is tempting to interpret this change in neural state as reflecting a form of movement planning. We suggest instead that this observed change in neural state could equally reflect an evolving decision. Indeed, the state of motor areas appears to continuously track belief state during decision-making tasks [49,50]. Although these results are often interpreted as reflecting partially formed or blended motor plans, our theory suggests instead that intermediate states might reflect a single control policy that is optimal given a partially formed belief about movement goals—effectively hedging against possible future fluctuations in belief or changes of mind [34] after the movement has begun. All procedures were approved by the Johns Hopkins University School of Medicine Institutional Review Board. All subjects provided written informed consent prior to participating. 24 adult (18–40 y/o, 11 female), right-handed, neurologically healthy subjects were recruited for this study. Subjects were seated at a glass-surfaced table. Their right forearm was supported by a plastic cradle equipped with pressurized air vents to allow frictionless planar arm movements. Subjects' arms were obstructed from view by a mirror positioned above the table surface, through which an LCD monitor (60Hz) displayed movement targets and the position of the index finger in a veridical horizontal plane. The index finger was tracked at 130Hz using a Flock-of-Birds magnetic tracker (Ascension Technology, VT, USA). A total of 10 subjects participated in Experiment 1. On each trial, subjects were required to position the cursor inside a start circle (10mm diameter). After 300ms, a sequence of four tones spaced 500ms apart was initiated (Fig. 1A). Synchronous with the first tone, a single target (25mm diameter) appeared at one of eight possible target locations, positioned uniformly on a circle of radius 0. 08m (Fig. 1B). Subjects were required to initiate a ‘shooting’ movement through the target, synchronous with the onset of the fourth tone. Movement onset was detected based on the first time that the tangential velocity of the cursor exceeded 0. 02m/s. In order to be successful, subjects were required to initiate movement within ±100ms of the onset of the fourth tone and move the center of the cursor through some part of the target region. An on-screen graphic displayed peak velocity after each trial and subjects were asked to keep this above a shown threshold that corresponded to 0. 9ms-1. On successful trials, subjects were rewarded with a “success” tone and points towards a cumulative score. On-screen text following each trial indicated whether subjects had initiated their movement too early or too late. 1s after movement onset, subjects were able to begin the next trial by returning to the start circle. In an initial familiarization session prior to the main experiment, all subjects received extensive training (>500 trials) at timing their movement initiation accurately. During the main experiment, the target was jumped to a different location in 30% of trials, at a random time (between 150ms and 550ms) prior to the fourth tone. The direction of the new target location differed by either ±45°, ±90° or ±135° from the original (Fig. 1B), and this difference was randomly selected on each jump trial. Subjects performed approximately 2000 trials total, divided into blocks of 100 trials. The full experimental session, including occasional breaks, lasted approximately 3 hours. Some subjects performed the main experiment across two separate sessions on different days. Experiment 2 followed the same pattern as Experiment 1, except that there were only 2 potential target locations. Six new subjects (4 Female) performed 500 trials (across 5 consecutive blocks) with a 45° separation between potential target locations (±22. 5° relative to straight-ahead), and 500 trials with a 135° separation (±67. 5° relative to straight-ahead). The order of target configurations (45° separation first or 135° separation first) was counterbalanced across subjects. The same basic setup was used in Experiment 3. Eight subjects participated in this experiment (3 Female), two of whom had also participated in Experiment 1. Subjects performed two main sessions, each consisting of 8 blocks of 100 trials. The No Barrier session was similar to Experiment 1, with 8 potential targets and the target jumping on 30% of trials, except that all jumps were ±45° in magnitude. The Barrier session was identical to the No Barrier session, except that a series of virtual barriers was introduced in the workspace (Fig. 5A). These barriers encouraged subjects to move in a straight line towards each target and prohibited intermediate movements. The barrier configuration effectively created a 10mm wide channel within which subjects could move freely. On trials in which subjects entered the barrier region, the barrier turned red, an unpleasant tone was played, and the subject received no score on that trial. Subjects were also verbally encouraged to avoid contacting the barrier. All subjects performed one session with barriers present and one session without and the order of sessions was counterbalanced across subjects. Barriers were also present during the second half of the initial familiarization session in which there were no target jumps. Position and velocity data were smoothed using a 2nd-order Savitzky-Golay filter with half width 54ms. We computed the time of movement onset based on the latest time that the smoothed tangential velocity was less than 0. 02m/s prior to the peak tangential velocity (note that this differed slightly from the onset time calculated online that determined the success or failure feedback given to subjects about the timing of their movements during the experiment). We expected that the initial direction of movement would depend on the amount of time available to revise the movement plan prior to movement initiation. We therefore computed, for each trial, the re-preparation time (rPT) —the duration between the time of the target jump and the time of movement onset. We computed the initial reach direction based on the direction of the tangential velocity 100ms after movement onset. All jump trials were transformed into a common reference frame such that the initial target was located at 0°, and the target jumped in a positive direction. Trials in which the hand failed to move further than 5cm from the start location were excluded from the analysis. In a small number of trials, movements were excessively curved in a way that did not permit a well-defined estimate of the reach direction 100ms after movement onset. This was often associated with failure to keep the hand stationary prior to movement initiation. We identified and eliminated excessively curved movements as follows: we computed the rate of change of estimated movement direction with respect to measurement time, dθ^dt=θ^ (100+Δ) −θ^ (100−Δ) 2Δ, (1) where θ^ (t) is the estimated reach direction at time t and Δ=1130 ms. Based on behavior in non-jump trials, we set a threshold on the absolute value of this rate of 1. 3°/ms. Of all non-jump trials, 99% fell within this range. As a result of this exclusion procedure, an average of 4. 5±3. 1 trials per subject were excluded in Experiment 1. These excluded trials were distributed similarly across each of the possible jump types (F (2,18) = 3. 15; p = 0. 07). An average of 4. 7±3. 3 trials per subject were excluded in Experiment 2, and an average of 2. 75±3. 0 trials per subject were excluded in Experiment 3, also not depending on the condition (Experiment 2, F (1,5) =. 03; p = 0. 8; Experiment 3, F (1,7) = 0. 02; p =. 88). In total, we excluded less than 3% of all trials on the basis of excessive curvature. In order to quantify the timecourse of the change in initial reach direction for comparison across conditions and across subjects, we assumed that the initial reach direction followed a sigmoidal relationship with available re-preparation time: θ=S (rPT) =A1+e− (rPT−t50) τ. (2) We assumed that A was equal to the actual jump amplitude. This function therefore contained two free parameters: a slope parameter τ that characterized the timescale over which gradual changes in reach direction occurred, and a latency parameter t50 which acted to shift the sigmoid along the time axis. An important feature of the data is the presence of uncertainty not just in the estimated reach direction, but also in the estimated rPT. In the presence of such uncertainty, an ordinary least squares fitting approach significantly overestimated τ. We therefore adopted a maximum likelihood approach that specifically accounted for the uncertainty in the rPT. Specifically, we assumed Gaussian noise both in the reach direction, due to either execution variability or measurement noise (s. d. σθ), and in the estimated re-preparation time, due to either variability on the part of the subject, uncertainty in our estimate of the movement onset time, or experimental error in controlling the precise target presentation time (s. d. σt). The likelihood for each observation was consequently given by Li∝∫exp[− (e−t) 22σt2− (θi−S (e; t50, τ) ) 22σθ2]de, (3) where e reflects possible values for the noise in the measured value of the rPT. We set σθ equal to the mean standard deviation of initial movement directions on non-jump trials across all subjects (σθ = 10. 7). We set σt = 10 ms since this value was found to lead to robust performance on pilot data. This likelihood was evaluated by trapezoidal integration over e. Not all subjects consistently generated data with rPTs in the critical slope region of the sigmoid. Behavior for which this data was unavailable was thus equally consistent with a broad range of sigmoid parameters. We resolved this ambiguity by biasing the sigmoidal fit towards more shallow slopes through an additional term added to the log-likelihood: Λ=∑iLi+ατ. (4) Thus the estimated slope was the shallowest (longest duration) slope that was consistent with the data. Importantly, this approach was conservative since this ambiguity tended to occur during larger amplitude target jumps where behavior was expected to be more abrupt. We set α = 0. 02, based on fits to synthetic data. Finally, maximizing this likelihood yielded accurate parameter estimates on synthetic datasets, but was quite sensitive to outlying data points in real data. We therefore extended our parameter estimation procedure to make it more robust to outlying data points by supposing that each data point could have been generated by an alternative, uniform distribution, with a fixed likelihood L0. We identified the sigmoid parameters that maximized the likelihood of this mixture model using an expectation maximization algorithm [51,52]. Following this procedure, we rejected an average of 3. 9%±2. 3% of data points per subject from Experiment 1 as outliers and 1. 7±1. 5% of data points in Experiment 2, in neither case biased towards any particular condition (p>0. 05). In Experiment 3, the average outlier rejection rate across subjects was 3. 2%±1. 9% and was marginally but consistently greater in the barrier condition (2. 5%±1. 8% No Barrier, 3. 9%±1. 8% Barrier; F (1,7) = 5. 61; p<0. 05). Here, we consider the problem of selecting optimal actions in order to achieve a goal in the presence of uncertainty. We model the arm through a linear dynamical system in discrete time with state xt, and subject to time-varying controls ut: xt+1=Axt+But. (5) We characterize the goal of the task through an accuracy cost Jx that penalizes deviations from some goal state g at the end of the movement (time t = T). In addition to this accuracy cost, we assume an effort cost Ju that penalizes large motor commands. It is difficult to say a priori exactly what the form of this cost should be [53]. Following standard approaches [20], however, we assume that this effort cost is a quadratic function of the overall sequence of motor commands: Ju=∑twtut2. (6) In this equation, wt is a potentially time-varying weight. According to the optimal feedback control hypothesis [20], the motor system selects motor commands ut that minimize the sum of accuracy and effort costs: J=Jx (xT−g) +Ju. (7) The key novelty of our model is that the location of the goal state g is not precisely known. Specifically, we assume that the true goal is at one of two possible locations, g1 and g2. Supposing that p represents the belief that g1 is the true goal location, and (1−p) the corresponding belief for g2, we introduce an evidence variable r which reflects the perceived log-odds ratio between the two targets: r=log (p1−p). (8) Furthermore, we assume that the belief about the state of the target can vary over time. As is commonly assumed in decision-making models [3,26], we model rt as following a Gaussian random walk: rt+1∼N (rt, σr2). In models of decision-making, the stochastic nature of rt reflects a distribution over stimuli that the subject may have perceived in a given trial. In this case, however, the dynamics of rt reflect the subjects’ subjective prior expectations about how their belief might change in the future, after movement onset. We set rt to follow a random walk with zero drift, reflecting the fact that subjects should expect their beliefs to change, but are not biased to expect that they will change in any particular direction. Note that we do not attempt to explicitly model the actual evolution of rt through the movement in response to presented stimuli. Instead, we focus on the implications that the possibility of future evidence being accumulated during the movement will have for the choice of action at the start of the movement. The overall expected cost depends upon the ultimate belief about the target location, rt, at the end of the movement (time T), i. e. rT: E[J]=pTJx (xT−g1) + (1−pT) Jx (xT−g2) +Ju =11+e−rTJx (xT−g1) +11+erTJx (xT−g2) +Ju. (9) Our hypothesis is that subjects act to minimize the expected value of this cost. Solving this optimal control problem is not straightforward. Although the overall state of the system (combining the limb state xt and belief state rt into a single vector) has linear dynamics and Gaussian noise, the endpoint cost is a non-linear function of that state (Equation 9). This precludes usual solution methods for optimal control problems which require an endpoint cost that is quadratic in the state. We are therefore forced to rely on a dynamic programming approach [54] which severely limits the dimensionality of problems for which we can obtain a solution. We implemented a simplified model to demonstrate the key features of behavior predicted by this framework. We modeled the center-out reaching task with a single spatial dimension xt representing the angular position of the hand. We assumed that the motor command ut specified the instantaneous angular velocity of the hand, i. e. x˙t=ut. We assumed a fixed time horizon of 200ms. We set wt in Equation 9,8 to increase linearly from 0 at t = 0 to wMAX at t = T, to reflect the fact that achieving a given angular velocity requires a higher Cartesian velocity when the hand is further away from the start position and should therefore be more costly. The endpoint cost for each target was given by a step function with width a around the goal region. In order to determine the control policy that minimized the total expected cost (Equation 9) we discretized the state space (angular position discretization of 0. 1°, belief discretization of 0. 5, and time discretization of 10ms) and used dynamic programming [54] to find the optimal expected cost-to-go V (x, r, t) at each state and time. We used the value function at t = 0 to determine the optimal initial reach angle x0* for each possible initial belief r0, i. e. We manually selected model parameters (wMAX =. 001, a = 1, σr = 1) that yielded qualitatively similar predictions to actual subject behavior. Note that our aim here was not to provide a quantitative fit to the data but to demonstrate the feasibility of our theory to account for our observations—principally the interaction between target separation and the time course of intermediate movements. Finally, based on observed discrepancies between the data and the model (see Results and Discussion), we considered the possibility that the nature of the task may have created an asymmetry between the initial goal and the post-jump goal that is not captured in the basic form of the model. We accommodated some asymmetry within the model through an asymmetric cost function in which the cost function Jx for the initial target location was scaled relative to the post-jump target location: J=α1ptJx1 (x−g2) + (1−pt) Jx2 (x−g2) +Ju. (12) We set α1 = 10 in order to yield behavior that qualitatively matched observed behavior. | Two critical processes need to occur before a movement can be made: identification of the goal of the movement and selection and preparation of the motor commands that will be sent to muscles to generate the movement-in other words, what movement to make, and how to make it. It has long been thought that preparing motor commands is a time-consuming process, and theories advocating this view have pointed to instances where apparently the wrong motor commands are issued if insufficient time is available to prepare them. The usual pattern of these wayward movements is that they are intermediate between two potential targets. In this article we show how such intermediate movements can alternatively be viewed as reflecting an intelligent and deliberate decision about how to move, given uncertainty about task goals. Our theory is supported by experiments that show that intermediate movements only occur in conditions where they are advantageous. The implication of our theory is that the primary bottleneck to generating a movement is deciding on exactly what to do; deciding how to do it is rapid and automatic. | lay_plos |
Scene: The University cafeteria
Raj: Here's what I wonder about zombies. (Others all groan) What happens if they can't get any human flesh to eat? They can't starve to death, they're already dead.
Howard: You take this one. I spent an hour last night on how do vampires shave when they can't see themselves in the mirror?
Sheldon: Well-groomed vampires meet in pairs and shave each other. Case closed.
Raj: Yeah, okay, so, zombies.
Leonard: I guess it depends on the zombies, Raj. Are we talking slow zombies, fast zombies? Like, in 28 Days, if those zombies didn't eat, they starved.
Howard: You're thinking of 28 Days Later. 28 Days is where Sandra Bullock goes to rehab and puts the audience into an undead state
Raj: Hey, don't bag on Sandra Bullock! You think it makes you look cultured, but you just come off as bitter.
Leonard: Oh, Dr. Siebert, twelve o'clock.
Howard: Why's the president of the university slumming in the cafeteria?
Sheldon: Perhaps he's emulating Shakespeare's Henry V, who dressed as a commoner and mingled among them, to find out how he was being perceived by his subjects. Course, if he'd have read any of the thirteen hundred e-mails I've sent him on the subject of his administration, he could have saved himself the trouble.
Raj: Or maybe he heard it's Tator Tot Tuesday. That's why I'm here.
Dr. Seibert: Hey, there's my favorite geniuses! How are we doing today?
Sheldon: That depends, how much longer do you plan on fondling my shoulder?
Seibert: Sorry, Dr. Cooper, I forgot you have a touch phobia.
Sheldon: It's not a touch phobia, it's a germ phobia. If you'd like to go put on a pair of latex gloves, I'll let you check me for a hernia.
Seibert: Yeah. So, listen, fellas, who's up for a little party this Saturday night? Open bar, good eats, might even be a few pretty girls.
Raj: Sounds great!
Howard: I'm in!
Sheldon: Hold on. Just because the nice man is offering you candy, doesn't mean you should jump into his windowless van. What's the occasion?
Seibert: Just a little fund-raiser for the university.
Sheldon: Aha! The tear-stained air mattress in the back of the van.
Seibert: I understand your reticence, Dr. Cooper, and I sympathize, but the hard facts are, occasionally, we have to shake a few hands and kiss a few butts to raise money for our research.
Sheldon: I don't care, it's demeaning. And I refuse to be trotted out and shown off like a prize hog at the Texas State Fair. Which, by the way, is something you don't want to attend wearing a Star Trek ensign's uniform.
Seibert: All right, let me put it this way. You're gonna put on a suit, you're gonna come to this party, and you're gonna explain your research to a bunch of old people, or I swear to God, I'll blind you with a hot spoon, like they did to that little boy in Slumdog Millionaire.
Raj: Oh, you don't want that.
Seibert: So, Saturday night! It's gonna be off the hook.
Sheldon: Ugh!
Seibert: Get over it.
Raj: Oh, boy! Tator tots and a party invitation? What a great day! Credits sequence.
Scene: The apartment.
Penny: There you go.
Leonard: Are you sure this is right?
Penny: Yeah, just tuck that part in your pants; you'll be fine.
Howard: Okay, let's go smooch some rich, wrinkled tuckus.
Penny: Oh, Howard, I can't believe Bernadette let you go to a fancy party wearing a dickey.
Howard: Excuse me, my girlfriend doesn't pick out my clothes. My mother does.
Leonard: Oh. We should get going.
Howard: What about Sheldon?
Sheldon: Sheldon is not going.
Leonard: Really? What do we tell Siebert?
Sheldon: Tell him Dr. Cooper feels that the best use of his time is to employ his rare and precious mental faculties to tear the mask off nature and stare at the face of God.
Penny: Sheldon, it's Saturday night, you'll be doing laundry.
Sheldon: Don't tell him that, tell him the mask thing.
Scene: The party.
Howard: Hey, put your tie back in your pants.
Leonard: Thanks.
Raj: Nice place. Reminds me of my parents' house back in New Delhi.
Howard: You're kidding.
Raj: No. We are very wealthy. But the only difference is, we have more servants.
Leonard: More than this?
Raj: More than we can use. You see, in India, we don't make the mistake of letting our poor people have dreams.
Seibert: Ah. There's my band of brainiacs. Where's Dr. Cooper?
Leonard: He's tearing the mask off nature to look at the face of God.
Seibert: The board of directors insists he has a beautiful mind. I think he's just bananas. Come on, let me introduce you to one of the university's leading donors.
Raj: I think we were misled about the cute girls.
Seibert: Mrs. Latham, I'd like you to meet three of our outstanding young researchers. This is Dr. Leonard Hofstadter, Dr. Rajesh Koothrappali and Howard Wolowitz.
Mrs Latham: Well, what happened to you, Wolowitz, couldn't stick with it long enough to get your PhD?
Howard: I'm an engineer. Most engineers don't bother with a PhD. But you may be interested to know I designed the zero-gravity waste-disposal system for NASA.
Mrs Latham: Got it, you're a space plumber.
Howard: I'm gonna go hit the bar.
Mrs Latham: Tell me about these two.
Raj: Do him first.
Seibert: Dr. Hofstadter is representing our experimental physics program tonight. I think you'll really enjoy hearing about his fascinating work.
Mrs Latham: Right. Fascinate me.
Leonard: Uh.. b.. d.. uh.. uh..
Mrs Latham: They're cute when they're about to wet themselves, aren't they? I'll make it easy for you. When you arrive at the lab in the morning, what sort of machine do you turn on?
Leonard: Coffee maker?
Mrs Latham: All right, Dr. Kooth... uh, whatever it is, you're up.
Raj: It's Koothrappali. I have to tinkle.
Scene: The apartment.
Sheldon: And so, instead of bowing to pressure, and going to that pointless soiree, I stayed right here and did a load of whites.
Amy (on webcam): Well, normally I respect your macho rebellious attitude toward The Man, but, in this case, I think you've made a foolish mistake.
Sheldon: Unlikely. But make your case. Keeping in mind that your critical attitude is ruining our Saturday night together, and I'm not above minimizing your window.
Amy: Sheldon, like it or not, until you manage to upload your intelligence into a self-sustaining orbiting satellite, equipped with high-speed Internet and a cloaking device, you will be dependent on other members of the human race.
Sheldon: That's it. Prepare to be minimized.
Amy: I'm not finished. All scientists have to fund-raise, Sheldon. How do you think I paid for my lab? I went to Saudi Arabia and met with a prince who had an interest in neurobiology.
Sheldon: Your lab is funded by some Middle-Eastern dilettante?
Amy: Technically, Faisal is my fiance. But I do have a state-of-the-art two-photon microscope and a place to stay in Riyadh for the winter.
Sheldon: Well, that explains those puzzling camel race photos on your Facebook page.
Amy: And consider this, without you to make the case for the physics department, the task will fall to people like Leonard and Rajesh.
Sheldon: Are you trying to scare me? 'Cause you're succeeding.
Amy: Well, then prepare to be terrified. If your friends are unconvincing, this year's donations might go to, say, the geology department.
Sheldon: Oh, dear,not, not the dirt people!
Amy: Or worse, it could go to the liberal arts.
Sheldon: No!
Amy: Millions of dollars being showered on poets, literary theorists and students of gender studies.
Sheldon: Oh, the humanities!
Scene: The party.
Leonard: On the bright side, I don't think President Siebert will be making us go to any more fund-raisers.
Howard: It was so much easier at my bar mitzvah. The old people just came up to you, pinched your cheek and handed you a savings bond.
Raj: Oh, don't be such gloomy Gusses. Look at the size of these shrimp! At what point do we start calling them lobsters?
Leonard: Face it, Raj, we crashed and burned tonight.
Mrs Latham: Oh, you didn't do that badly.
Leonard: Mrs. Latham, the first machine I turn on in the morning is the helium-neon laser, 'cause it needs to warm up.
Mrs Latham: I no longer care, dear. But don't worry, I really enjoyed meeting you this evening.
Leonard: You're kidding. That was good for you? 'Cause I was sweating through my T-shirt.
Mrs Latham: Excellent! There's nothing I like better than making smart people feel ill at ease.
Leonard: Why?
Mrs Latham: Oh, I don't know, it's one of the fun things you get to do when you have lots of money. Watch. Hey! Who said you could eat that shrimp? See? Fun.
Sheldon (behind them): No, no, no, I'm just here for your money. I don't want to shake anyone's germy hands. Explain it to them, Siebert.
Scene: The apartment.
Sheldon (on phone): I must confess I don't understand you, President Siebert. First you say you want me to appear at your fund-raisers, but now you say you never want me to go anywhere near your fund-raisers. Forgive me, but that sounds like a mixed message. Here we go again. If there's simply no talking to me, why did you call? I'm sorry, someone's on the other line. Why don't you see if you can organize your thoughts, and we'll try again later. Cooper-Hofstadter residence. Go for Cooper. Good morning, Mrs. Latham. Well, yes, of course I remember you. A woman well past her prime seeking to augment her social status by doling out her late husband's ill-gotten gains. So, how much money are you going to give me? I'm not crazy, my mother had me tested. Well, if you're not going to give me money, then why are you calling? She wants to talk to you. Who's crazy now?
Leonard: Hello, Mrs. Latham. Yes, I live with him. I don't, I, I really don't know why. Tonight? Sure, that'd be great. Okay, I'll, I'll see you then. Bye. She wants to have dinner and talk about my research.
Sheldon: An entire dinner to talk about your research? Where you going, the drive-thru at Jack in the Box?
Leonard: Well, wherever we're going, she's sending a car to pick me up.
Sheldon: Okay, I see what's happening.
Leonard: What?
Sheldon: My stature intimidates her, so she's using you to get to me. Crafty old gal.
Leonard: Excuse me, but you are not the only distinguished scientist in this apartment. I've been published in peer-reviewed journals, I received a Dissertation of the Year award for experimental particle physics.
Sheldon: No, that can't be it. And since you seem to have forgotten, the reason we live together is we're best friends. And I got your back, Jack.
Secne: Mrs Latham's car.
Leonard: That was a great meal.
Mrs Latham: I'm glad you enjoyed it.
Leonard: The only time I eat this well is when my mom's in town and she takes me out to dinner.
Mrs Latham: Is that so?
Leonard: You kind of remind me of her. She enjoys making people uncomfortable, too.
Mrs Latham: Well, you remind me of a boy I dated in college.
Leonard: No kidding.
Mrs Latham: Sweet boy. Very smart. If only he'd had money.
Leonard: Yeah, um, so, hey, speaking of money, how are you feeling about helping the physics department get a cryogenic centrifugal pump and molecular sieve?
Mrs Latham: Well, I must say, you make a very persuasive case for it.
Leonard: Oh, good, good.
Mrs Latham: And I'm seriously considering taking it to the next level.
Leonard: Terrific. Great. What level is that? (She grabs him and kisses him) Okay, now you don't remind me of my mom.
[SCENE_BREAK]
Scene: The apartment.
Sheldon: I'm sorry, so, eventually, zombies are going to attack the rehab facility where Sandra Bullock is?
Penny: Yes, Sheldon. Keep watching.
Sheldon: You know, it's a shame, all that work she's doing to get sober, only to be torn apart and eaten alive.
Howard: Hey. How was dinner?
Leonard: Swell. I need a drink. Do we have any alcohol?
Sheldon: No. But we have potatoes, I could make you vodka. It'll take two weeks.
Penny: Leonard, are you okay?
Leonard: Um, I'm not sure.
Howard: What's going on?
Leonard: Well, Mrs. Latham said she was seriously considering donating money so we could get a cryogenic centrifugal pump.
Sheldon: Oh, wow!
Howard: Yes!
Leonard: Then she stuck her tongue down my throat.
Sheldon: Why?
Penny: Okay, we can't keep explaining everything. Read that book we got you.
Leonard: She hit on me.
Howard: Wait-wait-wait. Are you telling us that old lady wanted to have s*x with you in exchange for giving your department millions of dollars?
Leonard: I think so.
Howard: You lucky duck.
Penny: You're really a broken toy, aren't you?
Leonard: I was able to get out of there before anything else happened, but she wants to see me again tomorrow night.
Sheldon: Excellent! What are you planning to wear?
Leonard: What?
Sheldon: Penny, you're an expert on trading sexual favours for material gain, walk him through this.
Leonard: Well, n-no, hold on a second, I'm not going to sleep with her.
Sheldon: But we need a cryogenic centrifugal pump.
Leonard: Well, forget it! It's not gonna happen.
Sheldon: Well, come now, Leonard, this may be your only chance to make a real contribution to science!
Leonard: I repeat, not gonna happen.
Penny: What was all that about me trading sexual favours for material gain?
Sheldon: It was a compliment. I believe in giving credit where credit is due.
Scene: The apartment, the following night.
Sheldon (on phone): Okay, fine. I'll tell him. Leonard, Mrs. Latham's car is here for you.
Leonard: I won't be too late. I'm just gonna make a final pitch for the funding and then say good night.
Sheldon: Hold on, I have something for you.
Leonard: What's this?
Sheldon: Just a few things you may need tonight. There's, uh, baby oil, condoms and, uh, a little something I procured from the school of pharmacology. They say it is to Viagra as Viagra is to a green M&M.
Leonard: I am not going to have s*x with her.
Sheldon: Maybe this will overcome your reluctance. I went on the Internet and found a photograph of a 25-year-old Mrs. Latham to get your libido humming. Check out those saddle shoes. Rar!
Leonard: Are you insane? I'm not going to prostitute myself just so we can get some new equipment.
Sheldon: Oh, come on! Why not?
Leonard: Good night, Sheldon.
Sheldon: Given how much time you spend engaging in pointless self-abuse, you might consider, just this once, using your genitalia to actually accomplish something!
Penny: He still won't shag the old lady, huh?
Sheldon: No. But thank you for asking.
Scene: Mrs Latham's car.
Leonard: Hey. Hi.
Mrs Latham: Hello, Leonard. I hope you're hungry.
Leonard: I'm very hungry. For food, right?
Mrs Latham: Oh, I made you uncomfortable last night. I'm so sorry.
Leonard: No, that's okay.
Mrs Latham: No, it most certainly is not. Leonard, I'm making the donation to your department regardless of what happens between us.
Leonard: Really?
Mrs Latham: Well, of course. There's no quid pro quo here. You and your colleagues are doing remarkable research, and you deserve the money.
Leonard: Oh. Then what was last night about?
Mrs Latham: I took a shot, sue me.
Leonard: Oh.
Mrs Latham: You're a very handsome man, Leonard.
Leonard: Thank you.
Mrs Latham: It was foolish of me to think someone your age might ever be interested in someone like me.
Leonard: Oh, don't say that. You're a very attractive woman.
Mrs Latham: Oh, please.
Leonard: No, it's true.
Mrs Latham: Well, aren't you sweet. Just for the record, you'd remember a night with me for the rest of your life.
Leonard: I'm sure I would. But why, why, exactly?
Mrs Latham: You're a very smart man. How do you think I landed such a rich husband?
Leonard: I hadn't really given it much thought.
Mrs Latham: Well, think about it.
Leonard: Do you mean?
Mrs Latham: Yep. I'm that good.
Leonard: Oh, what the hell.
Scene: The stairwell. Leonard is returning home looking dishevelled.
Penny: Good morning, slut.
Leonard: What?
Penny: Oh, please, I recognize the walk of shame when I see it. All you're missing is a little smeared mascara and a purse with panties wadded up in it.
Sheldon: What's going on?
Leonard: Oh, nothing's going on. Excuse me.
Sheldon: Are you just getting home?
Leonard: Yeah.
Sheldon: That's a good sign, right?
Penny: Oh, yeah.
Sheldon: I'm so proud of you! You sold yourself out like a common streetwalker!
Leonard: No, I didn't do it for the money.
Sheldon: She stiffed you?
Penny: I believe that's what your roommate did to her.
Sheldon: What?
Penny: Again, read the book we gave you.
Leonard: No, I mean, I, I, I got the money first.
Sheldon: Smart. Get paid up front. Yeah, I think you have a real knack for gigolo work, Leonard.
Leonard: Uh, I'm gonna go lie down.
Sheldon: That's a good idea, get your rest. There are a lot more rich old ladies out there, and Daddy needs a new linear accelerator. And I thought he didn't learn anything from his relationship with you.
Penny: Hey!
Sheldon: Another compliment! Learn to recognize them.
Scene: The cafeteria.
Seibert: Ah, there he is! The man of the hour! He took one for the team!
Leonard: I didn't do it for the money!
Seibert: Keep telling yourself that, it makes it easier. Trust me, I know.
Raj: Cool, buddy! That's awesome!
Howard: How was she? | President Siebert invites the men to a fundraising event at Caltech. While Leonard, Howard and Raj are willing to attend, Sheldon feels that such events are beneath him. He changes his mind when Amy points out that, without his participation, any monies raised might go to programs other than physics. When he finally arrives, Siebert quickly regrets inviting him. Meanwhile, Leonard makes a good impression on the considerably older Mrs. Latham. The next day, she picks him up in her limousine, and makes it clear that she will only donate the money if Leonard has sex with her. He refuses, but everyone else urges him to go through with it. After he does so and she donates the money, Leonard receives a standing ovation at work. | summ_screen_fd |
This article is about the plant that is a symbol of Ireland. Many places, organisations, businesses, products, creative works etc. use the shamrock in their names. For other uses, see Shamrock (disambiguation) Not to be confused with four-leaf clover A shamrock A shamrock is a young sprig, used as a symbol of Ireland. Saint Patrick, Ireland's patron saint, is said to have used it as a metaphor for the Christian Holy Trinity.[1] The name shamrock comes from Irish seamróg [ˈʃamˠɾˠoːɡ], which is the diminutive of the Irish word for plant (seamair) and means simply "little plant" or "young plant".[2] Shamrock usually refers to either the species Trifolium dubium (lesser clover, Irish: seamair bhuí)[3] or Trifolium repens (white clover, Irish: seamair bhán). However, other three-leaved plants—such as Medicago lupulina, Trifolium pratense, and Oxalis acetosella—are sometimes called shamrocks. The shamrock was traditionally used for its medicinal properties[citation needed] and was a popular motif in Victorian times. Botanical species [ edit ] There is still not a consensus over the precise botanical species of clover that is the "true" shamrock. John Gerard in his herbal of 1597 defined the shamrock as Trifolium pratense or Trifolium pratense flore albo, meaning Red or White Clover. He described the plant in English as "Three leaved grasse" or "Medow Trefoile", "which are called in Irish Shamrockes".[4] The Irish botanist Caleb Threlkeld, writing in 1726 in his work entitled Synopsis Stirpium Hibernicarum or A Treatise on Native Irish Plants followed Gerard in identifying the shamrock as Trifolium pratense, calling it White Field Clover.[5] The botanist Carl von Linné in his 1737 work Flora Lapponica identifies the shamrock as Trifolium pratense, mentioning it by name as Chambroch, with the following curious remark: "Hiberni suo Chambroch, quod est Trifolium pratense purpureum, aluntur, celeres & promtissimi roburis" (The Irish call it shamrock, which is purple field clover, and which they eat to make them speedy and of nimble strength).[6][7] Linnaeus based his information that the Irish ate shamrock on the comments of English Elizabethan authors such as Edmund Spenser who remarked that the shamrock used to be eaten by the Irish, especially in times of hardship and famine. It has since been argued however, that the Elizabethans were confused by the similarity between the Irish (Gaelic) name for young clover seamróg, and the name for wood sorrel seamsóg.[8] The situation regarding the identity of the shamrock was further confused by a London botanist James Ebenezer Bicheno, who proclaimed in a dissertation in 1830 that the real shamrock was Oxalis acetosella or Wood Sorrel.[9] Bichino falsely claimed that clover was not a native Irish plant and had only been introduced into Ireland in the middle of the 17th century, and based his argument on the same comments by Elizabethan authors that shamrock had been eaten. Bicheno argued that this fitted the wood sorrel better than clover, as wood sorrel was often eaten as a green and used to flavour food. Bicheno's argument has not been generally accepted however, as the weight of evidence favours a species of clover. A more scientific approach was taken by English botanists James Britten and Robert Holland, who stated in their Dictionary of English Plant Names published in 1878, that their investigations had revealed that Trifolium dubium was the species sold most frequently in Covent Garden as shamrock on St. Patrick's Day, and that it was worn in at least 13 counties in Ireland.[10] Finally, detailed investigations to settle the matter were carried out in two separate botanical surveys in Ireland, one in 1893[11][12] and the other in 1988.[13] The 1893 survey was carried out by Nathaniel Colgan, an amateur naturalist working as a clerk in Dublin; while the 1988 survey was carried out by E. Charles Nelson, Director of the Irish National Botanic Gardens. Both surveys involved asking people from all across Ireland to send in examples of shamrock, which were then potted up and allowed to flower, so that their botanical species could be identified. The results of both surveys were very similar, showing that the conception of the shamrock in Ireland had changed little in almost a hundred years. The results of the surveys are shown in the table below. Medicago lupulina flowers in Ireland from May to October[14] and so is not in flower on flowers in Ireland from May to Octoberand so is not in flower on St. Patrick's Day The results show that there is no one "true" species of shamrock, but that Trifolium dubium (Lesser clover) is considered to be the shamrock by roughly half of Irish people, and Trifolium repens (White clover) by another third, with the remaining fifth split between Trifolium pratense, Medicago lupulina, Oxalis acetosella and various other species of Trifolium and Oxalis. None of the species in the survey are unique to Ireland, and all are common European species, so there is no botanical basis for the widespread belief that the shamrock is a unique species of plant that only grows in Ireland. Early references [ edit ] The word shamrock derives from seamair óg or young clover, and references to semair or clover appear in early Irish literature, generally as a description of a flowering clovered plain. For example, in the series of medieval metrical poems about various Irish places called the Metrical Dindshenchus, a poem about Tailtiu or Teltown in Co. Meath describes it as a plain blossoming with flowering clover (mag scothach scothshemrach).[15] Similarly, another story tells of how St. Brigid decided to stay in Co. Kildare when she saw the delightful plain covered in clover blossom (scoth-shemrach).[16] However, the literature in Irish makes no distinction between clover and shamrock, and it is only in English that shamrock emerges as a distinct word. The first mention of shamrock in the English language occurs in 1571 in the work of the English Elizabethan scholar Edmund Campion. In his work Boke of the Histories of Irelande, Campion describes the habits of the "wild Irish" and states that the Irish ate shamrock: "Shamrotes, watercresses, rootes, and other herbes they feed upon".[17] The statement that the Irish ate shamrock was widely repeated in later works and seems to be a confusion with the Irish word seamsóg or wood sorrel (Oxalis).[8] There is no evidence from any Irish source that the Irish ate clover, but there is evidence that the Irish ate wood sorrel. For example, in the medieval Irish work Buile Shuibhne (The Frenzy of Sweeney), the king Sweeney, who has gone mad and is living in the woods as a hermit, lists wood sorrel among the plants he feeds upon.[18] The English Elizabethan poet Edmund Spenser, writing soon after in 1596, described his observations of war-torn Munster after the Desmond Rebellion in his work A View of the Present State of Ireland. Here shamrock is described as a food eaten as a last resort by starving people desperate for any nourishment during a post-war famine: Anatomies of death, they spake like ghosts, crying out of theire graves; they did eat of the carrions.... and if they found a plott of water cresses or shamrockes theyr they flocked as to a feast for the time, yett not able long to contynewe therewithall.[19] The idea that the Irish ate shamrock is repeated in the writing of Fynes Moryson, one-time secretary to the Lord Deputy of Ireland. In his 1617 work An itinerary thorow Twelve Dominions, Moryson describes the "wild Irish", and in this case their supposed habit of eating shamrock is a result of their marginal hand-to-mouth existence as bandits. Moryson claims that the Irish "willingly eat the herbe Schamrock being of a sharpe taste which as they run and are chased to and fro they snatch like beasts out of the ditches." The reference to a sharp taste is suggestive of the bitter taste of wood sorrel.[20] What is clear is that by the end of the sixteenth century the shamrock had become known to English writers as a plant particularly associated with the Irish, but only with a confused notion that the shamrock was a plant eaten by them. To a herbalist like Gerard it is clear that the shamrock is clover, but other English writers do not appear to know the botanical identity of the shamrock. This is not surprising, as they probably received their information at second or third hand. It is notable that there is no mention anywhere in these writings of St. Patrick or the legend of his using the shamrock to explain the Holy Trinity. However, there are two possible references to the custom of "drowning the shamrock" in "usquebagh" or whiskey. In 1607, the playwright Edward Sharpham in his play The Fleire included a reference to "Maister Oscabath the Irishman... and Maister Shamrough his lackey".[21] Later, a 1630 work entitled Sir Gregory Nonsence by the poet John Taylor contains the lines: "Whilste all the Hibernian Kernes in multitudes, /Did feast with shamerags steeved in Usquebagh."[22] Link to St. Patrick [ edit ] St. Patrick depicted with shamrock in detail of stained glass window in St. Benin's Church, Wicklow, Ireland Traditionally, shamrock is said to have been used by Saint Patrick to illustrate the Christian doctrine of the Holy Trinity when Christianising Ireland in the 5th century. The first evidence of a link between St Patrick and the shamrock appears in 1675 on the St Patrick's Coppers or Halpennies. These appear to show a figure of St Patrick preaching to a crowd while holding a shamrock, presumably to explain the doctrine of the Holy Trinity.[23] In pagan Ireland, three was a significant number and the Irish had many triple deities, which could have aided St Patrick in his evangelisation efforts.[24][25] Patricia Monaghan states that "There is no evidence that the clover or wood sorrel (both of which are called shamrocks) were sacred to the Celts". However, Jack Santino speculates that "The shamrock was probably associated with the earth and assumed by the druids to be symbolic of the regenerative powers of nature... Nevertheless, the shamrock, whatever its history as a folk symbol, today has its meaning in a Christian context. Pictures of Saint Patrick depict him driving the snakes out of Ireland with a cross in one hand and a sprig of shamrocks in the other."[26] Roger Homan writes, "We can perhaps see St Patrick drawing upon the visual concept of the triskele when he uses the shamrock to explain the Trinity".[27] Why the Celts to whom St Patrick was preaching would have needed an explanation of the concept of a triple deity is not clear (two separate triple goddesses are known to have been worshipped in pagan Ireland). The first written mention of the link does not appear until 1681, in the account of Thomas Dineley, an English traveller to Ireland. Dineley writes: The 17th day of March yeerly is St Patricks, an immoveable feast, when ye Irish of all stations and condicions were crosses in their hatts, some of pinns, some of green ribbon, and the vulgar superstitiously wear shamroges, 3 leav'd grass, which they likewise eat (they say) to cause a sweet breath.[28] There is nothing in Dineley's account of the legend of St. Patrick using the shamrock to teach the mystery of the Holy Trinity, and this story does not appear in writing anywhere until a 1726 work by the botanist Caleb Threlkeld.[5] Threlkeld identifies the shamrock as White Field Clover (Trifolium pratense album ) and comments rather acerbically on the custom of wearing the shamrock on St. Patrick's Day: This plant is worn by the people in their hats upon the 17. Day of March yearly, (which is called St. Patrick's Day.) It being a current tradition, that by this Three Leafed Grass, he emblematically set forth to them the Mystery of the Holy Trinity. However that be, when they wet their Seamar-oge, they often commit excess in liquor, which is not a right keeping of a day to the Lord; error generally leading to debauchery. The Rev Threlkeld's remarks on liquor undoubtedly refer to the custom of toasting St. Patrick's memory with "St. Patrick's Pot", or "drowning the shamrock" as it is otherwise known. After mass on St. Patrick's Day the traditional custom of the menfolk was to lift the usual fasting restrictions of Lent and repair to the nearest tavern to mark the occasion with as many St. Patrick's Pots as they deemed necessary. The drowning of the shamrock was accompanied by a certain amount of ritual as one account explains:[29][30] Shamrock on an Irish Defence Forces UN beret "The drowning of the shamrock" by no means implies it was necessary to get drunk in doing so. At the end of the day the shamrock which has been worn in the coat or the hat is removed and put into the final glass of grog or tumbler of punch; and when the health has been drunk or the toast honoured, the shamrock should be picked out from the bottom of the glass and thrown over the left shoulder. The shamrock is still chiefly associated with Saint Patrick's Day, which has become the Irish national holiday, and is observed with parades and celebrations worldwide. The custom of wearing shamrock on the day is still observed and depictions of shamrocks are habitually seen during the celebrations. Symbol of Ireland [ edit ] Drawing of the medal awarded to the First Magherafelt Volunteers for skill with broadsword showing shamrocks. As St. Patrick is Ireland's patron saint, shamrock has been used as a symbol of Ireland since the 18th century, in a similar way to how a rose is used for England, thistle for Scotland and daffodil for Wales. The shamrock first began to change from a symbol purely associated with St. Patrick to an Irish national symbol when it was taken up as an emblem by rival militias, during the turbulent politics of the late eighteenth century. On one side were the Volunteers (also known as the Irish Volunteers), who were local militias in late 18th century Ireland, raised to defend Ireland from the threat of French and Spanish invasion when regular British soldiers were withdrawn from Ireland to fight during the American Revolutionary War.[31] On the other side were revolutionary nationalist groups, such as the United Irishmen. Among the Volunteers, examples of the use of the shamrock include its appearance on the guidon of the Royal Glin Hussars formed in July 1779 by the Knight of Glin, and its appearance on the flags of the Limerick Volunteers, the Castle Ray Fencibles and the Braid Volunteers.[32][33] The United Irishmen adopted green as their revolutionary colour and wore green uniforms or ribbons in their hats, and the green concerned was often associated with the shamrock. The song The Wearing of the Green commemorated their exploits and various versions exist which mention the shamrock. The Erin go bragh flag was used as their standard and was often depicted accompanied by shamrocks, and in 1799 a revolutionary journal entitled The Shamroc briefly appeared in which the aims of the rebellion were supported.[34] Since the 1800 Acts of Union between Britain and Ireland the shamrock was incorporated into the Royal Coat of Arms of the United Kingdom, depicted growing from a single stem alongside the rose of England, and the thistle of Scotland to symbolise the unity of the three kingdoms. Since then, the shamrock has regularly appeared alongside the rose, thistle and (sometimes) leek for Wales in British coins such as the two shilling and crown, and in stamps. The rose, thistle and shamrock motif also appears regularly on British public buildings such as Buckingham Palace. Throughout the nineteenth century the popularity of the shamrock as a symbol of Ireland grew, and it was depicted in many illustrations on items such as book covers and St. Patrick's Day postcards. It was also mentioned in many songs and ballads of the time. For example, a popular ballad called The Shamrock Shore lamented the state of Ireland in the nineteenth century.[35] Another typical example of such a ballad appears in the works of Thomas Moore whose Oh the Shamrock embodies the Victorian spirit of sentimentality. It was immensely popular and contributed to raising the profile of the shamrock as an image of Ireland:[36] Oh The Shamrock - Through Erin's Isle, To sport awhile, As Love and Valor wander'd With Wit, the sprite, Whose quiver bright A thousand arrows squander'd. Where'er they pass, A triple grass Shoots up, with dew-drops streaming, As softly green As emeralds seen Through purest crystal gleaming. Oh the Shamrock, the green immortal Shamrock! Chosen leaf Of Bard and Chief, Old Erin's native Shamrock! Rose thistle and shamrock motif on gate pillar at Buckingham Palace Irish American Music sheet St Patrick's Day postcard 1912 Throughout the nineteenth and twentieth centuries, the shamrock continued to appear in a variety of settings.[37] For example, the shamrock appeared on many buildings in Ireland as a decorative motif, such as on the facade of the Kildare Street Club building in Dublin, St. Patrick's Cathedral, Armagh, and the Harp and Lion Bar in Listowel, Co. Kerry. It also appears on street furniture, such as old lamp standards like those in Mountjoy Square in Dublin, and on monuments like the Parnell Monument, and the O'Connell Monument, both in O'Connell Street, Dublin. Shamrocks also appeared on decorative items such as glass, china, jewellery, poplin and Irish lace. Belleek Pottery in Co. Fermanagh, for example regularly features shamrock motifs. Lamppost in Mountjoy Square, Dublin, early 20th Century Design on Harp and Lion Bar Listowel, Co. Kerry Work by Belleek Pottery, which often features shamrock motifs 2d Map of Ireland: the first Irish postage stamp featured the shamrock The shamrock is used in the emblems of many state organisations, both in the Republic of Ireland and Northern Ireland. Some of these are all-Ireland bodies, (such as Tourism Ireland)[38] as well as organisations specific to the Republic of Ireland (such as IDA Ireland)[39] and Northern Ireland (such as Police Service of Northern Ireland). The Irish Postal Service An Post, regularly features the shamrock on its series of stamps. The airline Aer Lingus uses the emblem in its logos, and its air traffic control call sign is "SHAMROCK". An Aer Lingus aircraft with a shamrock on its tail fin The shamrock has been registered as a trademark by the Government of Ireland.[40] In the early 1980s, Ireland defended its right to use the shamrock as its national symbol in a German trademark case, which included high-level representation from taoiseach Charles Haughey. Having originally lost, Ireland won on appeal to the German Supreme Court in 1985.[41] It has become a tradition for the Irish Taoiseach to present a bowl of shamrocks in a special Waterford Crystal bowl featuring a shamrock design to the President of the United States in the White House every St. Patrick's Day.[42] Shamrock is also used in emblems of UK organisations with an association with Ireland, such as the Irish Guards. Soldiers of the Royal Irish Regiment of the British Army use the shamrock as their emblem, and wear a sprig of shamrock on Saint Patrick's Day. Shamrock are exported to wherever the regiment is stationed throughout the world. Queen Victoria decreed over a hundred years ago that soldiers from Ireland should wear a sprig of shamrock in recognition of fellow Irish soldiers who had fought bravely in the Boer War, a tradition continued by British army soldiers from both the north and the south of Ireland following partition in 1921. The coat of arms on the flag of the Royal Ulster Constabulary George Cross Foundation was cradled in a wreath of shamrock.[43] The shamrock also appears in the emblems of a wide range of voluntary and non-state organisations in Ireland, such as the Irish Farmers Association,[44] the Boy Scouts of Ireland association, Scouting Ireland[45] Irish Girl Guides,[46] and the Irish Kidney Donors Association.,[47] In addition many sporting organisations representing Ireland use the shamrock in their logos and emblems. Examples include the Irish Football Association (Northern Ireland), Irish Rugby Football Union, Swim Ireland, Cricket Ireland, and the Olympic Council of Ireland. A sprig of shamrock represents the Lough Derg Yacht Club Tipperary, (est. 1836). The shamrock is the official emblem of Irish football club Shamrock Rovers. Oxalis regnellii. This flowering Shamrock was purchased at a grocery store. It is a South American species of wood sorrel Use overseas [ edit ] Shamrock commonly appears as part of the emblem of many organisations in countries overseas with communities of Irish descent. Outside Ireland, various organisations, businesses and places also use the symbol to advertise a connection with the island. Flag of St. Patrick's Battalion of the Mexican army reconstructed from description of Jon Riley. The flag has since become an emblem of Irish nationalism See also [ edit ] References [ edit ] Bibliography [ edit ] CLEVELAND, Ohio -- Soon, shamrocks will be sprouting and green beer will be flowing everywhere in celebration of St. Patrick's Day on Tuesday, March 17. It's a day that many will celebrate with pub crawls, parades, green socks and silly hats. Long before St. Patrick's Day was a holiday for revelry, it was a feast day in the Catholic church. The modern version of the holiday is celebrated beyond the United States, especially in countries with large Irish populations. How much do you know about the origins of the holiday? Do you secretly wonder if there really was a St. Patrick, and do you know how to make a proper Irish toast? Wonder no more; here's everything you need to know about the origins and lore surrounding St. Patrick's Day. The information came from the following websites: Time and Date.com Celebrating St. Patrick's Day Encyclopedia Britannica Huffington Post Irish Central Wiki How Your Irish Q. What is St. Patrick's Day? A. It's a global celebration of Irish culture on or around March 17. It particularly remembers St Patrick, one of Ireland's patron saints, who is credited with spreading Christianity in Ireland during the fifth century. The anniversary of St. Patrick's death on March 17 became a feast day in the Catholic Church. Q. What's the truth about his life? Did he really drive the snakes out of Ireland? A: The snakes story is a legend, according to the Encyclopedia Britannica's website. Here's part of the entry: "Born in Roman Britain in the late 4th century, he was kidnapped at the age of 16 and taken to Ireland as a slave. He escaped but returned about 432 to convert the Irish to Christianity. By the time of his death on March 17, 461, he had established monasteries, churches, and schools. Many legends grew up around him--for example, that he drove the snakes out of Ireland and used the shamrock to explain the Trinity. Ireland came to celebrate his day with religious services and feasts." Q. How did the holiday become so popular in this country? A: Irish immigrants to the United States turned St. Patrick's Day into secular celebration of all things Irish. Boston held its first St. Patrick's Day parade in 1737, followed by New York City in 1762. Chicago has dyed its river green to mark the holiday since the 1960s. Q: Does the rest of the world celebrate it? A: Yes, especially in countries with large Irish populations, such as Australia, Canada and the United Kingdom. Q. What are some good things to eat on this holiday? A. Feast on Irish brown bread, Irish stew and potato soup. Other traditional Irish foods include bangers and mash, bacon (boiled ham) and cabbage, stew, Shepherd's Pie, potato bread and black pudding. Q. Why are there shamrocks everywhere I look? A. The most common St. Patrick's Day symbol is the shamrock. The shamrock is the leaf of the clover plant and a symbol of the Holy Trinity. According to legend, St. Patrick used the shamrock to explain the Holy Trinity to pagans. "The wearing of the green" refers to the custom of wearing green clothes or a shamrock, the national flower of Ireland, in your lapel. Q. What's "drowning the shamrock"? A. That's the custom of putting the shamrock that's been worn on the lapel on St. Patrick's Day into the last drink of the night. Q. What's a leprechaun? A. As part of Irish mythology and folklore, leprechauns are a kind of fairy. They are mischievous, intelligent folk who are harmless, although they sometimes play tricks on humans. They also play tin whistles, fiddles and other Irish traditional instruments. It is said that every leprechaun has a pot of gold, hidden deep in the Irish countryside. To protect the leprechaun's pot of gold, the Irish fairies gave them magical powers to grant three wishes or to vanish into thin air if captured by a human. Q. What's a good St. Patrick's Day toast? A. "May the roof above us never fall in, and may we friends beneath it never fall out." Q. What does "erin go bragh" mean? A. "Ireland forever" in Gaelic, the traditional language of Ireland. Q. I want to impress my friends with some other Gaelic phrases. What can I learn in 5 minutes? A: Try "slainte" (good health or cheers!), or "beannachtai na feile padraig oraibh" (St. Patrick's Day blessing upon you!) Q. Got any suggestions for traditional Irish music I can add to my iPod? A. Celtic, folk and traditional Irish pub songs will help get you in the St. Patrick's Day spirit! Look for compilation CDs of traditional Irish songs or download some individual songs by The Chieftains, The Dubliners or other Irish bands. | We all know what a shamrock looks like, but-speaking in scientific terms-just what is it? A clover? A wood sorrel? Some other type of plant? Unfortunately, there's no single answer: The shamrock is the stuff of legend, not science, the Smithsonian reports. Over the centuries, a number of different plants have been called the "true" shamrock. It was identified with the clover in the 16th century (and on Wikipedia today), but there are many types of clover. And in the 19th century, a London botanist insisted that a plant called the wood sorrel was the real shamrock. Near the end of that century, an amateur naturalist decided to investigate, asking people from across Ireland to identify the "real" thing. The most popular responses were the yellow clover and the white clover, though the red clover and black medick were also called shamrocks. In the 1980s, a scientist tried the experiment again and got comparable results, with the yellow clover taking the top spot. And that's the one most often sold to tourists, the Smithsonian notes. As for the significance of the shamrock, even that isn't quite clear: Legend says that St. Patrick compared its three leaves to the Holy Trinity, but that story first appeared in writing centuries later. Regardless, you can take part in tradition by "drowning the shamrock," the Cleveland Plain Dealer reports: After wearing it on your lapel, place it in the night's final drink on St. Patrick's Day. And if that's a pint of Guinness, you'll be in good company, according to WalletHub: Some 13 million pints of the stuff will be downed in celebration. | multi_news |
The binding of proteins can shield DNA from mutagenic processes but also interfere with efficient repair. How the presence of DNA-binding proteins shapes intra-genomic differences in mutability and, ultimately, sequence variation in natural populations, however, remains poorly understood. In this study, we examine sequence evolution in Escherichia coli in relation to the binding of four abundant nucleoid-associated proteins: Fis, H-NS, IhfA, and IhfB. We find that, for a subset of mutations, protein occupancy is associated with both increased and decreased mutability in the underlying sequence depending on when the protein is bound during the bacterial growth cycle. On average, protein-bound DNA exhibits reduced mutability compared to protein-free DNA. However, this net protective effect is weak and can be abolished or even reversed during stages of colony growth where binding coincides – and hence likely interferes with – DNA repair activity. We suggest that the four nucleoid-associated proteins analyzed here have played a minor but significant role in patterning extant sequence variation in E. coli. The binding of proteins to DNA can alter DNA mutability. This has been explored most extensively in relation to nucleosomes. On the one hand, there is evidence that nucleosomes affect the ability of at least some repair enzymes to detect or gain access to their target lesions [1], [2]. Typically, nucleosomes appear to impede rather than facilitate repair, for example through steric hindrance or by altering DNA topology, which many repair enzymes exploit to recognize their targets [2]–[4]. Human uracil glycosylases, for instance, operate with 3- to 9-fold reduced efficiency in vitro when uracil needs to be removed in a nucleosome context compared to free DNA [5]. On the other hand, histone binding can also lower mutability by reducing the risk of lesion formation. In yeast, nucleosomal DNA exhibits reduced rates of cytosine deamination [6], probably because the nucleosome conformation reduces the amount of time the DNA spontaneously spends in a single-stranded state [6], which is associated with an elevated risk of deamination [7]. Being in close contact with a protein surface can also more directly inhibit mutagenic processes, as illustrated at the sub-nucleosome scale by the observation that residues facing the histone core have a lower propensity to form pyrimidine dimers [8]. Nucleosomes, therefore, can exert pleiotropic and contravening effects on sequence mutability, both interfering with repair and conferring physical protection. Although bacteria do not encode histones they do express a variety of DNA-binding proteins. Some, collectively known as nucleoid-associated proteins (NAPs), are abundant in the cell and fulfil both architectural and regulatory roles [9]. For a subset of NAPs, there is evidence from mutation-specific reporter strains that their presence too can affect sequence mutability. Notably, the DNA-protection during starvation (Dps) protein, expressed at high levels in stationary phase [10] and during oxidative stress [11] in E. coli, reduces the incidence of double strand breaks as well as C∶G to A∶T transversions [11], [12]. Similarly, small acid soluble proteins (SASPs), present in the spores of Bacillus subtilis, limit the formation of certain lesions including pyrimidine dimers [13]. Part of the protective effect may be indirect and global. Dps, for example, sequesters iron and thereby reduces the formation of mutagenic agents [14]. But there is also evidence for localized protection, where protein binding alters DNA mutability in and around the binding footprint, through direct physical interaction or by virtue of modulating repair dynamics [15], [16]. As NAPs constitute a diverse class of proteins, variously able to wrap, bridge, or bend DNA, it may come as no surprise that not all NAPs necessarily reduce lesion formation. The abundant DNA-binding protein Fis, for example, appears to promote the induction of (specifically) pyrimidine dimers [16], likely because its binding locally alters DNA curvature, making it more conducive to dimer formation [17]. In short, biochemical studies provide strong evidence that a number of different NAPs can affect DNA mutability in a number of different ways, variously able to confer localized protection, remodel DNA topology and thereby render DNA more prone to mutations, or – like Dps – modulate mutation risk systemically. Despite strong in vitro (and short-term lab culture) evidence from both eukaryotic and prokaryotic systems that protein occupancy can affect mutation dynamics, our knowledge of how repair and protective effects trade off to influence mutability in vivo and, ultimately, pattern segregating and fixed variation in natural populations remains rudimentary. Several genome-scale studies of polymorphism and divergence patterns in eukaryotes have found rates of evolution to vary with nucleosome occupancy [18]–[24] and a recent analysis of multiple cancer genomes uncovered striking correlations between different chromatin modifications and regional mutation rates [25]. However, it remains largely unclear whether correlations with nucleosome occupancy and chromatin status reflect differential repair efficacy, mutational liability or – especially where inter-specific comparisons are concerned – selection, for example for adequate nucleosome positioning. In fact, when it comes to repair, it is difficult to say whether we should have strong a priori expectations. Despite the stark differences in repair efficacy observed in vitro, histones and other DNA-binding proteins might be rather permeable barriers to repair in vivo where chromatin remodelers and polymerases regularly evict these proteins, provide opportunities for repair [26] and therefore potentially negate occupancy effects [27]. One approach to revealing how repair and protection affect heterogeneous mutability across the genome is to contrast the evolution of neutrally evolving sequences (thus by-passing selection as a potential confounder) bound under different physiological conditions (e. g. exponential versus stationary phase growth). The idea here is simple: being refractory to repair only matters if the relevant repair machinery is actually active at the time when the focal protein is bound. Conversely, protection will be of greater importance whenever the risk of mutagenesis is higher. Thus, in principle, analyzing physiologically specific binding events might allow us to look beyond the net impact on mutability and reveal insights into the relative sway of protective and repair effects. In practice, nucleosome binding profiles – at least in budding and fission yeast - change little when entering stationary phase, during heat shock or even in cells undergoing meiosis [28]–[30]. The binding landscapes of some bacterial NAPs, however, change much more radically throughout growth [9], [31], [32]. In this study, we therefore analyze data from a series of Chip-Seq experiments in E. coli where binding profiles of four NAPs (H-NS, Fis, IhfA and IhfB) were determined on a genome-wide scale [31], [32]. Relating NAP binding profiles assayed during different stages of the E. coli growth cycle to patterns of sequence evolution across 54 E. coli strains, we present evidence for weak but significant growth stage-specific effects of protein occupancy on mutability that reveal a dynamic balance between protective and repair effects. Next, we assessed mutability for sequence bound by a specific NAP at a defined stage of the growth cycle (during mid-exponential, late exponential, transition to stationary, and/or stationary phase). The most striking pattern to emerge is that, for C∶G to T∶A transitions in particular, mutability appears to change systematically with the time of binding (Figure 2). This is the case for all NAPs considered (note that Fis is only found at detectable levels during mid- and late exponential phase). Overall, regions bound later during the growth cycle seem to experience successively reduced mutability. Although these trends are intriguing, systematic biases may suggest a causal growth phase-specific association between protein occupancy and mutability when, in fact, there is none. For example, binding regions might differ by growth stage with regard to their expression level and hence opportunities to benefit from transcription-coupled repair. To establish whether there is an independent effect of binding status on mutability we trained a series of Random Forest classifiers to discriminate between at-risk 4-fold synonymous sites that had experienced a change and those that had not. Along with growth phase-specific NAP binding status, the classifiers were provided with features such as expression, genomic location, and regional GC content (see Methods for a full list of features) that might alternatively account for a difference in mutability between bound and unbound sequence. Note that some of these features relate to strand-specific processes (replication and transcription); we therefore trained a total of 12 (rather than six) classifiers, considering, for example, C to T changes on the transcribed strand as distinct from C to T changes on the non-transcribed strand. For each classifier, we then asked whether the predictive power of the classifier dropped when NAP binding status was randomized across sites, repeating the randomization plus classification procedure 50 times to establish significance. A significant drop in classifier performance means that NAP binding contains information relevant for predicting mutability that cannot be reproduced from any of the other variables, either alone or in combination. Focusing on those mutation categories where Figure 1 suggested a possible role for NAP binding, we find that, upon randomizing NAP occupancy, classifier performance drops significantly for C to G, G to C, C to T and T to C changes (Figure S2). Interestingly, NAP binding is not predictive of either A to G or G to A mutability, suggesting strand-specific effects. Overall, our results suggest that NAP binding is a weak predictor of net mutation risk, particularly when compared to other features in the analysis. For example, location on leading versus lagging strand has a considerably stronger influence on mutability (not shown), consistent with results from a recent mutation accumulation experiment [34]. However, as we argue below, this weak net effect of NAP binding conceals a more dynamic picture of the interplay between E. coli chromatin and mutability. Having established that binding is an independent, albeit weak predictor of mutability at 4-fold synonymous sites for a subset of mutation types, we wondered why mutability should be relatively higher for DNA bound during exponential growth. There are a number of scenarios that might explain this pattern: one simple explanation might be that there is an interaction between a protective effect of NAP binding and the timing of mutations. Imagine all mutations occurred during stationary phase so that the protective effect would be mediated exclusively by stationary phase binding. In this scenario, regions bound during exponential phase should behave the same as unbound sequence. However, we may still observe a signature of protective binding if early-bound regions are also bound in stationary phase. Such overlap in binding sites is indeed observed, with temporally more remote growth phases sharing increasingly fewer binding sites (Figure S3). A second possibility is that binding is more dynamic during exponential phase where protein occupancy is constantly disturbed by polymerases so that earlier growth phases contain a greater fraction of bound sites that spend a considerable amount of time in an unbound state, and therefore only benefit partially from the protective effect of binding. Two observations argue against these two models as satisfactory explanations. First, sequence bound throughout all stages of growth largely shows intermediate mutability (Figure S4). If stationary phase binding had been the only important variable, we would have expected mutability comparable to that in stationary phase. More importantly, regions bound exclusively early (during mid- and/or late-exponential but not later) or exclusively late (during transition to stationary and/or stationary phase but not earlier) show the most dramatic differences in mutability. Crucially, mutability in early-bound DNA commonly exceeds that of unbound sequence (Figure 3). If the only effect of NAP binding were to protect DNA from mutagenic processes, we would not expect mutation risk to ever exceed that of the unbound state, regardless of the timing of binding. Keeping in mind that the limited number of exclusively early-/late-bound sites precludes comprehensive confounder control, we suggest that protein occupancy during exponential growth actually impedes repair (or facilitates mutation, perhaps by interfering with polymerase processivity during replication). If NAP binding does, as suggested, interfere with repair and repair activity mainly occurred during exponential phase, that might explain why we observe elevated mutability during that stage of E. coli growth. Many key repair processes are indeed physically coupled to replication (3′-5′ exonucleolytic proof-reading of the DNA polymerase) or closely coupled in time (methyl-directed mismatch repair) and are down-regulated in stationary phase cells [35]. However, a limited number of repair enzymes are mainly expressed during stationary phase, including the alkylguaninetransferase Ada, which counteracts alkylation damage, the mismatch uracil glycosylase Mug, which reduces the incidence of C∶G to T∶A transitions and the very short patch VSP repair system (see below) [36]. Our hypothesis predicts that lesions specifically targeted by these enzymes should run counter to the overall trend and exhibit higher (not lower) mutability when bound during stationary phase. Is this the case? Testing this hypothesis is not trivial, the principal challenge being to identify mutations that have occurred (and should have been repaired by one of these enzymes) during stationary phase. Frequently, a variety of mutational processes, associated with diverse repair pathways, may have given rise to an observed nucleotide change. Pertinently, C∶G to T∶A changes can be caused by cytosine deamination to uracil, alkylation, or oxidation, result from deamination of 5-methylcytosine (5-meC) to thymine and even occur as a downstream consequence of UV-induced pyrimidine dimerization [37], [38]. Changes originating from 5-meC deamination are unique among these lesions because we can identify them based on the sequence context in which they occur. In E. coli, 5-meC is generated with high specificity and efficiency [39], [40] at the second cytosine of CCWGG motifs by the DNA cytosine methyltransferase Dcm. The T∶G mismatches that result from 5me-C deamination are repaired by the VSP repair pathway, which, importantly, is almost exclusively active during stationary phase [35], [41] and recognizes T∶G lesions outside the CCWGG context or U∶G mismatches created by cytosine deamination with much reduced efficiency [39], [42]. By implication, C to T changes that occurred in a CCWGG context are enriched for events that failed to be repaired during stationary phase whereas C to T changes outside that context more likely represent failures by other repair pathways such as methyl-directed mismatch or base excision repair, which are most active during exponential growth [35], [43]. We therefore compared C to T changes that occurred in a CCWGG context with changes in a control context (CCWHH) chosen to preserve local nucleotide neighbourhood. Focussing on exclusively late versus exclusively early bound sequences, which afford the greatest discriminatory power, we find that the control contexts exhibit the mutability behaviour observed previously (refractory effects of NAP occupancy during exponential phase, Figure 4). However, as predicted, changes that occurred in the CCWGG context show the opposite pattern, i. e. higher mutability for sequence bound during stationary phase. Observing higher mutability in late-bound regions inside the CCWGG context but lower mutability outside of it is unlikely to occur by chance (permutation test: P = 0. 013, see Methods). Once again, it is important to highlight that sites exclusively bound early or late are rare so that we cannot control systematically for potential confounders. However, on current evidence, this finding supports the hypothesis that NAP binding enhances mutability by interfering with repair pathways in a lesion- and growth phase-specific manner. It has been known for some time that both the rate [44] and spectrum [45] of mutations vary with growth stage in laboratory populations of E. coli and other bacteria, consistent with substantial physiological variability in the production of endogenous mutagens, differential activity of mutagenic processes such as replication or recombination, and variable expression of DNA repair enzymes. We argue that taking this physiological variability into account is crucial to understand the impact of DNA-binding proteins on sequence evolution in natural populations. This is because binding has pleiotropic effects on mutability, is capable of both protecting DNA from mutagens and interfering with lesion repair, so reducing or enhancing mutability in a dynamic fashion that depends on the physiological state of the cell. The analysis presented above suggests that the net effect of NAP binding on mutability is relatively weak and NAP binding is a comparatively poor predictor of within-genome differences in mutability. However, one should not jump to the conclusion that NAPs are largely irrelevant determinants of mutation dynamics on a mechanistic level. As indicated by the comparison between sites bound exclusively during exponential or stationary phase (Figure 3), and consistent with biochemical evidence [16], NAPs can exert countervailing effects on DNA mutability, elevating mutation risk under one condition but reducing it in another. We suggest that, over the bacteria' s life cycle, and evolutionary time, these forces partially counteract each other, leading to a relatively small net difference between NAP-bound and NAP-free sequence. Note also, that our analysis is very conservative. Some of the confounders might in fact be mechanistically linked to NAP binding. For example, it has been suggested that Fis binding changes repair dynamics at the tyrT locus through altering transcription patterns [16]. We would attribute such an effect to transcription rather than NAP binding therefore understating the significance of NAP binding. Our analysis also suggests that only a subset of mutation processes are affected by NAP binding. Given the plethora of lesion processes, some of which may be more, some less affected by DNA curvature and protein binding, it is not surprising that not all mutation types behave in a similar fashion. However, it is interesting to ask why we specifically observe an effect for C to G, G to C, T to C, and C to T changes. C∶G to G∶C transversions are rare and there is considerable uncertainty as to which lesions predominantly give rise to these mutations in the wild [46]. Guanines damaged by oxidative and alkylation processes can lead to C∶G to G∶C mutations [38], [47], but it is hard to see why NAP binding should preferentially reduce the incidence of oxidative lesions leading to C∶G to G∶C mutations but not affect mutability for C∶G to A∶T transversions, which are frequently derived from oxidatively damaged bases. Perhaps differences in lesion repair provide a more likely link to NAP binding. C∶C mismatches, which – if unrepaired – would yield C∶G to G∶C transversions, exhibit intrinsically high mobility compared to other mispaired bases [48]. It is conceivable that these mismatches are stabilized in the context of NAP binding, facilitating their detection and repair. However, this is speculative and we currently have no convincing model why NAP binding specifically affects C∶G to G∶C mutability. Reduced rates of C to T transitions might be owing to an effect of NAP binding on cytosine deamination dynamics, analogous to what has been proposed for nucleosomes in eukaryotes [6]. Comparing base-specific mutability in E. coli with base-specific substitution rates at 4-fold synonymous sites in yeast reveals that C∶G to T∶A transitions, the most common type of nucleotide replacement, are less likely to occur in a protein-bound context in both taxa (Figure S5). Although it remains unclear to what extent nucleosome-related substitution dynamics in yeast [20], [23] and other eukaryotes [21] are modulated by selection, recent data [6] suggest that elevated C∶G to T∶A rates in nucleosomal DNA principally reflect mutational input rather than selection. That does not automatically imply, of course, that protective effects in E. coli and eukaryotes are mediated by the same mechanism. However, it is tempting to speculate that both NAPs and nucleosomes reduce C∶G to T∶A mutability by limiting the amount of time the bound DNA spends in a vulnerable single-stranded state [6]. Such a model, where protection is not contingent on a specific protein-DNA binding conformation, is attractive because we observe consistent trends across proteins that differ substantially in how they bind to DNA and affect its topology [9]. Beyond C∶G to T∶A changes, there seems to be limited agreement in mutability trends between yeast and E. coli (Figure S5). However, this comparison is preliminary and a comprehensive assessment of convergent mutability will require a thorough, confounder-controlled analysis of mutability patterns in yeast. We extracted all complete E. coli and Shigella genomes available in GenBank. Excluding a number of genomes known to be of inferior quality [49], we arrived at a final set of 54 E. coli/Shigella genomes (Table S1). These genomes were aligned along with the genome of E. fergusonii using progressiveMauve [50]. We then only considered alignment blocks (locally collinear blocks in Mauve terminology) present across all 55 genomes. Further, we only considered blocks that did not overlap non-unique regions of the E. coli K-12 MG1655 genome. This is because binding calls from the Chip-Seq experiments only considered reads that uniquely mapped to the MG1655 genome so that – if we included non-unique regions – we would consider them unbound although they might in fact be bound. Non-unique portions of the E. coli genome were defined by subjecting the MG1655 genome to in silico digestion into overlapping windows of 300 nucleotides (off-set by 10 nt). These pseudo-reads were mapped against the MG1655 genome using GEM (http: //sourceforge. net/apps/mediawiki/gemlibrary/index. php? title=The_GEM_library) allowing up to 2 mismatches. In cases where pseudo-reads mapped to more than one genomic locale, these locales were excluded from downstream analysis. This procedure eliminates only a small fraction of the MG1655 genome (∼2%). Polymorphic sites were extracted using Mauve and filtered to obtain a set of sites where none of the species contained a gap and polymorphic status was not caused by a difference in E. fergusonii, i. e. we required a polymorphic site amongst the E. coli/Shigella genomes. Locally collinear blocks were concatenated, homomorphic positions removed and a phylogenetic tree constructed using RAxML 7. 2. 8 [51], [52] with 100 rapid bootstraps followed by thorough maximum likelihood search. The topology of the best tree (Figure S6) closely resembles the topologies of previously reported multi-strain E. coli trees [33], [53]. Considering E. coli genomes in a phylogenetic context, we reconstructed the history of nucleotide changes using the baseml algorithm in PAML [54]. The tree obtained above was used as a guide tree. We then applied the following filters to obtain a set of high confidence changes: a) only a single event had to be evoked to explain the distribution of states through the phylogeny, b) the reconstructed ancestral state had a posterior probability > = 0. 9, c) the change is not dependent on topology downstream of poorly supported (bootstrap support <98%) nodes (see Figure S6). These filters, along with requiring coverage across all 55 genomes, are designed to reduce the impact of false inferences caused by inter-strain recombination/horizontal gene transfer as well as sequencing and alignment errors. Genome-wide binding profiles for four nucleoid-associated proteins (H-NS, Fis, IhfA, and IhfB) were obtained from two publications (REFs. 29 and 30). The authors determined binding profiles at four points during colony growth: during mid-exponential (OD = 0. 5, Prieto, pers. comm.), late exponential (OD = 0. 8–0. 9), transition to stationary (OD = 1. 8–2), and stationary phase (after 24 hours). In order to establish whether NAP occupancy has an effect on mutability that can be separated from potential confounding factors we took the following approach: We trained Random Forest (RF) classifiers [55] to separate instances where a certain change at a 4-fold synonymous site (e. g. C to T) had occurred along the E. coli phylogeny from instances where the nucleotide at risk (C in our example) had not experienced a change. The (very large) class of unchanged nucleotides was randomly sub-sampled to obtain a sample exactly five times larger than the (smaller) changed-nucleotide class, which was kept intact. All 12 possible nucleotide changes were classified independently. The settings of the RF classifier were as follows: forest size was set to a very large value (10,000 trees) to avoid limiting predictive accuracy; the maximum tree depth was slightly decreased from the default setting (unlimited depth) to 10, for reasons of computational efficiency. While RF is considered robust to choice of parameters, one particular parameter may influence its predictive ability somewhat, namely the number of features considered at each node [55], here called K. Therefore we optimized K to maximize the average cross-validation accuracy of the RF models across the 12 nucleotide changes, yielding K = 2 as the optimum (Figure S7). A higher (5∶1) RF weight was put on the minority class (mutated nucleotides) than on the majority class (unchanged nucleotides) to counter the imbalance in the number of sampled nucleotides. Other RF parameters were left at the default values. 2-fold synonymous sites were included when analyzing transitions. The classifier was provided with a number of features potentially predictive of mutability such as the location of the corresponding gene relative to the origin of replication and its expression level at a certain stage of growth (see Table S2 for a full list of features). We recoded growth phase-specific NAP binding into four categories, bound throughout the growth cycle (always), bound during mid-exponential or mid- and late exponential phase (early), bound during stationary or stationary and transition to stationary phase (late) or not bound at any time point (never). While this approach discards more complex but potentially genuine binding behaviour, it captures the majority of binding profiles observed in the data (see Table S3) and reduces the number of binding categories, hence increasing power. We then assessed classifier performance in the presence of all features, including NAP binding, as the area under the curve (AUC). We then repeated the classification process 50 times, each time randomly shuffling NAP binding status across residues. The approximately normal distribution of AUC values from these randomized runs was then compared to the AUC derived from observed data by finding a Z-score to establish whether considering NAP binding improves classifier performance beyond confounding factors. A one-tailed P value was then derived from the Z-score. A one-tailed test is appropriate here because the only relevant outcome of the test is a decrease in the AUC score upon randomization. Note that it does not matter whether NAP binding is positively or negatively correlated with mutation risk; the non-randomized AUC score should always be higher. With 10% false positives expected at the P<0. 1 threshold, corresponding to 0. 6 false positive results from the 6 mutation types (3 couples) that were found associated with protein binding in our univariate odds ratio test (Figure 1), and 4 mutation types testing positive below the P<0. 1 threshold in the RF randomization test, we estimate a false discovery rate of FDR = 0. 6/4 = 15%. To establish whether the pattern depicted in Figure 4 (higher mutability across NAPs for late-bound residues in CCWGG contexts but lower mutability for late-bound residues in control contexts) is likely to arise by chance, we adopted the following strategy: we preserved the total number of mutations that occurred within each NAP+context combination (e. g. H-NS+control context) but redistributed mutations randomly to the late- or early-bound category according to the number of contexts in each category. Repeating this procedure 100,000 times for all six NAP+context combinations, we asked how likely we are to observe all NAP+context pairs to conform to the observed pattern. All classifier analyses were carried out within the Weka machine learning suite [56] and a customized version of the FastRandomForest classifier (http: //fast-random-forest. googlecode. com/). All statistical analyses were conducted in R [57]. | Mutations can be more or less likely to occur depending on whether DNA is naked or bound by proteins. On the one hand, DNA-binding proteins can shield the DNA from certain mutagenic processes. On the other hand, the very same proteins can interfere with efficient DNA repair. In this study, we reconstruct the history of mutations across 54 E. coli genomes and ask whether mutation risk is higher or lower in regions occupied by proteins that help organize bacterial DNA into chromatin. Intriguingly, we find that the effect of binding depends on its timing. When we consider genomic regions bound during stationary phase, we observe that binding is associated with lower mutation risk for some mutation classes compared to naked DNA, albeit weakly. However, when binding occurs during exponential phase, bound regions actually experience more mutations on average. We argue that this is because, during exponential phase, the major effect of binding is that it interferes with efficient DNA repair, whereas in stationary phase - when many repair pathways are inactive - the protective effect of binding dominates. Our results suggest that the four DNA-binding proteins considered here have a small but significant growth phase-specific effect on mutation dynamics in E. coli. | lay_plos |
By. Sarah Dean. An Australian jihadist has sent sickening threats against his former country after a warrant was issued for his arrest by the Australian Federal Police. Mohamed Elomar and Khaled Sharrouf, from Sydney, are fighting with al-Qaeda off-shoot Islamic State of Iraq and the Levant, in Syria, and will be arrested on terrorism-related charges if they ever return to Australia. 'Did you think I would leave your country without leaving a surprise. Fireworks coming up soon keep a close eye,' Elomar wrote on Twitter, The Australian claims. Scroll down for video. Disgusting: Mohamed Elomar, who is believed to have left Australia last year to fight in Syria, is shown holding up the decapitated heads of two men. After one Twitter user told him not to come back to Australia, he replied: 'No u idiot injured, dnt worry wouldn't want to go bk u should be more worried about wats coming to australia! [sic]' This comes after Daily Mail Australia revealed a disturbing photo of Elomar holding up two decapitated heads for the camera in Syria. The picture was uploaded on Friday by his friend, convicted terrorist Sharrouf, on Twitter. In the sickening image, Elomar is seen smiling - with a pair of sunglasses tucked into his shirt - as he holds up two decapitated heads by the victims' hair. Elomar wrote threats to ASIO on Twitter claiming he had been injured but would soon be fighting again. Despite being from Sydney originally, Elomar claimed that Australians should be worried about what is coming to the country. Eloma, also sent tweets aimed directly at ASIO on Tuesday, claiming he had been injured but would soon be fighting again. 'ASIO dnt worry I'll be Bak in direct conflict very soon, just need abit time for my knee injury suffered against Jash Al hoor,' he wrote. He added: 'Don't worry ASIO there is plenty of work for you guys coming up.' Criticisng his former country he said: 'He wrote Australian zionist Jews killing men and woman and children in Gaza is okay. Thumbs up Australia.' The 29-year-old's fighting name uses the world Australia and he used Australia's koala bear in one Tweet about his terrorist group ISIS. On Tuesday he compared the terrorist activity in Syria to the war on Gaza. He claimed people 'need to be stopped' and said there is a war on Islam. Another disturbing image, uploaded on Friday by Sharrouf, shows the heads of alleged Syrian solders being impaled on metal railings. Like something out of the 18th century, the heads were left on show in a sickening display that aims to cause terror and fear. The photos were shown alongside disgusting tweets such as 'bucket full of heads any1 in aus want some organs please dont be shy to ask I would love to assist u with body parts [sic]'. His disturbing rant continued 'few more heads how lovely bludy amazing stuff abuhafs u keep cutting those infidel throats but the last 1 is mine! [sic]' Sharrouf previously spent time in jail for his part in a foiled plot to blow up targets in Sydney and Melbourne. He is thought to have flown to Syria with Elomar late last year on his brother Mustafa's passport. Disturbing: Sharrouf also posted a photo of two men placing severed heads on a metal railing. Elomar's wife Fatima, 29, is still in Sydney with their four children. She appeared in court in Sydney on July 8 charged with 'preparing for incursions into a foreign state with the intention of engaging in hostile activities'. Fatima's lawyer, Zali Burrows, said on Tuesday that Elomar's arrest warrants for terrorism was not a surprise. However, she told the Sydney Morning Herald that she doubts the veracity of the Twitter posts, saying Elomar did not use the social network. She also claimed the photo of Elomar with severed heads may have been photoshopped. Elomar is also the brother of Ahmed Elomar, who was arrested in Lebanon for alleged terrorist links in 2007 and then sentenced to jail in Sydney in June for brutally bashing a police officer in the Hyde Park riots. Fatima Elomar hides under a burqa as she emerges from the Downing Centre court complex with her lawyer Zali Burrows (right) and four children after appearing on terrorism related charges. Mohomed Elomar (right) used to be a champion boxer. Here he is seen fighting Pises Buachai at the Entertainment Centre in Sydney, Wednesday, March 7, 2007. Elomar won the fight. Ahmed Elomar (right) punches Denchai Tiabkoon of Thailand in 2005 during a boxing match. Ahmed Elomar won the match to win the IBO Asia Pacific Featherweight Championship Title. Rioter Ahmed Elomar was sentenced to at least two and a half years in jail for the attack that happenned on September 15, 2012. The former champion boxer hit an officer with a pole during a confrontation in Sydney’s angry Islamic march as part of a global protest against the anti-Islamic YouTube clip The Innocence of Muslims. Judge Donna Woodburne said he had had 'agitated the crowd' as part of a group of 'aggressive men'. Ahmed Elomar's barrister Greg James QC told the Sydney District Court that his client was suffering an 'impaired intellectual state'. The two brothers, Ahmed and Mohamed, used to be champion boxers, Ahmed has competed in more than 20 professional bouts. The convicted terrorist said he wanted to cut 'infidel throats' He directed his tweets to people in Australia and joked about organ donation. Convicted terrorist Khaled Sharrouf has threatened the Australian Federal Police and his countrymen on his Twitter account. In 2007, their father Mamdouh Elomar slammed the clerics who he believes radicalised his sons in Sydney. Mr Elomar told The Sun-Herald that he urged his children to stay away from Sheik Mohammed at the Global Islamic Youth Centre. 'Sheiks like Feiz ruin people,' Mr Elomar said. 'He is not a sheik; he is brainwashing all these children. I know my religion, so I can tell him when he is wrong, but these kids believe everything he says and think it's their religion. Someone needs to stop him. 'Today our kids are either at one extreme, partying or using drugs, or at another extreme with their religion. I don't teach my children to hate non-Muslims. I have taught them to love everyone.' Sydney's angry Islamic marched in September 2012 as part of a global protest against the anti-Islamic YouTube clip The Innocence of Muslims. Supporters of Ahmed Elomar leaving the Central Local Court in Sydney, on September 18, 2012. On Thursday night Attorney General George Brandis's spokesman said that 'if real, these photos are evidence of serious crimes against Australian law and possible war crimes.' Daily Mail Australia has contacted AFP for further comment. Sharrouf has also taunted Australian police via his social media account two weeks ago, boasting that he would'slaughter' Australians. Those tweets came as the nation's Director General of Security David Irvine revealed 'tens of people' had already returned to Australian shores from fighting alongside suspected terrorist organisations. Speaking to media earlier this month, Attorney-General George Brandis said engagement with the Islamic community was an important strategy 'to keep Australia free from terrorism'. 'The imams who are faith leaders, who are influential and respected opinion members in their communities, are integral to our goal to saving young men - it is almost always young men - in their communities from being radicalised,' he said. Mr Irvine added a majority of the 60 Australians they were aware of fighting with Islamic radicals were siding against the government with Al-Qaeda off-shoots. 'We have some tens of people that have already returned [from the Middle East], we have probably another 150 we're looking at here in Australia who have inclinations to support those two extremist movements,' he said. When asked if the ones who had returned to Australia were being actively monitored, Mr Irvine said 'I'm not saying anything further'. On July 14, Sharrouf fired off a tweet to the AFP and Australians, saying: 'you cowards I am running to my death I want martyrdom thats [sic] why I am blessed u rock spiders'. Just half an hour earlier, he claimed 'Australia belongs to the muslims not infidels like you' as well as boasting of his evasion of police. 'u can't stop and trust me if I wanted to attack aus I could have easily,' Sharrouf said. He also added: 'I love to slaughter [Australians] & ALLAH LOVEs when u dogs r slaughtered'. These threatening tweets are part of a more disturbing picture of Sharrouf painted by his Twitter account. Some of the content is so graphic that Daily Mail Australia has chosen not to publish the posts. On July 14, Sharrouf tweeted he loved to slaughter Australians to the AFP. He said it would have been easy for him to attack Australia if he wanted. Sharrouf's account is littered with photos of him posing with weapons and military trucks as well as a young child brandishing a rifle | Khaled Sharrouf and Mohamed Elomar have warrants out for their arrests. Sharrouf posted a photo of Elomar holding up severed heads. Elomar has now threatened Australia with a terrorist attack. He's the brother of Ahmed Elomar, jailed in June for bashing police officer. Mohamed Elomar has a wife and four children in Sydney. Mohamed and Ahmed used to be champion boxers. Mohamed and Sharrouf are fighting for an al-Qaeda off-shoot in Syria. | cnn_dailymail |
Angus T. Jones, the "Two and a Half Men" actor who dismissed his show as "filth" and begged people to stop watching, has found another supporter -- and possibly a new gig -- in Charlie Sheen. "My former nephew is welcome at the Goodson Anger Management home anytime," Sheen told ABCNews.com, inviting Jones to appear on his FX series, "Anger Management," in which he plays therapist Charles Goodson. The former "Two and a Half Men" star also said, "It is radically clear to me that the show is cursed," and called Jones' video testimonial against the show a "Hale-Bopp-like meltdown." Jones apologized for bashing the show late Tuesday, telling the Hollywood Reporter, "Without qualification, I am grateful to and have the highest regard and respect for all of the wonderful people on 'Two and Half Men' with whom I have worked and over the past 10 years who have become an extension of my family." He said he regretted any indifference or disrespect that his remarks might have implied, and called his "Two and a Half Men" gig an "extraordinary opportunity of which I have been blessed." RELATED: Angus T. Jones 'Cool and Calm' After 'Two and a Half Men' Furor Jones' 15-minute-long sit down with pastor Christopher Hudson, who hosts the online show ForeRunner Chronicles, went viral after Hudson posted it to YouTube Monday. While most of the video is devoted to Jones' testimonial about becoming a Seventh-Day Adventist, he goes on a tirade about "Two and a Half Men," where he's played happy-go-lucky kid Jake Harper since 2003. "Jake from 'Two and a Half Men' means nothing," he says. "He is a non-existent character. If you watch 'Two and a Half Men,' please stop watching 'Two and a Half Men.' I'm on 'Two and a Half Men' and I don't want to be on it." He goes on, "Please stop watching. Please stop filling your head with filth, please. People say it's entertainment... the fact that it's entertainment... do some research on the effect of television on your brain and you'll have a decision to make." "It's bad news," he adds, shaking his head. "A lot of people don't like to think about how deceptive the enemy is." That "enemy" has made him rich. According to the Hollywood Reporter, the 19-year-old actor earns about $350,000 per episode, and received a raise along with co-stars Jon Cryer and Ashton Kutcher when the show was renewed for a tenth season in May. "Two and a Half Men" tapes about 20 episodes per season and has been on for 10 seasons. Representatives for CBS and Warner Bros., the studio that produces "Two and a Half Men," have yet to comment on the video. Hudson said he spoke with Jones on Monday and the actor is "doing very well." "He's maintaining very well," Hudson told ABCNews.com. "From speaking to him, he was very cool and calm and he's doing very well." Angus T. Jones Not Due on 'Two and a Half Men' Set for Weeks What happens now? "Two and a Half Men" managed to muddle out of an epic drama with Charlie Sheen two years ago when Sheen's erratic behavior led to CBS and Warner Bros. booting their star from what was TV's No. 1 sitcom. Sheen went on a media tour afterwards, slamming the show and its creator Chuck Lorre. By all accounts, it was a PR nightmare. Matt Belloni, news director of The Hollywood Reporter, doesn't think the same thing will happen with Jones. He noted that no studio would want to fire an actor for expressing his religious beliefs. But, he said it's likely that Lorre and the show's producers will ask Jones if he wants out. "Nobody wants someone who doesn't want to be there," he said. "He's paid very well, they treat him like a professional, it's not like there's some raging feud happening on the set like there was with Charlie Sheen. I could see them working something out where he leaves gracefully." Jones has time before he has to show his face on set. A source close to "Two and a Half Men" told ABCNews.com that Jones does not appear in the next two episodes being filmed. His character recently joined the Army and hasn't appeared often this season. WATCH: 'Two and a Half Men' Star Calls Show 'Filth' Angus T. Jones plans to fulfill his contractual obligations to Two and a Half Men and will exit the show after this season -- unless studio Warner Bros. Television asks him to leave earlier, sources tell The Hollywood Reporter. Jones, 19, apologized Tuesday for making incendiary comments about his hit CBS sitcom in a video posted online by the Fremont, Calif.-based Forerunner Christian Church. Jones called the show "filth," begged viewers to stop watching it and declared that he wants to quit. "You cannot be a true, God-fearing person and be on a television show like that," said Jones, who has starred on all 10 seasons of the ribald sitcom and makes about $350,000 an episode. PHOTOS: Hollywood's Memorable Mea Culpas The appearance of the video Monday caught Men producers and Warner Bros. executives off guard, and series co-creator Chuck Lorre was said to have been furious. On Tuesday, Jones turned to publicist Eddie Michaels, who issued a statement three hours after being hired on behalf of the actor, apologizing to Lorre, Warner Bros. chief Peter Roth and his cast and crew. "I am grateful to and have the highest regard and respect for all of the wonderful people on Two and Half Men, with whom I have worked over the past 10 years and who have become an extension of my family," Jones said in the statement, which did not address his opposition to the racy content in the show. Behind the scenes, Jones' reps at the Paradigm agency and his attorney Geoffry Oblath were said to be in discussions with Warner Bros. about the actor's statements and whether he wants to continue to work on the show or quit before his contract ends in May. (Warners insiders say they were surprised by the public statement.) But sources now say that despite Jones' professed belief that Men is "filth," he has decided he wants to stay with the sitcom and honor his deal. The only question now is whether Warner Bros. and Lorre will let him. A Warner Bros. rep is declining comment on whether the studio or Lorre has accepted Jones' apology and will allow him to stay on Men. Michaels, Jones' publicist, tells THR that "conversations are ongoing about his future role on the show." Jones is working under a one-year contract forged near the end of last season. At the time, CBS initially had wanted to renew the show for two additional seasons but ultimately got one year, partly because Jones wanted this to be his final season so he could attend college. His character joined the Army at the end of last season, so he does not appear in every episode of the current season 10, and he is not scheduled to appear in the final two episodes to be filmed before the holiday hiatus. "My character does Skype calls. He only does one-scene Skype calls," Jones told E! Online in October. "It's easy, but it's boring." STORY: Charlie Sheen Breaks Silence on Angus T. Jones 'Meltdown,' Says 'Two and a Half Men' Is 'Cursed' Jones' comments present a tricky personnel issue for Men producers. His jabs at the show likely would violate an anti-disparagement clause if one appears in his contract -- many TV talent deals contain language prohibiting actors from declaring war on their shows in the press -- but it's unlikely that Warner Bros. would abruptly fire the actor for the transgression, as it fired former Men star Charlie Sheen when his public behavior turned adversarial in 2011. That's an even less likely scenario here because of Jones' age (he's basically grown up on this volatile set) and because his comments were religious in nature. Employers tend to tread carefully when moves could be interpreted as being made in response to an employee expressing his or her religious beliefs. The studio could wish to simply ride out Jones' contract, including him in the show in a diminished capacity and paying him what he's owed. That plan, however, could lead to some awkward moments when Jones returns to the set in early 2013 to perform alongside those whose work he has trashed. Further complicating matters, it's far from clear whether Men will be back for an 11th season next fall, and both financial and creative considerations are in play. Despite dips from last year's boosted numbers when Ashton Kutcher replaced Sheen amid much fanfare, Men is averaging a formidable 3.7 rating this season among adults 18-to-49 (4.7 with Live+7), ranking it behind only Modern Family and The Big Bang Theory among comedies. (The show fetches $250,000 for a 30-second spot.) And overseas, Men remains a power, though ratings have begun to slip. In Australia, for instance, the once-dominant comedy now draws 460,000 to 700,000 viewers, compared with 700,000 to 1.2 million for Big Bang. On Canada's CTV, Men hovers below 3 million viewers, compared with 4.3 million for Big Bang. STORY: Angus T. Jones Apologizes for 'Two and a Half Men' Slam Syndication deals for Men were renegotiated when Sheen was replaced, so the show isn't quite as lucrative for Warner Bros. as it once was, especially for how expensive it is to produce. Sources close to the show say Lorre -- in consultation with Warner Bros.' Bruce Rosenblum and CBS' Les Moonves -- ultimately will decide whether to pull the plug. Scott Roxborough contributed to this report. Charlie Sheen It's Chuck Lorre's Fault Angus Lost His Mind Charlie Sheen -- It's Chuck Lorre's Fault Angus T. Jones Lost His Mind EXCLUSIVE isn't done blasting "" over thebrouhaha -- now he's targeting show creator... telling TMZ it's Lorre's fault Angus has suffered "an emotional tsunami."As we reported, Charlie has already called Angus' infamous YouTube confessional a "Hale-Bopp-like meltdown" -- referencing the Heaven's Gate cult suicide -- but that's just the beginning.Charlie tells us, "Obviously, not having been there for some time, the Angus T. Jones that I knew and still love is not the same guy I saw on YouTube yesterday."He adds, "I dare anyone to spend ten years in the laugh-track that is Chuck Lorre's hive of oppression and not suffer some form of an emotional tsunami."As we first reported, Angus has already issued a semi-apology for the video -- in which he calls "Men" filth -- telling us, "I apologize if my remarks reflect me showing indifference to and disrespect of my colleagues." Jones' character Jake enlisted in the army at the end of season 9 and has only appeared in a few episodes of the current season. "He won't be in every episode," our source says of the remaining season 10 episodes. "How much they use him will be up to [creator] Chuck Lorre. The intention wasn't ever for him to be in every ep this year anyway. Now it might be less." Jones and his costars Jon Cryer and Ashton Kutcher all have contracts through the end of this season and CBS has yet to pick up the show for season 11. Should Two and a Half Men be renewed, it's "unlikely" that Jones will return as a series regular. "You might see him for a few guest spots," our source reveals, adding that it's "likely" the 19-year-old will choose to go to college next year. "Part of the reason Angus is so wary of the [TV] industry and so involved with the church is that he's seen what's happened to Charlie [Sheen] front and center," the source says. "He doesn't want to end up like that. Who would?" As for Jones' reaction to the media storm the video has elicited, our source says he is "shocked" that it's become such a big deal, explaining, "He really didn't think anyone would care that much. He has never been the one to get attention." Angus T. Jones Is Not Leaving Two and a Half Men: Source The Half is back!Ever since Angus T. Jones bashed Two and a Half Men in a now-viral video, it begged the question: Will the 19-year-old actor return to the hit show?If he has it his way, he will."Angus expects to report to work after the holiday break in January," says a source close to the star. "He intends to honor his contract through the end of the season."Jones, who called the show "filth" and urged viewers in a video interview on a religious website to stop watching, issued an apology Tuesday night, saying he has the "highest regard" for the "wonderful people" on the show.Although Jones is not featured in an episode that tapes next week, he intends to show up on schedule after the break, the source says.In the meantime, the source adds, "Angus is feeling positive and he is concentrating on spending some downtime with family and friends." | Not surprisingly, Angus T. Jones probably won't return to Two and a Half Men, which he recently referred to as "filth," if the show is renewed for an 11th season, sources confirm to E!. His character-the "half" referred to in the title-joined the Army at the end of season nine and hasn't been in many episodes this season anyway. It's "unlikely" he'd be back as a regular for a hypothetical 11th season, a source says, though he may do guest spots. (Both People and the Hollywood Reporter note that Jones is expected to finish out his contract for this season, assuming execs will have him.) How will he fill all his newfound free time? Former Men co-star Charlie Sheen has an idea, telling ABC News Jones is welcome on his new show, Anger Management, any time. Sheen also referred to Jones' "filth" comments as a "Hale-Bopp-like meltdown," but didn't seem to blame Jones, noting that "the show is cursed." He also told TMZ, "I dare anyone to spend 10 years in the laugh-track that is Chuck Lorre's hive of oppression and not suffer some form of an emotional tsunami." Of course, there's the question of whether someone who thinks Two and a Half Men is "filth" would think any more highly of Sheen's sitcom. Plus, E!'s source notes, Sheen's own messy exit from Men and ensuing drama is "part of the reason Angus is so wary of the industry and so involved with the church.... He doesn't want to end up like that. Who would?" Jones recently apologized for his comments, which his family blames on the teen's church. | multi_news |
CROSS-REFERENCE TO RELATED APPLICATIONS This application is a continuation-in-part of Postal et al. U.S. application Ser. No. 09/288,764, filed Apr. 8, 1999 now U.S. Pat. No. 6,247,931 B1 which is a continuation-in-part of Postal et al. U.S. application Ser. No. 08/878,995, filed Jun. 19, 1997, now U.S. Pat. No. 5,931,672. The disclosures of patents 6,247,931 B1 and 5,931,672 are hereby incorporated herein by reference. STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT Not Applicable BACKGROUND OF THE INVENTION 1. Technical Field The present invention relates to a dental tool assembly having a head that imparts oscillatory motion to a desired dental treatment device coupled to the assembly. More particularly, the present invention relates to a drive mechanism for a dental tool assembly, the drive mechanism having a rotating drive shaft that engages a first end of a driven shaft to rotate the driven shaft in an oscillatory manner. The present invention also relates to a bearing assembly for a dental tool drive mechanism, the bearing assembly supports the drive shaft for rotary motion using a bushing shape to reduce friction while also aligning the drive shaft. A dental tool is coupled to a second end of the driven shaft and is thereby rotationally oscillated. 2. Description of the Related Art Dental tool assemblies, such as prophy angles and drills, which impart an oscillatory rotary motion to a dental treatment device coupled thereto are known in the art. In particular, such assemblies typically have a driving mechanism comprising a drive shaft with a rotation axis that is perpendicular to the rotation axis of a driven shaft to which the dental treatment device is coupled. The drive shaft of prior art driving mechanisms has an element positioned eccentric to its rotation axis and extending towards the driven shaft to engage a slot in the driven shaft. Rotation of the drive shaft thus imparts an oscillatory rotation to the driven shaft. For example, U.S. Pat. No. 1,711,846 to Heilbom shows a dental filing device having a drive shaft perpendicularly oriented with respect to a file holder. A crank pin, mounted on a crank disc on an end of the drive shaft adjacent the file holder, engages within a bore in the file holder. The crank pin is positioned on the crank disc eccentric to the rotation axis of the drive shaft. Thus, rotation of the drive shaft rotates the eccentrically positioned stud, thereby causing the file holder to rotate in an oscillatory manner. Similarly, the dental instrument in U.S. Pat. No. 2,135,933 to Blair has a rotary drive shaft with an eccentrically positioned stud that engages within a slot of a piston to which a massage tip is coupled. Rotation of the drive shaft causes oscillatory rotation of the massage tip. Another massage tool that imparts oscillatory motion to a head spindle to which a massage cup or brush is coupled is shown in U.S. Pat. No. 4,534,733 to Seigneur in et al. In the Seigneur in Patent, the stud that engages the head spindle is mounted eccentric to the rotation axis of the drive shaft, but is inclined to extend across the rotation axis. The portion of the stud that is aligned with the rotation axis of the drive shaft is also aligned with the rotation axis of the head spindle. The dental tool shown in U.S. Pat. No. 4,460,341 to Nakanishi also has a guide pin mounted eccentric to the rotation axis of a drive shaft and engaging within a slot of a driven shaft to which a dental treatment device is coupled. In all of the above-described dental tool assemblies, a stud or pin extends into a slot to drive the element to which the dental treatment device is coupled. Because the treatment device typically must be driven at very high speeds (e.g., the recommended speed of a standard prophy angle at approximately 6,000 rotations per minute), there is a risk of the stud or pin breaking off during use. Moreover, manufacturing of the drive shaft and driven shaft is complicated by the necessity of forming a stud and a slot that are shaped for ready, secure engagement such that rotation of the drive shaft causes oscillatory rotation of the driven shaft. Additionally, some of the drive shafts of the above-described patents also impart reciprocatory axial motion to the driven shaft along the longitudinal shaft of the driven shaft. When such axial motion is not desired, the driven shaft should be locked with respect to the housing in which the drive shaft and driven shaft are positioned, and thus locked with respect to the rotation axis of the drive shaft. Typically, such locking is accomplished by locking the driven element with respect to the housing such as by inter-engagement of stepped portions and/or flanges. However, such locking imparts substantial stresses against the housing and driven shaft. Another drawback of the above-described devices is that they are typically formed from metal and are reusable. The sterilization process necessary in order to reuse the device is typically costly and time consuming. It therefore has been desirable to provide disposable dental tool assemblies that are used only once and therefore need not be sterilized. Such tools typically are made from plastic. Because plastics are generally not as strong as metals, the driving mechanism used in the above-described devices cannot be used because of the inherent weakness of the stud. Therefore, the driving mechanisms of disposable dental tools typically have interengaging gears, such as shown in U.S. Patent No. 5,571,012 to Witherby et al. Because gears are used, the same reciprocatory rotary motion provided by the non-disposable tools cannot be achieved. However, such oscillating movement is desired for a number of reasons. The back and forth reciprocating motion provided by non-disposable dental tool assemblies permits greater speeds to be used and greater pressure to be applied than rotary type devices that do not oscillate, and also may massage the gums of the patient. Additionally, oscillatory movement generates less heat than a full rotational action. Moreover, the risks of hitting undercuts, cutting or tearing soft tissue, and splattering of agents applied by the treatment tool are reduced if not substantially eliminated. Another problem faced by any drive mechanism is how to provide support and alignment for the moving parts. There are many types of bearings that may be used for this purpose. Due to the size, construction methods, and materials used in prophy angles, journal or sleeve style bearing configurations are generally used. An example of such a bearing is shown in U.S. Pat. No. 5,340,310 issued to Bifulk on Aug. 23, 1994. As shown in FIGS. 3, 4 and 5 thereof, a housing having a cylindrical bore with fingers spaced from and extending axially parallel to the bore are provided for receiving a bushing having a square cross-section such that the bushing abuts turned in portions of the fingers. The fingers are flexible such that they bend outwardly allowing the bushing to be pushed past the turned in portions yet bend back to capture the bushing therein. Another example of a drive mechanism is shown in U.S. Pat. No. 5,931,672 issued to R. Postal on Aug. 3, 1999. The mechanism disclosed therein uses a series of flanges and a latch that includes a position retaining surface that contacts the outer periphery of at least one of the flanges. The extreme outer edge of the flanges provide axial alignment for the drive shaft. In use, this arrangement is susceptible to heat build-up on the various bearing surfaces. This problem is compounded by the difficulty in lubricating the bearing surfaces during assembly of the components. Therefore it is desirable to have a bearing surface that is less susceptible to heat build-up and is easily lubricated during assembly. It is also desirable to have a bearing arrangement that is less complex to assemble. The present invention addresses these desires for improving the bearing arrangement in prophy angle drive mechanisms. BRIEF SUMMARY OF THE INVENTION It is therefore an object of the present invention to provide a disposable dental tool assembly having a driving mechanism that imparts oscillatory rotary motion to a dental treatment device mounted on the assembly and to achieve this with a structure that can be economically, and reliably implemented in plastic to allow for disposability and the attendant avoidance of the spreading of infection. It is a related object of the present invention to provide a driving mechanism having a drive shaft and a driven shaft each having driving surfaces shaped to engage each other and ride along each other such that rotation of the drive shaft causes oscillatory rotation of the driven shaft. It is a further object of the present invention to provide a dental tool assembly having driving and driven elements that are stabilized with respect to each other against relative movement in a given direction. It is another object of the present invention to provide a dental tool assembly having a drive shaft that is coupled to a driven element such that the drive shaft imparts only oscillatory motion to the driven element without also imparting axial motion to the driven element. These and other objects of the present invention are accomplished in accordance with the principles of the present invention by providing a dental tool assembly having a rotating drive shaft that engages a driven shaft to impart oscillatory rotary motion to the driven shaft. The drive shaft and driven shaft are positioned transverse to each other. The drive shaft has a driving surface at its distal end that is shaped to engage a driven surface on a side of the driven shaft adjacent the drive shaft. Because of the manner in which the distal end is shaped, a stud or guide pin, such as used in the prior art, is no longer needed. Specifically, the driving surface is a cutaway, curved portion of an enlarged end of the drive shaft, and the driven surface is a cut-away side portion of the driven shaft. The cut-away portions of each shaft are shaped to interengage with substantially no play there between such that they are in continuous contact during rotation of the driving shaft. Because of the shapes of the cut-away portions, rotation of the driving shaft causes oscillatory rotation of the driven shaft. The drive shaft and driven shaft are positioned within a housing. In order to prevent relative movement of the shafts with respect to the housing, a plurality of locking mechanisms are provided. First, the drive shaft is provided with a longitudinally extending pin aligned with the rotation axis of the drive shaft. The driven shaft is provided with a slot through which the pin is passed. The slot is shaped so that oscillatory rotation of the driven shaft is not inhibited by the pin, yet axial movement of the driven shaft along its rotation axis is prevented. Another locking mechanism for the drive shaft is provided in the form of at least one flange extending radially from the drive shaft and engaging a radially inwardly extending flange on the inner surface' of the housing. The driven shaft is provided with a rearwardly positioned pin that fits within a bore in the housing to lock the driven shaft in the desired position for oscillation. In the alternative, a bearing assembly acting as both a thrust bearing and a retainer for holding the drive shaft in place is characterized in that it includes a bushing having an inwardly facing frustoconical surface acting in concert with a journal attached to the drive shaft and having a contact surface in contact with said frustoconical surface, said bushing also having a snap-fit arrangement on a peripheral surface thereof. BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWING These and other features and advantages of the present invention win be readily apparent from the following detailed description of the invention, the scope of the invention being set out in the appended claims. The detailed description will be better understood in conjunction with the accompanying drawing, wherein like reference characters represent like elements throughout the various views of the drawing and: FIG. 1 is an elevational, partially cut-away view of a dental tool assembly formed in accordance with the principles of the present invention; FIG. 2A is a cross-sectional view of the distal end of the dental tool assembly of FIG. 1 along line 2 — 2, with the driven shaft in the rest position; FIG. 2B is a cross-sectional view of the distal end of the dental tool assembly of FIG. 1 along line 2 — 2 with the drive shaft rotated 90° from the position shown in FIG. 2A; FIG. 3 is an elevational view of a drive shaft formed in accordance with the principles of the present invention; FIG. 4 is a perspective view of the drive shaft of FIG. 2; FIG. 5 is a perspective view of the drive shaft of FIGS. 3 and 4, rotated to another position; FIG. 6 is an end view of the drive shaft of FIG. 3; FIG. 7 is an elevational view of a driven shaft formed in accordance with the principles of the present invention; FIG. 8 is a plan view of the driven shaft of FIG. 7; FIG. 9 is a cross-sectional view along line 9 — 9 of the driven shaft of FIG. 8; FIG. 10 is a perspective view of the driven shaft of FIGS. 7-9; FIG. 11 is an elevational view of a driven shaft similar to that of FIG. 7 but with straight transverse walls of the driven surface; FIG. 12 is a plan view of the driven shaft of FIG. 11; FIG. 13 is a perspective view of a plastic driving mechanism of the dental tool assembly incorporating a flexible driving member; FIG. 14 is a perspective view of a plastic driving mechanism of the dental tool assembly incorporating a cylindrical driving member; FIG. 15 is a cross-sectional view of the dental tool assembly of FIG. 14 along lines 15 ; FIG. 16 is a perspective view of a plastic driving mechanism of the dental tool assembly incorporating a cylindrical driving member with shaft; FIG. 17 is a perspective view of a plastic driving mechanism of the dental tool assembly incorporating multiple cam driving mechanism; FIG. 18 is a perspective view of a plastic driving mechanism of the dental tool assembly incorporating a lobed member; FIG. 19 is a top view of the dental tool assembly of FIG. 18; FIG. 20 is a perspective view of the lobed member of FIG. 18; FIG. 21 is a perspective view of a plastic driving mechanism of the dental tool assembly incorporating a wedge-shaped member; FIG. 22 is a top view of the dental tool assembly of FIG. 21; FIG. 23 is a perspective view of a plastic driving mechanism of the dental tool assembly incorporating a multiple gear driving mechanism; FIG. 24 is a cross-sectional view of the dental tool assembly of FIG. 23 along lines 24 showing the engagement of the driving surfaces of partial gear and follower toothed gears; FIG. 25 is a perspective view of a plastic driving mechanism of the dental tool assembly incorporating a rack and pinion driving mechanism; FIG. 26 is a perspective view of a plastic driving mechanism of the dental tool assembly incorporating a multiple cam driving mechanism; FIG. 27 is a top view of the dental tool assembly in FIG. 26; FIG. 28 is a perspective view of a plastic driving mechanism of the dental tool assembly incorporating a multiple gear driving mechanism; FIG. 29 is a side view of the multiple gear driving mechanism; FIG. 30 is a perspective view of a plastic driving mechanism of the dental tool assembly incorporating an electromechanical operator; FIG. 31 is a perspective view of a plastic driving mechanism of the dental tool assembly incorporating a magnetic-mechanically coupled multiple clutch driving mechanism; FIG. 32 is a perspective view of a plastic driving mechanism of the dental tool assembly incorporating a spring like driving mechanism; FIG. 33 is a perspective view of a plastic driving mechanism of the dental tool assembly incorporating a piston driving mechanism. FIG. 34 is a view like FIG. 1 showing an alternative embodiment of a bearing assembly for rotational support of a drive shaft of the drive mechanism; FIG. 35 is an enlarged view, partially in section, showing a bushing and its journal on a drive shaft; FIG. 36 is a view similar to FIG. 35 including a housing shown in section for illustrating a snap-fit arrangement of the bushing in the housing; and FIG. 37 is an axial view of the bearing assembly taken along line 37 — 37 of FIG. 34. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS A dental tool assembly 10, formed in accordance with the principles of the present invention, is shown in FIG. 1. Dental tool assembly 10 includes a housing 12 having a proximal end 14 and a distal end 16, with main body portion 18 extending there between. Proximal end 14 is coupled to a dental tool hand piece (not shown) known in the art. Distal end 16 has a side opening 20 at which a desired dental treatment device (not shown) is coupled. It will be understood that any dental treatment device known in the art may be used. However, the preferred embodiment of the dental tool assembly shown in the FIGS. is a prophy angle to which a prophy cup or brush is coupled to apply prophy paste. Housing 12 is hollow such that first and second channels 22, 24 are formed therein for housing driving mechanism 28. First, longitudinal channel 22 is formed within main body portion 18 and extends from proximal end 14 to distal end 16 along longitudinal axis 23 of main body portion 18. Second, transverse channel 24 extends across the distal end 16 of housing 12 and opens at side opening 20 of housing 12. Longitudinal axis 25 of transverse channel 24 is transverse and preferably substantially perpendicular to longitudinal axis 23 of housing 12. Driving mechanism 28 includes a drive shaft 30 and a driven shaft 40. Drive shaft 30 is housed in first channel 22 and has a longitudinal rotation axis 31 which prefer-ably corresponds to longitudinal axis 23 of main body portion 18. A proximal end 32 of drive shaft 30 preferably extends beyond proximal end 14 of housing 12 for connection to a rotary unit (not shown), such as a motor, for rotating drive shaft 30, as known in the art. Distal end 34 of drive shaft 30 extends toward, and preferably partially into, second channel 24. Driven shaft 40 is housed in second channel 24 and has a longitudinal rotation axis 41 which preferably corresponds to longitudinal axis 25 of transverse channel 24. Driven shaft 40 preferably has a coupling element 42 extending therefrom through side opening 20 and out of housing 12. A desired dental treatment device, selected from those known in the art such as a prophy cup or brush, may be coupled to coupling element 42. Drive shaft 30 and driven shaft 40 have driving surfaces that are shaped to interengage each other to result in a camming action that translates rotation of drive shaft 30 into oscillatory rotation of driven shaft 40 substantially without play between the driving surfaces, as will now be described. As shown in FIGS. 2A, 2 B, and 3 - 6, drive shaft 30 has a drive surface 50 (which functions essentially as a cam) at distal end 34. Preferably drive surface 50 has a substantially conical cam surface, with cone axis 51 being at a preferably 45° angle with respect to rotation axis 31, as may be observed in FIG. 3. The conical shape is readily appreciated with reference to FIGS. 2A, 2 B, and 3 - 6. The tip 52 of conical drive surface 50 preferably is aligned with rotation axis 31 so that a longitudinal surface portion 54 of conical surface 50 is aligned with rotation axis 31 and a transverse surface portion 56 of conical surface 50 is substantially perpendicular, i.e., at a 90° angle, with respect to rotation axis 31 and thus with respect to longitudinal surface portion 54 : As may be seen in FIGS. 2 B and 3 - 6, conical surface 50 is formed to one side of rotation axis 31. Conical surface 50 may be formed by cutting away a portion of an enlarged region 30 a of shaft 30, thus leaving a flange-like section 58 at distal end 34. Driven shaft 40 (which essentially functions as a cam follower), shown in isolation in FIGS. 7-10, has a driven surface 60 along its side (i.e., extending along rotation axis 41 of driven shaft 40 ). The elevational view of FIG. 7 is similar to the view of driven shaft 40 in FIG. 1, except that driven shaft 40 is shown with driven surface 60 facing upward, rather than downward as in FIG. 1. Typically, driven surface 60 is formed as a cut-away portion of a side of driven shaft 40. Driven surface 60 has alternating hills 62 and valleys 64. Preferably, two hills 62 are provided opposite each other with a valley 64 between adjacent, juxtaposed sides of opposed hills 62, thus spacing hills 62 apart. Viewed another way, the upwardly extending sides of the opposite valleys 64 are joined to form hills 62. Hills 62 and valleys 64 are shaped to conform to the shape of drive surface 50 such that drive surface 50 is in continuous contact with driven surface 60 with substantially no play there between as drive shaft 30 rotates during operation of dental tool assembly 10. Specifically, valleys 64 of driven surface 60 are conically cut-away such that conical drive surface 50 may be engaged therewith such that transverse surface portion 56 and distal surface portions adjoining transverse surface portion 56 of conical drive surface 50 are in close contact with the surfaces of a valley 64. Because opposite sides of conical drive surface 50 are at an approximately 90° angle with respect to each other and valleys 64 are shaped to conform to conical drive surface 50 with hills 62 formed at the sides of valleys 64, peaks 66 of hills 62 are preferably also at an approximately 90° angle with respect to each other. The contour of driven surface 60 may be better understood from a review of the elevational views of FIGS. 8 and 10. The camming action of the present invention, which permits rotation of drive shaft 30 to cause oscillatory rotation of driven shaft 40 as a result of the interaction of the shapes of driving surfaces 50, 60, will now be described. When drive surface 50 engages a valley 64 of driven surface 60, driven shaft 40 is in a rest position (i.e., driven surface 60 completely faces drive surface 50 and proximal end 14 of housing 12, rather than a side of housing 12, as shown in FIG. 1 ). As drive shaft 30 rotates about rotation axis 31, drive surface 50 moves along driven surface 60 until drive surface 50 engages a hill 62. As described above, and as may be seen in FIG. 8, the peaks 66 of opposite hills 62 are positioned substantially 180° apart with the bottoms 65 of valleys 64 approximately 90° from each peak 66. Thus, when drive surface 50 has rotated 90° from a rest position in contact with valley 64 (such as shown in cross-sectional view 2 A), drive surface 50 comes into contact with adjacent hill 62. When transverse surface portion 56 of drive surface 50 contacts peak 66 of an adjacent hill 62, peak 66 is also transverse to rotation axis 31 such that driven shaft 40 is rotated 90° about its rotation axis 41 from its rest position. It is noted that peaks 66 are at an approximately 90° angle with respect to each other, as may be seen in FIG. 2B, and longitudinal and transverse portions 54, 56 of drive surface 50 are also at an approximately 90° angle with respect to each other, as may be appreciated with reference to FIGS. 1, 2 B, and 3. Thus, when transverse portion 56 of drive surface 50 contacts a peak 66 to rotate driven shaft 40, longitudinal portion 54 is in contact with the opposite peak 66. As drive surface 50 continues to be rotated upon rotation of drive shaft 30, drive surface 50 contacts the next valley 64 (opposite the first mentioned valley), returning driven shaft 40 to the rest position. Further rotation of drive shaft 30 brings drive surface 50 into contact with the next hill 62 (opposite the first-mentioned hill), thereby rotating driven shaft 40, in the same manner as described above but in the opposite direction, 90° about rotation axis 41. Thus, driven shaft 40 oscillates a total of 90°, performing a quarter turn in opposite directions from a rest position. As may be seen in the plan view of FIG. 8, transverse side walls 68 of driven surface 60 are curved. However, in order to provide greater clearance between side walls 68 and drive shaft 30 (particularly the outer walls of enlarged region 30 a extending substantially parallel to rotation axis 31 ) substantially straight side walls 68 a may, instead, be provided, as shown in FIGS. 11 and 12. Straight side walls 68 a extend, from the widest portions of valleys 64, along the periphery of driven surface 60 substantially perpendicular to rotation axis 41 of driven shaft 40. Because typically only oscillatory rotation, without axial reciprocation, of driven shaft 40 is desired, it is desirable to fix drive shaft 30 with respect to driven shaft 40. In accordance with the principles of the present invention, drive shaft 30 is provided with an axially extending pin 70 that is substantially aligned with rotation axis 31. Driven shaft 40 is provided with a corresponding slot 72, which may extend completely through driven shaft 40, as shown in FIGS. 2A, 2 B, and 7 - 10. It will be understood that slot 72 need not extend completely through driven shaft 40, as shown, as long as sufficient engagement between pin 70 and slot 72 is achieved. The axial extent of slot 72 along rotation axis 41 of driven shaft 40 is selected to provide a substantially close fit with the diameter of pin 70 to prevent axial reciprocation of driven shaft 40 along axis 41. However, the transverse extent of slot 72 (in a direction perpendicular to axis 41 ) is selected such that 90° rotation of driven shaft 40 with respect to drive shaft 30 (45° rotation of driven shaft 40 in each direction from the rest position) is permitted without causing shifting of either shaft 30, 40 from respective axes 23, 25 of housing 12. In order to prevent movement of shafts 30, 40 from their proper positions within channels 22, 24 of housing 12, position retaining elements are provided as follows. In order to prevent axial shifting of drive shaft 30 along axis 31, drive shaft 30 is provided with at least one radially extending stop flange 80. As shown in FIGS. 1 and 3 - 5, preferably a proximal flange 82 and a distal flange 84 are provided. Flange 58 may also be considered to perform the same function as that of flanges 82 and 84 and thus may be considered a stop flange 80 as well. Housing 12 is provided with a latch 86 (inserted after assembly in order to maintain the parts of dental tool assembly 10 in place) having a position retaining surface 89 extending radially inwardly from the walls of channel 22. Position retaining surface 89 is positioned adjacent and along a retaining surface 83 of proximal flange 82 to prevent proximal axial movement of drive shaft 30 towards proximal end 14 of housing 12. Additional position retaining surfaces may be provided extending radially inwardly from the inner walls of channels 22 to engage proximal position retaining surfaces on flanges 58 and 84 as well. It will be understood that the position retaining surfaces formed on housing 12 need, not be in the form of a latch, but may be in any other form, such as a radially inwardly extending shoulder, that provides a sufficient surface area for engaging a proximal face of at least one of the flanges 80 on drive shaft 30. Moreover, the position retaining surfaces on housing 12 must be securely fixed to housing 12 along axis 23 to prevent movement of drive shaft 30 along axis 23. In order to secure axial alignment of driven shaft 40 with axis 25, a positioning pin 90 may be provided at a rear, inner end of driven shaft 40 to fit within bore 92 at a rear end of channel 24 of housing 12, as shown in FIG. 1. Pin 90 not only serves to maintain proper alignment of driven shaft 40 during use, but also facilitates alignment of driven shaft 40 in housing 12 during assembly. Preferably, to assemble dental tool assembly 10, driven shaft 40 is first positioned in housing 12, with pin 90 fitting within bore 92 such that rotation axis 41 of driven shaft 40 is properly aligned with longitudinal axis 25 of channel 24. Driven shaft 40 is rotated into its rest position such that driven surface 60 faces proximal end 14 of housing 12. Drive shaft 30 may then be inserted into channel 22, with pin 70 extending into slot 72 of driven shaft 40. Latch 86 then is positioned such that position retaining surface 89 faces position retaining surface 83 to maintain drive shaft 30 in its proper position along longitudinal axis 31 of channel 22. Dental tool assembly 10 then is ready for coupling with the desired hand piece. An alternative embodiment of the present invention is illustrated in FIG. 13. In this embodiment, the inventive plastic driving mechanism 128 is driven by a rotating member 194, which serves as a source of rotary input power. Rotating member 194 is a simple elongated driving shaft, of the same configuration as the driving shaft in a conventional rotating prophy angle, and thus, like the other inventive embodiments of the invention may be easily substituted in existing dental apparatus in wide use at dentist's offices. Rotating member 194 is coupled to a flexible driving member 106 by any suitable coupling 107. During use, flexible driving member 106 is rotated in the direction indicated by arrows 109. The part of flexible driving member 106 closer to rotating member 194 rotates along a horizontal longitudinal oscillatory axis 108. Similarly, the distal portion of flexible driving member 106 rotates along a vertical oscillatory axis 111. In accordance with this embodiment of the invention, flexible driving member 106 is maintained in a curved configuration. As can be seen in FIG. 13, flexible driving member 106 has a thickness 113 which is much smaller than its width 115. Because of the flexible driving member 106 is maintained in a curved configuration as illustrated in FIG. 13, rotation of the proximal portion of flexible driving member 106 at a constant speed causes a snap in the angular rotation of the distal end of flexible driving member 106. This irregularity in angular speed amounts to a sort of stall during which gum tissue has an opportunity to resume an unstressed configuration. In many respects, the effect is similar to that achieved by the reciprocating motion of the prophy angle, which stresses the gum tissue in one direction, then reverses direction, relieving the stress and lessening the likelihood of tissue damage that would be more likely if one continued to apply stress and high-speed in one direction, as would be the case in the typical rotary prophy angle drive mechanism. Flexible driving member 106 is maintained in the curved configuration illustrated in FIG. 13 by housing, the same as in a curved tubular housing 117, which is illustrated in dashed lines in FIG. 13. More particularly, it is noted that in accordance with this embodiment of the invention, a rubber prophy angle 119, which functions as a tooth scrubbing surface and is of conventional design snaps onto a plastic support member 142. Plastic support member 142 is mounted for rotation within a secular mouth 121, which is configured to receive the disk shaped top of member 142, thus securely holding member 142 and allowing only rotation in the direction illustrated by arrow 123, in response to power input to the system by rotary member 194. The intermittent nature of the motion in the embodiment illustrated in FIG. 13 may be improved by introducing a measure of friction between member 142 and mouth 121. FIGS. 14 and 15 show an alternative embodiment of the inventive plastic driving mechanism 228. FIG. 14 is partially in exploded perspective as will be apparent from the following description. In this embodiment, plastic driving mechanism 228 is driven by a rotary input 294, of the type conventionally incorporated in a dental tool power source that might be used by a dentist to power a drill or other similar instrument. The output of rotary input 294 is coupled by a coupling mechanism 207 to a cylindrical driving member 209. Cylindrical driving member 209 defines a drive surface 250 comprising a figure-eight shaped groove. As can be seen in FIG. 14, this groove extends around cylindrical driving member 209 twice, crossing over itself at point 211 to define the figure-eight shape. For the sake of clarity of illustration, cylindrical driving member 209 is shown outside of sleeve 213. During operation, cylindrical driving member 209 is positioned within sleeve 213. Sleeve 213 has secured within it a cam following nub 215 which is positioned within groove-shaped drive surface 250, as illustrated in FIG. 15, during operation of the inventive plastic driving mechanism 228. As can be seen from FIGS. 14 and 15, as cylindrical driving member 209 is rotated, because it is fixed in position within a suitable housing structure, it tends to pull sleeve 213 in a reciprocating motion along the axial direction as illustrated by arrow 217. This reciprocating motion is. coupled to support 219. A drive member 221 is mounted for rotary movement in the directions of arrow 222 on support 219 by a shaft 223. Finally, prophy support 242 is coupled by a shaft 225 to drive member 221. As cylindrical driving member 209 is rotated in the direction indicated by arrow 205, nub 215 is pulled in the directions indicated by arrow 217, resulting in identical movement by the sleeve 213. This reciprocating movement is coupled to shaft 225 causing prophy support 242 and prophy angle 227 to reciprocate in the directions indicated by arrow 229 and achieve the desired action of cleaning the tooth without damage to the gums. Referring to FIG. 16, yet another inventive plastic driving mechanism 328 for achieving reciprocating motion in a prophy angle is shown. Generally, in this embodiment, the prophy angle is supported for rotary movement in a housing in much the same manner of the embodiments previously described. Rotary motion is converted into a periodic push which rotates the prophy angle against the prophy support. When the push is released, the prophy angle snaps back into its original position. More particular, cylindrical driving member 309 is rotated in the direction indicated by arrow 311. Rotation of cylindrical member 309 results in shaft 313 moving in a circular path. Shaft 313 is secured to cylindrical member 309. Periodically, shaft 313 bears against a shaft 315 which is secured to the prophy angle 317. When shaft 313 bears against shaft 315 on prophy angle 317, it moves shaft 315 and rotates prophy angle 317 in the direction indicated by arrow 319. When this occurs, the spring 321 in a groove 323 in prophy angle 317 is compressed by a stock 325 which is rigidly secured with respect to housing 327 within which the drive member as illustrated in FIG. 16 is contained. As shaft 313 continues to move in a circular path, eventually it is rotated away from shaft 315, releasing shaft 315 and allowing spring 321 to expand, driving prophy angle 317 in the direction of arrow 327, thus resulting in reciprocating movement. Referring to FIGS. 17 a - 17 c, yet another inventive plastic driving mechanism 428 for achieving reciprocating movement in a prophy angle is illustrated. Here the driving mechanism 428 is driven by a drive shaft 412 with rotary motion in the direction of arrow 413. At the end of drive shaft 412 is a driving cam 414. Drive shaft 412 is retained in position in a space 424 in housing 432 by a disc shaped position retaining element 482 having a centered hole 415 as shown in FIG. 17 a, and a second disc shaped position retaining element 484 having an off-centered hole 421 to accept drive shaft 412 as shown in FIG. 17 b. The proximal end of drive shaft 412 extends partially through a circumferential shaped driven cam 416 mounted for rotation in housing 432. Prophy angle support 419 can be detachably mounted into housing 432 by an annular ridge 422 Ridge 422 snappingly engages an annular groove 425. A coupling element 429 connects circumferential shaped driven cam 416 to support 419 in housing 432. A spring like member 431 springingly positions cam 416 in housing 432. Rotary motion is converted into reciprocating motion when driving shaft 412 rotates, causing the driving cam 414 to bear against circumferential shaped driven cam 416 in the direction as shown by arrow 450. As shown in FIG. 17 c, the result is to impart the forward portion of a reciprocating motion to coupling member 429 which is coupled to driven cam 416 and prophy angle support 419. Spring-like member 431, attached to driven cam 416, after a time becomes fully extended, moving in groove 452. When the peak 433 of driven cam 416 is passed by driving cam 414, as shown in dash-dot lines in FIG. 17 c, spring-like member 431 springs back causing driven cam 416 to return to its rest position illustrated in solid lines in FIG. 17 c. Driving shaft 412 then continues its rotational cycle in the direction of arrow 413 until the pushing and springing back of driven cam 416 is completed. As driven cam 416 repeatedly returns to its rest position, prophy angle support 419 rotates in the direction indicated by arrow 434, resulting in the desired reciprocating motion of prophy angle 429. Turning to FIGS. 18-20, still yet another mechanism for achieving reciprocating movement in a prophy angle is illustrated. In this embodiment, reciprocating motion is achieved in the inventive plastic driving mechanism 510 by applying rotary motion to a lobed member 512. Lobed member 512 includes a pair of lobes 514 and 516. As lobed member 512 rotates in the direction of arrow 519, lobes 514 and 516 follow circular paths but are separated from each other by 180 degrees. In a fashion similar to that of the previous embodiments, a prophy angle support 518 on which a prophy angle 520 is mounted, is supported for reciprocating motion within a housing 522. Prophy angle support 518 includes an elongated cam follower 524 which is alternately acted on by lobe 514 in the direction of arrow 526, and then by lobe 516 in the direction indicated by arrow 528. More particularly, when lobe 514 bears against elongated cam follower 524, prophy angle support 518 is moved in the direction indicated by arrow 530. Alternatively, when lobe 516 bears against elongated cam follower 524, prophy angle support 518 is moved in the direction indicated by arrow 532. The result of this alternating action is the desired inventive reciprocating motion indicated by arrow 534, as illustrated in FIG. 19. Turning to FIGS. 21-22, reciprocating motion is achieved through the use of a wedge-shaped member attached to the prophy support. More particular, the inventive plastic driving mechanism 610 comprises a rotating shaft 612 which has a pair of studs 614 and 616 attached to it. A wedge-shaped member 618 is secured to the top of prophy support 620. Wedge-shaped member 618 includes a pair of side wedge surfaces 622 and 624. As shaft 612 rotates in the direction of arrow 626, alternatively stud 616 bears against surface 624, driving it in the direction of arrow 628, followed by stud 614 bearing against surface 624 driving it in the direction of arrow 630. In accordance with the present invention, it is also contemplated that gears may be used to achieve reciprocating motion in a dental tool driven by a rotary power source. Referring to FIGS. 23-24, an inventive plastic driving mechanism 710 is provided with an input shaft 712 which is rotated, thus rotating a partial gear 714 which comprises about 75 degrees of a circular gear and has the appearance of a pie slice. As input shaft 712 is rotated in the direction of arrow 716, partial toothed gear 714 alternately engages smaller follower toothed gears 718 and 720 for a short period of time in the overall cycle of rotation of shaft 712. When follower toothed gear 718 is rotated, it rotates worm gear 722 to which it is attached by coupling shaft 724. This results in worm gear 722 engaging gear 726, causing gear 726 and prophy angle support 728 to which it is secured, to rotate in the direction of arrow 730. Similarly, when follower toothed gear 720 is rotated, it rotates worm gear 732 to which it is attached by coupling shaft 734. This results in worm gear 732 engaging gear 726, causing gear 726 and prophy angle support 728 to rotate in the direction of arrow 740. Referring to FIG. 25, yet another method for achieving the inventive reciprocating motion in a plastic driving mechanism 810 constructed in accordance with the present invention. In accordance with this embodiment, a drive shaft 812 is rotated in the direction indicated by arrow 814. This results in rotating disk 816 in the same direction. A pin 818 is mounted on disk 816. As disk 816 rotates, pin 818 follows a circular path. The result is to impart a reciprocating motion to coupling member 820 which is coupled to a rack 822 having a plurality of teeth 824 on it. Rack 822 is supported for sliding movement in the direction indicated by arrow 823 between a pair of support members 825. In this manner, rack 822 is given a reciprocating motion. Teeth 824 mesh with teeth 826 on pinion 828, causing reciprocating motion in pinion 828. The result is to achieve the desired reciprocating motion as indicated by arrow 829 in prophy angle support 830. Turning next to FIGS. 26 and 27, yet another mechanism for achieving reciprocating motion is shown. Here the inventive reciprocating plastic driving mechanism 910 is driven by a drive shaft 912 with rotary motion in the direction of arrow 914. At the end of drive shaft 912 are a pair of cams 916 and 918. Prophy angle support 920 includes a pair of cams 922 and 924. When cam 916 bears against cam 922 it urges prophy angle support 920 in the direction indicated by arrow 926. Similarly, when cam 918 bears against cam 924 it urges prophy angle support 920 in the direction indicated by arrow 928. Because cams 916 and 918 are positioned on shaft 912 at 180 degrees with respect to each other, they are bearing against cams 922 and 924 at different times, and this causes reciprocating motion in prophy angle support 920. Still yet another approach is illustrated in FIGS. 28 and 29. In this embodiment, power is provided to the inventive reciprocating plastic driving mechanism 1010 by a drive shaft 1012 which is rotated in the direction of arrow 1014. The end of shaft 1012 has a pair of partial pie-shaped toothed gears 1016 and 1018 which have teeth that mesh with teeth on a conical gear 1020. As can be seen in FIG. 28, as shaft 1012 rotates, gear 1018 causes follower gear 1020 to rotate in the direction of arrow 1022 when the teeth of gear 1020 engage gear 1018. At other times, when the teeth of gear 1016 engage the teeth of gear 1020, gear 1020 is caused to rotate in the direction indicated by arrow 1024, resulting in reciprocating motion of gear 1020 and prophy angle support 1026. Referring to FIG. 30, an electromechanical approach to the problem of providing reciprocating motion by an inventive reciprocating plastic driving mechanism 1120 is illustrated. The same may be done using an electromechanical operator 1112 to directly. provide reciprocating motion. Alternatively, a simpler electromechanical operator may be used which only provides for movement in one direction, with movement in the opposite direction being provided by a spring biased arrangement of the type illustrated and described in connection with FIG. 17, above. Turning next to FIG. 31, in accordance with the present invention it is contemplated that magnetic coupling may be used to relieve the stress applied to the gums during continuous motion. Such a magnetic coupling may simply comprise a magnetic clutch. In particular, the inventive reciprocating plastic driving mechanism 1210 is provided with a driving clutch member 1212 which is magnetically coupled to a driven clutch member 1214 to achieve a magnetic-mechanical connection between the two magnet members. Driving clutch member 1212 and driven clutch member 1214 may also be made of plastic, as such materials are inexpensive and widely available. As alluded to above, the invention contemplates the fabrication of all the embodiments of the invention in plastic, although substitution of other materials is possible. In any case, the inventive structures are conFig.d in a manner that provides for durability, even in relatively inexpensive and weak plastic materials. When driving clutch member 1212 is rotated, a driven clutch member 1214 is caused to rotate because of the magnetic-mechanical connection, thus resulting in a transfer of power. Clutch member 1214, in turn, is coupled to the prophy angle support 1216 in order to rotate the prophy angle 1218. Such a magnetic clutch will release if tension applied to the gums becomes too great. Such a mechanism can be used in combination with any of the reciprocating plastic driving mechanisms described in this application to achieve an additional measure of protection. In addition, magnetic coupling may be used in place of the various forms of mechanical coupling to achieve the desired reciprocating motion in the various embodiments disclosed herein. Turning next to FIG. 32, still yet another approach is illustrated. In accordance with this approach, a reciprocating plastic driving mechanism 1310 includes a plastic spring like member 1312 mounted for rotation on a support 1314 and coupled by a living hinge 1316 to a prophy angle support 1318 as illustrated. The far end 1320 of the spring like member 1312 is acted on by a stud 1322 mounted on a rotating member 1324. When rotating member 1324 rotates, stud 1322 impacts far end 1320, causing the other end to displace the position of living hinge 1316 causing movement of prophy angle support 1318 in the direction indicated by arrow 1326. Because member 1312 is a spring, when the impact is over, prophy angle support 1318 moves in the opposite direction, thus resulting in reciprocating motion. Turning to FIG. 33, because dental tools often have air pressure as a primary source of power, and it is this air pressure which is used to drive a dentist's drill, using a converter which converts air pressure into rotary motion, the possibility also exists to achieve reciprocating motion from air pressure directly. The same can be achieved in a reciprocating plastic driving mechanism 1410 by a number of means, including the use of a piston 1412 in a cylinder 1414. The air pressure drives the piston 1412 in the direction indicated by arrow 1416 against the action of a spring 1418 which is compressed by the movement of the piston 1412. Pressure maybe released by a vent 1420 causing spring 1418 to push the piston 1412 back in the direction indicated by arrow 1422. Piston 1412 is coupled by link 1424 to a prophy angle support 1426. The result is that link 1424 couples the reciprocating motion to prophy angle support 1426 resulting in reciprocating motion of prophy angle 1428. Referring to FIG. 34, an alternative embodiment for providing longitudinal axial positioning is provided. This embodiment also provides an alternative means of assembling dental tool assembly 1510. A bearing assembly 1549 includes a bearing journal 1551 and a bushing 1553 that cooperate in holding drive shaft 1530 in axial alignment holding drive surface 1550 and driven surface 1560 in contact with each other. Bushing 1553 is held in position within housing 1512 by means of a snap-fit arrangement. This embodiment replaces stop flange 80 and latch 86, as shown in FIG. 1. Referring now to FIGS. 35 to 37, the relationship between drive shaft 1530, journal 1551, and bushing 1553 is shown, with bushing 1553 being shown in section. Drive shaft 1530 includes a bearing journal 1551 having an outside diameter greater than the outside diameter of drive shaft 1530. Bearing journal 1551 may be integral with drive shaft 1530 or formed separately and attached to drive shaft 1530 by any suitable means. Journal 1551 includes a rearward contact surface 1555 part of which forms a contact area with bushing 1553. Surface 1555 may be any shape, preferably in an appropriate shape to reduce friction and increase reliability. Bushing 1553 is generally disk-shaped and has an inwardly facing frustoconical surface 1557. frustoconical surface 1557 has intrinsic characteristics in this configuration. Surface 1557 aids in centering drive shaft 1530 as it passes through bushing 1553 and also promotes a snug fit without being overly tight while simultaneously reducing the contact surface area with journal contact surface 1555, all desirable characteristics. At the same time, bushing 1553 presents a very simple configuration that is readily produced, especially in plastic. Referring now to FIG. 36, bearing assembly 1549 is shown in position within housing 1512 which is shown in section. For retaining bushing 1553 in position, a retaining structure on its periphery includes a ridge 1559 extending radially inwardly from an inside wall 1561 of housing 1512. Ridge 1559 is preferably integral with housing 1512 and may be any suitable shape but is preferably radiased. In order to ensure that bushing 1553 is held firmly in position a collar 1563 extends radially outwardly from bushing 1553 and is preferably integral therewith. The inside diameter of ridge 1559 is preferably less than the outer diameter of collar 1563 thereby forming an interference fit between the two forming what is commonly referred to as a snap-fit. The snap-fit arrangement described above is only one possible arrangement and similar arrangements are also contemplated by the present invention. By way of example and not limitation, a reduced diameter area in the outer diameter of bushing 1553 could be used to form a snap-fit arrangement with ridge 1559. In order to aid the preferred arrangement during assembly a chamfer 1565 is formed on an a leading edge of outer diameter of bushing 1553. Housing 1512 may include a reduced diameter section 1567 at the point where bushing 1553 is to be positioned. A receiving area 1569 formed between ridge 1559 and reduced diameter section is sized to receive collar 1563. The arrangement described above holds bushing 1553 in place which in turn holds drive shaft 1530 in place acting through journal 1551 for normal operation of dental tool assembly 1510. Housing 1512 also includes a raised portion 1571 extending outwardly from its outer surface and is preferably integral therewith. Raised portion 1571 is sized and positioned to aid a user in gripping dental tool assembly 1510, which may include a single or multiple spiral arrangement. Bearing assembly 1549 as described above has additional benefits in that it greatly simplifies assembly of dental tool assembly 1510. During assembly, driven shaft 1540 is properly positioned within housing 1512 and then drive shaft 1530 is inserted into housing 1512 in its proper position. Bushing 1553 is then introduced to the open end of housing 1512 with drive shaft 1530 sliding through a central aperture 1573 of bushing 1553. The fit between bushing 1553 and drive shaft 1530 is preferably toleranced so as to minimize contact between the two components yet allow for a minimal increase in diameter of drive shaft 1530 to form journal 1551 thereon. Once inserted into the end of housing 1512 bushing 1553 is then pushed down into its final position with the snap-fit occurring at the end of insertion. This then holds drive shaft 1530 and driven shaft 1540 in reasonably permanent position within housing 1512 which is the in use position for dental toll assembly 1510. While the foregoing description and drawings represent the preferred embodiments of the present invention, it will be understood that various additions, modifications and substitutions may be made without departing from the spirit and scope of the present invention as defined in the accompanying claims. In particular, it will be clear that the present invention may be embodied in other specific forms, structures, arrangements, proportions, and with other elements, materials, and components, without departing from the spirit or essential characteristics thereof. For example, although housing 12 is in the form of a prophy angle, driving mechanism 28 may be used in any other desired dental tool assembly, or any other motorized device that requires oscillating rotary motion of an output end. One skilled in the art will appreciate that the invention may be used with many modifications of structure, arrangement, proportions, materials, and components and otherwise, used in the practice of the invention, which are particularly adapted to specific environments and operative requirements without departing from the principles of the present invention. The presently disclosed embodiments are therefore to be considered in all respects as illustrative and not restrictive, the scope of the invention being indicated by the appended claims, and not limited to the foregoing description. | A transmission for changing rotary motion into angularly reciprocating motion and adapted to be mounted on the output end of a dental power unit of the type having a rotary drive output and used to drive a dental tool. The transmission is comprised of a support member, a driving resinous member supported by the support member for rotary motion, and a driven resinous member supported by the support member for reciprocating angular movement by the support member. The transmission subassembly is formed by the driving resinous member, driven resinous member and support member and adapted to be mounted on the output end of the dental power unit with a rotary drive output mechanically coupled to the driving resinous member. A driving cam surface is disposed on a portion of the driving resinous member. A driven cam surface is disposed on a portion of the driven resinous member. The driving cam surface is in contact with the driven cam surface during at least a portion of the cycle of rotation of the driving cam surface. The driven cam surface is configured and dimensioned to be driven by the driving cam surface in a positive angular direction during one part of the cycle and is driven by the driving cam surface in a negative angular direction during another part of the cycle. Angularly reciprocating motion is thereby imparted to the dental tool as the driving cam surface and driven cam surface engage each other. | big_patent |
SECTION 1. SHORT TITLE. This Act may be cited as the ``Stop Trading on Congressional Knowledge Act''. SEC. 2. NONPUBLIC INFORMATION RELATING TO CONGRESS. (a) Securities Transactions.--Section 10 of the Securities Exchange Act of 1934 is amended by adding at the end the following: ``(c) Nonpublic Information Relating to Congress.--Not later than 270 days after the date of enactment of this subsection, the Commission shall by rule prohibit any person from buying or selling the securities of any issuer while such person is in possession of material nonpublic information, as defined by the Commission, relating to any pending or prospective legislative action relating to such issuer if-- ``(1) such information was obtained by reason of such person being a Member or employee of Congress; or ``(2) such information was obtained from a Member or employee of Congress, and such person knows that the information was so obtained. ``(d) Nonpublic Information Relating to Other Federal Employees.-- ``(1) Rulemaking.--Not later than 270 days after the date of enactment of this subsection, the Commission shall by rule prohibit any person from buying or selling the securities of any issuer while such person is in possession of material nonpublic information derived from Federal employment and relating to such issuer if-- ``(A) such information was obtained by reason of such person being an employee of an agency, as such term is defined in section 551(1) of title 5, United States Code; or ``(B) such information was obtained from such an employee, and such person knows that the information was so obtained. ``(2) Material nonpublic information.--For purposes of this subsection, the term `material nonpublic information' means any information that an employee of an agency (as such term is defined in section 551(1) of title 5, United States Code) gains by reason of Federal employment and that such employee knows or should know has not been made available to the general public, including information that-- ``(A) is routinely exempt from disclosure under section 552 of title 5, United States Code, or otherwise protected from disclosure by statute, Executive order, or regulation; ``(B) is designated as confidential by an agency; or ``(C) has not actually been disseminated to the general public and is not authorized to be made available to the public on request.''. (b) Commodities Transactions.--Section 4c of the Commodities Exchange Act (7 U.S.C. 6c) is amended by adding at the end the following: ``(h) Nonpublic Information Relating to Congress.--Not later than 270 days after the date of enactment of this subsection, the Commission shall by rule prohibit any person from buying or selling any commodity for future delivery while such person is in possession of material nonpublic information, as defined by the Commission, relating to any pending or prospective legislative action relating to such commodity if-- ``(1) such information was obtained by reason of such person being a Member or employee of Congress; or ``(2) such information was obtained from a Member or employee of Congress, and such person knows that the information was so obtained. ``(i) Nonpublic Information Relating to Other Federal Employees.-- ``(1) Rulemaking.--Not later than 270 days after the date of enactment of this subsection, the Commission shall by rule prohibit any person from buying or selling any commodity for future delivery while such person is in possession of material nonpublic information derived from Federal employment and relating to such commodity if-- ``(A) such information was obtained by reason of such person being an employee of an agency, as such term is defined in section 551(1) of title 5, United States Code; or ``(B) such information was obtained from such an employee, and such person knows that the information was so obtained. ``(2) Material nonpublic information.--For purposes of this subsection, the term `material nonpublic information' means any information that an employee of an agency (as such term is defined in section 551(1) of title 5, United States Code) gains by reason of Federal employment and that such employee knows or should know has not been made available to the general public, including information that-- ``(A) is routinely exempt from disclosure under section 552 of title 5, United States Code, or otherwise protected from disclosure by statute, Executive order, or regulation; ``(B) is designated as confidential by an agency; or ``(C) has not actually been disseminated to the general public and is not authorized to be made available to the public on request.''. SEC. 3. AMENDMENT TO THE RULES OF THE HOUSE OF REPRESENTATIVES REGARDING SECURITIES TRADING BASED ON NONPUBLIC INFORMATION. Rule XXIII (known as the ``Code of Official Conduct'') of the Rules of the House of Representatives is amended by redesignating clause 18 as clause 19 and by inserting after clause 17 the following new clause: ``18. A Member, Delegate, Resident Commissioner, officer, or employee of the House shall not-- ``(a) disclose material nonpublic information relating to any pending or prospective legislative action relating to any publicly-traded company if that Member, Delegate, Resident Commissioner, officer, or employee has reason to believe that the information will be used to buy or sell the securities of such publicly-traded company based on such information; or ``(b) disclose material nonpublic information relating to any pending or prospective legislative action relating to any commodity if that Member, Delegate, Resident Commissioner, officer, or employee has reason to believe that the information will be used to buy or sell such commodity for future delivery based on such information.''. SEC. 4. TIMELY REPORTING OF SECURITIES TRANSACTIONS. (a) Amendment.--Section 103 of the Ethics in Government Act of 1978 is amended by adding at the end the following subsection: ``(l) Within 90 days after the purchase, sale, or exchange of any stocks, bonds, commodities futures, or other forms of securities that are otherwise required to be reported under this Act and the transaction of which involves at least $1000 by any Member of Congress or officer or employee of the legislative branch required to so file, that Member, officer, or employee shall file a report of that transaction with the Clerk of the House of Representatives in the case of a Representative in Congress, a Delegate to Congress, or the Resident Commissioner from Puerto Rico, or with the Secretary of the Senate in the case of a Senator.''. (b) Effective Date.--The amendment made by subsection (a) shall apply to transactions occurring on or after the date that is 90 days after the date of the enactment of this Act. SEC. 5. REGISTRATION OF POLITICAL INTELLIGENCE FIRMS. (a) Definitions.--Section 3 of the Lobbying Disclosure Act of 1995 (2 U.S.C. 1602) is amended-- (1) in paragraph (2)-- (A) by inserting after ``lobbying activities'' both places such term appears the following: ``or political intelligence activities''; and (B) by inserting after ``lobbyists'' the following: ``or political intelligence consultants''; and (2) by adding at the end the following new paragraphs: ``(17) Political intelligence activities.--The term `political intelligence activities' means political intelligence contacts and efforts in support of such contacts, including preparation and planning activities, research and other background work that is intended, at the time it is performed, for use in contacts, and coordination with the political intelligence activities of others. ``(18) Political intelligence contact.-- ``(A) Definition.--The term `political intelligence contact' means any oral or written communication (including an electronic communication) to or from a covered executive branch official or a covered legislative branch official, the information derived from which is intended for use in analyzing securities or commodities markets, that is made on behalf of a client with regard to-- ``(i) the formulation, modification, or adoption of Federal legislation (including legislative proposals); ``(ii) the formulation, modification, or adoption of a Federal rule, regulation, Executive order, or any other program, policy, or position of the United States Government; or ``(iii) the administration or execution of a Federal program or policy (including the negotiation, award, or administration of a Federal contract, grant, loan, permit, or license). ``(B) Exception.--The term `political intelligence contact' does not include a communication that is made by or to a representative of a media organization if the purpose of the communication is gathering and disseminating news and information to the public. ``(19) Political intelligence firm.--The term `political intelligence firm' means a person or entity that has 1 or more employees who are political intelligence consultants to a client other than that person or entity. ``(20) Political intelligence consultant.--The term `political intelligence consultant' means any individual who is employed or retained by a client for financial or other compensation for services that include one or more political intelligence contacts.''. (b) Registration Requirement.--Section 4 of that Act (2 U.S.C. 1603) is amended-- (1) in subsection (a)(1)-- (A) by inserting after ``whichever is earlier,'' the following: ``or a political intelligence consultant first makes a political intelligence contact,''; and (B) by inserting after ``such lobbyist'' both places such term appears the following: ``or consultant''; (2) in subsection (a)(2), by inserting after ``lobbyists'' both places such term appears the following: ``or consultants''; (3) in subsection (a)(3)(A)-- (A) by inserting after ``lobbying activities'' each place such term appears the following: ``and political intelligence activities''; and (B) in clause (i), by inserting after ``lobbying firm'' the following: ``or political intelligence firm''; (4) in subsection (b)(3), by inserting after ``lobbying activities'' both places such term appears the following: ``or political intelligence activities''; (5) in subsection (b)(4), by inserting after ``lobbying activities'' the following: ``or political intelligence activities''; (6) in subsection (b)(4)(C), by inserting after ``lobbying activity'' the following: ``or political intelligence activity''; (7) in subsection (b)(5), by inserting after ``lobbying activities'' both places such term appears the following: ``or political intelligence activities''; (8) in subsection (b)(6), by inserting after ``lobbyist'' both places such term appears the following: ``or political intelligence consultant''; (9) in subsection (c)(1), by inserting after ``lobbying contacts'' the following: ``or political intelligence contacts''; (10) in subsection (c)(2)-- (A) by inserting after ``lobbying contact'' the following: ``or political intelligence contact''; and (B) by inserting after ``lobbying contacts'' the following: ``and political intelligence contacts''; and (11) in subsection (d)(1), by inserting after ``lobbying activities'' both places such term appears the following: ``or political intelligence activities''. (c) Reports by Registered Political Intelligence Consultants.-- Section 5 of the Lobbying Disclosure Act of 1995 (2 U.S.C. 1604) is amended-- (1) in subsection (a), by inserting after ``lobbying activities'' the following: ``and political intelligence activities''; (2) in subsection (b)(2)-- (A) in the matter preceding subparagraph (A), by inserting after ``lobbying activities'' the following: ``or political intelligence activities''; (B) in subparagraph (A)-- (i) by inserting after ``lobbyist'' the following: ``or political intelligence consultant''; and (ii) by inserting after ``lobbying activities'' the following: ``or political intelligence activities''; (C) in subparagraph (B), by inserting after ``lobbyists'' the following: ``or political intelligence consultants''; and (D) in subparagraph (C), by inserting after ``lobbyists'' the following: ``or political intelligence consultants''; (3) in subsection (b)(3)-- (A) by inserting after ``lobbying firm'' the following: ``or political intelligence firm''; and (B) by inserting after ``lobbying activities'' both places such term appears the following: ``or political intelligence activities''; and (4) in subsection (b)(4), by inserting after ``lobbying activities'' both places such term appears the following: ``or political intelligence activities''. (d) Disclosure and Enforcement.--Section 6 of the Lobbying Disclosure Act of 1995 (2 U.S.C. 1605) is amended-- (1) in paragraph (3)(A), by inserting after ``lobbying firms'' the following: ``, political intelligence consultants, political intelligence firms,''; (2) in paragraph (7), by inserting after ``lobbying firm'' the following: ``, or political intelligence consultant or political intelligence firm,''; and (3) in paragraph (8), by inserting after ``lobbying firm'' the following: ``, or political intelligence consultant or political intelligence firm,''. (e) Rules of Construction.--Section 8 of the Lobbying Disclosure Act of 1995 (2 U.S.C. 1607) is amended in subsection (b) by inserting after ``lobbying contacts'' the following: ``, or political intelligence activities or political intelligence contacts,''. | Stop Trading on Congressional Knowledge Act - Amends the Securities Exchange Act of 1934 and the Commodities Exchange Act to direct both the Securities and Exchange Commission (SEC) and the Commodity Futures Trading Commission (CFTC) to prohibit purchase or sale of either securities or commodities for future delivery by a person in possession of material nonpublic information regarding pending or prospective legislative action if the information was obtained: (1) knowingly from a Member or employee of Congress; (2) by reason of being a Member or employee of Congress; and (3) other federal employees. Amends the Code of Official Conduct of the Rules of the House of Representatives to prohibit designated House personnel from disclosing material nonpublic information relating to any pending or prospective legislative action relating to either securities of a publicly-traded company or a commodity if such personnel has reason to believe that the information will be used to buy or sell the securities or commodity based on such information. Amends the Ethics in Government Act of 1978 to require formal disclosure of certain securities and commodities futures transactions to either the Clerk of the House of Representatives or the Secretary of the Senate. Amends the Lobbying Disclosure Act of 1995 to subject to its registration, reporting, and disclosure requirements political intelligence activities, contacts, firms, and consultants. | billsum |
SECTION 1. SHORT TITLE; TABLE OF CONTENTS. (a) Short Title.--This Act may be cited as the ``Hunting, Education, and Recreational Development Act'' or the ``HEARD Act''. (b) Table of Contents.--The table of contents for this Act is: Sec. 1. Short title; table of contents. Sec. 2. Findings and purpose. Sec. 3. Definitions. Sec. 4. Disposal. Sec. 5. Lands to provide or increase recreational and other opportunities. Sec. 6. Public availability of information on land potentially available for disposal. Sec. 7. Recreation and Public Purposes Act. Sec. 8. Limitations for administrative costs. Sec. 9. Recording. SEC. 2. FINDINGS AND PURPOSE. (a) Findings.--Congress finds the following: (1) The total Federal estate exceeds more than 635,000,000 acres. (2) The Federal Government owns parcels of varying size interspersed with or adjacent to private, State, and tribal lands throughout the United States, making many of these parcels difficult to manage and more appropriate for disposal. (3) The Bureau of Land Management identifies certain lands potentially available for disposal in revisions to resource management plans. (4) Existing law does not require the Bureau of Land Management to dispose of identified lands on a regular or frequent basis. As a result, lands identified as potentially available for disposal under valid resource management plans are rarely disposed of by the Bureau of Land Management. (5) The Forest Service has several authorities to dispose of Federal lands, but such authorities are rarely used. (b) Purposes.--The purposes of this Act are-- (1) to provide for the orderly disposal of certain Federal lands; (2) to benefit education through the sales of such lands and research focused on natural resource issues at educational institutions; (3) to consolidate Federal lands to achieve better management; and (4) to provide for the acquisition of certain lands to provide or increase recreational and other purposes. SEC. 3. DEFINITIONS. As used in this Act: (1) Hunting.--The term ``hunting'' means use of a firearm, bow, or other authorized means in the lawful-- (A) pursuit, shooting, capture, collection, trapping, or killing of wildlife; or (B) attempt to pursue, shoot, capture, collect, trap, or kill wildlife. (2) Land grant university.--The term ``land grant university'' means a land grant university-- (A) established under the Act of July 2, 1862 (known as the ``First Morrill Act''; 12 Stat. 503, chapter 130; 7 U.S.C. 301 et seq.); (B) established under the Act of August 30, 1890 (known as the ``Second Morrill Act''; 26 Stat. 419, chapter 841; 7 U.S.C. 321 et seq.); or (C) described in section 533(a)(1) of the Equity in Educational Land-Grant Status Act of 1994 (part C of title V of Public Law 103-382). (3) Recreational fishing.--The term ``recreational fishing'' means the lawful-- (A) pursuit, capture, collection, or killing of fish; or (B) attempt to pursue, capture, collect, or kill fish. (4) Recreational off-highway vehicles.--The term ``recreational off-highway vehicle'' means a motorized off- highway vehicle designed to travel on four or more tires, intended by the manufacturer for recreational use by one or more persons and having all of the following characteristics: (A) A steering wheel for steering control. (B) Foot controls for throttle and service brake. (C) Non-straddle seating. (D) Maximum speed capability greater than 30 miles per hour. (E) Gross vehicle weight rating no greater than 3,750 pounds. (F) Less than 80 inches in overall width, exclusive of accessories. (G) Engine displacement equal to or less than 61 cubic inches for gasoline fueled engines. (H) Identification by means of a 17-character personal or vehicle information number. (5) Recreation and public purposes act.--The term ``Recreation and Public Purposes Act'' means the Act entitled ``An Act to authorize acquisition or use of public lands by States, counties, or municipalities for recreational purposes'', approved June 14, 1926 (43 U.S.C. 869 et seq.). (6) Recreational shooting.--The term ``recreational shooting'' means any form of sport, training, competition, or pastime, whether formal or informal, that involves the discharge of a rifle, handgun, or shotgun, or the use of a bow. (7) Secretary concerned.--The term ``Secretary concerned'' means-- (A) the Secretary of the Interior, in reference to lands under the jurisdiction of that Secretary; and (B) the Secretary of Agriculture, in reference to lands under the jurisdiction of that Secretary. (8) Special account.--The term ``special account'' means the account in the Treasury of the United States established under this Act. (9) Unit of local government.--The term ``unit of local government'' means the governing body of each, community, county, municipality, city, town, or township created pursuant to State law with boundaries interspersed with or adjacent to Federal lands. SEC. 4. DISPOSAL. (a) Disposal.--In accordance with this Act, and other applicable law, and subject to valid existing rights, the Secretary concerned is authorized to dispose of Federal land. (b) Reservation for Local Public Purposes.--Not less than 30 days before the offering of lands for sale or exchange pursuant to subsection (a), States or the unit of local government in whose jurisdiction the lands are located may elect to obtain any such lands for local public purposes pursuant to the provisions of the Recreation and Public Purposes Act. Pursuant to any such election, the Secretary concerned shall retain the elected lands for conveyance to the States or such unit of the local government in accordance with the provisions of the Recreation and Public Purposes Act. (c) Selection.-- (1) Joint selection required.--The Secretary concerned and the unit of local government in whose jurisdiction lands referred to in subsection (a) are located shall jointly select lands to be offered for sale or exchange under this section. The Secretary concerned shall coordinate land disposal activities with the unit of local government concerned. Land disposal activities of the Secretary concerned shall be consistent with local land use planning and zoning requirements and recommendations. (2) Offering.--(A) The Secretary concerned shall make the first offering of land as soon as practicable after land has been selected in accordance with this subsection. (B) The Secretary of the Interior shall dispose of not less than 10 percent of lands currently identified by the Bureau of Land Management for disposal as of the date of the enactment of this Act in each of the first 8 years after the date of the enactment of this Act, for a total of 80 percent of such lands by the end of the eighth year after the date of the enactment of this Act. (C) The Secretary of the Interior shall dispose of not less than 20 percent of lands identified by the Bureau of Land Management for disposal in any resource management plan amendment made after the date of the enactment of this Act in each of the 4 years after such an amendment is made, for a total of 80 percent of such lands by the end of the fourth year after the date of such amendment. (D) The Secretary of Agriculture shall dispose of not less than 10 percent of lands currently identified by the Forest Service for disposal as of the date of the enactment of this Act in each of the first 8 years after the date of the enactment of this Act, for a total of 80 percent of such lands by the end of the eighth year after the date of the enactment of this Act. (E) The Secretary of Agriculture shall dispose of not less than 20 percent of lands identified by the Forest Service for disposal in any resource management plan amendment made after the date of the enactment of this Act in each of the 4 years after such an amendment is made, for a total of 80 percent of such lands by the end of the fourth year after the date of such amendment. (F) Private landowners with inholdings interspersed with or adjacent to Federal land being disposed of shall have the first right of refusal for the purchase of land sold or exchanged under this Act. (d) Disposition of Proceeds.-- (1) Land sales.--Of the gross proceeds of sales of land under this subsection in a fiscal year shall be made available as follows: (A) Fifteen percent shall be paid directly to the State where the sale takes place for use to supplement the education of students in kindergarten through grade 12, to supplement public support of institutions of public higher education, and to supplement State agricultural and natural resource agencies. (B) Fifteen percent shall be paid directly to the one or more land grant universities within the boundaries of the State of which the revenue is derived for the purposes of providing agricultural and natural resources research, extension, teaching and infrastructure. (C) Ten percent shall be paid directly to the one or more counties within the boundaries of which the revenue is derived with 50 percent of those revenues going to a county extension office. (D) Ten percent shall be deposited in a special account created in the Treasury of the United States for use pursuant to the provisions of paragraph (3). (E) The remainder shall be deposited into the General Fund of the Treasury. (2) Payments.-- (A) In general.--Amounts paid to land grant universities under subsection (B) shall be in addition to any other payments of public support. (B) Payments in lieu of taxes.--A payment to a county under subsection (C) shall be in addition to a payment in lieu of taxes received by the county under chapter 69 of title 31, United States Code. (3) Availability of special account.-- (A) In general.--Amounts deposited in the special account may be expended by the Secretary concerned for-- (i) any of the purposes described in section 5; and (ii) deferred maintenance, repairs, and capital improvements. (B) Procedures.--The Secretary concerned shall coordinate the use of the special account with States, the unit of local government in whose jurisdiction the lands are located, and other interested persons, to ensure accountability and demonstrated results. (C) Investment of special account.--All funds deposited as principal in the special account shall earn interest in the amount determined by the Secretary of the Treasury on the basis of the current average market yield on outstanding marketable obligations of the United States of comparable maturities. Such interest shall be added to the principal of the account and expended according to the provisions of paragraph (3). SEC. 5. LANDS TO PROVIDE OR INCREASE RECREATIONAL AND OTHER OPPORTUNITIES. (a) Acquisitions.-- (1) Definition.--For purposes of this subsection, the term ``recreational beneficial land'' means land or an interest in land, the acquisition of which the United States would, in the judgment of the Secretary concerned provide an opportunity-- (A) for hunting, recreational fishing, recreational shooting, recreational off-highway vehicles, or other recreational purposes; or (B) to achieve better management of public land through consolidation of Federal ownership. (2) Concurrence.--Before initiating efforts to acquire land under this subsection, the Secretary concerned shall obtain the concurrence of each affected State and unit of local government within whose jurisdiction the lands are located, including appropriate planning and regulatory agencies, and with other interested persons, concerning the necessity of making the acquisition, the potential impacts on State and local government, and other appropriate aspects of the acquisition. Concurrence under this paragraph is in addition to any other consultation required by law. (3) In general.--After the consultation process has been completed in accordance with paragraph (3), the Secretary concerned may acquire, with the proceeds of the special account, recreational beneficial land and interests in recreational beneficial land. Lands may not be acquired by eminent domain or condemnation or without the consent of the owner thereof. Funds made available from the special account may be used with any other funds made available under any other provision of law or any other non-Federal matching funds provided by a nongovernmental organization. (b) Determination of Fair Market Value.--The fair market value of land or an interest in land to be acquired by the Secretary concerned under this section shall be determined pursuant to section 206 of the Federal Land Policy and Management Act of 1976 and shall be consistent with other applicable requirements and standards. Fair market value shall be determined without regard to the presence of a species listed as threatened or endangered under the Endangered Species Act of 1973 (16 U.S.C. 1531 et seq.). (c) Payments in Lieu of Taxes.--Subparagraph (H) of section 6901(1) of title 31, United States Code, is amended by inserting ``or the Hunting, Education, and Recreational Development Act'' after ``the Southern Nevada Public Land Management Act of 1998''. (d) Limitation.--The total land acreage acquired annually under this Act shall not exceed the total Federal land acreage disposed of annually under this Act. SEC. 6. PUBLIC AVAILABILITY OF INFORMATION ON LAND POTENTIALLY AVAILABLE FOR DISPOSAL. (a) Bureau of Land Management.--The Bureau of Land Management, shall make publicly available, including on the Internet at http:// www.blm.gov/wo/st/ en/prog/planning/planning_overview/lands_potentially0.html, or any successor website, all public lands managed by the agency potentially available for disposal as identified in agency resource management plans. (b) Forest Service.--The Forest Service, shall make publicly available, including on the Internet, all public lands managed by the agency identified for disposal as identified in agency land and resource management plans. SEC. 7. RECREATION AND PUBLIC PURPOSES ACT. (a) In General.--Upon request by a grantee of lands within a local county that are subject to a lease or patent issued under the Recreation and Public Purposes Act, the Secretary concerned may transfer the reversionary interest in such lands to other non-Federal lands. The transfer of the reversionary interest under this section shall only be made to lands of equal value, except that with respect to States or a unit of local government an amount equal to the excess (if any) of the fair market value of lands received by the unit of local government over the fair market value of lands transferred by the unit of local government shall be paid to the Secretary concerned and shall be treated under subsection (d)(1) of section 4 as proceeds from the sale of land. For purposes of this subsection, the fair market value of lands to be transferred by States or a unit of local government may be based upon a statement of value prepared by a qualified appraiser. (b) Terms and Conditions Applicable to Reversionary Interest.-- Other non-Federal lands selected under this subsection by a grantee described in subsection (a) shall be subject to the activities defined as permissible under parts 2920 and 2930 of title 43, Code of Federal Regulations, shall be permissible. SEC. 8. LIMITATIONS FOR ADMINISTRATIVE COSTS. Amounts deposited in the special account created by this Act shall be expended by the Secretary concerned for reimbursement of-- (1) costs incurred by the local offices of the Bureau of Land Management and the Forest Service in arranging sales, conveyances, or exchanges under this Act; and (2) reimbursement of any other costs associated with this Act including investigations, reports, appraisals, surveys, and clearances. SEC. 9. RECORDING. The Secretary concerned shall record all final sales, conveyances and exchanges under this Act with the county within whose jurisdiction the lands are located. | Hunting, Education, and Recreational Development Act or the HEARD Act This bill authorizes the Department of the Interior and the Department of Agriculture (USDA) to dispose of federal lands under their respective jurisdictions by offering them for sale or exchange to units of local government in accordance with this bill. Before the offering of lands for sale or exchange, states or the unit of local government in whose jurisdiction the lands are located may elect to obtain any such lands for local public purposes pursuant to the Recreation and Public Purposes Act. Interior or USDA, as appropriate, shall retain the elected lands for conveyance to such states or unit of local government in accordance with that Act. The bill prescribes requirements for disposition of the gross proceeds of the sales of lands under this bill, including that: 15% be paid to the state where the sale takes place to be used to supplement the education of students in kindergarten through grade 12, to supplement public support of institutions of public higher education, and to supplement state agricultural and natural resource agencies; and 10% of such proceeds be deposited in a special account to be created in the Treasury which may be used for the acquisition of recreational beneficial lands and interests (providing an opportunity for hunting, recreational fishing, recreational shooting, recreational off-highway vehicles, or other recreational purposes, or to achieve better management of public lands through consolidation of federal ownership). | billsum |
(CNN) The shooting of Rep. Steve Scalise and four others at a baseball practice for Republican members of Congress on Wednesday morning in Alexandria, Virginia, was always going to quickly turn to politics. Rep. Chris Collins, one of President Donald Trump's most prominent congressional supporters, insisted that the shooting was directly tied to anti-Trump rhetoric from the left. "I can only hope that the Democrats do tone down the rhetoric," Collins said on a local radio station in upstate New York. "The rhetoric has been outrageous -- the finger-pointing, just the tone and the angst and the anger directed at Donald Trump, his supporters. Really, then, you know, some people react to things like that. They get angry as well. And then you fuel the fires." Collins also said in the same interview he would have his gun "in my pocket from this day forward." That pivot from police incident to politics happened rapidly around 11:15 a.m., when CNN confirmed that the alleged shooter was James T. Hodgkinson of Illinois. A quick scan of his social media presence -- Facebook and Twitter -- suggested that he was strongly opposed to Trump and was a supporter of the 2016 presidential candidacy of Vermont Sen. Bernie Sanders, an independent who ran as a Democrat. Hodgkinson also apparently volunteered for Sanders campaign in Iowa during the 2016 campaign. Sanders condemned the shooting in a statement issued Wednesday afternoon. "I have just been informed that the alleged shooter at the Republican baseball practice is someone who apparently volunteered on my presidential campaign," Sanders said in a statement. "I am sickened by this despicable act. Let me be as clear as I can be. Violence of any kind is unacceptable in our society and I condemn this action in the strongest possible terms." JUST WATCHED Bernie Sanders on shooting: I am sickened Replay More Videos... MUST WATCH Bernie Sanders on shooting: I am sickened 01:12 The Belleville News-Democrat, the local paper in the community where Hodgkinson reportedly lived, showed a photo of him holding a "Tax the Rich" sign in a protest outside a local post office. The newspaper described Hodgkinson this way "The shooter was James T. Hodgkinson of Belleville, who belonged to a number of anti-Republican groups, including one called 'Terminate the Republican Party.'" Police officials would not comment on any motive for the shooting or whether Hodgkinson was targeting Republicans. But CNN's Dana Bash reported that the shooting was deliberate and not a random act. Trump made no mention of politics in a brief statement just before noon eastern time. "We are strongest when we are unified and we work together for the common good," Trump said. JUST WATCHED President Trump reacts to attack (full remarks) Replay More Videos... MUST WATCH President Trump reacts to attack (full remarks) 04:15 House Speaker Paul Ryan and Minority Leader Nancy Pelosi both gave speeches of unity to applause on the floor of the House of Representatives. "An attack on one of us is an attack on all of us," Ryan said. None of that stopped some conservatives from concluding that Hodgkinson was aiming to injure Republicans specifically, and that he was driven by a liberal culture that glorifies violence against GOPers. Hill's reference is to a controversial production of Shakespeare's "Julius Caesar" by the Delacorte Theater in New York's Central Park. In it, the Caesar character bears a striking resemblance to Trump. Obviously, if you know history, Caesar is assassinated by his peers, including his best friend Brutus. Hill was far from the only conservative to cast the shooting in a very political light. "NBC mentions shooter's social media page, BUT WON'T TELL US WHAT IT SAYS. (Bernie Sanders & Democratic Socialism)," tweeted Ann Coulter "A @BernieSanders supporter did shooting spree: James T. Hodgkinson who pushed a http://Change.org petition to appt indep counsel." tweeted Laura Ingraham. "This could be the first political rhetorical terrorist attack," Illinois Republican Rep. Rodney Davis, who was at the practice, told CNN's Brianna Keilar Wednesday morning before Hodgkinson had been publicly identified as the alleged shooter. JUST WATCHED Lawmaker on shooting: Political hate to blame Replay More Videos... MUST WATCH Lawmaker on shooting: Political hate to blame 01:07 Former House Speaker Newt Gingrich, appearing on Fox News, called the shootings "part of a pattern" and blamed "an increasing intensity of hostility on the left." He said conservative college students are afraid they'll be beaten on campus. "The intensity is very real, whether it's a so-called comedian holding up the President's head covered in blood, or right here in New York City, a play that shows the President being assassinated, or it's Democratic leading national politicians using vulgarity because they can't find any common language to talk," he said. And Michael Caputo, a former Trump adviser, was even more blunt in an interview with a Buffalo radio station. "For nine months, Democratic Party leaders have lied, regularly calling me and my friends traitors, so forgive me if I'm not more tender with their karma in Alexandria," Caputo said Others were less direct in tying Hodgkinson's apparent politics to this incident but did suggest that that the partisan political atmosphere clearly had something to do with the shooting. JUST WATCHED Virginia governor: Too many guns on the street Replay More Videos... MUST WATCH Virginia governor: Too many guns on the street 00:47 Virginia Gov. Terry McAuliffe, a Democrat, echoed Davis' sentiment. "I do think that things have become very partisan and very hardened in the country today," he said. "We have to work together to get things done and we're the greatest nation in the world and there has been too much raw discourse today that is pulling people apart." But McAuliffe also mentioned that "there are too many guns on the streets," a common theme for gun control advocates in the wake of attacks like these involving guns. Rep. Mo Brooks, the Alabama Republican and another one of the people at the baseball field when the shooting happened, dismissed the idea that he might rethink his staunch support of the 2nd Amendment in the wake of this shooting. "The Second Amendment right to bear arms is to ensure that we always have a Republic," Brooks said. "What we just saw here is one of the bad side effects of someone not exercising those rights properly." Georgia Rep. Barry Loudermilk, a Republican from Georgia who was on the field when the shooting happened, suggested that members of Congress should be allowed to carry guns. "I think we need to look at some reciprocity for members here but we also need to look at security details," Loudermilk told CNN's Ashley Killough. Until we know more about Hodgkinson's motive -- assuming we can find it out since he has now died from wounds he suffered -- it's difficult to reach hard and fast conclusions about why, allegedly, he did what he did. But a man with a gun shooting at members of Congress will always be political. That Hodgkinson was an outspoken critic of Trump makes it even harder to keep away from politics. The question now is where the political debate goes from here. Does the focus land on better protecting members of Congress in public? Tamping down the viciousness of political rhetoric? Gun control? Something else? Congress Baseball Shooter Is a Trump Hater... Called to 'Destroy' Him Congress Baseball Shooter Is a Trump Hater, Called to 'Destroy' Trump EXCLUSIVE The man suspected of opening fire at a congressional baseball practice is 66-year-old James T. Hodgkinson... and judging by his social media, he REALLY hates Donald Trump. Hodgkinson's Facebook page is loaded with anti-Trump posts. He's also a staunch Bernie Sanders supporter. Also notable, Hodgkinson is also anti-Hillary Clinton, with several posts about her as well. Among the posts on his page, there's one that stands out from March which says, "Trump is Traitor. Trump Has Destroyed Our Democracy. It's Time to Destroy Trump & Co." 9:30 AM PT -- Sanders just released a statement on Hodgkinson, saying, "I have just been informed that the alleged shooter at the Republican baseball practice is someone who apparently volunteered on my presidential campaign. I am sickened by this despicable act. Let me be as clear as I can be. Violence of any kind is unacceptable in our society and I condemn this action in the strongest possible terms." When a gunman ambushed a practice for a charity congressional baseball game in Alexandria, Va., on Wednesday, at least five people were injured. And, witnesses have suggested, that number might have been higher had members of the U.S. Capitol Police not been there — as part of House Majority Whip Steve Scalise’s security detail — to fire back. “Without the Capitol Hill police, it would have been a massacre,” Kentucky Republican Senator Rand Paul told the press. As some speculate about whether partisan politics motivated the gunman — who reportedly asked one of the congressmen if Democrats or Republicans were playing — it can be noted that the U.S. Capitol Police force, which is charged with protecting Congress, was established after one particular assault that took place in the polarized political climate that was 1828. At the time, “Jacksonian” politicians (supporters of Andrew Jackson, who had nearly become president in 1824) had secured a majority in Congress after the midterm elections. Sitting President John Quincy Adams could have expected he was going to have to fight Jackson for the White House in the upcoming 1828 presidential election — but he probably didn’t expect his son to get into an actual physical fight with a Jacksonian before Election Day. Get your history fix in one place: sign up for the weekly TIME History newsletter Russell Jarvis, a journalist for a pro-Jackson newspaper The Washington Telegraph, attended a New Year’s Eve celebration at the White House with his wife and Boston relatives, when John Adams Jr., the president’s son and personal secretary, made what Jarvis later described as a “grievous insult to the ladies of my family.” Here’s how a book that chronicled the political and social scene in mid-19th century Washington, D.C., Perley’s Reminiscences of 60 Years in the National Metropolis, described the contretemps: Mr. Jarvis introduced them courteously, and they then passed on into the East Room. Soon afterward they found themselves standing opposite to Mr. John Adams, who was conversing with the Rev. Mr. Stetson. “Who is that lady?” asked Mr. Stetson. “That,” replied Mr. John Adams, in a tone so loud that the party heard it, “is the wife of one Russell Jarvis, and if he knew how contemptibly he is viewed in this house they would not be here.” The Bostonians at once paid their respects to Mrs. Adams and withdrew, Mr. Jarvis having first ascertained from Mr. Stetson that it was Mr. John Adams who had insulted them. A few days afterward Mr. Jarvis sent a note to Mr. John Adams, demanding an explanation, by a friend of his, Mr. McLean. Mr. Adams told Mr. McLean that he had no apology to make to Mr. Jarvis, and that he wished no correspondence with him. Jarvis is said to have been waiting for the right moment to seek revenge when he saw Adams in the Capitol Rotunda in April 1828. He said he asked the junior Adams if he would apologize, and when he didn’t, “I was excited by his continued refusal, and by a recollection of the offense, to commit an assault upon his person, which consisted merely in pulling his nose and slapping one side of his face, with my open hand,” Jarvis recalled, according to a personal account of the incident published in the newspaper Niles’ Register. Jarvis’s actions have been described as “all standard and approved provocations for a duel,” according to the biography of presidential families America’s Royalty: All the Presidents’ Children, “however, John Quincy Adams’s disapproval for dueling was made evident when he responded for his son by sending a message to Congress…requesting that Congress provide funds to secure the way between the president’s office and Congress so that future incidents could be prevented.” Congress passed the act creating the U.S. Capitol Police on May 2, 1828. Scalise is popular among his constituents. In November, he was reelected with 75 percent of the vote. His seat has long been occupied by Republicans: It’s one of the reddest districts in the country, and it spans much of boot-shaped Louisiana’s “toe,” which includes parts of the New Orleans suburbs. According to The Times-Picayune, Trump’s controversial first few months in office, as well as the drama over the Obamacare repeal bill, don’t “appear to be dampening support” for the congressman, who raised a record $1.6 million in early 2017. Scalise’s colleague, Alabama Representative Mo Brooks, told CNN and other reporters that Scalise was one of five people shot at the early-morning practice, where lawmakers were preparing to compete in the annual baseball game Thursday. The competition between Democratic and Republican lawmakers, which raises money for D.C.-area charities, is typically held at Nationals Park in Washington’s southeast. Republican lawmakers were fine-tuning their fielding miles away Wednesday morning at Alexandria, Virginia’s Eugene Simpson Stadium Park, where local Little League teams play. Scalise has been on the team for years. In 2015, The New York Times reported that “at shortstop, [he] is easily the G.O.P.’s best offensive player.” Scalise was on the field, playing second base, when the gunman opened fire. “I hear another bam and I realize there is an active shooter,” Brooks said. “At the same time, I hear Steve Scalise, over near second base, scream. He was shot.” Colleagues credited his attendance at the practice for saving more people from harm because, as a member of House leadership, he has a security detail. CBS News’s Nancy Cordes reported that those officers ran toward Scalise while the gunman was firing and managed to “bring that shooter down with their pistols.” Two were hit themselves, and the shooter was also taken to the hospital. He later died. “If they had not been here it probably would have been far worse,” Ohio Representative Brad Wenstrup told CBS News. (CNN) Louisiana's Steve Scalise is the 9th member of Congress to be shot while in office and one of two dozen to be targeted by attackers since 1789, according to a 2011 Congressional Research Service report. Scalise, the third-ranking Republican in the House, was shot in the hip by a man wielding a rifle at an early-morning practice for the GOP baseball team in advance of the scheduled Congressional Baseball Game tomorrow. He was one of five people injured -- including two members of the US Capitol Police force -- in the shooting. The Louisiana congressman was, according to a statement released by his office, undergoing surgery Wednesday morning but in stable condition. He is the first member of Congress to be shot since January 2011 when then Arizona Rep. Gabrielle Giffords, a Democrat, was shot in the head at a congressional event in Tucson. "My heart is with my former colleagues, their families & staff, and the US Capitol Police - public servants and heroes today and every day," Giffords tweeted in the immediate aftermath of the shooting. My heart is with my former colleagues, their families & staff, and the US Capitol Police- public servants and heroes today and every day. Six people were killed in the Arizona shooting that targeted Giffords. She returned to Congress in August 2011, but resigned from the chamber in January 2012. Other attacks were not included in the report, such as the 1998 shooting inside the Capitol when a gunman stormed into the building and shot and killed two Capitol Hill police officers. That gunman had a history of mental illness. Tourists leave the Capitol on a stretcher after the violence and chaos caused by the shootings that claimed the lives of US Capitol Police officers John Gibson and Jacob J. Chestnut in 1998. Prior to the attack on Giffords, it had been three decades since a member of Congress had been attacked. In November 1979, a woman with a knife got into Ted Kennedy's Senate office. She was stopped by the US Secret Service prior to reaching Kennedy; a Secret Service officer was "slightly wounded" according to CRS. Almost a year to the day prior, California Rep. Leo Ryan was shot and killed at an airstrip in Guyana after he and two dozen others traveled to investigate the Jonestown cult headed by Jim Jones, which was based there. Ryan, along with four others -- including three journalists -- were murdered. Jackie Speier, who now holds Ryan's seat, was on that trip as a staffer to the Congressman. She was shot five times but lived. (Jones as well as 908 of his followers committed mass suicide by poisoning on the same day Ryan was killed.) Jackie Speier, an aide to Congressman Leo Ryan, being taken from a plane at Georgetown on November 19, 1978, after its arrival from Jonestown where Speier was shot five times and Ryan and four others were ambushed and killed by members of the People's Temple. JUST WATCHED Jonestown survivor: 'I was shot five times' Replay More Videos... MUST WATCH Jonestown survivor: 'I was shot five times' 02:31 A decade earlier, New York Sen. Robert Kennedy, then a leading candidate for the Democratic presidential nomination, was assassinated in Los Angeles following his victory in the June California primary. Photos: Behind the Picture: RFK's Assassination, 1968 Behind the Picture: RFK's Assassination, 1968 – A wounded Paul Schrade, a regional director of the United Auto Workers Union, labor chair of Robert Kennedy's campaign and one of five other people shot by Sirhan Sirhan, on the floor of the kitchen at the Ambassador Hotel, June 5, 1968. Hide Caption 1 of 6 Photos: Behind the Picture: RFK's Assassination, 1968 Behind the Picture: RFK's Assassination, 1968 – Senator Robert Kennedy gives a speech at the Ambassador Hotel in Los Angeles before his assassination, June 1968. Hide Caption 2 of 6 Photos: Behind the Picture: RFK's Assassination, 1968 Behind the Picture: RFK's Assassination, 1968 – Sen. Kennedy gives a speech at the Ambassador Hotel in Los Angeles before his assassination, June 1968. Hide Caption 3 of 6 Photos: Behind the Picture: RFK's Assassination, 1968 Behind the Picture: RFK's Assassination, 1968 – "Heading for his victory speech in the Ambassador Hotel ballroom, Robert Kennedy stops in the kitchen to shake hands. A few minutes later the gunman was waiting for him in the corridor just outside the kitchen." Hide Caption 4 of 6 Photos: Behind the Picture: RFK's Assassination, 1968 Behind the Picture: RFK's Assassination, 1968 – A less-famous image of Sen. Robert Kennedy and Ambassador Hotel employee Juan Romero moments after RFK was shot by Sirhan Sirhan, June 1968. Hide Caption 5 of 6 Photos: Behind the Picture: RFK's Assassination, 1968 Behind the Picture: RFK's Assassination, 1968 – A young Robert Kennedy supporter registers disbelief after his shooting by Sirhan Sirhan, June 1968. Hide Caption 6 of 6 Rep. Kenneth Roberts shown here being carried down the Capitol steps after Puerto Rican nationalists opened fire in the Capitol Building, shouting "Free Puerto Rico." The incident involving the most members of Congress happened on March 1, 1954 when a group of Puerto Rican nationalists entered the House press gallery and opened fired. Reps. Clifford Davis (Tennessee), Alvin Bentley (Michigan), Ben Jensen (Iowa), George Hyde Fallon (Maryland) and Kenneth Roberts (Alabama) were injured -- although all five recovered. At issue was the ongoing US control of Puerto Rico Charles Orear, 50, a restaurant manager from St. Louis, said in an interview Wednesday that he became friendly with James T. Hodgkinson, whom law enforcement officials identified as the shooter, during their work in Iowa on Sen. Bernie Sanders’s presidential campaign. Orear said Hodgkinson was a passionate progressive and showed no signs of violence or malice toward others. “You’ve got to be kidding me,” Orear said when told by phone. “I met him on the Bernie trail in Iowa, worked with him in the Quad Cities area.” Orear described Hodgkinson as a “quiet guy” who was “very mellow, very reserved” when they stayed overnight at a Sanders’s supporter home in Rock Island, Ill., after canvassing for the senator. “He was this union tradesman, pretty stocky, and we stayed up talking politics,” he said. “He was more on the really progressive side of things.” The Post reached out to Orear after seeing that he liked one of Hodgkinson’s Facebook posts. Former Speaker Newt Gingrich (R-Ga.) on Wednesday linked a growing trend of "hostility" on the left to violent incidents like the shooting that morning at congressional Republicans' baseball practice. "It's part of a pattern," Gingrich said on Fox News. "An increasing intensity of hostility on the left." Gingrich pointed to the violence and intimidation toward conservatives on college campuses as another sign of the left's hostility toward Republicans and President Trump. ADVERTISEMENT "Look, I talk to college students regularly," Gingrich continued, "who say that if they are openly for Trump, they get threatened." "I've had college students tell me they get threatened with being beaten up, some of them get death threats," he said. Gingrich then targeted comedian Kathy Griffin as an example of a member of the left advocating violence against Trump and other Republicans. Griffin was fired from CNN's New Year's Eve program after she posted a photo of her holding up a dummy head of Trump covered in ketchup. "The intensity is very real, whether it's a so-called comedian holding up the president's head covered in blood, or right here in New York City, a play that shows the president being assassinated, or it's Democratic leading national politicians using vulgarity because they can't find any common language to talk," Gingrich argued. He likely was referring to Central Park’s summer Shakespeare production of “Julius Caesar” that appears to portray the slain Roman dictator as Trump and to Sen. Kirsten Gillibrand Kirsten Elizabeth GillibrandChris Murphy’s profile rises with gun tragedies Overnight Energy: Dems take on Trump's chemical safety pick Dems lambaste Trump’s ‘outrageous’ EPA chemical safety pick MORE (D-N.Y.), who recently slammed the president for not keeping his promises using the expression "f--- no." Close Get email notifications on Kevin McDermott daily! Your notification has been saved. There was a problem saving your notification. Whenever Kevin McDermott posts new content, you'll get an email delivered to your inbox with a link. Email notifications are only sent once a day, and only if there are new matching items. Virginia Gov. Terry McAuliffe raised the issue of gun control during a news conference Wednesday, after five people were injured in an early-morning shooting at a baseball field in Alexandria where Republican lawmakers had been practicing for an annual charity game. “Let me say this: I think we need to do more to protect all of our citizens. I have long advocated — this is not what today is about, but there are too many guns on the street,” the Democratic governor said, when asked if anything more needs to be done to protect politicians. “Background checks, shutting down gun show loopholes — that’s not for today’s discussion, but it’s not just about politicians. We worry about this every day for all of our citizens.” The incident — which sent five people, including House Majority Whip Steve Scalise, to the hospital — began to spur questions about gun rights and gun control policies, while members of both parties condemned the violence and called for unity. A House subcommittee had been scheduled to discuss a controversial proposal to ease regulations of gun silencers on Wednesday, but the hearing was cancelled in the wake of the shooting. Asked if the incident had changed his stance on gun control, Alabama Republican Rep. Mo Brooks, who was at the baseball practice but was uninjured, told reporters he continues to support Second Amendment rights. “As with any constitutional provision in the Bill of Rights, there are adverse aspects to each of those rights that we enjoy as people. And what we just saw here is one of the bad side effects of someone not exercising those rights properly,” Brooks said. “I’m not changing my position on any of the rights we enjoy as Americans,” he added. “With respect to this particular shooter, I’d really like to know more about him — whether he was an ex-felon, by way of example, who should have had possession of a firearm — I’d like to know other things about his background before I pass judgment.” | James Hodgkinson, the 66-year-old Illinois man police say shot and wounded multiple people during a GOP congressional baseball practice Wednesday, was a former volunteer for the Bernie Sanders campaign, Politico reports. Sanders condemned the shooting "in the strongest possible terms," calling it a "despicable act." "Violence of any kind is unacceptable in our society," he said. "Real change can only come through nonviolent action, and anything else runs against our most deeply held American values." Here's what else you need to know about Wednesday's shooting in Virginia: In addition to frequently posting pro-Sanders messages to Facebook, Hodgkinson also posted anti-President Trump and anti-Hillary Clinton statements, TMZ reports. In one post, he called Trump a "traitor" who "has destroyed our democracy." Charles Orear got to know Hodgkinson while working on the Sanders campaign in Iowa, telling the Washington Post he was "very mellow, very reserved" and "really progressive." Newt Gingrich tied the shooting to "an increasing intensity of hostility on the left," using Kathy Griffin as an example, the Hill reports. It was a sentiment echoed by others, including Ann Coulter and Republican Rep. Chris Collins, who blamed the "outrageous" rhetoric of Democrats, according to CNN. The Atlantic has a quick biography on Rep. Steve Scalise, the majority whip from Louisiana who was injured in the shooting. He was instrumental to getting the House health care bill passed and is said to be "the GOP's best offensive player" on the baseball field. CNN reports Scalise is only the ninth member of Congress since 1789 to be shot while in office. Sen. Rand Paul says if it weren't for US Capitol Police officers, who were at the practice as security for Scalise, the shooting would've been a "massacre." Time has a history of the Capitol Police, which was formed following a fight between the son of President John Quincy Adams and a supporter of Andrew Jackson. In March, neighbors complained to police that Hodgkinson was shooting his gun too close to their homes, but officers found he wasn't doing anything illegal, the St. Louis Post-Dispatch reports. Hodgkinson has past arrests for battery, fleeing from police, and resisting arrest. Finally, Virginia Gov. Terry McAuliffe says "there are too many guns on the street" and work needs to be done to increase background checks and close the gun show loophole, though that's a conversation for another day, according to Time. | multi_news |
ROCHESTER, N.H. (AP) — Journalist James Foley had worked in a number of conflict zones in the Middle East, but the danger didn't stop him from doing the job he loved. A ribbon is tied to a tree outside the home of American freelance journalist James Foley, on Tuesday Aug. 19, 2014, in Rochester, N.H. A video by Islamic State militants that purports to show the killing... (Associated Press) FILE - In this Friday, May 27, 2011, file photo, journalist James Foley poses for a photo during an interview with The Associated Press, in Boston. A video by Islamic State militants that purports to... (Associated Press) FILE - In this Friday, May 27, 2011, file photo, journalist James Foley responds to questions during an interview with The Associated Press, in Boston. A video by Islamic State militants that purports... (Associated Press) Rev. Paul Gousse from Our Lady of the Holy Rosary leaves after meeting with the family of American freelance journalist James Foley in Rochester, N.H., Tuesday Aug. 19, 2014. The White House said Tuesday... (Associated Press) EDS NOTE: GRAPHIC CONTENT - This undated image shows a frame from a video released by Islamic State militants Tuesday, Aug. 19, 2014, that purports to show the killing of journalist James Foley by the... (Associated Press) Captured and held for six weeks while covering the uprising in Libya, he knew the risks when he went to Syria two years ago to cover the escalating violence there. Foley was snatched again in Syria in November 2012 when the car he was riding in was stopped by four militants in a battle zone that Sunni rebel fighters and government forces were trying to control. On Tuesday, two U.S. officials said they believe Foley was the person executed by Islamic State militants in a video posted online. The officials spoke on the condition of anonymity because they weren't authorized to discuss the video by name. Foley's family confirmed his death on a webpage created to rally support for him. His mother, Diane Foley, said in a statement on the webpage he "gave his life trying to expose the world to the suffering of the Syrian people." At Foley's family home in Rochester, a light burned yellow in a center upstairs window and a yellow ribbon adorned a tree at the foot of the driveway. The Rev. Paul Gousse, of Our Lady of the Holy Rosary, where the Foleys are parishioners, spent about 45 minutes at the house but left without commenting. Foley, 40, and another journalist were working in the northern province of Idlib in Syria when they were kidnapped near the village of Taftanaz. After Foley disappeared, while contributing video for Agence France-Presse and the media company GlobalPost, his parents became fierce advocates for him and all those kidnapped in war zones. They held regular prayer vigils and worked with the U.S. and Syrian diplomatic corps to get whatever scraps of information they could. Diane Foley, asked in January 2013 if her son had reservations about going to Syria, said softly: "Not enough." She wrote Tuesday on the family's webpage, "We thank Jim for all the joy he gave us. He was an extraordinary son, brother, journalist and person." Foley had seen the dangers to journalists up close. Upon his release from Libya and return to the United States, he recalled in an interview with The Associated Press seeing a colleague, South African photographer Anton Hammerl, killed by forces loyal to Libyan leader Moammar Gadhafi. He tried to pull his friend's body out of harm's way but was turned back by heavy fire. "I'll regret that day for the rest of my life. I'll regret what happened to Anton," Foley said. "I will constantly analyze that." Foley also covered the war in Afghanistan but called the Libyan fighting the worst he had ever experienced to that point. Foley grew up in New Hampshire and studied history at Marquette University. He later taught in Arizona, Massachusetts and Chicago before switching careers to become a journalist, which he viewed as a calling. "Journalism is journalism," Foley said. "If I had a choice to do Nashua (New Hampshire) zoning meetings or give up journalism, I'll do it. I love writing and reporting." ___ Associated Press writers Lara Jakes and Julie Pace contributed to this report from Washington. This is a developing story and will be updated as new information becomes available. BOSTON, Mass. — The extremist Islamic State claims to have executed journalist James Foley, who has been missing since November 2012 when he was kidnapped while reporting in Syria. Video of Foley depicting his beheading was uploaded to YouTube on Tuesday afternoon and later removed. The FBI on Wednesday morning told the Foley family they believed the video was authentic, but were continuing a longer process of official authentication. Shortly thereafter, National Security Council spokesperson Caitlin Hayden announced that “the US Intelligence Community" had "reached the judgment that this video is authentic." In remarks delivered shortly before 1 p.m. Wednesday, President Barack Obama said the world is "appalled by the beheading of journalist James Foley" and said the United States will do whatever is needed to pursue "justice." The video appeared following a threat made last week, GlobalPost CEO Phil Balboni told NBC on Wednesday. The video asserts that the killing of Foley is in retaliation for recent airstrikes by the United States against IS militants in northern Iraq. In it, Foley, kneeling next to an apparent IS militant, makes comments against the US for its actions. The militant also shows on camera journalist Steven Joel Sotloff, who went missing in Syria a year ago, and issues a direct challenge to President Obama that Sotloff's fate will depend on the president's "next move." As of 7 a.m. local time on Wednesday, Foley's family in New Hampshire had no confirmation from the US government of Jim's death. Late on Tuesday evening the family acknowledged there was a small chance the video may still prove to be fake, but nonetheless released the following statement: "We have never been prouder of our son and brother Jim. He gave his life trying to expose the world to the suffering of the Syrian people. We implore the kidnappers to spare the lives of the remaining hostages. Like Jim, they are innocents. They have no control over American government policy in Iraq, Syria or anywhere in the world. "We thank Jim for all the joy he gave us. He was an extraordinary son, brother, journalist and person. Please respect our privacy in the days ahead as we mourn and cherish Jim." Earlier Tuesday, Philip Balboni, GlobalPost CEO and co-founder, made the following statement: "On behalf of John and Diane Foley, and also GlobalPost, we deeply appreciate all of the messages of sympathy and support that have poured in since the news of Jim’s possible execution first broke. We have been informed that the FBI is in the process of evaluating the video posted by the Islamic State to determine if it is authentic.... We ask for your prayers for Jim and his family." In the video purporting to show Foley's beheading, the militant next to him speaks with a British accent. The press office for UK Prime Minister David Cameron issued the following statement early Wednesday: "If true, the brutal murder of James Foley is shocking and depraved. The Prime Minister is returning to Downing Street this morning. He will meet with the Foreign Secretary and senior officials from the Home Office, Foreign Office and the agencies to discuss the situation in Iraq and Syria and the threat posed by ISIL (Islamic State of Iraq and the Levant) terrorists." More from GlobalPost: Britain is searching for Foley killer GlobalPost, for whom Foley had reported in Syria, mounted an extensive international investigation from November 2012 to determine who kidnapped Foley and where he was being held. Significant research was undertaken throughout the Middle East, including along the Syria-Turkish border, in Lebanon, in Jordan and in other locations. “Although GlobalPost’s investigation at one point led us to believe that James was being held by the Syrian government, we later were given strong reason to believe he was being held by Islamic militants in Syria," Balboni said. "We withheld this information at the request of the family and on the advice of authorities cooperating in the effort to protect Jim. GlobalPost, working with a private security company, has amassed an enormous amount of information that has not been made public.” Foley was on a freelance assignment for GlobalPost when he was abducted in northern Syria on Nov. 22, 2012. He was on his way to the Turkish border when he was stopped by a group of armed men. Foley reported for GlobalPost from Libya and Afghanistan before traveling to Syria in the early days of the now long-running civil war that has taken the lives of more than 170,000. Foley’s last article for GlobalPost detailed the growing frustration with the war among civilians in Aleppo. The fact of Foley’s kidnapping was revealed publicly for the first time by his parents, John and Diane Foley of Rochester, New Hampshire, on Jan. 1, 2012, two months after his abduction. The only other American whose identity has been publicly revealed is Austin Tice, a freelance reporter for the McClatchy News Service and the Washington Post, who was kidnapped in August 2012. While covering the Libyan civil war in 2011, Foley and two other journalists, American Claire Gillis and Spaniard Manu Brabo, endured a 44-day captivity in April and May of that year at the hands of then Libyan strongman Col. Muammar Gaddafi. A fourth journalist, South African Anton Hammerl, was killed when the journalists were captured by Gaddafi fighters near Benghazi in eastern Libya. Foley later returned to Libya to cover Gaddafi’s fall and eventual death. Foley and GlobalPost correspondent Tracey Shelton were at the scene of Gaddafi’s capture in October 2011. United States government officials confirmed that a video released by ISIS Tuesday shows the cold-blooded execution of journalist James Foley, of New Hampshire, who has been missing for nearly two years. Watch the report Images: James Foley in Syria Foley, a freelance photojournalist, was taken by an organized gang after leaving an Internet café in Binesh, Syria, on Thanksgiving Day in 2012. Photos: Who is ISIS? Shortly after the video's release, GlobalPost CEO and co-founder Philip Balboni also released a statement and said, "On behalf of John and Diane Foley, and also GlobalPost, we deeply appreciate all of the messages of sympathy and support that have poured in since the news of Jim's possible execution first broke. We ask for your prayers for Jim and his family." Foley was working with GlobalPost when he went missing in 2012. The video begins with Obama explaining his decision to order airstrikes in Iraq before switching to a man dressed in an orange jumpsuit kneeling with a person dressed in black by his side. The man, who United States government officials confirmed Tuesday night to be Foley, then delivers a statement saying the United States government is his killer. "For what will happen to me is only a result of their complacency and criminality," ABC News reported the Foley said in the video. "I wish I had more time. I wish I could have the hope of freedom of seeing my family once again, but that ship has sailed. I guess all in all, I wish I wasn't American." Seconds later, ABC News reported, the person dressed in black takes out a knife and identifies himself as being with ISIS. "Today your military air force has attacked us daily," the person said. "Your strikes have caused causalities amongst Muslims. Any attempt to deny the Muslims their right to live in safety under Islamic caliphate will result in the bloodshed of your people." The video then shows Foley being beheaded. Another journalist, Steven Sotloff, is also seen dressed in an orange jumpsuit in the video. "The life of this American citizen, Obama, depends on your next decision," the person dressed in black said. Foley was kidnapped once before while he was reporting in Benghazi, Libya, and was held for 44 days before the fall of dictator Muammar Gaddafi. Outside Foley's Rochester, New Hampshire, home, a yellow ribbon was tied around a tree in the driveway, and a priest was seen entering the home Tuesday night. "We have never been prouder of our son Jim," Diane Foley, James Foley's mother said on the Facebook page Free James Foley. "He gave his life trying to expose the world to the suffering of the Syrian people. We implore the kidnappers to spare the lives of the remaining hostages. "Like Jim, they are innocents. They have no control over American government policy in Iraq, Syria or anywhere else in the world. "We thank Jim for all the joy he gave us. He was an extraordinary son, brother, journalist and person. Please respect our privacy in the days ahead while he mourn and cherish Jim." United States Sen. Jeanne Shaheen (D-NH) released a statement Tuesday night calling Foley an accomplished journalist who devoted his life to the freedom of press. "Everyone who knew him recognized his dedication to his work and his commitment to sharing his eye-witness reporting of world events," Shaheen said in the statement. "His murder was a cowardly act of terrorism and underscores the threat that ISIL poses to the freedoms we hold dear. My thoughts are with the Foley family and everyone who knew and loved James, both in New Hampshire and around the world" "If anyone needed further evidence of the utter inhumanity of Islamic terrorism, this is it," Scott Brown said in a statement. "ISIS is pure evil, and they must be stopped." Neighbors are also offering prayers to Foley's family after learning of his execution by ISIS. Profile: James Foley, US journalist beheaded by Islamic State James Foley was reporting in Syria when he was captured in 2012 American journalist James Foley, who has been murdered by the Islamic State militant group, has been described as a "brave and tireless journalist". A video released on Tuesday shows his beheading. He was abducted in Syria in November 2012, while reporting on the civil war for Agence France-Press (AFP) and US media company GlobalPost. On his way through northern Syria, in Idlib province, his vehicle was stopped by militants. He was never seen again. Continue reading the main story “ Start Quote I'm drawn to the front line naturally” End Quote James Foley It is estimated that about 20 journalists are missing in Syria, according to the US Committee to Protect Journalists. Mr Foley, 40, came from the US city of Rochester in New Hampshire. He was a teacher in Arizona, Massachusetts and Chicago before switching to journalism in the mid-2000s. After graduating from the Medill School of Journalism, he began his work in the field. His curiosity about the reality of places like Iraq, where his brother was serving as an officer in the US air force, led him to embed himself with US troops there. James Foley's capture in Libya had a profound impact on him, but did not deter him from going to Syria James Foley (in the white helmet) was reporting in Libya when he was first captured in 2011 Libya capture ordeal In 2011, he went to Libya to cover the uprising against Col Muammar Gaddafi, embedding himself with rebel fighters. "The idea with the Libyan revolution is, it's journalists embedding with rebels, and that's essentially what we did," he recalled. In April 2011, Mr Foley and three other journalists were ambushed by Gaddafi's forces. Continue reading the main story “ Start Quote A brave and tireless journalist with a passion for the Syrian cause” End Quote Clarissa Ward CBS News Photojournalist Anton Hammerl was killed, while Mr Foley and the others were detained. "A soldier's pressing your face into the bed of a truck, bleeding from the scalp - it's the worst kind of shock," Mr Foley said afterwards. For 18 days, nobody knew whether he was still alive. His parents, Diane and John Foley, campaigned for his release, holding prayer vigils and working with US and Syrian diplomatic teams to gather information. "I can only assume that he's charmed his guards with his ready laugh...and is now drinking tea and smoking cigarettes with them" - Mr Foley's friend, Clare Morgana Gillis, 2013 James Foley's mother says he was "an extraordinary son" After six weeks, Mr Foley was released. But the death of Anton Hammerl, his friend and colleague, had a profound impact on him. "I'll regret that day for the rest of my life. I'll regret what happened to Anton," he said. "I will constantly analyse that." A passion for journalism His experience of being captured did not deter him. "It doesn't always repel you. Sometimes... it draws you closer. Feeling like you've survived something - it's a strange sort of force that you are drawn back to," he said. Continue reading the main story “ Start Quote There's an amazing reach for humanity in these places.” End Quote James Foley Following his ordeal, he was eager to cover the situation in Syria, saying he was "drawn to the drama of the conflict and trying to expose untold stories." He began reporting the violence committed by forces loyal to Syrian President Bashar al-Assad, before he was captured in November 2012. Max Fisher of the Vox news website has said he will celebrate James Foley's "dedication to truth and understanding". Penny Sukraj, the widow of journalist Anton Hammerl, who was close friends with Mr Foley, said: "He was passionate about getting out there and telling the stories about the most vulnerable people and the effects of the different conflicts and wars that had ravaged their lives." "He lived and breathed the conflict journalism that he was involved with and not for any self-glorification purpose at all," she added. Freelance journalists are particularly at risk in troubled areas, without few guarantees of their safety. But James Foley had a passion for it nonetheless: "There's extreme violence, but there's a will to find who these people really are. And I think that's what's really inspiring about it." Tributes to James Foley "Jim gave his life trying to expose the world to the suffering of the Syrian people" - James Foley's mother, Diane Foley. "Everybody, everywhere, takes a liking to Jim as soon as they meet him. Men like him for his good humour and tendency to address everyone as "bro" or "homie" or "dude" after the first handshake. Women like him for his broad smile, broad shoulders, and because, well, women just like him." - Clare Morgana Gillis, American journalist who was detained with James Foley in Libya. Syrian activists in Kafranabel show their support for James Foley "James Foley was my middle school teacher in a very poor neighborhood and all I want to say is thank you Mr. Foley you helped shape me into the man I am today. He was also our basketball coach, lead us to the district championship. This man always preached being open and respectful to other cultures. It's unfortunate that his openness lead to this." - Reddit user wheelchaircharlie. "I'll remember James Foley in Antakya. He had some problems with his video camera…'Brother,' he said, 'I can only pay you in whiskey.' His gear was spread out across a small Turkish coffee table. All of it in terrible shape. It had seen a lot of use. I told him he was brave for working with such cranky gear. 'It's all I need,' he said, with a big smile." - Ben C. Solomon, a journalist with The New York Times. BAGHDAD/EDGARTOWN Mass. U.S. President Barack Obama expressed revulsion on Wednesday at the beheading of an American journalist by Islamist militants and vowed the United States would do what it must to protect its citizens as international condemnation of the insurgents grew. Not long after Obama called Islamic State a "cancer" with a bankrupt ideology, the Pentagon said U.S. aircraft conducted 14 air strikes in the vicinity of Iraq's Mosul Dam, destroying or damaging militants' Humvees, trucks and explosives. Islamic State posted a video on Tuesday that purported to show the beheading of journalist James Foley in revenge for U.S. air strikes in Iraq. It prompted widespread horror that could push Western powers into further action against the group. Britain's prime minister cut short his vacation as UK intelligence tried to identify Foley's killer, while France called for international coordination against the Islamist militants fighting in Syria and Iraq. U.S. officials said on Wednesday that intelligence analysts had concluded that the Islamic State video, titled "A Message to America," was authentic. It also showed images of another U.S. journalist, Steven Sotloff, whose fate the group said depends on how the United States acts in Iraq. The gruesome video presented Obama with bleak options that could define American involvement in Iraq and the public reaction to it, potentially dragging him further into a conflict he built much of his presidency on ending. Obama called the beheading of Foley "an act of violence that shocked the conscience of the entire world" and said the militants had killed innocent civilians, subjected women and children to torture, rape and slavery and targeted Muslims, Christians and religious minorities. "So ISIL speaks for no religion. Their victims are overwhelmingly Muslim, and no faith teaches people to massacre innocents. No just God would stand for what they did yesterday and what they do every single day," Obama said in brief comments to reporters in Edgartown, Massachusetts, where he has been vacationing. He said he had spoken with Foley's family. "ISIL has no ideology of any value to human beings. Their ideology is bankrupt." U.S. Secretary of State John Kerry said the United States would "never back down in the face of such evil. "ISIL and the wickedness it represents must be destroyed, and those responsible for this heinous, vicious atrocity will be held accountable," Kerry said in a statement. INTELLIGENCE SERVICES British anti-terrorist police began an investigation of the video, in which Foley's killer spoke with a London accent. Apparently a British national, the killer is just one of hundreds of European Muslims drawn to join Islamic State in Iraq and Syria, who authorities say pose a security threat to U.S. and European interests if they return home from the Middle East. The video showed a high level of technical proficiency and the use of a British voice may have been intended to make its contents clear to audiences in the United States, Islamic State's declared enemy. Foreign Secretary Philip Hammond said he was not surprised to hear the British accent and that large numbers of British nationals were fighting in Iraq and Syria. "Our intelligence services will be looking very carefully on both sides of the Atlantic at this video to establish its authenticity, to try to identify the individual concerned and then we will work together to try to locate him," Hammond told Sky news. France said it wanted the permanent members of the U.N. Security Council and regional countries, including Arab states and Iran, to coordinate action against Islamic State. President Francois Hollande called for an international conference to discuss how to tackle the group. U.N. Secretary-General Ban Ki-moon condemned "the horrific murder of journalist James Foley, an abominable crime that underscores the campaign of terror the Islamic State of Iraq and the Levant continues to wage against the people of Iraq and Syria," U.N. spokesman Stephane Dujarric said. Iraqi Foreign Minister Hoshiyar Zebari urged the world to back his country against Islamic State, which he described as a threat to the world, not just to the minority ethnic groups whose members it has killed in Iraq. Germany and Italy said they were ready to send arms to bolster the military capabilities of Iraqi Kurds fighting Islamic State in northern Iraq. Sending arms into conflict zones is a major departure for Germany, which has often shied away from direct involvement in military conflicts since World War Two due to its Nazi past. The video's message was unambiguous, warning of greater retaliation to come against Americans following nearly two weeks of U.S. air strikes that have pounded militant positions and halted the advance of Islamic State, which until this month had captured a third of Iraq with little resistance. Foley, 40, was kidnapped on Nov. 22, 2012, in northern Syria, according to GlobalPost. He had earlier been kidnapped and released in Libya. Sotloff, who appeared at the end of the video, went missing in northern Syria while reporting in July 2013. He has written for TIME among other news organizations. On Facebook, Foley's mother, Diane Foley, said: "We have never been prouder of our son Jim. He gave his life trying to expose the world to the suffering of the Syrian people. "We implore the kidnappers to spare the lives of the remaining hostages. Like Jim, they are innocents. They have no control over American government policy in Iraq, Syria or anywhere in the world." The video was posted after the United States resumed air strikes in Iraq this month for the first time since the end of the U.S. occupation in 2011. U.S. Senator John McCain, a Republican, said Foley's death should serve as a turning point for Obama in his deliberations over how to deal with Islamic State. "First of all, you've got to dramatically increase the air strikes. And those air strikes have to be devoted to Syria as well," McCain said in a telephone interview. ISLAMIC STATE Islamic State, which has declared a caliphate in the parts of Iraq and Syria it controls, opened the video with a clip of Obama saying he had authorized strikes in Iraq. The words "Obama authorizes military operations against the Islamic State effectively placing America upon a slippery slope towards a new war front against Muslims" appeared in English and Arabic on the screen. It showed black and white aerial footage of air strikes with text saying: "American aggression against the Islamic State." A man identified as Foley, head shaven and dressed in an orange outfit similar to uniforms worn by prisoners at the U.S. detention camp in Guantanamo Bay, is seen kneeling in the desert next to a man holding a knife and clad head to toe in black. "I call on my friends, family and loved ones to rise up against my real killers, the U.S. government, for what will happen to me is only a result of their complacency and criminality," the kneeling man says. The man next to him, in a black mask, speaks in a British accent and says, "This is James Wright Foley, an American citizen, of your country. As a government, you have been at the forefront of the aggression towards the Islamic State." ISLAMIC ARMY "Today your military air force is attacking us daily in Iraq. Your strikes have caused casualties amongst Muslims. You are no longer fighting an insurgency. We are an Islamic army, and a state that has been accepted by a large number of Muslims worldwide." Following his statement, he beheads the kneeling man. At the end of the video, words on the side of the screen say, "Steven Joel Sotloff," as another prisoner in an orange jumpsuit is shown on screen. "The life of this American citizen, Obama, depends on your next decision," the masked man says. University of Virginia political scholar Larry Sabato said the killing was like the beheading of American journalist Daniel Pearl in Pakistan in 2002. He said it could help bolster a perception among Americans that the United States will have to be more aggressive in dealing with Islamic State militants. Syria has been the most dangerous country for journalists for more than two years. At least 69 other journalists have been killed covering the conflict there and more than 80 journalists have been kidnapped in Syria. The U.S.-based Committee to Protect Journalists estimates that approximately 20 journalists are currently missing in Syria. Many of them are believed to be held by Islamic State. As well as taking territory, Islamic State has seized a number of oil wells in northern Iraq. The government in Baghdad said it was troubled by reports that Islamic State was smuggling oil to export markets and warned that the purchase of such supplies could help the group fund its operations. (Additional reporting by Stephanie Nebehay in Geneva, Oliver Holmes and Tom Perry in Beirut, Sabine Siebold in Berlin, Costas Pitas and William James in London, Louis Charbonneau at the United Nations and John Irish in Paris; Writing by Giles Elgood and Jim Loney; Editing by David Stamp and Dan Grebler) Twitter is taking down images and video of the beheading of U.S. journalist James Foley by Islamic militants. "We have been and are actively suspending accounts as we discover them related to this graphic imagery," Twitter (TWTR, Tech30) CEO Dick Costolo said. In a video posted by the Islamic State in Iraq and Syria (ISIS) on YouTube, Foley is seen kneeling next to a man dressed in black. Foley reads a message, presumably scripted by his captors, that his "real killer'' is America. The U.S. said Wednesday the video was authentic. ISIS has carried out many killings, including beheadings, as part of its effort to establish an Islamic caliphate that stretches from Syria into Iraq. In many cases, ISIS -- which refers to itself as the Islamic State -- videotapes the murders and posts them online. CNN is not airing the video showing Foley's death and threats to kill another journalist, Steven Sotloff. Related: Twitter co-founder live-tweets protests in Ferguson Foley's family used social media to appeal for privacy, and to urge people not to watch the video, or share it -- a call echoed by many Twitter users Wednesday. A Twitter spokesperson declined to comment on the number of accounts it had suspended, or provide further details. Twitter says it removes imagery of deceased individuals under certain circumstances. "Immediate family members and other authorized individuals may request the removal of images or video of deceased individuals, from when critical injury occurs to the moments before or after death, by sending an email to [email protected]," company policy states. "When reviewing such media removal requests, Twitter considers public interest factors such as the newsworthiness of the content and may not be able to honor every request." A spokesperson for YouTube said it was company policy to ban content showing "gratuitous violence, hate speech and incitement to commit violent acts," and it removes such videos when they're flagged by users. "We also terminate any account registered by a member of a designated Foreign Terrorist Organization and used in an official capacity to further its interests," the spokesperson said. | The mother of the American journalist beheaded by ISIS has urged the militant group to free other Americans it has threatened to kill. "We have never been prouder of our son Jim. He gave his life trying to expose the world to the suffering of the Syrian people," Diane Foley writes in a post at the Free James Foley Facebook group. "We implore the kidnappers to spare the lives of the remaining hostages," she writes. "Like Jim, they are innocents. They have no control over American government policy in Iraq, Syria, or anywhere in the world." American officials have confirmed that a video released by the militants shows the execution of Foley, who was kidnapped in Syria in November 2012, WCVB reports. Meanwhile, media outlets are offering some details on his background: The 40-year-old native of Rochester, NH, worked for AFP and GlobalPost; the latter says it mounted a huge investigation and "amassed an enormous amount of information that has not been made public" after his disappearance. His early career was as a teacher all over the US, the BBC reports. His brother was an Air Force officer in Iraq, where Foley "embedded himself" with troops, the BBC notes. Afterward, he headed to Libya in 2011. "The idea with the Libyan revolution is, it's journalists embedding with rebels, and that's essentially what we did," he said. There, he was detained after an ambush by Moammar Gadhafi's fighters. A colleague was killed, but Foley was released after six weeks. "Feeling like you've survived something-it's a strange sort of force that you are drawn back to," he said-and indeed, despite the Libyan incident, he next traveled to Syria, where he wanted "to expose untold stories," the BBC reports. There, he was stopped by militants while driving through a contested area, the AP notes. He had been working with another journalist in Idlib, a province in northern Syria. In the ISIS video, another person, believed to be missing American journalist Steven James Sotloff, is shown, and the militant who beheaded Foley warns that his life depends on President Obama's next decision, reports Reuters. Twitter has since taken down the images, notes CNN, and Foley's friends and family have started a campaign urging people not to view or share the video. | multi_news |
Angel: "You've had vision."
Cordy: "Boy! Howdy! - Doyle kissed me just before he died and he passed it on to me!" Cordy in her new apartment..
Cordy: "I'm not giving up this apartment!"
Angel: "It's haunted!"
Cordy: "It's rent controlled!" Cordy talking to Aura in the phone: "Yeah, I have a room-mate, but it's cool. I never see him. (Her can of rootbeer slides across the end-table and Cordy covers up the receiver) Hey! Phantom Dennis, put that back!" Night, the office. Cordelia is putting on some lipstick using one of the lobby windows as a mirror.
Angel: "You look nice." Cordy jumps and spins around, then looks back at her reflection in the window. There is a streak of lipstick from the corner of her mouth halfway up her cheek.
Cordy pulls out a tissue: "And now I look like the Joker."
Angel: "Sorry."
Cordy fixes her lipstick: "Hopefully I'm still too young and carefree for a heart attack. Would it kill you to hum a little tune when sneaking up on people?"
Angel looking at a file: "I don't hum. I'm confused here. Why is Mrs. Bensen filed under 'P'?"
Cordy: "That's not a 'P' that's an 'F'. Or is it an 'R'?"
Angel: "I don't know. Maybe we can be a little less - young and carefree with the filing?"
Cordy: "Oh, it's an 'F'. I remember now." Angel blinks and scratches his neck: "All right, so... (Chuckles) Why is it Mrs. Bensen is filed under 'F'?"
Cordy: "Because she is from France. - Remember what a pain she was?"
Angel: "Yeah. It made me what to drink a lot."
Cordy: "Well, that's the French for you."
Wesley walks into the office: "Hello. I was just in the neighborhood, patrolling with my new Bavarian fighting axe (Holds up an axe), when I suddenly thought 'perhaps Cordelia has had a vision', perhaps you need my help in the battle against evil."
Angel: "We seem to be evil free at the moment."
Wesley: "I also packed along a 'Word Puzzle 3-D' if either of you has the nerve to take me on."
Cordy: "Gee, Wesley, I'd love to, but unlike you, I'm not in my 80s quite yet."
Wesley: "If shaking your booty at the latest trendy hot spot is your idea of a life, then call me... (Two glamorous girls walk in) ...sick with envy."
Sarina to Wesley: "Hi. I'm Sarina. Nice axe."
Wesley laughs: "Uh, ah, no, this old thing..." Wesley swings the axe around and embeds it in the wall beside him. Angel looks at him, then over at Sarina, but doesn't say anything.
Sarina to Cordy: "We're late. Wilson practically had to have phone s*x with the manager at Lounge La Brea to get us in."
Cordy turns around: "Okay, okay. How do I look?"
Sarina: "Like you always do. Wilson won't be able to take his eyes off of you."
Angel: "Who's Wilson?"
Sarina: "Christopher." Angel looking at Cordy who turns away: "Christopher Wilson."
Emily: "Wilson Christopher."
Wesley: "No. The ethno-archeologist from Brandies?"
Sarina: "The fashion photographer from LA who's been seeing Cordelia. Third times the charm. (Turns towards Wesley) And that Hugh Grant thing is really starting to work for me."
Angel walks over to Cordy: "So, ahem (chuckles) You've been seeing someone. How come I didn't know?"
Cordy: "Because I'm ashamed of you. Not to mention how you'd embarrass me by giving him the third degree."
Emily: "Your boss could give me the third degree anytime."
Sarina to Emily: "You're a sucker." Cordelia puts a hand to her forehead and drops to the floor behind the desk. Angel quickly knocks some letters to the floor: "Ah, Cordelia, grab that file. Sorry." Wesley runs over and starts to gather the letters: "Oh, not to worry. Heh, heh, whoopsie." Angel turns to the two girls standing between them and the desk: "So, La Brea. Sounds like that could be an evening (Wesley straightens up and stands next to Angel) with all sorts of evening type... I - I heard the bands there are..."
Sarina: "They don't have any bands."
Angel: "Which I like. Because if it's too loud..."
Emily amused: "Want to come?"
Angel: "Oh, I think I may be busy."
Cut to Cordy writhing on the floor. We get a flash of her vision.
Angel: "Beside, ah, I, uhm, I don't lounge all that well."
Wesley laughs: "Good one! Oh, yes. No, he's ah, he's no lounger (puts an arm around Angel) this one."
Sarina to Emily: "The good ones are always gay. (Wesley lets go of Angel) Cor, tick, tock." Cordelia pulls herself back to her feet and Angel turns towards her.
Angel: "So, that client I'm supposed to be meeting tonight, what's he like again?"
Cordy: "Like a big baby (flash to her vision) hatching from a big egg with really large hands, in need of a manicure. (Writes an address on a piece of paper and hands it to Angel) You're meeting him here. Okay." Sarina as the three girls leave the office: "Are my girls ready to party?" Wesley as Angel goes to get his coat: "I don't suppose you need any help, slaying the big baby creature, do you? Not that an evening alone with word puzzle isn't plenty exhilarating in it's own right. (Angel passes him and hands him the address. Wesley looks at it a big grin spreading over his face) Right." Wesley hurries after Angel then turns back to sling his bag over his shoulder and try to pull the axe from the wall. Angel watches from the lobby through one of the windows as Wesley pulls the axe free, falling to the floor in the process.
Cut to Wesley looking at the address on the paper, 23 Cabrillo. He lowers the paper to reveal a mail box with that address. Angel pulls out a sword and Wesley has the axe in his right hand and a loaded crossbow in his left as they walk up the steps to the door. Angel nods at him. Wesley kicks in the door and jumps through it weapons held at the ready. Angel tries to follow but bounces off an invisible wall. Wesley to an older couple sitting watching TV: "Don't move a muscle. - Demon spawn! (The man blinks at him) Cowards, don't make me thrash it out of you. Where do you lay your eggs? In the cellar?" Angel looks over at the neighboring house and sees the thing from Cordy's vision hatching through one of the windows: "Wesley..."
Wesley: "In the bedroom?"
Angel: "Yeah, that's right, termites lay their eggs anywhere, such as next door." Angel motions to the next house. Wesley backs up and looks out through the door, weapons still trained on the older couple, who are just sitting there looking at him.
Wesley lowers his weapons: "Oh."
Angel: "And we fight termites, where ever they may roam." Angel and Wesley leave, then Wesley turns back to the older couple: "Sorry about the door." Intro. We see the bedroom window from the outside. There is orange slime spattered on the glass. We hear the demon baby scream and the sounds of Angel's sword and Wesley's axe. We see shadows of Wesley and Angel fighting the thing. One of them gets thrown against the wall next to the window and the plaster on the outside breaks. Camera pans over to the closed door and we see Wesley flying through it with a scream. He picks himself up, axe still in hand and runs up the stairs and back in through the broken door.
Wesley: "You'll pay for that." More sounds of fighting, then Angel steps through the door spattered with gore, sword in one hand, sheath in the other. Wesley follows him, looking disheveled, and also spattered with gore.
Wesley: "That was bracing." Angel wipes his sword off on his sleeve: "Yeah. Baby just hatched. Wouldn't want to run into him when he grows up and gets his drivers license."
Wesley laughs: "And thank you very much, Cordelia, for sending us to the wrong house. Another five minutes and that thing would have been loose in the world."
Angel sheathes his sword: "Huh, well, it all worked out."
Wesley: "This time. (Looks back at the broken door) Maybe we should clean up...(turns back to Angel) You think a Tahval demon leaves a hefty security deposit?"
Angel turns to go: "I'm sure of it."
Wesley: "Well, it's not my place, Cordelia works for you, but she doesn't seem to be paying attention to her duties lately."
Angel: "She's had a lot to deal with. I mean, Doyle's death, inheriting his visions, she's young. She's still trying to find her way in the world."
Wesley: "But we're not in the world. Demon hunters like us have a higher calling." Angel motions with a finger to his cheek: "You, uh..." Wesley mirrors his motion and wipes some gook of his cheek: "Thanks. I mean, no one is more fond about Cordelia than I, but if she wants to go gad-abouting with those doxies..." Angel walks past him with a smile: "I think they liked you."
Wesley follows him: "Really! I - I didn't mean doxy in the sexual promiscuous sense, exactly. I - I... You don't think sticking the axe in the wall put them off?" Angel as they walk by a lady in a bathrobe with curlers in her hair: "That was charming."
Wesley: "What about the fact that they thought we were gay?"
Angel: "Adds mystery."
Cut to the La Brea Lounge.
Bartender hands Sarina a drink: "Here you go."
Sarina: "Thanks."
Bartender: "Don't mention it."
Sarina: "I won't. But I'll tip for it. (Turns to a guy) Jason, mullah." Jason smiles at her and hands her a bill. Sarina hands it to the bartender.
Jason: "I'm not saying it was worthy of a kiss." Sarina kissed Jason as Emily comes over.
Emily: "I'm bored."
Sarina: "Emily, do you know what it takes to get in here?"
Emily: "Still bored."
Sarina: "Well, Cordy's not." Camera pans to show Cordy and Wilson sitting on a couch talking.
Wilson: "So you left Sunnydale and came to LA. What was that like?"
Cordy: "Like skydiving without a parachute (Wilson chuckles) except for the smashing your body to bits part. - Actually, no, it was like that, too. Oh, and that guy that was supposed to be here when you arrive..."
Wilson: "The guy?"
Cordy: "With the big bag of fame and fortune."
Wilson: "Oh, that guy."
Cordy: "So, what happened to him?"
Wilson: "He comes and goes. He's sort of fleeting that way."
Cordy: "Well, if you see him will you tell him to fleet my way?"
Wilson laughs and nods: "Thank you."
Cordy: "For what?"
Wilson: "For making me *not* hate dating. - Sarina is really something. I'm going to send her flowers (Cordy looks at him) for introducing you to me."
Cordy: "I knew that. (Laughs) God, for the first time I like LA. In high school I knew my place and, okay, it was a haughty place, and may be I was a *tad* shallow."
Wilson: "Oh, hey, nobody feels like they belong here. I mean that's the point of LA to make you feel as insecure as possible."
Cordy: "You don't feel that way."
Wilson: "Sure I do."
Cordy: "Right. You're doing what you came to LA to do. You're photographing all these gorgeous, famous people. Where is the insecure?"
Wilson: "In the pictures, which are further proof that everybody is having a real life, except me. I'm the guy behind the camera, watching and recording life, not - living it - each and every moment - like you."
Cordy: "Wow, I'm living life? Look at me."
Wilson: "Look at you."
Cut to Cordy unlocking the door to her apartment.
Cordy: "Well, here we are. (Turns around in the open door) Thank you for the club, and for driving me home, and listening to my entire life's story. I think I may have left out a couple of weeks in early 1987, but..."
Wilson: "I had a great time, and you didn't talk too much. I want to know everything about you."
Cordy: "Me, too. I mean, about you, not... because I already know (Wilson steps over the threshold) about me..." Cordy trails off as he kisses her.
Wilson: "Call you tomorrow?"
Cordy: "Uhm, you don't have to..."
Wilson laughs: "Call you?"
Cordy: "Go home? I mean, right away? It's still early (Wilson looks at his watch) in Australia. (They laugh) Will you come in?" Wilson walks in and looks around: "Nice." Cordy closes the door and dims the lights: "Yeah, compared to my old apartment, it's Buckingham Place."
Wilson: "You live alone, right?" The lights go bright again. Cordy goes to turn them back down: "In the sense that I'm the only one living here that's actually alive."
Wilson: "That was a yes, I think." Cordy as the lights brighten again: "Wiring. Old buildings."
Wilson: "Well, no worries. Besides I - I like looking at you."
Cordy: "Look the truth is that my dating game skills are kind of rusty. You're the first person I've had over in a long... well, - ever. (They laugh) So, I'm open to suggestions."
Wilson: "Music?"
Cordy: "Right. Music." She turns the radio on but it switches from the mellow music that was playing to some rousing Polka.
Wilson: "That's some jaunty Polka." Cordy laughs as she reaches back to turn the radio back off.
Wilson: "Oh, I know, I know, the wiring."
Cordy laughs: "How about some tea?"
Wilson: "That be great."
Cordy: "Okay." Cordy once she gets into the kitchen: "All right, Dennis, *knock it off*! This is the one guy, I've actually liked in a long time, and if you keep killing the mood, I'll kill *you*! (Gets the teakettle) All right, empty threat, you being a ghost and already dead and all. (Fills the kettle with water and sets it on the stove) But I'll do something *worse*! I'll play 'Evita' around the clock. (Puts her hands on her hips) The one with *Madonna*!"
Wilson walks up behind her: "Who are you talking to?"
Cordy spins around: "Uhm...my ghost. I have a ghost. He's jealous. (Grimaces and laughs) Kidding. The apartment's great, but things are always breaking and, uhm, and I have no one to complain to, so sometimes *just* to keep myself company I talk to myself." Wilson leans forward and kisses her. When he pulls back Cordy stares at him for a moment then leans in and kisses him.
Cut to the darkened bedroom and more kissing. Then they are laying on the bed and we get still more kissing and a glimpse of a bare back.
Cut to Cordy waking up the next morning.
Cordy turns around: "Wilson?" The bed beside her is empty and she reaches for the alarm clock. It's 10:47 am.
Cordy pushing herself up: "Uh-oh, somebody is going to be late for work." She pushes the blanket aside and stares at her hugely pregnant belly.
Cut to Cordy sitting up dressed in bed.
Cut to Angel and Wesley walking up to Cordy's door (they are under some kind of roof, so no direct sunlight).
Angel: "What time is it?"
Wesley: "Quarter past noon."
Angel: "I left two messages, she should have called back. (Bangs his fist on her door) Cordelia!"
Wesley: "Maybe she unplugged her phone. Or she slept somewhere else. (Wesley turns to go) Well, I guess we should... (Angel turns the knob, breaking the lock and walks in) break down her door and trespass."
Angel looks around the apartment: "Cordelia? -I'm getting a bad feeling here."
Wesley closes the door: "I thought it was just me."
Angel: "This isn't like her."
Wesley: "Avoiding her responsibilities? Lately it seems quite like her. (Angel opens the bedroom door) I'm sure it'll all work out once we...(The door swings open and we see them stare at Cordy sitting up in bed) Mother of God."
Cordy not looking at them: "Angel?"
Angel comes in: "It's alright. We're here."
Cordy: "I'm ready to wake up now. I - I don't seem to be - waking up. - Help me." Angel sits down on the edge of the bed: "We're going to. What do you remember?"
Cordy close to crying: "Well, we went to the club. And Wilson and I just sort of hid out on this couch and we talked and talked and then he drove me home and I asked him in. - He was really nice. And we ah, - you know? - It was normal. He was normal and it was safe, it was - it was all really safe!"
Angel: "Shh, shh. It's okay. Have you talked to Wilson?"
Cordy: "No. I haven't talked to anyone. What would I say to him? 'I had a really great time. I think you left something at my place'? I don't think this is right." Angel reaches for her cordless phone and holds it out to her: "What ever is happening to you, he may have some answers."
Cordy: "I can't."
Angel: "Just dial his number and I'll talk to him." Cordy takes the phone and dials: "Oh, God. I'm being punished."
Wesley: "You're certainly not being punished. This is... We'll get to the bottom of it." Angel takes the phone and hears: "The number you have reached has been disconnected, there is no new number at this time."
Angel hangs up: "He's not answering right now. I want you to rest, and we're going to handle this. Hey! You're not alone."
Cordy looking at her belly: "That's sort of the problem, isn't it? (Angel and Wesley look at each other) Could you - just leave me alone for now?"
Angel gets up: "We're going to be right outside." He and Wesley leave and close the door. The box of tissues floats up from the night stand and one of the tissues floats over to Cordy who takes it, blows her nose and blots at her eyes. The blanket moves up to her neck and Cordy sighs.
Cut to Angel hanging up the phone: "Thanks."
Wesley: "Any luck with your contact?"
Angel: "Wilson's home and business phones have been disconnected, no unlisted numbers, no forwarding addresses, no criminal record."
Wesley: "Well, that's something."
Angel sighs: "I'm guessing it's some kind of procrea-parasitic demon."
Wesley: "Hmm, a demon who can only reproduce by implanting a human woman with its seed. Yes, I've heard of such entities. - The human mothers..."
Angel: "Rarely survive labor, and the ones that do, wish they hadn't."
Wesley: "If she's that pregnant in one night, she could give birth at any moment."
Angel: "We have to move fast. You're gonna have to see what's inside her."
Wesley shocked: "I beg your pardon?"
Angel with a smile: "Pre-natal exam, Wesley."
Wesley: "Oh, of course. And what about you?"
Angel: "I'm going to find Daddy."
Cut to the La Brea Lounge. Angel walks up to the bar and clears his throat.
Bartender: "I didn't see you."
Angel: "I get that a lot."
Bartender: "What can I get you?"
Angel: "I need some help."
Bartender: "I'm kind busy."
Angel: "Yeah, I know. I won't take much of your time. A friend of mine was here last night. Her name is Cordelia. Big smile, really pretty?"
Bartender: "Yeah, we get a lot of that." Angel pulls out some money and lays it on the bar.
Bartender: "What's this?" Angel looks down at the bill under his hand then back up at the guy: "Probably an insult. I'm guessing that you're serving drinks day and night to jerks that think that they can buy anything."
Bartender comes around the bar: "Yeah, that be a good guess."
Angel: "One of those jerks hurt my friend. I need to find him, fast."
Bartender: "Who was it?"
Angel: "Wilson Christopher. (Bartender nods) Look, I want to know who his friends are, where they hang..."
Bartender: "Pretty much where ever Sarina tells them to."
Angel: "Sarina.'
Bartender: "Yeah. You know her?"
Angel: "Yeah."
Bartender: "They travel in packs. The guys have the money, the girls have the pretty. The girls decide what club's the flavor of the month and Sarina rules the girls."
Angel turns to go: "Thanks."
Bartender: "So, you're her boyfriend?"
Angel: "No. I'm family."
[SCENE_BREAK]
Cut to a hospital by Pershing Square. Cut to the waiting room.
Pregnant woman to Cordy: "Do you know what it is? (Cordy looks at her in shock) Boy or girl?" Wesley comes to sit down next to Cordy: "Shouldn't be long. (Leans forward and whispers) I told them that it was rather urgent."
Woman: "You're carrying low. I bet it's..." She reaches over towards Cordy and Cordy jumps in her chair freaked: "Shut up! Don't touch me!"
Cut to the doctor examining Cordy: "You're what, eight and a half months along?"
Wesley aside to Cordy: "Feels like only yesterday, doesn't it?"
Doctor: "Well, I see you left a lot of blanks on the patient information form. It would help to have the name of your previous doctor."
Cordy: "You're the only doctor we've been to..."
Wesley jumps in: "in California. We just moved here from England."
Doctor: "Lovely country. So, how are you feeling?"
Cordy: "I'm as big as a house, everything hurts, I..."
Doctor: "That's all normal at this stage. And once your little one comes out, which will probably be in no time, you'll feel a lot better."
Cordy: "God, it's a nightmare." The doctor and the nurse stare at Cordy.
Wesley takes Cordy's hand: "Hold on."
Doctor: "All right, Mrs. Penborn, why don't you lie back and see what's baking in the oven?" Cordy lies back with a sigh and the doctor and the nurse start the ultra sound.
Doctor: "Have you folks settled on a name yet? It's the hardest part for a lot of people. (Wesley standing next to Cordy giving her a reassuring smile. Doctor looks at the monitor) Hmm, looks like somebody is having twins."
Cordy and Wesley: "Twins?!?" Doctor still looking at the screen: "No, there is a third heartbeat."
Nurse stares: "There is another one." Wesley goes to look over the doctor's shoulder.
Doctor: "Five... six (Cordy stares at her belly and shakes her head) oh, my God!"
Cordy: "What is it? What's wrong?"
Doctor: "I - I'm sure it's nothing. But I - I'd like to withdraw - a little of the amniotic fluid just, just to make sure that everything is, ah... shipshape. (Cordy narrows her eyes at him) So, nurse, if you would prep Mrs. Penborn right away?" Wesley stares at the screen then looks over at Cordy.
Cut to Angel stepping out of an elevator in to a dimly lit hallway. He walks up to a door and knocks.
Angel: "Sarina?"
Sarina: "Just leave it outside."
Angel: "Sarina, it's Angel, Cordelia's friend. Can I come in?"
Sarina: "Okay." Angel walks in and looks around: "Sarina?" The apartment is dark lit only by some candles. Angel walks further and sees Sarina with her back to him lighting another candle.
Angel: "Sarina?" Sarina still with her back to him: "The light hurts my eyes lately."
Angel: "I know the feeling."
Sarina: "I thought you were the liquor store. (Lifts a bottle to her lips) I'm almost dry. (Drinks) I know what you're thinking. I shouldn't, right? (Turns around. She is as pregnant as Cordy) It'll hurt the baby? (Looks at her belly) I hope!"
Cut to the clinic.
Doctor syringe in hand: "I need to tell you that there is a point 5% chance of miscarriage from the procedure. Now, it's a very small risk..."
Cordy: "I'll take it."
Doctor: "Now, you'll feel a pinch. Just count backwards from five, and we'll be done."
Cordy: "5 - 4 - 3 - 2 - 1 - 1 - 1 - 1!" The doctor withdraws the needle and stares at the fluid: "All done. (Hands it to the nurse) Now, that wasn't too bad, was it? Now we'll just run a few tests..." The nurse turns back around staring at the syringe in horror: "Dr. Wasserman?" The syringe cracks and the nurse drops it and jumps back with a scream. Doctor looks at the syringe on the floor as the fluid starts to eat through the floor: "This is, uhm... (he and the nurse leave the room) Excuse me." Wesley bends down to get a closer look at the hole in the floor then looks over at Cordy, his mouth hanging open.
Cordy: "You saw what's inside of me?"
Wesley: "I think we should find Angel."
Cordy: "Wesley, please just tell me!"
Wesley doesn't look at her: "Cordelia..."
Cordy: "Do they look healthy?" Wesley stares at her.
Cut to Angel and Sarina.
Sarina: "It's like it's not real, but it is. Right? It's really happening?"
Angel blinks: "It's real. It's happening to Cordelia, too."
Sarina: "Oh, God. - I can't reach Jason. He's gone."
Angel: "So is Wilson." Sarina sits down on a couch: "I didn't know this would happen."
Angel: "But you knew something."
Sarina: "Yeah, I knew, I knew, I knew the guys, - Jason and Nick, and then Wilson wanted to meet Cordelia. I don't know. (Sighs) I knew something wasn't right. Their money..."
Angel: "What about their money?"
Sarina: "It's stupid. It kind of - smelled. (Sighs) I mean, *really* smelled. And sometimes the guys were like - jumpy. But this town, - you know? Everything is fake. Things are weird and you stop asking questions. - You sure this is really happening?" Angel sits down on the edge of the table in front of her: "Do you have someone you can call?"
Sarina: "I Call?"
Angel: "Family."
Sarina shakes her head: "No. No one. The guys seemed like they liked that. Wilson asked about Cordelia and I told him that she didn't have anybody either."
Angel: "Sarina, where can I find..." Sarina bends forward and screams. Blends into Cordy leaning forward and screaming. Wesley holds her as they ride down in the elevator to Angel's apartment.
Wesley leads her out: "It's all going to work out. You'll see, everything's going to be just fine."
Cordy: "I know it will be." Wesley leads her into Angel's bedroom: "There's a brave girl. I'm sure Angel won't mind if you rest in here. Just relax, get comfortable, well, ah, ha ,ha, as comfortable as is possible at any rate. (He covers her up with a blanket) Should you need anything, anything at *all*... I'll just..."
Cordy looks hard at him: "You're afraid."
Wesley: "What?"
Cordy: "You're afraid of what's inside of me. You think it's horrible. You think that I won't be able to handle it. That if I find out, I'll do something - bad."
Wesley: "Cordelia, the truth is I haven't yet formulated a theory. I need time to analyze the ultrasound and weigh the data, and - and the moment there is anything concrete to report..."
Cordy rubbing her belly: "There is 7 of them. There is 7 of his children - growing inside of me. They are talking to me. They're talking all at once. (To her belly) I can't understand." Wesley sits down on the edge of the bed: "Cordelia, I know how difficult this must be for you..."
Cordy: "No! You don't know."
Wesley: "All right."
Cordy whispers: "You don't know what it's like to be a partner in creation."
Wesley: "I - I - I just meant..."
Cordy: "Wesley..."
Wesley: "Yes?"
Cordy whispers: "They're not human."
Wesley whispers: "I imagine that's true."
Cordy: "But, I mean... that could be okay, right? I mean, look at Angel. He's not human. And Doyle, he wasn't either..."
Wesley: "Shh. Shh."
Cordy: "I mean, not totally. (Whispers) He was good." Wesley blots her forehead with a handkerchief: "Shh. Shh." Cordy sinks back into the cushions and closes her eyes with a sigh. Wesley pulls the blanket up to her chin, looks at her for a moment. He turns away to find Angel standing in the door to the bedroom. They look at each other for a moment then Wesley follows Angel into the main apartment.
Wesley: "Any luck locating Wilson?"
Angel: "Not yet. But I did find Cordelia's friend Sarina. She's a victim, too. As big as Cordelia. Wilson's rich buddies are in on it. (Starts flipping through a phone book) Four of them, maybe more, I don't know how many women they've impregnated." Wesley shows him a piece of paper: "And it's worse than you know. The ultra sound results. Seven heartbeats, at least, maybe more. And with multiple pregnancies..."
Angel: "Someone is raising an army."
Wesley: "An army of what?"
Angel: "Good question. We need to find the demon fathers." Wesley sees what section of the yellow pages Angel is looking at: "Gun clubs? Guns can kill them? Well, I say, that makes it easier."
Angel: "Sarina said Wilson and his buddies hang out at some private gun club. Guns and Cigars. She doesn't know where exactly. While I find them you should be narrowing down the species. Maybe we can figure out a way to terminate this without hurting her."
Wesley: "And if we can't?" Angel rips out a page and sighs: "Then we need to know what to do once they're born." Wesley follows Angel out of the living room: "Yes, well, it mustn't come to that. The odds of her even surviving are..." They stop as the enter the kitchen area and see Cordy standing in front of the open refrigerator gulping down blood out of a clear container. Some of it runs down her chin.
Angel swallows: "I don't think I ever realized just how disgusting that was. Get her back to bed."
Wesley: "Yes."
Angel: "Maybe order her a pizza or something."
Wesley: "Good idea."
Angel: "Uh..." Cordy puts the half-empty container back in the fridge, wipes the blood on her mouth on her sleeve and walks past them.
Cordy: "I was hungry."
Cut to Wilson shooting a pistol at the gun club. He takes his ear protection off and turns to find Angel standing behind him. Wilson takes off the safety glasses: "You shouldn't sneak up on people like that in here. (Drops out the empty magazine and puts in a new one) That's how accidents happen."
Angel: "Speaking of accidents. I'm a friend of Cordelia Chase."
Wilson: "This is a private club. Featured word - 'private'."
Angel: "You don't talk to me, I'll kick your ass. Featured word - 'ass'."
Wilson: "Angel, right? Her boss? (Turns and points his gun at Angel's neck) She told me all about you, man." Angel grabs his gun hand, making him drop the gun, then twists him around and wraps one arm around his throat: "Yeah, well, somehow, I doubt that. (Angel throws him against the wall) You're human. Then you can't be the father. (Wilson tries to get past Angel) So you and your friends are just a link. (grabs Wilson by the front of his shirt and pushes him up against a pillar) Huh? How does it work? Those things come to term, she'll die. You do know that?"
Wilson: "So? I'm not telling you anything."
Angel: "Was so hoping you'd say that." Angel hauls back and hits Wilson with a hard right. Wilson hits back, Angel blocks and hits him again, knees him in the stomach, then drops him on the floor with a left hook. Wilson's buddies come in just as Angel gets down to pull Wilson up and hit him again.
Nick: "Hey! Uhmm, - someone's mommy didn't teach him to play nice."
Wilson picks himself up: "You have no idea what you're dealing with."
Cut to Wesley comparing the picture from the ultra sound to some engravings of a demon in one of Angel's books using a magnifying glass.
Wesley: "Eeesh!" He jumps when he looks up to see Cordy standing next to him.
Cordy looking at the engraving: "That's him, isn't it?" She picks up the book to get a closer look.
Wesley: "I ask that you not overreact. Keep in mind that oft times these 16th century engravers tended to exaggerate. (Looks worriedly at Cordy's face) Cordelia? I - I know it seems dire, but now that we've identified the species, there is every chance that we will be able to stop what's happening to you. (Cordy looks at him) That's right. We mustn't lose hope." Cordy closes the book and slams it against the side of Wesley's head. He staggers against the table.
Cordy: " You're not going to hurt my babies. (She hits him again and he drops to the floor) No one is going to hurt *my* babies."
Cut to Angel and Wilson's buddies at the gun club.
Angel: "You know, I'm starting to get the big picture here. You guys proxy for big daddy demon, he imbues you with his life force or whatever it is you're implanting in these women."
Jason: "He's trouble finding his own dates. We just - help him out a little, that's all."
Wilson: "Shut up, Jason."
Angel: "And you get what in return, fame, money, success? That's it, isn't it? How else would losers like you get ahead? I mean, you'd have to become procreative surrogates for a vile demonic entity."
Jason with a smirk: "Well, mostly, I do it for the s*x."
Wilson: "Welcome to Los Angeles. There are worse things to be in business with." Angel gets right into Wilson's face: "Where is he? Where is this demon you worship?" Wilson smiles as Nick pulls a gun from under his sweater: "Even if we did tell you where to find him, it wouldn't matter, since you're about to have an accident." Wilson pushes Angel back, catches the gun Nick throws him and shoots Angel three times in the chest. Angel doubles over groaning. The guys' smirks disappear when Angel straightens up in vamp face. Angel kicking the gun out of Wilson's hand: "I really don't like it when people shoot me." Angel proceeds to beat the crap out of the four guys. At the end he kicks Wilson backward through a glass door then steps through after him and puts his foot on his face.
Angel: "Now you're going to tell me what I need to know."
Cut to Cordy walking into a deserted warehouse. The other pregnant girls are coming as well. They look at each other while we hear the sounds of their babies talking to them.
Cut to a phone booth. Cut to the phone ringing in Angel's apartment. Wesley pushes himself up off the floor holding his head. Cut to Angel in the phone booth digging bullets out of his chest with his fingers while he is waiting for some one to answer the phone. Cut to Wesley picking up the phone.
Wesley: "Hello?"
Angel: "Yeah, Wesley, it's Angel." Wesley picks his glasses off the floor: "Oh, Angel, thank God."
Angel: "I found Wilson. Whatever it is Cordelia is carrying around inside her, he's not the father."
Wesley: "I know. It's a Hacksaw beast, an inner earth demon. Oh, this is all my fault."
Angel: "How is that your fault?"
Wesley: "Cordelia ran off. I tried to stop her. She became insanely protective when I identified the Hacksaw as the father of her - her, ah, (picks up the open book) I fear she may have gone off to rendezvous with it."
Angel: "She has. Miliken Industrial Park in Reseda."
Wesley: "What?"
Angel: "That's where Wilson and his friends built their shrine."
Wesley flipping through the book: "How does Cordelia know that?"
Angel: "She's telepathically linked to its unborn. That's how it's controlling Cordelia."
Wesley: "Of course, a psychic umbilical cord. The Hacksaw's telepathic connection is what's sustaining its unborn spawn."
Angel: "So, all we have to do is cut the cord."
Wesley: "We slay this demon and poof! No more evil pregnancies. Well, this is good news. We can end this without harming the women. There is just one tiny problem."
Angel: "What's that?"
Wesley reading through book: "Well, I don't wish to use the words 'impossible to kill', but fire won't kill it, decapitation won't, - and it's really huge."
Angel: "Wesley, can you shoot straight?"
Wesley: "Beg your pardon?"
Cut to the girls changing into white robes. Led by Cordy they walk up some steps, then down into a big vat filled with some yellow-brown liquid. Wesley steps around a corner and sees them: "Cordelia!" He walks up to the edge of the vat: "Come out of there this instant! (Looks at the other women) All of you please!"
Cordy: "We don't expect you to understand."
Wesley: "I understand. (Walks up the steps to stand on the wide concrete rim of the vat, and crouches down beside Cordy) You'll die unless you come with me, and that is the most vile smelling filth I've ever had the displeasure of inhaling. Now don't make me come in there after you."
Cordy: "We serve our master."
Wesley: "Please come before..." The ground begins to shake with thundering footsteps. They all look at the huge Hacksaw demon stepping through a hole in the wall.
Demon: "Who is the interloper to think you could disturb the birth of my children? Who are you?"
Wesley stands up: "Wesley Wyndham-Pryce, rogue demon hunter. (Puts up his fists) And I'm here to fight you, Sir, to the death, - preferably yours."
Demon: "You?"
Wesley: "As a heathen I wouldn't expect you to be familiar with the biblical story of David and Goliath. But I assure you it's of particular relevance to this situation."
Demon: "You said you came here to do battle, then lets fight and be done." Wesley looks around then slowly inches closer to the demon on the rim of the vat: "Yes, well, - as a point of courtesy, - I like to get to know my opponents, before I engage them in mortal combat. Do you, ah - do you have any hobbies?"
Demon: "Enough talk. I'll come to you." Angel rolls a gas tank down a ramp drawing everyone's attention.
Angel walking down the ramp: "Sorry I'm late to the baby shower. Brought a little gift." Angel picks up the tank, spins and throws it like a discus up at the Hacksaw, who catches it easily out of the air. We see a label 'Liquid Nitrogen' on the front of the tank. Wesley pulls out a gun and shoots a hole in the tank. The Hacksaw drops the tank and a stream of liquid nitrogen shoots out of the hole up at him. The demon screams in the fog. The women in the vat scream, their bellies slowly deflating. Wesley and Angel stare as the demon turns into a Popsicle. The girls stop screaming. Cordy looks around then walks up the steps out of the vat. She walks up to Wesley, who backs a couple steps away from her, then grabs a big pulley hanging on a rope and swings it into the Hacksaw demon, shattering its frozen body.
Cordy: "I really hate dating."
Cut to Angel's office. Wesley is dusting Cordy's desk and computer. The board above the coffee machine has 'Welcome back Cordelia' written on it. Angel walks in and drops some magazines on her desk.
Angel: "She likes magazines. I got a few, you know, for when she comes back."
Wesley picks them back up: "Excuse me, I did just neaten this up for her." The door opens and Cordy come in.
Angel: "Cordelia. Hi. You look great."
Wesley: "Marvelous!"
Angel: "I mean it's only been two days. You didn't have to come in so soon."
Wesley: "Yes, if you need more time, Angel can manage. I've been helping out a little... (Angel rolls his eyes up as Wesley bends over the coffee machine) ..Someone forgot to close the filter again."
Angel leans towards Cordy: "Of course if you're ready to come back..."
Cordy smiles: "I'm fine. I had this great audition today for Max Crax, you know, the little crackers?"
Angel: "That's terrific."
Wesley: "Yes."
Angel: "Because, you know, a cracker is something everyone can.."
Wesley: "Eat."
Angel: "Eat."
Cordy with a big smile: "This producer was so nice. He said that I'm his first choice. - We're going out to dinner tonight." The guys look at each other.
Angel: "Uh-huh, tonight?" Cordy nods without looking up from her desk.
Wesley: "Well, best to get back on the horse, I suppose. If he seems..."
Cordy gushing: "He is so sweet. He says that all I have to do is let him impregnate me with his demon master's seed, and I've got the part. (Looks back over her shoulder and smiles at the guys, who look at each other then back at her) Guys, I appreciate all the concern, but I'm *okay*. I mean, it was an ordeal, but I got through it, - and I'm a lot stronger than those loser demon surrogates thought."
Angel: "I'm starting to learn that."
Cordy: "I learned something, too. I learned, uhm, - men are evil? Oh, wait, - I knew that. I learned that LA is full of self-serving phonies. No, - had that one down, too. Uh... s*x is bad?"
Angel with a smile: "We all knew that."
Cordy: "Okay. I learned that I have two people I trust absolutely with my life. - And that part's new." Wesley takes a deep breath, then looks away dabbing his handkerchief at his eye: "Uh, some, uh - allergies." Cordy smiles at Angel, who looks back at her trying not to laugh. | After sleeping with a photographer she had seen a few times, Cordelia wakes up to find herself extremely pregnant; she and several other women are carrying the spawn of a Haxil Beast that uses men as sexual surrogates. Angel and Wesley must find a way to break the demon's psychic control over its human incubators before they deliver-an ordeal likely to be lethal to the hosts. | summ_screen_fd |
Forty-two years after founding one of the nation’s most influential evangelical megachurches, the Rev. Bill Hybels told his congregation Tuesday night that he would step down from the helm of Willow Creek Community Church six months ahead of schedule. His departure comes less than a month after a Chicago Tribune investigation disclosed that Hybels had been the subject of inquiries by church leaders into claims that he ran afoul of church teachings by engaging in inappropriate behavior with women in his congregation — including employees — allegedly spanning decades. The inquiries had cleared Hybels. At times choking back tears, Hybels told the somber crowd at a hastily called meeting at the church’s main campus in South Barrington that, while he continued to enjoy support from within his congregation, the controversy was proving to be a distraction from the church’s mission and work. Referring to his wife, he said, “It has been extremely painful for Lynne and I to see this controversy continue to be a distraction.” He also announced that he would not lead the church’s Global Leadership Summit, an annual event featuring leaders from business, government, entertainment and churches hosted by the Willow Creek Association, a nonprofit dedicated to leadership development, where Hybels had planned to focus his energy after retirement. While saying he believes he has been cleared of all of the accusations, he apologized to the church for how he handled himself. He said he regretted that he reacted in anger when the accusations were made public. “I apologize to you, my church, for a response that was defensive instead of one that invited conversation and learning,” he said. Hybels had previously called the allegations against him “flat-out lies.” He also had said that the multiyear effort by former senior leaders of the church to push for an independent investigation was nothing but “collusion.” On Tuesday, he said he intended to reflect on what has happened and how he reacted. “I feel the need to look deep inside myself and determine what God wants to teach me through all of this,” Hybels said. “I have complete peace about this decision and I’m not going to rush this process. Your prayers would be much appreciated during this upcoming season of reflection.” Hybels also said that while the accusations against him have been misleading and “some entirely false,” he apologized for making choices that put him in situations that could be misconstrued. “I too often placed myself in situations that would have been far wiser to avoid,” he said, adding: “I was naive about the dynamics those situations created. … I commit to never putting myself into similar situations again.” He has said since May 2012 that he would step down in October of this year. Willow Creek, which hosts more than 25,000 worshippers a weekend at its main campus in South Barrington and seven satellite sites, made history in evangelical circles last October by naming a woman to succeed Hybels as lead pastor. Heather Larson, the executive pastor, will take over as chief executive of the megachurch while Steve Carter will become the lead teaching pastor, effective Tuesday. “This is going to take time for all of us to process,” Larson said. “This is not the end of the story. It’s not the end of Bill’s story. It’s not the end of Willow’s story, and it’s certainly not the end of God’s story.” Larson added that leaders would be working on a way to appropriately honor the Hybels family. Hybels’ abrupt departure comes days after one of his accusers, as well as a former teaching pastor and a former longtime elder, posted personal blog entries explaining why they made their concerns public when they did. Vonda Dyer, a former leader of the church’s vocal ministry, who told the Tribune that Hybels kissed and caressed her stomach in an overseas hotel suite 20 years ago, said she stayed silent for decades because she had confronted Hybels privately and believed her encounters were isolated incidents. “I believe the women who have come forward because our stories are so similar,” she wrote. “For the sake of the other women and for the sake of the church, I cannot stay silent.” Betty Schmidt, a current member and former elder, said that current elders had misrepresented what she told them when she met with them 15 months ago. “Women need to know that if they muster the courage to tell their stories church leadership will listen with compassion and fairness,” Schmidt wrote. “That has not happened here. Yet. I hope and pray it will.” The Rev. John Ortberg, pastor of Menlo Park Church in California and a former teaching pastor at Willow Creek, said that women must feel safe coming forward if they have been abused by church leaders. “In a family, all voices should be heard, and every story should be told,” Ortberg wrote. “This should happen in a setting where there is a balance of power and independent judgment can be made about their accounts. The women cannot and must not be silenced.” The announcement left many in the mostly full sanctuary stunned and saddened. After Carter invited the elders and the Hybels family to the stage for a closing prayer, some members of the congregation slowly trickled out of the auditorium. Others stayed in their seats, processing the news. Jim Tofilon said he believes Hybels was a victim of the #MeToo bandwagon. “It’s very destructive,” said Tofilon, who has attended the church since 1991. “It worked. It destroyed an old man’s life. Nothing good came out of it. I hope people making the accusations feel satisfied.” His friend John Stob said that when he received the invitation to the service he knew it “was not going to be a good news night.” “It felt like walking into a funeral home,” said Stob, who has attended the church for 28 years. But after the announcement, he said it felt a little different. “I feel sad and hopeful and proud to be part of this church,” he said. Natalie Sum said she has attended the church since 1984. She was disappointed that the elders didn’t more thoroughly address how they investigated the allegations. While she was sad to see Hybels step down early, she agreed with his decision. A Tribune investigation examined allegations of inappropriate behavior by Willow Creek Community Church Pastor Bill Hybels, documented through interviews with current and former church members, elders and employees, as well as hundreds of emails and internal records. (Erin Hooley / Chicago Tribune) A Tribune investigation examined allegations of inappropriate behavior by Willow Creek Community Church Pastor Bill Hybels, documented through interviews with current and former church members, elders and employees, as well as hundreds of emails and internal records. (Erin Hooley / Chicago Tribune) Last October, the Rev. Bill Hybels stood before worshippers at his packed sanctuary and made a stunning announcement. After 42 years building northwest suburban Willow Creek Community Church into one of the nation’s most iconic and influential churches, Hybels was planning to step down as senior pastor. “I feel released from this role,” he said, adding that he felt called to build on Willow Creek's reach across 130 countries with a focus on leadership development, particularly in the poorest regions of the world. After introducing his successors, he invited church elders onstage at the expansive church to lay hands on them and pray. What much of the church didn’t know was that Hybels had been the subject of inquiries into claims that he ran afoul of church teachings by engaging in inappropriate behavior with women in his congregation — including employees — allegedly spanning decades. The inquiries had cleared Hybels, and church leaders said his exit had nothing to do with the allegations. An investigation by the Chicago Tribune examined those allegations and other claims of inappropriate behavior by Hybels, documented through interviews with current and former church members, elders and employees, as well as hundreds of emails and internal records. The alleged behavior included suggestive comments, extended hugs, an unwanted kiss and invitations to hotel rooms. It also included an allegation of a prolonged consensual affair with a married woman who later said her claim about the affair was not true, the Tribune found. Elders of the church — appointed members who oversee Willow Creek’s administration and pastor — had conducted the reviews after claims about Hybels came to their attention more than four years ago. Pushing for the investigation were two former teaching pastors and the wife of a longtime president of the Willow Creek Association, a nonprofit organization related to the church. Some of those pressing for more scrutiny say the church’s prior investigation had shortcomings in their opinion and at least three leaders of the association’s board resigned over what they believed was an insufficient inquiry. A humanitarian aid agency also chose not to renew its sponsorship of the church’s Global Leadership Summit over concerns about the association’s process for reviewing complaints about senior leaders. Hybels sat down with the Tribune for a lengthy interview this week and at times grew emotional as he flatly denied doing anything improper and dismissed the allegations against him as lies spun with the intent of discrediting his ministry. The pastor said he has built his church with a culture of open conversation, strength and transparency, and said he could not understand why a group of former prominent members of his church — some of them onetime close friends — have “colluded” against him. An investigation by the Chicago Tribune examined allegations of inappropriate behavior by Willow Creek Senior Pastor Bill Hybels, documented through interviews with current and former church members, elders and employees, as well as hundreds of emails and internal records. (Chicago Tribune) (Chicago Tribune) “This has been a calculated and continual attack on our elders and on me for four long years. It’s time that gets identified,” he told the Tribune. “I want to speak to all the people around the country that have been misled … for the past four years and tell them in my voice, in as strong a voice as you’ll allow me to tell it, that the charges against me are false. There still to this day is not evidence of misconduct on my part. “I have a wife and kids and grandkids,” he added, praising the elders for their work to look into the allegations. “My family has had enough and they want the record clear. And they feel strongly supportive of me saying what I have to say to protect my family and clear my family’s name as well.” In the case of the alleged affair, the wife of the association’s outgoing president said the woman confided in her, expressing regret and misgivings. She later denied the alleged affair when contacted by an elder investigating the matter, according to internal documents and interviews. Hybels also denied the alleged affair during an initial inquiry in 2014. The elders said they believed him. Elders have a vital oversight role at Willow Creek. Among their duties is to “carry the ultimate responsibility and authority to see that the church remains on a true biblical course,” the church’s website says. That includes an annual review of the senior pastor, and “confronting those who are contradicting biblical truth or continuing in a pattern of sinful behavior.” Last year, elders retained a Chicago law firm that specializes in workplace issues to look into allegations against Hybels involving three women. According to communications from the law firm reviewed by the Tribune, that investigation was also to include any other evidence “of sex-related sin, whether conducted or condoned by Bill Hybels,” and be limited to his time as a church minister. So far this year, two women have told the Tribune that they had been contacted by an elder to participate in a review. One of those women, Vonda Dyer, declined to participate, citing concerns about the process. Dyer, a former director of the church’s vocal ministry who often traveled with Hybels and whose husband also worked at Willow, told the Tribune that Hybels called her to his hotel suite on a trip to Sweden in 1998, unexpectedly kissed her and suggested they could lead Willow Creek together. She said she hoped Hybels would acknowledge his alleged behavior was wrong and look to God for forgiveness. “I would love for him to experience that kind of redemption,” she said. Erin Hooley / Chicago Tribune Vonda Dyer, a former member and employee of Willow Creek Community Church in South Barrington, at her home in Lewisville, Texas, on March 18, 2018. Vonda Dyer, a former member and employee of Willow Creek Community Church in South Barrington, at her home in Lewisville, Texas, on March 18, 2018. (Erin Hooley / Chicago Tribune) (Erin Hooley / Chicago Tribune) Asked about Dyer’s allegations, Hybels told the Tribune that they are false and that he never did anything inappropriate with her. He had invited her to a conference area of his hotel suite, he said, to discuss adding a song to church programming. He said he was unsure why Dyer would now make the claim. “I’ve never had an unkind word or a falling-out of any kind” with Dyer, Hybels said. “I’ve never had a cross conversation with her. Then, in the last four weeks, a story from (1998) with untrue allegations, pops up right at the same time that these other ones are being molded together to discredit my ministry. And I’m like, how convenient.” The church’s highest-ranking elder, Pam Orr, said she is confident that the church’s inquiries were thorough and reliable. “We felt really good about the conclusions that we came to, and then put the matter to rest,” Orr told the Tribune in an interview. She said the board hired a “very qualified” outside lawyer to conduct an investigation and that he came back with the same conclusion. She said the church was not presented with any clear evidence that Hybels had behaved inappropriately. The board of the Willow Creek Association, a nonprofit founded by Hybels that trains Christian leaders around the globe, also considered investigating the allegations that Hybels had behaved inappropriately, but ultimately dropped the matter, internal documents show. Three association board members resigned, after arguing to the board at the time that they believed the elders’ review had been inadequate. Many of the women who spoke with the Tribune were loath to come forward for fear of betraying a man who had encouraged their leadership in a way that no other pastor had before and undermining a ministry that has transformed thousands of lives. But when they heard there were other women who had similar stories to tell, even in the last year, they said their silence could not last. “That was a bit of a tipping point for me,” said Nancy Beach, the church’s first female teaching pastor and a prominent leader in the evangelical community. She recounted more than one conversation or interaction she felt was inappropriate during moments alone with Hybels over the years. “He changed my life. I wouldn’t have the opportunities I’ve had,” she added. “I know that. I’m very clear on that. I credit him for that. But then there’s this other side.” Humble beginnings One of the nation’s most influential pastors, Hybels grew Willow Creek from a group of zealous 20-somethings inside a Palatine movie theater to one of the largest megachurches in the U.S., hosting more than 25,000 worshippers at its main campus in South Barrington and seven satellite sites any given weekend. The Willow Creek Association has expanded Hybels’ vision to more than 11,000 churches worldwide that share Willow’s core philosophies. From the beginning, he has affirmed women in leadership, tapping them to serve as elders, key volunteers and teaching pastors. Last October, Willow Creek made history in evangelical circles by naming a woman as lead pastor or effectively as chief executive of the megachurch. “I feel so conflicted about the whole situation because I’m so protective of the reputation of the church, not just here but globally,” Beach said. “But I have confidence that the truth matters. Even though he’s 66 years old, there are still young women in his path. I certainly wouldn’t want one of my daughters or anyone else to be in this kind of situation.” The #MeToo movement has spurred women across industries and academia to break their silence about sexual harassment or abuse. In the church community, many say there is a higher standard for religious leaders. “In the Christian world, a consensual affair is still an extremely serious offense,” Beach said. Beach has known Hybels since he arrived on his Harley-Davidson more than 45 years ago at her church in the northwestern suburb of Park Ridge. The intrepid youth pastor had a magnetic effect on the teens. “His leadership horsepower did captivate me, and we have a lot in common in terms of our gift mix,” said Beach, now 60. “We’re both communicators, both leaders.” When Hybels and others set out to start their own church in the Palatine movie theater three years later, Beach and many others from the youth group eventually joined them. Within a year, the church had grown to 1,000 people, many of them spiritual seekers who never had set foot inside a church. Beach served as a key volunteer. She joined the full-time staff in 1984 to oversee the artistic elements of the worship services, and 10 years later she was preaching on a regular basis. In 1992, Hybels expanded Willow’s reach around the world by establishing the Willow Creek Association. He rose to national prominence, eventually serving as a spiritual adviser to President Bill Clinton around the time of the Monica Lewinsky scandal. At least twice a year, a team traveled overseas to host conferences or coach church leaders. During these travels, Hybels scheduled side trips on his own, sometimes to coach, sometimes to catch his breath, Beach said. In 1999, he asked Beach to tack two extra days on to a European trip and meet him on the coast of Spain to coach a church, she said. With two young children and a working husband at home, Beach didn’t want to extend the trip but said she also didn’t want to disappoint her boss. But during their two days there, work took a backseat to leisurely walks, long dinners and probing personal conversations, she said. Over a three-hour dinner, she said he told her that she needed to loosen up and take more emotional risks. He asked her what her most attractive body part was, then told her it was her arms, she said. It also wasn’t the first time he talked about how unhappy he was in his marriage, she recalled. “I’m thinking, ‘As a good friend, I’m going to be a sounding board for him,’ which is totally inappropriate on my part, but I didn’t see it that way at the time,” she said. “I knew him since I was 15. He was my pastor. In all those years, nothing inappropriate had happened with him and me.” But something had changed, she recalled. After dinner, Beach said Hybels invited her to his hotel room for a glass of wine. Before she left, she recalls him giving her an awkwardly long embrace. “He would always say, ‘You don’t know how to hug. That’s not a real hug.’ So it was like a lingering hug that made me feel uncomfortable. But again, I’m trying to prove that I’m this open person.” The next day, Beach recalled, Hybels didn’t seem happy. They didn’t have any more long conversations and flew separate flights home. A week later, he asked Beach to stay after a management team meeting and suggested they not tell anyone about what happened in Spain, she said. “I was so embarrassed. I was like ‘Oh, no. We’re fine.’ And I never did,” she said. “I didn’t tell my husband until recently when all this stuff came out. I just put it in the category of ‘That was really strange.’” She did tell church elders in 2016 about the alleged incident but later declined to cooperate with an inquiry that she believed didn’t meet the criteria of a truly independent investigation. In the years to come, Hybels occasionally invited Beach to his house after midweek worship services to catch up, she said, adding that she stopped going when she realized he invited her only when his wife was away. Hybels, in the interview with the Tribune, insisted that he does not give hugs and denies doing anything inappropriate with Beach, at times bringing his hand down on a table in frustration. Beach had been a close friend, he said, and was a strong enough leader in his church that she would have had the freedom to tell him at the time that she was offended by something he did. He said he recalled not giving Beach latitude to do as much teaching at Willow Creek as she might have liked but said he did not know whether that had triggered her making allegations against him. Regardless, he insisted he did nothing wrong. “When (the allegation) surfaced in 2016, I was like, no, who twisted that one?” Hybels told the Tribune. “I don’t talk about women’s appendages. But there was chatter mostly from other women around (Beach), and I probably said people say they wish they could wear the same outfits you do. That it got brought up as potentially something sexual is maddening.” Erin Hooley / Chicago Tribune Vonda Dyer and her husband, Scott Dyer, at Bent Tree Bible Fellowship in Carrollton, Texas, on March 18, 2018. Vonda Dyer and her husband, Scott Dyer, at Bent Tree Bible Fellowship in Carrollton, Texas, on March 18, 2018. (Erin Hooley / Chicago Tribune) (Erin Hooley / Chicago Tribune) ‘It felt like a proposition’ Raised in rural Iowa in a conservative Christian community that eschewed the idea of women in the pulpit, Vonda Dyer discovered a whole new world at Willow Creek when she came east to attend Wheaton College. She was immediately drawn to Willow’s contemporary sound and approach to evangelism and volunteered on the vocal team. She eventually became a full-time employee in 1997. She met and married her husband, Scott, a youth music pastor also at Willow. Both became part of Hybels’ travel team and accompanied him on more than a dozen trips. But Vonda Dyer said she made it into Hybels’ inner circle and accompanied him on more trips. Since Hybels spent most of his summers at a second home in South Haven, Mich., he occasionally took Dyer and others out on his sailboat, Dyer said. On one such excursion with another female colleague, she said he joked that any woman who drops the winch handle had to give the men on the boat a “blowjob.” Dyer told her husband at the time, an account that he confirmed recently to the Tribune. Hybels denied making the remark, calling it “disgusting.” Scott Dyer said Hybels coached men to avoid being alone with any woman besides the man’s spouse — known as the Billy Graham Rule, highlighted recently by Vice President Mike Pence. Yet Hybels didn’t seem to abide by that rule when it came to Vonda, said her husband of now more than 26 years. Hybels invited Dyer to meet alone several times, they said. “I trusted her character entirely, so I knew nothing would happen,” Scott Dyer said. “But I was like, that feels like a violation of what you’ve told everybody. … I was uncomfortable with it, and I voiced that to her.” On one international trip, Hybels invited Vonda Dyer alone to his hotel room with explicit instructions to exclude her husband who was there too, the Dyers said. On another trip, Hybels called her up to his room and answered the door, freshly showered, wearing slacks with no shirt and just staring at her, she said. He made a casual remark, she said, before she returned downstairs, wondering why she had been called there in the first place. Her husband remembers being told by Vonda about that as well. “It was these situations that were not enough to say that it crossed a major line,” she said, “but enough to make you go, ‘Whoa, what was that?’” Hybels denied that alleged incident occurred. Vonda Dyer said Hybels did cross a line in Sweden in February 1998. Dyer was getting ready to go to bed when Hybels summoned her to his room. Her roommate at the time said in an interview with the Tribune that she remembers picking up the phone and relaying Hybels’ message. Dyer recounted that she went to Hybels’ room where he poured wine and invited her to stretch out on the couch while he sat in a separate chair. She said she presumed it would be a quick chat when he told her that he had taken Ambien, a sleep aid. The conversation quickly turned uncomfortable, she said, when he started complimenting her appearance and criticizing her husband, and suggested they lead Willow together. She said he came over, put his hands on her waist, caressed her stomach and kissed her. “He told me what he thought about how I looked, very specifically, what he thought about my leadership gifts, my strengths,” she said. She recalled Hybels told her she was “sexy.” “That was the night that he painted a picture of what great leaders we would be. We could lead Willow together.” “It felt like a proposition,” she recently told the Tribune. She immediately told him he should stop and go to bed, she recalled. As she left his hotel suite and pulled the door shut, she recalled bursting into tears, still clutching the doorknob. “The Holy Spirit spoke to me: ‘Get out of here,’” she said. “All I heard the Holy Spirit say to me is, ‘If you stay in this room, you will be destroyed.’” The next morning at breakfast, Dyer said Hybels approached her and asked whether anything had happened that would prompt her to tell the elders. She said she recounted the details and told him if he did it again, she would report it. Though Dyer was contacted independently by one elder this year, she has never shared details of what happened with current elders or church investigators, because she didn't think the church would take her allegations seriously. Hybels told the Tribune he never kissed or touched Dyer. “I don’t even want to dignify... I have never touched another woman’s stomach other than my wife. Why in the world would I touch Vonda Dyer’s stomach?” he said. He said he has a strict protocol for taking sleep aids such as Ambien because he never wants to be out of control, and characterized the rest of Dyer’s story as completely false. “This has reached a point that I can’t sit silently by and listen to these allegations anymore,” he said. “I will dispute what she said to my dying breath. She is telling lies.” Dyer recalled that she told her husband about what had happened in Hybels’ hotel suite soon after she returned, which he confirmed to the Tribune. But she said she did not tell church officials at the time, confident she had sufficiently admonished Hybels. She did confide at the time in her “small group” — a quartet of church women who met regularly to support one another’s spiritual journeys. One of those in the group was Betty Schmidt, an original elder at Willow Creek and current member, who confirmed being told about the unwanted kiss in Sweden. As time went on, Dyer watched Hybels and how women acted around him. By 2000, she remembers that she started to suspect he was flirting, if not trying to seduce others too. She said she confronted him and, after listing the specific women, told him to knock it off. He didn’t deny it, she said. “Understood,” she remembers him saying. Hybels told the Tribune he did not recall the conversation. Two years later, she was terminated. She has not alleged any connection between her termination and her confrontation with Hybels and has not taken legal action. When told of Vonda Dyer’s story, church elders said they did not know of it. “We can only act upon what’s brought to our attention,” Orr, the highest-ranking elder, said. Dyer said she is speaking up now because she does not want Hybels’ behavior to continue what she believes is damaging the church. “It is God who saves and redeems and heals,” she said, “but he wants none of this behavior in his people, in his church.” An inquiry begins In the fall of 2013, Leanne Mellado was planning to move to Colorado with her husband, Jimmy, the longtime president of the Willow Creek Association. Amid the goodbyes, a friend asked Leanne Mellado for a private conversation. Something had happened with Hybels, Mellado recalled the woman said. The friend arrived at the Mellados’ home, curled up on the couch in the fetal position and began to sob, Mellado recalled in an interview. Mellado said the woman told her things had started after a meeting at Hybels’ home in Inverness, when the pastor pulled her in for an extended hug, which left her feeling awkward. The relationship progressed through intimate communication over email, the woman said. Mellado told the Tribune that the woman told her the two eventually had consensual encounters, including oral sex. Leanne Mellado and the woman exchanged a series of emails. After seven months, Mellado said she decided that the time had come to tell the elders. It was up to the elders to investigate, uncover the truth and protect everyone, Mellado believed, including this woman. She had an additional concern. Her husband’s new employer, Compassion International, helped sponsor Willow Creek’s Global Leadership Summit. It would be irresponsible, she said, for the charity to renew that sponsorship without making sure the woman’s allegation had been properly vetted. Leanne Mellado emailed the woman in late March 2014 saying it was time for light to shine on what had happened. But the woman did not want to cooperate. “I hope you understand. But if it comes to forcing me, I will be silent,” the woman wrote in an email reviewed by the Tribune. “I feel I should not have trusted you.” The woman did not respond to Tribune requests for comment. But by April that year, Mellado had shared her concerns with Willow Creek’s highest-ranking elder at the time, Brian Johnson. Johnson, who did not respond to requests for comment, told Mellado in text messages that he found 1,150 emails between the woman and Hybels, but was not able to read them. He then alerted other elders about the situation, said Pam Orr and Rob Campbell, two elders at that time. The woman urged Mellado to drop it because the board did not have the woman’s firsthand story, “which for all anybody knows could be a made-up lie,” she wrote in an email. By then it was too late. Mellado had shared the allegations with Johnson. In addition, she also had sought pastoral counsel from John and Nancy Ortberg, former teaching pastors at the church. Nancy Ortberg was then on the board of the Willow Creek Association. Ortberg reached out to Johnson to emphasize the need for a “fair and thorough investigative process … that has high integrity which protects all parties in pursuit of what is true.” But Ortberg and Mellado would allege later that the elders’ review was not as thorough as they had expected. On April 6, 2014, a Sunday, Johnson and another elder asked Hybels about the alleged affair, and he denied it, Campbell said. Though Hybels offered his electronic devices and financial records for review, the elders were unable to read the emails. Hybels told the Tribune that his email had been hacked twice in the last 20 years, so he made sure his messages weren’t archived to prevent sensitive pastoral matters from leaking out. Also on April 6, Orr contacted the woman, who also denied any affair. Later that day, the woman wrote an apologetic email to Mellado. “Some of what I told you happened.. the insinuations, the flirting. But there is no truth to the other things,” she wrote in an email, adding that she had invented the rest because she was angry with Hybels and the church. Hybels remembers meeting two elders backstage that day after he preached, and being told he was being accused of having an affair with a woman he describes as a friend. “It was shocking to me,” he told the Tribune. “I told them in the first 30 seconds of hearing it, ‘This is a lie. There is no truth to this.’” Hybels said he knew the woman was angry with him at the time for not giving her a job. He said the woman showed up on the doorstep of his home the following night sobbing and apologized for having lied to Mellado about Hybels, adding that she had considered taking her own life. “It came like a meteor out of the sky,” he said of the allegation. “To this day, I cannot understand. I have no way of knowing what was going on in her mind.” Less than a week later, Hybels emailed Nancy Ortberg and told her the woman had “made it all up,” Ortberg said. Ortberg, though, was unsatisfied. She and other members of the association board pushed for an independent investigation. Ortberg recalled that her frustrations with the pastor mounted while the initial allegations of an affair were being considered by the Willow Creek Association board. She recalled that during that debate, Hybels told her that the woman at the center of the inquiry was suicidal. He said he continued to offer her counseling — a clear conflict, in Ortberg’s view, for someone in Hybels’ position. The elders, though, didn’t share Ortberg’s alarm because, Campbell said, Hybels was fulfilling his pastoral duty and had kept elders informed every time the woman reached out to him. After hearing this, Ortberg renewed contact with another woman who, eight years earlier, had confided in Ortberg about her own allegedly inappropriate encounter with Hybels. The woman told Ortberg about hugs that went on too long and flirty emails and texts using what she said was the code word “moon” — a reference to a time they had been on Hybels’ boat alone gazing at a full moon in the night sky, Ortberg said. The woman also told Ortberg in 2014 that she had stripped naked on his boat and swam in front of Hybels, Ortberg said. The woman did not allege sexual contact, Ortberg said. Ortberg took notes — reviewed by the Tribune — on the conversation, in 2014. Ortberg says she sent the notes to her board colleagues and tried to persuade the woman to talk with a private investigator. But the woman balked in an emailed reply, saying she did not want to be singled out as an accuser. What she allegedly did on the boat, she said, did not compare to Hybels’ alleged extended and consensual affair with another woman. “The main reason: if (the first woman) does not come clean there is clearly no case — and having my name out there associated with him, as little a deal as it is comparatively, does not do any good other than me looking foolish,” she wrote in an email reviewed by the Tribune. The woman did not respond to Tribune requests for comment. Hybels scoffed when asked by the Tribune about that alleged incident on the boat, saying he did recall the woman swimming at night near his boat, but denied doing anything improper or maintaining an inappropriate relationship with her that included coded messaging. The word moon was not a code word and instead was a reference to leisure time in South Haven, he said. “When the moon would come up over the trees she thought that was the most wonderful manifestation of God’s beautiful creative hand,” Hybels said. “Look at the moon, everybody. This became a thing. She’s a religious, spiritual person.” Once again, Ortberg said her pleas ended in frustration. Church elders informed the association in late 2014 that they considered the matter closed. Without any clear evidence, they had found nothing indicating an improper relationship, elders said. In December, the association board decided to drop the matter, too. Erin Hooley / Chicago Tribune Willow Creek Community Church on Jan. 13, 2018, in South Barrington. Willow Creek Community Church on Jan. 13, 2018, in South Barrington. (Erin Hooley / Chicago Tribune) (Erin Hooley / Chicago Tribune) Mounting concerns For Ortberg and two other board members, the decision was the last straw. Ortberg, along with Jon Wallace, president of Azusa Pacific University, and Kara Powell, executive director of a research center at Fuller Theological Seminary, resigned from the association board in January 2015, later citing what they deemed an inadequate review. “It is our firm belief that leaders should be open to examination of and accountability for our actions,” Wallace and Powell said in a joint statement provided to the Tribune earlier this month. Ortberg told the Tribune that the board’s decision not to pursue another inquiry was, in her opinion, a “complete abdication of fiduciary responsibility,” and left the board vulnerable to litigation if the allegations were proved true. Soon after, there was more fallout from the board’s decision. Compassion International chose not to renew a long-standing sponsorship of the Willow Creek Association’s Global Leadership Summit. “The decision was made, in part, as a result of Compassion’s concerns over WCA’s process for reviewing complaints regarding Willow Creek Community Church senior leadership,” the organization said in a statement. Still, for much of 2015 and 2016, Leanne Mellado and Nancy Ortberg would, together and separately, continue to seek more accountability. It was in this period that Mellado reached out to Hybels’ wife, whom she considered a close friend, to ensure she knew about the allegations swirling around her husband. For nine months, John Ortberg tried unsuccessfully to set up a meeting between Hybels and the four of them. Hybels said he would meet with them as a group only if he could do one-on-one meetings first. Concerned Hybels may try to intimidate them in individual meetings, they refused, Ortberg said. Hybels said the Mellados and Ortbergs are at the center of what he describes as the collusion against him, describing the couples as a kind of “vacuum cleaner” pulling in false accusations. Both couples denied orchestrating a campaign to bring Hybels down by gathering false claims to bring against him. The last four years have been painful, they said. “It’s absolutely not the case,” John Ortberg said. “This information came to us in a way that was unlooked for, unwanted, and it put us in a terrible situation. To say I was motivated to find a problem couldn’t be further from the truth.” He added, “I love Willow Creek dearly.” Pam Orr, the leading Willow Creek elder, said she realized that those pushing for continued investigation were not going to drop the matter unless the elders did something drastic. “By 2016, it had become clear there was a whisper campaign,” Orr said. “It was the overall persistence. Their claim was that we hadn’t done a thorough investigation.” In August 2016, five elders gathered with the Mellados, Ortbergs, Beach and Schmidt for a frank conversation about their concerns. Beach disclosed her alleged hug in Spain. Schmidt disclosed Hybels’ alleged kiss with another woman in Sweden, but didn’t share Dyer’s name. In March 2017, Lynne Hybels wrote to Leanne Mellado, saying she had been shocked and disoriented by the allegation of an affair, and had eventually talked with the woman who Mellado said made the claim. Lynne Hybels wrote that the woman again had denied the affair. “I believed her,” Lynne Hybels wrote, lamenting what she said were breaches in confidence and asking Mellado to “drop this battle, or whatever it is.” Lynne Hybels did not respond to messages seeking comment. Erin Hooley / Chicago Tribune Founding and Senior Pastor Bill Hybels preaches during a service at Willow Creek Community Church on Jan. 13, 2018, in South Barrington. Founding and Senior Pastor Bill Hybels preaches during a service at Willow Creek Community Church on Jan. 13, 2018, in South Barrington. (Erin Hooley / Chicago Tribune) (Erin Hooley / Chicago Tribune) Hiring outside attorney By then, Willow Creek elders had taken a more dramatic step, hiring an outside attorney, Jeffrey Fowler of Laner Muchin in Chicago, a law firm that specializes in workplace issues. Fowler reached out to the Mellados and Ortbergs requesting their participation in a renewed investigation. The Mellados and Ortbergs brought on as an adviser Basyle Tchividjian, a former sex crimes prosecutor in Florida and founder of a nonprofit group the helps victims of sexual abuse and abuse of power by clergy members. Tchividjian later outlined for Fowler what he viewed as deficiencies in Willow Creek church’s earlier handling of the Hybels situation, calling it a “cursory examination of Pastor Hybels’ electronic devices, finances and travel records,” Tchividjian said the Mellados and Ortbergs would participate in Laner Muchin’s investigation only if it was, in their view, “thorough, objective, and independent.” In an interview with the Tribune, Fowler said his work led to no findings of misconduct, even if the investigation was somewhat hampered by not having the full cooperation of many involved in the matter. “After looking at thousands of documents, after interviewing 29 people, and doing as much as I possibly could, I concluded that there was no basis for believing that Pastor Hybels had engaged in a pattern and practice of misconduct, and to the extent any specific incident had been raised with me, I concluded that his actions in those instances were not inappropriate,” Fowler said. In April 2017, Fowler closed his investigation, clearing Hybels. The elders declined to release a full copy of any final report to the Mellados and Ortbergs, and a copy was not provided to the Tribune. Just weeks ago, another woman who alleged Hybels made improper contact met with Fowler and Orr to hear the results of the investigation of her claims. The woman, who asked not to be identified, said she told church leaders last summer that Hybels had put her in several uncomfortable situations, which included remarks about how she looked in her clothes and an invitation to a hotel room. Hybels denied anything improper occurred and provided emails that he said showed he discouraged the woman’s suggestions to go to his hotel room for a glass of wine. The woman also described witnessing an episode with another woman on a boat in South Haven, where she said Hybels had suggestively touched the woman’s bare leg. During the course of the investigation, she said, Fowler asked her to identify the model of the boat and presented a pair of images. He told her later that he questioned the validity of her account because she had failed to identify Hybels’ boat correctly, she said. Willow Creek founding pastor Bill Hybels steps down amid misconduct allegations Weeks after revelations that Willow Creek Community Church investigated misconduct allegations against founding pastor Bill Hybels, the megachurch minister announced his early retirement from the congregation Tuesday night. Hybels, 66, already presented a new leadership structure in October for the Christian church he started in Palatine in 1975, saying he would step down in October 2018. But in the wake of an exhaustive Chicago Tribune report that outlined misconduct allegations — which he vociferously denies — Hybels made his retirement immediate on Tuesday. “This decision was mine and mine alone after a lot of prayer,” an emotional Hybels said in a hastily announced Willow Creek gathering. Hybels cited the distraction from the misconduct allegations as “hindering our elders and church staff,” claiming “some in the wider Christian community continue to be confused and conflicted. “In recent times, I have been accused of many things I simply did not do,” he said. Among the allegations leveled in the March 23 Tribune report were “inappropriate behavior” with women in his congregation dating back to the 1990s, including “suggestive comments, extended hugs, an unwanted kiss and invitations to hotel rooms,” as well as “an allegation of a prolonged consensual affair with a married woman who later said her claim about the affair was not true,” the newspaper reported. On Tuesday, Hybels said he had been “naive about the dynamics those situations created,” but again denied wrongdoing. “I realize now that in certain settings and circumstances in the past I communicated things that were perceived in ways I did not intend, at times making people feel uncomfortable,” he said. The church previously acknowledged it hired a law firm to conduct an investigation, saying they determined the allegations were not credible. “Simply put, there was nothing to report,” the church said in a statement explaining why the investigation was not announced publicly. Willow Creek, now based in South Barrington, has grown to eight Chicago-area locations and is one of the largest evangelical churches in the country. Leaders say it draws 25,000 attendees each week at its locations in Chicago, Glenview, South Barrington, Crystal Lake, Huntley, Lincolnshire and Wheaton. In his October announcement, Hybels said he planned to devote more attention to the Global Leadership Summit, which he launched in 1995 to train faith leaders. But he said Tuesday he wouldn’t be taking part in the summit, which has featured several well-known figures over the years, including former President Bill Clinton and U2’s lead singer Bono. Two new positions were created to replace Hybels, the church said last fall. Prominent pastor Bill Hybels announced Tuesday he is stepping down from his Chicago-area megachurch Willow Creek, just weeks after the Chicago Tribune published allegations of misconduct from several women. Hybels, who with his wife co-founded one of the nation’s largest churches in 1975, was a spiritual adviser to President Bill Clinton around the time of the Monica Lewinsky scandal. He told the church publicly last year that he was planning to step down in October, but he resigned Tuesday, saying he would be a distraction to the church’s ministry. Some members of his congregation shouted “No!” in response to his decision, and the crowd gave him a standing ovation following his address. The stories surrounding Hybels have been part of a series of recent high-profile allegations of sexual misconduct among some evangelical leaders. [‘When you Google evangelicals, you get Trump’: High-profile evangelicals will meet privately to discuss their future] In March, the Chicago Tribune published allegations that Hybels made suggestive comments, extended hugs, an unwanted kiss and invitations to a staff member to hotel rooms. The newspaper also reported allegations of a consensual affair with a married woman, and the woman who said she had an affair later retracted her allegations. Hybels has denied all the allegations and said on Tuesday again that the church’s investigations found no evidence of misconduct. However, he told his congregation he felt attacked and wished he had responded differently. “I apologize to you, my church, for a response that was defensive instead of one that invited conversation and learning,” he said. He said that while most of the members of Willow Creek accepted the church’s findings of investigations, “some of the Christian community continue to be confused and conflicted.” The decision, he said, was his, with the approval of the church’s elders. Hybels also said that he “placed myself in situations that would be far wiser to avoid. I was naive … I commit to never putting myself in similar situations again.” He said he plans to seek counsel during his withdrawl but he plans to return to Willow Creek as his home church. The church conducted its own internal review, and hired an outside attorney who investigated the allegations and told the Tribune his work led to no findings of misconduct by Hybels. In the Tribune’s report, other high-profile evangelical leaders, including John and Nancy Ortberg, suggested that the church’s internal review of the allegations was inadequate. Hybels had said that those leaders had colluded, to which John Ortberg responded in a recent blog post, saying it was “untrue.” “It takes great courage for women to tell their stories,” wrote John Ortberg, who leads a megachurch in California. “In this case, the tremendous courage of several women has been met with an inadequate process that has left them without a refuge and with no way to be assured of a fair hearing.” Hybels has been hugely influential in the megachurch movement, helping to popularize what was known as “seeker-sensitive,” a consumer-friendly approach it has shied away from in the past decade. Willow Creek has also been known for adding women to significant leadership roles in the church. Last year, Willow Creek appointed two pastors to replace him: Heather Larson and Steve Carter. With Larson as a lead pastor, Willow Creek was described last year by Christianity Today as the only major evangelical megachurch with male-female lead pastors who aren’t married. She addressed the women in the congregation after Hybels’s address, saying recent media accounts did not match many congregants’ experience. “I know many of you are confused or frustrated,” she said. “We can respect someone’s story and stand up for our own. You are strong. You have your own voice.” In his address to his church, Hybels said he would also leave the board of the Willow Creek Association, a network of thousands of churches around the world, and he will no longer lead Willow Creek’s Global Leadership Summit, an annual event that draws popular speakers and authors. The church draws more than 25,000 people every weekend at its main campus and seven satellite sites, according to the Tribune. This story has been updated with additional comments from Tuesday. | A pastor and former spiritual adviser to President Clinton has stepped down from one of the largest evangelical churches in the country over allegations of misconduct with women. Bill Hybels, 66, planned to retire from the Chicago-area megachurch he founded 43 years ago in October. But at Willow Creek's Tuesday service in South Barrington, Hybels said allegations of suggestive comments, unwanted kissing, invitations to hotel rooms, and an extramarital affair, raised last month by the Chicago Tribune, had become an "extremely painful" distraction, though the church's own investigation found no wrongdoing, per the Tribune. "In recent times, I have been accused of many things I simply did not do," but "some in the wider Christian community continue to be confused and conflicted," Hybels said, announcing his resignation was effective Tuesday. Hybels did admit he "placed myself in situations that would be far wiser to avoid. I was naive" but "I realize now that... I communicated things that were perceived in ways I did not intend, at times making people feel uncomfortable," per the Washington Post and Chicago Sun-Times. He said, "I feel the need to look deep inside myself and determine what God wants to teach me through all of this," adding, "I have complete peace about this decision." Two successors will now take over: Executive pastor Heather Larson will become chief executive while Steve Carter will become lead teaching pastor, reports the Sun-Times. One of 25,000 worshipers across Willow Creek's Illinois campuses wasn't happy with Tuesday's news, blaming the #MeToo movement, per the Tribune. "It worked. It destroyed an old man's life. Nothing good came out of it," he said. | multi_news |
TECHNICAL FIELD [0001] This application relates in general to a method, apparatus, and article of manufacture for providing adaptive medical therapies utilizing a neural network based learning engine, and more particularly to a method, apparatus, and article of manufacture for implementing an implantable cardiac device having adaptive treatment therapies utilizing a neural network based learning engine. BACKGROUND [0002] Implantable cardiac device have recently become increasingly commonplace in providing cardiac therapies to patients in need of constant monitoring of heart conditions that require immediate treatment. These cardiac devices typically provide a single therapy, or at most a few different therapies, to a patient depending upon a small set of observable parameters regarding the condition of a patient's heart. As a result of the more widespread use of such devices, it is becoming more evident that the operation of these devices need to be customized to provide a more optimal set of therapies to any given patient. [0003] The computational capabilities inherent within implantable devices has increased along with the general increase in computational technology during this same time period. However, performing more complex computations also results in increased power consumption on any given computational platform, including the devices within implantable systems. As a result, many data processing strategies have not been readily utilized in these devices as the computational requirements of such systems has typically been too complex to be realistically utilized. SUMMARY [0004] This application relates in general to a method, apparatus, and article of manufacture for providing adaptive medical therapies utilizing a neural network based learning engine. One possible embodiment of the present invention is a system providing adaptive medical therapies utilizing a neural network based learning engine to a cardiac patient. The system includes a cardiac devices module for providing adaptive medical therapies to the patient, an artificial neural network processing module for training and validating the operation of a neural network, and a communications link between the cardiac device module and the artificial neural network processing module. The cardiac device module includes a cardiac devices data collection module for collecting patient data associated with the cardiac health state of the patient's heart, a cardiac therapy module for applying corrective medical therapies to the patient's heart upon detection of undesired health conditions, and a runtime neural network module for processing collected patient data to determine the corrective medical therapies to be applied using the cardiac therapy module. The artificial neural network processing module includes a cardiac neural network training module for processing collected patient data to determine a set of operating coefficients used by the artificial neural network when determining optimal treatment therapies, a cardiac device interface module for receiving collected patient data from the cardiac device module and for transmitting the set of operating coefficients associated used by the artificial neural network when determining optimal treatment therapies and a collected patient data history data store for maintaining all of the patient collected data history and treatment therapies. The cardiac device runtime neural network module and the neural network training module implement identical networks of nodes. BRIEF DESCRIPTION OF THE DRAWINGS [0005] FIG. 1 illustrates an example embodiment of a cardiac device system having adaptive treatment therapies utilizing a neural network based learning engine according to one possible embodiment of the present invention. [0006] FIG. 2 illustrates an example embodiment of a cardiac device module that is part of an overall system for providing adaptive treatment therapies utilizing a neural network based learning engine according to one possible embodiment of the present invention. [0007] FIG. 3 a illustrates an example embodiment of an artificial neural network server module for use with a cardiac device module that is part of an overall system for providing adaptive treatment therapies utilizing a neural network based learning engine according to one possible embodiment of the present invention. [0008] FIG. 3 b illustrates a cardiac device processing system according to an example embodiment of the present invention. [0009] FIG. 4 illustrates a computing system that may be used to construct various computing systems that may be part of a distributed processing and communications system according to one embodiment of the present invention. [0010] FIG. 5 illustrates another example embodiment of a cardiac device module showing data flow through a system for providing adaptive treatment therapies utilizing a neural network based learning engine according to one possible embodiment of the present invention. [0011] FIG. 6 illustrates another example embodiment of an artificial neural network server module for use with a cardiac device module showing data flow through a system for providing adaptive treatment therapies utilizing a neural network based learning engine according to one possible embodiment of the present invention. DETAILED DESCRIPTION [0012] This application relates in general to a method, apparatus, and article of manufacture for providing adaptive medical therapies utilizing a neural network based learning engine. In the following detailed description of exemplary embodiments of the invention, reference is made to the accompanied drawings, which form a part hereof, and which is shown by way of illustration, specific exemplary embodiments of which the invention may be practiced. These embodiments are described in sufficient detail to enable those skilled in the art to practice the invention, and it is to be understood that other embodiments may be utilized, and other changes may be made, without departing from the spirit or scope of the present invention. The following detailed description is, therefore, not to be taken in a limiting sense, and the scope of the present invention is defined only by the appended claims. [0013] Throughout the specification and claims, the following terms take the meanings explicitly associated herein, unless the context clearly dictates otherwise. The term “connected” means a direct connection between the items connected, without any intermediate devices. The term “coupled” means either a direct connection between the items connected, or an indirect connection through one or more passive or active intermediary devices. The term “circuit” means either a single component or a multiplicity of components, either active and/or passive, that are coupled together to provide a desired function. The term “signal” means at least one current, voltage, or data signal. Referring to the drawings, like numbers indicate like parts throughout the views. [0014] FIG. 1 illustrates an example embodiment of a cardiac device system having adaptive treatment therapies utilizing a neural network based learning engine according to one possible embodiment of the present invention. The cardiac device system includes a cardiac device 200 electronically connected to a patient's heart 100 using electrical connections 101 to obtain data associated with a collection of observable parameters related to the health of the heart 100 as well as provide a collection of medical therapies as needed to maintain a healthy operation for the heart. The cardiac device 200 contains a set of data operating parameters associated with providing these collection of medical therapies that are used to control the operation of the cardiac device when it provides these collection of medical therapies to the heart 100 based upon the collection of observable parameters measured by the cardiac device 200. [0015] The data operating parameters associated with providing the collection of medical therapies may be adjusted periodically to provide a patient with a more optimal set of therapies for a given set of observed parameters for the operation of the patient's heart 100. Types of therapies may include, but is not limited to: pacing at physiological rates to correct a heartbeat that is too slow (pacemaker therapy: PM), pacing at physiological rates to correct for dissynchrony between the contractions of the left and right ventricles (heart failure therapy or cardiac resynchronization therapy: HF/CRT), pacing at high rates to break cycles of tachycardia (anti-tachycardia pacing: ATP), and shock therapy to break tachycardia, particular fibrillation (defibrillation shock or shock). For PM and HF/CRT, the cardiac device typically provides a continuous series of pulses, low voltage and narrow in time, just strong enough to stimulate the heart into contraction at controlled times or intervals. For ATP, the cardiac device typically provides a burst or set of bursts of narrow pulses at higher voltages. For shock, the cardiac device typically provides a high-voltage pulse of characteristic width and shape. In the case of cardiac devices having ATP and/or shock therapy available, the devices are typically designed to respond with appropriate therapy when tachycardia or fibrillation is detected. The therapies may be applied to the atria or the ventricles of the heart, or both, as indicated by the underlying condition of the heart. [0016] One skilled in the art will recognize that many therapy parameters are typically provided for adjusting corrective therapies to best treat the condition of individual patients. Examples may include but not be limited to, the lower rate limit (LRL), atrio-ventricular (AV) delay, maximum tracking rate (MTR), pulse width, and pulse voltage for PM and HF/CRT; the burst cycle length (BCL), number of pulses per burst, number of burst per attempt, change in BCL per burst, and pulse width and voltage for ATP; and energy, polarity, waveform (monophasic or biphasic), and duty cycle (percentage of discharge time spent in one polarity of a biphasic waveform) for shock. The values of the applicable therapy parameters, in conjunction with the patient's observed parameters, are provided to an ANN processing system. Values stored in cardiac device 200 are provided to the ANN processing system 300 over communications link 400. Observed parameters for the patient may be associated with past therapy attempts, both those existing before the therapy and those existing in response to this therapy, or may include general trending information in the patient's history, not directly associated with a particular therapy attempt. The ANN processing system 300 then determines an optimized set of applicable therapy parameters and communicates them to the cardiac device 200 over communications link 400. [0017] The communications link 400 may consist of any electrical communications link between the processing devices within the cardiac device 200 and the ANN processing system 300. One skilled in the art will recognize that such a link may be constructed using an RF communications link, an IR communications link, an electrical communications link, an audio based communications link, or any other communications link that provides digital communications between these devices without deviating from the spirit and scope of the present invention as recited within the attached claims. [0018] FIG. 2 illustrates an example embodiment of a cardiac device module that is part of an overall system for providing adaptive treatment therapies utilizing a neural network based learning engine according to one possible embodiment of the present invention. The cardiac device module 200 includes a set of processing modules to perform the operations associated with collecting data, providing therapies and communicating with the ANN processing system 300. These processing modules include a cardiac data interface module 201, a cardiac therapy module 202, a cardiac device data collection module 203, a cardiac neural network runtime module 204, an ANN processing system interface module 205 and a local data storage module 210. [0019] The cardiac data interface module 201 interfaces the cardiac device module 200 to the patient's heart 100 to collect data regarding the current health of the heart. This data may include but is not limited to a collection of observable parameters such as values for an A Rate, a V Rate, an A Rate dispersion, a V Stability, an AV pattern, an NSR template, an arrhythmia template, a detected or classified depolarization morphology, a measure of patient activity such as derived from a Minute Ventilation (MV) sensor and/or an accelerometer, a measure of hemodynamic function such as derived from a hemodynamic sensor, a number of past attempts required to treat a given observed condition, an identification of a particular therapy that provided an effective treatment of an observed condition, an identification of a particular therapy that provided an ineffective treatment of an observed condition, an age of a patient, an identification of medications known to be taken by the patient, etc and other environmental factors, including patient's age, gender, and vital statistics at last observed exertion. [0020] The cardiac therapy module 202 provides the medical therapies by cardioverting/defibrillating a heart. This module obtains its operating instructions from the cardiac neural network runtime module 204 to provide either no therapy when no therapy is warranted or when noise is observed, to provide one or more therapies from the collection of medical therapies as defined previously in an optimal or most efficient manner depending upon the collection of observable parameters. [0021] The cardiac device data collection module 203 collects values observed by the cardiac data interface module 201 immediately preceding and during an event that gives rise to the cardiac therapy module 202 providing therapies to a patent. These collected values are stored within the local data storage module 210 for later use. These values are typically transmitted to the ANN processing system to update the operating parameters associated with the therapies; however, such collected values may also be used by various cardiac device modules during the operation of the cardiac device 200. For example, the collected values may include an indication of the last successful therapy to correct a particular observed condition. This last successful therapy may be utilized as a first attempted therapy if the particular observed condition recurs at a later date and time. [0022] The cardiac neural network runtime module 204 and ANN processing system interface module 205 provide a mechanism for determining a course of treatment to deliver an optimal therapy to a patient for a given set of observed conditions. In the system according the present invention, a run-time only version of an artificial neural network is included within the cardiac device 200. The runtime version of the neural network accepts input data associated with observed conditions of a patient's heart and generates a particular therapy or sequence of therapies to be utilized to correct the observed condition. The runtime module is contrasted with the neural network training module found within the ANN processing system 300 that analyzes the data collected in the cardiac device data collection module 203 to determine a particular therapy or sequence of therapies to be utilized. [0023] The artificial neural network disclosed herein corresponds to a generic neural network of no particular topology for the network of nodes contained therein. The neural network typically utilizes a form of competitive learning for the operation of the nodes within the network. Within these learning networks, a large number of data vectors are distributed in a high-dimensional space. These data vectors represent known values for experimental data that typically reflect a probability distribution of the input observed data. From this probability distribution representation, predictions for unknown values for similar input data may be determined. [0024] In all of these learning networks, the networks are typically presented a set of input data that possesses a corresponding set of results data. From these data values, the network of nodes “learns” a relationship between the input data and its corresponding results data. In this process, the probability distribution relationship is estimated using the multi-dimensional network of nodes. This relationship is represented within a set of artificial neural network coefficients for a particular topology of nodes. [0025] One skilled in the art will recognize that competitive learning networks include a nearly infinite number of network topologies that may be used to represent a particular probability distribution relationship without deviating from the spirit and scope of the present invention as recited within the attached claims. In addition, artificial neural networks may utilize various well-known algorithm architectures, including hard-competitive learning (i.e. “winner-take-all” learning), soft competitive learning without a fixed network dimensionality, and soft competitive learning with a fixed network dimensionality, to specify an artificial neural network according to the present invention as recited within the attached claims. Each of these algorithm architectures represents the same probability distribution relationship; however each of the various algorithm architectures better optimize corresponding processing parameters which are often mutually exclusive with each other. These parameters include error minimization or the minimization of an expected quantization error, entropy maximization for the reference vectors used within a network, and topology-preserving or feature mapping architectures that attempt to map high-dimensional inputs signals onto lower-dimensional structures in a manner that attempts to preserve similar relationships found within the original data within the post-mapping data. As such, any of these types of algorithm architectures may be used to construct an artificial neural network without deviating from the spirit and scope of the present invention as recited within the attached claims. [0026] The ANN processing system interface module 205 provides the data communications functions between the cardiac device 200 and the ANN processing system 300 over the communications link 400. This module transfers the collected data from the data storage module 210 to the ANN processing system 300. This module 205 also accepts parameters used by the cardiac neural network runtime module 204 from the ANN processing system 300 after updated parameters are generated. [0027] FIG. 3 a illustrates an example embodiment of an artificial neural network server module for use with a cardiac device module that is part of an overall system for providing adaptive treatment therapies utilizing a neural network based learning engine according to one possible embodiment of the present invention. The artificial neural network processing system 300 comprises a training module 303, a prediction module 305, and a database of network node coefficients 310. The training module 303 is used with a set of collected data that possesses a corresponding set of observed parameter values obtained by the cardiac device module 200 to generate a set of network node coefficients that represent a probability distribution relationship for an observed parameter data—observed results data set for a particular neural network topology and algorithm architecture. The training module 303 includes a data learning input module that receives the observed parameter data—observed results data set generated using the learning process described above. The training module 303 also includes an ANN node training module that processes the observed parameter data—observed results data set to generate the coefficients used to specify the probability distribution relationship and an ANN coefficient storage module 310 for storing the coefficients that have been previously generated for later use. [0028] The data processing within the training module 303 may proceed in a batch processing fashion in which all of the vectors within the observed parameter data—observed therapy data set are processed at a single time. In such a process, the observed parameter data—observed therapy data set is received by the interface module 301 from the cardiac device 200, processed by the training module 303, and the generated coefficients are placed within the database 310 by the prediction module 305. Alternatively, the observed parameter data—observed therapy data set may be processed as a sequence of smaller data sets in which the observed parameter data 315 -observed therapy parameter 316 data set data values are generated at different times. In such a process, the training module 303 uses the previously stored coefficients retrieved by the storage module along with a new small data set provided by the input module 312 to generate an updated set of coefficients as shown in FIG. 3 b. These updated coefficients may be once again stored within the database 310 for use at a later time. [0029] FIG. 3 b illustrates an artificial neural network 331 implemented as a set of processing modules according to the present invention. Once an artificial neural network 331 has been trained as discussed above, a prediction module 321 may be used to predict, or classify, a particular test data value 325. The prediction module 321 includes a data prediction input module 322, an ANN prediction module 323, and an ANN output module 324. The data prediction input module 322 receives the input test data generated as described above for use in the prediction module. The ANN prediction module 323 receives and utilizes the network coefficient values for the neural network from the ANN coefficient database 332 to predict the possible result for the probability distribution relationship specified within the neural network. This output value 326 is used by the ANN prediction module 305 to determine all possible values for a given observed therapy parameter, to determine an output set of therapy parameter value. This slope value is then output for later use in ranking and classifying the individual therapies used to determine preferred course of therapy for any given observed condition. [0030] The operation of the ANN processing system 300 typically involves an operator connecting the ANN processing system 300 to the cardiac device 200 and controlling the operation of the devices using a monitor 341 and keyboard. The user interacts with a user interface module 304 to instruct the devices to transfer collected data from the cardiac device, to input additional patient data, to train the neural network and generate an updated set of neural network node coefficients, and to upload the updated set of values to the cardiac device 200. [0031] FIG. 4 illustrates a computing system that may be used to construct various computing systems that may be part of a distributed processing and communications system according to one embodiment of the present invention. In an exemplary embodiment of a ANN processing system 400, computing system 400 is operative to provide a neural network training and data collection system. Those of ordinary skill in the art will appreciate that the neural network training and data collection system 400 may include many more components than those shown with reference to a computing system 300 shown in FIG. 3. However, the components shown are sufficient to disclose an illustrative embodiment for practicing the present invention. As shown in FIG. 3, neural network training and data collection system 300 is connected to a cardiac device 200, or other devices as needed. Those of ordinary skill in the art will appreciate that a network interface unit 410 includes the necessary circuitry for connecting neural network training and data collection system 400 to a network of other computing systems, and is constructed for use with various communication protocols including the TCP/IP protocol. Typically, network interface unit 410 is a card contained within neural network training and data collection computing system. [0032] ANN processing system 400 also includes processing unit 412, video display adapter 414, and a mass memory, all connected via bus 422. The mass memory generally includes RAM 416, ROM 432, and one or more permanent mass storage devices, such as hard disk drive 428, a tape drive 438, CD-ROM/DVD-ROM drive 426, and/or a floppy disk drive. The mass memory stores operating system 420 for controlling the operation of ANN processing system 400. It will be appreciated that this component may comprise a general purpose server operating system as is known to those of ordinary skill in the art, such as UNIX, MAC OS™, LINUX™, or Microsoft WINDOWS NT®. Basic input/output system (“BIOS”) 418 is also provided for controlling the low-level operation of processing system 400. [0033] The mass memory as described above illustrates another type of computer-readable media, namely computer storage media. Computer storage media may include volatile and nonvolatile, removable and non-removable media implemented in any method or technology for storage of information, such as computer readable instructions, data structures, program modules or other data. Examples of computer storage media include RAM, ROM, EEPROM, flash memory or other memory technology, CD-ROM, digital versatile disks (DVD) or other optical storage, magnetic cassettes, magnetic tape, magnetic disk storage or other magnetic storage devices, or any other medium which can be used to store the desired information and which can be accessed by a computing device. [0034] The mass memory also stores program code and data for providing a software development and neural network analysis and training system. More specifically, the mass memory stores applications including neural network based training program 430, programs 434, and similar analysis tool applications 436. ANN processing program 400 includes computer executable instructions which are executed to perform the logic described herein. [0035] ANN processing system 400 also comprises input/output interface 424 for communicating with external devices, such as a mouse, keyboard, scanner, or other input devices not shown in FIG. 4. Likewise, ANN processing system 400 may further comprise additional mass storage facilities such as CD-ROM/DVD-ROM drive 426 and hard disk drive 428. Hard disk drive 428 is utilized by ANN processing system 400 to store, among other things, application programs, databases, and program data used by ANN processing system application program 430. The operation and implementation of these databases is well known to those skilled in the art. [0036] FIG. 5 illustrates another example embodiment of a cardiac device module showing data flow through a system for providing adaptive treatment therapies utilizing a neural network based learning engine according to another example embodiment of the present invention. As discussed above with respect to FIG. 3, a cardiac data collection module 501 obtains data from electrical signals measured on a patient's heart 100. This data may include any number of observable parameters including values for an A Rate, a V Rate, an A Rate dispersion, a V Stability, an AV pattern, an NSR template, an arrhythmia template, a detected or classified depolarization morphology, a measure of patient activity such as derived from a Minute Ventilation (MV) sensor and/or an accelerometer, a measure of hemodynamic function such as derived from a hemodynamic sensor, a number of past attempts required to treat a given observed condition, an identification of a particular therapy that provided an effective treatment of an observed condition, an identification of a particular therapy that provided an ineffective treatment of an observed condition, an age of a patient, an identification of medications known to be taken by the patient, etc. This collected data may be passed to a cardiac data storage module 510 for storage into a local data store 511 for later use after being transmitted to the ANN processing system 300 by a ANN system interface module 512. [0037] This data may also be passed to a cardiac data transformation module 502 that combines one or more of the above collected data values into a set of one or more health indications for use by the artificial neural network runtime module 503. For example, the collected data values may be combined to generate a health state of the patient's heart 100 that possesses various values such as good, average and bad, poor or similar state classifications. These classifications may then be used by the ANN runtime module 503 to generate an appropriate therapy, if any, is to be applied to the patent's heart. [0038] Of course, one skilled in the art will recognize that the transformation module 502 may be eliminated in its entirety and the collected data used by the ANN runtime module 503 without deviating from the spirit and scope of the present invention as recited within the attached claims. The elimination of the transformation module 502 may increase the complexity of the neural network used within the cardiac device as well and the processing needed to utilize such a network. However, the use of the transformation module 502 reduces the amount of data available to the ANN runtime module, and as such may reduce the accuracy of the prediction function associated with selecting an optimal therapy. [0039] The ANN runtime module 503 uses the data received from the transformation module 502 to generate commands to a therapy module 504 that provides the course of therapies provided to a patient. As discussed above, these may include pacing, ATP, low energy therapy and max energy therapy depending upon the observed data. [0040] FIG. 6 illustrates another example embodiment of an artificial neural network server module for use with a cardiac device module showing data flow through a system for providing adaptive treatment therapies utilizing a neural network based learning engine according to another possible embodiment of the present invention. The ANN processing system receives collected patient data from the cardiac device 200 through a cardiac device interface module 601 for use in training an artificial neural network resident both in the cardiac device as well as the ANN processing system 300. Typically, this operation occurs in a medical facility on a periodic basis as a patient seeks to optimize the operation of the cardiac device module 200. The frequency of these update operations may vary from patient to patient depending upon a number of factors including the nature of the cardiac problems of the particular patient, any changes in the nature of the cardiac problems of the particular patient, and the effectiveness of the adaptive treatment therapies provided by the cardiac device module for the particular patient. [0041] The collected patient data is passed to an ANN patient data processing module 602 which performs preprocessing and data transformation similar to processing that occurs in the cardiac device module 200. Once the data has been preprocessed as needed, the data is passed to an artificial neural network module 604 to update and train the neural network. This neural network module 604 includes any training and validation modules needed to calculate the coefficients associated with the nodes within the neural network. The form and operation of the neural network module exactly matches the nodes in the runtime neural network located within the cardiac device module 200. The artificial neural network module processes the collected patient data along with other data received from a therapy data store 610 using a patient data module 605 that defined the course of therapy used within the cardiac device 200. The patient data module 605 may also receive data from an operator of the ANN processing system 300 for use in training the neural network in the neural network module 604. [0042] The coefficients associated with the nodes of the neural network are passed to an ANN processing network update module 603. This module 603 formats and passes the data updates to the cardiac device module 200 for use in providing adaptive therapies to patients. The update module 603 also interacts with the cardiac device interface module 601 to perform the data communications with the cardiac device module 200 over the communications link 400. [0043] The sequence of possible therapies to be applied by the cardiac device module 200 may include any number of possible treatment options that are applied using a sequence of criteria. The rules and control over when any of the possible treatment options are applied to a patient is embedded within the operation of the artificial neural network. The runtime module in the cardiac device implements these possible treatment options through the application of the artificial neural network to patient data collected by the cardiac device module 200. One skilled in the art will recognize that a number of possible variations on the treatment options as applied to a given patient may arise utilizing the artificial neural network as recited herein within the attached claims without deviating from the spirit and scope of the present invention. [0044] FIG. 4 illustrates an example of a suitable operating environment in which the invention may be implemented. The operating environment is only one example of a suitable operating environment and is not intended to suggest any limitation as to the scope of use or functionality of the invention. Other well known computing systems, environments, and/or configurations that may be suitable for use with the invention include, but are not limited to, personal computers, server computers, hand-held or laptop devices, multiprocessor systems, microprocessor-based systems, programmable consumer electronics, network PCs, minicomputers, mainframe computers, distributed computing environments that include any of the above systems or devices, and the like. [0045] The invention may also be described in the general context of computer-executable instructions, such as program modules, executed by one or more computers or other devices. Generally, program modules include routines, programs, objects, components, data structures, etc. that perform particular tasks or implement particular abstract data types. Typically the functionality of the program modules may be combined or distributed as desired in various embodiments. [0046] Processing devices attached to a communications network typically includes at least some form of computer readable media. Computer readable media can be any available media that can be accessed by these devices. By way of example, and not limitation, computer readable media may comprise computer storage media and communication media. Computer storage media includes volatile and nonvolatile, removable and non-removable media implemented in any method or technology for storage of information such as computer readable instructions, data structures, program modules or other data. Computer storage media includes, but is not limited to, RAM, ROM, EEPROM, flash memory or other memory technology, CD-ROM, digital versatile disks (DVD) or other optical storage, magnetic cassettes, magnetic tape, magnetic disk storage or other magnetic storage devices, or any other medium which can be used to store the desired information and which can be accessed by processing devices. [0047] Communication media typically embodies computer readable instructions, data structures, program modules or other data in a modulated data signal such as a carrier wave or other transport mechanism and includes any information delivery media. The term “modulated data signal” means a signal that has one or more of its characteristics set or changed in such a manner as to encode information in the signal. By way of example, and not limitation, communication media includes wired media such as a wired network or direct-wired connection, and wireless media such as acoustic, RF, infrared and other wireless media. Combinations of any of the above should also be included within the scope of computer readable media. [0048] Additionally, the embodiments described herein are implemented as logical operations performed by a programmable processing devices. The logical operations of these various embodiments of the present invention are implemented (1) as a sequence of computer implemented steps or program modules running on a computing system and/or (2) as interconnected machine modules or hardware logic within the computing system. The implementation is a matter of choice dependent on the performance requirements of the computing system implementing the invention. Accordingly, the logical operations making up the embodiments of the invention described herein can be variously referred to as operations, steps, or modules. [0049] While the above embodiments of the present invention describe a distributed processing for providing adaptive medical therapies utilizing a neural network based learning engine, one skilled in the art will recognize that the use of a particular computing architecture for a display computing system and a web server are merely example embodiments of the present invention. It is to be understood that other embodiments may be utilized and operational changes may be made without departing from the scope of the present invention as recited in the attached claims. [0050] As such, the foregoing description of the exemplary embodiments of the invention has been presented for the purposes of illustration and description. They are not intended to be exhaustive or to limit the invention to the precise forms disclosed. Many modifications and variations are possible in light of the above teaching. It is intended that the scope of the invention be limited not with this detailed description, but rather by the claims appended hereto. The present invention is presently embodied as a method, apparatus, and article of manufacture for providing an ANN processing system 400 for providing adaptive medical therapies utilizing a neural network based learning engine. | A cardiac device system for implementing a cardiac device having adaptive treatment therapies utilizing a neural network based learning engine includes an implantable cardiac device module and an external data processing system for specifying the operating characteristics of the cardiac device module. Both the cardiac device module and the external processing system possess an artificial neural network to specify the operation of the cardiac device module as it provides adaptive treatment therapies. The external data processing system includes a complete neural network module that trains and validates the operation of the neural network to match the optimal treatment options with a received set of collected patient data. In contrast, a runtime neural network module that only provides real time operation of the neural network using collected patient data is located within the cardiac device module. The cardiac device module and the external processing module communicate with each other to pass collected patient data from the cardiac device module to the external processing system when the operation of the neural network is to be updated. The cardiac device module and the external processing module also communicate with each other to pass operating coefficients for the neural network back from the external processing system to the cardiac device module once these coefficients are updated. | big_patent |
Mycobacterial pathogens are the causative agents of chronic infectious diseases like tuberculosis and leprosy. Autophagy has recently emerged as an innate mechanism for defense against these intracellular pathogens. In vitro studies have shown that mycobacteria escaping from phagosomes into the cytosol are ubiquitinated and targeted by selective autophagy receptors. However, there is currently no in vivo evidence for the role of selective autophagy receptors in defense against mycobacteria, and the importance of autophagy in control of mycobacterial diseases remains controversial. Here we have used Mycobacterium marinum (Mm), which causes a tuberculosis-like disease in zebrafish, to investigate the function of two selective autophagy receptors, Optineurin (Optn) and SQSTM1 (p62), in host defense against a mycobacterial pathogen. To visualize the autophagy response to Mm in vivo, optn and p62 zebrafish mutant lines were generated in the background of a GFP-Lc3 autophagy reporter line. We found that loss-of-function mutation of optn or p62 reduces autophagic targeting of Mm, and increases susceptibility of the zebrafish host to Mm infection. Transient knockdown studies confirmed the requirement of both selective autophagy receptors for host resistance against Mm infection. For gain-of-function analysis, we overexpressed optn or p62 by mRNA injection and found this to increase the levels of GFP-Lc3 puncta in association with Mm and to reduce the Mm infection burden. Taken together, our results demonstrate that both Optn and p62 are required for autophagic host defense against mycobacterial infection and support that protection against tuberculosis disease may be achieved by therapeutic strategies that enhance selective autophagy. Autophagy is a fundamental cellular pathway in eukaryotes that functions to maintain homeostasis by degradation of cytoplasmic contents in lysosomes [1]. During autophagy, protein aggregates or defective organelles are sequestered by double-membrane structures, called isolation membranes or phagophores, which mature into autophagosomes capable of fusing with lysosomes. Autophagy was previously considered a strictly non-selective bulk degradation pathway. However, recent comprehensive studies have highlighted its selective ability. Selective autophagy depends on receptors that interact simultaneously with the cytoplasmic material and with the autophagosome marker microtubule-associated protein 1 light chain 3 (Lc3), thereby physically linking the cargo with the autophagy compartment [2,3]. Different selective autophagy pathways are classified according to their specific cargo; for example, mitophagy is the pathway that degrades mitochondria, aggrephagy targets misfolded proteins or damaged organelles, and xenophagy is directed against intracellular microorganisms. Recent studies have firmly established xenophagy as an effector arm of the innate immune system [4–6]. The xenophagy pathway targets microbial invaders upon their escape from phagosomes into the cytosol, where they are coated by ubiquitin. These ubiquitinated microbes are then recognized by selective autophagy receptors of the Sequestosome 1 (p62/SQSTM1) -like receptor (SLR) family, including p62, Optineurin (OPTN), NDP52, NBRC1, and TAX1BP1 [5]. In addition to targeting microbes to autophagy, SLRs also deliver ubiquitinated proteins to the same compartments. It has been shown that the processing of these proteins into neo-antimicrobial peptides is important for elimination of the pathogen Mycobacterium tuberculosis in macrophages [7]. M. tuberculosis (Mtb) is the causative agent of chronic and acute tuberculosis (Tb) infections that remain a formidable threat to global health, since approximately one-third of the human population carry latent infections and 9 million new cases of active disease manifest annually. Current therapeutic interventions are complicated by increased incidence of multi-antibiotic resistance of Mtb and co-infections with Human Immunodeficiency Virus (HIV). Despite decades of extensive research efforts, the mechanisms of how Mtb subverts the host’s innate immune defenses are incompletely understood, which poses a bottleneck for developing novel therapeutic strategies [8]. Because of the discovery of autophagy as an innate host defense mechanism, the potential of autophagy-inducing drugs as adjunctive therapy for Tb is now being explored [9]. Many studies have shown that induction of autophagy in macrophages by starvation, interferon-γ (IFN-γ) treatment, or by autophagy-inducing drugs, promotes maturation of mycobacteria-containing phagosomes and increases lysosome-mediated bacterial killing [7,10–12]. Furthermore, it has been shown that the ubiquitin ligase Parkin and the ubiquitin-recognizing SLRs p62 and NDP52 are activated by the escape of Mtb from phagosomes into the cytosol [13,14]. Subsequently, the ubiquitin-mediated xenophagy pathway targets Mtb to autophagosomes [13,14]. Parkin-deficient mice are extremely vulnerable to Mtb infection [14]. However, a recent study has questioned the function of autophagy in the host immune response against Mtb, since mutations in several autophagy proteins, with the exception of ATG5, did not affect the susceptibility of mice to acute Mtb infection [15]. The susceptibility of ATG5-deficient mice in this study was attributed to the ability of ATG5 to prevent a neutrophil-mediated immunopathological response rather than to direct autophagic elimination of Mtb. In the same study, loss of p62 did not affect the susceptibility of mice to Tb, despite that p62 has previously been shown to be required for autophagic control of Mtb in macrophages [7,15]. These different reports suggest that Mtb is able to suppress autophagic defense mechanisms and that the host requires strong autophagy induction to overcome such bacterial virulence mechanisms [12]. Taken together, the role that autophagy plays in Tb is complex and further studies are required to determine if pharmacological intervention in this process is useful for a more effective control of this disease. In this study, we utilized zebrafish embryos and larvae to investigate the role of selective autophagy during the early stages of mycobacterial infection, prior to the activation of adaptive immunity. Zebrafish is a well-established animal model for Tb that has generated important insights into host and bacterial factors determining the disease outcome [16,17]. Infection of zebrafish embryos with Mycobacterium marinum (Mm), a pathogen that shares the majority of its virulence factors with Mtb, results in the formation of granulomatous aggregates of infected macrophages, considered as a pathological hallmark of Tb [17–19]. Using a combination of confocal imaging in GFP-Lc3 transgenic zebrafish larvae and correlative light and transmission electron microscopy, we have previously shown that double membrane autophagic vesicles capture single Mm bacteria and fuse with larger Mm-containing degradative compartments [20,21]. Furthermore, we found that the DNA-damage regulated autophagy modulator Dram1 is upregulated during infection by the central Myd88-NFκB innate immunity signaling pathway and protects the zebrafish host against Mm infection by a p62-dependent mechanism [21]. However, the role of p62 and other SLRs in host defense against Mm remains to be further elucidated. p62 is known to function cooperatively with OPTN in xenophagy of Salmonella enterica [22–24]. Both these SLRs are phosphorylated by Tank-binding kinase 1 (TBK1) and bind to different microdomains of ubiquitinated bacteria as well as interacting with Lc3 [23,25]. While several studies have implicated p62 in autophagic defense against Mtb, OPTN has thus far not been linked to control of mycobacterial infection [7,13,24–26]. We found gene expression of p62 and optn to be coordinately upregulated during granuloma formation in zebrafish larvae [27], and set out to study the function of these SLRs by CRISPR/Cas9-mediated mutagenesis. We found that either p62 or Optn deficiency increased the susceptibility of zebrafish embryos to Mm infection, while overexpression of p62 or optn mRNAs enhanced Lc3 association with Mm and had a host-protective effect. These results provide new in vivo evidence for the role of selective autophagy as an innate host defense mechanism against mycobacterial infection. Phagosomal permeabilization and cytosolic escape of Mtb is known to induce the STING-dependent DNA-sensing pathway, resulting in ubiquitination and targeting of bacteria to autophagy [13]. We have previously shown that this pathway is also functional in zebrafish larvae infected with Mm and that a failure to induce autophagy reduces host resistance [21]. However, it had not been formally demonstrated that Mm bacteria are ubiquitinated in this model. To examine whether ubiquitin interacts with Mm and Lc3 during infection of zebrafish, we infected embryos at 28 hours post fertilization (hpf) and performed immunostaining for ubiquitin at 1,2, and 3 days post infection (dpi), time points at which the early stages of tuberculous granuloma formation can be observed (Fig 1A). This process of granuloma formation is known to be induced by infected macrophages, which attract new macrophages that subsequently also become infected [28]. Developing granulomas also attract neutrophils and usually contain extracellular bacteria released by dying cells [29]. Using the zebrafish Tg (CMV: GFP-map1lc3b) autophagy reporter line [30], hereafter referred to as GFP-Lc3, we observed that around 3% and 9% of mCherry-labelled Mm clusters are targeted by GFP-Lc3 at 1 and 2 dpi, respectively, which increases to non-quantifiable levels at 3 dpi because of the increasing numbers and size of granulomas (Fig 1B and 1C). Mm is known to be rapidly phagocytosed after intravenous infection and replicate inside zebrafish macrophages [31,32]. To confirm that the GFP-Lc3 targeting of Mm clusters occurs in macrophages, we used mCrimson-labelled Mm to infect double transgenic embryos with the GFP-Lc3 marker and the macrophage-specific and membrane-localized mpeg1: mCherryF marker [33] (Fig 1D). The results of GFP-Lc3 imaging were confirmed by Western blot, showing that LC3-II protein levels–indicative of autophagosome formation–gradually increased during Mm infection compared to uninfected controls (Fig 1E). Using a FK2 ubiquitin antibody, which can recognize monoubiquitinated cell surface molecules as well as polyubiquitin chains, we observed that ubiquitin co-colocalized with approximately 4% and 10% of the Mm clusters at 1 and 2 dpi, respectively (Fig 1F and 1G). Ubiquitin staining strongly increased by 3 dpi, not only in association with Mm clusters but also in the surrounding tissue (Fig 1F). In agreement, we observed by Western blot detection that Mm infection increased general levels of protein ubiquitination at 3 dpi (Fig 1H). In addition, we found that ubiquitin and GFP-Lc3 co-localized at Mm clusters (Fig 1I). Ubiquitin has been found to associate with cytosolic bacteria as well as with damaged membranes of phagosomes [34]. While we could not distinguish between these possibilities, we observed several instances of a tight association between ubiquitin signal and Mm bacteria, suggestive of direct ubiquitination of the bacteria (Fig 1F (inset) and Fig 1I). Collectively, these data demonstrate that Mm clusters are marked by ubiquitin and that overall ubiquitination levels are increased during infection in the zebrafish model, which coincides with autophagic targeting of bacteria. Since ubiquitinated bacteria are targets for members of the sequestosome-like receptor family, we compared the protein sequences of its members p62, Optn, Calcoco2 (Ndp52), Nbrc1, and Tax1bp1 between human, zebrafish and other vertebrates, showing a high overall degree of conservation (S1A Fig). We focused this study on two members of the family, p62 and optn, which are transcriptionally induced during Mm infection of zebrafish based on published RNA sequencing data [27] and show strong similarity with their human orthologues in the ubiquitin-binding domains (UBA in p62 and UBAN in Optn) and Lc3 interaction regions (LIR) (S1B Fig). With the aim to investigate the functions of Optn and p62 in anti-mycobacterial autophagy, we utilized CRISPR/Cas9 genome editing technology to generate mutant zebrafish lines. We designed short guide RNAs for target sites at the beginning of coding exons 2 of the optn and p62 genes, upstream of the exons encoding the ubiquitin and Lc3 binding regions, such that the predicted effect of CRISPR mutation is a complete loss of protein function (Fig 2A). A mixture of sgRNA and Cas9 mRNA was injected into zebrafish embryos at the one cell stage and founders carrying the desired mutations were outcrossed to GFP-Lc3 zebrafish (Fig 2B). The established optn mutant allele carried a 5 nucleotides deletion at the target site, which we named optnΔ5n/Δ5n (ZFIN allele name optnibl51) (Fig 2C). The p62 mutant allele carried an indel resulting in the net loss of 37 nucleotides, which we named p62Δ37n/Δ37n (ZFIN allele name p62ibl52) (Fig 2C). The homozygous mutants were fertile and produced embryos that did not exhibit detectable morphological differences compared with embryos produced by their wild-type (optn+/+ or p62+/+) siblings (S1C Fig). Furthermore, no significant deviation from the Mendelian 1: 2: 1 ratio for +/+, +/- and -/- genotypes was observed when the offspring of heterozygous incrosses were sequenced at 3 months of age (Fig 1D). Western blot analysis using anti-OPTN and anti-p62 C-terminal antibodies confirmed the absence of the proteins in the respective mutant lines (Fig 2D). In addition, quantitative PCR (Q-PCR) analysis revealed approximately 4. 5-fold reduction of optn mRNA in the optnΔ5n/Δ5n larvae and 10-fold reduction of p62 mRNA in the p62Δ37n/Δ37n larvae, indicative of nonsense-mediated mRNA decay (Fig 2E). Collectively, the optnΔ5n/Δ5n and p62Δ37n/Δ37n mutant zebrafish produce no functional Optn or p62, respectively, and the loss of these ubiquitin receptors does not induce detectable developmental defects that could interfere with the use of the mutant lines in infection models. To analyze the effects of Optn or p62 deficiency on autophagy, we performed Lc3 Western blot detection on whole embryo extracts and imaged GFP-Lc3 signal in vivo (Fig 3A). Differences in the levels of the cytosolic (Lc3-I) and membrane-bound (Lc3-II) forms of Lc3 or effects on GFP-Lc3 puncta accumulation can be due to altered autophagosome formation or differences in autophagosome degradation. To distinguish between these possibilities, we examined Lc3-II/Lc3-I levels and GFP-Lc3 accumulation in the absence or presence of Bafilomycin A1 (Baf A1), which is an inhibitor of vacuolar H+ ATPase (V-ATPase) that blocks autophagosome degradation by inhibiting the fusion with lysosomes [35,36]. First, we performed a dose range assay to determine the effect of Baf A1 on Lc3-II accumulation in zebrafish embryos. Results showed that after 12 h of incubation, a dosage of 100nM resulted in Lc3-II accumulation without affecting the Lc3-I level (S2A Fig). The accumulation of Lc3-II in the presence of Baf A1 indicates that autophagic flux is occurring in the zebrafish embryos. Because a higher dosage of Baf A1 additionally increased the Lc3-I level (S2A Fig), we utilized a dosage of 100nM to test Lc3-II accumulation in WT and mutant embryos not carrying the GFP-Lc3 reporter (Fig 3B). No differences in Lc3-II accumulation were observed between optn+/+ and optnΔ5n/Δ5n embryos or between p62+/+ and p62Δ37n/Δ37n embryos in the absence of Baf A1 (Fig 3C). However, Baf A1 treatment induced significantly higher accumulation of Lc3-II in WT embryos than in optn or p62 mutant embryos (Fig 3C). In agreement, Baf A1 treatment resulted in accumulation of GFP-Lc3 puncta in optn or p62 mutants, but a significantly higher GFP-Lc3 accumulation was detected in the corresponding WT controls (Fig 3D and 3E). The function of Optn and p62 as ubiquitin receptors implies that these proteins are degraded themselves during the process of autophagy. Therefore, we asked if p62 protein levels are affected in optn mutants or, vice versa, if p62 mutation impacts Optn protein levels. Western blot analysis showed accumulation of p62 and Optn protein in wildtype embryos in response to Baf A1 treatment, confirming that these ubiquitin receptors are substrates for autophagy under basal conditions (S2B Fig). Levels of p62 protein were reduced in optnΔ5n/Δ5n embryos compared with optn+/+, both in absence or presence of Baf A1 (S2B Fig). This difference was not due to a transcriptional effect, since p62 mRNA levels were not significantly different between optn+/+ and optnΔ5n/Δ5n embryos (Fig 2E). Similarly, levels of Optn protein were reduced in p62Δ37n/Δ37n embryos compared with p62+/+ in absence or presence of Baf A1 (S2B Fig), and again this was not associated with a difference in mRNA expression (Fig 2E). In conclusion, the absence of either of the ubiquitin receptors, Optn or p62, leads to reduced protein levels of the other ubiquitin receptor, which cannot be restored by blocking lysosomal degradation. Furthermore, loss of either of the receptors leads to lower levels of Lc3-II and GFP-Lc3 accumulation when lysosomal degradation is blocked, suggesting reduced activity of the autophagy pathway in the optn and p62 mutants. Next, we asked if optn or p62 mutations would affect the resistance of zebrafish embryos to mycobacterial infection. We injected Mm into embryos via the caudal vein at 28 hpf to measure infection burden at 3 dpi (Fig 4A). The infection data showed that optn or p62 mutant embryos were hypersusceptible to Mm infection compared with their WT controls, culminating in an increase of the Mm fluorescent signal of 2. 8 and 2. 9 times, respectively (Fig 4B). Western blot analysis was performed to determine if mutation of p62 or optn affected the protein levels of the other SLR during infection. The level of p62 was approximately 60% lower in optn mutants and a similar reduction was observed for Optn in p62 mutants, indicating that reduced levels of the other SLR may contribute to the hypersusceptibility phenotype of the mutant lines (S2C Fig). In addition, we examined whether transient knockdown of optn or p62 would phenocopy the infection phenotype of the mutant lines. We injected optn or p62 antisense morpholino (MO) oligonucleotides into the one cell stage of embryos and collected injected individuals at 1–3 dpi for confirmation of the knockdown effect by reverse transcription polymerase chain reaction (RT-PCR) and Western blot (S3A–S3E Fig). Despite that strong reduction of mRNA and protein levels was observed only until 1 dpi, the analysis of Mm infection burden at 3 dpi showed that transient knockdown of optn or p62 led to similar increases of the Mm infection burden as had been observed in the mutant lines (Fig 4C). These results suggests that differences in the resistance to Mm are already determined early in the infection process. As a further control for the specificity of the optn and p62 mutant phenotypes, we demonstrated that injection of the corresponding mRNA rescued the hypersusceptibility of the mutants to Mm infection (Fig 4D). Since OPTN and p62 are known to function cooperatively in xenophagy of Salmonella enterica [22–24], we next asked if double deficiency of Optn and p62 in zebrafish resulted in an increased infection burden compared to single mutation of either optn or p62. No additive effect on the infection burden was observed when p62 MO was injected into optn mutant embryos or optn MO into p62 mutant embryos (Fig 4E). Because Mm depends on the RD1 virulence locus to escape from phagosomes [37,38], infection with ΔRD1 mutant bacteria is not expected to trigger selective autophagy. In agreement, infection with ΔRD1 Mm led to similar infection burdens in optn and p62 mutants as in their wild type siblings (Fig 4F). Taken together, our data demonstrate that both Optn and p62 are required for controlling Mm infection and that loss of either of these ubiquitin receptors cannot be compensated for by the other receptor in this context. Furthermore, these data show that Optn and p62 specifically target Mm bacteria that have a functional RD1 virulence locus mediating phagosomal escape. Having established that mutation of either optn or p62 results in increased Mm infection burden, we investigated if the inability of mutant embryos to control infection might be associated with altered inflammatory responses or is due to a reduction in the targeting of mycobacteria to autophagy. Therefore, we first examined the expression levels of 4 key markers of inflammation, tnfa, il1b, cxcl11aa, and cxcl8a (il8), which were selected based on previous expression profiling of Mm infection [27]. The analysis showed that the increased infection burden of p62 and optn mutants was not associated with differences in the expression of these inflammatory genes (S4 Fig). Next, we investigated the autophagic targeting of mycobacteria in the mutants by examining the association of GFP-Lc3 with Mm at 1 dpi. Mm has formed small infection foci at this time point, which could be manually scored as positive or negative for GFP-Lc3 association. In wildtype (WT) embryos 5–6% of these infection foci were positive for GFP-Lc3 (S5A and S5B Fig). The percentage of GFP-Lc3 positive Mm clusters was approximately 50% lower in the optn or p62 mutant embryos compared with their WT controls, but differences were not statistically significant due to the relatively low number of these GFP-Lc3 association events (S5A and S5B Fig). We continued to examine GFP-Lc3 targeting to Mm at 2 dpi and found that mutation of optn or p62 resulted in significantly decreased GFP-Lc3 co-localization with Mm clusters (Fig 5A, 5B and 5C). In addition, we used GFP-Lc3-negative mutant and WT larvae for Western blot analysis of Lc3-II protein levels in response to infection. We found that Mm infection increased Lc3-II protein levels approximately 3- to 5-fold in WT (optn+/+ and p62+/+) larvae at 3 dpi, whereas this induction level was approximately 50% lower in the optn and p62 mutant larvae (Fig 5D). Mm-infected mutant embryos also showed reduced Lc3-II accumulation in the presence of Baf A1 (S5C Fig). Despite the reduced GFP-Lc3 targeting, we could still detect Lysotracker-positive Mm clusters in the optn and p62 mutant larvae, indicating that the phago-lysosomal pathway is not defective in these mutants (S5D Fig). Taken together, these data support the hypothesis that Optn and p62 are required for autophagic defense against mycobacterial infection. To further test the hypothesis that Optn and p62 mediate autophagic defense against Mm, we generated full-length optn and p62 mRNAs in vitro and injected these into embryos at the one cell stage, resulting in ubiquitous overexpression (Fig 6A). The increase in Optn or p62 protein levels following mRNA injection was verified by Western blot analysis (Fig 6B). Overexpression of optn or p62 mRNAs significantly reduced Mm infection burden at 3 dpi compared to the control groups (Fig 6C). Furthermore, injection of optn or p62 mRNAs carrying deletions in the sequences encoding the ubiquitin binding domains or Lc3 interaction regions did not lead to a reduction of the Mm infection burden compared with the control groups (Fig 6C and S6A Fig). Thus, we conclude that optn or p62 overexpression protects against Mm infection in a manner dependent on the interaction of the Optn and p62 proteins with both ubiquitin and Lc3. Since overexpression of optn or p62 mRNAs resulted in decreased Mm infection burden, we postulated that elevation of the Optn or p62 protein levels would result in increased targeting of Mm to autophagy by these ubiquitin receptors, in a manner dependent on the functions of the Lc3 interaction (LIR) and ubiquitin binding domains (UBAN/UBA). To test this hypothesis, we injected the full-length mRNAs, or mRNAs generated from deletion constructs lacking these domains, and quantified GFP-Lc3-positive and GFP-negative Mm infection foci at 1 dpi and 2 dpi (Fig 7A–7C, S6B and S6C Fig). The results showed that overexpression of full-length optn or p62 mRNAs significantly increased the percentage of GFP-Lc3-positive Mm clusters at 2 dpi, compared with the control groups (Fig 7B and 7C). Conversely, injection of optn ΔUBAN, optn ΔLIR, p62 ΔUBA and p62 ΔLIR mRNAs did not increase the association of GFP-Lc3 with Mm clusters (Fig 7B and 7C). Similar results could be observed as early as 1 dpi (S6B and S6C Fig). In conclusion, our combined results demonstrate that Optn and p62 can target Lc3 to Mm and that increasing the level of either of these receptors promotes host defense against this mycobacterial pathogen. Members of the family of sequestosome (p62/SQSTM1) -like receptors (SLRs) function in autophagic host defense mechanisms targeting a range of intracellular pathogens, including Salmonella, Shigella, Streptococci, Listeria, Mycobacteria, and Sindbis virus [5,13,14,39]. These discoveries inspired investigations into autophagy modulators as host-directed therapeutics for treatment of infectious diseases, including Tb [9,40,41]. However, the relevance of autophagic defense mechanisms for host resistance against Mtb infection has recently been questioned [15,42]. This indicates that there are significant gaps in our understanding of the interaction between components of the autophagy pathway and mycobacterial pathogens, emphasizing the need for more research in animal models of Tb [12]. Here, we have studied the function of two SLR family members in the zebrafish Mm infection model. We show that selective autophagy mediated by p62 and Optn provides resistance against mycobacterial infection in the context of our in vivo infection model that is representative of the early stages of Tb granuloma formation [17,19]. Our findings support the host-protective role of p62 in Tb by autophagic targeting of Mycobacteria, in line with previous in vitro studies [13,14]. Importantly, we also present the first evidence linking Optn to resistance against Mycobacteria, expanding our understanding of the function of SLRs in host defense against intracellular pathogens. The zebrafish embryo and larval Tb model provides the opportunity to image critical stages of the mycobacterial infection process, from the initial phagocytosis of Mm by macrophages up to the early stages of Tb granuloma formation [43]. The model is representative of miliary Tb, where the infection is disseminated to multiple organs of the host. The embryonic and larval stages of the zebrafish allow us to study the contribution of innate immunity to host defense, since they lack a matured adaptive immune response at this time point of development [17]. We therefore used this model to study the importance of autophagic defense mechanisms during innate host defense against mycobacterial infections. In this study, we successfully generated p62 and optn loss-of-function zebrafish mutant lines using CRISPR/Cas9 technology. Besides its role in host defense, p62 is a stress-inducible protein that functions as a signalling hub in diverse processes like amino acid sensing and the oxidative stress response [44]. Defects in autophagy pathways caused by mutations in OPTN have been associated with human disorders like glaucoma, Paget disease of bone, and amyotrophic lateral sclerosis [24,45]. Despite the important functions reported for p62 and OPTN in cellular homeostasis, the mutant fish lines we generated are viable and fertile. The absence of either p62 or Optn resulted in reduced protein levels of the other ubiquitin receptor, even when lysosomal degradation was blocked. The mechanism responsible for this cross-talk currently remains unknown. In addition, loss of either of the receptors leads to lower levels of Lc3-II and GFP-Lc3 accumulation when lysosomal degradation is blocked, indicating that mutation of either p62 or optn is sufficient to reduce activity of the autophagy pathway in zebrafish larvae. Therefore, we could use these mutant lines to gain a better understanding of the role of p62, Optn, and selective autophagy in host defense against mycobacterial infection. Genetic links between autophagy pathway genes and susceptibility to Tb in human populations support the function of autophagy in innate host defense against Mtb [46]. However, the contribution of autophagy as a direct anti-mycobacterial mechanism has recently been challenged, since macrophage-specific depletion of a number of autophagy genes, including p62, did not affect the outcome of disease in a mouse model of Tb [15,42]. A possible explanation for these findings, as suggested by the authors of this study, is that Mtb, like other successful intracellular pathogens, could have evolved virulence mechanisms that subvert or exploit autophagic defense mechanisms employed by the host [47]. In case of one of the autophagy genes, ATG5, macrophage-specific depletion increased Mtb infection in mice by over-activating inflammation rather than by impairing autophagic processes [15]. It is therefore conceivable that modulating the activity of SLRs could also affect inflammation. Indeed, OPTN has been implicated in inflammatory bowel disease and both p62 and OPTN are involved in regulation of inflammatory signaling downstream of NF-κB [48–52]. Through a process that involves polyubiquitination of regulatory proteins, both p62 and OPTN can modulate the activity of the IKK kinase complex that activates NFκB [48,49]. We therefore investigated the expression of several inflammatory cytokine and chemokine genes that are targets of NFκB signaling. The upregulation of these genes during Mm infection was unaffected by optn and p62 mutation. Nevertheless, we cannot rule out the possibility that more subtly altered inflammatory responses in p62 and optn mutants could explain (part of) the increase in mycobacterial burden observed in zebrafish hosts, while the beneficial role for autophagic defense mechanisms targeting the bacteria might be limited. To investigate the possible role of Optn and p62 in anti-mycobacterial autophagy, we quantified the association between GFP-Lc3 and Mm under loss-of-function and gain-of-function conditions of both receptors. During Mm infection in zebrafish, GFP-Lc3 puncta are frequently detected in close vicinity of intracellular bacteria, but only a small percentage of Mm bacteria are found inside vesicles with autophagic morphology as determined by electron microscopy [20,21]. In agreement, only 3–5% of the bacteria co-localized with autophagic vesicles one day after a systemic infection of WT zebrafish embryos with mycobacteria. Although the number of GFP-Lc3 positive bacterial clusters rises over the next two days, the percentage of bacteria targeted by autophagy at any distinct time point remained relatively low (e. g. ~10% at 2 dpi). According to these results, the host only employs autophagic defense mechanisms against a small proportion of the invading mycobacteria during early stages of the infection, either because there is no greater need, or because the pathogens are indeed effectively suppressing this response. It is important to note though that GFP-Lc3 association with Mm is a transient process [20], which means that the percentage of bacteria that encounter autophagic defenses throughout the early infection process might be much higher. Strikingly, the percentage of bacteria labeled by ubiquitin closely resembled the percentage of bacteria targeted by autophagy, and we were able to detect clear colocalization between ubiquitin and GFP-Lc3 at bacterial clusters. Upon loss-of-function of either p62 or Optn, the co-localization between bacteria and autophagic vesicles decreased and the bacterial burden increased. Conversely, overexpression of either ubiquitin binding receptor increased autophagic targeting of bacteria and resulted in lower bacterial burdens, both of which required the presence of functional Lc3 and ubiquitin binding domains. Taken together, we conclude that autophagic targeting of Mm by p62 and Optn indeed provides protection against infection in the zebrafish model. Mm and Mtb share the RD1 virulence locus, which encodes the ESX1 secretion system that is required for bacterial translocation from phagosomes into the cytosol [37,38,53] The autophagy machinery can directly entrap cytosolic bacteria that become ubiquitinated after phagosomal escape, or it can delay the escape process by membrane repair of permeabilized and ubiquitinated phagosomes [54]. In agreement, we found that optn and p62 mutants and their WT siblings were equally susceptible to infection with the escape incompetent RD1 mutant strain. While our results implicate SLRs in the autophagic defense against cytosolic Mm, another mechanism for targeting these bacteria to degradative vesicles has also been reported. The RD1-dependent phagosomal escape of Mm in bone-marrow derived macrophages triggered bacterial ubiquitination and subsequent targeting to LAMP1-positive vesicles in an ATG5-independent manner [38]. Besides this autophagy-independent sequestration, Mm bacteria were also observed inside classical double-membraned autophagosomes, suggesting that SLR-mediated targeting also occurs in this model. It is possible that autophagy-dependent and -independent routes of targeting cytosolic Mm also occur in zebrafish and that actin polymerization might protect a subset of bacteria against sequestration, as has been found in the study of macrophages [38]. We observed acidification of part of the Mm-containing vesicles in both WT and SLR mutant zebrafish, but the experimental tools are currently lacking to further investigate the fate of Mm after sequestration in autophagic or other compartments. Because mycobacteria are known for being able to largely tolerate the acidic environment that results from lysosome fusion [35], it is possible that the major contribution of autophagy to bacterial killing might not be made by xenophagic delivery of Mm to acidic lysosomes, but by SLR-mediated delivery of ubiquitinated peptides that have strong anti-microbial activity against mycobacteria [7]. In summary, our findings on Mm infection in zebrafish confirm that p62 mediates ubiquitin-dependent autophagic targeting of mycobacteria in an in vivo model for early stages of Tb granuloma formation. We also provide the first evidence that the SLR family member Optn is involved in autophagic targeting of ubiquitinated mycobacteria. The effects of optn and p62 mutation cannot be entirely separated, since we found that mutation of one SLR reduced protein levels of the other. Nevertheless, our data suggest that Optn and p62 have non-redundant and possibly cooperative functions in defense against Mm. This is consistent with the distinct functions of these SLRs in the selective autophagy response to Salmonella Typhimurium and Listeria monocytogenes [22,23,55]. Furthermore, a direct interaction between the two SLRs has been demonstrated in a study showing that tumor-suppressor HACE1, a ubiquitin ligase, ubiquitinates OPTN and promotes its interaction with p62 to form the autophagy receptor complex [56]. While expression of major inflammatory markers was unaffected by p62 or Optn deficiency, we have shown that the autophagic targeting of Mm by these ubiquitin-binding receptors forms an important aspect of innate host defense against Tb in the zebrafish model. Our results are therefore especially important for the development of new treatment strategies for Tb patients with a compromised adaptive immune system–such as in HIV-coinfection. Based on these results, selective autophagy stimulation remains a promising strategy for development of novel anti-Tb therapeutics. Zebrafish lines in this study (S1 Table) were handled in compliance with local animal welfare regulations as overseen by the Animal Welfare Body of Leiden University (License number: 10612) and maintained according to standard protocols (zfin. org). All protocols adhered to the international guidelines specified by the EU Animal Protection Directive 2010/63/EU. The generation of zebrafish optnibl51 and p62ibl52 mutant lines was approved by the Animal Experimention Committee of Leiden University (UDEC) under protocol 14198. All experiments with these zebrafish lines were done on embryos or larvae up to 5 days post fertilization, which have not yet reached the free-feeding stage. Embryos were grown at 28. 5°C and kept under anesthesia with egg water containing 0. 02% buffered 3-aminobenzoic acid ethyl ester (Tricaine, Sigma) during bacterial injections, imaging and fixation. Single guide RNAs (sgRNAs) targeting the second coding exon of zebrafish optn (ENSDART00000014036. 10) and the third coding exon of p62 (ENSDART00000140061. 2) were designed using the chop-chop website [57]. To make sgRNAs, the template single strand DNA (ssDNA) (122 bases) was obtained by PCR complementation and amplification of full length ssDNA oligonucleotides. Oligonucleotides up to 81 nucleotides were purchased from Sigma-Aldrich using standard synthesis procedures (25 nmol concentration, purification with desalting method) (S2 and S3 Tables). The pairs of semi-complimentary oligos were annealed together by a short PCR program (50 μL reaction, 200uM dTNPs, 1 unit of Dream Taq polymerase (EP0703, ThermoFisher); PCR program: initial denaturation 95°C/3 minute (min), 5 amplification cycles 95°C/30 Second (s), 55°C/60 s, 72°C/30 s, final extension step 72°C/15 min) and subsequently the products were amplified using the primers in S2 Table with a standard PCR program (initial denaturation 95°C/3 min, 35 amplification cycles 95°C/30 s, 55°C/60 s, 72°C/30 s, final extension step 72°C/15 min). The final PCR products were purified with Quick gel extraction and PCR purification combo kit (00505495, ThermoFisher). The purified PCR products were confirmed by gel electrophoresis and Sanger sequencing (Base Clear, Netherlands). For in vitro transcription of sgRNAs, 0. 2 μg template DNA was used to generate sgRNAs using the MEGA short script ®T7 kit (AM1354, ThermoFisher) and purified by RNeasy Mini Elute Clean up kit (74204, QIAGEN Benelux B. V., Venlo, Netherlands). The Cas9 mRNA was transcribed using mMACHINE® SP6 Transcription Kit (AM1340, Thermo Fisher) from a Cas9 plasmid (39312, Addgene) (Hrucha et al 2013) and purified with RNeasy Mini Elute Clean up kit (74204, QIAGEN Benelux B. V., Venlo, Netherlands). A mixture of sgRNA and Cas9 mRNA was injected into one cell stage WT embryos (sgRNA 150 pg/embryo and Cas9 mRNA 300 pg/embryo). The effect of CRISPR injection was confirmed by PCR and Sanger sequencing. Genomic DNA was isolated from an individual embryo (2 dpf) or small pieces of the tail fin tissue of adults (>3 months) by fin clipping. Embryos or tissue samples were incubated in 200 μL 100% Methanol at -20°C overnight (O/N), then methanol was removed, and remaining methanol was evaporated at 70°C for 20 min. Next, samples were incubated in 25 μL of TE buffer containing 1. 7 μg/μL proteinase K at 55°C for more than 5 h. Proteinase K was heat inactivated at 80°C for 30 min, after which samples were diluted with 100 μL of Milli-Q water. Genotyping was performed by PCR-amplification of the region of interest using the primers in S5 Table followed by Sanger sequencing to identify mutations (Base Clear, Netherlands). Embryos or larvae were anaesthetised with Tricaine (Lot#MKBG4400V, SIGMA-ALDRICH) and homogenised with a Bullet-blender (Next-Advance) in RIPA buffer (#9806, Cell Signalling) containing a protein inhibitor cocktail (000000011836153001, cOmplete, Roche). The extracts were then spun down at 4°C for 10 min at 12000 rpm/min and the supernatants were frozen for storage at -80°C. Western blot was performed using Mini-PROTEAN-TGX (456–9036, Bio-Rad) or 18% Tris-Hcl 18% polyacrylamide gels, and protein transfer to commercial PVDF membranes (Trans-Blot Turbo-Transfer pack, 1704156, Bio-Rad). Membranes were blocked with 5% dry milk (ELK, Campina) in Tris buffered saline (TBS) solution with Tween 20 (TBST, 1XTBS contains 0. 1% Tween 20) buffer and incubated with primary and secondary antibodies. Digital images were acquired using Bio-Rad Universal Hood II imaging system (720BR/01565 UAS). Band intensities were quantified by densitometric analysis using Image Lab Software (Bio-Rad, USA) and values were normalised to actin as a loading control. Antibodies used were as follows: polyclonal rabbit anti-OPTN (C-terminal) (1: 200, lot#100000; Cayman Chemical), polyclonal rabbit anti-p62 (C-terminal) (PM045, lot#019, MBL), polyclonal rabbit anti Lc3 (1: 1000, NB100-2331, lot#AB-3, Novus Biologicals), Anti mono-and polyubiquitinated conjugates mouse monoclonal antibody (1: 200; BML-PW8810-0100, lot#01031445, Enzo life Sciences), Polyclonal actin antibody (1: 1000,4968S, lot#3, Cell Signaling), Anti-rabbit IgG, HRP-Linked Antibody (1: 1000,7074S, Lot#0026, Cell Signaling), Anti-mouse IgG, HRP-linked Antibody (1: 3000,7076S, Lot#029, Cell Signaling). optn and p62 splice blocking MOs were purchased from Gene Tools. For MO sequences see S4 Table. MOs were diluted in Milli Q water with 0. 05% phenol red and 0. 1 pmol optn or 0. 5 pmol p62 MO was injected in a volume of 1 nL into the one cell stage of embryos as previously described [21]. The knockdown effect was validated by RT-PCR and Western blot. Infections of zebrafish embryos were performed by microinjection into the blood island at 28 hpf as previously described [58], using the mCherry-labeled Mm 20 strain, the mCrimson-labeled Mm M strain, and the mCherry labeled Mm ΔRD1 strain [59]. The injection dose was 200 CFU for experiments with WT Mm bacteria and 400 CFU for infections with the ΔRD1 mutant. Before the injection, embryos were manually dechorionated around 24 hpf. Approximately 5 min before bacterial injections, zebrafish embryos were brought under anaesthesia with tricaine. Infected embryos were imaged using a Leica MZ16FA stereo fluorescence microscopy with DFC420C camera, total fluorescent bacterial pixels per infected fish were determined on whole-embryo stereo fluorescent micrographs using previously described software [60]. Fixed or live embryos were mounted with 1. 5% low melting agarose (140727, SERVA) and imaged using a Leica TCS SPE confocal microscope. For quantification of basal autophagy, fixed uninfected 4 dpf larvae were imaged by confocal microscopy with a 63x water immersion objective (NA 1. 2) in a pre-defined region of the tail fin to detect GFP-LC3-positive vesicles (Fig 3D and 3E). The number of GFP-Lc3 vesicles per condition was quantified using Fiji/ImageJ software (Fig 3D and 3E). For quantification of the autophagic response targeted to Mm clusters (Fig 1B and 1C, S4A, S4B, S6A and S6B Figs), live or fixed infected embryos were viewed by confocal microscopy with a 63x water immersion objective (NA 1. 2) and the number of Mm clusters that were targeted by GFP-Lc3 puncta in the tail region were counted manually. The same approach was used to quantify Ubiquitin targeting to Mm clusters (Fig 1E and 1F). To quantify the percentage of GFP-Lc3+ Mm clusters, we imaged the entire caudal hematopoietic tissue (CHT) region of 2 dpi larvae (confocal microscopy; 40X water immersion objective with NA 1. 0) and stitched multiple images together to manually count the number of Mm clusters positive for GFP-Lc3 out of the total number of clusters (Figs 5B, 5C, 7B and 7C). Embryos (1,2, 3 dpi) were fixed with 4% PFA in PBS and incubated overnight with shaking at 4ᵒC. After washing the embryos three times briefly in PBS with 0. 8% Triton-x100) (PBSTx), the embryos/larvae were digested in 10 μg/ml proteinase K (000000003115879001, SIGMA-ALDRICH) for 10 minutes at 37ᵒC. Subsequently, the embryos were quickly washed, blocked with PBSTx containing 1% Bovine serum albumins (BSA) (A4503-100g, SIGMA-ALDRICH) for 2h at room temperature and incubated overnight at 4ᵒC in mono-and polyubiquitinated conjugates mouse monoclonal antibody (1: 200; BML-PW8810-0100; Enzo lifes Siences), diluted in the blocking buffer. Next, embryos were washed three times in PBSTx, incubated for 1 h in blocking buffer at room temperature, incubated for 2 h at room temperature in 1: 200 dilution of Alexa Fluor 488 or 633 goat anti-mouse (Invitrogen) in blocking buffer, followed with three times washes in PBSTx for imaging. optn (ENSDART00000014036. 10, Ensembl) and p62 (ENSDART00000140061. 2, Ensembl) cDNAs were amplified from 3 dpf WT embryos by PCR (primers in S5 Table) and ligated into a vector using the Zero-blunt cloning PCR kit (450245, Invitrogen). The sequence was confirmed by Sanger sequencing (BaseClear, Netherlands), after which optn and p62 cDNAs were subcloned into a pCS2+ expression vector. optn ΔUBAN cDNA was produced by in vitro transcription of optn-pCS2+ constructs digested by Sca1 (R3122, NEB), which excludes the region encoding the UBAN protein domain. optn ΔLIR cDNA was amplified from optn-pCS2+ constructs by designed primers (S5 Table), excluding the LIR protein domain. The PCR products were gel purified by Quick gel Extraction PCR Purification Combo Kit (K220001, Invitrogen) and the two fragments and pCS2+ plasmid were digested by BamH1 (R0136S, NEB) and EcoR1 (R0101S, NEB), after which the two fragments were ligated into pCS2+ plasmid by T4 DNA ligase. p62 ΔUBA cDNA was obtained from a p62-pCS2+ construct by Nco1 (R0193S, NEB) digestion and religation, which excludes the region encoding the UBA protein domain. p62 ΔLIR cDNA was obtained from a p62-pCS2+ construct by NcoN1 digestion and religation. Optn mRNA, optn ΔUBAN, and optn ΔLIR mRNA was generated using SP6 mMessage mMachine kit (Life Technologies) from Kpn1 or Sac1 (R0156S, NEB) digested optn–pCS2+ constructs. RNA purification was performed using the RNeasy Mini Elute Clean up kit (QIAGEN Benelux B. V., Venlo, Netherlands). In vitro transcription of p62, p62 ΔUBA, and p62 ΔLIR was performed using mMESSAGE mMACHINE T3 Transcription Kit (AM1348, Thermo Fisher) and purified using the RNeasy MiniElute Cleanup kit (QIAGEN Benelux B. V., Venlo, Netherlands). All mRNAs were injected into one cell stage embryos at a dose of 100 pg in a volume of 1 nL, and the overexpression effects of optn or p62 were validated by Q-PCR and Western blot. Total RNA was extracted using Trizol reagent (15596026, Invitrogen) according to the manufacturer’s instructions and purified with RNeasy Min Elute Clean up kit (Lot: 154015861, QIAGEN). RNAs were quantified using a NanoDrop 2000c instrument (Thermo Scientific, U. S). Reverse transcription reaction was performed using 0. 5 μg of total RNA with iScript cDNA synthesis kit (Cat: #170–8891, Bio-Rad). The mRNA expression level was determined by quantitative real-time PCR using iQSYBR Green Supermix (Cat: 170–8882, Rio-Rad) and Single color Real-Time PCR Detection System (Bio-Rad, U. S) as previously described [61]. All primers are listed in S5 Table. Statistical analyses were performed using GraphPad Prism software (Version 5. 01; GraphPad). All experimental data (mean ± SEM) was analyzed using unpaired, two-tailed t-tests for comparisons between two groups and one-way ANOVA with Tukey’s multiple comparison methods as a posthoc test for comparisons between more than two groups. (ns, no significant difference; *p < 0. 05; **p < 0. 01; ***p < 0. 001). To determine whether the offspring of F1 heterozygous mutants follows Mendelian segregation, the obtained data was analysed with a Chi-square test (ns, no significant difference). | Tuberculosis is a serious infectious disease that claims over a million lives annually. Vaccination provides insufficient protection and the causative bacterial pathogen, Mycobacterium tuberculosis, is becoming increasingly resistant to antibiotic therapy. Therefore, there is an urgent need for novel therapeutic strategies. Besides searches for new antibiotics, considerable efforts are made to identify drugs that improve the immune defenses of the infected host. One host defense pathway under investigation for therapeutic targeting is autophagy, a cellular housekeeping mechanism that can direct intracellular bacteria to degradation. However, evidence for the anti-mycobacterial function of autophagy is largely based on studies in cultured cells. Therefore, we set out to investigate anti-mycobacterial autophagy using zebrafish embryos, which develop hallmarks of tuberculosis following infection with Mycobacterium marinum. Using red-fluorescent mycobacteria and a green-fluorescent zebrafish autophagy reporter we could visualize the anti-mycobacterial autophagy response in a living host. We generated mutant and knockdown zebrafish for two selective autophagy receptors, Optineurin and p62, and found that these have reduced anti-bacterial autophagy and are more susceptible to infection. Moreover, we found that increased expression of these receptors enhances anti-bacterial autophagy and protects against infection. These results provide new evidence for the host-protective function of selective autophagy in mycobacterial disease. | lay_plos |
Introduction North Korea's systematic violation of its citizens' human rights and the plight of North Koreans trying to escape their country have been well documented in multiple reports issued by governments and other international bodies. The Bush Administration initially highlighted and later de-emphasized Pyongyang's human rights record as its policy on nuclear weapons negotiations evolved. Congress has consistently drawn attention to North Korean human rights violations on a bipartisan basis. On several occasions, Congress has criticized the executive branch for its approach to these issues, through tough questioning of Administration witnesses during multiple hearings and through written letters of protest to high-level officials. Although the Obama Administration has indicated that it will seek to continue the nuclear negotiations, it has not indicated how the issue of human rights will be addressed. The passage of the North Korean Human Rights Act of 2004 ( H.R. 4011 ; P.L. 108 - 333 ; and 22 U.S.C. 7801.) and its reauthorization in 2008 ( H.R. 5834, P.L. 110 - 346 ) serve as the most prominent examples of legislative action on these issues. The legislation both reinforced some aspects of the Bush Administration's rhetoric on North Korea and expresses dissatisfaction with other elements of its policy on North Korea. The reauthorization bill explicitly criticizes the implementation of the original law and reasserts Congressional interest in adopting human rights as a major priority in U.S. policy toward North Korea. U.S. attention to North Korean human rights and refugees is complicated by the geopolitical sensitivities of East Asia. China is wary of U.S. involvement in the issue and chafes at any criticism based on human rights. South Korea also had reservations about a more active U.S. role, particularly in terms of refugees, although the current Lee administration in Seoul has been more amenable to such efforts. Both want to avoid a massive outflow of refugees, which they believe could trigger instability or the collapse of North Korea. U.S. executive branch officials worry that criticism of how Seoul and Beijing approach North Korea's human rights violations could disrupt the multilateral negotiations to deal with Pyongyang's nuclear weapons programs. North Korean refugees seeking resettlement often transit through other Asian countries, raising diplomatic, refugee, and security concerns for those governments. The Role of Human Rights in U.S. Policy Toward North Korea under the Bush Administration In the first several years of the Bush Administration, high-level officials, including the President and Secretary of State, publicly and forcefully criticized the regime in Pyongyang for its human rights practices. As efforts to push forward the Six-Party talks accelerated in 2007, the Administration did not propose any negotiations with North Korea over human rights but asserted that human rights is one of several issues to be settled with North Korea after the nuclear issue is resolved. The Six-Party Agreement of February 13, 2007, calls for the United States and North Korea to "start bilateral talks aimed at resolving bilateral issues and moving toward full diplomatic relations." Prior to the Agreement in 2007, the Bush Administration held that it would not agree to normalization of diplomatic relations with North Korea until there was progress on human rights (presumably including refugees) and other issues. However, after the signing of the agreement in February 2007, some observers say that former Assistant Secretary of State for East Asian and Pacific Affairs Christopher Hill focused exclusively on a satisfactory settlement of the nuclear issue. The North Korean Human Rights Act of 2004 The 108 th Congress passed by voice vote, and President Bush signed, the North Korean Human Rights Act of 2004 (NKHRA). The legislation authorized up to $20 million for each of the fiscal years 2005-2008 for assistance to North Korean refugees, $2 million for promoting human rights and democracy in North Korea and $2 million to promote freedom of information inside North Korea; asserted that North Koreans are eligible for U.S. refugee status and instructs the State Department to facilitate the submission of applications by North Koreans seeking protection as refugees; and required the President to appoint a Special Envoy to promote human rights in North Korea. The act also expressed the sense of Congress that human rights should remain a key element in negotiations with North Korea; all humanitarian aid to North Korea shall be conditional upon improved monitoring of the distribution of food; support for radio broadcasting into North Korea should be enhanced; and that China is obligated to provide the United Nations High Commissioner for Refugees (UNHCR) with unimpeded access to North Koreans inside China. Some hail the NKHRA as an important message that human rights will play a central role in the formulation of U.S. policy towards North Korea. Passage of the legislation was driven by the argument that the United States has a moral responsibility to stand up for human rights for those suffering under repressive regimes. Advocates claim that, in addition to alleviating a major humanitarian crisis, the NKHRA will ultimately enhance stability in Northeast Asia by promoting international cooperation to deal with the problem of North Korean refugees. Critics say the legislation risks upsetting relations with South Korea and China, and ultimately the diplomatic unity necessary to make North Korea abandon its nuclear weapons program through the Six-Party Talks. Further, they insist that the legislation actually worsens the plight of North Korean refugees by drawing more attention to them, leading to crackdowns by both North Korean and Chinese authorities and reduced assistance by Southeast Asian countries concerned about offending Pyongyang. Selected Implementation Progress While the passage of the NKHRA raised the profile of congressional interest in North Korean human rights and refugee issues, many of the activities had existing authorizations already in place. The State Department had programs directed toward raising awareness of North Korean human rights issues as well as providing some assistance to vulnerable North Korean refugees through other organizations. Korean-language broadcasting by Radio Free Asia (RFA) and Voice of American (VOA) pre-dated the passage of the law. However, some activities appear to have been enhanced as a result of the law's enactment, particularly the admission of North Korean refugees for resettlement in the United States. Human Rights Reports required by the act have outlined steps taken by the State Department and other executive branch bodies to promote human rights in North Korea. The State Department has not requested funding explicitly under the NKHRA, but officials assert that the mission of the NKHRA is fulfilled under a number of existing programs. For democracy promotion in North Korea, the State Department's Democracy, Human Rights, and Labor (DRL) Bureau gives grants to U.S.-based organizations: in the FY2008 budget, DRL requested $1 million for North Korea human rights programs, as well as $1 million for media freedom programs. DRL also considers several other programs, such as those under the National Endowment of Democracy account specific to North Korea, as fulfilling part of the NKHRA's mission. The Special Envoy attended three international Freedom House conferences organized to raise awareness of human rights conditions in North Korea in 2005-2006. The U.S. government has also sponsored and supported United Nations resolutions condemning North Korea's human rights abuses. Refugee Resettlement The NKHRA appears to have had the greatest impact in the area of refugee admissions. As of January 2009, the United States had accepted 71 North Korean refugees for resettlement from undisclosed transit states. The first refugees—four women and two men—were accepted in May 2006. The State Department's Population, Refugees, and Migration (PRM) Bureau annually provides funds for UNHCR's annual regional budget for East Asia, which includes assistance for North Korean refugees, among other refugee populations. PRM funds international organizations such as UNHCR and the International Committee for the Red Cross. Radio Broadcasting into North Korea The NKHRA calls on the Broadcasting Board of Governors (BBG) to "facilitate the unhindered dissemination of information in North Korea" by increasing the amount of Korean-language broadcasts by RFA and VOA. (A much more modest amount has been appropriated to support independent radio broadcasters.) The hours of radio broadcasts into North Korea, through medium- and short-wave, were modestly increased beginning in 2006, and original programming was added in 2007. The BBG currently broadcasts to North Korea ten hours per day: RFA broadcasts three and one-half hours of original programming and one and one-half hours of repeat programming, and VOA broadcasts four hours of original and one hour of repeat programming with news updates each day. In FY2008, the BBG's budget request included $8.1 million to implement the 10-hour broadcast schedule, and the FY2009 request includes $8.5 million to maintain this schedule. Content includes news briefs, particularly news involving the Korean peninsula, interviews with North Korean defectors, and international commentary on events happening inside North Korea. The BBG cites an InterMedia survey of escaped defectors that indicates that North Koreans have some access to radios, many of them altered to receive international broadcasts. The BBG continues to explore ways to expand medium wave broadcast capability into North Korea. VOA is broadcast from BBG-owned stations in Tinian, Thailand, and the Philippines, and from leased stations in Russia and Mongolia. RFA is broadcast from stations in Tinian and Saipan and leased stations in Russia and Mongolia. Reauthorization Bill The reauthorization bill renews funding that expired in FY2008, reasserts key tenets of the legislation, and criticizes the pace of the executive branch implementation of the original law. It also cites the small number of resettlements of North Korean refugees and the slow processing of such refugees overseas. Funding is reauthorized through 2012 at the original levels of $2 million annually to support human rights and democracy programs, $2 million annually to promote freedom of information to North Koreans, and $20 million annually to assist North Korean refugees. It also requires additional reporting—portions of which can be classified as necessary—on U.S. efforts to process North Korean refugees, along with reporting from the Broadcasting Board of Governors on progress toward achieving 12 hours per day of broadcasting Korean language programming. Focus on Special Envoy The role and activities of the Special Envoy for Human Rights in North Korea (per the reauthorization bill, now the "Special Envoy for North Korean Human Rights Issues") have garnered particular attention from Congress. Jay Lefkowitz, appointed as the Special Envoy by President Bush nearly five months after the law was enacted in August 2005, was criticized for accepting the job as a part-time position while maintaining his legal career in New York. The reauthorization bill stipulates that the Special Envoy be an ambassador-level position—which requires confirmation by the Senate—and expresses the sense of Congress that the position should be full-time. Whereas the original legislation was vague on whether the refugee-specific provisions fell under the Envoy's responsibilities, the reauthorization bill includes the sense of Congress that the Envoy should "participate in policy planning and implementation" on North Korean refugee issues. The Obama Administration has not yet selected its Special Envoy. Lefkowitz's role varied in its public profile: at times he was an active and vocal advocate for human rights issues, and at other times he faded from public view. He attended international conferences dedicated to raising awareness of human rights abuses in North Korea and testified at multiple congressional hearings. As the Korean-U.S. Free Trade Agreement was negotiated, he raised questions about labor practices at the Kaesong complex, an industrial park located in North Korea in which a consortium of South Korean firms employ North Korean labor. Lefkowitz's visibility declined particularly in 2007 as the Bush Administration renewed its effort on nuclear negotiations. His statements occasionally sparked controversy: in January 2008, he gave a speech at a Washington think tank in which he criticized the denuclearization talks and voiced doubt that North Korea would ever give up its nuclear weapons. In response, then-Secretary of State Condoleezza Rice said, "Jay Lefkowitz has nothing to do with the Six-Party Talks... he certainly has no say in what American policy will be in the Six-Party Talks." In his final report to Congress in January 2009, Lefkowitz criticized some aspects of U.S. handling of refugee admissions and critiqued the North Korean policies of the South Korean, Chinese, and Japanese governments. Complications with Refugee Provisions Implementation Challenges Some observers contend that good-faith implementation of NKHRA's refugee provisions may be counterproductive. They argue that the legislation on North Korean refugee admissions could send a dangerous message to North Koreans that admission to the United States as a refugee is assured, encouraging incursions into U.S. diplomatic missions overseas. State Department officials say that given the tight security in place at U.S. facilities abroad, unexpected stormings could result in injury or death for the refugees. Secondly, granting of asylum status to North Korean refugees involves a complex vetting process that is further complicated by the fact that the applicants originate from a state with which the United States does not have official relations. In congressional hearings, State Department officials have cautioned that effective implementation of the NKHRA depends on close coordination with South Korea, particularly in developing mechanisms to vet potential refugees given the dearth of information available to U.S. immigration officials on North Koreans. Funding Concerns Some government officials and NGO staff familiar with providing assistance to North Korean refugees say that funding explicitly associated with the NKHRA is problematic because of the need for discretion in reaching the vulnerable population. Refugees are often hiding from authorities, and regional governments do not wish to draw attention to their role in transferring North Koreans, so funding is labeled under more general assistance programs. In addition, many of the NGOs that help refugees do not have the capacity to absorb large amounts of funding effectively because of their small, grass roots nature. A Ready Alternative for Resettlement South Korea remains the primary destination for North Korean refugees. In addition to automatically granting South Korean citizenship, the South Korean government administers a resettlement program and provides cash and training for all defectors. According to press reports, over 14,000 defectors from North Korea have resettled in the South since the conclusion of the Korean War in 1953, including over 2,500 in 2007 alone. The South Korean system of accepting refugees is faster and more streamlined than the U.S. process. Changes in South Korea's Approach? As part of its policy of increasing economic integration and fostering better ties with North Korea, South Korea until recently refrained from criticizing Pyongyang's human rights record and downplayed its practice of accepting North Korean refugees. Lee Myung-bak's election as South Korea's president in December 2007, however, appeared to usher in a new approach: Lee's administration has tied assistance from the South to North Korean progress on denuclearization and openly criticized North Korea's human rights situation. In years past, South Korea had usually abstained from voting on United Nations resolutions calling for improvement in North Korea's human rights practices. At the U.N. Human Rights Council meeting in March 2008, however, South Korea voted for a similar resolution. Lee has also conditioned fertilizer and food aid on improved access to the North's distribution systems to ensure that such aid is not going only to Pyongyang's elite and military. This approach has contributed to a considerable chill in North-South relations since Lee took office. A joint statement from President Bush and President Lee in August 2008 urged progress in improving North Korea's human rights, the first time such a mention appeared. | As the incoming Obama Administration conducts a review of U.S. policy toward North Korea, addressing the issue of human rights and refugees remains a priority for many members of Congress. The passage of the reauthorization of the North Korean Human Rights Act in October 2008 (P.L. 110-346) reasserted congressional interest in influencing executive branch policy toward North Korea. In addition to reauthorizing funding at original levels, the bill expresses congressional criticism of the implementation of the original 2004 law and adjusts some of the provisions relating to the Special Envoy on Human Rights in North Korea and the U.S. resettlement of North Korean refugees. Some outside analysts have pointed to the challenges of highlighting North Korea's human rights violations in the midst of the ongoing nuclear negotiations, as well as the difficulty in effectively reaching North Korean refugees as outlined in the law. Further, the law may complicate coordination on North Korea with China and South Korea. At this point, it remains unclear what focus the Obama Administration will place on human rights and refugees as it takes over the nuclear negotiations. For more information, please see CRS Report RL34189, North Korean Refugees in China and Human Rights Issues: International Response and U.S. Policy Options, coordinated by [author name scrubbed]. | gov_report |
Photo Elon Musk, the chief executive of Tesla Motors, has now responded in detail to the account of my test drive of his Model S electric car, using the company’s new East Coast Superchargers, that was published in The Times on Feb. 10. His broadest charge is that I consciously set out to sabotage the test. That is not so. I was delighted to receive the assignment to try out the company’s new East Coast Supercharger network and as I previously noted in no way anticipated – or deliberately caused – the troubles I encountered. The test was initially proposed by Tesla to Times editors, and the company arranged the timing, which came during a cold snap on the East Coast. It is fair to say that when I set out I did not fully appreciate how much of an effect the freezing temperatures would have on the travel range of the car. Since 2009, I have been the Washington bureau reporter responsible for coverage of energy, environment and climate change. I have written numerous articles about the auto industry and several vehicle reviews for the Automobiles pages. (In my 16 years at The Times I have served as White House correspondent, Washington editor, Los Angeles bureau chief and a political correspondent.) Before I set out in the Model S, I did speak with the company’s chief technology officer, J B Straubel, about the charging network and some of the car’s features and peculiarities. Neither he nor the Tesla representative who delivered the car to me provided detailed instructions on maximizing the driving range, the impact of cold weather on battery strength or how to get the most out of the Superchargers or the publicly available lower-power charging ports along the route. About three hours into the trip, I placed the first of about a dozen calls to Tesla personnel expressing concern about the car’s declining range and asking how to reach the Supercharger station in Milford, Conn. I was given battery-conservation advice at that time (turn off the cruise control; alternately slow down and speed up to take advantage of regenerative braking) that was later contradicted by other Tesla personnel. I was on the phone with a Tesla engineer in California when I arrived, with zero miles showing on the range meter, at the Milford Supercharger. Photo Beginning early in the morning of my second day with the car, after the projected range had dropped precipitously while parked overnight, I spoke numerous times with Christina Ra, Tesla’s spokeswoman at the time, and Ted Merendino, a Tesla product planner at the company’s headquarters in California. They told me that the loss of battery power when parked overnight could be restored by properly “conditioning” the battery, a half-hour process, which I undertook by sitting in the car with the heat on low, as they instructed. That proved ineffective; the conditioning process actually reduced the range by 24 percent (to 19 miles, from 25 miles). It was also Tesla that told me that an hour of charging (at a lower power level) at a public utility in Norwich, Conn., would give me adequate range to reach the Supercharger 61 miles away, even though the car’s range estimator read 32 miles – because, again, I was told that moderate-speed driving would “restore” the battery power lost overnight. That also proved overly optimistic, as I ran out of power about 14 miles shy of the Milford Supercharger and about five miles from the public charging station in East Haven that I was trying to reach. To reiterate: Tesla personnel told me over the phone that they were able to monitor the state of the battery. It was they who cleared me to leave Norwich after an hour of charging. I spoke at some length with Mr. Straubel and Ms. Ra six days after the trip, and asked for the data they had collected from my drive, to compare against my notes and recollections. Mr. Straubel said they were able to monitor “certain things” remotely and that the company could store and retrieve “typical diagnostic information on the powertrain.” Mr. Straubel said Tesla did not store data on exact locations where their cars were driven because of privacy concerns, although Tesla seemed to know that I had driven six-tenths of a mile “in a tiny 100-space parking lot.” While Mr. Musk has accused me of doing this to drain the battery, I was in fact driving around the Milford service plaza on Interstate 95, in the dark, trying to find the unlighted and poorly marked Tesla Supercharger. He did not share that data, which Tesla has now posted online, with me at the time. Here are point-by-point responses to specific assertions Mr. Musk has made: • “As the State of Charge log shows, the Model S battery never ran out of energy at any time, including when Broder called the flatbed truck.” The car’s display screen said the car was shutting down, and it did. The car did not have enough power to move, or even enough to release the electrically operated parking brake. The tow truck driver was on the phone with Tesla’s New York service manager, Adam Williams, for 15 or 20 minutes as he was trying to move the car onto a flatbed truck. • “The final leg of his trip was 61 miles and yet he disconnected the charge cable when the range display stated 32 miles. He did so expressly against the advice of Tesla personnel and in obvious violation of common sense.” The Tesla personnel whom I consulted over the phone – Ms. Ra and Mr. Merendino – told me to leave it connected for an hour, and after that the lost range would be restored. I did not ignore their advice. • “In his article, Broder claims that ‘the car fell short of its projected range on the final leg.’ Then he bizarrely states that the screen showed ‘Est. remaining range: 32 miles’ and the car traveled ‘51 miles’ contradicting his own statement (see images below). The car actually did an admirable job exceeding its projected range. Had he not insisted on doing a nonstop 61-mile trip while staring at a screen that estimated half that range, all would have been well. He constructed a no-win scenario for any vehicle, electric or gasoline.” The phrase “the car fell short of its projected range” appeared in a caption with an accompanying map; it was not in the article. What that referred to (and admittedly could have been more precise) was that the car fell short of the projected range, 90 miles, that it showed when I parked it overnight at a hotel in Groton, Conn. Tesla is correct that the car did exceed the projected range of 32 miles when I left Norwich, as I was driving slowly, and it gave me hope that the Tesla employee I’d consulted was correct that the mileage lost overnight was being restored. It wasn’t enough, however, to get to Milford. • “On that leg, he drove right past a public charge station while the car repeatedly warned him that it was very low on range.” If there was a public charging station nearby, no one made me aware of it. The Tesla person with whom I was in contact located on the Internet a public charging station in East Haven, Conn., and that is the one I was trying to reach when the car stalled in Branford, about five miles shy of East Haven. • “Cruise control was never set to 54 m.p.h. as claimed in the article, nor did he limp along at 45 m.p.h. Broder in fact drove at speeds from 65 m.p.h. to 81 m.p.h. for a majority of the trip, and at an average cabin temperature setting of 72 F.” I drove normally (at the speed limit or with prevailing traffic) when I thought it was prudent to do so. I do recall setting the cruise control to about 54 m.p.h., as I wrote. The log shows the car traveling about 60 m.p.h. for a nearly 100-mile stretch on the New Jersey Turnpike. I cannot account for the discrepancy, nor for a later stretch in Connecticut where I recall driving about 45 m.p.h., but it may be the result of the car being delivered with 19-inch wheels and all-season tires, not the specified 21-inch wheels and summer tires. That just might have affected the recorded speed, range, rate of battery depletion or any number of other parameters. Tesla’s data suggests I was doing slightly more than 50 over a stretch where the speed limit was 65. The traffic was heavy in that part of Connecticut, so cruise control was not usable, and I tried to keep the speed at 50 or below without impeding traffic. Certainly, and as Tesla’s logs clearly show, much of my driving was at or well below the 65 m.p.h. speed limit, with only a single momentary spike above 80. Most drivers are aware that cars can speed up, even sometimes when cruise control is engaged, on downhill stretches. • “At the point in time that he claims to have turned the temperature down, he in fact turned the temperature up to 74 F.” I raised and lowered the cabin heat in an effort to strike a balance between saving energy and staying somewhat comfortable. (It was 30 degrees outside when I began the trip, and the temperature plunged that night to 10 degrees.) Tesla jumped to the conclusion that I claimed to have lowered the cabin temperature “at 182 miles,” but I never wrote that. The data clearly indicates that I sharply lowered the temperature setting – twice – a little over 200 miles into the trip. After the battery was charged I tried to warm the cabin. • “The charge time on his second stop was 47 minutes, going from —5 miles (reserve power) to 209 miles of Ideal or 185 miles of E.P.A. Rated Range, not 58 minutes as stated in the graphic attached to his article. Had Broder not deliberately turned off the Supercharger at 47 mins and actually spent 58 mins Supercharging, it would have been virtually impossible to run out of energy for the remainder of his stated journey.” According to my notes, I plugged into the Milford Supercharger at 5:45 p.m. and disconnected at 6:43 p.m. The range reading was 185 miles. • “For his first recharge, he charged the car to 90%. During the second Supercharge, despite almost running out of energy on the prior leg, he deliberately stopped charging at 72%. On the third leg, where he claimed the car ran out of energy, he stopped charging at 28%. Despite narrowly making each leg, he charged less and less each time. Why would anyone do that?” I stopped at 72 percent because I had replenished more than enough energy for the miles I intended to drive the next day before fully recharging on my way back to New York. In Norwich, I charged for an hour on the lower-power charger, expressly on the instructions of Tesla personnel, to get enough range to reach the Supercharger station in Milford. • “The above helps explain a unique peculiarity at the end of the second leg of Broder’s trip. When he first reached our Milford, Conn., Supercharger, having driven the car hard and after taking an unplanned detour through downtown Manhattan to give his brother a ride, the display said “0 miles remaining.” Instead of plugging in the car, he drove in circles for over half a mile in a tiny, 100-space parking lot. When the Model S valiantly refused to die, he eventually plugged it in. On the later legs, it is clear Broder was determined not to be foiled again.” I drove around the Milford service plaza in the dark looking for the Supercharger, which is not prominently marked. I was not trying to drain the battery. (It was already on reserve power.) As soon as I found the Supercharger, I plugged the car in. The stop in Manhattan was planned from the beginning and known to Tesla personnel all along. According to Google Maps, taking the Lincoln Tunnel into Manhattan (instead of crossing at the George Washington Bridge) and driving up the West Side Highway added only two miles to the overall distance from Newark, Del., to Milford, Conn. Neither I nor the Model S ever visited “downtown Manhattan.” • “When I first heard about what could at best be described as irregularities in Broder’s behavior during the test drive, I called to apologize for any inconvenience that he may have suffered and sought to put my concerns to rest, hoping that he had simply made honest mistakes. That was not the case.” Mr. Musk not only apologized, he said the charging stations should be 60 miles closer together and offered me a second test drive when additional stations were built. Matt Quinn is CTO of TIBCO, the parent company of LogLogic, which provides log management software. When you think of “Big data,” you rarely think of log data. It just doesn’t have much sex appeal: It’s what IT uses to monitor applications, compliance, and security. But we got a reminder this week that log data truly is valuable big data. After New York Times writer John Broder published a review bashing Tesla’s new Model S, Tesla CEO Elon Musk stepped forward Wednesday with what he said was log data generated by the car during Broder’s test drive, and the data clearly refutes the writer’s account. Log data and log data management make the world a little bit more honest by giving us hard facts about what’s really happening in the moment, at the most granular level of detail imaginable. Beyond the intrinsic value in data-as-it-happens, log data immediately becomes a historical record that can prove or disprove our hunches and, at times, fabrications. Whether Musk or Broder are giving us the facts isn’t the overarching point. With this story, log data hit the mainstream consciousness and will be an expectation in the future. “Where’s the log data?” is now the question that can close the argument and help us make the decision about what to believe and where to put our resources. The Tesla case isn’t the first time we’ve seen log data make headlines. It was in the spotlight recently when MIT was accused of releasing its network logs to law enforcement without a subpoena, providing the information used to accuse Aaron Swartz of downloading scientific journals for public release. Swartz’s lawyers worked to suppress the highly detailed logs because they knew these ‘golden records’ were powerful, detailed evidence of what happened. The Swartz case and its unfortunate outcome is a harbinger of what’s to come with log data. Log data was also a key component of the case against Martha Stewart in 2004. In 2006, Florida Circuit Court Judge Elizabeth Maass, writing on the e-discovery challenge, said that electronic data “… are the modern-day equivalent of the paper trail.” Those cases have laid the legal groundwork for log data, but there’s a rapidly changing volume, variety, and velocity of data that makes it much harder to keep up with. We’re globalizing and automating at a rapid rate, leaving the world more vulnerable to problems on a scale we haven’t seen before, like sporadic, vexing outages in enormous data centers, sophisticated digital fraud launched from anywhere in the world, and even computer viruses that can ‘sleep’ for periods of time to avoid detection before waking up and wreaking havoc. But everything, as Musk knows well as Broder found out, leaves a data trail. Managing log data is big data without the hype of needles in haystacks. Log data used on the fly stops fraudulent credit card transactions before they happen for retailers like RadioShack, brings up another server before the website goes down for Hamleys in the UK, and demonstrates that we’re following regulations at the detailed level for headset manufacturer Plantronics. Log data is the cold, hard, fast-moving, fast-accumulating truth. Tesla versus New York Times will fade into history, but the technology Musk is pointing to is the future. This week, the world was introduced to a key big data story whether they knew it or not. [Top image credit: Maksim Kabakou /Shutterstock] | A New York Times auto reviewer has issued a point-by-point rebuttal to Tesla's claims that he deliberately set out to make the Model S electric car look bad. In one of the standout accusations, based on the car's driving logs, Tesla's Elon Musk wrote that John Broder "drove in circles for over half a mile in a tiny, 100-space parking lot" in an attempt to run down the battery. How on earth might Broder explain that? Pretty easily, it turns out: "I drove around the Milford service plaza in the dark looking for the Supercharger, which is not prominently marked. I was not trying to drain the battery. (It was already on reserve power.) As soon as I found the Supercharger, I plugged the car in." Adds Simon Dumenco at AdAge: "Stay tuned for, surely, more drama to come-including return fire from Musk-and hopefully a TV-movie dramatization of this epic CEO-vs.-reporter, blog-vs.-blog battle." But while this might seem over the top, the real story here is how the spat illustrates the growing importance of log data, writes Matt Quinn at VentureBeat. "This week, the world was introduced to a key big data story whether they knew it or not." | multi_news |
Understanding the theoretical foundations of how memories are encoded and retrieved in neural populations is a central challenge in neuroscience. A popular theoretical scenario for modeling memory function is the attractor neural network scenario, whose prototype is the Hopfield model. The model simplicity and the locality of the synaptic update rules come at the cost of a poor storage capacity, compared with the capacity achieved with perceptron learning algorithms. Here, by transforming the perceptron learning rule, we present an online learning rule for a recurrent neural network that achieves near-maximal storage capacity without an explicit supervisory error signal, relying only upon locally accessible information. The fully-connected network consists of excitatory binary neurons with plastic recurrent connections and non-plastic inhibitory feedback stabilizing the network dynamics; the memory patterns to be memorized are presented online as strong afferent currents, producing a bimodal distribution for the neuron synaptic inputs. Synapses corresponding to active inputs are modified as a function of the value of the local fields with respect to three thresholds. Above the highest threshold, and below the lowest threshold, no plasticity occurs. In between these two thresholds, potentiation/depression occurs when the local field is above/below an intermediate threshold. We simulated and analyzed a network of binary neurons implementing this rule and measured its storage capacity for different sizes of the basins of attraction. The storage capacity obtained through numerical simulations is shown to be close to the value predicted by analytical calculations. We also measured the dependence of capacity on the strength of external inputs. Finally, we quantified the statistics of the resulting synaptic connectivity matrix, and found that both the fraction of zero weight synapses and the degree of symmetry of the weight matrix increase with the number of stored patterns. One of the fundamental challenges in neuroscience is to understand how we store and retrieve memories for a long period of time. Such long-term memory is fundamental for a variety of our cognitive functions. A popular theoretical framework for storing and retrieving memories in recurrent neural networks is the attractor network model framework [1–3]. Attractors, i. e. stable states of the dynamics of a recurrent network, are set by modification of synaptic efficacies in a recurrent network. Synaptic plasticity rules specify how the efficacy of a synapse is affected by pre- and post-synaptic neural activity. In particular, Hebbian synaptic plasticity rules lead to long-term potentiation (LTP) for correlated pre- and post-synaptic activities, and long-term depression (LTD) for anticorrelated activities. These learning rules build excitatory feedback loops in the synaptic connectivity, resulting in the emergence of attractors that are correlated with the patterns of activity that were imposed on the network through external inputs. Once a set of patterns become attractors of a network (in other words when the network “learns” the patterns), upon a brief initial activation of a subpopulation of neurons, the network state evolves towards the learned stable state (the network “retrieves” a past stored memory), and remains in that state after removal of the external inputs (and hence maintains the information in short-term memory). The set of initial network states leading to a memorized state is called the basin of attraction, whose size determines how robust a memory is. The attractor neural network scenario was originally explored in networks of binary neurons [1,2], and then extended from the 90s to networks of spiking neurons [4–7]. Experimental evidence in different areas of the brain, including inferotemporal cortex [8–11] and prefrontal cortex [12–14], has provided support for the attractor neural network framework, using electrophysiological recordings in awake monkeys performing delayed response tasks. In such experiments, the monkey has to maintain information in short-term (working) memory in a ‘delay period’ to be able to perform the task. Consistent with the attractor network scenario, some neurons exhibit selective persistent activity during the delay period. This persistent activity of ensembles of cortical neurons has thus been hypothesized to form the basis of the working memory of stimuli shown in these tasks. One of the most studied properties of attractor neural network as a model of memory is its storage capacity, i. e. how many random patterns can be learned in a recurrent network of N neurons in the large N limit. Storage capacity depends both on the network architecture and on the synaptic learning rule. In many models, the storage capacity scales with N. In particular, the Hopfield network [1] that uses a Hebbian learning rule has a storage capacity of 0. 138N in the limit of N → ∞ [15]. Later studies showed how the capacity depends on the connection probability in a randomly connected network [16,17] and on the coding level (fraction of active neurons in a pattern) [18,19]. A natural question is, what is the maximal capacity of a given network architecture, over all possible learning rules? This question was answered by Elizabeth Gardner, who showed that the capacity of fully connected networks of binary neurons with dense patterns scales as 2N [20], a storage capacity which is much larger than the one of the Hopfield model. The next question is what learning rules are able to saturate the Gardner bound? A simple learning rule that is guaranteed to achieve this bound is the perceptron learning rule (PLR) [21] applied to each neuron independently. However, unlike the rule used in the Hopfield model, the perceptron learning rule is a supervised rule that needs an explicit “error signal” in order to achieve the Gardner bound. While such an error signal might be available in the cerebellum [22–24], it is unclear how error signals targeting individual neurons might be implemented in cortical excitatory synapses. Therefore, it remains unclear whether and how networks with realistic learning rules might approach the Gardner bound. The goal of the present paper is to propose a learning rule whose capacity approaches the maximal capacity of recurrent neural networks by transforming the original perceptron learning rule such that the new rule does not explicitly use an error signal. The perceptron learning rule modifies the synaptic weights by comparing the desired output with the actual output to obtain an error signal, subsequently changing the weights in the opposite direction of the error signal. We argue that the total synaptic inputs (‘local fields’) received by a neuron during the presentation of a stimulus contain some information about the current error (i. e. whether the neuron will end up in the right state after the stimulus is removed). We use this insight to build a field dependent learning rule that contains three thresholds separating no plasticity, LTP and LTD regions. This rule implements basic biological constraints: (a) it uses only information local to the synapse; (b) the new patterns can be learned incrementally, i. e. it is an online rule; (c) it does not need an explicit error signal; (d) synapses obey Dale’s principle, i. e. excitatory synapses are not allowed to have negative weights. We studied the capacity and the size of the basins of attraction for a binary recurrent neural network in which excitatory synapses are endowed with this rule, while a global inhibition term controls the global activity level. We investigated how the strength of external fields and the presence of correlations in the inputs affect the memory capacity. Finally, we investigated the statistical properties of the connectivity matrix (distribution of synaptic weights, degree of symmetry). We simulated a network of N binary (McCulloch-Pitts) neurons, fully-connected with excitatory synapses (Fig 1A). All the neurons feed a population of inhibitory neurons which is modeled as a single aggregated inhibitory unit. This state-dependent global inhibition projects back onto all the neurons, stabilizing the network and controlling its activity level. At each time step, the activity (or the state) of neuron i (i = 1…N) is described by a binary variable si ∈ {0,1}. The state is a step function of the local field vi of the neuron: s i = Θ (v i - θ), (1) where Θ is the Heaviside function (Θ (x) = 1 if x > 0 and 0 otherwise) and θ is a neuronal threshold. The local field vi represents the overall input received by the neuron from its excitatory and inhibitory connections (Fig 1B). The excitatory connections are of two kinds: recurrent connections from within the excitatory population, and external inputs. The recurrent excitatory connections are mediated by synaptic weights, denoted by a matrix W whose elements wij (the weight of the synapse from neuron j to i) are continuous non-negative variables (wij ∈ [0, ∞); wii = 0). In the following, and in all our simulations, we assume that the weights are initialized randomly before the training takes place (see Materials and Methods). Therefore, in the absence of external inputs, the local field of each neuron i is given by: v i = ∑ j = 1 N w i j s j - I 0 (s →), (2) where I 0 (s →) represents the inhibitory input. For the sake of simplicity, we simulated a synchronous update process, in which the activity of each neuron si is computed from the local field vi at the previous time step, and all updates happen in parallel. The network was designed so that, in absence of external input and prior to the training process, it should spontaneously stabilize itself to some fixed overall average activity level f (fraction of active neurons, or sparseness), regardless of the initial conditions. In particular, we aimed at avoiding trivial attractors (the all-off and all-on states). To this end, we model the inhibitory feedback (in absence of external inputs) as a linear function of the overall excitatory activity: I 0 (s →) = H 0 + λ (∑ i = 1 N s i - f N). (3) The parameters H0 and λ can be understood as follows: H0 is the average inhibitory activity when the excitatory network has the desired activity level f, i. e. when ∑ i = 1 N s i = f N; λ measures the strength of the inhibitory feedback onto the excitatory network. This expression can be interpreted as a first-order approximation of the inhibitory activity as a function of the excitatory activity around some reference value fN, which is reasonable under the assumption that the deviations from fN are small enough. Indeed, by properly setting these two parameters in relation to the other network parameters (such as θ and the average connection strength) it is possible to achieve the desired goal of a self-stabilizing network. In the training process, the network is presented a set of p patterns in the form of strong external inputs, representing the memories which need to be stored. We denote the patterns as { ξ μ → } (where μ = 1…p and ξ i μ ∈ { 0,1 }), and assume that each entry ξ i μ is drawn randomly and independently. For simplicity, the coding level f for the patterns was set equal to the spontaneous activity level of the network, i. e. ξ i μ = 1 with probability f, 0 otherwise. During the presentation of a pattern μ, each neuron i receives an external binary input x i = X ξ i μ, where X denotes the strength of the external inputs, which we parameterized as X = γ N. In addition, the external input also affects the inhibitory part of the network, eliciting a response which indirectly downregulates the excitatory neurons. We model this effect as an additional term H1 in the expression for the inhibitory term (Eq 3), which therefore becomes: I (x →, s →) = H 0 + H 1 ∑ i = 1 N x i f N X + λ (∑ i = 1 N s i - f N), (4) The general expression for the local field vi then reads: v i = ∑ j = 1 N w i j s j + x i - I (x →, s →). (5) In the absence of external fields, xi = 0 for all i, and thus Eqs 4 and 5 reduce to the previous expressions Eqs 3 and 2. The goal of the learning process is to find values of wij’s such that the patterns { ξ μ → } become attractors of the network dynamics. Qualitatively, this means that, if the training process is successful, then whenever the network state gets sufficiently close to one of the stored patterns, i. e. whenever the Hamming distance d = ∑ i = 1 N ∣ ξ i μ − s i ∣ between the current network state and a pattern μ is sufficiently small, the network dynamics in the absence of external inputs should drive the network state towards a fixed point equal to the pattern itself (or very close to it). The general underlying idea is that, after a pattern is successfully learned, some brief external input which initializes the network close to the learned state would be sufficient for the network to recognize and retrieve the pattern. The maximum value of d for which this property holds is then called the basin of attraction size (or just basin size hereafter for simplicity); indeed, there is generally a trade-off between the number of patterns which can be stored according to this criterion and the size of their basin of attraction. More precisely, the requirement that a pattern ξ μ → is a fixed point of the network dynamics in the absence of external fields can be reduced to a condition for each neuron i (cfr. Eqs 4 and 5): ∀ i: Θ (∑ j = 1 N w i j ξ j μ - I (0 →, ξ μ →) - θ) = ξ i μ. (6) This condition only guarantees that, if the network is initialized into a state s → = ξ μ →, then it will not spontaneously change its state, i. e. it implements a zero-size basin of attraction. A simple way to enlarge the basin size is to make the requirement in Eq 6 more stringent, by enforcing a more stringent constraint for local fields: ∀ i: { ∑ j = 1 N w i j ξ j μ - I (0 →, ξ μ →) > θ + f N ϵ if ξ i μ = 1 ∑ j = 1 N w i j ξ j μ - I (0 →, ξ μ →) < θ - f N ϵ if ξ i μ = 0, (7) where ϵ ≥ 0 is a robustness parameter. When ϵ = 0, we recover the previous zero-basin-size scenario; increasing ϵ we make the neurons’ response more robust towards noise in their inputs, and thus we enlarge the basin of attraction of the stored patterns (but then fewer patterns can be stored, as noted above). In the training phase, the network is presented with patterns as strong external fields xi. Patterns are presented sequentially in random order. For each pattern μ, we simulated the following scheme: Step 1: The pattern is presented (i. e. the external inputs xi are set to X ξ i μ). A single step of synchronous updating is performed (Eqs 1,4 and 5). If the external inputs are strong enough, i. e. γ is large enough, this updating sets the network in a state corresponding to the presented pattern. Step 2: Learning occurs. Each neuron i may update its synaptic weights depending on 1) their current value w i j t, 2) the state of the pre-synaptic neurons, and 3) the value of the local field vi. Therefore, all the information required is locally accessible, and no explicit error signals are used. The new synaptic weights w i j t + 1 are set to: wijt+1 = { wijt−ηsj, if θ0<vi<θwijt+ηsj, if θ<vi<θ1wijt, otherwise, (8) where η is the learning rate, and θ0 and θ1 are two auxiliary learning thresholds set as θ 0 = θ - (γ + ϵ) f N (9) θ 1 = θ + (γ + ϵ) f N. (10) We refer to this update scheme as the “three-threshold learning rule” (3TRL). After some number of presentations, we checked whether the patterns are learned by presenting a noisy version of these patterns, and checking whether the patterns (or network states which are very close to the patterns) are fixed points of the network dynamics. When N ≫ 1, γ is large enough, and H1 = fX, the update rule described by Eq 8 is essentially equivalent to the perceptron learning rule for the task described in Eq 7. This can be shown as follows (see also Fig 2 for a graphical representation of the case f = 0. 5 and ϵ = 0): when a stimulus is presented, the population of neurons is divided in two groups, one for which xi = 0 and one for which xi = X. The net effect of the stimulus presentation on the local field has to take into account the indirect effect through the inhibitory part of the network (see Eq 4), and thus is equal to −fX for the xi = 0 population and to (1 − f) X for the xi = X population. Before learning, the distribution of the local fields across the excitatory population, in the limit N → ∞, is a Gaussian whose standard deviation is proportional to N, due to the central limit theorem; moreover, the parameter H0 is set so that the average activity level of the network is f, which means that the center of the Gaussian will be within a distance of order N from the neuronal threshold θ (this also applies if we use different values for the spontaneous activity level and the pattern activity level). Therefore, if X = γ N is large enough, the state of the network during stimulus presentation will be effectively clamped to the desired output, i. e. s i = ξ i μ for all i. This fact has two consequences: 1) the local field potential can be used to detect the desired output by just comparing it to the threshold, and 2) each neuron i will receive, as its recurrent inputs {sj}j ≠ i, the rest of the pattern { ξ j μ } j ≠ i. Furthermore, due to the choice of the secondary thresholds θ0 and θ1 in Eqs 9 and 10, the difference between the local field and θ0 (or θ1) during stimulus presentation for the xi = 0 population (or xi = X, respectively) is equal to the difference between the local field and θ − f N ϵ (or θ + f N ϵ, respectively) in the absence of external stimuli, provided the recurrent inputs are the same. Therefore, the value of the local field vi during stimulus presentation in relation to the three thresholds θ, θ0 and θ1 is sufficient to determine whether an error is made with respect to the constraints of Eq 7, and which kind of error is made. Following these observations, it is straightforward to map the standard perceptron learning rule on the 4 different cases which may occur (see Fig 2), resulting in Eq 8. In Fig 3 we demonstrate the effect of the learning rule on the distribution of the local field potentials as measured from a simulation (with f = 0. 5 and ϵ = 1. 2): the initial distribution of the local fields of the neurons, before the learning process takes place and in the absence of external fields, is well described by a Gaussian distribution centered on the neuronal threshold θ (see Fig 3A) with a standard deviation which scales as N. During a pattern presentation, the resulting distribution becomes a bimodal one; before learning takes place, the distribution is given by the sum of two Gaussians of equal width, centered around θ 0 + f N ϵ and θ 1 − f N ϵ (Fig 3B). The left Gaussian corresponds to the cases where xi = 0 and the right one to the cases where xi = X. Having applied the learning rule, we observe that the depression region (i. e. the interval (θ0, θ) ) and the potentiation region (i. e. (θ, θ1) ) gets depleted (Fig 3C). In the testing phase, when the external inputs are absent, the left and right parts of the distribution come closer, such that the distance between the two peaks is equal to at least 2 ϵ f N (Fig 3D). This margin between the local fields of the ON and OFF neurons makes the attractors more robust. Since our proposed learning rule is able to mimic (or approximate, depending on the parameters) the perceptron learning rule, which is known to be able to solve the task posed by Eq 7 whenever a solution exists, we expect that a network implementing such rule can get close to maximal capacity in terms of the number of memories which it can store at a given robustness level. The storage capacity, denoted by α = p/N, is measured as a ratio of the maximum number of patterns p which can successfully be stored to the number of neurons N, in the limit of large N. As mentioned above, it is a function of the basin size. We used the following definition for the basin size: a set of p patterns is said to be successfully stored at a size b if, for each pattern, the retrieval rate when starting from a state in which a fraction b of the pattern was randomized is at least 90%. The retrieval rate is measured by the probability that the network dynamics is able to bring the network state to an attractor within 1% distance from the pattern, in at most 30 steps. The distance between the state of the network and a pattern μ is measured by the normalized Hamming distance 1 N ∑ i = 1 N ∣ s i − ξ i μ ∣. Therefore, at coding level f = 0. 5, reaching a basin size b means that the network can successfully recover patterns starting from a state at distance b/2. Fig 4A shows the maximal capacity as a function of the basin size for a simulated network of N = 1001 neurons. We simulated many pairs of (α, ϵ) with different random seeds, obtaining a probability of success for each pair. The red line shows the points for which the probability of successful storage is 0. 5, and the error bars span 0. 95 to 0. 05 success probability. The capacity was optimized over the robustness parameter ϵ. The maximal capacity (the Gardner bound) in the limit of N → ∞ at the zero basin size is αc = 2 for our model (see Materials and Methods for the calculation), as for a network with unconstrained synaptic weights [20]. In Fig 4A, we also compare our network with the Hopfield model. Our network stores close to the maximal capacity at zero basin size, at least eleven times more than the Hopfield model. Across the range of basin sizes, 3TLR achieves more than twice the capacity that can be achieved with the Hopfield model. The enlargement of the basin of attraction was achieved by increasing the robustness parameter ϵ. We computed the maximal theoretical capacity as a function of ϵ at N → ∞ (see Materials and Methods) and compared it to our simulations, and to the maximal theoretical capacity of the Hopfield network. The results are shown in Fig 4B. For any given value of ϵ, the cyan curve shows the maximum α for which the success ratio with our network was at least 0. 5 across different runs. The difference between the theory and the experiments in our model can be ascribed to several factors: the finite size of the network; the choice of the finite learning rate η, and the fact that we imposed a hard limit on the number of pattern presentations (see number of iterations in Table 1), while the perceptron rule for excitatory synaptic connectivity is only guaranteed to be optimal in the limit of η → 0, with a number of presentations inversely proportional to η [25]. Note that the correspondence between the PLR and the 3TLR is only perfect in the large γ limit, and is only approximate otherwise, as can be shown by comparing explicitly the synaptic matrices obtained by both algorithms on the same set of patterns (see Materials and Methods). A crucial ingredient of the 3TLR is having a strong external input which effectively acts as a supervisory signal. How strong do the external fields need to be? How much does the capacity depend on this strength? To answer these questions, we measured the maximum number of stored patterns as a function of the parameter γ which determines the strength of external fields as X = γ N. This parameter, in fact, determines how far the two Gaussian distributions of the local field are; as shown in Fig 2, the distance between the two peaks of the distribution is X. For large enough γ, the overlap of these two distributions is negligible and the capacity is maximal; but as we lower γ, the overlap increases, causing the learning rule to make mistakes, i. e. when it should potentiate, it depresses the synapses and vice versa. In our simulations with N = 1001 neurons in the dense regime f = 0. 5 at a fixed epsilon ϵ = 0. 3, we varied γ and computed the maximum α that can be achieved with a fixed number of iterations (1000). The capacity indeed gradually decreases as γ decreases, until it reaches a threshold, below which there is a sharp drop of capacity (see Fig 5). With the above values for the parameters, this transition occurs at γ ≈ 2. 4. The 3TLR can also be adapted to work in a sparser regime, at a coding level lower than 0. 5. However, the average activity level of the network is determined by H0, and their relationship also involves the variance of the distribution of the synaptic weights when f ≠ 0. 5 (see Materials and Methods). During the learning process, the variance of the weights changes, which implies that the parameter H0 must adapt correspondingly. In our simulations, this adaptation was performed after each complete presentation of the whole pattern set. In practice, this additional self-stabilizing mechanism could still be performed in an unsupervised fashion along with (or in alternation with) the learning process. Using this adjustment, we simulated the network at f = 0. 2 and compared the results with the theoretical calculations. As shown in Fig 6, we can achieve at least 70% of the critical capacity across different values of the robustness parameter ϵ. We also investigated numerically the effect of correlations in the input patterns. The PLR is able to learn correlated patterns as long as a solution to the learning problem exists. As the 3TLR approximates the PLR, we expect the 3TLR to be able to learn correlated patterns as well. As a simple model of correlation, we tested patterns organized in L categories [26,27]. Each category was defined by a randomly generated prototype. Prototypes were uncorrelated from category to category. For each category, we then generated p/L patterns independently with a specified correlation coefficient c with the corresponding prototype. We show in Fig 7 the results of simulations with L = 5, f = 0. 2 and ϵ = 3. The figure shows that the learning rule reaches a capacity that is essentially independent of c, in the range 0 ≤ c ≤ 0. 75. We next investigated the statistical properties of the connectivity matrix after the learning process. Previous studies have shown that the distribution of synaptic weights in perceptrons with excitatory synapses becomes at maximal capacity a delta function at zero weight, plus a truncated Gaussian for strictly positive weights [25,28–30]. Our model differs from this setting because of the global inhibitory feedback. Despite this difference, the distribution of weights in our network bear similarities with the results obtained in these previous studies: the distribution exhibits a peak at zero weight (‘silent’, or ‘potential’ synapses), while the distribution of strictly positive weights resembles a truncated Gaussian. Finally, the fraction of silent synapses increases with the robustness parameter (see Fig 8). We have also computed the degree of symmetry of the weight matrix. The symmetry degree is computed as the Pearson correlation coefficient between the reciprocal weights in pairs of neurons. We observe a general trend towards an increasingly symmetric weight matrix as more patterns are stored, for all values of the robustness parameter ϵ (see Fig 9). The 3TLR can be framed in the setting of the classic Bienenstock-Cooper-Munro (BCM) theory [34,35], with additional requirements to adapt it to the attractor network scenario. The original BCM theory uses firing-rate units, and prescribes that synaptic modifications should be proportional to (1) the synaptic input, and (2) a function ϕ (v) of the total input v (or, equivalently, of the total output). The function ϕ (v) is subject to two conditions: (1) ϕ (v) ≥ 0 (or ≤ 0) when v > θ (or < θ, respectively); (2) ϕ (0) = 0. The parameter θ is also assumed to change, but on a longer time scale (such that the changes reflect the statistics of the inputs); this (metaplastic) adaptation has the goal of avoiding the trivial situations in which all inputs elicit indistinguishable responses. This (loosely specified) framework ensures that, under reasonable conditions, the resulting units become highly selective to a subset of the inputs, and has been mainly used to model the developmental stages of primary sensory cortex. The arising selectivity is spontaneous and completely unsupervised: in absence of further specifications, the units become selective to a random subset of the inputs (e. g. depending on random initial conditions). Our model is defined on simpler (binary) units; however, if we define ϕ (v) = Θ (v − θ) Θ (θ1 − v) − Θ (θ − v) Θ (v − θ0), then ϕ behaves according to the prescriptions of the BCM theory. Furthermore, we have essentially assumed the same slow metaplastic adaptation mechanism of BCM, even though we have assigned this role explicitly to the inhibitory part of the network (see Materials and Methods). On the other hand, our model has additional requirements: (1) ϕ (v) = 0 when v < θ0 or v > θ1, (2) plasticity occurs during presentation of external inputs, which in turn are strong enough to drive the network towards a desired state. The second requirement ensures that the network units become selective to a specific subset of the inputs, as opposed to a random subset as in the original BCM theory, and thus that they are able to collectively behave as an attractor network. The first requirement ensures that each unit operates close to critical capacity. Indeed, these additional requirements involve extra parameters with respect to the BCM theory, and we implicitly assume these parameters to also slowly adapt according to the statistics of the inputs during network formation and development. A variant of the BCM theory, known as ABS rule [36,37] introduced a lower threshold for LTD, analogous to our θ0, motivated by experimental evidence; however, a high threshold for LTP, analogous to our θ1, was not used there, or—to our knowledge—in any other BCM variant. The idea of stopping plasticity above some value of the ‘local field’ has been introduced previously to stabilize the learning process in feed-forward networks with discrete synapses [38–40]. Our study goes beyond these previous works in generalizing such a high threshold to recurrent networks, and showing that the resulting networks achieve close to maximal capacity. In vitro experiments have characterized how synaptic plasticity depends on voltage [41] and firing rate [42], both variables that are expected to have a monotonic relationship with the total excitatory synaptic inputs received by a neuron. In both cases, a low value of the controlling variable leads to no changes; intermediate values lead to depression; and high values to potentiation. These three regimes are consistent with the three regions for v < θ1 in Fig 2. The 3TLR predicts that a fourth region should occur at sufficiently high values of the voltage and/or firing rates. Most of the studies investigating the dependence of plasticity on firing rate or voltage have not reported a decrease in plasticity at high values of the controlling variables, but these studies might have not increased sufficiently such variables. To our knowledge, a single study has found that at high rates, the plasticity vs rate curve is a decreasing function of the input rate [43]. Another test of the model consists in comparing the statistics of the synaptic connectivity with experimental data. As it has been argued in several recent studies [25,28,30,44,45], networks with plastic excitatory synapses are generically sparse close to maximal capacity, with a connection probability that decreases with the robustness of information storage, consistent with short range cortical connectivity [46,47]. Our network is no exception, though the fraction of silent synapses that we observe is significantly lower than in models that lack inhibition. Furthermore, network that are close to maximal capacity tends to have a connectivity matrix that has a significant degree of symmetry, as illustrated by the over-representation of bidirectionally connected pairs of neurons, and the tendency of bidirectionally connected pairs to form stronger synapses than unidirectionally connected pairs as observed in cortex [47,48], except in barrel cortex [49]. Again, the 3TLR we have proposed here reproduces this feature (Fig 9), consistent with the fact that the rule approaches the optimal capacity. Our network uses the simplest possible single neuron model [50]. One obvious direction for future work would be to implement the learning rule in a network of more realistic neuron models such as firing rate models or spiking neuron models. Another potential direction would be to understand the biophysical mechanisms leading to the high threshold in the 3TLR. In any case, we believe the results discussed here provide a significant step in the quest for understanding how learning rules in cortical networks can optimize information storage capacity. The main equations of the network, the neuron model, the learning rule, and the criteria for stopping the learning algorithm are outlined in the Results section, Eqs 1–7. We present here additional details about network simulations. | Recurrent neural networks have been shown to be able to store memory patterns as fixed point attractors of the dynamics of the network. The prototypical learning rule for storing memories in attractor neural networks is Hebbian learning, which can store up to 0. 138N uncorrelated patterns in a recurrent network of N neurons. This is very far from the maximal capacity 2N, which can be achieved by supervised rules, e. g. by the perceptron learning rule. However, these rules are problematic for neurons in the neocortex or the hippocampus, since they rely on the computation of a supervisory error signal for each neuron of the network. We show here that the total synaptic input received by a neuron during the presentation of a sufficiently strong stimulus contains implicit information about the error, which can be extracted by setting three thresholds on the total input, defining depression and potentiation regions. The resulting learning rule implements basic biological constraints, and our simulations show that a network implementing it gets very close to the maximal capacity, both in the dense and sparse regimes, across all values of storage robustness. The rule predicts that when the total synaptic inputs goes beyond a threshold, no potentiation should occur. | lay_plos |
Drug resistance represents one of the main problems for the use of chemotherapy to treat leishmaniasis. Additionally, it could provide some advantages to Leishmania parasites, such as a higher capacity to survive in stress conditions. In this work, in mixed populations of Leishmania donovani parasites, we have analyzed whether experimentally resistant lines to one or two combined anti-leishmanial drugs better support the stress conditions than a susceptible line expressing luciferase (Luc line). In the absence of stress, none of the Leishmania lines showed growth advantage relative to the other when mixed at a 1: 1 parasite ratio. However, when promastigotes from resistant lines and the Luc line were mixed and exposed to different stresses, we observed that the resistant lines are more tolerant of different stress conditions: nutrient starvation and heat shock-pH stress. Further to this, we observed that intracellular amastigotes from resistant lines present a higher capacity to survive inside the macrophages than those of the control line. These results suggest that resistant parasites acquire an overall fitness increase and that resistance to drug combinations presents significant differences in their fitness capacity versus single-drug resistant parasites, particularly in intracellular amastigotes. These results contribute to the assessment of the possible impact of drug resistance on leishmaniasis control programs. Leishmaniasis, a neglected tropical parasitic disease that is prevalent in 98 countries spread across three continents, is caused by protozoan parasites belonging to the genus Leishmania [1]; visceral leishmaniasis (VL), caused by species of the Leishmania donovani complex, is a lethal disease if left untreated. The recommended first-line therapies for VL include: i) pentavalent antimonials (meglumine antimoniate and sodium stibogluconate), except in some regions in the Indian subcontinent where there are significant areas with drug resistance [2]; ii) the polyene antibiotic amphotericin B or the liposomal amphotericin B formulation AmBisome; iii) the aminoglycoside paromomycin; and iv) the oral drug miltefosine. Although WHO [1,3] recommended the use of either a single dose of AmBisome or combinations of anti-leishmanial drugs in order to reduce the duration and toxicity of treatment, to prolong the therapeutic life-span of existing drugs and to delay the emergence of resistance, recent experimental findings have demonstrated the ability of Leishmania to develop experimental resistance to different drug combinations [4]. The emergence and spread of Leishmania antimonial-resistant parasites have led to a high rate of antimonial failure in India [5] and have raised questions about the selection and propagation risk of drug resistant parasites [6,7]. The spread of drug-resistant parasites in the field probably depends on their transmission potential, which is influenced by, among other factors, the relative fitness of drug-resistant versus drug-susceptible parasites. Previous results have demonstrated that the acquisition of drug resistance could have an impact on parasite fitness, which could in turn influence other important biological properties involved in the regulation of proliferation and differentiation of parasites [6,8]. As defined previously [6,9], the fitness of Leishmania parasites can be measured by: i) capacity to survive, grow and generate infective metacyclic forms in the vector, ii) capacity to survive and grow in the mammalian host, and iii) capacity of transmission between the host and the vector. Throughout its natural life cycle, Leishmania encounters adverse conditions that include: i) nutrient starvation and acidification of the medium, conditions that induce metacyclogenesis [10], ii) heat shock, when the parasite moves from growth at 28°C in the sandfly to 37°C inside the mammalian host macrophage, and iii) reactive oxygen species (ROS) and reactive nitrogen species (RNS), when it is phagocytized by macrophages of the host [11,12]. Leishmania has evolved a broad spectrum of mechanisms to protect itself against these host defenses, including: i) enzymes that detoxify ROS and RNS [13], ii) use of thiols as antioxidant defenses [14], and iii) inhibition of the host' s oxidative defense mechanisms [15]. However, it is not strictly true that all of the above increased the fitness capacity of all L. donovani resistant strains, as some strains showed little or no difference in their in vivo survival capacity compared to antimony-sensitive strains [16]. As suggested, other factors such as the genetic background of parasites could be important in the in vivo fitness capacity of L. donovani isolates that are clinically resistant to antimonials [16]. In this work, we evaluate whether L. donovani lines that are experimentally resistant to single anti-leishmanial drugs [amphotericin B (AmB), miltefosine (MIL), paromomycin (PMM) and trivalent antimony (SbIII) ] and to drug combinations (AmB-MIL, AmB-PMM, AmB-SbIII, MIL-PMM and SbIII-PMM), present any advantages in their ability to bear the different stress conditions with respect to susceptible cells expressing the luciferase gene (Luc gene). For this purpose, we have studied the susceptibility of mixed promastigote populations under different stress conditions and their ability to infect and survive in mouse peritoneal macrophages. The results of this study using parasites that are experimentally resistant to single and multi-drug combinations are discussed in relation with their potential impact on future leishmaniasis control programs. L. donovani promastigotes (MHOM/ET/67/HU3) and the previously described derivative resistant lines A, M, P, S, AM, AP, AS, MP, and SP (resistant to AmB, MIL, PMM, SbIII, AmB+MIL, AmB+PMM, AmB+SbIII, MIL+PMM and SbIII+PMM, respectively) [4] were grown at 28°C in an RPMI 1640-modified medium (Invitrogen) supplemented with 20% heat-inactivated fetal bovine serum (hiFBS, Invitrogen). L. donovani with the luciferase gene integrated into the parasite genome (Luc line) was grown in the same conditions. Phothinus pyralis luciferase gene (luc) was amplified from vector pX63NEO-3Luc [17] by PCR using the primers LucNcoIF 5’-GACGCCCATGGATGGAAGACGCCAAAAACAT-3’ and LucNotIR 5’-GACGTAGCGGCCGCTTACAATTTGGACTTTCCGC-3’ including (in bold) NcoI and NotI restriction sites, respectively. The luc gene was then cloned into the NcoI-NotI sites of vector pLEXSY-hyg2 (Jena bioscience, Jena, Germany) which harbor a marker gene for selection with hygromycin-B (hyg gene). The vector generated was denominated pLEXSYHyg-Luc. In this construct, sequences of the 18S rRNA gene flanked the luc and hyg genes. Following linearization with SwaI, stationary promastigotes were transfected with 3 μg of linearized pLEXSYHyg-Luc plasmid to integrate the luc and hyg genes into the 18S rRNA (ssu) locus by homologous recombination, using a previously described protocol [18]. Twenty-four hours after transfection, the culture medium was supplemented with 25 μg/mL of hygromycin-B. Hygromycin-resistant parasites were usually selected after 7 days. After establishing the transgenic parasites, they were plated onto 1. 5% agar plates containing culture medium plus 100 μg/mL hygromycin-B. After 10 days incubating at 28°C, clones were selected on agar plates and further propagated in liquid RPMI-modified medium supplemented with 100 μg/mL hygromycin-B. Integration of the expression cassette into the ssu locus was confirmed by PCR using genomic DNA from the wild-type (WT) and transgenic strains of L. donovani (Luc line) as a template. For this purpose, we used primer pairs ssu forward primer F3001 5’-GATCTGGTTGATTCTGCCAGTAG-3’ and 5’ utr (aprt) reverse primer A1715 5’-TATTCGTTGTCAGATGGCGCAC-3’, and primer pairs hyg forward primer A3804 5’-CCGATGGCTGTGTAGAAGTACTCG-3’ and ssu reverse primers 3002 5’-CTGCAGGTTCACCTACAGCTAC-3’. Promastigotes and amastigotes isolated as described previously [19], were resuspended in HBS buffer (21 mM HEPES, 0. 7 mM Na2HPO4,137 mM NaCl, 5 mM KCl, and 6 mM D-glucose, pH 7. 1) supplemented with 25 μM cell-permeable DMNPE-luciferin. After 15 minutes at room temperature, aliquots of this suspension (100 μL/well) were distributed into 96-well white polystyrene microplates. Luminescence was recorded with an Infinite F200 microplate reader (Tecan Austria GmbH, Austria). To measure the luciferase activity of intracellular amastigotes contained within infected cells, the Luciferase Assay System (Promega, Madison, Wis) was used according to the instructions of the manufacture. Luminescence was measured in the Infinite F200 microplate reader immediately after mixing. Log-phase promastigotes from the control (WT) and A, M, P, S, AM, AP, AS, MP and SP resistant lines were mixed in a 1: 1 ratio with log-phase promastigotes from the Luc line (1x106 mixed parasites/mL). Parasite density was microscopically determined every 24 h for a total of 144 h using Neubauer count chambers in order to monitor the growth. Parasite density in the WT line plus Luc line mixture was used as a control. Also, we evaluated the growth of each line in these mixed populations after 144 h of incubation, determining the luminescence as a measure of the cellular density of the Luc line. All growth experiments with the different parasite lines were performed in triplicate. In parallel, the mixed populations at a 1: 1 ratio (4x106 mixed parasites/mL) were sub-cultured every 48 h (logarithmic phase) and luminescence measured at the end of the first and second sub-culture. Promastigotes from the WT and resistant lines were mixed with the Luc line in a 1: 1 ratio (4x106 mixed parasites/mL), and the proliferation of resistant parasites exposed to different stress conditions (late stationary growth phase, starvation and heat shock plus pH modification) was compared to the Luc line by measuring the luminescence intensity. The mixture of the WT and Luc lines was used as a control for all experiments. For experiments studying intracellular amastigotes, mouse peritoneal macrophages were obtained as described previously [20] and plated at a density of 3 x 104 or 3 x 105 macrophages/well in 96-well white polystyrene microplates or 24-well tissue culture chamber slides, respectively, in an RPMI 1640 medium supplemented with 10% hiFBS, 2 mM glutamate, penicillin (100 U/mL) and streptomycin (100 μg/mL). Promastigotes from resistant or WT lines were mixed with the Luc line 1: 1 ratio and maintained in culture for 6 days. Afterwards, the mixed populations of stationary phase cultures were used to infect macrophages at a macrophage/parasite ratio of 1: 10. Six hours after infection at 35°C and 5% CO2, extracellular parasites were removed by washing with serum-free medium. Infected macrophages were maintained in culture medium at 37°C with 5% CO2 for 24 h and 96 h. To determine the infection index (% infection x amastigotes/macrophages), infected macrophages maintained in 24-well plates were fixed for 30 min at 4°C with 2. 5% paraformaldehyde in PBS buffer (1. 2 mM KH2PO4,8. 1 mM Na2HPO4,130 mM NaCl, and 2. 6 mM KCl, adjusted to pH 7), and permeabilized with 0. 1% Triton X-100 in PBS for 30 min. Intracellular parasites and macrophages were detected by nuclear staining with ProLong Gold antifade reagent plus DAPI. To determine the intracellular proliferation profile of each line, infected macrophages maintained in 96-well plates were lysed and then the luminescence measured using the Luciferase Assay System (Promega). Eight-week-old male BALB/c mice were purchased from Charles River Breeding Laboratories and maintained in our Animal Facility Service under pathogen-free conditions. They were fed a typical rodent diet and given drinking water ad libitum. These mice were used to collect primary peritoneal macrophages. All experiments were performed according to National/EU guidelines regarding the care and use of laboratory animals in research. Approval for these studies was obtained from the Ethics Committee of the Spanish National Research Council (CSIC, file CEA-213-1-11). Statistical comparisons between groups were performed using Student’s t-test. Differences were considered significant at a level of p<0. 05. The firefly luciferase (Luc) [21] has proved to be a useful reporter gene for monitoring gene expression [22] and quantifying Leishmania infections in macrophages and animal models, with the overall aim of probing host-microbe interactions [23,24]. To assess the feasibility of using bioluminescence as a quantitative indicator of parasite proliferation, studies were performed to correlate bioluminescence with parasite number. For this purpose, the Luc gene was amplified by PCR and cloned into pLEXSY-hyg2. The LUC-expressing vector was electroporated into L. donovani parasites which were then selected in the presence of hygromycin-B. To test whether luciferase activity correlated well with parasite number, 4-fold serial dilutions were prepared and their luciferase activity measured. An excellent linear correlation was observed between the number of transgenic promastigotes and the luminescence intensity (S1 Fig). Transfectant parasites that overexpress luciferase (from now on, Luc line) were also tested for their ability to infect macrophages. Stationary-phase recombinant promastigotes were used to infect mouse peritoneal macrophages. Intracellular Leishmania infection was observed microscopically after DAPI staining and no significant differences were noted in the infectivity of the Luc line versus the WT line (S2A Fig). Furthermore, an excellent correlation was observed between amastigote numbers and luminescence intensity (S2B Fig). Collectively, these results strongly suggest that the Luc line constitutes a valuable tool for assessing the viability and dynamics of mixed populations. The promastigote number was evaluated every day for 6 days so that the growth features of each resistant line, or the WT line mixed in a 1: 1 parasite ratio with the Luc line, could be studied and compared. We found that all mixed populations showed a similar growth profile as the control (WT+Luc) (Fig 1A). Moreover, the luminescence values were similar for each of these mixed populations after the 6th day of culture (Fig 1B). These results clearly indicate that the Luc line was present in the same ratio in all mixed populations and, therefore, there was no predominance of one line over another line under these conditions. To evaluate whether there was predominance of any resistant lines over the susceptible Luc line, promastigotes from resistant lines and the Luc line were mixed in a 1: 1 parasite ratio and grown without stress or exposure to different stresses. To assess the growth recovery, the luminescence intensity of mixed populations was determined in all cases after 48 h of culture in standard conditions. The WT plus Luc lines (WT+Luc) mixture was used as a control. The total number of mixed parasite cultures shows no significant differences between WT+Luc and the resistant lines+Luc in the different stress conditions, ranging indistinctly between 22-32x107 mixed parasites/mL. Within the mammalian host, Leishmania promastigotes differentiate into amastigotes and multiply predominantly inside macrophages, where they are exposed to stress, including starvation, acidic pH, high temperatures (heat shock) and ROS and RNS production [11,26]. To determine whether intracellular amastigotes from resistant lines were able to displace in vitro intracellular amastigotes from the susceptible Luc line, macrophages were infected with mixed populations of promastigotes taken from 6 day-old cultures where, as shown in Fig 1, no differences were observed in proliferation and the Luc line ratio. The infection index and luminescence of intracellular amastigotes were determined at 24 and 96 h post-infection to assess their infectivity and survival rates in mouse peritoneal macrophages. The infection indexes were similar in all cases, with values ranging for 24 h between 183±22 and 234±28, and for 96 h between 182±27 and 250±34. The results showed that in the early stage of macrophage infection (24 h) all the resistant lines, except the M line, had a significant predominance over the Luc line compared to the control (Fig 5A). The different lines showed a range of luminescence between 31 and 63% from SP+Luc and S+Luc populations, respectively, compared to the control (Fig 5A). These results could be due to: i) a higher percentage of metacyclic parasites, ii) tolerance to oxidative stress, and/or iii) tolerance to acidic pH and high temperatures of the resistant lines compared to the susceptibility of the Luc line. Also, in the late stage of infection (96 h), the resistant lines, again with the exception of the M line, were able to fully benefit from their initial advantage (Fig 5B). They showed a range of luminescence between 27 and 68% from SP+Luc and A+Luc populations, respectively, compared to the luminescence produced by the control (Fig 5B). Additionally, there were no significant differences on predominance of resistant lines between 24 and 96 h, with the exception of the S line (p<0. 05). These results suggest that the predominance of some resistant lines over the Luc line is the result of a higher infection rate during the initial stage which reflects as a higher survival rate of intracellular amastigotes within macrophages. Leishmania have successfully adapted to different environments for thousands of years and developed a highly flexible nature. Their survival capacity mainly relies on their ability to suppress oxidative outbursts of the host defense mechanism [15] and on a unique oxidant-protective redox metabolism, where thiols play a key role in antioxidant defenses [14]. In this regard, we have previously described that the resistant lines, except the M and S lines, had higher non-protein thiol levels than the WT line [4], which could contribute to a greater parasite survival rate within the host macrophages. It has been demonstrated that some L. donovani strains resistant to antimonials have a more variable and markedly higher capacity of in vivo infection compared to antimony susceptible Leishmania strains [16]. Strains resistant to antimony also have a higher metacyclogenic capacity [27] and have specifically evolved extra mechanisms to manipulate their host cells in order to avoid antimony-induced stress [28]. Such adaptations would not only improve the parasites survival capacity when stressed by antimony, but would also favor their survival in drug-free conditions. Since SbIII, AmB and MIL kill Leishmania through a common cell death pathway to achieve apoptosis, strains resistant to one or more of these drugs could develop tolerance to apoptosis, which would grant them a higher survival rate in macrophages, as we have observed with our Leishmania resistant lines (Fig 5B). In conclusion, the experiments using our transgenic Leishmania luc line have clearly demonstrated and validated the fact that Leishmania lines experimentally resistant to individual and combinatorial anti-leishmanial drugs have an increased fitness compared to Leishmania susceptible lines, probably as a consequence of their metabolic adaptations which all converge on the higher tolerance to stress conditions, as recently described [25]. Subsequently, they also have a better chance of survival. However, although this approach using promastigotes for assessing the viability and dynamics of mixed populations have important advantages, their use on intracellular amastigotes has some methodological limitations. Therefore, the emergence and spread of drug-resistant parasites in the field will probably result in a greater competitive fitness cost with respect to susceptible parasites, plus negative effects on the chemotherapy strategies used to control leishmaniasis. | Chemotherapy is currently the only treatment option for leishmaniasis, a neglected tropical disease produced by the protozoan parasite Leishmania. However, first-line drugs have different types of limitations including toxicity, price, efficacy and mainly emerging resistance. The WHO has recently recommended a combined therapy in order to extend the life expectancy of these compounds. The emergence and spread of Leishmania antimonial-resistant parasites have led to a high rate of antimonial failure in India and have raised questions about the selection and propagation risk of drug resistant parasites. The spread of drug-resistant parasites in the field probably depends on their transmission potential, which is influenced by, among other factors, the relative fitness of drug-resistant versus drug-susceptible parasites. In light of this, we have designed experimental studies to determine whether Leishmania donovani parasites resistant to single and combinations of anti-leishmanial drugs present any advantages in their ability to bear the different stress conditions versus a susceptible L. donovani line. Our results suggest that resistant parasites acquire an overall fitness increase and that resistance to drug combinations presents significant differences in their fitness capacity, particularly in intracellular amastigotes. | lay_plos |
This invention relates generally to surgical instruments and surgical techniques and, more particularly, to eye surgery and to phacoemulsification apparatus and methods for their use. This application is a continuation-in-part application of application Ser. No. 11/069,773, filed Mar. 1, 2005, which claims priority from provisional application Ser. No. 60/613,645, filed Sep. 27, 2004 and which also claims priority from provisional application Ser. No. 60/828,599, filed Oct. 6, 2006 all of which are incorporated herein by reference. BACKGROUND OF THE INVENTION A common ophthalmological surgical technique is the removal of a diseased or injured lens from the eye. Earlier techniques used for the removal of the lens typically required a substantial incision to be made in the capsular bag in which the lens is encased. Such incisions were often on the order of 12 mm in length. Later techniques focused on removing diseased lenses and inserting replacement artificial lenses through as small an incision as possible. For example, it is now a common technique to take an artificial intraocular lens (IOL), fold it and insert the folded lens through a relatively small incision, allowing the lens to unfold when it is properly positioned within the capsular bag. Techniques and instruments have also been developed to accomplish the removal of the diseased lens through an equally small incision. One such technique is known as phacoemulsification. A typical phacoemulsification system includes a handpiece having a tip sized to fit through a small incision. Within the tip a hollow needle is vibrated at ultrasonic frequencies in order to fragment the diseased lens into small enough particles to be aspirated from the eye. Commonly, an irrigation sleeve is mounted around the needle through which irrigating liquids are infused into the eye to flush the lens particles created by the vibrations. Often the needle is hollow and forms a pathway to aspirate the irrigating fluid and lens particles from the eye. In this way both aspiration and irrigation are performed by a single instrument requiring only a single incision. It is extremely important to properly infuse liquid during such surgery. Maintaining a sufficient amount of liquid prevents collapse of certain tissues within the eye and attendant injury or damage to delicate eye structures. As an example, endothelial cells can easily be damaged during such collapse and this damage is permanent because these cells do not regenerate. One of the benefits of using as small an incision as possible during such surgery is to minimization any leakage of liquid during and after surgery to prevent tissue collapse. Separate flow paths are required for the infusing and aspirating functions to be carried out properly. This requires the use of separate lengths of flexible tubing extending from the handpiece to the flow system control module. Typically these tubing lengths are on the order of 200 to 250 cm. Because the aspiration and irrigation tubes both go from the handpiece to the control module they often become tangled with one another, making manipulation of the handpiece more difficult. While this invention is principally described with reference to eye surgery and the use of instruments and techniques for phacoemulsification, instruments requiring separate fluid flow paths, such as for aspiration and irrigation, are known in other surgical arts as well. For example, some instruments used in liposuction (the shaping and removal of adipose tissue by breaking up the tissue and aspirating the tissue particles) also are designed to be used with separate fluid flow lines providing aspiration and irrigation. Multichannel tubing is well represented in the prior art. U.S. Pat. Nos. 6,287,290, 6,527,761 and 6,709,401 teach and describe methods, systems and kits for lung volume reduction which utilize catheters having coaxial tubes or tubes with coextensive multiple channels for introduction such expedients as gas for inflating a balloon attached to the catheter, guide channels for the introduction of other catheters and as aspiration channels. U.S. Pat. No. 6,143,373 teaches and describes a catheter system and method for injection of a liquid embolic composition and a solidification agent for the injection of a liquid and a solidifying agent to close off aneurysm. The multiple lumens are used for the injection of different liquids into the circulatory system. U.S. Pat. No. 6,066,130 teaches and describes a system for delivering laser energy in which, in one embodiment, a liquid and a guide wire are fed through separate channels in a single catheter. U.S. Pat. No. 6,013,048 teaches and describes an ultrasonic assisted liposuction system including an instrument used in liposuction, and the irrigation and aspiration functions of the instrument. The need thus exists for aspiration/irrigation tubing apparatus and connectors that can be connected to existing surgical handpieces and control consoles without modifying the handpieces. A further need exists for such apparatus which allows a surgeon to manipulate the handpiece without kinking the aspiration/irrigation tubing. Further, a need exists for such tubing and connectors to be made available in inexpensive and disposable versions. While the following describes a preferred embodiment or embodiments of the present invention, it is to be understood that these descriptions are made by way of example only and are not intended to limit the scope of the present invention. It is expected that alterations and further modifications, as well as other and further applications of the principles of the present invention will occur to others skilled in the art to which the invention relates and, while differing from the foregoing, remain within the spirit and scope of the invention as herein described and claimed. Where means-plus-function clauses are used in the claims such language is intended to cover the structures described herein as performing the recited functions and not only structural equivalents but equivalent structures as well. For the purposes of the present disclosure, two structures that perform the same function within an environment described above may be equivalent structures. BRIEF DESCRIPTION OF THE DRAWINGS These and further objects and characteristics of the present invention will become apparent upon consideration of the following drawings, in which: FIG. 1 illustrates a prior art surgical irrigation and aspiration apparatus and its associated tubing; FIG. 2 is an enlarged view of both ends of the irrigation tube of FIG. 1, showing the connectors that secure the tube to the surgical handpiece and the irrigation solution supply bottle; FIG. 3 is an enlarged view of the control module cassette of FIG. 1 ; FIG. 4 is an enlarged view of the cassette of FIG. 3 showing the tubing ends taped in place; FIG. 5 illustrates the use of a prior art handpiece with separate irrigation and aspiration tubes attached thereto; FIG. 6 is an lateral elevational view of an adaptor embodying elements of the present invention; FIG. 7 is a view along line 7 - 7 of FIG. 6 ; FIG. 8 is a partial sectional view of a second embodiment of an adaptor embodying the present invention; FIG. 9 is a partial sectional view of the adaptor of FIG. 8 attached to a handpiece; FIG. 10 is a partial sectional and elevational view showing a third embodiment of an adaptor used to connect coaxial tubing to the control cassette; FIG. 11 illustrates a prior art liposuction instrument utilizing separate irrigation and aspiration lines; FIG. 12 is an axial cross-sectional view of a double-lumen irrigation/aspiration tube; FIG. 13 is a lateral sectional view of the tube of FIG. 12 ; FIG. 14 is a schematic/partial sectional view of a handpiece connector adaptor for use with the tube shown in FIG. 12 ; FIG. 15 is a schematic/partial sectional view of a cassette connector adaptor for use with the tube shown in FIG. 12 ; FIG. 16 is a cross-sectional view of a double-lumen tube with the second lumen formed on the exterior of the tube; FIG. 17 is a partial sectional view of a connector adaptor for the tube of FIG. 16 ; and FIG. 18 is a partial sectional view showing the connection of the tube of FIG. 16 to the connector adaptor of FIG. 17. DETAILED DESCRIPTION OF THE INVENTION Referring now to FIG. 1, the numeral 10 indicates generally a prior art phacoemulsification apparatus consisting of a handpiece 12, a flexible, tubular aspiration line 14, flexible tubular irrigation lines 16 and 16 ′ and a control cassette 18. Control cassette 18 provides a single control apparatus to connect a supply of irrigation solution to a phacoemulsification handpiece and to complete a path from the handpiece to an aspiration chamber for collecting the aspirated fluid, particles and the like. Electrical line 28 provides electrical power to handpiece 12. Referring now to FIG. 2, an enlarged view of prior art irrigation line 16 is shown. Typically, irrigation line 16 has a male end connector 20 which is inserted into an irrigation connector port on handpiece 12 in a liquid-tight friction fit. FIG. 2 also illustrates a typical irrigation fluid supply connector 22, used to connect line 16 ′ to a container of sterile irrigating solution, such as a flexible plastic bag or the like. Referring now to FIG. 3, an enlarged view of prior art control cassette 18 is shown demonstrating the connection to cassette 18 of irrigation line 16 (to the handpiece), aspiration line 14 (from the handpiece) and irrigation line 16 ′ (from the solution supply container). Referring now to FIG. 4, lines 14, 16 and 16 ′ are shown secured to prior art cassette 18 with a length of adhesive tape 30 used to secure lines 14, 16 and 16 ′ to cassette 18 in an attempt to keep them from separating from the cassette, tangling or kinking. Referring now to FIG. 5, a prior art handpiece 32 is shown being hand held by a surgeon 34 with aspiration line 14 and irrigation line 16 attached. Aspiration line 14 and irrigation line 16 are attached at one end to handpiece 32 and at the other end to control cassette 18. However, in between these attachment points both aspiration line 14 and irrigation line 16 are separate. During surgery, efforts must be made to prevent tubes 14, 16 from kinking and tangling. FIG. 5 shows handpiece 32 as it is held typically during surgery. As can be seen in FIG. 5, lines 14, 16 are separate and must be moved by the surgeon each time the handpiece 32 is moved. Handpiece 32 shown in FIG. 5 is typified by the model 8065 817 801 handpiece sold by Alcon. In a first preferred embodiment of the present invention, a pair of connecting tubes are disposed one within the other to carry out the aspiration and irrigation functions without the snags and tangles experienced when separate tubes are used. As a part of the invention, adaptors are provided to connect the coaxial tubes to existing handpieces. Referring now to FIG. 6, the numeral 36 identifies a tubing-and-adaptor apparatus constructed in accordance with the present invention. A tubing assembly 38 has an inner tube 40 disposed within an outer tube 42 with both tubes 40 and 42 manufactured from flexible material such as silicone. Tubes 40, 42 will be referred to throughout as “coaxial” even though, strictly speaking, the axes of the tubes are not required to coincide. Referring to FIG. 7 a cross-section of tubes 40, 42 is shown, illustrating their relative dimensions. Typically a prior art irrigation tube has an inner diameter of about 3.0 mm and an outer diameter of about 5.0 mm, while a typical prior art aspiration tube has an inner diameter of about 1.0 mm and an outer diameter of about 4.0 mm. In a preferred embodiment of the present invention, aspiration tube 40 has the same inner and outer diameters as the prior art tube and thus has a cross-sectional area of about 7.1 mm 2 available for fluid flow. Irrigation tube 42 has an inner diameter of about 7.0 mm and an outer diameter of about 9.0 mm, and a cross-sectional area of about 38.5 mm 2. When aspiration tube 40 is placed within irrigation tube 42 and the cross-sectional area measured by the inner diameter of irrigation tube 42 is subtracted from the cross-sectional area measured by the outer diameter of aspiration tube 40 there is a cross-sectional area of about 25.9 mm 2 available for irrigation flow, or 18.8 mm 2 more than with a conventional irrigation tube. This creates a flow volume 3.6 times greater than that of a prior art irrigation tube, making possible increased irrigation flow while at the same time keeping the irrigation and aspiration tubes from becoming tangled. FIG. 6 shows tubing assembly 38 attached to an adaptor 44 constructed to allow tubing assembly 38 to be attached to a conventional phacoemulsification handpiece. Adaptor 44 has a first, generally horizontal and tapered hollow plug 46 having a first, open end 48 with plug 46 tapering outwardly from end 48 to a break 50 and, thereafter, tapering inwardly to a second open end 52. Integrally formed with adaptor 44 is a collar 54 within which second end 52 is disposed. A plug channel 56 extends through plug 46 from first end 48 to second end 52. Integral with and depending from plug 46 is a port leg 58 comprising a first, downwardly depending leg segment 60 and a second leg segment 62 extending at substantially a right angle to segment 60 and terminating in a port collar 64. A port channel 66 begins at and extends through port collar 64, segment 62 and segment 60 terminating in a connector block 68. A connector tube 70, fluid-tightly attached to connector block 68 extends through and past collar 54. As seen in FIG. 6, tube assembly 38 is connected to adaptor 44 in the following manner. Inner tube 40 is fluid tightly fit to connecting tube 70 while outer tube 42 is inserted into collar 54 and is frictionally and fluid tightly attached to tapered portion 52 of plug 46. In this fashion two separate fluid-tight flow paths are created. The first flow path extends from opening 48 and plug 46 through collar 54 and to outer tube 42. The second flow path begins at port collar 64 and extends through channel 66, connecting block 68 and straight connecting tube 70 to inner tube 40. When connected to a suitable handpiece, plug 48 is inserted into the port on the handpiece through which the irrigating solution is directed while port 58 forms an attachment point for a plug on the handpiece through which aspiration occurs. Referring now to FIG. 8, the numeral 74 identifies a second adaptor or connector having a plug assembly 76 having a first cylindrical section 78 preferably formed as a right cylindrical section and a second or formed integrally with a second plug section 80 larger in diameter than section 78 and having a tapered inner wall 82 formed therewithin. As seen in FIG. 8, outer tube 42 fits liquid tightly about the outer diameter of first section 78 and abuts against second section 80. Inner tube 40 is attached to a straight tube section 84 which protrudes from plug assembly 76. The configured plug assembly 76 forms a pair of flow channels, the first of which is a relatively large cylindrical flow channel 86 having a first right cylindrical cross section 88 and a second flow section with a frustoconical cross section 90, which tapers outwardly toward an opening 92 through plug section 80. The second flow path is defined by a tube 84 which is inserted, fluid tightly into inner tube 40. Referring now to FIG. 9, the numeral 94 identifies a handpiece constructed to receive the connector and tube assembly shown in 74. Handpiece 94 has a first end 96 terminating in a hollow nipple 98 tapered outwardly from end 100 to body 102 of handpiece 94. Handpiece 94 also has a central cannula or channel 104 extending from end 96 toward tip 106. As seen in FIG. 9, adaptor and tube assembly 74 is attachable to end piece 94 by inserting the free end of straight tube 84 into cannula 104 while, at the same time, securing plug 86 to tapered end 96 in a fluid-tight fit. Thus, as seen in FIG. 9, a path for aspirating liquids is formed by tip 106, cannula 104, and inner tube 40 while a flow path from infusing liquid is formed by outer tube 42, end 96 and the channel formed through end 96. Referring now to FIG. 10, the numeral 108 illustrates a preferred method for connecting tube 42 to a control cassette 110. A cassette adaptor 112 similar in construction to adaptor 74 has an irrigation inlet port 114 and an aspiration outlet port 116. A first extension tube 118 has a plug 120 at one end sized to fit irrigation outlet port 122 on cassette 110. Tube 118 is also sized to allow a fluid-tight fit to irrigation port 114. In similar fashion, a second extension tube 124 has a plug 126 at one end sized to fit aspiration inlet port 128 on cassette 110. Tube 126 is also sized to allow a fluid-tight fit to aspiration port 116. One extension tube found to be useful in making the foregoing connections is the Nipro Extension Tube No. EX5-60AC which may be cut and the male connector ends used to make the connections to the cassette. Referring now to FIG. 11, the numeral 128 identifies a prior art surgical handpiece used in liposuction, having a body 130 and a hollow, ultrasonically-vibrating cannula 132. An aspiration line 134 draws fluid and tissue particles through hollow cannula 132, while irrigating solution is supplied via irrigation tube 136 through an irrigation channel formed in cannula 132. Electrical power is supplied to the vibrating motor in body 130 via electrical line 138. Applying the principles of the present invention to handpiece 128, an adaptor (not shown) is designed to fit liquid-tightly at one end to aspiration port 140 and irrigation port 142 and at another end to coaxial tubes such as those shown in FIGS. 6 and 7 and as described herein. Referring now to FIG. 12, the numeral 142 identifies a double-lumen tube having an outer wall 144 and an inner wall 146 integral with and extending axially along an interior surface 148 of tube 142. Inner wall 146 has a first, exterior surface 150 and a second, interior surface 152. First surface 150 and a first portion 154 of interior surface 148 define a first, or irrigation lumen 156, while second surface 152 and a second portion 158 of interior surface 148 define a second, or aspiration lumen 160. Tube 142 is preferably connected to a phacoemulsification handpiece or a control cassette such that first lumen 156 and second lumen 160 become part of two separate flow paths. A preferred method of making such a connection is to provide adaptors that will connect with tube 142 and are also configured to connect with selected handpieces, control cassettes or other flow components. One such connection system is shown in FIGS. 13 through 15. Referring now to FIG. 13, an end of tube 142 configured to connect to an adaptor is shown in lateral cross-section. End 162 is intended to be attached to an adaptor for connection to either a phacoemulsification handpiece or a control cassette as described below. To do so, inner wall 146 is cut or foreshortened to form a setback 164 extending from end 162 to inner wall end 166. Referring now to FIG. 14 a preferred handpiece adaptor 168 is shown having a port collar 170 communicating with a port channel 172 which, in turn, connects with a connection block 174 to form a flow path for aspiration of emulsified lens fragments. A collector tube 176 is fluid-tightly attached to block 174 and extends past a plug 178 into tube 142. Tube 142 is held liquid-tightly to adaptor 168 at a shoulder 180 of plug 178. As seen in FIG. 14, collector tube 176 has a first, straight segment 182, a second, angled segment 184 and a third straight segment 186 sized and shaped to fit liquid-tightly into second lumen 160. Setback 164 is selected to allow a sufficient portion of third segment 182 to extend into second lumen 160 to form a liquid-tight fit. First lumen 156 communicates with a hollow plug 188 having an open end 190 communicating with a plug channel 192 to form an irrigation flow path. Referring now to FIG. 15, a preferred cassette adaptor 194 is shown having a plug assembly 196 having a first cylindrical section 198 preferably formed as a right cylindrical section and a second or formed integrally with a second plug section 200 larger in diameter than section 198 and having a tapered inner wall 202 formed therewithin. As seen in FIG. 15, tube 142 fits liquid tightly about the outer diameter of first section 198 and abuts against second section 200. A connector tube 204 has a first, straight section 206 that extends through plug 200 and into tube 142. A second, angled section 208 is formed integrally with first section first section 206 and terminates in a third, straight section 210, which is sized and shaped to fit liquid-tightly into second lumen 160. The configured plug assembly 196 forms a pair of flow channels, the first of which is a relatively large cylindrical flow channel 212 having a first right cylindrical cross section 214 and a second flow section with a frustoconical cross section 216, which tapers outwardly toward an opening 218 through plug section 200. The second flow channel is formed by lumen 160 and tube 204. Referring now to FIG. 16 the numeral 220 identifies a double-lumen tube having a first cylindrical lumen 222 with an outer wall 224 and a first flow channel 226. A second lumen 228 is formed integrally and coextensively with first lumen 222 such that lumen 228 is not within first flow channel 226. Second lumen 228 has an outer wall 230 which, with a segment 232 of outer wall 224 forms a second flow channel 234. Referring now to FIG. 17, the numeral 236 identifies a sectional view of a connector adaptor to connect to tube 220. Connector 236 has an inlet end 238 formed as part of a solid body 240. A first flow passage 242 begins at end 238 and extends through body 240, and a second flow passage 244 extends through body 240. A first nipple 246 is formed on end 238 and serves as the connector for passage 242, and a second nipple 248 is formed on end 238 and serves as the connector for passage 244. Referring now to FIG. 18, tube 220 is shown connected to connector 236, with first lumen 222 frictionally and liquid-tightly fit to first nipple 246 and second lumen 228 frictionally and liquid-tightly fit to second nipple 248. When so connected, first flow channel 226 communicates with first flow passage 242 and second flow channel 234 communicates with second flow passage 244. It is anticipated that a separately-configured adaptor will be supplied to fit each existing surgical handpiece and will be supplied with selected lengths of tubing constructed in accordance with the invention variations described herein. tubing. | An irrigation and aspiration tubing system for use with surgical handpieces and irrigation fluid supplies has a flexible tube with first and second lumens formed integrally along its length, with a first lumen used for transporting irrigation fluid to the handpiece and a second lumen used for aspiration of fluid and emulsified particles from a surgical site. The cross-sectional area of the first lumen is selected to provide a cross-sectional area available for fluid flow in excess of the cross-sectional area of a standard surgical irrigation tubes. The system also includes at least one adaptor to allow the tubing to be attached to known surgical handpieces. Preferably, a second adaptor is also provided allowing attachment to sources of irrigating fluid and aspiration vacuum. One said lumen is formed integral with either the interior or exterior surface of the flexible tube. | big_patent |
High-throughput RNA sequencing enables quantification of transcripts (both known and novel), exon/exon junctions and fusions of exons from different genes. Discovery of gene fusions–particularly those expressed with low abundance– is a challenge with short- and medium-length sequencing reads. To address this challenge, we implemented an RNA-Seq mapping pipeline within the LifeScope software. We introduced new features including filter and junction mapping, annotation-aided pairing rescue and accurate mapping quality values. We combined this pipeline with a Suffix Array Spliced Read (SASR) aligner to detect chimeric transcripts. Performing paired-end RNA-Seq of the breast cancer cell line MCF-7 using the SOLiD system, we called 40 gene fusions among over 120,000 splicing junctions. We validated 36 of these 40 fusions with TaqMan assays, of which 25 were expressed in MCF-7 but not the Human Brain Reference. An intra-chromosomal gene fusion involving the estrogen receptor alpha gene ESR1, and another involving the RPS6KB1 (Ribosomal protein S6 kinase beta-1) were recurrently expressed in a number of breast tumor cell lines and a clinical tumor sample. The transcriptome comprises the set of all transcripts in a cell and their quantity at a specific stage and time. RNA-Seq enables hypothesis-neutral investigation of the expression of the transcripts including non-coding RNA and viruses [1]. RNA-Seq provides advantages over microarray technology such as the detection of novel transcripts (both truly novel as well as those arising from alternative splicing) and sensitivity over a greater range of expression [2]. Methods to more comprehensively analyze RNA sequencing data are being developed, with particular focus on normalization of differential gene expression, annotation of the transcriptome, and characterization of the splicing junctions [3]–[12]. Paired-end RNA-Seq further enhances quantification of alternative transcripts [13]–[16]. Analysis of tissue and single-cell-specific RNA is revealing cellular gene expression diversity and phenotypy [17]–[19]. Gene fusions arise from mutations including translocations, deletions, inversions, or trans-splicing. Fusion genes are thought to cause tumorigenesis by over-activating proto-oncogenes, deactivating tumor suppressors, or altering the regulation and/or splicing of other genes which lead to defects in key signaling pathways [20]. Fused RNAs are found to occur in significantly higher frequency in cancer than in matched benign samples and may be potential biomarkers [21]. For example, 95% of patients with clinical chronic myeloid leukemia (CML) express the BCR-ABL gene fusion in their leukemia cells due to a reciprocal translocation between the long arms of chromosomes 9 and 22 [22], [23]. BCR-ABL is also found to be a factor in 30% to 50% of adult acute lymphoblastic leukemia cases [24]. Imatinib is a specific tyrosine kinase inhibitor targeting BCR-ABL and is an effective treatment for CML [25], [26]. Gene fusions are also detected repeatedly in other tumors. Examples include ETV6-NTRK3 in mesoblastic nephroma, congenital fibrosarcoma, and breast carcinoma [27]–[29]. MYB-NFIB in head and neck tumors [30], TMPRSS2-ERG/ETS in prostate cancer [31]–[34], and EML4-ALK in lung cancer [33], [35]. Most lung tumors with ALK rearrangements are shown to shrink and stabilize when patients are given the ALK inhibitor Crizotinib [36]. Hypothesis-neutral gene fusion detection with RNA-Seq was recently demonstrated by different groups [37]–[46]. For example, the FusionSeq software uses paired-end reads to find candidate fusions, and applies a set of filtration modules to remove false positive candidates [41]. FusionSeq applies misalignment filters for large- and small-scale homology, low complexity repetitive regions, and mitochondrial genes particularly considering reads that fall on SNP regions or on RNA edited transcripts that may cause misalignments. deFuse guides a dynamic programming based spliced read detection module with paired-end alignments [42]. Both of these methods reply upon paired-end alignments as the initial evidence and apply spliced read mapping on the candidate regions. PERAlign relies upon mapping spliced reads to the whole genome first and then guiding them with paired-end alignments [38]. In this study, we describe a new method which considers spliced-read and paired-end alignments independently from each other, enabling detection of fusions from single fragment or paired-end experiments. We also introduce techniques for mapping of spliced-reads to a suffix array based virtual gene fusion reference with annotation-aided pairing rescue and methods for quality assessment of alignments and splice junctions. We tested our analysis tool by calling the exon/exon junctions and gene fusions from data generated by sequencing three paired-end RNA-Seq libraries, each with two technical replicates. We also compared our results to TopHat and FusionSeq software on the MCF-7 sample. Next we validated candidate MCF-7 gene fusions using TaqMan® assays and showed that 90% of the calls were valid and over 65% were specific to MCF-7. We also identified what appears to be an early breakpoint bias at the 5′ fused genes. Finally, we surveyed a subset of MCF-7 and UHR fusions on a panel of breast cancer cell lines and discovered evidence for recurrence. We prepared strand-specific, paired-end RNA libraries from the Universal Human Reference (UHR), the Human Brain Reference (HBR), and the breast cancer cell line MCF-7 using the Total RNA-Seq kit from Applied Biosystems. These RNA libraries were sequenced using ligation-based high throughput SOLiD™ system [47]. Fragments were gel-selected for insert sizes between 100–200 base pairs (Figure S1 in Text S1). Using a new transcriptome alignment pipeline in which each pair of reads is mapped to genome, junction, exon and filter references and paired with a pairing quality value (PQV), we obtained total of 580 million read pairs that were confidently mapped to the human genome (Table S1 in Text S2). Histograms of gene expression showed a wide range of distribution, and average R2 correlation of gene expression between replicates ranged from 0. 95 to 0. 96 (Figures S2, S3 in Text S1). Splice junctions were discovered by combining three approaches: (1) BRIDGE evidence found by paired-end reads in which the forward read maps on an exon and the reverse read maps on another exon with a PQV above a confidence threshold; (2) SPAN evidence found by single reads (of paired-end reads) in which the read alignment spans the breakpoint of a set of known and putative splice junctions; (3) Fusion SPAN evidence found by fusion alignments spanning hypothetical breakpoints of any two exons discovered using the SASR aligner which assesses all exon-exon combinations in the genome (Figure 1). Using this strategy, for each sample, we identified an average of 133,000 RefSeq and 15,315 non-RefSeq (putative) splice junctions and 5 to 56 candidate fusion breakpoints (Table 1 and Table S1 in Text S2). To assess the performance of mapping quality values generated with the system, we compared fold-change ratio (Log2 [UHR/HBR]) of gene RPKM values with gene expression assays from the MicroArray Quality Control (MAQC) project [48], [49]. We compared correlation of four different PQV (1,10,20 and 40) thresholds with the assays (Figure S4 in Text S1). Pearson correlations of data from TaqMan assays with that of the data from the SOLiD system were not significantly different between PQV thresholds. The slope (m) of the regression fits, however, was significantly affected by the threshold settings. As PQV is increased from 1 to 40, the slope increased dramatically from 0. 77 to 0. 88, indicating significantly greater accuracy compared to a “gold standard” qPCR method. The increase in accuracy is likely a result of increased specificity. Essentially, the log ratio dynamic range increases with increasing PQV settings (Figure S5 in Text S1). RPKM distributions show an increase in low-end signal for lower PQV. If this increase in “sensitivity” represents additional noise, it can contribute to a loss of accuracy in the fold change calculations. The increase in the low end suggests that these reads may be spurious (Figure S6 in Text S1). In order to find optimal filters for detecting splice junctions with our combined approach, we compared three quality thresholds using data from the UHR and HBR barcoded libraries: (1) one SPAN evidence (1-SR), (2) two unique SPAN evidences (2-SR), and (3) one BRIDGE and one SPAN evidences (1-PE-1-SR). In addition to these tested thresholds, we applied default filter of choosing only primary alignments with PQV>10. The results, as illustrated in Figure 2, suggest an increased number of false positives for 1-SR evidence even though it may have greater sensitivity. 1-PE-1-SR threshold reduced false positives especially for fusions, generating less calls than 2-SR threshold. For the analyses described later in the text, 1-PE-1-SR threshold was chosen for calling splice junctions and 2-PE-2-SR for calling gene fusions. On average, 82% of junctions identified in the libraries were present in the RefSeq database. 84% of these known junctions and 26% of the putative junctions were shared between at least two of the three libraries (Figure S7 in Text S1). The highest number of library-specific known junctions was observed in HBR, and the highest number of library-specific putative junctions was observed in MCF-7. Next, we formulated a Junction Confidence Value (JCV) and investigated its utility to identify true versus false junctions. Details of JCV and its formulation are explained in supplementary methods in Text S1. One type of false positive fusion junction is likely called between highly expressed exons for which the random chance of encountering a misalignment or mispairing is elevated. Homology between highly expressed genes would also increase this type of false positive. We created JCV to test the quantity and quality of BRIDGE evidences when compared to an ‘error expectation metric’. This metric is defined as the estimation of the null hypothesis of encountering a random junction between the two exons. Increasing JCV increased known/putative junction ratio which was predictive of the false discovery rate and at the same time distinguished significant number of novel junctions to either lower or higher score bins (Figure 3A). Known/putative ratios ranged from 0. 15 for JCV cutoff of 0; 3 for JCV cutoff of 50 and 16 for JCV cutoff of 100. In order to test the sensitivity of JCV, we simulated 1,000,000 junctions based on a combination parameter model of true junction expression ratio and false junction misalignment ratio. True positive rate (TPR) and false positive rate (FPR) were calculated by comparing whether a called junction was real (Figures S8, S9, S10 in Text S1). These simulations showed that JCV was predictive of true junction calls and higher JCV thresholds resulted in much less FPR and slightly less TPR. We performed a separate simulation of gene fusion detection using reads from a DH10B (E. coli) DNA sequencing experiment where introns, exons and a gene model were simulated to make the data similar to RNA-Seq experiments. Our algorithm was able to detect 86 out of 93 simulated fusions in this experiment (Figure S11 in Text S1). Next, we ran TopHat (v1. 3. 0) on the MCF-7-1 dataset by using default paired-end parameters for color space. TopHat reported 124,236,156 mapped reads of which 33,634,800 were properly paired whereas LifeScope reported 404,901,929 mapped reads, of which 300,341,259 pairs were mapped to the same chromosome and 158,050,096 were properly paired (Table S1 in Text S2). Of note, TopHat allows 2 mismatches on mapped reads by default and does not report pairs of reads mapped across different chromosomes. Next we identified appropriate score threshold for calling TopHat junctions. For each junction found, TopHat reports a score which corresponds to the number of reads that span the junction. TopHat reported 1,391,319 total junctions without any score filter and with score>5 threshold this number reduced to 53,402 (Figure S12 in Text S1). We used TopHat candidate junctions with score>5 for comparison to RefSeq known and Lifescope candidate junctions (Figure 3B). There is some evidence that score>10 may yield more specific results for TopHat (Figure S12 in Text S1). Of note, known (RefSeq) junctions called by TopHat dropped from 129,316 (score>0) to 106,962 (score>5). These results suggest that TopHat detects a large number of putative novel junctions yet is not as sensitive when distinguishing false positives. LifeScope detected 15,074 putative novel junctions between known exons of the same gene that weren' t called by TopHat. We could distinguish that more than half of these ‘LifeScope-only’ junctions were likely true positives by looking at their JCV; 3,520 had JCV = 0 and 8,481 had JCV = 100 with the rest having scores between 0 and 100. Using the combined BRIDGE&SPAN approach described above on the UHR sample, we called and validated previously reported gene fusions including BCR-ABL1, GAS6-RASA3, ARFGEF2-SULF2, NUP214-XKR3 and BAT3-SLC44A4 [37], [39], [40]. These fusions were not described in the literature for HBR, and as expected were not identified in the HBR samples sequenced. In MCF-7, a total of 40 putative fusions were identified in the first sequencing run (50×25 paired-end), of which 26 were detected again in a second run (75×35 paired-end) out of a total 56 fusion calls (Table S2 in Text S2). We also analyzed first MCF-7 sequencing dataset using FusionSeq (Sboner et. al.). Six of the forty gene fusions identified by LifeScope were also called by this software. FusionSeq' s confidence value (RESPER) for these calls ranged from 1. 15 to 4. 53. Of importance, the ribosomal filter and single read validation module of FusionSeq (version 0. 7) did not handle color space data or data with different read length pairs adding to 5807 total calls with RESPER>1 (Table S3 in Text S2). Based on the calls from the first MCF-7 sequencing experiment, we prepared 40 TaqMan fusion assays and run them on the UHR, HBR, and MCF-7 samples along with the prostate cell line PC-3 as an additional control. 36 (90%) of the fusions were validated with the assays and 25 (63%) were found to be specific to MCF-7 and UHR (Table 2 and Table S4 in Text S2). To note, 19 of these “specific” fusions were called with our algorithms in the second run of MCF-7. JCV values correlated with whether a fusion was called again, and also with the number of unique start points (Figure S13 in Text S1). Real-time PCR Cycle Threshold (CT) values showed that each of the MCF-7 gene fusions was expressed in UHR with around ten-fold less expression (Table 2). This suggests that MCF-7 or one of its parent or sister cell lines is very likely part of the UHR pool. According to the information provided by the supplier, UHR RNA is prepared from a pool of ten different cancer cell lines, one of which is an ‘adenocarinoma, mammary gland’. MCF-7 is an adenocarcinoma cell line from mammary gland. From the RNA-Seq calls, nine of the MCF-7 gene fusions were detectable in UHR with ∼200 million confidently mapped reads whereas these fusions were detectable in MCF-7 with only ∼80 million reads. It is likely that deeper sequencing of the UHR pool would have identified the remaining fusions. Many MCF-7 fusions were between genes in three bands of Chr 1, Chr 17 and Chr 20 (Figure 4). These bands were previously described as rearrangement “hot-spots” [50]. Of the total 11 inter-chromosomal or inverted intra-chromosomal fusions, five had premature stop codons (not in frame), while six were in frame. Two of the fusions were alternatively spliced including the fusion from the second exon of ESR1 to the sixth and seventh exon of C6orf97, and the fusion from the first and second exon of ADAMTS19 to the tenth exon of SLC27A6. We also found several new intra-chromosomal gene fusions mostly between adjacent or neighboring genes (Table S4 in Text S2). We observed an enrichment of fusions for which the breakpoints were in the first intron of a gene, a similar bias explained also in Inaki et al., 2011. This pattern was not observed for the UHR and HBR samples (Figure 5A–B). On average, first introns in the RefSeq database (hg18) constitute 22% of a gene. We asked whether the large intron size alone might explain the breakpoint bias at the 5′ introns. We used a parametric bootstrap approach to test the hypothesis that gene fusions are more likely to occur towards the 5′ end of a gene; for example, after the first exon. Assuming that the breakpoint was in the middle of the intron following the fused exon, we considered breakpoints for 23 fusions from Table 2 (omitting multiple splices for ESR1 and ADAMTS19). We simulated 100,000 gene fusion locations in these 23 genes from a uniform distribution within the gene. We normalized the location of the real gene fusions by gene length (defined as the distance between the start of the first exon and the end of the last exon). We calculated the mean fusion location of the 23 genes, in the observed fusions and in the simulated fusions. In the real fusions, the mean insert location was 0. 2587 (26% of the length of the gene, Figure 5C). In 100,000 simulated sets of 23 fusions, the mean was 0. 5 and the standard deviation was 0. 06. Only three in 100,000 of the simulated sets of fusions had a value less than 0. 2587. Thus the observed location of the gene fusions is statistically significantly biased towards the 5′ end of the gene, with a p-value estimated at 3×10−5. To test recurrence, we selected 24 fusions from UHR and MCF-7 and investigated their expression in 20 cancer cell line samples (Figure 6). UHR fusions BCR-ABL1 and BAT3-SLC44A4 were found expressed in the myelogenous leukemia cell line K562 but with eightfold higher expression than in UHR. GAS6-RASA3 fusion was expressed only in UHR. Most of the fusions in MCF-7 were also expressed at a low level in the Du4475 cell line with a higher CT value (>35 for most cases). Both MCF-7 and Du4475 cell lines are traced to a 69/70-year old Caucasian female from Georgetown, but contamination, mixing, mislabeling, or differences in culturing may have caused the observed expression. Two of the intra-chromosomal gene fusions were expressed in multiple samples: ESR1-C6ORF97 and RPS6KB1-TMEM49. The first of these fusions, between the estrogen receptor alpha gene ESR1 and its neighboring gene C6ORF97 on Chr 6 was expressed in two other ER+ breast cancer cell lines in addition to UHR, MCF-7, and Du4475. This fusion may have occurred due to an inversion or rearrangement, as normally the ESR1 gene is downstream of C6ORF97 on the genome (on the same strand, 128,831 base pairs apart); yet the fusion junction was observed to be from the second exon of ESR1 to the sixth exon of C6ORF97, in the reverse order of expected transcription. We noted that these two genes were considerably expressed in MCF-7 (RPKM 16 and 46 average), though not expressed at all in HBR (RPKM<0. 5), and weakly expressed in UHR (RPKM 1 and 1. 8). The second recurring fusion, RPS6KB1-TMEM49, was found expressed in four cancer cell lines including HCC2157 and HelaS3. We further tested the presence of 24 candidate fusions in cDNA from 48 Clinical Samples of Normal and Breast Tumors (Origene). We found ESR1-C6ORF97 expressed in one ER+ tumor, and none of the other fusions were expressed. RNA-Seq allows interrogation of known and novel transcript expression and discovery of gene fusions. We describe a new suffix array algorithm to find fusion breakpoint spanning reads in a hypothesis-neutral fashion. We combine this algorithm with a new paired-end mapping approach to detect gene fusions sensitively and reliably. Our mapping method works with a predefined set of exon boundaries which is readily available for the human genome from RefGene or Ensembl databases. To detect novel splicing sites different from the known junctions, one can first find novel expressed islands of reads with tools such as TopHat [8], and next add the predicted novel exons to the gene model, prior to using our tool. Other de novo assembly algorithms, as long as they generate de novo exon boundaries, and mapped back to genome coordinates, may also be used with our tool [51]–[54]. By sequencing and analyzing the MAQC samples UHR and HBR, and the breast cancer cell line MCF-7, we validated 25 gene fusions specific to MCF-7 and UHR. Of these, five were not in frame and had premature stop codons. These fusions might still deploy a negative constraint on the fused genes by increasing non-sense mediated decay (NMD) [55]. In addition, several gene fusions that occur at the genomic level might not have been detected by messenger RNA sequencing (mRNA) because their pre-cursor mRNAs would have been degraded by the NMD mechanism. Such fusions may be identified by DNA-level sequencing. Of the 29 intra-chromosomal fusions called in MCF-7 in this study, 12 were not described in investigated literature (Table S4 in Text S2). Interestingly, most of the adjacent MCF-7 gene fusions did not fit the standard definition of “read-through” since they did not occur between last and first exons of the fused genes and in some cases they occurred in inverse order of expected transcription. This indicates that these fusions may had arisen due to trans-splicing or structural mutations such as deletions or inversions. These hypotheses may be tested by directly sequencing the DNA from these regions. By surveying cancer cell lines with TaqMan assays, we observed that two of the MCF-7 fusions involving adjacent genes, ESR1-C6ORF97 and RBS6KB1-TMEM49 were expressed recurrently. Fusions of the ESR1 gene may disrupt estrogen signaling pathways and thus events involving this gene may be significant. RBS6KB1-VMP1 fusion was described as a recurrent event recently by another group [45]. VMP1 is another name for TMEM49. Amplification of the RPS6KB1 loci (Ribosomal protein S6 kinase beta-1) was described in other breast cancers as an oncogene event [56]. Still, it is possible these recurrent fusions arise only in immortalized cell lines rather than being driver mutations. In fact, the ESR1 fusion tested positive in only one of the 48 clinical breast samples, while the RPS6KB1 fusion was not expressed in any of them. Of interest, six of the fusions originated on the band 17q23, which was previously identified as a common region of amplification in cancer [57]. In many of the MCF-7 fusions, the first or early introns of the 5′ genes harbored the gene fusion breakpoint. A similar pattern was observed in prostate cancer: the complete exon-1 of TMPRSS2 was identified to fuse with ETV1 or ERG as one of the most recurrent rearrangements [31]. Recent studies on prostate cancer found extended breakpoints at the androgen receptor binding sites possibly due to LINE-1-induced ORF or topoisomerase-II beta. These enzymes, when co-recruited with an androgen receptor, were linked to increased chromosomal translocations of the TMPRSS2, ETV1, and ERG genes [58], [59]. Presence of the early 5′ breakpoints in MCF-7 genes suggest that recurrent double-stranded breaks may occur in breast tumors at the gene promoter and early splicing sites due to factors not mediated by the androgen receptor. In conclusion, we presented a novel method of splice and fusion detection from RNA-Seq data. We sequenced MCF-7, UHR and HBR, and demonstrated high specificity in finding splices and fusions de novo. We further showed that two of the MCF-7 gene fusions are expressed recurrently in a number of tumor cell lines. Reads were aligned to a reference using the Mapreads module of the BioScope 1. 3 and LifeScope 2. 0 software (http: //www. lifetechnologies. com/lifescope). Four fasta references were used for increased throughput and accuracy: (1) genomic reference, (2) junction reference, (3) exon reference, and (4) filter reference (Figure 1B). Filter reference contained polyA, polyC, polyG, polyT, ribosomal RNAs, tRNAs, LINE, SINE, LTR and satellite repeats, rRNA, scRNA and snRNAs, as well as adaptor, barcode, and primer sequences. In our experiments, most reads filtered to ribosomal RNAs and merged adaptor-barcode-primer sequences. When aligning reads to the genome, two mismatches were allowed on the seed, and alignments were extended when possible based on a dynamic scoring function. The junction reference library was generated from a list of known and putative exon-exon pairs within RefSeq transcripts and contained approximately two million fasta entries. Reverse reads in our experiments were shorter than forward reads (25 vs 50, or 35 vs 75). To increase the mapping rate for the shorter reverse reads, they were additionally aligned to an ‘exon reference’ by allowing three mismatches on the seed. This exon reference contained each known exon as a separate reference entry. An exon rescue step was performed for reads where one pair was mapped within a gene and its pair was unaligned, by aligning the unmapped read within the downstream exons of the same gene with up to six mismatches. The genome, exon, junction, and rescued alignments were merged to generate a single set of alignments for the forward and reverse tags separately. Reads that aligned confidently to the filter reference were subtracted from these alignments. A final pairing step was performed to find most probable alignment pairs and assign a pairing quality value (for formulas see Methods in Text S1). These final paired alignments were put in a genome-coordinate BAM file which represents the summary of mapped alignments except for fusion alignments found by SASR. For reads that were admissible as a candidate to be spliced on a fusion junction (see Methods in Text S1 for admission criteria), we performed a suffix array search as follows. A read was defined to provide evidence of a splice junction between an exon X and exon Y if and only if (1) exon X maps to the prefix of the read, (2) exon Y maps to the suffix of the read and (3) the sum of the two map lengths is equal to the length of the read. For 50-bp long reads (or 49 colors plus a leading base), the suffix data structure was simply a list (an array) of all suffixes of length 10 through 38 from all exons. The suffixes were stored in lexicographically increasing order. A string s = s1s2…sm is lexicographically (i. e. alphabetically) less than a string t = t1t2…tn if s1<t1 or s1 = t1, and string s2s3…sm is lexicographically less than string t2t3…tn. Each suffix was represented compactly by a pair of integers (an integer and a byte in the implementation): an index to the relevant exon in the input exon list, and the length of the suffix. Such a data structure is called a suffix array [60]. Because of the lexicographic order proper, all suffixes that start with any given decamer were consecutive in such a list. Therefore, one may quickly find all matching suffixes with a binary search into the suffix array. Once the list of exons that mapped to the prefix and suffix of the read were identified, it could be determined whether the read provided evidence for a unique junction. A read was considered to be a SPAN evidence for a junction X-Y between two exons if it was already junction mapped or if it was discovered by SASR as described above. A paired-end read was considered BRIDGE evidence for a junction X-Y if one read of the pair mapped to exon X and the other mapped to exon Y with PQV>10. Candidate junctions were stored, each with a count of evidence, number of unique start points and corresponding PQV, in a sparse, directed graph. In this graph, exons corresponded to nodes, and SPAN and BRIDGE evidences corresponded to two types of edges between nodes. After all evidence was collected, junctions were called by evaluating each candidate and assigning a junction confidence value. At least one and two unique evidences of each type were respectively required to call same-gene and different-gene junctions (fusion). Exons could partially overlap, allowing for junctions with different donor and acceptor sites to be counted as alternative splices as long as at least two alternatives were detected. Genes with overlapping annotations were not counted towards a gene fusion if the evidence was ambiguous. Human Breast Adenocarcinoma (MCF-7) Total RNA and FirstChoice® Human Brain Reference RNA (HBR) were obtained from Ambion. Universal Human Reference Total RNA (UHR) was obtained from Stratagene. Oligo (dT) selection was performed twice by using MicroPoly (A) Purist™ kit (Ambion) according to the manufacturer' s recommendations. After polyA selection, 500 ng polyA RNA was fragmented using RNase III. 50 ng fragmented RNA was then subjected to hybridization and ligation using the SOLiD Total RNA-Seq Kit (Ambion) according to the manufacturer' s instructions. Duplicate libraries, with three different insert sizes (100–200 bp, 100–300 bp, 150–250 bp), were generated from HBR and UHR RNAs. A total of 12 libraries were multiplexed using the SOLiD RNA Barcoding Kit (Applied Biosystems) and pooled at an equi-molar ratio. Two libraries were made from same lot of MCF-7 polyA RNA with standard insert size (100–200 bp). The final purified products were quantitated using a NanoDrop® instrument, and the size range of the products was confirmed by Bioanalyzer™ instrument analysis. The samples were then diluted and used for emulsion PCR. Libraries were sequenced utilizing 50 or 75 bp forward and 25 or 35 bp reverse paired-end sequencing chemistry on the SOLiD system [47]. TaqMan probes and primers were designed for selected fusion targets. For each putative fusion call, the target region for assay design was composed of 200 bases around the fusion point: the first 100 from the 5′ gene exon and the second 100 from the 3′ gene exon. If either of the exons was smaller than 100 bases, the entire exon was taken but no bases from a further exon were used. Therefore, any target region had a maximum of 200 bases. These target sequences were then used to select TaqMan assay probes and primers which were ordered from Applied Biosystems. These assays were used to validate the novel fusion candidates in Universal Human Reference RNA sample (Stratagene), MCF-7 RNA (Ambion), Human Brain Reference RNA (Ambion), and a no template control sample. cDNAs were generated from 2. 5 ug total RNA from each sample using the High Capacity cDNA Archive Kit and protocol (Applied Biosystems). The resulting cDNA products were diluted twenty-fold and four replicates were run for each gene for each sample in a 384-well format plate on 7900HT Fast Real-Time PCR System (Applied Biosystems). 24 selected fusion targets (Figure 6) were screened across 20 cancer cell line RNAs and negative template control (NTC, Table S5 in Text S2) using TaqMan probe and primers. Real-time PCR reactions were run as described above. The same selected 24 fusion targets were also screened in 48 breast cancer clinical samples (Origene) using TaqMan probe and primers. cDNAs were generated from 2 ng total RNA from each sample using the High Capacity cDNA Archive Kit and protocol (Applied Biosystems). The resulting cDNA was subjected to a 16-cycle PCR amplification followed by real-time PCR reaction using the manufacturer' s TaqMan PreAmp Master Mix Kit Protocol (Applied Biosystems). Preamplifed cDNA products were diluted twentyfold and four replicates were run for each gene for each sample in a 384-well plate on a 7900HT Fast Real-Time PCR System (Applied Biosystems). | Advances in sequencing technology are enabling detailed characterization of RNA transcripts from biological samples. The fundamental challenge of accurately mapping the reads on transcripts and gleaning biological meaning from the data remains. One class of transcripts, gene fusions, is particularly important in cancer. Some gene fusions are prominent markers in leukemia, prostate, and other cancers and putatively causative in certain tumor types. We present a set of new RNA-Seq analysis techniques to map reads, and count expression of genes, exons and splicing junctions, especially those that give evidence of gene fusions. These tools are available in a software package with a straightforward graphical user interface. Using this software, we called and validated several gene fusions in a breast cancer cell line. By testing the presence of these fusions in a larger population of tumor cell lines and clinical samples, we found that two of them were expressed recurrently. | lay_plos |
To generate energy efficiently, the cell is uniquely challenged to co-ordinate the abundance of electron transport chain protein subunits expressed from both nuclear and mitochondrial genomes. How an effective stoichiometry of this many constituent subunits is co-ordinated post-transcriptionally remains poorly understood. Here we show that Cerox1, an unusually abundant cytoplasmic long noncoding RNA (lncRNA), modulates the levels of mitochondrial complex I subunit transcripts in a manner that requires binding to microRNA-488-3p. Increased abundance of Cerox1 cooperatively elevates complex I subunit protein abundance and enzymatic activity, decreases reactive oxygen species production, and protects against the complex I inhibitor rotenone. Cerox1 function is conserved across placental mammals: human and mouse orthologues effectively modulate complex I enzymatic activity in mouse and human cells, respectively. Cerox1 is the first lncRNA demonstrated, to our knowledge, to regulate mitochondrial oxidative phosphorylation and, with miR-488-3p, represent novel targets for the modulation of complex I activity. In eukaryotes, coupling of the mitochondrial electron transport chain to oxidative phosphorylation (OXPHOS) generates the majority of ATP that fulfils cellular energy requirements. The first enzyme of the electron transport chain, NADH: ubiquinone oxidoreductase (complex I), catalyses the transfer of electrons from NADH to coenzyme Q10, pumps protons across the inner mitochondrial membrane and produces reactive oxygen species (ROS). Mammalian mitochondrial complex I dynamically incorporates 45 distinct subunits into a ~ 1 MDa mature structure (Vinothkumar et al., 2014; Guerrero-Castillo et al., 2017). It is known that oxidatively damaged subunits can be exchanged in the intact holo-enzyme (Dieteren et al., 2012), but how this process may be regulated is poorly understood. The efficiency and functional integrity of OXPHOS are thought to be partly maintained through a combination of tightly co-ordinated transcriptional and post-transcriptional regulation (Mootha et al., 2003; van Waveren and Moraes, 2008; Sirey and Ponting, 2016) and specific sub-cytoplasmic co-localisation (Matsumoto et al., 2012; Michaud et al., 2014). The nuclear encoded subunits are imported into the mitochondria after translation in the cytoplasm and their complexes assembled together with the mitochondrially encoded subunits in an intricate assembly process (Perales-Clemente et al., 2010; Lazarou et al., 1793; Vogel et al., 2007). Mitochondrial biogenesis is co-ordinated first transcriptionally from both genomes (Scarpulla et al., 2012), and then post-transcriptionally by regulatory small noncoding RNAs such as microRNAs (miRNAs) (Dumortier et al., 2013; Li et al., 2012). Recently, SAMMSON a long noncoding RNA (lncRNA) was found to bind p32 and, within mitochondria, enhanced the expression of mitochondrial genome-encoded polypeptides (Leucci et al., 2016). Nuclear-encoded and cytosol-located lncRNAs have not yet been implicated in regulating mitochondrial OXPHOS (Vendramin et al., 2017) despite being surprisingly numerous and often found localised to mitochondrion- and ribosome-adjacent portions of the rough endoplasmic reticulum (van Heesch et al., 2014). It is here, on the ribosome, that turnover of miRNA-targeted mRNAs frequently occurs during their translation (Tat et al., 2016). Here we describe a novel mammalian conserved lncRNA, termed Cerox1 (cytoplasmic endogenous regulator of oxidative phosphorylation 1). Cerox1 regulates complex I activity by co-ordinately regulating the abundance of at least 12 complex I transcripts via a miRNA-mediated mechanism. Cerox1 knockdown decreases the enzymatic activities of complexes I and IV. Conversely, elevation of Cerox1 levels increases their enzymatic activities, halves cellular oxidative stress, and protects cells against the cytotoxic effects of the complex I inhibitor rotenone. To our knowledge, Cerox1 is the first lncRNA modulator of normal mitochondrial energy metabolism homeostasis and cellular redox state. The miRNA-dependency of Cerox1 and the regulation of associated OXPHOS transcripts are supported by: (i) direct physical interaction of miR-488–3p with Cerox1 and complex I transcripts; (ii) decrease or increase in Cerox1 and complex I transcripts following miR-488–3p overexpression or inhibition, respectively; (iii) miR-488–3p destabilisation of wildtype Cerox1, but not a Cerox1 transcript containing a mutated miR-488–3p miRNA recognition element (MRE) seed region; and, (iv) absence of the OXPHOS phenotypes either in cell lines deficient in microRNA biogenesis or when Cerox1’s predicted miR-488–3p response element is mutated. The miRNA-dependent role of Cerox1 illustrates how RNA-interaction networks can regulate OXPHOS and that lncRNAs represent novel targets for modulating OXPHOS enzymatic activity. Cerox1 was selected for further investigation from among a set of central nervous system-derived polyadenylated long non-coding RNAs identified by cDNA sequencing (GenBank Accession AK079380,2810468N07Rik) (Carninci et al., 2000; Ponjavic et al., 2007). Mouse Cerox1 is a 1. 2 kb, two exon, intergenic transcript which shares a bidirectional promoter with the SRY (sex determining region Y) -box 8 (Sox8) gene (Figure 1A). A human orthologous transcript (CEROX1, GenBank Accession BC098409) was identified by sequence similarity and conserved synteny (60–70% nucleotide identity within alignable regions, Figure 1B, C). Both mouse and human transcripts have low protein coding potential (Materials and methods, Figure 1—figure supplement 1A) and no evidence for translation from available proteomic datasets. Four human or mouse data types supported CEROX1 as having an important organismal role. First, its promoter shows a greater extent of sequence conservation than the adjacent SOX8 promoter and its exons are conserved among eutherian mammals (Figure 1B). Second, from expression data, its levels in primary tissues and cells are exceptionally high, within the top 13% of a set of 879 lncRNAs with associated cap analysis of gene expression (CAGE) clusters (Figure 1D, E). Expression is particularly high in neuroglia, neural progenitor cells and oligodendrocyte progenitors (Bergmann et al., 2015; Mercer et al., 2010; Zhang et al., 2014) (Figure 1—figure supplement 1B). Cerox1 is notable in its higher expression in the adult brain than 64% of all protein coding genes. Cerox1 expression is also developmentally regulated. For example, it is a known marker gene for type one pre-haematopoietic stem cells in the dorsal aorta (Zhou et al., 2016) and its expression is high in mouse embryos beyond the 21 somite stage (https: //dmdd. org. uk/). High expression of Cerox1 in the brain was confirmed using quantitative real-time PCR (qPCR) for both mouse and human orthologous transcripts (Figure 1—figure supplement 1C, D). Third, single nucleotide variants are significantly associated both with CEROX1 expression and anthropomorphic traits, measured in the UK Biobank. These lie within an 85 kb interval encompassing the CEROX1 locus, and 5’ regions of SOX8 and LMF1 genes. For example, rs3809674 is significantly associated with standing and sitting heights; arm fat-free mass (left or right); arm predicted mass (left or right); trunk fat-free or predicted mass; and whole body fat-free mass (p<5×10−8, Supplementary file 1); this variant is also a CEROX1 expression quantitative trait locus (eQTL) for 30 GTEx tissues (p≤0. 05), some with absolute normalised effect sizes reaching 0. 83 (Figure 1—figure supplement 1E). Genetically determined expression change in CEROX1 therefore could explain, in part, variation in these anthropomorphic traits. These variations affect a large fraction of the human population (minor allele frequency of 28% in 1000 Genomes data). An alternative interpretation that rs3809674 (and linked variants) act on anthropomorphic traits through LMF1, rather than CEROX1, is consistent with this variant being an eQTL for LMF1, but is not consistent with the known protein function of LMF1, a lipase maturation factor, because the associated anthropomorphic traits relate only to fat-free mass. A summary-data-based Mendelian randomization analysis that uses rs3809674 as an instrumental variable, predicts an effect of CEROX1 gene expression on glioma risk (Melin et al., 2017). Finally, it has recently been demonstrated in a mouse model of haematopoietic lineage differentiation that Cerox1 depletion may impair the contributions of stem cell or B cell differentiation to haematopoiesis (Delás et al., 2019). In mouse neuroblastoma (N2A) cells the most highly expressed Cerox1 transcript (Figure 1—figure supplement 1F, G) is enriched in the cytoplasmic fraction (Figure 1F) with a short half-life of 36 ± 16 mins (Figure 1—figure supplement 1H) and is mainly associated with the ribosome-free fraction (Figure 1—figure supplement 1I). Expression of Cerox1 was manipulated by transient overexpression or shRNA-mediated knockdown. Twelve shRNAs were tested for the ability to knock-down Cerox1 (Figure 2—figure supplement 1A). One of these (sh92) decreased expression levels by greater than 60%, with the next best shRNA (sh1159) decreasing expression by approximately 40%. As expected from their sharing of the Cerox1-Sox8 bidirectional promoter, CRISPR-mediated activation and inhibition of the Cerox1 locus was not specific as it led also to changes in Sox8 expression (Figure 1A, Figure 2—figure supplement 1B). However, decreasing Cerox1 levels in N2A cells by shRNA or transient overexpression had no effect on the expression of neighbouring genes (Figure 2—figure supplement 1C). In contrast, Cerox1 overexpression led to differential expression of 286 distal genes (q < 0. 05, Bonferroni multiple testing correction; Supplementary file 2), of which an unexpected and large majority (83%; 237) were upregulated (p<10−6; binomial test). Our attention was immediately drawn to the considerable (≥20 fold) enrichment of the mitochondrial respiratory chain gene ontology term among upregulated genes (Figure 2A). The mitochondrial electron transport chain (ETC) consists of five multi-subunit complexes encoded by approximately 100 genes of which only 13 are located in the mitochondrial genome. The 15 ETC transcripts that show statistically significant differential expression after Cerox1 overexpression are nuclear encoded (Figure 2B, C) with the greatest changes observed by qPCR for complex I subunit transcripts (Figure 2—figure supplement 1D). Twelve of 35 nuclear encoded complex I subunits or assembly factors transcripts increased substantially and significantly (>40%) following Cerox1 overexpression; we consider these to be gene expression biomarkers for Cerox1 activity in the mouse N2A system (Figure 2C). In the reciprocal Cerox1 knock-down experiment, all 12 were reduced in abundance using sh92, three significantly, with a concordant pattern observed for the less effective shRNA, sh1159 (Figure 2—figure supplement 1E, F). Taken together, these results indicate that Cerox1 positively and co-ordinately regulates the levels of many mitochondrial complex I transcripts. Increased abundance of OXPHOS subunit transcripts, following Cerox1 overexpression, was found to elevate protein levels. Western blots using reliable antibodies for the key complex I catalytic core proteins NDUFS1 and NDUFS3 showed approximately 2. 0-fold protein level increases that surpassed their ~ 1. 4 fold transcript level changes (median 2. 4 and 1. 4-fold increases [p=0. 0013 and 0. 002], respectively; Figure 2D). Cerox1 transcript abundance is thus coupled positively to OXPHOS transcript levels and to their availability for translation, resulting in an amplification of the amount of protein produced. In summary, protein subunits of the same complex (Complex I) that are sustained at high abundance and with long half-lives (Dörrbaum et al., 2018; Mathieson et al., 2018; Schwanhäusser et al., 2011) (Figure 2—figure supplement 1G), and whose transcripts are stable (Schwanhäusser et al., 2011; Friedel et al., 2009; Sharova et al., 2009; Tani et al., 2012) (Figure 2—figure supplement 1H) and also have very high copy numbers in the cell (Schwanhäusser et al., 2011; Cao et al., 2017), can be increased two-fold, and co-ordinately, by the simple expediency of increasing the level of this single abundant lncRNA. These large effects on protein and mRNA copy number are associated with both metabolic and cellular phenotypes. Ten metabolites are significantly different in N2A cells overexpressing Cerox1 (Figure 2E). These cells show a significant increase in the reduced glutathione to oxidized glutathione ratio (GSH: GSSG, p=1. 3×10−3, Figure 2D Inset) indicative of a more favourable cellular redox state. Cerox1-overexpressing cells also exhibited a 43% reduction in cell cycle activity, yet without a change in the proportion of live/dead cells or a deviation from normal cell cycle proportions (Figure 2—figure supplement 1I, J, K). Cerox1 levels thus affect the overall timing of cell division. Increased translation of some complex I transcripts leads to increased respiration (Shyh-Chang et al., 2013) and, more specifically, to an increase in the enzymatic activity of complex I (Alvarez-Fischer et al., 2011). To address this hypothesis we used oxidative phosphorylation enzyme assays to investigate whether changes in expression to a subset of subunits lead to a change in enzyme activity and oxygen consumption. Indeed, complex I and complex IV enzymatic activities increased substantially after Cerox1 overexpression (by 22%, p=0. 01; by 50%, p=0. 003, respectively; 2-tailed Student’s t-test; Figure 3A). Such rate increases for two of the eukaryotic cell’s most abundant and active enzymes were unexpected. We next measured oxygen consumption under these conditions using a Seahorse XFe24 Analyzer. These complexes’ more rapid catalytic rates resulted, unexpectedly, in large increases in: (i) overall basal oxygen consumption (by 85%), (ii) ATP-linked oxygen consumption (by 107%) and, (iii) maximum uncoupled respiration (by 59%; p=5×10−4, p=1×10−4, p=4×10−3, respectively, 2-tailed Student’s t-test; Figure 3B). These increases in enzyme activities and mitochondrial respiration are expected to produce persistent and substantial increases beyond the already very high basal rate of ATP formation (Rich, 2003), due to the long-half lives of the Cerox1 sensitive complex I protein subunits (Dörrbaum et al., 2018; Mathieson et al., 2018; Schwanhäusser et al., 2011). Conversely, after sh92-mediated Cerox1 knockdown complex I and complex IV enzymatic activities decreased significantly (by 11%, p=0. 03% and 19%, p=0. 02, respectively; Figure 3C), with concomitant large decreases in basal oxygen consumption (53%), ATP linked oxygen consumption (52%) and maximal uncoupled respiration (61%; p=0. 011, p=0. 034, p=0. 042 respectively, 2-tailed Student’s t-test; Figure 3D). These observed changes in enzymatic activity were not due to changes in mitochondria number because the enzymatic activities of complexes II, III and citrate synthase (Figure 3A, B), and the mitochondrial-to-nuclear genome ratio (Figure 3—figure supplement 1A, B), each remained unaltered by changes in Cerox1 levels. These data indicate that Cerox1 can specifically and substantially regulate oxygen consumption and catalytic activities of complex I and complex IV in mouse N2A cells. Complex I deficient patient cells experience elevated ROS production (Pitkanen and Robinson, 1996). In Cerox1 knockdown N2A cells ROS levels were increased significantly, by almost 20% (p=4. 2×10−6; Figure 4A). Conversely, in cells overexpressing Cerox1, ROS production was nearly halved (p=3. 5×10−7; Figure 4A), and protein carbonylation, a measure of ROS-induced damage, was reduced by 35% (p=1×10−3; Figure 4B). Knock-down of Cerox1 resulted in a 6. 6% increase in protein carbonylation compared to the control (p=0. 05, data not shown). The observed Cerox1-dependent reduction in ROS levels is of particular interest because mitochondrial complex I is a major producer of ROS which triggers cellular oxidative stress and damage, and an increase in ROS production is a common feature of mitochondrial dysfunction in disease (Murphy, 2009). We next demonstrated that the increased activities of complex I and complex IV induced by Cerox1 protect cells against the deleterious effects of specific mitochondrial complex inhibitors, specifically rotenone and sodium azide (complex I and complex IV inhibitors, 37% and 58% respectively, p<0. 01); conversely, Cerox1-knockdown cells were significantly more sensitive to rotenone and exposure to heat (12%, p<0. 001% and 11%, p<0. 01 respectively Figure 4C). Cells overexpressing Cerox1 and treated with rotenone, a complex I inhibitor, exhibited no significant difference in protein carbonylation (data not shown). Taken together, these results indicate that elevation of Cerox1 expression leads to decreased ROS production, decreased levels of oxidative damage to proteins and can confer protective effects against complex I and complex IV inhibitors. Due to their positive correlation in expression and cytoplasmic localisation we next considered whether Cerox1 regulates complex I transcripts post-transcriptionally by competing with them for the binding of particular miRNAs. To address this hypothesis, we took advantage of mouse Dicer-deficient (DicerΔ/Δ) embryonic stem cells that are deficient in miRNA biogenesis (Nesterova et al., 2008). We first tested Cerox1 overexpression in wildtype mouse ES cells and showed that this, again, led to an increase in transcript levels, specifically of six complex I subunits (Figure 5A), of which four had previously shown significant changes in N2A cells after Cerox1 overexpression (Figure 2C). In contrast, overexpression in DicerΔ/Δ cells failed to increase levels of these transcripts (Figure 5A). These results indicate that Cerox1’s ability to alter mitochondrial metabolism is miRNA-dependent. Four miRNA families (miR-138–5p, miR-28/28–5p/708–5p, miR-370–3p, and miR-488–3p) were selected for further investigation based on the conservation of their predicted binding sites (MREs) in both mouse Cerox1 and human CEROX1 (Figure 5B). All five MREs conserved in mouse Cerox1 and human CEROX1 for N2A-expressed miRNAs (Figure 5B Figure 5—figure supplement 1A) were mutated by inversion of their seed regions. This mutated Cerox1 transcript failed to alter either complex I transcript levels or enzyme activities when overexpressed in mouse N2A cells (Figure 5C, D). This indicates that Cerox1’s molecular effects are mediated by one or more of these MREs. If so, then the overexpression of these miRNAs in turn would be expected to deplete Cerox1 RNA levels. Indeed, overexpression of each miRNA reduced Cerox1 levels (Figure 5E). Overexpression of the tissue-restricted miRNA miR-488–3p (Figure 5—figure supplement 1B, C; Landgraf et al., 2007; Isakova and Quake, 2018) caused the greatest depletion of the Cerox1 transcript (Figure 5E) indicating that this MRE is likely to be physiologically relevant. Dual fluorescent RNA in situ hybridization of miR-488–3p and Cerox1 indicates the proximity of these non-coding RNAs within the N2A cell (Figure 5F) and that both Cerox1 and miR-488–3p are localised in the cytoplasm (Figures 1F and 5F). CEROX1 transcripts are predominantly (94%) localised to ribosomes (van Heesch et al., 2014), as is the destabilisation by microRNAs of mRNAs as they are being translated (Tat et al., 2016). Together, these observations imply that Cerox1 and mitochondrial protein mRNAs are targets of miR-488–3p on ribosomes within the rough ER that forms a network around mitochondria (Wu et al., 2017; Eskelinen, 2008). Our previous results showed that Cerox1 abundance modulates complex I activity and transcripts (Figures 2–4) and that miR-488–3p has the greatest effect in decreasing Cerox1 transcript levels (Figure 5E). To determine whether miR-488–3p modulates complex I transcript levels we overexpressed and inhibited miR-488–3p in N2A cells (Figure 6A, B). Results showed that miR-488–3p modulates these transcripts’ levels, with overexpression leading to a significant downregulation of all 12 Cerox1-sensitive complex I transcripts (Figure 6A), whilst, conversely, miR-488–3p inhibition leads to increased expression for 10 of 12 transcripts, of which 4 (Ndufa2, Ndufb9, Ndufs4 and Ndufs1) were significantly increased (Figure 6B). To determine whether the single predicted miR-488–3p MRE in Cerox1 is required to exert its effects on complex I we created a Cerox1 transcript containing three mutated nucleotides within this MRE (Figure 6C). As expected for a bona fide MRE, these substitutions abrogated the ability of miR-488–3p to destabilise Cerox1 transcript in a luciferase assay (Figure 6D). Importantly, these substitutions also abolished the ability of Cerox1, when overexpressed, to elevate complex I transcript levels (Figure 6E), and to enhance complex I enzymatic activity (Figure 6F). The latter observation is important because not all bona fide miRNA-transcript interactions are physiologically active (Bassett et al., 2014). Finally, direct physical interaction between Cerox1 and miR-488–3p was confirmed by pulling-down transcripts with biotinylated miR-488–3p (Figure 6G). This experiment also identified 10 of 12 complex I transcripts tested as direct targets of miR-488–3p binding. These included transcripts not predicted as containing a miR-488–3p MRE, as expected from the high false negative rate of MRE prediction algorithms (Mazière and Enright, 2007; Yue et al., 2009; Tabas-Madrid et al., 2014). Also as expected, the two negative control transcripts, which are not responsive to Cerox1 transcript levels and have no predicted MREs for miR-488–3p, failed to bind miR-488–3p. Considered together, these findings indicate that: (i) Cerox1 can post-transcriptionally regulate OXPHOS enzymatic activity as a miRNA decoy, and (ii) of 12 miR-488–3p: Nduf transcript interactions that were investigated, all 12 are substantiated either by responsiveness to miR-488–3p through miRNA overexpression or inhibition (Figure 6A, B), or by direct interaction with a biotinylated miR-488–3p mimic (Figure 6G). Consequently, our data demonstrates that miR-488–3p directly regulates the transcript levels of Cerox1 and at least 12 nuclear encoded mitochondrial complex I subunit genes (31% of all) and indirectly modulates complex I activity (Figure 6F) in N2A cells. Fewer than 20% of lncRNAs are conserved across mammalian evolution (Necsulea et al., 2014) and even for these functional conservation has rarely been investigated. In our final set of experiments we demonstrated that CEROX1, the orthologous human transcript, is functionally equivalent to mouse Cerox1 in regulating mitochondrial complex I activity. Similar to mouse Cerox1, human CEROX1 is highly expressed in brain tissue, is otherwise ubiquitously expressed (Figure 1—figure supplement 1B), and is enriched in the cytoplasm of human embryonic kidney (HEK293T) cells (Figure 7A). CEROX1 is expressed in human tissues at unusually high levels: it occurs among the top 0. 3% of all expressed lncRNAs (Figure 7B) and its average expression is higher than 87. 5% of all protein coding genes (GTEx Consortium, 2017). Its expression is highest within brain tissues, particularly within the basal ganglia and cortex (Figure 7C). Importantly, mitochondrial complexes’ I and III activities increased significantly following CEROX1 overexpression in HEK293T cells (Figure 7D). CEROX1 overexpression had a greater effect on complex I activity than the mouse orthologous sequence and also increased the activity of complex III, rather than complex IV activity, in these cells. In addition to these observed increases in enzyme activity, basal respiration increased by 35%, ATP-linked respiration increase by 31% and maximum uncoupled respiration increased by 31% (p=0. 02, p=0. 04, p=0. 01 respectively, 2-tailed Student’s t-test; Figure 7E). The latter distinction could reflect the differences in miRNA pools between mouse and human cell lines and/or the presence of different MREs in the lncRNA and human OXPHOS transcripts. Strikingly, either reciprocal expression of mouse Cerox1 in human HEK293T cells or human CEROX1 in mouse N2A cells, recapitulates the previously observed increase in complex I activity (Figure 7F). This effect of mouse Cerox1 overexpression in mouse N2A cells is greater than for human CEROX1 overexpression in these cells. The role of both Cerox1 and CEROX1 in modulating the activity of mitochondrial complex I has thus been conserved over 90 million years since the last common ancestor of mouse and human. Cerox1 is the first evolutionarily conserved lncRNA to our knowledge that has been demonstrated experimentally to regulate mitochondrial energy metabolism. Its principal location in N2A cells is in the cytoplasm (Figure 1F) where it post-transcriptionally regulates the levels of mitochondrial OXPHOS subunit transcripts and proteins by decoying for miRNAs (Figure 5—figure supplement 1), most particularly miR-488–3p. This microRNA shares with Cerox1 an early eutherian origin and elevated expression in brain samples (Landgraf et al., 2007), and it previously was shown to alter mitochondrial dynamics in cancer cells (Yang et al., 2017). Changes in Cerox1 abundance in vitro alter mitochondrial OXPHOS subunit transcript levels and, more importantly, elicit larger changes in their protein subunits levels, leading to unexpectedly large changes in mitochondrial complex I catalytic activity. The observed changes in catalytic activity are in line with the degree of change seen in diseases exhibiting mitochondrial dysfunction (Schapira et al., 1990; Ritov et al., 2005; Andreazza et al., 2010). Overexpression of Cerox1 in N2A cells increases oxidative metabolism, halves cellular oxidative stress and enhances protection against the complex I inhibitor rotenone. The effect of Cerox1 on complex I subunit transcript levels can be explained by their sharing MREs with Cerox1, and subsequent competition for miRNA binding, most notably for miR-488–3p, which buffers the OXPHOS transcripts against miRNA-mediated repression. Multiple RNA transcripts have been experimentally shown to compete with mRNAs for binding to miRNAs, thereby freeing the protein coding mRNA from miRNA-mediated repression (Cesana et al., 2011; Karreth et al., 2011; Sumazin et al., 2011; Tay et al., 2011; Karreth et al., 2015; Tan et al., 2014). It has been experimentally demonstrated that this miRNA: RNA regulatory crosstalk can initiate rapid co-ordinate modulation of transcripts whose proteins participate within the same complex or process (Tan et al., 2014). Physiological relevance of this crosstalk mechanism remains incompletely understood. Furthermore, mathematical modelling (Ala et al., 2013; Figliuzzi et al., 2013; Martirosyan et al., 2016) and experimental investigation (Bosson et al., 2014; Denzler et al., 2014) of the dynamics and mechanism of endogenous transcript competition for miRNA binding have resulted in contrasting conclusions. Current mathematical models do not take full account of miRNA properties, such as the repressive effect not being predictable from its cellular abundance (Mullokandov et al., 2012), intracellular localisation such as at the rough ER (Stalder et al., 2013), loading on the RNA-induced silencing complex (RISC) (Flores et al., 2014), or AGO2’s phosphorylation status within the RISC (Golden et al., 2017). The conclusions of experiments have also assumed that all miRNA target transcripts that contain the same number and affinity of miRNA binding sites are equivalent, that steady-state measurements are relevant to repression dynamics, and that observations for one miRNA in one experimental system are equally applicable to all others (Smillie et al., 2018). Considered together, our lines of experimental evidence indicate that miRNA-mediated target competition by Cerox1 substantially perturbs a post-transcriptional gene regulatory network that includes at least 12 complex I subunit transcripts. This is consistent with the expression level of miR-488–3p (Kozomara and Griffiths-Jones, 2014) and the high in vivo expression of both Cerox1 and OXPHOS transcripts (Schwanhäusser et al., 2011; Vogel et al., 2010). Human CEROX1 levels, for example, exceed those of all complex I subunit transcripts (those in Figure 6a) in both newly-formed and myelinating oligodendrocytes (Forrest et al., 2014). Cerox1 could maintain OXPHOS homeostasis in cells with sustained high metabolic activity and high energy requirements. Such cells occur in the central nervous system, in which Cerox1 levels are high (Sokoloff, 1977), and in haematopoiesis where depletion of Cerox1 results in B cell depletion or myeloid enrichment (Delás et al., 2019). Our experiments demonstrate that post-transcriptional regulation of a subset of complex I subunits by Cerox1 leads to elevated oxygen consumption. How consumption increases when there is a higher abundance of only a subset of OXPHOS transcripts remains unclear. However, this phenomenon has been observed previously in mouse dopaminergic neurons (Alvarez-Fischer et al., 2011) and primary mouse embryonic fibroblasts and pinnal tissues (Shyh-Chang et al., 2013). Our observation of increased enzymatic activity may relate to the formation, by the complexes of the respiratory chain, of higher order supercomplexes (Schägger and Pfeiffer, 2000; Genova and Lenaz, 2014). Alternatively, the observed increases in OXPHOS activity may reflect some subunits of the complex I holo-enzyme (including NDUFS3 and NDUFA2) being present as a monomer pool and therefore being available for direct exchange without integration into assembly intermediates (Dieteren et al., 2012). This monomer pool facilitates the rapid swapping out of oxidatively damaged complex I subunits (Dieteren et al., 2012). It is thus possible that Cerox1-mediated expansion of the monomer pool thereby improves complex I catalysis efficiency (Figure 8). However, we note that overexpression of Cerox1 results in the differential expression of 286 genes, most (83%) of which are upregulated. These genes’ transcripts will be both the targets of miR-488–3p decoying by Cerox1 (Figure 6) and those whose upregulation is secondary to Cerox1 (and miR-488–3p) mediated effects, for example relating to the observed changes in cellular metabolism and proliferation (Figure 2—figure supplement 1I, J, K). More efficient ETC enzymatic activity might be relevant to mitochondrial dysfunction, a feature of many disorders that often manifests as decreases in the catalytic activities of particular mitochondrial complexes. A decrease in catalytic activity can result in elevated ROS production, leading to oxidative damage of lipids, DNA, and proteins, with OXPHOS complexes themselves being particularly susceptible to such damage (Musatov and Robinson, 2012). Parkinson’s and Alzheimer’s diseases both feature pathophysiology associated with oxidative damage resulting from increased ROS production and a cellular energy deficit associated with decreased complex I and IV activities (a reduction of 30% and 40%, respectively) (Schapira et al., 1990; Keeney et al., 2006; Canevari et al., 1999). A deficiency in complexes II, III and to a lesser extent complex IV, has also been described in Huntington disease (Mochel and Haller, 2011). Currently no effective treatments exist that help to restore mitochondrial function despite demonstration that a 20% increase in complex I activity protects mouse midbrain dopaminergic neurons against MPP+, a complex I inhibitor and a chemical model of Parkinson’s disease (Alvarez-Fischer et al., 2011). We note that the highest expression of CEROX1 occurs primarily in the basal ganglia (Figure 7C inset) regions of which are specifically vulnerable to the progressive neurological disorders Parkinson’s (substantia nigra pars compacta) and Huntington’s diseases (striatum: caudate and putamen). The specific energy demands of these neurons may make them particularly susceptible to damage due to an energy deficit. For instance, the dopaminergic neurons of the substantia nigra, which are especially sensitive to degeneration in Parkinson’s disease, have unusually large axonal arbours that require tight regulation of cellular energy to maintain (Bolam and Pissadaki, 2012). In addition, the medium spiny neurons of the striatum, which preferentially degenerate in Huntington disease, exhibit a high degree of axonal collateralization – a morphological trait which implies high cellular energy consumption for its maintenance (Parent and Parent, 2006) – therefore causing these cells to be vulnerable to decreased cellular ATP production. CEROX1’s ability to increase mitochondrial complex I activity might be recapitulated pharmacologically to restore mitochondrial function, as an exemplar of therapeutic upregulation of gene expression (Wahlestedt, 2013). The lncRNA transcripts were assessed for coding potential using the coding potential calculator (Kong et al., 2007), PhyloCSF (Lin et al., 2011) and by mining proteomics and small open reading frame resources for evidence of translation (Wilhelm et al., 2014; Kim et al., 2014a; Bazzini et al., 2014). A lack of protein-coding potential for human CEROX1 (also known as RP11-161M6. 2, LMF1-3) is supported by a variety of computational and proteomic data summarised in LNCipedia (Volders et al., 2015). Expression data from somite-staged mouse embryos were acquired from ArrayExpress (E-ERAD-401 - Strand-specific RNA-seq of somite-staged second generation genotypically wild-type embryos of mixed G0 lineage from the Mouse Genetics Project/DMDD). Genome wide associations were performed on the UK Biobank data as described in Canela-Xandri et al. (2018) using data from up to 452,264 individuals. Total RNA from twenty normal human tissues (adipose, bladder, brain, cervix, colon, oesophagus, heart, kidney, liver, lung, ovary, placenta, prostate, skeletal muscle, small intestine, spleen, testes, thymus, thyroid and trachea) were obtained from FirstChoice Human Total RNA Survey Panel (Invitrogen). Total RNA from twelve mouse tissues (bladder, brain, colon, heart, kidney, liver, pancreas, skeletal muscle, small intestine, stomach and testis) were obtained from Mouse Tissue Total RNA Panel (Amsbio). RNA from cell lines was extracted using the RNeasy mini kit (Qiagen) according to the manufacturer’s instructions, using the optional on column DNase digest. cDNA synthesis for all samples was performed on 1 μg of total RNA using a QuantiTect Reverse Transcription kit (Qiagen) according to the manufacturer’s instructions. RNA was extracted from samples used for the detection of miRNAs using the miRNeasy mini kit (Qiagen) according to the manufacturer’s instructions (with on column DNase digest). All RNA samples were quantified using the 260/280 nm absorbance ratio, and RNA quality assessed using a Tapestation (Agilent). RNA samples with an RNA integrity number (RIN) >8. 5 were reverse transcribed. 1 μg of total RNA from the miRNA samples were reverse transcribed using the NCode VILO miRNA cDNA synthesis kit. Expression levels were determined by real-time quantitative PCR, using SYBR Green Master Mix (Applied Biosystems) and standard cycling parameters (95°C 10 min; 40 cycles 95°C 15 s, 60°C 1 min) followed by a melt curve using a StepOne thermal cycler (Applied Biosystems). All amplification reactions were performed in triplicate using gene specific primers. Multiple reference genes were assessed for lack of variability using geNorm (Vandesompele et al., 2002). Human expression data were normalised to TUBA1A and POLR2A, whilst mouse expression data were normalised to Tbp and Polr2a. Oligonucleotide sequences are provided in Supplementary file 4. Mouse Neuro-2a neuroblastoma cells (N2A; RRID: CVCL_0470; ECACC 89121404) and human embryonic kidney (HEK293T; RRID: CVCL_0063; ECACC 12022001) were sourced from the European authenticated cell culture collection. HEK293T cells were confirmed by STR profiling and cell lines were tested monthly for mycoplasma contamination. Cells were grown at 37°C in a humidified incubator supplemented with 5% CO2. Both cell lines were grown in Dulbecco’s modified Eagle medium containing penicillin/streptomycin (100 U/ml, 100 ug/ml respectively) and 10% fetal calf serum. Cells were seeded at the following densities: six well dish, 0. 3 × 106; 48 well dish, 0. 2 × 104; T75 flask 2. 1 × 106. We had three reasons for the choice of HEK293T cells for this experiment. First, they are of neural crest ectodermal origin (Lin et al., 2014) and therefore have a number of neural cell line characteristics in that they express neuronal markers (Shaw et al., 2002). Second, they are in use as a cell culture model for neurodegenerative diseases such as Parkinson’s disease (Falkenburger et al., 2016; Schlachetzki et al., 2013). Third, HEK293T cells are a widely used cell line to interrogate human mitochondrial biochemistry, and exhibit complex I dependent respiration (Kim et al., 2014b). Mouse embryonic stem cells and dicer knock-out embryonic stem cells were maintained as described previously (Nesterova et al., 2008). Cells were counted using standard haemocytometry. For flow cytometry the cells were harvested by trypsinization, washed twice with PBS and fixed in 70% ethanol (filtered, −20°C). The cell suspension was incubated at 4°C for 10 min and the cells pelleted, treated with 40 μg/ml RNase A and propidium iodide (40 μg/ml) for 30 min at room temperature. Cells were analysed using a FACSCalibur (BD-Biosciences) flow cytometer. Branched chain DNA probes to Cerox1, Malat1 and mmu-miR-488–3p were sourced from Affymetrix. The protocol was preformed according to the manufacturer’s instructions using the QuantiGene ViewRNA miRNA ISH cell assay kit (QVCM0001) for adherent cells. The following parameters were optimised: cells were fixed in 4% formaldehyde for 45 min and a 1: 2000 dilution of the protease was optimal for the N2A cells. Cells were imaged using an Andor Dragonfly confocal inverted microscope, and images acquired using an Andor Zyla 4. 2 plus camera. Cells were fractionated into nuclear and cytoplasmic fractions in order to determine the predominant cellular localization of lncRNA transcripts. Briefly, approximately 2. 8 × 106 cells were collected by trypsinization, washed three times in PBS and pelleted at 1000 g for 5 min at 4°C. The cell pellet was resuspended in 5 volumes of lysis buffer (10 mM Tris-HCl, pH 7. 5,3 mM MgCl2,10 mM NaCl, 5 mM EGTA, 0. 05% NP40, and protease inhibitors [Roche, complete mini]) and incubated on ice for 15 min. Lysed cells were then centrifuged at 2000 g for 10 min at 4°C, and the supernatant collected as the cytoplasmic fraction. Nuclei were washed three times in nuclei wash buffer (10 mM HEPES, pH 6. 8,300 mM sucrose, 3 mM MgCl2,25 mM NaCl, 1 mM EGTA), and pelleted by centrifugation at 400 g, 1 min at 4°C. Nuclei were extracted by resuspension of the nuclei pellet in 200 μl of nuclei wash buffer containing 0. 5% Triton X-100 and 700 units/ml of DNase I and incubated on ice for 30 mins. Nucleoplasm fractions were collected by centrifugation at 17 000 g for 20 min at 4°C. RNA was extracted as described above, and RNA samples with RIN values > 9. 0 used to determine transcript localisation. To determine the stability of the lncRNA transcripts, cells were cultured to ~50% confluency and then transcription was inhibited by the addition of 10 μg/ml actinomycin D (Sigma) in DMSO. Control cells were treated with equivalent volumes of DMSO. Transcriptional inhibition of the N2A cells was conducted for 16 hr with samples harvested at 0 hr, 30 mins, 1 hr, 2 hr, 4 hr, 8 hr and 16 hr. RNA samples for fractionation and turnover experiments were collected in Trizol (Invitrogen) and RNA purified and DNAse treated using the RNeasy mini kit (Qiagen). Reverse transcription for cellular localisation and turnover experiments was performed as described earlier. The 5’ and 3’ ends of Cerox1 and CEROX1 were identified by 5’ and 3’ RACE using the GeneRacer Kit (Invitrogen) according to manufacturer’s instructions. As an overexpression/transfection control the pCAG-EGFP backbone was used (RRID: Addgene_89684). The EGFP was removed from this backbone, and all full length lncRNAs were cloned into the pCAG backbone. For cloning into the pCAGs vector, PCR primers modified to contain the cloning sites BglII and XhoI sites were used to amplify the full length mouse Cerox1, whilst human CEROX1 and the mouse 5x MRE mutant were synthesized by Biomatik (Cambridge, Ontario), and also contained BglII and XhoI sites at the 5’ and 3’ ends respectively. All other MRE mutants were produced using overlapping PCR site directed mutagenesis to mutate 3 bases of the miRNA seed region. All purified products were ligated into the prepared backbone and then transformed by heat shock into chemically competent DH5α, and plated on selective media. All constructs were confirmed by sequencing. Short hairpin RNAs specific to the transcripts were designed using a combination of the RNAi design tool (Invitrogen) and the siRNA selection program from the Whitehead Institute (Yuan et al., 2004). Twelve pairs of shRNA oligos to the target genes and β-galactosidase control oligos were annealed to create double-stranded oligos and cloned into the BLOCK-iT U6 vector (Invitrogen), according to the manufacturer’s instructions. miRNA expression constructs were generated and cloned into the BLOCK-iT Pol II miR RNAi expression vector (Invitrogen) according to the manufacturer’s instructions. miRNA inhibitors were sourced from Ambion and used according to manufacturer’s instructions. Transfection efficiency was initially assessed by FACS, and the optimised transfection protocol used for all further assays (6: 1 transfection reagent to DNA ratio). One day prior to transfection cells were either seeded in six well dishes (0. 3 × 106 cells/well), or in T75 flasks (2. 1 × 106 cells/flask). Twenty-four hours later cells in six well dishes were transfected with 1 μg of shRNA, miRNA or overexpression construct and their respective control constructs using FuGENE 6 (Promega) according to the manufacturer’s guidelines. Cells in T75 flasks were transfected with 8 μg of experimental or control constructs. Transfected cells were grown for 48-72 hrs under standard conditions, and then harvested for either gene expression studies or biochemical characterisation. Efficacy of the overexpression and silencing constructs was determined by real-time quantitative PCR. Transcripts for the luciferase destabilisation assays were cloned into the pmirGLO miRNA target expression vector (Promega) and assayed using the dual-luciferase reporter assay system (Promega). miRCURY LNA biotinylated miRNAs (mmu-miR-488–3p and mmu-negative control 4) were purchased from Exiqon, and direct mRNA-miRNA interactions were detected using a modified version of Orom and Lund (2007) and enrichment of targets was detected by qPCR. MREs were predicted using TargetScan v7. 0 (Agarwal et al., 2015; RRID: SCR_010845) in either the 3’UTR (longest annotated UTR, ENSEMBL build 70; RRID: SCR_002344) or the full length transcript of protein coding genes, and across the entire transcript for lncRNAs. The average expression across 46 human tissues and individuals according to the Pilot one data from the GTEx Consortium (Lonsdale et al., 2013; RRID: SCR_013042; 98) was computed for both protein-coding genes and intergenic lncRNAs from the Ensembl release 75 annotation (Flicek et al., 2014). We used the normalised number of CAGE tags across 399 mouse cells and tissues from the FANTOM5 Consortium (http: //fantom. gsc. riken. jp; Kawaji et al., 2014) as an approximation of expression levels for protein-coding genes and intergenic lncRNAs from the Ensembl release 75 annotation. If multiple promoters were associated with a gene, we selected the promoter with the highest average tag number. Conserved sequence blocks in the lncRNA sequences were identified using LALIGN (Goujon et al., 2010). Microarray analysis was performed on 16 samples (four overexpression/four overexpression controls; four knock-down/four knock-down controls), and hybridizations were performed by the OXION array facility (University of Oxford). Data were analysed using the web-based Bioconductor interface, CARMAweb (Rainer et al., 2006). Differentially expressed genes (Bonferroni corrected p-value<0. 05) were identified between mouse lncRNA overexpression and control cells using Limma from the Bioconductor package between the experimental samples and the respective controls. Microarray data are accessible through ArrayExpress, accession E-MATB-6792. Metabolites were extracted from 6-well plates by washing individual wells with ice-cold PBS and addition of cold extraction buffer (50% methanol, 30% acetonitrile, 20% water solution at −20°C or lower). Extracts were clarified and stored at −80°C until required. LC-MS was carried out using a 100 mm x 4. 6 mm ZIC-pHILIC column (Merck-Millipore) using a Thermo Ultimate 3000 HPLC inline with a Q Exactive mass spectrometer. A 32 min gradient was developed over the column from 10% buffer A (20 mM ammonium carbonate), 90% buffer B (acetonitrile) to 95% buffer A, 5% buffer B. 10 μl of metabolite extract was applied to the column equilibrated in 5% buffer A, 95% buffer B. Q Exactive data were acquired with polarity switching and standard ESI source and spectrometer settings were applied (typical scan range 75–1050). Metabolites were identified based upon m/z values and retention time matching to standards. Quantitation of metabolites was carried out using AssayR (Wills et al., 2017). Data were normalised using levels of 9 essential amino acids (histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan, valine), and errors propagated, in order to account for cell count differences. Total protein was quantified using a BCA protein assay kit (Pierce). 10 μg of protein was loaded per well, and samples were separated on 12% SDS-PAGE gels in Tris-glycine running buffer (25 mM Tris, 192 mM glycine, 0. 1% SDS). Proteins were then electroblotted onto PVDF membrane (40V, 3 hr) in transfer buffer (25 mM Tris-HCl, 192 mM glycine, 20% methanol), the membrane blocked in TBS-T (50 mM Tris-HCl, 150 mM NaCl, 0. 1% Tween 20) with 5% non–fat milk powder for 1 hr. The membrane was incubated with primary antibodies overnight at 4°C with the following dilutions: anti-NDUFS1 (RRID: AB_2687932; rabbit monoclonal, ab169540,1: 30,000), anti-NDUFS3 (RRID: AB_10861972; mouse monoclonal, 0. 15 mg/ml, ab110246), or anti-alpha tubulin loading control (RRID: AB_2241126; mouse monoclonal, ab7291,1: 30,000). Following incubation with the primary antibodies, blots were washed 3 × 5 min, and 2 × 15 mins in TBS-T and incubated with the appropriate secondary antibody for 1 hr at room temperature: goat anti-rabbit HRP (RRID: AB_2536530; Invitrogen G-21234) 1: 30,000; goat anti-mouse HRP (RRID: AB_2617137; Dako P0447) 1: 3000. After secondary antibody incubations, blots were washed and proteins of interested detected using ECL prime chemiluminescent detection reagent (GE Healthcare) and the blots imaged using an ImageQuant LAS 4000 (GE Healthcare). Signals were normalised to the loading control using ImageJ (Schneider et al., 2012). Cell lysates were prepared 48 hr post-transfection, by harvesting cells by trypsinisation, washing three times in ice cold phosphate buffered saline followed by centrifugation to pellet the cells (two mins, 1000 g). Cell pellets were resuspended to homogeneity in KME buffer (100 mM KCl, 50 mM MOPS, 0. 5 mM EGTA, pH 7. 4) and protein concentrations were determined using a BCA protein assay detection kit (Pierce). Cell lysates were flash frozen in liquid nitrogen, and freeze-thawed three times prior to assay. 300–500 μg of cell lysate was added per assay, and assays were normalised to the total amount of protein added. All assays were performed using a Shimadzu UV-1800 spectrophotometer, absorbance readings were taken every second and all samples were measured in duplicate. Activity of complex I (CI, NADH: ubiquinone oxidoreductase) was determined by measuring the oxidation of NADH to NAD+ at 340 nm at 30°C in an assay mixture containing 25 mM potassium phosphate buffer (pH 7. 2), 5 mM MgCl2,2. 5 mg/ml fatty acid free albumin, 0. 13 mM NADH, 65 μM coenzyme Q and 2 μg/ml antimycin A. The decrease in absorbance was measured for 3 mins, after which rotenone was added to a final concentration of 10 μM and the absorbance measured for a further 2 mins. The specific complex I rate was calculated as the rotenone-sensitive rate minus the rotenone-insensitive rate. Complex II (CII, succinate dehydrogenase) activity was determined by measuring the oxidation of DCPIP at 600 nm at 30°C. Lysates were added to an assay mixture containing 25 mM potassium phosphate buffer (pH 7. 2) and 2 mM sodium succinate and incubated at 30°C for 10 mins, after which the following components were added, 2 μg/ml antimycin A, 2 μg/ml rotenone, 50 μM DCPIP and the decrease in absorbance was measured for 2 mins. Complex III (CIII, Ubiquinol: cytochrome c oxidoreductase) activity was determined by measuring the oxidation of decylubiquinol, with cytochrome c as the electron acceptor at 550 nm. The assay cuvettes contained 25 mM potassium phosphate buffer (pH 7. 2), 3 mM sodium azide, 10 mM rotenone and 50 μM oxidized cytochrome c. Decylubiquinol was synthesized by acidifying decylubiquinone (10 mM) with HCl (6M) and reducing the quinine with sodium borohydride. After the addition of 35 μM decylubiquinol, the increase in absorbance was measured for 2 mins. Activity of Complex IV (CIV, cytochrome c oxidase) was measured by monitoring the oxidation of cytochrome c at 550 nm, 30°C for 3 min. A 0. 83 mM solution of reduced cytochrome c was prepared by dissolving 100 mg of cytochrome c in 10 ml of potassium phosphate buffer, and adding sodium ascorbate to a final concentration of 5 mM. The resulting solution was added into SnakeSkin dialysis tubing (7 kDa molecular weight cutoff, Thermo Scientific) and dialyzed against potassium phosphate buffer, with three changes, at 4°C for 24 hr. The redox state of the cytochrome c was assessed by evaluating the absorbance spectra from 500 to 600 nm. The assay buffer contained 25 mM potassium phosphate buffer (pH 7. 0) and 50 μM reduced cytochrome c. The decrease in absorbance at 550 nm was recorded for 3 mins. As a control the enzymatic activity of the tricarboxylic acid cycle enzyme, citrate synthase (CS) was assayed at 412 nm at 30°C in a buffer containing 100 mM Tris-HCl (pH 8. 0), 100 μM DTNB (5,5-dithiobis[2-nitrobenzoic acid]), 50 μM acetyl coenzyme A, 0. 1% (w/v) Triton X-100 and 250 μM oxaloacetate. The increase in absorbance was monitored for 2 mins. The following extinction coefficients were applied: complex I (CI), ε = 6220 M−1 cm−1, CII, ε = 21,000 M−1 cm−1; CIII, ε = 19,100 M−1 cm−1; CIV, ε = 21,840 M−1 cm−1 (the difference between reduced and oxidised cytochrome c at 550 nm); CS, ε = 13,600 mM−1 cm−1. Cellular oxygen consumption rate was determined using the Seahorse XFe24 Analyzer (Agilent). Cells were plated on poly-L-lysine coated XFe24 microplates at 50,000 cells per well and incubated at 37°C and 5% CO2 for 24 hr. Cells were transfected with an overexpression (pCAG-Cerox1 or pCAG-CEROX1) or the Cerox1 shRNA silencing construct and assayed 24–36 hr later. Cells were washed three times with Seahorse Assay media (Agilent), supplemented with 10 mM glucose and 2 mM pyruvate. After 30 min of pre-incubation in a non-CO2 37°C incubator, cells were entered into the analyser for oxygen consumption rate measurements. After basal respiration measurements, 1 μM of oligomycin was injected to inhibit ATP synthase, then 0. 2 μM of carbonyl cyanide 4- (trifluoromethoxy) phenylhydrazone (FCCP) was injected to uncouple respiration. Finally 1 μM each of rotenone and antimycin A were injected to inhibit complex I and complex III respectively. Data was normalised to total cellular protein content using the sulforhodamine B assay (Skehan et al., 1990). Basal respiration, ATP linked respiration and maximum uncoupled respiration were calculated from the normalised data using the Agilent Seahorse XF calculation guidelines. Briefly, basal respiration = measurement 3 (last rate measurement before oligomycin injection) – measurement 10 (minimum respiration after injection of rotenone/antimycin A); ATP-linked respiration = measurement 3 – measurement 4 (minimum rate measurement after oligomycin injection); maximum uncoupled respiration = measurement 7 (maximum rate after FCCP injection) – measurement 10. Hydrogen peroxide production was assessed as a marker of reactive oxygen species generation using the fluorescent indicator Amplex Red (10 μM, Invitrogen) in combination with horseradish peroxidise (0. 1 units ml−1). Total amount of H2O2 produced was normalised to mg of protein added. Protein carbonylation was assessed using the OxyBlot protein oxidation detection kit (Merck Millipore), and differential carbonylation was assessed by densitometry. The cell stress assay was performed on cells seeded in 48 well plates, and assayed 12 hr later by the addition of (final concentration): rotenone (5 μM), malonate (40 μM), antimycin A (500 μM), oligomycin (500 μM), sodium azide (3 mM), NaCl (300 mM), CaCl2 (5. 4 mM) for 1 hr. Cells were heat shocked at 42°C and UV irradiated using a Stratlinker UV Crosslinker for 10 min (2. 4 J cm−2). Cell viability was assessed by the addition of Alamar Blue (Invitrogen) according to the manufacturer’s instructions. | Animal cells generate over 90% of the energy they need within small structures called mitochondria. Converting food into energy requires many different proteins and cells control the relative amounts of the proteins in mitochondria to ensure this process is efficient. To make more of a given protein, the cell must copy the DNA of the gene that encodes it into another molecule known as a messenger RNA, before reading the instructions in the messenger RNA to build the protein. However, this is not the only way that a cell uses molecules of RNA. A second group of RNAs called long non-coding RNAs (or lncRNAs) can help regulate the production of proteins in complex ways, and each lncRNA can have an effect across multiple genes. Some lncRNAs, for example, stop a third group of RNAs - microRNAs - from blocking certain messenger RNAs from being read. Sirey et al. set out to answer whether a lncRNA might help to co-ordinate the production of the many proteins needed by mitochondria. In experiments with mouse cells grown in the laboratory, Sirey et al. identified a lncRNA called Cerox1 that can co-ordinate the levels of at least 12 mitochondrial proteins. A microRNA called miR-488-3p suppresses the production of many of these proteins. By binding to miR-488-3p, Cerox1 blocks the effects of the microRNA so more proteins are produced. Sirey et al. artificially altered the amount of Cerox1 in the cells and showed that more Cerox1 leads to higher mitochondria activity. Further experiments revealed that this same control system also exists in human cells. Mitochondria are vital to cell survival and changes that affect their efficiency can be fatal or highly debilitating. Reduced efficiency is also a hallmark of ageing and contributes to conditions including cardiovascular disease, diabetes and Parkinson's disease. Understanding how mitochondria are regulated could unlock new treatment methods for these conditions, while a better understanding of the co-ordination of protein production offers other insights into some of the most fundamental biology. | lay_elife |