This review summarizes recent advances and current gaps in understanding of innate immunity to human immunodeficiency virus (HIV) infection, and identifies key scientific priorities to enable application of this knowledge to the development of novel prevention strategies (vaccines and microbicides). It builds on productive discussion and new data arising out of a workshop on innate immunity against HIV held at the European Commission in Brussels, together with recent observations from the literature.Increasing evidence suggests that innate responses are key determinants of the outcome of HIV infection, influencing critical events in the earliest stages of infection including the efficiency of mucosal HIV transmission, establishment of initial foci of infection and local virus replication/spread as well as virus dissemination, the ensuing acute burst of viral replication, and the persisting viral load established. They also impact on the subsequent level of ongoing viral replication and rate of disease progression. Modulation of innate immunity thus has the potential to constitute a powerful effector strategy to complement traditional approaches to HIV prophylaxis and therapy. Importantly, there is increasing evidence to suggest that many arms of the innate response play both protective and pathogenic roles in HIV infection. Consequently, understanding the contributions made by components of the host innate response to HIV acquisition/spread versus control is a critical pre-requisite for the employment of innate immunity in vaccine or microbicide design, so that appropriate responses can be targeted for up- or down-modulation. There is also an important need to understand the mechanisms via which innate responses are triggered and mediate their activity, and to define the structure-function relationships of individual innate factors, so that they can be selectively exploited or inhibited. Finally, strategies for achieving modulation of innate functions need to be developed and subjected to rigorous testing to ensure that they achieve the desired level of protection without stimulation of immunopathological effects. Priority areas are identified where there are opportunities to accelerate the translation of recent gains in understanding of innate immunity into the design of improved or novel vaccine and microbicide strategies against HIV infection. Rational approaches to HIV vaccine design have so far focused principally on the induction of virus-specific antibody or T cell responses. Results from large-scale clinical trials of both antibody- and T cell-targeted immunogens have given largely disappointing results ,2 and alThe classical application of innate immunity in vaccine design has been in an adjuvant role: innate immune responses are stimulated at the time of vaccination to promote the induction of adaptive response(s) capable of mediating protection on subsequent pathogen encounter . The neeUnderstanding the contributions made by different innate host resistance mechanisms and innate responses to HIV acquisition and disease progression is a critical pre-requisite for the rational design of novel prophylactic and therapeutic strategies focusing on innate immunity: this will inform the selection of responses to target for up- or down-modulation by vaccination or microbicides. There is also an important need to understand the mechanisms via which innate responses are triggered, so that these can be selectively exploited or inhibited in vaccine or microbicide design. Finally, strategies for achieving the desired modulation of innate functions will need to be developed and subjected to rigorous testing to ensure that they achieve the desired level of protection without stimulation of immunopathological effects. Given that many components of the innate response mediate pleiotropic functions and can both inhibit HIV infection and exert immunomodulatory effects that may enhance viral replication, it is critical to assess whether these opposing outcomes can be dissected and mapped to functionally distinct effector pathways or sites within a given soluble factor, thereby providing a basis for their selective exploitation in prophylactic or therapeutic strategies.The following sections discuss current understanding of the roles of different components of innate immunity in protection or pathogenesis in HIV infection and of how the activation of innate responses is stimulated and regulated, together with the knowledge gaps and priorities for research. Components of the innate response are considered in the sequence in which they may be invoked in combating infection: as mucosal HIV exposure occurs; local foci of infection are established; and as more widespread viral dissemination takes place Figure .The observation that following heterosexual transmission of HIV, the viral quasispecies generated in acute infection is frequently derived from a single infecting virion , providein vitro under conditions designed to mimic those encountered during sexual transmission of HIV through formation of amyloid fibrils that capture and focus virus onto target cells [The molecular mode of action of many of these recently-discovered factors remains to be elucidated, with indications from published data highlighting substantial diversity in potency and mode of interaction with HIV. For example, SEVI, a small semen cationic peptide, enhances HIV infection et cells -9. Underin vivo is not clear, although local elevations in α-defensin levels during genital tract infections are associated with enhanced HIV acquisition [Defensins are also small cationic peptides. They are produced by epithelial cells and leukocytes and are involved in combating infection with a broad range of bacteria, fungi and viruses, including HIV . Mechaniuisition ,23. Analet al., submitted). How ps20 regulates cell adhesion and HIV infection is not known. In addition to their ability to regulate HIV infection, whey acidic proteins are recognised for their anti-inflammatory activity, e.g. they are able to suppress lipopolysaccharide (LPS)-stimulated production of cytokines like tumour necrosis factor (TNF)α [et al., unpublished). SLPI has also been noted to suppress the enzyme activation-induced cytidine deaminase (AID) in epithelial cells [Whey acidic proteins have traditionally been associated with broad antimicrobial activity at portals of pathogen entry and are identified to be under strong selection pressure, which is a hallmark of innate immunity . Two of r (TNF)α ; and ps2al cells . AID is al cells . Epithelal cells . SLPI inal cells . The molal cells ,34). Maial cells .in vivo is currently unclear. Likewise type 1 IFN induction following HIV transmission as infection is established and begins to spread, and its production at subsequent stages of infection could also have opposing effects - this is discussed further below.Type I IFNs are innate cytokines that also possess direct anti-HIV activity  and, likin vitro model systems are critical. In addition, development of specific assays for the accurate measurement of these secreted innate factors will enable their regulation and expression pattern in mucosal tissue during acute and chronic infection to be assessed. Together, this will provide a platform for considering the potential exploitation of these innate immune mediators in novel prophylactic or therapeutic strategies.Taken together these data highlight that innate immune mediators present at local sites of infection can exert a significant HIV regulatory effect through physical interaction with the virus, competitive binding to cell-surface entry proteins, triggering of signals that alter the permissiveness of target cells and/or immunomodulatory activities Figure . At presHuman cells express a number of proteins that block cross-species transmission of retroviruses . Some ofHIV restriction factors are constitutively expressed at baseline levels in many cell types, but their expression can be rapidly up-regulated by type 1 IFNs -49. Up-rper se, or provided a surrogate marker for IFN induction, which was mediating its effects via a variety of mechanisms. This question is of importance in the context of understanding and exploiting innate immune effector mechanisms in therapeutic strategies. A recent study in a murine model system showed that type 1 IFN-mediated suppression of the replication of hepatitis B virus  was not dependent on APOBEC3 expression [A key question, yet to be answered, is whether increased expression of endogenous restriction factors could prevent HIV infection or limit virus replication in acute/early infection. Interestingly, a recent study suggested that APOBEC3G expression can be modified by vaccination. Rectal mucosal immunization of macaques with SIV antigens and CCR5 peptides, linked to the 70 kD heat shock protein, showed a progressive increase in APOBEC3G mRNA in PBMCs which was maintained for at least 17 weeks. Mucosal challenge with simian immunodeficiency virus (SIV) resulted in a significant increase in APOBEC3G mRNA in CD4+CCR5+ cells in the circulation and draining iliac lymph nodes in immunized animals (which did not become infected) compared to un-immunised animals, consistent with an association between APOBEC3G expression and protection from infection . Howeverpression . A systeThe low efficiency of heterosexual HIV transmission may be due not only to the physical and immunological barriers to infection at genital mucosal surfaces (discussed above), but also to difficulties encountered by the virus in establishing initial foci of infection at the local mucosal site and undergoing subsequent spread. A full understanding of the earliest virus-host cell interactions that take place in infection and the role of local innate responses in blocking or amplifying initial virus replication is paramount to enable the development of prophylactic strategies to intervene at this critical stage of infection where the window of opportunity for virus eradication is still open.The first cells with which the virus interacts at the genital mucosa may include DCs, macrophages and CD4+ T cells. Conventional (c)DCs in the submucosa expressing the C-type lectin dendritic cell-specific, intercellular adhesion molecule-grabbing non-integrin (DC-SIGN) are hypothesized to play a key role in HIV dissemination to CD4+ T cells due to their ability to capture and internalize virions via DC-SIGN and mediate trans-infection of CD4+ T cells, either at the mucosal infection site or following migration into draining lymphoid tissues. Some macrophages in mucosal tissues also express DC-SIGN and, although they do not migrate into lymph nodes, may contribute to local HIV transmission to CD4+ T cells . HIV intLangerhans cells located in the mucosal epithelium express an alternative capture receptor, Langerin. In contrast to virions captured by DC-SIGN, HIV-1 captured by Langerin is internalised into Birbeck granules and degraded . Unlike Acquisition of HIV infection is known to be enhanced by the presence of other sexually transmitted infections. Langerhans cell activation may be only one of a number of mechanisms involved, others including breach of the physical mucosal barrier to infection, the presence of larger-than-normal numbers of CD4+ T cells, macrophages and DCs in the subepithelium, and the heightened state of activation of these cells. In the resting state, the paucity of CD4+ T cells in the submucosa may be one of the factors that restricts the establishment of foci of HIV infection. Recent studies in the SIV macaque model have suggested that production of macrophage inflammatory protein (MIP)-3α at mucosal infection sites may attract plasmacytoid (p)DCs and other inflammatory cells, which in turn help to recruit additional CD4+ T cells through production of chemokines such as MIP1α and β . These pin vivo. HIV is no exception: it exploits DC subsets not only to facilitate the initial establishment of infection and local virus spread as described above, but also to enhance systemic virus replication and impair host control of infection.DCs play a key role in the orchestration of innate and adaptive responses, responding to the presence of infection by initiating soluble factor production and engaging in cross-talk with other cell types to induce and regulate an immune response that will mediate pathogen control with minimum immunopathology. Given the central role of DCs in the host immune response, many viruses subvert DC functions to promote their persistence in vitro [HIV can infect both cDCs and pDCs, and has been found to initiate infection in both DC subsets more efficiently than in other cell types (including macrophages and CD4+ T cells) in vitro . Howeverin vitro . Nonethein vitro , thus siin vitro . Second,In vitro studies have shown that pDCs are very rapidly activated following contact with HIV, upregulating MHC and costimulatory molecules, producing high levels of type 1 IFNs and other cytokines and acquiring increased T cell stimulatory capacity [in vitro findings would predict a rapid widespread activation of pDCs and subsequent maturation of cDCs as systemic HIV spread occurs: a picture which fits well with observations made in vivo.capacity . HIV sticapacity . By contcapacity . The reacapacity . These iin vitro, and impairs their response to TLR7 and TLR9 ligation [The increase in plasma viral titres in acute HIV-1 infection (AHI) is associated with an ordered sequence of elevations in systemic levels of multiple cytokines and chemokines . The earligation ,77. Thisin vitro (discussed above), it seems likely that these antiviral cytokines also contribute to control of HIV replication in vivo, although the extent to which they constrain the acute viral burst and the mechanisms by which they mediate this are important issues which require investigation. In addition to their antiviral effects type 1 IFNs and other innate cytokines such as IL-15 also possess potent immunostimulatory properties, mediating immune activation both directly and indirectly (via induction of other cyokines/chemokines) - hence they may contribute to control of viral replication indirectly, by activating other protective immune responses. However they may simultaneously act to enhance HIV replication by driving widespread immune activation; further, type 1 IFNs promote apoptosis, so they may also contribute to CD4+ T cell loss in HIV infection [in vivo remains unclear - although the fact that HIV has not evolved to avoid or inhibit pDC activation suggests that on balance the early innate cytokine production and ensuing immune activation may be advantageous for virus replication. In support of this, administration of IL-15 to rhesus macaques during the acute phase of SIV infection resulted in enhancement of NK cell and SIV-specific CD8+ T cell responses at peak viremia and a reduction in the number of SIV-infected cells in lymph nodes, but despite this, establishment of higher persisting viral loads and enhanced disease progression [Given the ability of type 1 IFNs to inhibit HIV replication nfection . Which ogression . A recengression . In earlgression .Analysis of the extent and kinetics of type 1 IFN induction and immune activation stimulated during SIV infections of their natural non-human primate hosts, which unlike HIV infection in humans generally do not lead to the development of AIDS, has also helped to give insight into what may consititute more protective versus pathogenic aspects of the early immune response. Most studies suggest that pDC activation, type 1 IFN production and immune activation occur in the acute phase of both pathogenic and non-pathogenic SIV infections, but non-pathogenic infections are distinguished by the fact that the acute-phase immune activation resolves very rapidly even though substantial virus replication continues after the transition to chronic infection -85. By cin vivo impairments in DC-T cell interactions can be overcome by pre-priming of HIV-specific T cell responses.Although HIV stimulates pDC activation, hence promoting a state of generalised immune activation that permits widespread high-level virus replication, increasing evidence suggests that it also modifies both pDC and cDC functions in order to concurrently reduce or impair the activation of virus-specific T cell responses. Although cDCs are not directly activated by HIV, exposure to HIV impairs their maturation in response to other stimuli and promotes production of IL-10 and induction of a regulatory T (Treg) cell response . In addiNK and NKT cells are innate lymphocyte populations that can be rapidly activated in response to infection and are capable of mediating potent effector and immunoregulatory functions. As such, they warrant consideration in HIV vaccine design, where modulation of events taking place in the earliest stages of infection is paramount. A better understanding of the NK and NKT cell responses activated following HIV infection and their contributions to control of viral replication and/or to immunopathological immune activation is thus a current priority.ex vivo (degranulation and cytokine production) at this time, a property that is sustained throughout early infection. Interesting changes take place in the peripheral blood NK cell subset composition during AHI. The frequency of CD56bright (regulatory) NK cells decreases (perhaps due in part to recruitment into lymph nodes), whilst the frequency of CD56dim (effector) NK cells increases [In AHI, peripheral blood NK cells become activated and increase in frequency as the acute burst of viral replication occurs, prior to the maximal expansion of CD8+ T cells . NK cellncreases . Notablyncreases . The mecin vitro; and the observation that HIV has evolved strategies for modulating ligand expression on the surface of the cells it infects so as to minimize both CD8+ T cell and NK cell activation but maximise NK cell inhibition suggests that NK cells also mediate antiviral activity in vivo [in vitro [in vivo: co-expression of HLA-Bw480I with KIR3DS1 or certain inhibitory alleles of KIR3DL1 has been found to be associated with low-level viremia in early HIV infection and also with delayed disease progression [KIR3DL alleles and viral loads in SIV-infected rhesus macaques [There is increasing evidence to suggest that NK cells make a significant contribution to containment of viral replication in HIV-infected individuals. NK cells are able to control HIV replication  in vivo ,100. Notin vitro . Geneticgression -104. Likmacaques . NK cellmacaques ,107, an macaques ,109. AltHLA-Bw480I subjects respond strongly to stimulation with HLA-deficient cells in vitro [First, why do some NK cells, e.g. those expressing KIR3DS1 or certain KIR3DL1 allotypes, seem to be particularly protective in HIV infection? If the reasons for this were understood, it may be possible to design strategies to induce NK cells in people who do not express these receptors or their ligands to mediate better control of HIV replication . It is hypothesized that the activating receptor KIR3DS1 interacts with a specific ligand on HIV-infected cells that results in efficient triggering of effector functions. However the nature of this ligand and reasons for the efficacy of NK stimulation via KIR3DS1 remain unclear. Whether HIV-infected cells can express ligands for other activating KIRs also remains to be determined. KIR3DL1 is an inhibitory receptor, and is hypothesized to act during NK cell development/functional maturation to permit NK cells to acquire particularly powerful effector functions. In line with this, KIR3DL1+ NK cells from in vitro . HoweverSecond, although recent studies have begun to give insight into the systemic activation of NK cells in HIV infection, relatively little is known about the NK cell populations present at other sites, such as the genital mucosa, the gut and lymph nodes, and the roles they may be playing in combating local HIV replication and/or mediating immunopathological effects. Of particular interest is the recently-described IL-22-producing NK population in the gut, which may play an important role in mucosal defence and local immunoregulation .KIR3DS1+ compared to KIR3DS1- individuals, independent of expression of HLA-Bw480I [KIR3DS1 and HLA-Bw480I progress to AIDS slowly, KIR3DS1 homozygosity in the absence of HLA-Bw480I expression is associated with accelerated disease progression [in vivo importance of NK cell-mediated immunopathologic effects, the mechanisms by which they are mediated and the principal NK cell subset(s) involved is a priority for future work. This information will determine the feasibility of designing vaccine strategies to stimulate protective but not immunopathological NK responses, or to down-modulate immunopathological NK cell-mediated activity [Third, it is critical to understand whether NK cells have immunopathological effects in addition to their putative protective functions in acute and early HIV infection. NK cells may contribute to immunopathological CD4+ T cell destruction, e.g. a role for NKp44-expressing NK cells in mediating lysis of uninfected CD4+ T cells expressing a gp41 peptide-induced NKp44 ligand has been suggested . NK cellA-Bw480I ; and altgression . This coactivity .NKT cells are innate lymphocytes with properties of both NK cells and T cells, e.g. they express both a T cell receptor and markers characteristic of NK cells. Some NKT cells express relatively invariant TCRs that interact with ligands presented by the non-classical class I molecule CD1d, whilst others  express a much wider range of TCRs that interact with ligands presented by classical class I molecules.et al, unpublished) the frequency of circulating CD4+ NKT cells declines, likely as a consequence of virus infection, whilst CD8+ NKT cells increase in frequency, potentially as a result of antigen-driven expansion. Identification of the ligands recognized by NKT cells on HIV-infected cells is a priority for future studies, as is further analysis of the potential for employing these cells in an effector role in HIV vaccine design.Relatively little is known about NKT cell responses in acute and early HIV infection and the roles that these cells may be playing in protection and/or immunopathology. CD3+CD56+ NKT cells may contribute to control of HIV replication by mediating cytolysis of infected cells and/or via production of β-chemokines and other soluble factors; and both CD3+CD56+ and invariant NKT cells may mediate immunoregulatory effects that contribute to the initiation/enhancement of HIV-specific immune responses. Conversely, NKT cells may also mediate detrimental effects via promotion of immune activation and viral replication. Recent studies have shown that during acute SIV  and alsoAs discussed above, there is increasing understanding of the important role played by innate responses in both promoting and inhibiting HIV transmission, the establishment, dissemination and amplification of infection and the level at which viral replication is subsequently contained Table . AlthougThe simplest method of employing innate defences for the control of HIV replication would be to directly administer innate antiviral effector molecules or the protective components thereof prophylactically in the form of microbicides, or therapeutically at a systemic level. Likewise protection could also be afforded by direct administration of agents able to block the activity of innate pathways that promote HIV replication. For example, recombinant forms of the active components of WAPs such as SLPI and Elafin, which are associated with reduced HIV transmission, could potentially be employed as microbicides or therapeutic agents; whilst blocking the activity of WFDC1/ps20, which promotes HIV spread, with monoclonal antibodies or peptide antagonists could represent an "anti-immunopathological" strategy.An extension of this approach would be to directly administer substances known to modulate the effector activity of innate subsets. For example, HIV-specific monoclonal antibodies known to trigger antibody-dependent cell-mediated viral inhibition (ADCVI) responses by NK cells, macrophages or other effector cells could administered topically as part of a microbicide strategy, or be infused therapeutically in early HIV infection - an approach supported by the observation that the ability of one HIV-specific antibody, b12, to confer passive protection in a macaque vaginal simian/human immunodeficiency virus (SHIV) challenge model was dependent in part on its CD16-binding capacity . ConversAs the concept of using vaccines to stimulate the production of specific antibodies and memory T cell populations is well-established, the most straightforward approach to developing innate response-modulating vaccines may be to design vaccines to prime adaptive responses that will modulate innate effector functions in infected individuals. For example, vaccines could be developed to induce HIV-specific antibodies capable of mediating ADCVI. Notably, there was an inverse correlation between serum ADCVI antibody titres and HIV infection rate in the Vax 004 vaccine study . Alternahigh T cells that produced IL-10 in a HIVp24-specific fashion and could suppress the proliferation of HIVp24-specific CD4+CD25- T cells in vitro could be detected early after infection with HIV [high T cell frequencies and the frequency of activated (HLA-DR+) CD4+ T cells in early infection [Another possibility may be to develop vaccines or microbicides that can expand populations of regulatory T (Treg) cells that will dampen immune activation in HIV-infected individuals and reduce viral replication and spread. One study showed that CD4+CD25with HIV , suggestnfection , althougA more radical concept would be to use vaccines to modulate innate subsets/activity directly. One of the properties classically thought to distinguish innate and adaptive responses is that the former lack memory: but recent studies have challenged this view, providing evidence that NK cell responses can exhibit features characteristic of adaptive responses including "memory" -122. OneAnother mechanism by which vaccines could induce long-lasting alterations in innate responses that would not require the existence of innate memory would be if the vaccine persisted and provided a source of continuous or repeated stimulation. There are a number of potential pitfalls to this approach, as chronic stimulation can have detrimental effects on immune functions; also maintenance of a generalized state of immune activation could enhance HIV acquisition/replication. However the possibility of providing an intermittent stimulus to retain selective innate responses at an elevated level remains worth exploring with caution.Given the potential (discussed in previous sections) for innate responses to mediate immunopathogenic effects in acute/early HIV infection, it is critically important that vaccination strategies designed to confer protection via modulation of innate responses are thoroughly tested to determine the full spectrum of innate functions altered and the potential for detrimental consequences. This is especially key where modulation of innate responses at mucosal sites is attempted, given the documented capacity of microbicide-induced modulation of local immune activation to enhance acquisition of infection (reviewed in ).The effects of novel innate response-modulating vaccine strategies should ideally be tested in non-human primate immunodeficiency virus infection models prior to human administration. With this in mind, it is important that reagents appropriate for analyzing innate responses in non-human primates are developed, and the innate responses naturally activated in response to immunodeficiency virus infection characterized. Whilst work carried out to date in SIV-infected rhesus macaques indicates that there are many parallels between the innate responses activated here and in acute HIV-1 infection, there may also be some critical inter-species differences that may limit cross-species testing of certain vaccination strategies; this needs to be explored.An as yet poorly-exploited avenue for gaining insight into the effects of vaccination strategies on innate responses and how these in turn may impact on protection is to analyze how vaccines that are currently in human trials may be modulating innate responses, how durable these effects are, and whether there is any impact on innate responses/functions during acute/early infection in vaccinated individuals who subsequently become infected. Given the difference in kinetics of activation of innate responses and the adaptive responses on which vaccine trials typically focus, this may require inclusion of extra post-vaccination sampling timepoints during vaccine trial design.Innate immunity plays important roles in mediating defence against HIV acquisition, control of infection following virus transmission and containment of virus replication during both the acute and chronic phases of infection. Conversely, innate responses can also have detrimental effects at all stages of infection, promoting virus transmission and the establishment of infection, enhancing subsequent virus replication and spread and contributing to CD4+ T cell destruction and disease progression. Importantly, recent studies have shown these opposing protective and pathogenic effects are mediated by every major component of the innate immune system , prompting a need for more precise definition of the underlying molecular mechanisms so that they can be specifically up- or down-modulated by intervention strategies. Increasing evidence suggests that there may be potential to stimulate protective innate effector mechanisms to a level where HIV transmission and/or the establishment of disseminated infection is prevented. Likewise strategies for blocking generalised immune activation may also have prophylactic potential, and hold great promise as a novel therapeutic approach. The major challenge ahead lies in determining how these potentially important goals may be safely realized to yield novel infection and disease prevention strategies that will complement more traditional approaches. Some of the priorities for research in this area are summarised in Table ADCVI: antibody-dependent cell-mediated virus inhibition; AHI: acute HIV-1 infection; AID: activation-induced cytidine deaminase; AIDS: acquired immunodeficiency syndrome; APOBEC: apolipoprotein B editing complex, catalytic subunit (APOBEC); CCR: chemokine receptor; cDC: conventional DC (including myeloid DCs and other non-pDC types); DC: dendritic cell; DC-SIGN: dendritic cell-specific, intercellular adhesion molecule-grabbing non-integrin; GALT: gut-associated lymphoid tissue; HIV: human immunodeficiency virus; HLA: human leukocyte antigen; IDO: indoleamine 2,3-dioxygenase; IFN: interferon; IL: interleukin; IRF: interferon-regulatory factor; Ig: immunoglobulin; LARG: leukemia associated Rho guanine nucleotide-exchange factor; LPS: lipopolysaccharide; MHC: major histocompatibility complex; MIP: macrophage inflammatory protein; NF: nuclear factor; NK: natural killer; PBMC: peripheral blood mononuclear cell; pDC: plasmacytoid DC; PRR: pattern-recognition receptor; RIG-I: retinoic acid-inducible gene I; SEVI: semen-derived enhancer of virus infection; SIV: simian immunodeficiency virus; SHIV: simian/human immunodeficiency virus; SLPI: secretory leukocyte protease inhibitor; TLR: toll-like receptor; TNF: tumour necrosis factor; TRIM: tripartite motif; Treg: regulatory T; WAP: whey acidic protein; WFDC1: whey acidic protein four-disulfide core domain 1The authors declare that they have no competing interests.RJS, AV and PB organized the EUROPRISE workshop, where participants gave formal presentations and contributed to discussion. PB, RJS and AV drafted sections of the text, edited one another's contributions and read and approved the final manuscript.PB is a Reader in the Nuffield Dept of Clinical Medicine at the University of Oxford, UK whose research focuses on innate and T cell responses in persistent virus infections including HIV.RJS is Professor of Cellular and Molecular Infection at St George's, University of London, UK. His work focuses on development of new prevention technology for HIV including vaccines and microbicides.AV is a Senior Lecturer in the Department of Infectious Diseases at King's College London, UK whose research includes studies of virus/host interactions that regulate innate and adaptive T-cell immunity to HIV.


Little is known about the contribution of transcranial direct current stimulation (tDCS) to the exploration of memory functions. The aim of the present study was to examine the behavioural effects of right or left-hemisphere frontal direct current delivery while committing to memory auditory presented nouns on short-term learning and subsequent long-term retrieval.Twenty subjects, divided into two groups, performed an episodic verbal memory task during anodal, cathodal and sham current application on the right or left dorsolateral prefrontal cortex (DLPFC).Our results imply that only cathodal tDCS elicits behavioural effects on verbal memory performance. In particular, left-sided application of cathodal tDCS impaired short-term verbal learning when compared to the baseline. We did not observe tDCS effects on long-term retrieval.Our results imply that the left DLPFC is a crucial area involved in short-term verbal learning mechanisms. However, we found further support that direct current delivery with an intensity of 1.5 mA to the DLPFC during short-term learning does not disrupt longer lasting consolidation processes that are mainly known to be related to mesial temporal lobe areas. In the present study, we have shown that the tDCS technique has the potential to modulate short-term verbal learning mechanism. Memory is a key issue in cognitive neuroscience and probably constitutes one of the most complex cognitive functions. The human memory system comprises various memory subtypes controlled by complex cortico-subcortical networks . The preThe advent of functional neuroimaging techniques has brought with it an accumulation of evidence pointing to the involvement of the prefrontal cortex in the encoding and retrieval of verbal and non-verbal stimuli -4, and iWe applied transcranial direct current stimulation (tDCS) with the aim of examining hemispheric dominance during an auditory verbal memory task. The decision to modulate the right and left PFC separately was motivated by the hemispheric encoding/retrieval asymmetry (HERA) model, which is a process-specific description of experimental data provided by a large set of functional neuroimaging studies . The tDCOnly a paucity of the tDCS studies to date has explored the modulation of prefrontal areas during explicit memory tasks and to our knowledge, none of these used auditory presented nouns as stimuli. For example, a previous research that evaluated the effect of tDCS on a visual letter working memory task reported that anodal stimulation of the left DLPFC increased performance accuracy when compared with sham stimulation (baseline) on the same side . AnotherTo our knowledge, none of the published studies presented auditory verbal stimuli during tDCS application in order to test its modulatory effect on both short-term learning and subsequent long-term retrieval. We therefore sought to use the tDCS method to examine the question of relative hemispheric specialisation of the DLPFC in auditory verbal learning mechanism. We hypothesised in view of the findings of some neuroimaging studies ,8 verbalTwenty male volunteers (native Swiss-German) ranging in age from 19 to 26 years  were recruited for the experiment. All participants were university students with a similar level of education . According to the Annett-Handedness-Questionnaire  all subjThe participants were placed in a comfortable chair in front of a screen and two loudspeakers positioned at an angle of about 90 degrees in the horizontal plane and performed the experiment in a well-lit and quiet room. Volunteers were assigned to one of two groups each performing the same three stimulation blocks  in a randomised order. During the experiment, the participants fixated a small cross in the middle of the screen while single nouns were presented (loudness ~50 dB sound pressure level).The auditory stimuli consisted of 25 German nouns out of the VLMT test  were presented in randomized order across subjects and groups. In order to avoid ceiling effects, the original version of 15 semantically unrelated nouns was expanded to 25, controlling for word frequency (on the basis of a comprehensive search using the "google" search system)  and catePrior to each session, the subjects performed a German verbal intelligence (MWT A) and a short-term attention test (d2) with the intention of controlling for group homogeneity in task-relevant cognitive abilities. Each block  comprised the following trial sequence: (I) "VLMT short-term learning test (STL)", (II) "NVLT non-verbal learning test", (III) "Pause", (IV) "d2 attention test", (V) "VLMT long-term retrieval test (LTR)" and (VI) "Pause". During trial (I), the participants had to encode and immediately retrieve the auditory presented words of the VLMT test three times. Simultaneous sham, anodal or cathodal stimulation was delivered via a frontolateral electrode. Trial (II) was a nonverbal recognition test with the intention of avoiding active memory strategies until later retrieval (V). After a short pause (III) in which a silent cartoon was presented, a second d2 test followed (IV). The pause had the function of excluding after-effects of current delivery on cortical excitability/suppression before the later retrieval (LTR) was next (V). The d2 test was inserted to control attention during the entire experiment. Before starting the next block the participants had a second pause (20 minutes) (VI) designed to distract the participants by means of a silent cartoon before the next parallel form of the memory test was presented. Each of the three blocks had duration of 47 minutes. Thus the total duration of the experiment  was about 130 minutes. Figure 2 (7 cm × 4 cm). We choose a mastoid electrode with a larger surface  in order to reduce current density at the posterior-lateral brain side. Cathodal and anodal stimulation were delivered with a constant current of 1.5 mA. The baseline condition (sham) was performed without any tDCS influence. Stimulation was applied for a period of 5 minutes, with a linear fade in/fade out of 10 seconds and was congruent with the duration of the three encoding trials (see VLMT test). Anodal/cathodal/sham application was randomly controlled across subjects and groups. Both groups run through the same experimental setting but differed in stimulation side .The experiment was conducted with a transcranial direct current stimulator. Current was transferred by a saline-soaked pair of surface sponge electrodes and delivered by a battery-driven constant current stimulator . The electrodes were applied unilaterally (i.e. to the right or left hemisphere) at fronto-lateral location  and over the mastoid. This method of DLPFC localisation has been used in previous studies ,20-22 anThe MWT (Mehrfachwahl-Wortschatz-Intelligenz-Test) is a clinical test for assessing the verbal intelligence quotient. The entire test can be executed in about 300 seconds and allows fast screening of general verbal intellectual capacities.Each d2 short-term attention test had duration of 280 seconds. Score evaluation was based on the difference between the sum of correctly arranged items and the confusion errors . During The applied NVLT test  comprised 120 meaningless figures. Each figure was presented visually on the screen for 3 seconds. Eight figures were presented 5 times. The subject had to indicate by pressing a keyboard button whether the figures had been presented before or not. The performance scores were not further analysed because they were beyond the main interest of this study.For the purpose of further data analysis it is important that both groups were comparable in the following task-relevant variables: age, years of education, verbal intelligence and short-term attention. It was also relevant that both groups showed a similar level of achievement during sham stimulation and that attention was comparable between both groups during the entire experiment. To control for the influence of these variables, we statistically compared the two groups.Before subjecting age, years of education, intelligence/attention scores and VLMT performance during sham stimulation across both groups to parametrical statistical testing, we ascertained that data were normally distributed (Kolmogorov-Smirnov-test). T-tests for independent samples did not reveal significant differences in these control variables among groups. In addition we computed d2 scores in a 2 × 3 repeated-measure ANOVA looking for attention effects across groups among the three blocks. We tested the prerequisites for an analysis of variance, namely homogeneity of variances (Mauchly's test of sphericity) and normal data distribution (Kolmogorov-Smirnov-test). Neither the main effects "stimulation mode" (SM) and "group" (G) nor the interaction "stimulation mode" × "group" (SMG) reached significance. Thus we assumed a comparable attention level in both groups.2 = .72).Short-term learning was quantified by evaluating the total number of remembered words after the third encoding run , thus evidencing that tDCS application had a significant effect only in the LHG. To further elucidate the SM effect found in the LHG, we computed three t-tests (one-tailed) for dependent samples . The results of these post-hoc comparisons showed a significant result only for the Sham vs. Cathodal contrast  = 2.44, p = .018, one tailed; Bonferroni corrected p value = .016). Figure To further examine this interaction we computed two separate one-way ANOVAs, one for each group . This statistical analysis was applied to examine the significant interaction we found in the higher level 3 × 2 ANOVA. The outcome of this procedure revealed a significant SM effect in the left but not in the right hemisphere group  = 6.0, p = .037, etaWe recorded the correctly remembered words after the delay period as the index for long-term retrieval. We computed a repeated-measure 3 × 2 ANOVA with the factors SM and G. The analysis revealed no significant main  or interaction effects.The aim of this study was to examine the behavioural effects of right and left-hemisphere frontal direct current delivery while memorizing auditory presented words on short-term learning and subsequent long-term retrieval. Our results provide evidence for a short-term effect that appeared not to influence consolidation mechanisms. As a main result, we found that left-side cathodal tDCS application induced poorer performance than sham tDCS application in the same area. Our results demonstrate that the left DLPFC is a crucial area involved in short-term verbal learning mechanisms and that tDCS is a suitable method that permits to modulate verbal memory functions. In line with this, several functional imaging studies consistently showed prefrontal activation during committing to memory various types of stimuli  but the Only few tDCS studies to date have focussed on memory functions, and none of these used auditory presented verbal stimuli. Previous tDCS studies mainly collected behavioural data by performing verbal or non-verbal working memory tasks, disregarding other subtypes of memory functions. For example, by using visually presented verbal stimuli Fregni et al.  and alsoOur results suggest a pattern of material specific activation principally involving the left language-dominant hemisphere. Somewhat deviating from the HERA model , our resFinally, our data suggest that prefrontal direct current delivery did not affect the memory consolidation mechanism mainly known to be related to mesial temporal areas ,39. If tA methodological limitation of tDCS protocols is the low spatial resolution and the fact that the modulation of a particular brain area's response to a certain stimulation reflects a limited view of a large-scale functional network . ConsequThe aim of the present study was to examine hemispheric dominance while learning auditory presented nouns. We designed a study in which the participants memorized auditory presented verbal stimuli while direct current stimulation was delivered to the DLPFC with a view to examining the modulatory impact of this on short-term learning and long-term retrieval. We examined the behavioural effects of both left and right-side stimulation to gain more knowledge about the distinct or overlapping neural networks involved in learning verbal stimuli. To our knowledge, none of the studies that have applied tDCS to address issues in memory research have presented auditory verbal stimuli to test the effects of direct current on both short-term learning and subsequent long-term retrieval.Our results indicate that only cathodal tDCS elicits short-term behavioural effects on verbal memory performance. In particular, left-sided stimulation impaired memory performance compared with sham tDCS. The present study demonstrates that the left DLPFC plays a pivotal role while learning auditory presented verbal stimuli. It is remarkable that a complex cognitive function such as verbal memory can be modulated by external stimulation of the brain.The tDCS technique has a great potential for future applications. Due to the ease of utilisation, the tDCS method enables the testing of hypotheses on memory functions that emerging from basic neuroscience studies and neuroimaging protocols in humans with and without brain lesions . FurtherThe authors declare that they have no competing interests.SE designed the experimental paradigm, performed the statistical analysis and drafted the manuscript. MB contributed to the hypothesis, to the design and performed the data acquisition. BR contributed to the hypothesis, the design and performed the data acquisition. MM participated in the design/coordination of the study and contributed to the manuscript. LJ conceived of the study, contributed to the hypothesis, design, results, discussion and to the preparation of the manuscript. All authors read and approved the final manuscript.


Arabidopsis thaliana.Recent years have seen a dramatic increase in the use of mathematical modeling to gain insight into gene regulatory network behavior across many different organisms. In particular, there has been considerable interest in using mathematical tools to understand how multistable regulatory networks may contribute to developmental processes such as cell fate determination. Indeed, such a network may subserve the formation of unicellular leaf hairs (trichomes) in the model plant Arabidopsis, dimerization of the GL3-GL1 complex and cooperative sequential binding of GL3-GL1 to the CPC promoter are each independently sufficient for bistability.In order to investigate the capacity of small gene regulatory networks to generate multiple equilibria, we present a chemical reaction network (CRN)-based modeling formalism and describe a number of methods for CRN analysis in a parameter-free context. These methods are compared and applied to a full set of one-component subnetworks, as well as a large random sample from 40,680 similarly constructed two-component subnetworks. We find that positive feedback and cooperativity mediated by transcription factor (TF) dimerization is a requirement for one-component subnetwork bistability. For subnetworks with two components, the presence of these processes increases the probability that a randomly sampled subnetwork will exhibit multiple equilibria, although we find several examples of bistable two-component subnetworks that do not involve cooperative TF-promoter binding. In the specific case of epidermal differentiation in Arabidopsis epidermal cell differentiation subnetwork in particular, we illustrate how future experimental research may be guided by network structure analysis.Computational methods utilizing CRN-specific theorems to rule out bistability in small gene regulatory networks are far superior to techniques generally applicable to deterministic ODE systems. Using these methods to conduct an unbiased survey of parameter-free deterministic models of small networks, and the  The availability of high-throughput techniques for gene expression analysis and identification of promoter-transcription factor (TF) interactions has led to characterization of the intricate gene regulatory networks that govern organism behavior -3. TheseBistability in particular is ubiquitous in biological systems ranging from biochemical networks to ecosystems -16. In bArabidopsis thaliana. In Arabidopsis, trichomes differentiate from pluripotent epidermal cells by the action of regulatory proteins belonging to the R2R3-MYB  and basic helix-loop-helix (bHLH)  classes )Arabidopsis epidermal cell differentiation, we have shown that gene regulatory subnetworks consistent with established components and interactions may have the capacity for bistability. This capacity of course depends on the specific details of the network architecture. In particular, we found that dimerization of the active GL3-GL1 complex and cooperative sequential binding of GL3-GL1 to the CPC promoter are each independently sufficient to generate a bistable subnetwork, even in the absence of direct positive autoregulation. Both of these alternatives have some experimental support. Sequential binding of GL3-GL1 to the CPC promoter is plausible, as multiple TF binding sites in gene promoters are a general characteristic of eukaryotic gene expression [Applying the CRNT to the real-world example of pression . And whipression  either tpression , or thropression . BistabiArabidopsis is not in fact subserved by a bistable gene regulatory network. It may also be that some unknown extrinsic mechanism plays a role in Arabidopsis epidermal cell fate determination. A mechanism of this kind may render a bistable network irreversible by causing one equilibrium state to be inaccessible [If it can be determined that GL3-GL1 does not in fact dimerize or bind to two separate binding sites in the CPC promoter, and that it is in fact a stable complex, then the current view of the core trichome differentiation network  is a stand-alone DOS program that uses CRN theory ,44-47 tono, yes-, yes+; see Table no, yes) and, if so, whether the dimer-promoter complex is productive . Because this latter question is only relevant when dimer can form, there are 3 + 3.3 = 12 distinct one-component networks.One- and two-component subnetworks were generated in the Matlab programming environment . One-component networks are distinguished by whether TF monomer can bind the promoter and, if monomer binding does occur, whether the monomer-promotor complex results in transcription; thus there are three options with respect to monomer binding  are relabeled by exchanging the subscripts 1 and 2. The heterodimers P1P2 and P2P1 were assumed to be equivalent. These distinct possibilities for homodimer and heterodimer formation were subsequently instantiated multiple times to account for each possible pattern of monomer and dimer binding, resulting in 40,680 two-component networks.The generation of two-component networks involves enumerating all possibilities with regard to homodimer and heterodimer formation, being careful to avoid double counting of networks that are equivalent via symmetry, i.e., when all species (XDSG and GS performed all the computational and mathematical analyses. EG and GS were involved in the initial development of the concept. All authors contributed to the manuscript writing and have read and approved the final manuscript.The capacity for multistability in small gene regulatory networks: Supplementary Materials. Additional notes on the mathematical tools used and the role of positive and negative feedback in bistability. Also included are the SR graphs for the one component networks.Click here for file


This population-based cohort study evaluated the impact of primary androgen deprivation therapy (PADT) on cancer-specific and overall survival in elderly men with localized (T1-2) prostate cancer.Data of 89,877 men > 66 years with T1-2 prostate cancer diagnosed from 1992-2002 was obtained from the population-based Surveillance, Epidemiology, and End Results program (SEER) database. Exclusion criteria included men who either received definitive local therapy  within 180 days of diagnosis or had died during the same period, those with missing data or unknown cancer grade and those in whom ADT was started before cancer diagnosis. This left a group of 19,271 men of median age 77 years available for analysis; 7867 (41%) of these had received PADT in the form of luteinizing hormone-releasing hormone agonists or B/L orchiectomy as primary cancer therapy during the first 180 days following diagnosis whereas 11,404 (59%) had been conservatively managed during this period.Overall and cancer-specific survival was analyzed in these two groups using instrument variable analysis (IVA) technique. The instrumental variables used for the two treatment groups, i.e. PADT and conservative management were high PADT use area and low PADT use area, respectively. These corresponded to the top and bottom tertiles of PADT utilization areas which were calculated from the proportion of patients receiving PADT in each health service area.There were 11,045 deaths from all causes in the study group of which 1560 were prostate cancer-related deaths .P value: < 0.001) though no difference was seen in the overall survival. However, similar analysis done in the subset of patients with poorly differentiated cancer (Gleason score 8-10) showed a borderline improvement in cancer-specific survival in the PADT group as compared to the conservative management group  though again no significant difference was seen in the overall survival.Using IVA, patients in the PADT group had lower 10-year prostate cancer-specific survival  and no increase in 10-year overall survival  when compared with patients on conservative management. IVA in the subset of patients with moderately differentiated cancer (Gleason score 5-7) again revealed significantly poorer prostate cancer-specific survival in the PADT group as compared to the conservative management group (In patients receiving PADT and surviving at least three years (5826 patients), cancer-specific and overall mortality was lower in those receiving therapy for < 12 months as compared to those receiving therapy for longer periods (13-36 months), irrespective of grade of tumor.Based on the above findings, the authors concluded that PADT is not associated with improved survival when compared with conservative management in the majority of elderly men with localized cancer prostate.Although PADT for localized cancer prostate is not advocated as standard therapy by major guidelines,–3 its usA plausible explanation for the findings may be that when well/ moderately differentiated cells, which may never have harmed the patients overall survival are suppressed by instituting ADT, rapidly growing malignant clones may establish and overgrow. This would then increase the probability of death due to prostate cancer instead of a competing cause of death.In the light of the above findings, the fact that more data on PADT use in this setting is currently lacking and the increasing knowledge that ADT may be associated not only with impotency, gynecomastia, hot flashes etc., but also may increase the risk of fracture, diabetes mellitus, coronary heart disease and sudden cardiac death, the clin


A reversible cell labelling method has been developed for non-destructive and non-invasive cell labelling and purification. Our method uses high affinity single strand DNA (ssDNA) aptamers against surface exposed target molecules on cells. The aptamers are subsequently removed from the cell surface using DNase nuclease treatment. We exemplified our method by labelling human acute lymphoblastic leukemia cells with Qdot-ssDNA aptamers, and restoring them to the label-free condition by treatment with Benzonase. Binding of the fluorescent-aptamers to the cells was evaluated by measuring fluorescence intensity and was further confirmed using flow cytometry. Removal of the aptamers can be achieved in ~10 min by the DNase nuclease digestion. Incubation of cells with aptamers or with the nucleases results in no apparent damage to the cells and does not affect their growth rates. The latter were equivalent to the rates measured for the untreated cells. Our method provides an alternative to traditional antibody-based techniques and could be especially suitable for non-invasive reversible cell labelling and cell separations where maintaining native cell activity is needed. Separation of uniform phenotype cells whilst maintaining their native states is important both for post genome cell based studies and practical applications of those cells for regenerative medicine. A number of cell separation methods have been reported to date, such as density-gradient centrifugation, antibiotic screening and fluorescence-activated cell sorting (FACS). The latter gained significant popularity because of its simplicity and throughput. One of the problems of the FACS and other antibody-based labelling and separation techniques is that strong, often nearly irreversible conjugation between antibody and antigen molecules on cell surface may affect the function of the targeted cells and their interaction efficacy with other cells .As an alternative to antibodies, we developed nucleotide based probes capable of binding target cell antigens . Aptamer2 atmosphere in RPMI 1640 medium containing 10% heat-inactivated FBS, 100 IU/ml penicillin, and 100 μg/ml streptomycin .Human acute lymphoblastic leukemia cells, CCRF-CEM , were cultured at 37°C under a 5% CO5'-Biotin-labeled ssDNA was used as the target DNA aptamers for specific CCRF-CEM cell labelling, and random sequence ssDNA consisting of the same length and base composition as the target DNA aptamers were used as negative control DNA aptamers . SequencFigure Next, the 5'-Biotin of the ssDNA aptamers bound to the cells were labelled by addition of 0.1 μM Qdot-streptavidin conjugate . After 60 min incubation at 4°C, the cells were washed three times with the cold culture medium to remove excess free Qdot. These Qdot-aptamer labelled cells (aptamer cells) were then characterised by fow cytometry. To confirm the specificity of aptamer binding, both the target-specific aptamers and the negative control aptamers (irrelevant sequences) were used.6 cells in flow cytometry buffer  were measured by counting 30,000 events.Fluorescence measurements were made using an LSR II flow cytometer . Qdot fluorescence of samples initially containing 10To remove the attached Qdot-aptamer from cells, the aptamer cells were incubated in the pre-incubation culture medium containing 0.4 Unit/μL Benzonase nuclease  at 37°C for 10 min. After the incubation, the cells were washed three times with the cold culture medium, and were characterised by flow cytometry as described above.To evaluate the invasiveness of nuclease digestion, the growth rates of the three kinds of aptamer-treated cells were measured after Benzonase nuclease treatment: cells treated with target-specific aptamer, with the negative control aptamer treatment, and those with no aptamer treatment. Aptamer attachment and Benzonase nuclease treatment proceeded as described above, and then cultured in a medium for six days. The same number of non-treated intact cells was also cultured as control. In each sample, the number of cells collected by centrifugation was counted using a Burker-Turk counting chamber. The growth rate represented the cell number normalized by those as 100% when growth-measurement started (day 0).To this end we have developed and evaluated the procedure of non-destructive and reversible fluorescent labelling of live cells using DNA-aptamers (see Figure To evaluate possible damage to cells during labelling or nuclease digestion procedures we measured and compared the growth rates of intact and treated cells. Figure The conventional SELEX method is aimed at the development of DNA/RNA based drug-like molecules, and therefore aptamers need to be resistant to nuclease activity under physiological conditions, e.g. to plasma 3'-exonucleases ,9. Some In conclusion, we developed a reversible cell-labelling procedure in which live cells are labelled with fluorescent DNA aptamers, followed by their removal with nuclease treatment. Our labelling procedure produces no noticeable damage to the labelled cells. Our method is advantageous over traditional antibody labelling because it allows non-invasive cell separation and reversible cell labelling. It has a great promise for a variety of applications ranging from cell-based research to regenerative medicine.HT carried out whole experiments and participated in the design of the study and contributed to the drafting of the manuscript. YA carried out aptamer quality check and cell cultivation experiments. MS participated in the design of the study and the interpretation of results, and contributed to the drafting of the manuscript. KY conceived of the study, participated in its design and coordination and drafted the manuscript. All authors read and approved the final manuscript.The authors declare that they have no competing interests.


Communication between chloroplasts and the nucleus is one of the milestones of the evolution of plants on earth. Proteins encoded by ancestral chloroplast-endogenous genes were transferred to the nucleus during the endosymbiotic evolution and originated this communication, which is mainly dependent on specific transit-peptides. However, the identification of nuclear-encoded proteins targeted to the chloroplast lacking these canonical signals suggests the existence of an alternative cellular pathway tuning this metabolic crosstalk. Non-coding RNAS (NcRNAs) are increasingly recognized as regulators of gene expression as they play roles previously believed to correspond to proteins. Avsunviroidae family viroids are the only noncoding functional RNAs that have been reported to traffic inside the chloroplasts. Elucidating mechanisms used by these pathogens to enter this organelle will unearth novel transport pathways in plant cells. Here we show that a viroid-derived NcRNA acting as a 5′UTR-end mediates the functional import of Green Fluorescent Protein (GFP) mRNA into chloroplast. This claim is supported by the observation at confocal microscopy of a selective accumulation of GFP in the chloroplast of the leaves expressing the chimeric vd-5′UTR/GFP and by the detection of the GFP mRNA in chloroplasts isolated from cells expressing this construct. These results support the existence of an alternative signaling mechanism in plants between the host cell and chloroplasts, where an ncRNA functions as a key regulatory molecule to control the accumulation of nuclear-encoded proteins in this organelle. In addition, our findings provide a conceptual framework to develop new biotechnological tools in systems using plant chloroplast as bioreactors. Finally, viroids of the family Avsunviroidae have probably evolved to subvert this signaling mechanism to regulate their differential traffic into the chloroplast of infected cells. Chloroplasts are the hallmark organelle of photosynthetic eukaryotes The exchange of genetic information between the nucleus and the chloroplast has resulted in a close coordination in the activities of these two organelles during plant development which is mediated by both anterograde (nucleus-chloroplast) and retrograde (chloroplast-nucleus) signals High-throughput sequencing methods have unveiled a complex network of transcripts derived from the eukaryotic genome that include a large number of noncoding RNAs (ncRNAs) Pospiviroidae and the Avsunviroidae. Members of the family Avsunviroidae replicate and accumulate in the chloroplast and are the only functional RNAs that have been reported to traffic inside this organelle Viroids are small ncRNAs that infect plants N. benthamiana cells, thus supporting the existence of a novel signaling mechanism between the host cell and these organelles.Here we report that a viroid-derived ncRNA sequence acting as 5′UTR (vd-5′UTR) mediates the specific import of a functional GFP mRNA into the chloroplasts of Eggplant latent viroid (ELVd) Agrobacterium tumefaciens. The correct transcriptional fusion of the vd-5′UTR-GFP fragments was checked by DNA sequencing and by in silico translation of the chimeric mRNA  thus confirming that the GFP translated from the chimeric transcript vd-5′UTR/GFP accumulates specifically in this organelle.When the infiltrated plants were analyzed by confocal microscopy we observed that the unmodified GFP (used as a control) was uniformly distributed in the nucleus and cytoplasm of the examined cells , while tN. benthamiana cells expressing the GFP or vd-5′UTR/GFP constructs  The viroid-derived cDNA fragment was obtained by PCR, starting from the cDNA of ELVd (AN - AJ536613) Total proteins were extracted by grinding four leaf discs (5.5 mm diameter) in 100 µl of protein extraction buffer . The homogenate was centrifuged for 1 min at 12.000 rpm in a micro centrifuge and the supernatant collected. The supernatant (30 µl) was denatured and fractionated by 10% SDS-PAGE and transferred to Hybond™-P membranes . Membranes were blocked for 1 h  and incubated overnight at 4°C with an Anti-Green fluorescent protein (GFP) mouse IgG monoclonal antibody . Membranes were washed , incubated with ECL™ Peroxidase labeled anti-mouse antibody, and revealed by luminescence  according to the manufacturer instructions. A similar construct carrying unmodified GFP was used as control.N. benthamiana plants were infiltrated with the 5′UTR-GFP or GFP constructs as previously described The Chloroplasts were isolated from plants infiltrated with the vd-5′UTR/GFP or GFP constructs respectively, following a modification of a previous protocol N. benthamiana leaves using TRI reagent  according to the manufacturer instructions. RT-PCR analysis was performed as previously described The RNAs were extracted from isolated chloroplasts and


Application of virtual environment (VE) technology to motor rehabilitation increases the number of possible rehabilitation tasks and/or exercises. However, enhancing a specific sensory stimulus sometimes causes unpleasant sensations or fatigue, which would in turn decrease motivation for continuous rehabilitation. To select appropriate tasks and/or exercises for individuals, evaluation of physical activity during recovery is necessary, particularly the changes in the relationship between autonomic nervous activity (ANA) and sensory stimuli.We estimated the ANA from the R-R interval time series of electrocardiogram and incoming sensory stimuli that would activate the ANA. For experiments in real exercise, we measured vehicle data and electromyogram signals during cycling exercise. For experiments in virtual exercise, we measured eye movement in relation to image motion vectors while the subject was viewing a mountain-bike video image from a first-person viewpoint.For the real cycling exercise, the results were categorized into four groups by evaluating muscle fatigue in relation to the ANA. They suggested that fatigue should be evaluated on the basis of not only muscle activity but also autonomic nervous regulation after exercise. For the virtual exercise, the ANA-related conditions revealed a remarkable time distribution of trigger points that would change eye movement and evoke unpleasant sensations.For expanding the options of motor rehabilitation using VE technology, approaches need to be developed for simultaneously monitoring and separately evaluating the activation of autonomic nervous regulation in relation to neuromuscular and sensory systems with different time scales. It takes a long time for functional recovery in motor rehabilitation, and providing appropriate tasks and/or exercises during the progression of recovery is necessary to continue promoting motor rehabilitation with sufficient effectiveness, as well to motivate the patient. Current virtual reality (VR) and virtual environment (VE) technologies are now being applied to rehabilitation engineering  because However, enhancing or augmenting a specific sensory stimulus in a VE sometimes causes unpleasant sensations due to conflicts among sensory stimuli (sensory conflict theory ). This pPhysical activity mainly consists of several functional components with different time scales. Autonomic nervous activity (ANA) dominantly regulates the person's physical conditions after exercise or exercise-related sensory stimuli for several seconds. In contrast, muscle activity and sensory activity work within a few tens of milliseconds. When selecting appropriate tasks and/or exercises for individuals, we should consider the relationship between ANA and sensory stimuli.We conducted a feasibility study of using autonomic nervous regulation in response to several sensory stimuli, for real cycling and for virtual mountain biking using a first-person-view video image. We estimated the ANA from the R-R interval time series of electrocardiogram (ECG) and incoming sensory stimuli that would activate the ANA. To evaluate the exercise-related factors in real exercise, we measured vehicle data and electromyogram (EMG) signals during the cycling exercise. We measured the eye movement of the subject in relation to image motion vectors while he or she was viewing the first-person-view vection-inducing mountain-bike video images. Although muscle contractions generally elicit a strong demand on the ANA, visual stimuli are not always the strong demand for everyone. Accordingly, we carefully considered where and when the incoming stimuli and the ANA should be evaluated.Since autonomic nervous regulation should be evaluated after incoming stimuli, we focused on the specific sections before and after climbing a hill on a bicycle in the real world Figure  and the The subjects were volunteers and were informed of the risks involved and signed a consent form in advance, and were free to withdraw at any time during the experiment. For biosignal processing, a time interval of over a few minutes is necessary to estimate the ANA, even though the exercises or exercise-related sensory stimuli are very short events. A trial consisted of a series of events followed by enough rest to estimate the ANA.For the real exercise , the subFor the virtual exercise ,11, the At the sensory systems level, we used the correlation coefficient to derive the relationship between the external sensory stimuli and those responses due to different time scales. For evaluating the response to external sensory stimuli, we compared the behavior of ANA-related indices before and after the stimuli.ARV-trq. As a muscle fatigue-related index, we used the mean power frequency (MPF) from the EMG signals and calculated the correlation coefficient between ARV and MPF, γARV-MPF. These correlation coefficients were obtained from samples estimated with a sliding 50-ms interval every 25 ms during each pedal-stroke interval of 400 ms. Surveying the results from around 200 contractions for each trial, we selected five consecutive pedal strokes immediately before the hilltop during climbing and averaged γARV-trq and γARV-MPF. Grouping was done for every trial with γARV-MPF and ANA-related indices. We estimated the ANA-related indices for each phase from the R-R interval time series by using the continuous wavelet transform. For the real exercise, the focused frequency band was related to the respiratory sinus arrhythmia (RSA) [prRSA.In the real exercise, the strongest demand on ANA was from muscle contractions. We used the average rectified value (ARV) from the EMG signals as a muscle-force-related index ,12, thenia (RSA) , which hGMV-eye, every 10 s as the sensory response. Identifying the input visual stimuli for unpleasant sensations is difficult because they are relatively weak. We obtained a time-varying ANA-related indices for every frame (30 frame/s) with a 10-s interval from the R-R interval, the respiration, and the blood pressure time series by using the continuous wavelet transform. The focused frequency bands of the indices were 0.04–0.15 Hz (Mayer wave related low-frequency (LF) band) and 0.16–0.45 Hz (RSA related high-frequency (HF) band). The LF and HF components for five consecutive tasks were further normalized using the average LF and HF components estimated during a preceding rest period, respectively. We then defined some sensation section (SSS) on the basis of three ANA-related conditions [tg, by searching the local minimum of the LF component backwards in time. To screen the visually induced sensation before and after the video viewing  of the video images: GMV is a key technology in image data compression . We calcg Figure , we usedprRSA and γARV-MPF, we classified the 103 trials into four groups. First, we set the threshold at 20% of the average prRSA before climbing, estimating the median (21.3%) from all the trials. For a high-percentage prRSA (HRSA) before climbing, a large fluctuation occurred in the R-R interval before and after climbing, specifically during the rest periods [prRSA (LRSA) before climbing. Second, we used the γARV-MPF immediately before the hilltop because the samples for the positive γARV-MPF region showed the largest shift in prRSA in relation to climbing efforts. During the first half of a peal stroke, positive and negative γARV-MPF means increasing muscle activity and muscle fatigue, respectively [ARV-MPF means decreasing and disappearing muscle activity, respectively. We represent positive γARV-MPF as increasing or decreasing muscle activity (I/D) and negative γARV-MPF as fatigue or disappearing muscle activity (F/D).The participants in the real exercise were 13 healthy volunteers . Using  periods . A littlectively ,12. DuriprRSA and the sign of γARV-MPF for five consecutive pedal strokes immediately before the hilltop and plotted the results in a scatter graph , especially for the HRSA-I/D group. Contrary to our expectation for torque-assisted bicycles, the torque-assist supported the appearance of HRSA, but it was sometimes not enough for muscle fatigue.We categorized each trial into one of four groups on the basis of the median of h Figure . Table 1GMV-eye showed the similar behavior to the trigger points for the first task, but did not after the second task. That is, around the 71–80-s segment, the subjects' eyes relatively followed the camera motion for the first task (γGMV-eye = 0.4), but γGMV-eye decreased after the second task. This did not occur for the "non-unpleasant" group. Figure t-test, p < 0.05).Fifteen healthy men (21.9 ± 0.9 years) voluntarily participated in the virtual exercise. Nine experienced unpleasant sensations while watching the mountain-bike video and six did not, as classified by their total SSQ scores. The time distribution of the total 60 trigger points for each 10-s segment is shown in Figure A virtual environment increases the number of options for selecting approaches to motor rehabilitation. The options range from active exercise in the real world with muscle contractions to passive exercise in the virtual world with visual stimulation. To provide appropriate tasks and/or exercises for individuals undergoing motor rehabilitation, we focused on the impact of external sensory stimuli and autonomic nervous regulation as their responses.ARV-trq reflects a mismatch between muscle activity and pedal torque resulting from poor pedaling skills. This mismatch could increase muscular fatigue, resulting in strong requests for autonomic regulation after climbing, even with the power-assist on. However, γARV-trq did not significantly differ among the four groups (Table prRSA was not fully linked to muscular fatigue (Figure prRSA is necessary for preventing muscular fatigue in motor rehabilitation.Autonomic regulation is important in a series of repetitive exercises because it controls the cardiovascular system. During real exercise, the muscle sympathetic nerve activity activated by strong muscle contractions elicits autonomic nervous responses ,16. Thise Figure . Refer tIn contrast, the ANA was more difficult to distinguish during virtual exercise than during real exercise, according to the results for the first-person-view vection-inducing video images. We defined the ANA-related conditions and obtained a remarkable time distribution of trigger points that would evoke unpleasant sensations in ANA Figure . Around The level in the disturbance of autonomic regulation depends on the individual. Therefore, to provide appropriate tasks and/or exercises as recovery progresses, we need to simultaneously monitor and separately evaluate the neuromuscular and sensory systems and autonomic regulation as their responses. Appropriate tasks and/or exercises in motor rehabilitation will properly activate the ANA by neuromuscular and sensory systems: muscle contractions in real exercise and visual stimuli in virtual exercise are trigger factors. The autonomic nervous system receives many types of stimuli from different sensory systems with different time scales and seems to set individual priority for autonomic responses. Since the threshold between positive and negative effects would vary even for the same stimuli, depending on the behavior of autonomic nervous regulation, the differences between real and virtual exercises should be studied in terms of the ANA-related indices. We did a preliminary cross-validation study between real and virtual exercises for the same nine subjects, but we have not yet identified specific features. Further cross-validation studies should provide hints for designing continuous repetitive training or exercises for motor rehabilitation.We investigated the process of repetitive training or exercises to be used for continuous motor rehabilitation with sufficient effectiveness and motivation by comparing real and virtual exercises. The evaluated factors were muscle activity and vision properties depending on the type of task and exercises as well as the autonomic nervous activity estimated from the heart rate variability. Our results showed that fatigue in the real world should be evaluated on the basis of not only muscle activity but also autonomic nervous regulation after exercise. Moreover, unpleasant sensations in the virtual world should be checked first in terms of vision properties and then in terms of autonomic nervous regulation. To expand the options for motor rehabilitation using virtual environment technology, we need to develop approaches for simultaneously monitoring and separately evaluating the activation of autonomic nervous regulation in relation to neuromuscular and sensory systems with different time scales.


Ultrasound-guided vacuum-assisted breast biopsy technology is extremely useful for diagnostic biopsy of suspicious breast lesions and for attempted complete excision of appropriately selected presumed benign breast lesions.® excision procedures performed during seven separate sessions. Additionally, two of these 16 breast lesions were removed by open surgical excision. A histopathologic diagnosis of fibroadenoma and/or fibroadenomatous changes was confirmed at all lesion excision sites. Interval follow-up ultrasound imaging revealed no evidence of a residual lesion at the site of any of the 16 original breast lesions.A female patient presented with 16 breast lesions (eight within each breast), documented on ultrasound and all presumed to be fibroadenomas. Over a ten and one-half month period of time, 14 of these 16 breast lesions were removed under ultrasound guidance during a total of 11 separate 8-gauge MammotomeThis report describes an innovative approach of utilizing ultrasound-guided 8-gauge vacuum-assisted breast biopsy technology for assisting in achieving complete eradication of multiple bilateral fibroadenomas in a patient who presented with 16 documented breast lesions. As such, this innovative approach is highly recommended in similar appropriately selected patients. In aThe present report describes an innovative approach of utilizing an ultrasound-guided vacuum-assisted excision technique with the 8-gauge breast biopsy system described above -10 for aA 21 year-old, healthy, white female presented to our comprehensive breast health services facility with multiple bilateral breast lesions that were presumed to represent multiple bilateral fibroadenomas. She reported having previously undergone a surgical excision procedure of a fibroadenoma of each of her breasts at a local community hospital at a time of approximately three years prior to her current presentation.Prior to her current presentation at our facility, the patient was evaluated at an out-of-state university medical center and was noted to have multiple ultrasound lesions within both of her breasts. Due to the multiplicity of the bilateral breast lesions and the complexity of her particular case, the breast surgeon at that out-of-state university medical facility told the patient and her family that her "only realistic option" was for bilateral nipple-sparing mastectomies and expander-to-implant reconstruction.Upon her presentation to our facility, the patient reported that she had noticed an enlarging mass of her central right breast over the last two years and two smaller palpable masses of her left breast. On clinical breast examination, she had medium-sized breasts (36-C cup size). She had an old surgical scar extending from the 8 to 10 o'clock axis of the periareolar aspect of her right breast and similar surgical scar extending from the 10 to 12 o'clock axis of the periareolar aspect of her left breast. Most remarkably, she was noted to have a 10 cm × 7 cm × 5 cm clinically palpable mass encompassing her entire right breast and an overlying 1.5 cm clinically palpable mass in the in the 3 o'clock axis of the periareolar aspect of her right breast. Two palpable masses were also noted in her left breast, clinically measuring 3 cm in size in the 3 to 4 o'clock axis of the mid-breast field and 2 cm in size in the subareolar region. No axillary, supraclavicular, or cervical adenopathy was noted. The patient also reported being on hormonal contraception for the past five years. For the last 18 months, she was maintained on a combination transdermal contraception product containing norelgestromin (6 mg) and ethinyl estradiol (0.75 mg).Bilateral breast imaging was performed with both bilateral whole-breast ultrasound and bilateral breast MRI. Ultimately, a total of 16 separate breast lesions were identified on both ultrasound and MRI, consisting of eight lesions documented in each breast  was completely avoided in this particular case. Therefore, the presentation of this case nicely illustrates the contention of the present author that this ultrasound-guided 8-gauge vacuum-assisted excision technique should be considered as a viable alternative or as an adjunct to consideration of much more radical open surgical approaches when faced with the daunting task of removing multiple bilateral fibroadenomas. As such, this innovative approach is highly recommended in similar appropriately selected patients.The discontinuation of the patient's hormonal contraception appeared to be highly effective in successfully reducing the size of two of the left breast lesions (lesion number 9 and 14) and was therefore crucial in allowing for successful complete excision of these two lesions that were previously deemed as too large for consideration of attempted excision by the ultrasound-guided 8-gauge vacuum-assisted excision technique, based on the current author's previously published size and shape criteria for predicting successful lesion excision . The conFrequently, when a patient presents with a palpable event that represents a fibroadenoma, it is not uncommon to identified one or more additional lesions within the ipsilateral breast or contralateral breast on diagnostic breast ultrasound that represent other nonpalpable fibroadenomas candidates. This particular situation represents a scenario that is extremely appropriate for consideration of using an 8-gauge vacuum-assisted breast biopsy system for attempting ultrasound-guided excision of all presumed fibroadenoma candidates. Such an innovative and pro-active approach may allow for achieving complete eradication of multiple fibroadenomas candidates and avoiding later surgical excision of these lesions if their continued growth pattern were to later preclude future attempted ultrasound-guided 8-gauge vacuum-assisted excision. This approach would be particularly useful for those patients who you may suspect are inclined not to adhere to a regimented follow-up schedule for ongoing monitoring of such lesions that you may otherwise leave in place for such an ongoing monitoring strategy. Likewise, this approach would also be particularly useful for those patients on hormonal contraception in whom you are concerned about ongoing stimulation of the growth pattern of pre-existing fibroadenomas in order to prevent a situation in which a fibroadenoma which was previously amendable to attempted ultrasound-guided 8-gauge vacuum-assisted excision then becomes only removable by a surgical excision procedure. For obvious reasons, such an approach which maximizes cosmetic outcome by eliminating the necessity for creating major surgical scars on the breast is one that is of particular importance to young female patients and should be factored into the decision-making process of how to manage these patients.As a last point of interest, and has been previously discussed by the present author , the ultThis report describes, for the first time, an innovative approach of utilizing an ultrasound-guided vacuum-assisted excision technique with the previously mentioned 8-gauge breast biopsy system -10 for aThe author(s) declare that they have no competing interests.SPP was the surgeon who performed each of the ultrasound-guided 8-gauge vacuum-assisted excision procedures and the surgical excision procedure. He designed the current study and collected all the data. He organized, wrote, and edited all aspects of this manuscript. He approved the final version of this manuscript.


We report an interesting ocular finding of bilateral multiple coin-shaped epithelial lesions along with the confocal microscopy findings in a patient following an acute attack of erythema multiforme (EM) minor. A 30-year-old male presented with a history of watering and irritation in both eyes of three days duration. He was diagnosed to have EM minor and was on oral acyclovir. Slit-lamp examination revealed multiple coin-shaped epithelial lesions. Confocal microscopy showed a corresponding conglomerate of hyper-reflective epithelial lesions. The corneal lesions resolved over six weeks with oral steroids and acyclovir. An immunological mechanism is suspected. Ocular manifestations following Steven-Johnson's syndrome (SJS) is common and have been well-described in literature.2 However3 with predominant neutrophils (72%). Routine urine examination was normal. Blood and urine cultures were negative for bacteria and fungi. Chest X-ray was normal. Antinuclear antibody and rheumatoid factor were negative. He was on oral acyclovir 800 mg five times/day, started by his treating dermatologist, given for a total of seven days.A 30-year-old male presented to us with a history of watering and irritation in both eyes of three days duration. He was diagnosed to have EM minor following an attack of viral fever with skin eruptions on the face, arms and legs along with shallow, aphthous ulcers, one week back. There was no history of any drug intake leading to the fever and occurrence of skin eruptions. The erythrocyte sedimentation rate (ESR) was 42 mm/h and the total white blood count was 12,000 cells/mmOn examination, he had papular skin eruptions on the face, arms and legs with two shallow painful ulcers in the buccal mucosa . His bes+ T lymphocytes in the epidermis, induce apoptosis of scattered keratinocytes and lead to satellite cell necrosis.Erythema multiforme is an acute mucocutaneous hypersensitivity reaction characterized by a skin eruption, with or without oral or other mucous membrane lesions. EM has been classified into a number of different variants based on the degree of mucosal involvement and the nature and distribution of the skin lesions. EM major  is more severe, typically involving two or more mucous membranes with more variable skin involvement – which is used to distinguish it from SJS, where there is extensive skin involvement and significant mortality and morbidity of 5-15%. These feOn the other hand, EM minor typically affects no more than one mucosa, is the most common form and may be associated with symmetrical target lesions on the extremities. Herpes simplex virus-induced EM minor constitutes 15-60% of EM minor. The treatment is a combination of acyclovir and oral steroids.Ocular involvement in EM minor is rare. Unlike SJS, ocular findings in EM minor have not been very well-described in literature (Medline search).245 We re24In this case report, an interesting ocular finding of coin-shaped epithelial lesions with central clearing along with the confocal microscopy findings is reported in a patient following an acute attack of EM minor.


Cucurbita maxima trypsin inhibitor (CMTI-I) and glycerol at the AM1 level. An effective potential, combining non-bonding and polarization plus charge transfer (PLCT) terms, was introduced to evaluate the magnitude of the interaction between each amino acid and the ligand. In this case study, the nonbonding–PLCT non-compensation characterizes the stabilization energy of the association process in study. The main residues  with net attractive effects and Arg1 (with a net repulsive effect), responsible by the stability of protein-ligand complex, are associated with large nonbonding energies non-compensated by PLCT effects. The results obtained enable us to conclude that the present decomposition scheme can be used for understanding the cohesive phenomena in proteins.In this work, the partition method introduced by Carvalho and Melo was used to study the complex between  In vitro methods and mathematical models for enzymatic reactions [Computational methods are of great interest to evaluate binding affinities between proteins and ligands, with many applications in structure-based drug design (SBDD) . A compleactions –16 are tIn this work, the application of the SemiEmpirical Energy Based (SEEB) partition method introduced by Carvalho and Melo  to studyA non-covalent (no) association between a protein (P) with n amino acid residues and a ligand (L) can be represented by A general association process can be described by a hypothetical mechanism involving two steps see . In the In the second step, the rearranged species (P(rearr) and L(rearr)) associate each other preserving their geometries and originating the dimer (P:L). According to the SEEB formalism, the stabilization energy can be partitioned as:In In An effective pair-wise potential, combining non-bonding and PLCT contributions, is proposed in this work:In this context, the The transformation of the exact interaction energy decomposition  into theIn consistency with the SEEB formalism, the stabilization energy associated with step 2 can be also partitioned into strongly perturbed  were calculated using both Mulliken [where qce field  was usedMulliken  and MerzMulliken  schemes.In this work, the association of CMTI-I and glycerol was studied at an semiempirical level , AM1, usThe physically meaningful components of the complex CMTI-I glycerol stabilization energy, obtained using the modified SEEB formalism, are presented in The nonbonding interaction energies between the amino acids and the ligand are presented in −1. Three residues  are involved in specific hydrogen bonds with the hydroxyl groups of glycerol  have the most relevant contributions, which correspond to absolute values larger than 10 kJ molerol see . Arg5 isIn the complex in study, the most electropositive groups of glycerol are oriented in the opposite direction of this guanidinium-carboxylate salt-bridge see  and 4. T−1 and/or a nonbonding interaction with ligand larger in absolute value larger than 3.0 kJ mol−1 were included in the strongly perturbed region. Twelve residues  satisfied this requirement.Residue addictive effective PLCT terms, defined according to −1) for the stabilization energy. A fifth residue (Cys28) had been previously identified as relevant, because its nonbonding interaction energy with ligand is markedly negative (–15.2 kJ mol−1). However, this residue has an opposite effective PLCT energy of 15.8 kJ mol−1 and its overall contribution is near null.The global contributions of the amino acid residues for the stability of the CMTI-I:glycerol complex was evaluated using the effective pair-wise potential introduced in the previous section. The results obtained are presented in The partition into the strongly perturbed and bulk components is presented in −1 representing only 6.3 % of the total stabilization energy (−29.05 kJ mol−1) and the long-range bulk component . The residues are also divided between a strongly perturbed and a bulk region. This spatial partition was carried out according to the appropriate requirements previously discussed. In fact, the correspondent short-range bulk component  pair-wise energies can be assumed as physically meaningful components, which can provide an important contribution to better understanding of the protein-ligand association processes.