The multicolor banding (MCB/mBAND) technique provides a unique opportunity to characterize intrachromosomal rearrangements and to determine chromosomal breakpoints. Until recently, MCB probes have only been available for human and some murine chromosomes. Generation of MCB probes for chromosomes of other species, useful and required in many cytogenetics research fields, was limited by technical difficulties. MCB probes are established by chromosome microdissection followed by whole genomic DNA amplification. However, unambiguous identification of the target chromosome is required for MCB-probe establishment. Previously proposed protocols suggested G-banding staining or preliminary FISH with whole chromosome paints (WCP) as methods to identify the chromosome of interest.Here we present a complete workflow for MCB probe generation for those cases and species where chromosome morphology is too challenging to recognize target chromosomes by conventional methods and where WCP probes are not available. The workflow was successfully applied for murine chromosomes that are difficult to identify unambiguously. Additionally, we showed that glass-needle based microdissection enables establishment of a whole set of WCP paints by microdissection of individual chromosomes of a single metaphaseThe present method can be applied for generation of whole or region-specific DNA probes for species, where karyotyping of G-banded chromosomes is challenging due to similar chromosome morphology and/or chromosome banding patterns. Glass needle-based microdissection of chromosomes can be applied to establish chromosome specific probes of different species [Gorilla gorilla[Hylobates lar[Pan paniscus[The identification of chromosomes and chromosomal subregions can be a challenging task. While in clinical cytogenetics and in many mammalian species the available staining methods provide informative banding patterns along the chromosomes, there are many more species in which no such chromatin-related patterns of alternated light and dark bands can be induced; for example, G-banding is only reliable in higher vertebrates . Introdu species ,3. Besid species  for humaa gorilla, Hylobatbates lar and Pan  paniscus. However paniscus. To dist paniscus. HoweverPCP probes for MCB probe mixes are generated conventionally by glass needle microdissection ,10,11, wThe whole workflow for generating mcb probes for those cases, where chromosome morphology makes chromosome identification complicated, is presented in Figure At first we aimed to establish wcp probes for all mouse chromosomes to be later used in FISH-MD experiments. The strategy we applied was to dissect all chromosomes of the same metaphase one by one and to collect them into separate micropipettes, containing chromosome collection buffer . The collection buffer with a single dissected chromosome was then transferred into a tube with 5 μl of PCR-grade water. Further amplification was done using GenomePlex® Single Cell Whole Genome Amplification Kit (Sigma-Aldrich). The resulting 40 proto-wcp probes corresponding to 40 murine individual chromosomes were based on the amplification of DNA from a single dissected chromosome. These proto-wcp paints produced weak, but identifiable signals on one pair of chromosomes upon reverse-FISH back onto mouse metaphases . These proto-wcp probes were further used in FISH-MD experiments to generate wcp probes based on 20 dissected chromosomes per probe. Collection of multiple chromosomes aimed to improve the representation of all chromosomal regions in the generated library and to exclude the possibility of loss of any of the fragments while dissecting or collecting them into micropipettes. After this second round of microdissection 40 murine wcp paints were obtained. But as they were generated “blindly” it was not known which wcp corresponded to which chromosome. To assign these wcp probes, we performed several rounds of co-hybridization. Initially, we co-hybridized different wcp probes labeled with different fluorochromes onto control mouse metaphases to exclude the identical probes corresponding to the same pair of homologous chromosomes. When the doubles were excluded we got 19 unique wcp probes for autosomes and wcp probes for the X and Y chromosomes (which were identified earlier). To detect which wcp corresponded to which chromosome we have used several different approaches: 1) wcp probes were applied onto metaphases from WMP2 cell line known to contain easily identifiable metacentric fusion chromosomes characterized before by G-banding and FISH ,13; 2) wHence, in spite of the fact that it was not possible to reliably identify chromosomes in mouse metaphase spreads based on GTG-banding, we have successfully generated mcb probe sets for all 19 murine autosomes and the X chromosome. The only chromosome, for which we have failed to establish an mcb mix, was the Y-chromosome – all pcp probes for the Y-chromosome resulted in staining of the entire chromosome. This fact might be due to the enormously expended ampliconic portion of the mouse Y chromosome, taking up to 95% of the chromosome , so all We believe that the proposed workflow can be applied for chromosomes of other species as well in order to generate wcp, pcp and mcb probe sets. The approach of “blind” microdissection of all chromosomes of one single metaphase is especially helpful for obtaining chromosome-specific DNA libraries for the cases when chromosome isolation by flow sorting is hampered by similar size of chromosomes.One of the problems one may encounter while establishing wcp or pcp paints can be the selection of suitable DNA amplification method. For many years DOP-PCR with 6MW primer was a method of choice for amplifying microdissected material, and it is still conventionally used for amplification of human microdissected chromosomes. The anti-6MW DOP-PCR primer was proposed later  to help Application of mcb probes requires prehybridization with Cot1 DNA for the suppression of repetitive elements to block non-specific hybridization. The source of Cot1 DNA for the species of interest should be available, as Cot1 DNA is essential for the FISH-MD experiments with wcp paints, as well as for reverse-FISH tests and mcb probe applications. Due to the fact that all DNA which is dissected and collected into a micropipette is subjected to amplification, we believe it is also important to reduce the amount of Cot1 DNA used in FISH-MD experiments to avoid the excessive amplification of repetitive elements in the generated DNA library (the dissected fragments contain DNA from the hybridized wcp probe). The exact amount of Cot1 DNA used in FISH-MD and in reverse-FISH should be determined experimentally.Though glass needle based microdissection requires experience and is time consuming, it is still a method of choice for generating pcp paints as it works finer than other techniques and enables precise dissection and collection of target chromosome regions. It is even possible to dissect several different pcp libraries corresponding to different regions from the same chromosome, thus saving chromosome material. We were able to attribute “blindly” generated murine wcp paints by co-hybridizing them with other region specific and BAC probes available in our laboratory. For the chromosomes of other species these kinds of probes can be simply not available; we suggest that in such cases wcps can be identified by painting the previously G-banded chromosomes.We have demonstrated that generation of mcb probes is possible even in cases where chromosome morphology complicates chromosome recognition. The proposed approach of “blind” microdissection of all chromosomes of the same metaphase can be also helpful for establishing chromosome-specific DNA libraries for the species which chromosomes cannot be flow sorted due to their similar size.FISH-banding methods and mcb technique especially, open new opportunities for evolutionary cytogenetics studies. Recent application of some murine mcb paints in nine rodent species  has provMurine chromosome spreads of different origin were used for microdissection and FISH: mouse embryonic and adult fibroblasts cultures, short-term cultivation of murine spleen tissue and WMP2 cell line . WMP2 ce′-CCG ACT CGA GNN NNN NAT GTG G-3′) and the anti-6MW (5′-CCG TGA GCT CNN NNN NTA CAC C-3′) primer [Amplification of microdissected DNA was carried out either with DOP-PCR , or with) primer . 3 μl of) primer , or by P) primer . Before Mouse BAC probes were purchased from the Children’s Hospital Oakland Research Institute (CHORI), Oakland, USA. All BAC clone DNA was isolated, PCR-amplified and labeled as described .Hybridization, post-hybridization washes and detection steps were done as described previously . Image aDNA: Deoxyribonucleic acid; PCR: Polymerase chain reaction; DOP-PCR: Degenerate oligonucleotide-primed PCR.The authors declare that they have no competing interests.NK conceived the study, performed microdissection experiments and drafted the manuscript. ABH, KP, PS, WS and SR performed microdissection and FISH experiments. AC, VT and XF participated in the study design and protocol elaboration, and helped to draft the manuscript. All authors read and approved the final manuscript.Video 1. FISH-MD experiment. Murine chromosome 9 is identified by FISH with wcp 9 probe labeled with Spectrum Orange-dUTP (pointed with a white arrow). Microdissection is done using phase-contrast imaging as chromosomes are not counterstained. After chromosome region is dissected, it is transferred into a micropipette containing collection buffer. (AVI 16311 kb)Click here for file


Drosophila melanogaster. As our experimental design also involved the manipulation of the social environment within each full-sibling family, we were able to further test for the presence of genotype-by-environment interactions (GEIs) in these traits, which have the potential to compromise mate choice for genetic benefits. Our experimental manipulation of the social environment revealed plastic expression of both traits; males exposed to a rival male during the sensitive period of adult sexual maturation exhibited shorter mating latencies and longer copulation durations than those who matured in isolation. However, we found no evidence for GEIs, and no significant additive genetic variation underlying these traits in either environment. These results undermine the notion that the evolution of female choice rests on covariance between female preference and male displays, an expectation that underpins indirect benefit models such as the good genes and sexy sons hypotheses. However, our results may also indicate depletion of genetic variance in these traits in the natural population studied, thus supporting the expectation that traits closely aligned with reproductive fitness can exhibit low levels of additive genetic variance.A key assumption underpinning major models of sexual selection is the expectation that male sexual attractiveness is heritable. Surprisingly, however, empirical tests of this assumption are relatively scarce. Here we use a paternal full-sib/half-sib breeding design to examine genetic and environmental variation in male mating latency  and copulation duration in a natural population of  Sexual selection is often thought to favour the evolution of elaborate traits that provide honest signals of  genetic quality ,2. AccorAn important complication when assessing the genetic basis of sexual signals is that their expression can depend on the social and/or physical environment in which they are expressed -16, suchDrosophila [Drosophila species male sexual attractiveness – measured from mating latency – has been shown to be heritable [There is compelling evidence for the implication of indirect selection on female choice in osophila ,23-28, aeritable ,24,29. Heritable -32. A geeritable . NeverthD. melanogaster under varying social conditions. Specifically, we used a paternal full-sib/half-sib breeding design to generate pairs of full-sibling males that were exposed to one of two social environments; males were kept in isolation or in the company of another male during the period of early adulthood in which they reach sexual maturity. Our ensuing quantitative genetic analyses enabled us to test for genetic and environmental sources of variance underlying the expression of these traits, and for the presence of GEIs by assessing the interaction between social context and sire genotype. In this study we determine the level of genetic variation in mating latency and copulation duration in There are no ethical issues for this non-invasive study of an invertebrate species. This species is not an endangered or protected species. Adult flies were collected on private land with the permission of Howard Park and Houghton’s wineries and then returned to the laboratory.Drosophila melanogaster were collected in the Swan Valley region of Western Australia in May 2009 and maintained in the laboratory in mass populations of over 500 individuals with overlapping generations until the start of our experiment in January 2010. Populations were fed on a Drosophila medium containing water, oats, sugar, agar and baker’s yeast, with a sprinkling of dry yeast. Populations were kept on a 12:12hr light: dark cycle at 26°C. To generate the paternal full-sib/half-sib families for the experiment, we collected 100 virgin sires from the stock population and mated each of them to three virgin females at two-day intervals. Virgin flies were collected within 12 hours of eclosing from pupal cases and kept in separate vials for each sex for three days before the first mating. Each dam was removed from her respective laying vial three days after she had mated. From each full-sib family we collected four sons within 12 hours of eclosion and split them into two social environment treatments. These males  were either housed alone, or with an unrelated sexually mature (5-7 days old) ‘rival’ male from the stock population. All focal males were maintained in their respective social treatments for seven days during the sensitive period of adult sexual maturation. We then removed the rival male and transferred each focal male to a fresh vial with a 3-4 day old sexually mature virgin female from the stock population. We presented focal males individually to a single female to avoid potentially confounding effects of direct male-male competition on the traits measured (see below) [Adult e below) . Mating e below) ) and so Our final analysis comprised data from 91 sires, 194 dams and 663 sons . The effects of social environment on genetic variation in male mating behaviour were examined with general linear mixed-effects models using Type III sums of squares in STATISTICA 8.0 . Sire, aA and the coefficient of additive genetic variation, CVA) [P and CVR, respectively) following methods described in [A = √VA / Ẋ. Finally, we calculated standard errors for heritabilities, variance components and evolvability estimates using the jack-knife procedure [To estimate genetic parameters (including heritabilities) of the behavioural traits we conducted separate analyses within each social environment treatment group. For hypothesis testing, we used a mixed-model nested ANOVA with Type III sums of squares. Variance component estimation and subsequent analyses of genetic variation were conducted using restricted maximum-likelihood (REML) in a mixed-model fitted with the lmer function  ,42,43 anribed in , where Crocedure ,44. Males exposed to a rival male during sexual maturity exhibited significantly shorter mating latencies than those who matured in isolation (mean ± SE mating latencies: exposed to a rival male 46.53 ± 2.25 min., N = 341; in isolation = 62.45 ±2.74 min., N = 322; see Drosophila. They reported that increased copulation duration in response to a competitor was a consistent response across the four species studied, but ruled out sperm competition as the selective factor underlying this response as two of the four species had extremely low levels of remating. We found a significant effect of the social environment on both mating latency and copulation duration. Males that were exposed to a rival male during the period between eclosion and sexual maturity were more attractive to females and significantly increased their copulation duration compared to those reared in isolation. This effect was evident despite all males being tested individually in a single male mating arena with a random virgin female. The phenomenon of increased copulation duration in response to a rival male in the rearing or mating arena has been reported across many insect taxa and has been most commonly interpreted as a behavioural response to increased risk of sperm competition ,33,45,46D. melanogaster and its sibling species, D. simulans, has shown that males spend considerable amounts of time engaged in courtship, even to the detriment of female survival [Teleogryllus oceanicus, subdominant males have been shown to compensate for their lower status by up-regulating their investment in short-range cuticular hydrocarbons, which directly increases their mating success [One way in which males might decrease their latency to mating is to increase the effort and time spent engaged in direct courtship behaviour towards females. Previous work in survival -50. Anotsurvival -54. Cons success ,56.Drosophila [Drosophila, mating speed and courtship behaviour can exhibit either low or undetectable heritabilities due to “single event testing”, where the traits in focal individuals are measured just once rather than repeatedly. However, in their meta-analysis of repeatability of animal behaviours, Bell et al. [We partitioned total phenotypic variance into its genetic and environmental components and estimated the heritability and evolvability of mating latency and copulation duration. We found no significant additive genetic variation underlying mating latency or copulation duration in either treatment  and 3. Tosophila ,29. Nevel et al.  report nD. melanogaster, our ensuing estimates of heritability and coefficients of additive genetic variance may reflect both high environmental variance and low additive genetic variance in natural populations compared to their lab-adapted counterparts. Conner et al. [The problem of a potential underestimation of heritabilities may be further compounded when we consider that in order to measure focal male mating latency and copulation duration we used a random sample of females with which to measure these traits. Thus, any genetic or phenotypic variance in female preferences or female condition as well as sampling error surrounding the choice of females may inflate estimates of phenotypic variance in the traits investigated in the focal males, hence leading to an underestimation of heritability (see ,59 for ar et al. , for exa


Aberrant emotional salience attribution has been reported to be an important clinical feature in patients with schizophrenia. Real life stimuli that incorporate both positive and negative emotional traits lead to affective asymmetry such as negativity bias and positivity offset. In this study, we investigated the neural correlates of emotional salience attribution in patients with schizophrenia when affective asymmetry was processed. Fifteen patients with schizophrenia and 14 healthy controls were scanned using functional magnetic resonance imaging (fMRI) while performing an emotion judgment task in which two pictures were juxtaposed. The task consisted of responding to affective asymmetry condition  and affective symmetry conditions (positive and negative), and group comparisons were performed for each condition. Significantly higher activity in the medial prefrontal cortex and inferior frontal gyrus was observed for the ambivalent condition than for the other conditions in controls, but not in patients. Compared with controls, patients showed decreased activities in the dorsolateral prefrontal cortex, dorsal anterior cingulate cortex, insula, and putamen for the ambivalent condition, but no changes were observed for the neutral condition. Multiple prefrontal hypoactivities during salience attribution of negativity bias in schizophrenia may underlie deficits in the integrative processing of emotional information. Regional abnormalities in the salience network may be the basis of defective emotional salience attribution in schizophrenia, which is likely involved in symptom formation and social dysfunction. Salience refers to the state or quality of an item that makes it stand out relative to its neighbors It has been suggested that patients with schizophrenia experience a state of aberrant salience attribution In studying the neural bases of aberrant salience attribution in patients with schizophrenia, several structures are of particular interest. First, the dorsal anterior cingulate cortex (ACC) and insula have been identified as a salience network that functions to recognize the most relevant of several stimuli in order to guide behavior Real life situations are complex and can involve conflicting emotions, such as stimuli incorporating both positive and negative emotions together. When a person evaluates these complex stimuli, one emotion is disproportionately more influential in the holistic appraisal than the other emotion, producing affective asymmetry. The first example is “negativity bias,” which refers to a tendency for the negative system to respond more intensely than the positive system when evaluative input increases A previous neuroimaging study performed by our research group The aim of the current study was to investigate the neural correlates associated with emotional salience attribution in patients with schizophrenia when the positive and negative systems were co-activated with asymmetric manifestations. To explore these functional correlates, we used an emotion judgment task involving two pictures with similar or different emotions during event-related functional magnetic resonance imaging (fMRI). We made the following predictions based on previous findings. First, when two opposite emotions are induced simultaneously, both patients and controls will show a discrepancy in the degree of attention paid to each emotion (positive or negative), resulting in emotional salience attribution. Second, emotional salience attribution that affects the initial perception of stimuli with two opposite emotional traits will be associated with impaired processing of affective asymmetry in patients with schizophrenia. Third, when affective asymmetry is processed, compared with controls, patients will show altered activations in brain regions related to salience attribution, as well as affective asymmetry, such as the DLPFC, VLPFC, ACC, insula, striatum, amygdala, hippocampus, and parahippocampal gyrus.Fifteen patients with schizophrenia (eight men) and 14 normal controls (six men) participated in this study. Exclusive diagnosis of schizophrenia in patients and exclusion of any psychiatric disorders in controls were made using the Structural Clinical Interview for DSM-IV During fMRI, participants performed an emotion judgment task, in which nine positive, nine negative, and nine neutral images  from theMRI data were acquired on a 3T MR scanner . Twenty-eight contiguous 4.5-mm-thick axial slices covering the entire brain were collected using a single-shot, T2*-weighted echo planar imaging sequence depicting the blood-oxygenation-level-dependent (BOLD) signal . Axial 1.5-mm-thick T1-weighted images  were also collected.Spatial preprocessing and statistical analyses were performed using Statistical Parametric Mapping 8 (SPM8). Corrections for differences in slice acquisition time were performed using user-specified sequences. Head motion was corrected by realignment, and corrected images were co-registered to the T1-weighted image for each participant. T1-weighted images were normalized to the standard T1 template, and the resulting transformation matrices were applied to the co-registered functional images. Normalized images were smoothed with an 8-mm full-width-at-half-maximum Gaussian filter.t-tests and chi-square tests. Response rates were analyzed using the Generalized Linear Mixed Model Demographic and clinical data were compared between patients and controls by independent samples t-test on the whole brain volume was performed for screening group comparisons. In these two analyses, education level was used as a covariate, and the threshold was set at an uncorrected p<0.001 with more than 30 contiguous voxels. Then, a two-sample t-test after small volume correction (SVC) with a threshold at a family-wise error-corrected p<0.05 was performed in the a priori regions, which were related to affective asymmetry and salience attribution. The investigated regions and their coordinates  included the DLPFC  http://marsbar.sourceforge.net/). Correlations of regional percent signal changes with negativity bias and positivity offset scores were examined using regression analysis of GLM, in which p-values were adjusted for multiple correlations using a sequential Holm-Bonferroni procedure.Experimental trials for all emotional conditions and null trials were modeled separately for each condition minus the null events using a canonical hemodynamic response function and its first-order temporal derivative. The group and condition effects, as well as their interactions, were analyzed using ANOVA, and a two-sample As shown in Brain regions showing a significant main effect for group and condition are summarized in t-test in each condition are also described in Imaging results of screening two-sample To explore neural representations related to salience attribution during affective asymmetry in schizophrenia, fMRI was performed during the emotion judgment task in patients with schizophrenia and controls. For the ambivalent condition, both groups interpreted the emotional salience of the stimuli as a negative trait, which could be interpreted as ‘negativity bias.’ This result differed from our assumption that patients with schizophrenia would show a reduction in negativity bias, which we demonstrated in our behavioral study Despite the absence of significant behavioral differences in the ambivalent condition, remarkable group differences in the salience processing triggered by ambivalent stimuli were revealed in the medial prefrontal cortex and inferior frontal gyrus, which exhibited an interaction effect between group and condition. Significantly higher activity in the ambivalent condition than the other conditions was found in controls, but not in patients. The medial prefrontal cortex and inferior frontal gyrus have been proposed to be involved in emotional regulation The inferior frontal gyrus receives projections from the orbitofrontal cortex and subcortical areas such as the midbrain and amygdala, which are involved in processing motivational and emotional information The dorsal ACC, insula, and putamen were hypoactive in patients, relative to controls, for the ambivalent condition. These three regions are part of the salience network, which functions to identify the most relevant of several internal and extrapersonal stimuli to guide appropriate behavior Although both groups showed negativity bias in the current design, we were able to confirm our hypothesis of abnormally decreased activity in the affective asymmetry and salience related regions in patients. Compared with controls, patients showed similar negative responses in the ambivalent condition, but decreased negative or positive responses in the univalent conditions, suggesting that an underlying mechanism of negativity bias may be different between the two groups. There is evidence that patients with schizophrenia use a different prefrontal network and strategies in the executive control process for successfully performing a working memory task Meanwhile, patients had significantly lower nPnN response rates than controls for the neutral condition, suggesting that they have a tendency to impart emotional salience to neutral stimuli. While other emotional stimuli have characteristics of primary inducers that automatically and obligatorily elicit emotional responses, neutral stimuli have characteristics of secondary inducers, which are related to “thought,” “memories”, or “imagination” Several limitations of this study should be noted. First, the sample size was relatively small, which might explain why the expected correlations between regional activities and clinical measures were not found. Second, patients were all taking antipsychotic medication. Although the effects of antipsychotics on emotional responses are known to be negligible In summary, emotional salience attribution during the processing of negativity bias in patients with schizophrenia was associated with multiple prefrontal hypoactivities in the medial prefrontal cortex, inferior frontal gyrus, and DLPFC, which might be related to a deficit in integration of positivity and negativity. The salience network regions, such as the dorsal ACC, insula, and putamen also showed altered activities during the processing of negativity bias in patients, suggesting abnormal emotional salience attribution in schizophrenia. The neural basis of emotional salience attribution during the processing of positivity offset in schizophrenia was not clarified in this experiment in spite of definite behavioral evidence. These regional abnormalities may underlie defective emotional salience attribution in patients with schizophrenia, which likely influences both symptom formation and social dysfunction.Table S1Images from the International Affective Picture System used in the emotion judgment task.(DOCX)Click here for additional data file.Table S2Group comparison of reaction times.(XLSX)Click here for additional data file.Table S3Raw data of the response rates and reaction times for the emotion judgment task in each participant.(DOCX)Click here for additional data file.Table S4Brain regions showing significant main and interaction effects.(DOCX)Click here for additional data file.Table S5Decreased brain activation across the whole brain in patients with schizophrenia compared with controls (two-sample t-test).(DOCX)Click here for additional data file.Table S6Increased brain activation across whole brain in patients with schizophrenia compared with controls (two-sample t-test).(DOCX)Click here for additional data file.Table S7t-test after small volume correction.Percent signal changes in brain regions showing a significant group difference in two-sample (XLSX)Click here for additional data file.


Lychas mucronatus is one scorpion species widely distributed in Southeast Asia and southern China. Anything is hardly known about its venom components, despite the fact that it can often cause human accidents. In this work, we performed a venomous gland transcriptome analysis by constructing and screening the venom gland cDNA library of the scorpion Lychas mucronatus from Yunnan province and compared it with the previous results of Hainan-sourced Lychas mucronatus.Lychas mucronatus, which greatly increase the number of currently reported scorpion venom peptides. Interestingly, we also identified nineteen atypical types of venom molecules seldom reported in scorpion species. Surprisingly, the comparative transcriptome analysis of Yunnan-sourced Lychas mucronatus and Hainan-sourced Lychas mucronatus indicated that enormous diversity and vastly abundant difference could be found in venom peptides and proteins between populations of the scorpion Lychas mucronatus from different geographical regions.A total of sixteen known types of venom peptides and proteins are obtained from the venom gland cDNA library of Yunnan-sourced Lychas mucronatus from different geographical regions, which thoroughly reveals the fact that the venom peptides and proteins of the same scorpion species from different geographical regions are highly diversified and scorpion evolves to adapt a new environment by altering the primary structure and abundance of venom peptides and proteins.This work characterizes a large number of venom molecules never identified in scorpion species. This result provides a comparative analysis of venom transcriptomes of the scorpion  More than 400 million years of evolution does not make the scorpions alter their morphology. However, although this one of the oldest arachnid is highly conserved in shape all along, they occupy vast territory of the world from Africa to Asia, Australia and America. Such powerful adaptability mostly owes to their highly specialized venom apparatus which consists of the vesicle holding a pair of venom glands connected to the stinger used to inject the venom. The extIt is acknowledged that the Buthidae family possesses a very different venom arsenal comparing to other non-Buthidae families. MoreoveAndroctonus[Buthus[Mesobuthus[Parabuthus[Centruroides[Tityus[Lychas mucronatus belonging to the genus Lychas of the family Buthidae is one of these scorpion species lacking of concern. Lychas mucronatus is widely distributed in Southeast Asia and southern China including Hainan island[Lychas mucronatus from Hainan province. We performed its venom transcriptome analysis, and some new toxins were identified[Lychas mucronatus from Yunnan province  Generally, Lychas mucronatus is 40-65 mm long, carapace, mesosomal tergites and legs are yellow and blotched, metasoma segments are yellow to yellowish-brown from base to terminal, pedipalp, patella is predominantly dark, pedipalps manus is bright yellow with sparse, minute black spots and fingers are dark yellow brown; (2) Pedipalp fingers of males are curved, whereas those of female are straight; (3) Second segment of metasoma has 10 keels, and the third metasomal segment has 8 keels; (4) Sixth cutting edge on movable fingers of pedipalps has 3 granules; (5) Pectinal teeth number is 16-26 (frequently 19-23); (6) Metasoma of males has the same length as that of females. According to microscopic observation, both scorpion populations we collected possess the same characters mentioned above. So we convinced that two scorpion populations obtained from two provinces actually belong to the same species Lychas mucronatus.s Figure . Based oLychas mucronatus cDNA library was constructed with the same methods as Hainan-sourced Lychas mucronatus[Lychas mucronatus cDNA library possessed a 2.6 × 106 cfu/ml titer with more than 99% recombinant efficiency. In order to get more types of peptides and proteins, we selected 1000 random colonies for sequencing which have a total of 738 readable sequences. These EST sequences were then submitted into the dbEST (accession numbers: GT028570-GT029307). After processed by EGassembler online bioinformatics service[The Yunnan-sourced ucronatus. Both ofLychas mucronatus cDNA library can be got from our submitted work "Evolution of the scorpion venom arsenal inferred by comparative transcriptome and molecular phylogeny analyses" (unpublished data). The distribution of all ESTs in both populations was depicted in Figure We compared the consensual cluster sequences against SWISS-PROT and GenBank NCBI databases by BLAST algorithms for a functional classification of these unique sequences. 189 clusters 488 ESTs) showed homology to existent peptides or proteins , whereas the other 191 clusters (250 ESTs) hadn't good matches , accounting for approximately 46% of the total venom gland transcripts. All these sequences possess signal peptides. Most of these types have been identified at the protein level, whereas a few of them are only found at the transcript level. The particular comparison of these known toxin types from Yunnan Lychas mucronatus and previously identified known toxin types from Hainan Lychas mucronatus will be detailed in subjects below . This peptide shows homology to the beta-toxin Toxin CsEv1 identified from Centruroides sculpturatus. This homology may indicate that the most abundant 'long-chain' neurotoxin in the venom of Yunnan-sourced Lychas mucronatus can affect sodium channel activation by binding voltage-independently at site-4 of sodium channels. Although the most abundant NaTx transcripts (EU159276 and EU159292) from Hainan-sourced library have the same cysteine pattern as GT028621, obvious diversity can be observed in primary structures. Specially, GT028621 don't contain a basic amino acid residue at C-terminal region, which shows that C-terminal modification would not exist at the post-translational level.One cluster of putative β-NaTxs (GT028621 and GT028626) encoded by 16 transcripts is the most abundant NaTx cluster in Yunnan-sourced population library. This cluster contains two almost identical sequences differing by only two amino acids. The transcript GT028621 encodes a mature peptide of 63 amino acid residues with 8 cysteines which would form a disulfide bridges pattern of "C-C-CXLychas mucronatus, the NaTxs were distinct in primary structures  encoded for α-KTx. Among them, 9 clusters of putative α-KTxs are constrained by 3 disulfide bridges, and the other 2 clusters are packed with four disulfide bridges . They also represent the most abundant cluster of β-KTx in the venom of Yunnan-sourced Lychas mucronatus. Based on similar analysis as described before[There are two other neurotoxin types identified from Yunnan-sourced al region. We obtas Figure . The pried before,38, GT022+ channel toxins have been obtained from scorpion venoms[Lychas mucronatus. GT029194 has an 8 amino acid residues long pro-peptide and a mature peptide of 34 amino acid residues constrained by three disulfide bridges. FE193690 from Hainan-sourced population has the same primary structure as GT029194, which suggests that the calcine is conserved between two scorpion populations , Ponericin-like antimicrobial peptides (Plamp), bradykinin-potentiating peptide like (bpp like), Glycine-rich peptides and Anionic peptides.In Yunnan-sourced Mesobuthus martensii[Lychas mucronatus, which can effectively inhibit standard bacteria, especially gram-positive bacteria. From Yunnan-sourced Lychas mucronatus, we obtained 2 clusters (26 ESTs) of Scamp. Among them, GT028857 shared the same primary structure as mucroporin. Actually, both two clusters of Scamp obtained from Yunnan-sourced Lychas mucronatus have homologous transcripts in Hainan-sourced population venom  encoded for Plamp, which was a homologous toxin type of Ponericins identified from ant venom with a high activeness against Gram-positive and Gram-negative bacteria[From Yunnan-sourced  bacteria. Plamp hclusters  ESTs encMesobuthus martensii[Lychas mucronatus, we obtained 1 clusters (24 ESTs) encoded for this toxin type, represented by GT028786. GT028786 has almost identical primary structure like EU163895 from Hainan-sourced population , phospholipase A2 (PLA2), serine protease from scorpion venom (SPSV), metalloprotease, salivary protein, La1-like peptides and 8C toxin. LVPs are a group of long chain toxins shared high homology to those NaTxs but in defect of a cysteine. This structure makes LVPs form an interchain disulfide bridge and exert distinct biological activity on adipocyte lipolysis. 7 clustd Figure . PLA2, Stralasiae. But theTityus serrulatus venom[Lychas mucronatus venom because it's not affected by the extraction of venom and the dynamic expression of the gland or peptide maturation like proteomic approach[Lychas mucronatus populations.Scorpion venom is a complex mixture of biologically active peptides with diverse physiological effects. Most oftus venom. Since ttus venom. The powtus venom. We chos approach. The traScorpio maurus palmatus from four geographically isolated localities in Egypt was investigated. Both obvious morphological differences and protein molecular weights diversity were observed[Lychas mucronatus successively obtained from Hainan and Yunnan provinces were convinced to be the same species according to morphological analysis, our study conduct on the venom components of Lychas mucronatus by a transcriptome approach may fitly figure out the question.Some evidences already displayed the phenomenon of intraspecific diversity of scorpion venom peptides,26. Acco observed. But theLychas mucronatus was the same as Hainan-sourced Lychas mucronatus[The experimental methods for constructing the venom gland cDNA library of Yunnan-sourced ucronatus. 60 adulLychas mucronatus possesses a higher abundant and more diversified α-KTxs in venom and the homologous transcripts of the most abundant α-KTx cluster in the venom of Hainan-sourced Lychas mucronatus have a relatively low expressed level in the venom of Yunnan-sourced Lychas mucronatus. All these obvious diversity should be intricately related to scorpions' adaption in different environment which primarily concern the scorpions' interaction with their prey and predators. On the other hand, Hainan-sourced Lychas mucronatus possesses a higher abundance of NaTxs in venom. Because the NaTxs can cause severe neurotoxic symptoms after scorpion envenomation[Lychas mucronatus could be a more dangerous killer, which is more efficient for not only preying but also defensing itself from other predators.Almost all high-abundant toxin types were obtained from two scorpion populations. Great diversity was observed in these highly expressed venom compositions, especially neurotoxins and AMPs. Among neurotoxins, short chain α-KTx was highly expressed in Yunnan-sourced population, whereas long chain NaTx and β-KTx was highly expressed in Hainan-sourced population. Particularly, α-KTx in the venom of Yunnan-sourced population is not only 2-fold higher abundant than in Hainan-sourced population, but also more diversified in primary structures. GT028663 represents the most abundant α-KTx cluster in Yunnan-sourced population, but we can not find its homologous transcripts in the venom gland cDNA library of Hainan-sourced population. EU163848 represents the most abundant α-KTx cluster from Hainan-sourced population. Although EU163848 shows homology to GT028601 from Yunnan-sourced population, EU163848 obviously has a higher expression level. Taken together, Yunnan-sourced enomation, Hainan-Lychas mucronatus away from microbial infection, and it may also implicate other unascertained benefits for the survival of the population.According to the transcriptome analysis, we can conclude that both scorpion populations possess relatively high abundant AMPs. But it's unexpected that the abundance of AMPs in the venom of Hainan-sourced population reached remarkable 40% of all toxin-like peptides. Till now, more and more AMPs have been isolated and characterized,57. AMPsLychas mucronatus. In this biotope, Hainan-sourced Lychas mucronatus has enough prey. But this hot and humid environment may also bring powerful nature enemy and more pathogenicbacteria. In a word, the distinctive living environments probably relate to the adaptive evolution of two Lychas mucronatus populations.Hainan island locates in Southern China and is separated from mainland by Qiongzhou strait. Because surrounded by South China Sea, the climate of Hainan island is hot and humid all the year round. Yunnan locates in southwest China and is a mountainous region, whose rainfall is relatively less than Hainan province. Hainan island is an excellent living environment for Lychas mucronatus change their toxin types much in venom, but the primary structure and abundance of these toxin types change a lot which probably related to the adaption of Lychas mucronatus in different environment. To the best of our knowledge, this work is the first comprehensive comparative analysis of venom transcriptomes between two populations of the same scorpion species from different geographical regions. It provides accurate difference of venom peptides between Lychas mucronatus from Hainan and Yunnan. Sight is no longer restricted to only protein molecular weights and concentration, but also the detailed diversity of primary structure and abundance. Besides, because two scorpion populations are obtained successively from island and mainland, Lychas mucronatus could represents a useful model system for studying molecular evolution and biogeography. In addition, the geographical variability in the venom of the same species also provides some clues for producing of effective anti-venoms and understanding the symptomatology of envenomation.The dissimilarity of toxin-like peptides between two populations was showed in Figure Our work unravels a large number of venom molecules never identified in scorpion species. The result provides a comprehensive comparative analysis of venom transcriptomes of the same scorpion species from different geographical regions, which thoroughly reveals the fact that peptides and proteins of the same scorpion species from different geographical regions are highly diversified and the scorpion venom arsenal is a constantly evolving system to adapt the different biotopes. We can make a conclusion that there are far more peptides in one scorpion species venom than previous expectation considering of the geographical isolation.Lychas mucronatus were collected from Shidian county of Yunnan province in September 2008. Venom glands of 60 wild specimens were cut off 2 days after extraction of their venom by electrical stimulation[® mRNA Isolation Systems . SuperScript™ Plasmid System (Invitrogen) was used to construct a directional cDNA library from 6 μg mRNA. cDNA inserts were directionally cloned into the plasmids pSPORT 1 according to the supplier's instructions. The recombinant plasmids were transformed into electrocompenent cells.The Yunnan-sourced After growing the clones overnight in appropriate Luria Broth culture medium containing 100 μg/ml of ampicillin, random colonies were selected in order to obtain an unbiased overview of the venom gland transcriptome. After overnight culture, plasmid DNA was isolated using alkaline lysis method. Purified plasmids were single-pass sequenced on ABI 3730 automated sequencers .http://egassembler.hgc.jp/ with the default parameter[http://www.expasy.org/tools/blast/ and GenBank NCBI database http://www.ncbi.nlm.nih.gov/blast using BLAST algorithms with an e-value cut-off set to <10-4. After BLAST search, the unmatched clusters were further identified for open reading frames using the ORFfinder http://www.ncbi.nlm.nih.gov/projects/gorf/. Considering the extreme diversity of scorpion toxins, those clusters putative to encode venom peptides was re-examined manually to pick out individual different isoforms. All clusters were checked for the existence of signal peptides using the SignalP 3.0 program http://www.cbs.dtu.dk/services/SignalP/[Phred program were used to examine the trace files of sequenced clones, the cutoff Phred score was set to 40 as before,61. Vectparameter. The res/SignalP/. All the/SignalP/, and vie/SignalP/.Ruiming Zhao carried out cDNA library construction, participated in the bioinformatics analysis, and drafted the manuscript. Yibao Ma participated in the alignment analysis, and drafted the manuscript. Yawen He participated in the sequencing. Zhiyong Di completed the characterization of scorpion species. Yingliang Wu participated in the design and coordination of the study. Zhijian Cao participated in the design and coordination of the study, and drafted the manuscript. Wenxin Li conceived of the study, and participated in its coordination. All authors have read and approved the final manuscript.Lychas mucronatusAtypical venom molecules characterized from the scorpion Yunnan-sourced . The data represents nineteen novel types of venom peptides with special primary structures and unrevealed functions from the scorpion Yunnan-sourced Lychas mucronatus.Click here for file


The sea lamprey is an important model organism for investigating the evolutionary origins of vertebrates. As more vertebrate genome sequences are obtained, evolutionary developmental biologists are becoming increasingly able to identify putative gene regulatory elements across the breadth of the vertebrate taxa. The identification of these regions makes it possible to address how changes at the genomic level have led to changes in developmental gene regulatory networks and ultimately to the evolution of morphological diversity. Comparative genomics approaches using sea lamprey have already predicted a number of such regulatory elements in the lamprey genome. Functional characterisation of these sequences and other similar elements requires efficient reporter assays in lamprey. In this report, we describe the development of a transient transgenesis method for lamprey embryos. Focusing on conserved non-coding elements (CNEs), we use this method to investigate their functional conservation across the vertebrate subphylum. We find instances of both functional conservation and lineage-specific functional evolution of CNEs across vertebrates, emphasising the utility of functionally testing homologous CNEs in their host species. Petromyzon marinus, is a member of the jawless fish lineage (agnathans), the only extant vertebrate sister group to the jawed vertebrates (gnathostomes) in-situ hybridisation, morpholino-mediated gene knockdown and cell labelling The sea lamprey, cis-regulatory elements in early vertebrates are likely to have contributed significantly to the evolution of the vertebrate body plan. Whilst embryonic gene expression patterns can be informative as to the developmental changes underlying morphological evolution, the changes at the cis-regulatory level that are responsible for divergent gene expression have received less attention. There are a handful of studies where early vertebrate cis-regulatory evolution has been addressed through testing amphioxus or lamprey genomic sequences for enhancer activity in gnathostomes cis- and trans- regulatory state during vertebrate evolution, it is important also to test such elements in their host species cis-regulatory elements are uncovered The emergence and elaboration of cis-regulatory elements that were present in the last common ancestor of all vertebrates cis-regulatory elements (e.g. cis-regulatory elements across the vertebrate sub-phylum.Shared conserved non-coding elements (CNEs), have been identified through comparisons between lamprey and gnathostome genomic sequences, representing a collection of putative nts e.g. . Thus, let al. Lampetra japonica. In their study, circular plasmid constructs containing the GFP coding sequence downstream of either a viral promoter or upstream regulatory regions of medaka actin genes were injected into fertilised eggs before the first cleavage, resulting in mosaic GFP expression. Interestingly, muscle-specific actin promoters from medaka were able to drive GFP expression in the developing muscle of lamprey embryos. Whilst the long lamprey life-cycle impinges upon the likelihood of generating transgenic lines, this study illustrated the feasibility of assaying CNEs for enhancer activity in lamprey.Kusukabi In a study focusing on putative Hox-regulated enhancers conserved between jawed vertebrates and lamprey, we presented reporter expression patterns driven by two lamprey elements in lamprey embryos, verifying that ancient CNEs have broad functional conservation across vertebrates et al. CNEs were amplified from genomic DNA by PCR , 2 μl 10X I-SceI buffer, 0.2 μl 100X BSA and 400 ng of reporter plasmid , were incubated at 37°C for 40 minutes prior to being immediately injected into lamprey embryos during their first cell division (drop volume approximately 2–3 nl).For linearised plasmid injection, the pm3285_cfos_EGFP plasmid, consisting of the lamprey homolog of CNE 3285 cloned into the pGW_cfosEGFP vector, was linearised with KpnI (NEB), purified with a Qiagen PCR purification kit and eluted in distilled water. Lamprey embryos were obtained as described previously meis1/2 a and b probes performed according to published protocols meis2 locus, to drive reporter expression in lamprey embryos. These CNEs are highly conserved between jawed vertebrates and lamprey meis2 in zebrafish, which is broadly expressed in the central nervous system, particularly in the hindbrain We sought to develop a functional assay to test the ability of lamprey homologs of two CNEs, pm3285 and pm3299, associated with the c-Fos minimal promoter, which we used in the zebrafish assay, by injecting the pm3285_cfos_EGFP plasmid into lamprey embryos. The plasmid was linearised to increase the chance of genomic integration. Injection of this construct during the first cleavage resulted in a high death rate immediately post-injection, as well as during gastrulation, such that the frequency of injected embryos surviving through gastrulation was very low (−1) produced surviving embryos but none were expressing GFP. In a proportion of the survivors from the 100 ngµl−1 injections, GFP was expressed in a mosaic manner in the cranial ganglia and neurons of the spinal cord . Rather, the continued association of the enzyme with the digested construct may prevent its degradation or concatamerisation, thus increasing the probability of genomic integration In an effort to reduce mosaicism and increase embryo survival rate, we applied the I-SceI meganuclease-mediated transgenesis method to lamprey embryos. We reasoned that by increasing the probability of early genomic integration of the injected construct, the amount of DNA injected could also be lowered, lessening the toxic effect of exogenous DNA whilst decreasing mosaicism. This method utilises the rare-cutting I-SceI meganuclease and requires a construct in which the DNA fragment to be integrated is flanked by I-SceI recognition sites. The construct is digested with the meganuclease enzyme When pm3285 was tested using the meganuclease assay, the embryo survival was considerably higher than with linearised plasmid  and 35 embryos showed expression in the hindbrain or spinal cord, with 10 of these 35 also expressing GFP in the cranial ganglia . Repeat et al. (2003) The transient transgenic embryos obtained through the meganuclease approach display a range of mosaicism with respect to GFP expression in neurons. To compare mosaicism between the two transgenesis approaches, we categorised transient transgenics into two groups depending on whether they had more than or less than 50 GFP-positive neurons at stage 24. For the linearised plasmid injection, 0/5 embryos with neuronal GFP expression exhibited this expression in more than 50 neurons. In contrast, for the meganuclease approach, 9/35 embryos with neuronal expression had more than 50 neurons expressing GFP  and lamprey (pm) CNEs when tested in zebrafish and lamprey embryos. As the same CNE sequences were used in each assay, the expression patterns driven by them are directly comparable meis genes Click here for additional data file.Figure S2Multiple sequence alignment of CNE 3299 from vertebrate genomes. Primers used for amplification are highlighted in red.(PDF)Click here for additional data file.Figure S3Comparison of mosaicism from linearised plasmid and I-SceI meganuclease transgenisis approaches. Examples of stage 24–25 transient transgenic embryos obtained through each approach using the pm3285 enhancer are shown.(TIFF)Click here for additional data file.Table S1Minimal promoters tested in the lamprey reporter assay.(DOC)Click here for additional data file.


A typical Go/No-Go decision is thought to be implemented via the activation of direct and indirect pathways in the basal ganglia. Indeed, optogenetic activation of the direct pathway increased ambulation, whereas that of the indirect pathway induced freezing . StriataSpecifically, we show that: (i) Because D1 MSNs receive higher recurrent inhibition from FSIs  and D2 MThese observations help us to explain several experimental and behavioral findings involving the basal ganglia. The model suggest that under dopamine depletion conditions, even for weak cortical inputs, D2 MSNs activity is higher than D1 MSNs, which is consistent with the fact that Parkinson's disease (PD) patients have difficulty in initiating voluntary actions. We also observed that dopamine depletion reduced the parameter regime supporting D1 MSNs activation, by shifting the decision threshold towards lower cortical inputs. This suggests that under dopamine-depleted conditions, the striatum would require arbitration by the STN-GPe network, even for a low conflict task, providing a plausible explanation of increased reaction times in PD patients. Finally, increased activity in GPe under the influence of deep-brain stimulation (DBS) could also reduce the activity of FSIs on an average, thus shifting the decision threshold towards higher cortical input rates. Taken together, the model provides a mechanistic explanation of impulsive behavior in PD patients with DBS.


These responses after secondary exposures caused bacterial pneumonia and most deaths. Of the unexplained characteristics of the 1918–19 influenza pandemic, the extreme mortality rate among young adults  is the foremost. Lack of a coherent explanation of this and other epidemiologic and clinical manifestations of the pandemic contributes to uncertainty in preparing for future pandemics. Contemporaneous records suggest that immunopathologic responses were a critical determinant of the high mortality rate among young adults and other high-risk subgroups. Historical records and findings from laboratory animal studies suggest that persons who were exposed to influenza once before 1918  were likely to have dysregulated, pathologic cellular immune responses to infections with the A/H1N1 1918 pandemic strain. The immunopathologic effects transiently increased susceptibility to ultimately lethal secondary bacterial pneumonia. The extreme mortality rate associated with the 1918–19 pandemic is unlikely to recur naturally. However, T-cell–mediated immunopathologic effects should be carefully monitored in developing and using universal influenza vaccines. The influenza pandemic of 1918–19 was the most deadly single event in recorded history. Because of its unique severity and global effects, it is the prototype of a global natural disaster. In recent years, fears of recurrence of an influenza pandemic similar to that in 1918 have motivated planning, preparations, and allocations of resources by public health and other government agencies, nongovernmental organizations, medical care providers, pharmaceutical and medical device manufacturers, medical researchers, private businesses, and persons worldwide . During the 1918 pandemic, influenza infection rates were similar to those during other pandemics of the last century; and in most affected populations, overall mortality rates were <1%, and case-fatality rates were <3% . Th,In 1889–90, pandemic influenza (Russian flu) spread rapidly throughout the world, and from 1890 through the winter of 1900–01, widespread epidemics of influenza-like illness recurred  caused widespread illness but few deaths (3-day fever). The second wave  caused widespread illness and high mortality rates  viruses, persons affected during the first wave should have been protected from infection and, in turn, illness, secondary pneumonia, and death during the second wave. Protection from infection would have derived from neutralizing antibodies against the same or similar viral surface antigens .,There are conflicting reports regarding the immunologic susceptibility to infection during the second wave among persons who were infected during the first wave. Several reports of the experiences of localized groups  suggest that illness during the first wave protected from influenza during the second wave  pandemic strain in clinics, in ambulances, and on crowded open wards. However, during the lethal second wave, nurses and medical officers of the Australian Army had influenza-related illness rates similar to, but mortality rates lower than, any other occupational group  and Nauru (16%) (The 1918 influenza A (H1N1) pandemic strain is genetically similar to the novel pandemic (H1N1) 2009 strain. However, clinical expressions of infections in 2009 were much less severe than in 1918, e.g., mortality rates in 1918 were >100× higher than in 2009 . When persons were reexposed to the identical epitope in 1918, epitope-specific memory CD8+ T-cells produced excessive cytokines, chemokines, immune cell activation, and epithelial cell necrosis. The immunopathologic effects of the dysregulated T-cell response transiently increased susceptibility of infected hosts to respiratory bacterial strains against which they lacked protective antibodies.In contrast, persons who were first exposed in 1918 to the immunodominant T-cell epitope of hypothesized concern may have had primary T-cell responses that controlled virus replication without increasing susceptibility to bacterial invasion of the lower respiratory tract. Persons who had multiple prior exposures to influenza viruses and other respiratory infectious agents before 1918 had diversely partitioned memory CD8+ T-cell repertoires and extensive portfolios of bacterial strain–specific antibodies. Their immune responses to infection with the 1918 pandemic strain may have controlled virus replication without increasing their susceptibility to bacterial invasion.,,Modern genetic analyses have estimated that 3 distinct variants of influenza A (H1N1) viruses co-circulated in the early 1900s   remains a unique and unexplained characteristic of the 1918 pandemic (Persons born before 1901 (the last year of widespread circulation of the 1890 Russian flu pandemic strain) and after 1875 (the first year after widespread epidemics of a poorly characterized influenza-like illness) were 18–43 years of age in 1918. Worldwide, members of these birth cohorts had high influenza attack rates and were likely to die from secondary bacterial pneumonia during the 1918 pandemic period .Persons born before 1875 were >43 years of age in 1918. Before the 1918 pandemic period, they likely had been exposed to more heterosubtypes of influenza A and more respiratory bacterial strains than their younger counterparts. In general, during the pandemic period, middle-age and elderly adults had lower influenza attack rates and were less likely to die than their younger counterparts .Also in 1918, persons born after 1901 were less likely than those older to have been exposed to the 1890 pandemic strain or displaced by war-related activities. During the pandemic period, persons <17 years of age had relatively high influenza attack rates but relatively low mortality rates (except for infants) .We hypothesize that, soon after infection with the 1918 pandemic influenza strain, infected persons experienced a transient increase in susceptibility of the lower respiratory tract to invasion by bacteria to which they were immunologically naive. Thus, for example, those who were relatively new to their living or work environments  at the time they were infected with the 1918 pandemic strain had a relatively high risk of death from secondary bacterial pneumonia , followed by infection with pandemic (H1N1) 2009 virus, elicited severe immunopathogenic responses  strain depended on the number, nature, and diversity of prior infections with influenza virus and respiratory bacteria. Specifically, mortality rates during the lethal second wave were highest among persons with prior exposures to heterosubtypic influenza strains that enhanced immunopathogenic effects when a person was infected with the 1918 pandemic strain and had limited exposures to other respiratory infectious agents. In such persons, infection with the pandemic strain caused high viral loads, dysregulated and pathogenic cell mediated immune responses, and transient increases in susceptibility to invasive bacterial infections. If such influenza virus–infected hosts were subsequently exposed to bacterial strains to which they had no protective antibodies, they were at high risk of acquiring life-threatening secondary bacterial pneumonia.The unique circumstances that enabled the unprecedented mortality rates of the 1918 pandemic no longer exist on a global scale. For example, in modern times, even the most isolated communities  are interconnected through myriad commercial and sociopolitical activities. As a result, most populations are exposed to annual seasonal influenza viruses, and most young adults are exposed to numerous viral and bacterial respiratory pathogens. Thus, compared with the situation in 1918, adults in modern communities have more diversified immune repertoires against influenza strains and bacterial respiratory pathogens. The hypotheses presented may explain at least in part the relatively low mortality rate associated with pandemic (H1N1) 2009 virus. During the 2009 pandemic, many persons who died had underlying medical conditions, including obesity, asthma, cardiovascular diseases, diabetes, and pregnancy; histopathologic changes consistent with diffuse alveolar damage; and evidence of bacterial co-infections (Finally, the findings of this report are relevant to the research and development of a universal influenza vaccine. Candidate vaccines that contain antigens that are highly conserved across influenza A strains and strongly immunogenic must be closely monitored to ensure that T-cell–mediated immune responses to future seasonal and pandemic strains are protective but not pathogenic.


We report a case of probable contrast-induced neurotoxicity that followed a technically challenging cardiac catheterization in a 69-year-old woman. The procedure had involved the administration of a large cumulative dose of an iodinated, nonionic contrast medium into the innominate artery: twelve hours following the catheterization, the patient developed a seizure followed by a left hemiplegia, and an initial computed tomography (CT) scan showed sulcal effacement in the right cerebral hemisphere due to cerebral swelling. The patient's clinical symptoms resolved within 24 hours, and magnetic resonance imaging at 32 hours showed resolution of swelling. Contrast-induced neurotoxicity should be found in the differential diagnosis of acute neurological deficits occurring after radiological procedures involving iodinated contrast media, whether ionic or nonionic. The hazards associated with the contrast agents used for cardiac and cerebral angiography are well known. Aside from allergic reactions and renal toxicity, relatively common neurological complications are vasospasm related to the catheterization, intimal lesions, pseudoaneurysms, and embolism . An uncoCIN has been associated with the iodinated contrast media used for intravascular cerebral and cardiac angiography and for intrathecal injections . The mecThe clinical presentation of CIN ranges from headache and vomiting to seizures, dysarthria, hemiparesis, hemianopia, parkinsonism, transient global amnesia, and transient cortical blindness , 7. CIN 2.A 69-year-old right-handed woman underwent elective cardiac catheterization because of an abnormal stress test. She had previously undergone coronary artery bypass grafting and also had hypertension, diabetes mellitus, and renal impairment, with an estimated glomerular filtration rate of 49 mL/min/1.73 mThe cardiac catheterization was performed via a right common femoral artery approach, and injections were made into the left ventricle, the native vessels, and the venous graft—an innominate artery to right coronary artery greater saphenous vein graft. A stent was placed into the venous graft. The procedure was technically difficult and involved repeated injections into the innominate artery. A total of 320 mL of Iodixanol 320 mgI/mL, a dimeric nonionic contrast medium, was administered. The patient was observed to be clinically normal after the procedure. No general anesthetic had been administered during the procedure.Twelve hours later, the patient developed a left-sided motor seizure that became secondarily generalized. The seizure was terminated with lorazepam. Postictally, there was left-sided homonymous hemianopia, hemisensory loss, hemiparesis, and hemineglect. The patient was thought to have suffered an ischemic stroke involving the right middle cerebral artery territory. Unenhanced CT scanning performed within 1 hour showed sulcal effacement in the right cerebral hemisphere due to cerebral swelling; the changes were most marked in the high frontal and parietal lobes Figures . There wAn MRI at 32 hours from patient's symptoms showed no edema and no swelling or cortical enhancement on the postcontrast T1-weighted images (T1WIs) or on the fluid-attenuated inversion recovery T2-weighted images (FLAIR-T2) Figures . Two 1 mIt was subsequently determined that one year earlier the patient had experienced transient binocular blurred vision after a cardiac catheterization procedure at an outside hospital. At that time, an unenhanced computed tomography (CT) scan had shown questionable occipital lobe hypodensities that resolved within 24 hours.The differential diagnoses in this patient included contrast-induced neurotoxicity (CIN), stroke, seizure-induced brain swelling, and subarachnoid hemorrhage. The acute neurological deficits, the initial neuroimaging changes in the symptomatic hemisphere and the correlation of the resolution of the symptoms with the complete resolution of the CT changes made CIN the most likely diagnosis in this patient. The patient had also received a very high dose of iodinated contrast (320 ml of 320 mgI/mL Iodixanol)—the maximal recommended dose being 200 mL—into the symptomatic hemisphere.The initial clinical diagnosis was of a seizure secondary to acute right hemispheric ischemic stroke in the setting of a technically difficult cardiac catheterization involving the innominate artery. However, the involvement of the whole hemisphere with no definite vascular territory should exclude this diagnosis. Typically, in hyperacute stroke, the brain CT scan is normal or may show subtle areas of hypodensity and/or corticosubcortical blurring that are indicative of early infarction. There may also be a hyperdense middle cerebral artery sign indicative of thrombosis. The rapid improvement of the patient's symptoms also was atypical. The punctate small infarctions on MRI could not explain her significant transient neurological deficits. Another differential diagnosis was that of a first-onset of idiopathic seizure followed by a Todd's paralysis. However, the extent of swelling was too large for seizure-induced brain swelling, and there was a lack of corresponding changes on the MRI . SubaracCIN resulting from Iodixanol administration is extremely rare, and this is the second case reported to our knowledge . IodixanIt is important to keep in mind that various radiographic findings have been associated with CIN . InitialThe prognosis of CIN is excellent. Although there is no specific treatment for this condition, steroids and mannitol have been administrated in some cases . SeizureIn conclusion, CIN is a rare condition that should be taken into account in the differential diagnoses of acute focal or generalized neurological symptoms after procedures involving the use of intracarotid injection of iodinated contrast media, whether ionic or nonionic. Its recognition may be crucial for patient management. This syndrome can be differentiated from stroke on the basis of clinical and imaging findings.


Escherichia coli homologs onto metabolic and protein-protein interaction networks. These maps identified bacterial processes with components that were well-represented in the datasets. In summary this study highlights the potential of exploiting the economy of HTS platforms for metatranscriptomics.With the advent of high through-put sequencing (HTS), the emerging science of metagenomics is transforming our understanding of the relationships of microbial communities with their environments. While metagenomics aims to catalogue the genes present in a sample through assessing which genes are actively expressed, metatranscriptomics can provide a mechanistic understanding of community inter-relationships. To achieve these goals, several challenges need to be addressed from sample preparation to sequence processing, statistical analysis and functional annotation. Here we use an inbred non-obese diabetic (NOD) mouse model in which germ-free animals were colonized with a defined mixture of eight commensal bacteria, to explore methods of RNA extraction and to develop a pipeline for the generation and analysis of metatranscriptomic data. Applying the Illumina HTS platform, we sequenced 12 NOD cecal samples prepared using multiple RNA-extraction protocols. The absence of a complete set of reference genomes necessitated a peptide-based search strategy. Up to 16% of sequence reads could be matched to a known bacterial gene. Phylogenetic analysis of the mapped ORFs revealed a distribution consistent with ribosomal RNA, the majority from Bacteroides or Clostridium species. To place these HTS data within a systems context, we mapped the relative abundance of corresponding  Boosted by the advent of high through-put sequencing (HTS) platforms, metagenomics has emerged as a powerful approach for analyzing complex bacterial communities Choice of HTS platform has a significant impact on the yield of informative reads. In most recent RNA HTS (RNA-Seq) studies, mRNA are mapped onto reference genomes for a single species  Bioinformatics analyses of metatranscriptomics datasets are an intensive area of research and development. Previous approaches have focused on matching sequences to gene families as defined by knowledgebases such as the Clusters of Orthologous Groups (COG), Gene Ontology (GO) and SEED resources Here we are interested in exploring the feasibility of using the Illumina HTS platform to perform metatranscriptomics on a model gut microbiome. Our choice of experimental system is guided by the recent appreciation of the considerable role that commensal flora in the human gut play in human health and disease. For example, metagenomic surveys have already illuminated potential mechanisms by which inflammatory bowel diseases (IBD), metabolic syndrome and type 2 diabetes develop and persist Lactobacillus acidophilus, L. salivarius, Parabacteroides distasonis, Mucispirillum schaedleri, three members of Clostridium cluster XIV and a relatively uncharacterized species of Firmicutes. All but P. diastonis (a member of the phylum Bacteroidetes) and M. schaedleri (a member of the phylum Deferribacteres) are members of the phylum Firmicutes. Although reference genome sequences of strains related to the two Lactobacillus spp. are available, we note that the two reference genomes for L. acidophilus (strains 30SC and NCFM) display considerable genomic variation, with 2037 and 1864 putative open reading frames (ORFs), respectively. Since the objective of many research groups is to apply metatranscriptomic methods to complex environmental and mammalian-host-associated bacterial communities that will exhibit diverse previously uncharacterized species, we selected the altered ASF colonized NOD intestinal samples, as a relatively simple pilot, to examine our ability to agnostically assign HTS reads to samples for which no reference genome is available.To assess molecular protocols and computational analyses for Illumina-based metatranscriptomics, we sequenced RNA prepared from the flushed cecum and colon of age-matched NOD mice weeks prior to diabetes onset. The mice were the progeny of NOD animals that had been re-derived by embryo transfer into a completely germ-free environment and then colonized with an Altered Schaedler flora (ASF) containing eight known species http://www.ncbi.nlm.nih.gov/Traces/sra: Accession number: SRA051354). After filtering for HTS read quality, these samples produced 21.7 million single-end 76-nt reads , which selects for longer RNAs; and Invitrogen mirVANA , which also purifies microRNAs. In addition, we examined the use of the Invitrogen RiboMinus Bacterial kit to selectively deplete rRNA, thereby enriching the high complexity mRNA. Twelve combinations of RNA preparation conditions were used and the samples were multiplexed and sequenced in a single lane of an Illumina Genome Analyzer IIx flow cell. Sequence reads have been made available for download from the National Center for Biotechnological Information (NCBI) Sequence Read Archive . Although the three expected phyla were most often represented in the sequence dataset (>99% of reads), other phyla not representative of ASF species were detected, including Actinobacteria and Spirochaetes . Similarly, 16.8% of sequences were classified as Clostridiaceae, rather than the related families Lachnospiraceae (17.5%) or Ruminococcaceae (0.12%). We interpret these results as evidence that short reads (50–76 nt) obtained by shotgun sequencing are inherently noisy markers of phylogeny; since shotgun reads may be generated from any sub-sequence of an rRNA transcript, reads originating from moderately to highly conserved gene segments may not contain sufficient numbers of unique characters to distinguish among low-level phylogenetic groups.Because the mice used in this study were colonized with a defined set of bacteria, we could use the expected distribution of bacteria in the sample set to evaluate the utility of using relatively short rRNA reads to classify microorganisms. ASF comprises eight bacterial species representing three phyla  and five families . This strategy was more successful, with 1,234,400 of non-mouse-derived reads (32%) mapping to 237,570 unique bacterial transcripts. Most matches (>70–80% depending on sample) were high quality  of putative mRNA reads against the protein non-redundant database  while GF5525 contained five members, all from the order Clostridiales . Given their predicted relative expression, these hypothetical proteins merit further functional investigation. Among gene families that could be ascribed a putative function, eight were implicated in regulatory roles . The ability to map RNA-Seq data onto a defined set of gene families thus provides a surrogate for elucidating functional components from metatranscriptome data.We applied the Markov clustering algorithm (MCL) to the 237,570 unique transcripts identified from our 12 samples to define 12,784 gene families on the basis of sequence similarity scores (see Methods). Summing the RPKM values for each transcript in a class was used to derive the relative abundance of each gene family. The size of the 500 gene families with the highest number of transcripts assigned did not correlate well with their relative expression . This suTo examine the distribution of reads assigned to general functional categories, we assigned transcripts to COG categories on the basis of best BLAST matches to the COG database. RPKM values were then used to calculate the frequency of representation for each COG category. It should be noted in these analyses that as for the phylogenetic analyses, we observed consistency across samples . For manWe next examined functional representation within our datasets by representation of metabolic pathways. Previous analyses have focused on KEGG representations of metabolic pathways To demonstrate how a network-based approach may transcend traditional KEGG defined pathways, we constructed a subnetwork of enzymes for the top 100 expressed enzymes (in terms of RPKM) represented in the NOD503CecMN dataset . Within Nπ-phosphohistidine—sugar phosphotransferase (EC:2.7.1.69). Such differences may indicate subtle changes in the reliance of these key enzymes for directing flux within the network.Adopting a network approach facilitates the identification of nodes that mediate important roles within the network. Betweenness centrality is a metric that assesses the importance of the node to the network through determining the proportion of shortest path lengths that pass through that node Consistent with previous metagenomic analyses, these studies have highlighted the importance of metabolic activities involved in the production of biomass. More importantly, the use of the network for mapping metatranscriptomic datasets shows how groups of functionally related enzymes, differentially expressed across samples, can be readily identified.E. coli will undoubtedly preclude the identification of systems specific to particular taxa , many processes such as cell wall biogenesis, transcription and translation are well conserved throughout bacteria. We may therefore expect these analyses to yield insights into the activity of basic core processes. First, we identified E. coli homologs of each transcript on the basis of InParanoid-derived relationships E. coli gene was then generated from the sum of RPKM values of the transcripts that map to each gene. Correlation across samples varied (Spearman correlation coefficients 0.45–0.90) but was greatest between samples with the most mapped reads . As noted above, the focus on E. coli suggests that the relative expression of each gene may correlate with its relative level of conservation. This might imply that our mapping simply reflects underlying conservation biases and indeed we note some correlation between expression and conservation  embryo transfer into germ-free pseudo-pregnant females. The germ-free status of mice in the facility is maintained by housing in flexible-film isolators and monitored by qPCR analysis of fecal DNA preparations using pan-specific bacterial 16S primers, and culture of cecal contents under anaerobic (blood agar) and aerobic (Luria broth) culture conditions. In addition DNA staining of cecal contents with Sytox green is used to confirm the bacterial absence. The absence of parasites, bacteria and virus contamination was independently confirmed quarterly by shipment of sentinel mice for analysis at a commercial facility.Lactobacillus acidophilus (ASF 360), Lactobacillus murinus (ASF 361), Bacteroides distasonis, (ASF 519), Mucispirillum schaedleri (ASF 457), Eubacterium plexicaudatum (ASF 492), a Fusiform-shaped bacterium (ASF 356) and two Clostridium species . These ASF-colonized gnotobiotic mice were then bred in isolators to ensure no additional species were introduced. The presence of the ASF species was confirmed by species-specific bacterial qPCR Once GF status was confirmed as described, GF NOD mice were colonized by co-housing with gnotobiotic mice colonized with defined cultured bacterial species , cecum, and colon were collected individually, by scraping biomass from intestinal wall with a sterile scalpel. Specimens were placed in separate microcentrifuge tubes, frozen at −80C, and shipped to the laboratory of Dr. Frank on dry ice. Upon thawing, approximately 30 mg luminal biomass was suspended in two volumes (mass/vol) of phosphate buffered saline (pH7.4), producing a 100 µl slurry of biomass. Two RNA extraction protocols were compared: 1) Cell lysis in RNAzol B Reagent  followed by RNA purification using the RNeasy kit . Poor quality bases were removed by iterating a 5 nt window across the 5′ and 3′ ends of each sequence and removing nucleotides in windows with a mean quality score <20; iteration was stopped when the mean quality score was >20. Adaptor sequences were removed using Cross_match (http://www.phrap.org) to search a database of Illumina adaptor sequences. Following trimming and adaptor removal, reads with lengths less than 50 nt were discarded. Due to the poor performance obtained from applying the Ribosomal Database Project (RDP) classifier Sequencing was performed with the Illumina Genome Analyzer IIx (GaIIx) platform at the Center for Advanced Genomics . After deconvolution of the barcodes used for multiplexing, 21,680,028 76 bp reads were generated on a single flow cell. Reads are available for download from the National Center for Biotechnology Information (NCBI) Sequence Read Archive ; 2) the mouse genome; and 3) a database of 1078 bacterial genomes downloaded from the NCBI , using the software tool BWA To identify potential host contaminants, putative mRNA transcripts were mapped to: 1) a database of mouse derived transcripts (Ensemble release V.59 – −5). The Markov clustering algorithm (MCL - −3). Enzyme classification (EC) assignments were assigned by performing a BLASTP  search of the 246,538 transcripts against a database of 127,478 enzyme proteins annotated by SwissProt Version 57.0 E. coli homolog mapping was performed through BLAST-based similarity searches to identify the closest matching sequence in the set of E. coli K12 transcripts . The relative abundance of each COG category, EC number and E. coli homolog was derived from the sum of RPKM values for each transcript that maps to the specific category, number or homolog in question. The relative conservation of E. coli genes was generated through the systematic identification of putative orthologs of each E. coli gene in the set of 1078 bacterial genomes using the tool InParanoid http://www.mathworks.com/matlab).To generate gene family assignments for each transcript, we performed an all-vs-all BLAST search of the 236,769 unique transcripts identified from our 12 samples Click here for additional data file.Figure S2RPKM values of the top 500 largest gene families. The graphs indicate the RPKM values associated with each gene family as a function of the number of members associated with that family. For each sample we observe only weak correlation between the size of the family and its relative expression within the datasets.(EPS)Click here for additional data file.