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Background Early Intervention in Psychosis (EIP) services improve health outcomes for young people with psychosis in the medium-long term, but 25% of young people disengage in the first 12 months with costs to their mental health, families, society and health services. This study will evaluate the effectiveness of a team-based motivational engagement intervention, the Early Youth Engagement (EYE-2) intervention. Methods and design The EYE-2 trial is a cluster randomised controlled trial comparing the EYE-2 intervention plus standardised EIP service to standardised EIP service alone, with randomisation at the clinical team (cluster) level. The study aimed to enrol 950 young people (aged 14–35 years) with first episode psychosis in 10 teams per arm. Results The primary outcome is time to disengagement: days from the date of allocation to care coordinator to date of the last contact following either refusal to engage with an EIP team or lack of response to EIP contact for 3 consecutive months which will be analysed using a shared frailty model. Secondary outcomes are Health of the Nation Outcome Scale (HoNOS), Process of Recovery Questionnaire (QPR), DIALOG (a service user-reported measure of quality of life and treatment satisfaction) and service use outcomes which will be analysed using mixed effects regression models. Discussion This paper is the detailed statistical analysis plan for the EYE-2 trial. Any changes to, or deviations from, this plan will be described and justified in the final trial report. Trial registration ISRCTN 51629746. Prospectively registered on 7 May 2019. Date assigned 10 May 2019.

Background Early Intervention in Psychosis (EIP) services improve health outcomes for young people with psychosis in the medium–long term, but 25% of young people disengage in the first 12 months with costs to their mental health, families, society and the NHS. This study will evaluate the effectiveness, cost-effectiveness and implementation of a team-based motivational Early Youth Engagement (EYE-2) intervention. Method The study design is a cluster randomised controlled trial (RCT) with economic evaluation, comparing the EYE-2 intervention + standardised EIP service to standardised EIP service alone, with randomisation at the team level. A process evaluation will evaluate the delivery of the intervention qualitatively and quantitatively across contexts. The setting is 20 EIP teams in 5 sites: Manchester, South London, East Anglia, Thames Valley and Hampshire. Participants are young people (14–35 years) with first episode psychosis, and EIP staff. The intervention is the team-based motivational engagement (EYE-2) intervention, delivered alongside standardised EIP services, and supported by additional training, website, booklets and social groups. The comparator is the standardised EIP service. Both interventions are delivered by EIP clinicians. The primary outcome is time to disengagement (time in days from date of allocation to care coordinator to date of last contact following refusal to engage with EIP service, or lack of response to EIP contact for a consecutive 3-month period). Secondary outcomes include mental and physical health, deaths, social and occupational function, recovery, satisfaction and service use at 6, 12, 18 and 24 months. A 12-month within-trial economic evaluation will investigate cost-effectiveness from a societal perspective and from an NHS perspective. Discussion The trial will provide the first test of an engagement intervention in standardised care, with the potential for significant impact on the mental health and wellbeing of young people and their families, and economic benefits for services. The intervention will be highly scalable, supported by the toolkit including manuals, commissioning guide, training and resources, adapted to meet the needs of the diverse EIP population, and based on an in-depth process evaluation. Trial registration ISRCTN 51629746 prospectively registered 7th May 2019. Date assigned 10th May 2019.

Background:Cognitive behavioral therapy (CBT) is an efficacious intervention for generalized anxiety disorder (GAD). Digital CBT may provide a scalable means of delivering CBT at a population level. We investigated the efficacy of a novel digital CBT program in those with GAD for outcomes of anxiety, worry, depressive symptoms, sleep difficulty, wellbeing, and participant‐specific quality of life. Methods:This online, two‐arm parallel‐group superiority randomized controlled trial compared digital CBT with waitlist control in 256 participants with moderate‐to‐severe symptoms of GAD. Digital CBT (Daylight), was delivered using participants’ own smartphones. Online assessments took place at baseline (Week 0; immediately preceding randomization), mid‐intervention (Week 3; from randomization), post‐intervention (Week 6; primary endpoint), and follow‐up (Week 10). Results:Overall, 256 participants were randomized and intention‐to‐treat analysis foundDaylightreduced symptoms of anxiety compared with waitlist control at post‐intervention, reflecting a large effect size (adjusted difference [95% CI]: 3.22 [2.14, 4.31],d = 1.08). Significant improvements were found for measures of worry; depressive symptoms, sleep difficulty, wellbeing, and participant‐specific quality of life. Conclusion:Digital CBT (Daylight) appears to be safe and efficacious for symptoms of anxiety, worry, and further measures of mental health compared with waitlist control in individuals with GAD.

Background and objectives Cognitive behavioral therapy (CBT) is a first-line treatment for anxiety, but it is not widely available as clinical guidelines recommend. We examined the feasibility and efficacy of a novel smartphone-based fully automated digital CBT intervention, ‘Daylight™‘, to improve symptoms of Generalized Anxiety Disorder (GAD). Methods In this multiple-baseline design, 21 adults (20 F; mean age 43yrs. range 19–65yrs.) with moderate-to-severe symptoms of GAD were randomized to one of three baseline durations (2-, 4-, or 6-weeks) and then received access to digital CBT. Participants completed daily ratings of anxiety and worry, weekly measures of anxiety, depressive symptoms, and sleep, and measures of anxiety, worry, wellbeing, quality of life, CBT skill acquisition, and work performance at initial assessment prior to baseline randomization, post-intervention, and follow-up. Results Digital CBT was found to be feasible in terms of engagement, satisfaction, and safety. For preliminary efficacy, improvements were detected in daily and weekly outcomes of anxiety for most participants. Despite individual differences, significant improvements occurred with the introduction of digital CBT and not during baseline. Overall, 70% of participants no longer had clinically significant symptoms of GAD, 61% no longer had significant depressive symptoms, and 40% no longer had significant sleep difficulty at post-intervention. Limitations The study sample was recruited using the internet and was mostly female, limiting the generalizability of the findings. Conclusions Findings support the feasibility and efficacy of Daylight. Further examination in randomized controlled trials is now warranted.

In support of the construct validity of mindfulness questionnaires, meta-analytic reviews have reported that scores increase in mindfulness-based interventions (MBIs). However, several studies have also found increased mindfulness scores in interventions with no explicit mindfulness training, raising a question about differential sensitivity to change with treatment. We conducted a systematic review and meta-analysis of 37 randomized controlled trials in which mindfulness questionnaires were administered before and after an evidence-based MBI and a nonmindfulness-based active control condition. The central question was whether increases in mindfulness scores would be greater in the MBI than in the comparison group. On average, participants in MBIs showed significantly greater pre–post changes in mindfulness scores than were seen in active control conditions with no explicit mindfulness elements, with a small overall effect size. This effect was moderated by which mindfulness questionnaire was used, by the type of active control condition, and by whether the MBI and control were matched for amount of session time. When mindfulness facet scores were analyzed separately, MBIs showed significantly greater pre–post increases than active controls in observing, nonjudging, and nonreactivity but not in describing or acting with awareness. Although findings provide partial support for the differential sensitivity of mindfulness questionnaires to change with treatment, the nonsignificant difference in pre–post change when the MBI and control were matched for session time highlights the need to clarify how mindfulness skills are acquired in MBIs and in other interventions and whether revisions to mindfulness questionnaires would increase their specificity to changes in mindfulness skills., Public Significance Statement This review found that scores on mindfulness questionnaires increase more in mindfulness-based interventions than in interventions with no explicit mindfulness training, but not under all conditions. When the two treatments had equal session time, increases in mindfulness scores were similar. Nonmindfulness-based interventions may implicitly cultivate mindfulness skills, or the questionnaires may need revisions to increase their specificity to changes in mindfulness skills.

Compassion has received increasing societal and scientific interest in recent years. The science of compassion requires a tool that can offer valid and reliable measurement of the construct to allow examination of its causes, correlates, and consequences. The current studies developed and examined the psychometric properties of new self-report measures of compassion for others and for the self, the 20-item Sussex-Oxford Compassion for Others Scale (SOCS-O) and 20-item Sussex-Oxford Compassion for the Self Scale (SOCS-S). These were based on the theoretically and empirically supported definition of compassion as comprising five dimensions: (a) recognizing suffering, (b) understanding the universality of suffering, (c) feeling for the person suffering, (d) tolerating uncomfortable feelings, and (e) motivation to act/acting to alleviate suffering. Findings support the five-factor structure for both the SOCS-O and SOCS-S. Scores on both scales showed adequate internal consistency, interpretability, floor/ceiling effects, and convergent and discriminant validity.

Background Healthcare workers experience higher levels of work-related stress and higher rates of sickness absence than workers in other sectors. Psychological approaches have potential in providing healthcare workers with the knowledge and skills to recognise stress and to manage stress effectively. The strongest evidence for effectiveness in reducing stress in the workplace is for stress-management courses based on cognitive behavioural therapy (CBT) principles and mindfulness-based interventions (MBIs). However, research examining effects of these interventions on sickness absence (an objective indicator of stress) and compassion for others (an indicator of patient care) is limited, as is research on brief CBT stress-management courses (which may be more widely accessible) and on MBIs adapted for workplace settings. Methods/design This protocol is for two randomised controlled trials with participant preference between the two trials and 1:1 allocation to intervention or wait-list within the preferred choice. The first trial is examining a one-day CBT stress-management workshop and the second trial an 8-session Mindfulness-Based Cognitive Therapy for Life (MBCT-L) course, with both trials comparing intervention to wait-list. The primary outcome for both trials is stress post-intervention with secondary outcomes being sickness absence, compassion for others, depression symptoms, anxiety symptoms, wellbeing, work-related burnout, self-compassion, presenteeism, and mindfulness (MBCT-L only). Both trials aim to recruit 234 staff working in the National Health Service in the UK. Discussion This trial will examine whether a one-day CBT stress-management workshop and an 8-session MBCT-L course are effective at reducing healthcare staff stress and other mental health outcomes compared to wait-list, and, whether these interventions are effective at reducing sickness absence and presenteeism and at enhancing wellbeing, self-compassion, mindfulness and compassion for others. Findings will help inform approaches offered to reduce healthcare staff stress and other key variables. A note of caution is that individual-level approaches should only be part of the solution to reducing healthcare staff stress within a broader focus on organisational-level interventions and support. Trial registration ISRCTN Registry, ISRCTN11723441. Registered on 16 June 2017. Protocol Version 1: 24 April 2017. Trial Sponsor: Sussex Partnership NHS Foundation Trust (ResearchGovernance@sussexpartnership.nhs.uk). Electronic supplementary material The online version of this article (10.1186/s13063-018-2547-1) contains supplementary material, which is available to authorized users.

Given the extensive evidence base for the efficacy of mindfulness-based stress reduction (MBSR) and mindfulness-based cognitive therapy (MBCT), researchers have started to explore the mechanisms underlying their therapeutic effects on psychological outcomes, using methods of mediation analysis. No known studies have systematically reviewed and statistically integrated mediation studies in this field. The present study aimed to systematically review mediation studies in the literature on mindfulness-based interventions (MBIs), to identify potential psychological mechanisms underlying MBCT and MBSR's effects on psychological functioning and wellbeing, and evaluate the strength and consistency of evidence for each mechanism. For the identified mechanisms with sufficient evidence, quantitative synthesis using two-stage meta-analytic structural equation modelling (TSSEM) was used to examine whether these mechanisms mediate the impact of MBIs on clinical outcomes. This review identified strong, consistent evidence for cognitive and emotional reactivity, moderate and consistent evidence for mindfulness, rumination, and worry, and preliminary but insufficient evidence for self-compassion and psychological flexibility as mechanisms underlying MBIs. TSSEM demonstrated evidence for mindfulness, rumination and worry as significant mediators of the effects of MBIs on mental health outcomes. Most reviewed mediation studies have several key methodological shortcomings which preclude robust conclusions regarding mediation. However, they provide important groundwork on which future studies could build.

Structural variation (SV) is typically defined as variation within the human genome that exceeds 50 base pairs (bp). SV may be copy number neutral or it may involve duplications, deletions, and complex rearrangements. Recent studies have shown SV to be associated with many human diseases. However, studies of SV have been challenging due to technological constraints. With the advent of third generation (long-read) sequencing technology, exploration of longer stretches of DNA not easily examined previously has been made possible. In the present study, we utilized third generation (long-read) sequencing techniques to examine SV in the EGFR landscape of four haplotypes derived from two human samples. We analyzed the EGFR gene and its landscape (+/- 500,000 base pairs) using this approach and were able to identify a region of non-coding DNA with over 90% similarity to the most common activating EGFR mutation in non-small cell lung cancer. Based on previously published Alu-element genome instability algorithms, we propose a molecular mechanism to explain how this non-coding region of DNA may be interacting with and impacting the stability of the EGFR gene and potentially generating this cancer-driver gene. By these techniques, we were also able to identify previously hidden structural variation in the four haplotypes and in the human reference genome (hg38). We applied previously published algorithms to compare the relative stabilities of these five different EGFR gene landscape haplotypes to estimate their relative potentials to generate the EGFR exon 19, 15 bp canonical deletion. To our knowledge, the present study is the first to use the differences in genomic architecture between targeted cancer-linked phased haplotypes to estimate their relative potentials to form a common cancer-linked driver mutation.

Research into the effectiveness and mechanisms of mindfulness-based interventions (MBIs) requires reliable and valid measures of mindfulness. The 39-item Five Facet Mindfulness Questionnaire (FFMQ-39) is a measure of mindfulness commonly used to assess change before and after MBIs. However, the stability and invariance of the FFMQ factor structure have not yet been tested before and after an MBI; pre to post comparisons may not be valid if the structure changes over this period. Our primary aim was to examine the factor structure of the FFMQ-39 before and after mindfulness-based cognitive therapy (MBCT) in adults with recurrent depression in remission using confirmatory factor analysis (CFA). Additionally, we examined whether the factor structure of the 15-item version (FFMQ-15) was consistent with that of the FFMQ-39, and whether it was stable over MBCT. Our secondary aim was to assess the general psychometric properties of both versions. CFAs showed that pre-MBCT, a 4-factor hierarchical model (excluding the “observing” facet) best fit the FFMQ-39 and FFMQ-15 data, whereas post-MBCT, a 5-factor hierarchical model best fit the data for both versions. Configural invariance across the time points was not supported for both versions. Internal consistency and sensitivity to change were adequate for both versions. Both FFMQ versions did not differ significantly from each other in terms of convergent validity. Researchers should consider excluding the Observing subscale from comparisons of total scale/subscale scores before and after mindfulness interventions. Current findings support the use of the FFMQ-15 as an alternative measure in research where briefer forms are needed. (PsycINFO Database Record (c) 2016 APA, all rights reserved)

Compassion has long been regarded as a core part of our humanity by contemplative traditions, and in recent years, it has received growing research interest. Following a recent review of existing conceptualisations, compassion has been defined as consisting of the following five elements: 1) recognising suffering, 2) understanding the universality of suffering in human experience, 3) feeling moved by the person suffering and emotionally connecting with their distress, 4) tolerating uncomfortable feelings aroused (e.g., fear, distress) so that we remain open to and accepting of the person suffering, and 5) acting or being motivated to act to alleviate suffering. As a prerequisite to developing a high quality compassion measure and furthering research in this field, the current study empirically investigated the factor structure of the five-element definition using a combination of existing and newly generated self-report items. This study consisted of three stages: a systematic consultation with experts to review items from existing self-report measures of compassion and generate additional items (Stage 1), exploratory factor analysis of items gathered from Stage 1 to identify the underlying structure of compassion (Stage 2), and confirmatory factor analysis to validate the identified factor structure (Stage 3). Findings showed preliminary empirical support for a five-factor structure of compassion consistent with the five-element definition. However, findings indicated that the 'tolerating' factor may be problematic and not a core aspect of compassion. This possibility requires further empirical testing. Limitations with items from included measures lead us to recommend against using these items collectively to assess compassion. Instead, we call for the development of a new self-report measure of compassion, using the five-element definition to guide item generation. We recommend including newly generated 'tolerating' items in the initial item pool, to determine whether or not factor-level issues are resolved once item-level issues are addressed.

It is now well-known that proteins exist at equilibrium as ensembles of conformational states rather than as unique static structures. Here we review from an ensemble perspective important biological effects of such spontaneous fluctuations on protein allostery, function, and evolution. However, rather than present a thorough literature review on each subject, we focus instead on connecting these phenomena through the ensemble-based experimental, theoretical, and computational investigations from our laboratory over the past decade. Special emphasis is given to insights that run counter to some of the prevailing ideas that have emerged over the past 40 years of structural biology research. For instance, when proteins are viewed as conformational ensembles rather than as single structures, the commonly held notion of an allosteric pathway as an obligate series of individual structural distortions loses its meaning. Instead, allostery can result from energetic linkage between distal sites as one Boltzmann distribution of states transitions to another. Additionally, the emerging principles from this ensemble view of proteins have proven surprisingly useful in describing the role of intrinsic disorder in inter-domain communication, functional adaptation mediated by mutational control of fluctuations, and evolutionary conservation of the energetics of protein stability.

Pim-2 kinase is one of the three highly conserved Pim family members which are known to be involved in cell survival and cell proliferation. Here we demonstrate that like Pim-1, Pim-2 also phosphorylates the cell cycle inhibitor p21Cip1/WAF1 (p21) on Thr145 in vitro and in vivo. Overexpression of Pim-2 in HCT116 cells leads to the increased stability of p21 and results in enhanced levels of both exogenous and endogenous p21 proteins. Knockdown of Pim-2 expression via siRNA results in reduced level of endogenous p21, indicating that like Pim-1, Pim-2 is another legitimate p21 kinase. However, Pim-2 has no influence on the nuclear localization of p21 in HCT116 cells. In addition, Pim-2 is able to arrest the cell cycle at G1/S phase and inhibit cell proliferation through phosphorylation of p21 in HCT116 cells. These data suggest that Pim-2 phosphorylation of p21 enhances p21's stability and inhibits cell proliferation in HCT116 cells.

The importance of compassion is widely recognized and it is receiving increasing research attention. Yet, there is lack of consensus on definition and a paucity of psychometrically robust measures of this construct. Without an agreed definition and adequate measures, we cannot study compassion, measure compassion or evaluate whether interventions designed to enhance compassion are effective. In response, this paper proposes a definition of compassion and offers a systematic review of self- and observer-rated measures. Following consolidation of existing definitions, we propose that compassion consists of five elements: recognizing suffering, understanding the universality of human suffering, feeling for the person suffering, tolerating uncomfortable feelings, and motivation to act/acting to alleviate suffering. Three databases were searched (Web of Science, PsycInfo, and Medline) and nine measures included and rated for quality. Quality ratings ranged from 2 to 7 out of 14 with low ratings due to poor internal consistency for subscales, insufficient evidence for factor structure and/or failure to examine floor/ceiling effects, test-retest reliability, and discriminant validity. We call our five-element definition, and if supported, the development of a measure of compassion based on this operational definition, and which demonstrates adequate psychometric properties.

In contrast to other viral infections such as CMV, circulating frequencies of HIV-1-specific CD4+ T cells in peripheral blood are quantitatively diminished in the majority of HIV-1-infected individuals. One mechanism for this quantitative defect is preferential infection of HIV-1-specific CD4+ T cells, although

Bcl-2 proteins represent a rheostat that controls cellular viability. Obatoclax, a BH3-mimetic, has been designed to specifically target and counteract anti-apoptotic Bcl-2 proteins. We evaluated the biological effects of obatoclax on the anti-tumour activity of rituximab and chemotherapy agents. Obatoclax induced cell death of rituximab/chemotherapy-sensitive (RSCL), -resistant cell lines (RRCL) and primary tumour-cells derived from patients with B-cell lymphomas (N=39). Obatoclax also enhanced the activity of rituximab and had synergistic activity when combined with chemotherapy agents. The ability of Obatoclax to induce PARP cleavage varied between patient samples and was not observed in some RRCL. Inhibition of caspase activity did not affect obatoclax activity, suggesting the existence of caspase-independent death pathways. Autophagy was detected by LC3 conversion and/or electron microscopy in RRCL and in patient-derived tumour cells. Moreover, obatoclax activity was inhibited by Beclin-1 knockdown. In summary, obatoclax is an active Bcl-2 inhibitor that potentiates the activity of chemotherapy agents and, to a lesser degree, rituximab. Defining the molecular events triggered by obatoclax is necessary to further its clinical development and identify potential biomarkers that are predictive of response.

The serine/threonine kinase, PIM1, is involved in promoting cell survival in part by phosphorylation and inhibition of proapoptotic proteins. Apoptosis signaling kinase 1 (ASK1), a mitogen-activated protein kinase kinase kinase, is involved in the so-called stress-activated pathways that contribute to apoptotic cell death. Here we show that PIM1 phosphorylates ASK1 specifically on serine residue 83 (Ser83) both in vitro and in vivo and that PIM1 binds to ASK1 in cells by co-immunoprecipitation. Using H1299 cells, our results further demonstrate that PIM1 phosphorylation of ASK1 decreases its kinase activity induced by oxidative stress. PIM1 phosphorylation of ASK1 on Ser83 inhibited ASK1-mediated c-Jun N-terminal kinase phosphorylation as well as p38 kinase phosphorylation. Under H(2)O(2)-induced stress conditions that normally lead to apoptosis, these phosphorylation events were associated with inhibition of caspase-3 activation and resulted in reduced cell death. Moreover, knockdown of PIM1 in H1299 cells decreased phosphorylation of endogenous Ser83 of ASK1 and was associated with a decrease in cell viability after H(2)O(2) treatment. Taken together, these data reveal a novel mechanism by which PIM1 promotes cell survival that involves negative regulation of the stress-activated kinase, ASK1.

The presence of interleukin-2 (IL-2)-producing human immunodeficiency virus type 1 (HIV-1)-specific CD4 + T-cell responses has been associated with the immunological control of HIV-1 replication; however, the causal relationship between these factors remains unclear. Here we show that IL-2-producing HIV-1-specific CD4 + T cells can be cloned from acutely HIV-1-infected individuals. Despite the early presence of these cells, each of the individuals in the present study exhibited progressive disease, with one individual showing rapid progression. In this rapid progressor, three IL-2-producing HIV-1 Gag-specific CD4 + T-cell responses were identified and mapped to the following optimal epitopes: HIVWASRELER, REPRGSDIAGT, and FRDYVDRFYKT. Responses to these epitopes in peripheral blood mononuclear cells were monitored longitudinally to >1 year postinfection, and contemporaneous circulating plasma viruses were sequenced. A variant of the FRDYVDRFYKT epitope sequence, FRDYVDQFYKT, was observed in 1/21 plasma viruses sequenced at 5 months postinfection and 1/10 viruses at 7 months postinfection. This variant failed to stimulate the corresponding CD4 + T-cell clone and thus constitutes an escape mutant. Responses to each of the three Gag epitopes were rapidly lost, and this loss was accompanied by a loss of antigen-specific cells in the periphery as measured by using an FRDYVDRFYKT-presenting major histocompatibility complex class II tetramer. Highly active antiretroviral therapy was associated with the reemergence of FRDYVDRFYKT-specific cells by tetramer. Thus, our data support that IL-2-producing HIV-1-specific CD4 + T-cell responses can exert immune pressure during early HIV-1 infection but that the inability of these responses to enforce enduring control of viral replication is related to the deletion and/or dysfunction of HIV-1-specific CD4 + T cells rather than to the fixation of escape mutations at high frequencies.

Position-specific denatured-state thermodynamics were determined for a database of human proteins by use of an ensemble-based model of protein structure. The results of modeling denatured protein in this manner reveal important sequence-dependent thermodynamic properties in the denatured ensembles as well as fundamental differences between the denatured and native ensembles in overall thermodynamic character. The generality and robustness of these results were validated by performing fold-recognition experiments, whereby sequences were matched with their respective folds based on amino acid propensities for the different energetic environments in the protein, as determined through cluster analysis. Correlation analysis between structure and energetic information revealed that sequence segments destined for beta-sheet in the final native fold are energetically more predisposed to a broader repertoire of states than are sequence segments destined for alpha-helix. These results suggest that within the subensemble of mostly unstructured states, the energy landscapes are dominated by states in which parts of helices adopt structure, whereas structure formation for sequences destined for beta-strand is far less probable. These results support a framework model of folding, which suggests that, in general, the denatured state has evolutionarily evolved to avoid low-energy conformations in sequences that ultimately adopt beta-strand. Instead, the denatured state evolved so that sequence segments that ultimately adopt alpha-helix and coil will have a high intrinsic structure formation capability, thus serving as potential nucleation sites.

BACKGROUND: HIV specific T cells are putatively anergic in vivo. IL-2, a member of a class of cytokines that binds to receptors containing the common gamma chain (gammac) has been shown to reverse anergy. We examined the role of gammac cytokines in reversing HIV specific T cell anergy. METHODS: PBMC from untreated HIV-infected individuals were briefly exposed to a panel of gammac cytokines, and frequencies of gag specific T cells were enumerated by intracellular IFN-gamma flow cytometry. RESULTS: Of the gammac cytokines, brief exposure to IL-2, IL-15, or combined IL-15/IL-7 significantly enhanced (range 2-7 fold) the CD4(+) and CD8(+) T cell IFN-gamma responses to HIV gag, with IL-15 giving the greatest enhancement. The effects of cytokines were not due to enhanced proliferation of pre-existing antigen specific cells, but were due to a combination of enhanced cytokine production from antigen specific T cells plus activation of non-epitope specific T cells. CONCLUSIONS: These observations support the notion that a significant number of HIV specific T cells are circulating in an anergic state. IL-2, IL-7 and particularly IL-15 as an immune modulator to reverse HIV-1 specific T cell anergy should be investigated, with the caveat that non-specific activation of T cells may also be induced.

The Wiggle series are support vector machine–based predictors that identify regions of functional flexibility using only protein sequence information. Functionally flexible regions are defined as regions that can adopt different conformational states and are assumed to be necessary for bioactivity. Many advances have been made in understanding the relationship between protein sequence and structure. This work contributes to those efforts by making strides to understand the relationship between protein sequence and flexibility. A coarse-grained protein dynamic modeling approach was used to generate the dataset required for support vector machine training. We define our regions of interest based on the participation of residues in correlated large-scale fluctuations. Even with this structure-based approach to computationally define regions of functional flexibility, predictors successfully extract sequence-flexibility relationships that have been experimentally confirmed to be functionally important. Thus, a sequence-based tool to identify flexible regions important for protein function has been created. The ability to identify functional flexibility using a sequence based approach complements structure-based definitions and will be especially useful for the large majority of proteins with unknown structures. The methodology offers promise to identify structural genomics targets amenable to crystallization and the possibility to engineer more flexible or rigid regions within proteins to modify their bioactivity., Synopsis Proteins are not static entities in biology and are constantly changing their shape and form to perform their necessary biological roles. While we are intuitively aware of their constantly changing nature, we have little understanding of how their flexibility is encoded in the protein sequence. To address this knowledge gap, predictors were created to identify sequence patterns that dictate local regions to be flexible and serve a functional purpose. By combining protein dynamic modeling and machine learning techniques, the Wiggle predictor series were able to generalize the sequence-flexibility relationship for all proteins. With these predictors we are able to identify flexible regions of functional importance such as hinges, recognition loops, and catalytic loops using only sequence information. This work has important contributions to our understanding of the sequence-flexibility relationship and paves the road to identifying local sequence modulations that impact protein function without necessarily changing the structure.