Deubiquitinating enzyme amino acid profiling reveals a class of ubiquitin esterases.

The reversibility of ubiquitination by the action of deubiquitinating enzymes (DUBs) serves as an important regulatory layer within the ubiquitin system. Approximately 100 DUBs are encoded by the human genome, and many have been implicated with pathologies, including neurodegeneration and cancer. Non-lysine ubiquitination is chemically distinct, and its physiological importance is emerging. Here, we couple chemically and chemoenzymatically synthesized ubiquitinated lysine and threonine model substrates to a mass spectrometry-based DUB assay. Using this platform, we profile two-thirds of known catalytically active DUBs for threonine esterase and lysine isopeptidase activity and find that most DUBs demonstrate dual selectivity. However, with two anomalous exceptions, the ovarian tumor domain DUB class demonstrates specific (iso)peptidase activity. Strikingly, we find the Machado-Joseph disease (MJD) class to be unappreciated non-lysine DUBs with highly specific ubiquitin esterase activity rivaling the efficiency of the most active isopeptidases. Esterase activity is dependent on the canonical catalytic triad, but proximal hydrophobic residues appear to be general determinants of non-lysine activity. Our findings also suggest that ubiquitin esters have appreciable cellular stability and that non-lysine ubiquitination is an integral component of the ubiquitin system. Its regulatory sophistication is likely to rival that of canonical ubiquitination.

Structural basis for RING-Cys-Relay E3 ligase activity and its role in axon integrity.

MYCBP2 is a ubiquitin (Ub) E3 ligase (E3) that is essential for neurodevelopment and regulates axon maintenance. MYCBP2 transfers Ub to nonlysine substrates via a newly discovered RING-Cys-Relay (RCR) mechanism, where Ub is relayed from an upstream cysteine to a downstream substrate esterification site. The molecular bases for E2-E3 Ub transfer and Ub relay are unknown. Whether these activities are linked to the neural phenotypes is also unclear. We describe the crystal structure of a covalently trapped E2~Ub:MYCBP2 transfer intermediate revealing key structural rearrangements upon E2-E3 Ub transfer and Ub relay. Our data suggest that transfer to the dynamic upstream cysteine, whilst mitigating lysine activity, requires a closed-like E2~Ub conjugate with tempered reactivity, and Ub relay is facilitated by a helix-coil transition. Furthermore, neurodevelopmental defects and delayed injury-induced degeneration in RCR-defective knock-in mice suggest its requirement, and that of substrate esterification activity, for normal neural development and programmed axon degeneration.

Photocrosslinking Activity-Based Probes for Ubiquitin RING E3 Ligases.

Activity-based protein profiling is an invaluable technique for studying enzyme biology and facilitating the development of therapeutics. Ubiquitin E3 ligases (E3s) are one of the largest enzyme families and regulate a host of (patho)physiological processes. The largest subtype are the RING E3s of which there are >600 members. RING E3s have adaptor-like activity that can be subject to diverse regulatory mechanisms and have become attractive drug targets. Activity-based probes (ABPs) for measuring RING E3 activity do not exist. Here we re-engineer ubiquitin-charged E2 conjugating enzymes to produce photocrosslinking ABPs. We demonstrate activity-dependent profiling of two divergent cancer-associated RING E3s, RNF4 and c-Cbl, in response to their native activation signals. We also demonstrate profiling of endogenous RING E3 ligase activation in response to epidermal growth factor (EGF) stimulation. These photocrosslinking ABPs should advance E3 ligase research and the development of selective modulators against this important class of enzymes.

Interventions integrating health and academic interventions to prevent substance use and violence: a systematic review and synthesis of process evaluations.

BACKGROUND: Within increasingly constrained school timetables, interventions that integrate academic and health education to reduce substance use and violence may hold promise as a category of intervention that can positively affect both academic and health outcomes. There are no current systematic reviews exploring the effectiveness of such interventions or factors that affect their implementation. METHODS: A total of 19 bibliographic databases and 32 websites were searched. References were also extracted from the reference lists of included studies, and experts and authors were contacted to identify relevant studies. We included reports with no restrictions on language or date. References were screened on title/abstract and those not thus excluded were screened on full report. Data extraction and appraisal followed the Critical Appraisal Skills Programme, Evidence for Policy and Practice Information and Co-ordinating Centre and Cochrane tools. Extracted process data were qualitatively meta-synthesised for common themes. RESULTS: Seventy-eight thousand four hundred fifty-one unique references were identified, and 62 reports were included. A total of 16 reports (reporting on 15 studies of 12 interventions) evaluated process. Key facilitators of integrated academic and health curricula were supportive senior management and alignment of the intervention with school ethos; a positive teaching environment, including positive perceptions around the ability to be flexible in the adaptation and delivery of integrated academic and health curricula; positive pre-existing student and teacher attitudes towards intervention content; and parental support of interventions, largely through reinforcement of messaging at home. Important barriers were over-burdened teachers, with little time to learn and implement integrated curricula. CONCLUSION: Several useful facilitating and inhibiting factors linked to the implementation of interventions that integrate academic and health education for reduced substance use and/or violence were identified, providing tentative but insightful evidence of context-specific issues that may impact intervention success. However, overall, there is still a considerable gap in our understanding of how to achieve the successful implementation of these interventions.

Trivalent RING Assembly on Retroviral Capsids Activates TRIM5 Ubiquitination and Innate Immune Signaling.

TRIM5 is a RING domain E3 ubiquitin ligase with potent antiretroviral function. TRIM5 assembles into a hexagonal lattice on retroviral capsids, causing envelopment of the infectious core. Concomitantly, TRIM5 initiates innate immune signaling and orchestrates disassembly of the viral particle, yet how these antiviral responses are regulated by capsid recognition is unclear. We show that hexagonal assembly triggers N-terminal polyubiquitination of TRIM5 that collectively drives antiviral responses. In uninfected cells, N-terminal monoubiquitination triggers non-productive TRIM5 turnover. Upon TRIM5 assembly on virus, a trivalent RING arrangement allows elongation of N-terminally anchored K63-linked ubiquitin chains (N-K63-Ub). N-K63-Ub drives TRIM5 innate immune stimulation and proteasomal degradation. Inducing ubiquitination before TRIM5 assembly triggers premature degradation and ablates antiviral restriction. Conversely, driving N-K63 ubiquitination after TRIM5 assembly enhances innate immune signaling. Thus, the hexagonal geometry of TRIM5's antiviral lattice converts a capsid-binding protein into a multifunctional antiviral platform.

Truncation- and motif-based pan-cancer analysis reveals tumor-suppressing kinases.

A major challenge in cancer genomics is identifying "driver" mutations from the many neutral "passenger" mutations within a given tumor. To identify driver mutations that would otherwise be lost within mutational noise, we filtered genomic data by motifs that are critical for kinase activity. In the first step of our screen, we used data from the Cancer Cell Line Encyclopedia and The Cancer Genome Atlas to identify kinases with truncation mutations occurring within or before the kinase domain. The top 30 tumor-suppressing kinases were aligned, and hotspots for loss-of-function (LOF) mutations were identified on the basis of amino acid conservation and mutational frequency. The functional consequences of new LOF mutations were biochemically validated, and the top 15 hotspot LOF residues were used in a pan-cancer analysis to define the tumor-suppressing kinome. A ranked list revealed MAP2K7, an essential mediator of the c-Jun N-terminal kinase (JNK) pathway, as a candidate tumor suppressor in gastric cancer, despite its mutational frequency falling within the mutational noise for this cancer type. The majority of mutations in MAP2K7 abolished its catalytic activity, and reactivation of the JNK pathway in gastric cancer cells harboring LOF mutations in MAP2K7 or the downstream kinase JNK suppressed clonogenicity and growth in soft agar, demonstrating the functional relevance of inactivating the JNK pathway in gastric cancer. Together, our data highlight a broadly applicable strategy to identify functional cancer driver mutations and define the JNK pathway as tumor-suppressive in gastric cancer.

How Do Contextual Factors Influence Implementation and Receipt of Positive Youth Development Programs Addressing Substance Use and Violence? A Qualitative Meta-Synthesis of Process Evaluations.

OBJECTIVE: Positive youth development (PYD) often aims to prevent tobacco, alcohol, and drugs use and violence. We systematically reviewed PYD interventions, synthesizing process, and outcomes evidence. Synthesis of outcomes, published elsewhere, found no overall evidence of reducing substance use or violence but notable variability of fidelity. Our synthesis of process evaluations examined how implementation varied and was influenced by context. DATA SOURCE: Process evaluations of PYD aiming to reduce substance use and violence. Study Inclusion Criteria: Overall review published since 1985; written in English; focused on youth aged 11 to 18 years; focused on interventions addressing multiple positive assets; reported on theory, process, or outcomes; and concerned with reducing substance use or violence. Synthesis of process evaluations examined how implementation varies with or is influenced by context. DATA EXTRACTION: Two reviewers in parallel. DATA SYNTHESIS: Thematic synthesis. RESULTS: We identified 12 reports. Community engagement enhanced program appeal. Collaboration with other agencies could broaden the activities offered. Calm but authoritative staff increased acceptability. Staff continuity underpinned diverse activities and durable relationships. Empowering participants were sometimes in tension with requiring them to engage in diverse activities. CONCLUSION: Our systematic review identified factors that might help improve the fidelity and acceptability of PYD interventions. Addressing these might enable PYD to fulfill its potential as a means of promoting health.

Development of a framework for the co-production and prototyping of public health interventions.

BACKGROUND: Existing guidance for developing public health interventions does not provide information for researchers about how to work with intervention providers to co-produce and prototype the content and delivery of new interventions prior to evaluation. The ASSIST + Frank study aimed to adapt an existing effective peer-led smoking prevention intervention (ASSIST), integrating new content from the UK drug education resource Talk to Frank ( www.talktofrank.com ) to co-produce two new school-based peer-led drug prevention interventions. A three-stage framework was tested to adapt and develop intervention content and delivery methods in collaboration with key stakeholders to facilitate implementation. METHODS: The three stages of the framework were: 1) Evidence review and stakeholder consultation; 2) Co-production; 3) Prototyping. During stage 1, six focus groups, 12 consultations, five interviews, and nine observations of intervention delivery were conducted with key stakeholders (e.g. Public Health Wales [PHW] ASSIST delivery team, teachers, school students, health professionals). During stage 2, an intervention development group consisting of members of the research team and the PHW ASSIST delivery team was established to adapt existing, and co-produce new, intervention activities. In stage 3, intervention training and content were iteratively prototyped using process data on fidelity and acceptability to key stakeholders. Stages 2 and 3 took the form of an action-research process involving a series of face-to-face meetings, email exchanges, observations, and training sessions. RESULTS: Utilising the three-stage framework, we co-produced and tested intervention content and delivery methods for the two interventions over a period of 18 months involving external partners. New and adapted intervention activities, as well as refinements in content, the format of delivery, timing and sequencing of activities, and training manuals resulted from this process. The involvement of intervention delivery staff, participants and teachers shaped the content and format of the interventions, as well as supporting rapid prototyping in context at the final stage. CONCLUSIONS: This three-stage framework extends current guidance on intervention development by providing step-by-step instructions for co-producing and prototyping an intervention's content and delivery processes prior to piloting and formal evaluation. This framework enhances existing guidance and could be transferred to co-produce and prototype other public health interventions. TRIAL REGISTRATION: ISRCTN14415936 , registered retrospectively on 05 November 2014.

Cross-sectional study examining the prevalence, correlates and sequencing of electronic cigarette and tobacco use among 11-16-year olds in schools in Wales.

OBJECTIVES: To examine the prevalence and frequency of electronic (e)-cigarette use among young people in Wales, associations with socio-demographic characteristics, smoking and other substances and the sequencing of e-cigarette and tobacco use. DESIGN: A cross-sectional survey of school students in Wales undertaken in 2015. SETTING: 87 secondary schools in Wales. PARTICIPANTS: Students aged 11-16 (n=32 479). RESULTS: Overall, students were nearly twice as likely to report ever using e-cigarettes (18.5%) as smoking tobacco (10.5%). Use of e-cigarettes at least weekly was 2.7% in the whole sample, rising to 5.7% among those aged 15-16. Almost half (41.8%) of daily smokers reported being regular e-cigarette users. Regular e-cigarette use was more prevalent among current cannabis users (relative risk ratio (RRR)=41.82; 95% CI 33.48 to 52.25)), binge drinkers (RRR=47.88; 95% CI 35.77 to 64.11), users of mephedrone (RRR=32.38; 95% CI 23.05 to 45.52) and laughing gas users (RRR=3.71; 95% CI 3.04 to 4.51). Multivariate analysis combining demographics and smoking status showed that only gender (being male) and tobacco use independently predicted regular use of e-cigarettes (p<0.001). Among weekly smokers who had tried tobacco and e-cigarettes (n=877), the vast majority reported that they tried tobacco before using an e-cigarette (n=727; 82.9%). CONCLUSIONS: Since 2013, youth experimentation with e-cigarettes has grown rapidly in Wales and is now almost twice as common as experimentation with tobacco. Regular use has almost doubled, and is increasing among never and non-smokers. These data suggest that e-cigarette use among youth is an emerging public health issue, even though there remains no evidence that it represents a new pathway into smoking. Mixed methods longitudinal research is needed to explore why young people use e-cigarettes, and to develop interventions to prevent further increases in use.

Where next with theory and research on how the school environment influences young people's substance use?

Substance use (smoking, drinking and illicit drug use) remains, a serious problem for young people living in industrialised countries. There is increasing interest in interventions to modify the school, environment, addressing the multiple upstream determinants of young, people's health. This article provides an overview of current theory, about how secondary school environments influence young people's, substance use before focusing on the Theory of Human Functioning and, School Organisation. It critically examines the extent to which this, theory is substantiated by quantitative and qualitative evidence and, considers how the theory might be elaborated to better inform future, empirical research.

What is positive youth development and how might it reduce substance use and violence? A systematic review and synthesis of theoretical literature.

BACKGROUND: Preventing adolescent substance use and youth violence are public health priorities. Positive youth development interventions are widely deployed often with the aim of preventing both. However, the theorised mechanisms by which PYD is intended to reduce substance use and violence are not clear and existing evaluated interventions are under-theorised. Using innovative methods, we systematically searched for and synthesised published theoretical literature describing what is meant by positive youth development and how it might reduce substance use and violence, as part of a broader systematic review examining process and outcomes of PYD interventions. METHODS: We searched 19 electronic databases, review topic websites, and contacted experts between October 2013 and January 2014. We included studies written in English, published since 1985 that reported a theory of change for positive youth development focused on prevention of smoking, alcohol consumption, drug use or violence in out-of-school settings. Studies were independently coded and quality-assessed by two reviewers. RESULTS: We identified 16 studies that met our inclusion criteria. Our synthesis suggests that positive youth development aims to provide youth with affective relationships and diverse experiences which enable their development of intentional self-regulation and multiple positive assets. These in turn buffer against or compensate for involvement in substance use and violence. Existing literature is not clear on how intentional self-regulation is developed and which specific positive assets buffer against substance use or violence. CONCLUSIONS: Our synthesis provides: an example of a rigorous systematic synthesis of theory literature innovatively applying methods of qualitative synthesis to theoretical literature; a clearer understanding of how PYD might reduce substance use and violence to inform future interventions and empirical evaluations.

Electronic-cigarette use among young people in Wales: evidence from two cross-sectional surveys.

OBJECTIVES: To examine the prevalence of electronic(e)-cigarette use, prevalence of e-cigarette and tobacco use by age, and associations of e-cigarette use with sociodemographic characteristics, tobacco and cannabis use among young people in Wales. DESIGN: Data from two nationally-representative cross-sectional surveys undertaken in 2013-2014. Logistic regression analyses, adjusting for school-level clustering, examined sociodemographic characteristics of e-cigarette use, and associations between e-cigarette use and smoking. SETTING: Primary and secondary schools in Wales. PARTICIPANTS: Primary-school children aged 10-11 (n=1601) and secondary-school students aged 11-16 (n=9055). RESULTS: Primary-school children were more likely to have used e-cigarettes (5.8%) than tobacco (1.6%). Ever use of e-cigarettes remained more prevalent than ever use of tobacco until age 14-15. Overall, 12.3% of secondary-school students (aged 11-16) reported ever using e-cigarettes, with no differences according to gender, ethnicity or family affluence. The percentage of 'never smokers' reporting having used e-cigarettes was 5.3% at age 10-11 to 8.0% at age 15-16. The proportion of children who had ever used an e-cigarette and reported currently smoking increased from 6.9% among 10-11 year olds to 39.2% in 15-16 year olds. Only 1.5% (n=125) of 11-16 year-olds, including 0.3% of never smokers, reported regular e-cigarette use (use at least once a month). Current weekly smokers were 100 times more likely than non-smokers to report regular e-cigarette use (relative risk ratio (RRR=121.15; 95% CI 57.56 to 254.97). Regular e-cigarette use was also more likely among those who had smoked cannabis (RRR 53.03; 95% CI 38.87 to 80.65). CONCLUSIONS: Many young people (including never-smokers) have tried e-cigarettes. However, regular use is less common, and is associated with tobacco cigarette use. Longitudinal research is needed to understand age-related trajectories of e-cigarette use and to understand the temporal nature of relationships between e-cigarette and tobacco use.

HIV-1 evades innate immune recognition through specific cofactor recruitment.

Human immunodeficiency virus (HIV)-1 is able to replicate in primary human macrophages without stimulating innate immunity despite reverse transcription of genomic RNA into double-stranded DNA, an activity that might be expected to trigger innate pattern recognition receptors. We reasoned that if correctly orchestrated HIV-1 uncoating and nuclear entry is important for evasion of innate sensors then manipulation of specific interactions between HIV-1 capsid and host factors that putatively regulate these processes should trigger pattern recognition receptors and stimulate type 1 interferon (IFN) secretion. Here we show that HIV-1 capsid mutants N74D and P90A, which are impaired for interaction with cofactors cleavage and polyadenylation specificity factor subunit 6 (CPSF6) and cyclophilins (Nup358 and CypA), respectively, cannot replicate in primary human monocyte-derived macrophages because they trigger innate sensors leading to nuclear translocation of NF-κB and IRF3, the production of soluble type 1 IFN and induction of an antiviral state. Depletion of CPSF6 with short hairpin RNA expression allows wild-type virus to trigger innate sensors and IFN production. In each case, suppressed replication is rescued by IFN-receptor blockade, demonstrating a role for IFN in restriction. IFN production is dependent on viral reverse transcription but not integration, indicating that a viral reverse transcription product comprises the HIV-1 pathogen-associated molecular pattern. Finally, we show that we can pharmacologically induce wild-type HIV-1 infection to stimulate IFN secretion and an antiviral state using a non-immunosuppressive cyclosporine analogue. We conclude that HIV-1 has evolved to use CPSF6 and cyclophilins to cloak its replication, allowing evasion of innate immune sensors and induction of a cell-autonomous innate immune response in primary human macrophages.

The school environment and student health: a systematic review and meta-ethnography of qualitative research.

BACKGROUND: There is increasing interest in promoting young people's health by modifying the school environment. However, existing research offers little guidance on how the school context enables or constrains students' health behaviours, or how students' backgrounds relate to these processes. For these reasons, this paper reports on a meta-ethnography of qualitative studies examining: through what processes does the school environment (social and physical) influence young people's health? METHODS: Systematic review of qualitative studies. Sixteen databases were searched, eliciting 62,329 references which were screened, with included studies quality assessed, data extracted and synthesized using an adaptation of Noblit and Hare's meta-ethnographic approach. RESULTS: Nineteen qualitative studies were synthesised to explore processes through which school-level influences on young people's health might occur. Four over-arching meta-themes emerged across studies focused on a range of different health issues. First, aggressive behaviour and substance use are often a strong source of status and bonding at schools where students feel educationally marginalised or unsafe. Second, health-risk behaviours are concentrated in unsupervised 'hotspots' at the school. Third, positive relationships with teachers appear to be critical in promoting student wellbeing and limiting risk behaviour; however, certain aspects of schools' organisation and education policies constrain this, increasing the likelihood that students look for a sense of identity and social support via health-risk behaviours. Fourth, unhappiness at school can cause students to seek sources of 'escape', either by leaving school at lunchtime or for longer unauthorized spells or through substance use. These meta-themes resonate with Markham and Aveyard's theory of human functioning and school organisation, and we draw on these qualitative data to refine and extend this theory, in particular conceptualising more fully the role of young people's agency and student-led 'systems' in constituting school environments and generating health risks. CONCLUSION: Institutional features which may shape student health behaviours such as lack of safety, poor student-staff relationships and lack of student voice are amenable to interventions and should be the subject of future investigation. Future qualitative research should focus on health behaviours which are under-theorised in this context such as physical activity, sexual and mental health.

Inhibition of retroviral replication by members of the TRIM protein family.

The TRIM protein family is emerging as a central component of mammalian antiviral innate immunity. Beginning with the identification of TRIM5α as a mammalian post-entry restriction factor against retroviruses, to the repeated observation that many TRIMs ubiquitinate and regulate signaling pathways, the past decade has witnessed an intense research effort to understand how TRIM proteins influence immunity. The list of viral families targeted directly or indirectly by TRIM proteins has grown to include adenoviruses, hepadnaviruses, picornaviruses, flaviviruses, orthomyxoviruses, paramyxoviruses, herpesviruses, rhabdoviruses and arenaviruses. We have come to appreciate how, through intense bouts of positive selection, some TRIM genes have been honed into species-specific restriction factors. Similarly, in the case of TRIMCyp, we are beginning to understand how viruses too have mutated to evade restriction, suggesting that TRIM and viruses have coevolved for millions of years of primate evolution. Recently, TRIM5α returned to the limelight when it was shown to trigger the expression of antiviral genes upon recognition of an incoming virus, a paradigm shift that demonstrated that restriction factors make excellent pathogen sensors. However, it remains unclear how many of ~100 human TRIM genes are antiviral, despite the expression of many of these genes being upregulated by interferon and upon viral infection. TRIM proteins do not conform to one type of antiviral mechanism, reflecting the diversity of viruses they target. Moreover, the cofactors of restriction remain largely enigmatic. The control of retroviral replication remains an important medical subject and provides a useful backdrop for reviewing how TRIM proteins act to repress viral replication.

The effects of the school environment on student health: a systematic review of multi-level studies.

Health outcomes vary between schools and it is theorised that this may be partly attributable to variation in the school environment. Existing systematic reviews have not drawn authoritative conclusions because of methodological limitations in the review or studies available. We identified 42 multi-level studies, ten of which were judged of sufficient quality to narratively synthesize. There was consistent evidence that schools with higher attainment and attendance than would be expected from student intake had lower rates of substance use. Findings on the influence of smoking/alcohol policies were mixed. Three studies examined the health effects variously associated with school campus area and observability, year structure, school size and pupil-to-teacher ratio with mixed findings. The studies reviewed support the potential influence of the school environment on student health.

Social network influences on smoking, drinking and drug use in secondary school: centrifugal and centripetal forces.

We explore how school experiences and social networks structure young people's substance use in different institutional contexts. The concepts of 'selection' and 'influence' are situated within the context of bounded agency, counter-school cultures and Bourdieusian notions of capital. We employed individual and group interviews, network-mapping, and observations at two contrasting English secondary schools. Both schools were characterised by extended social network structures that appeared to influence patterns of substance use, although the mechanisms via which this occurred varied according to school context. At Grange House school (suburban context) a minority of students from disadvantaged families were alienated by the attainment-focused regime, marginalised by a strong peer-led centrifugal force pushing them outwards, and substance use was an alternative source of bonding and identity for these students. In contrast, at North Street a centripetal force operated whereby the majority of students were pulled towards highly-visible, normative markers of 'safe', 'road culture', such as cannabis use and gang-involvement, as they attempted to fit in and survive in an inner-city school environment. We conclude that health inequalities may be reproduced through these distinctive centrifugal and centripetal forces in different institutional contexts, and this should be the focus of quantitative examination in the UK and elsewhere.

Antibodies attenuate the capacity of dendritic cells to stimulate HIV-specific cytotoxic T lymphocytes.

BACKGROUND: Control of HIV is suggested to depend on potent effector functions of the virus-specific CD8(+) T-cell response. Antigen opsonization can modulate the capture of antigen, its presentation, and the priming of specific CD8(+) T-cell responses. OBJECTIVE: We have previously shown that opsonization of retroviruses acts as an endogenous adjuvant for dendritic cell (DC)-mediated induction of specific cytotoxic T lymphocytes (CTLs). However, in some HIV-positive subjects, high levels of antibodies and low levels of complement fragments coat the HIV surface. METHODS: Therefore we analyzed the effect of IgG opsonization on the antigen-presenting capacity of DCs by using CD8(+) T-cell proliferation assays after repeated prime boosting, by measuring the antiviral activity against HIV-infected autologous CD4(+) T cells, and by determining IFN-γ secretion from HIV-specific CTL clones. RESULTS: We find that DCs exposed to IgG-opsonized HIV significantly decreased the HIV-specific CD8(+) T-cell response compared with the earlier described efficient CD8(+) T-cell activation induced by DCs loaded with complement-opsonized HIV. DCs exposed to HIV bearing high surface IgG levels after incubation in plasma from HIV-infected subjects acted as weak stimulators for HIV-specific CTL clones. In contrast, HIV opsonized with plasma from patients exhibiting high complement and low IgG deposition on the viral surface favored significantly higher activation of HIV-specific CD8(+) T-cell clones. CONCLUSION: Our ex vivo and in vitro observations provide the first evidence that IgG opsonization of HIV is associated with a decreased CTL-stimulatory capacity of DCs.

CPSF6 defines a conserved capsid interface that modulates HIV-1 replication.

The HIV-1 genome enters cells inside a shell comprised of capsid (CA) protein. Variation in CA sequence alters HIV-1 infectivity and escape from host restriction factors. However, apart from the Cyclophilin A-binding loop, CA has no known interfaces with which to interact with cellular cofactors. Here we describe a novel protein-protein interface in the N-terminal domain of HIV-1 CA, determined by X-ray crystallography, which mediates both viral restriction and host cofactor dependence. The interface is highly conserved across lentiviruses and is accessible in the context of a hexameric lattice. Mutation of the interface prevents binding to and restriction by CPSF6-358, a truncated cytosolic form of the RNA processing factor, cleavage and polyadenylation specific factor 6 (CPSF6). Furthermore, mutations that prevent CPSF6 binding also relieve dependence on nuclear entry cofactors TNPO3 and RanBP2. These results suggest that the HIV-1 capsid mediates direct host cofactor interactions to facilitate viral infection.

School-related predictors of smoking, drinking and drug use: evidence from the Belfast Youth Development Study.

OBJECTIVE: To examine whether students' school engagement, relationships with teachers, educational aspirations and involvement in fights at school are associated with various measures of subsequent substance use. METHODS: Data were drawn from the Belfast Youth Development Study (n = 2968). Multivariate logistic models examined associations between school-related factors (age 13/14) and substance use (age 15/16). RESULTS: The two factors which were consistently and independently associated with regular substance use among both males and females were student-teacher relationships and fighting at school: positive teacher-relationships reduced the risk of daily smoking by 48%, weekly drunkenness by 25%, and weekly cannabis use by 52%; being in a fight increased the risk of daily smoking by 54%, weekly drunkenness by 31%, and weekly cannabis use by 43%. School disengagement increased the likelihood of smoking and cannabis use among females only. CONCLUSION: Further research should focus on public health interventions promoting positive relationships and safety at school.