RNA polymerase II stalling promotes nucleosome occlusion and pTEFb recruitment to drive immortalization by Epstein-Barr virus

Epstein-Barr virus (EBV) immortalizes resting B-cells and is a key etiologic agent in the development of numerous cancers. The essential EBV-encoded protein EBNA 2 activates the viral C promoter (Cp) producing a message of ~120 kb that is differentially spliced to encode all EBNAs required for immortalization. We have previously shown that EBNA 2-activated transcription is dependent on the activity of the RNA polymerase II (pol II) C-terminal domain (CTD) kinase pTEFb (CDK9/cyclin T1). We now demonstrate that Cp, in contrast to two shorter EBNA 2-activated viral genes (LMP 1 and 2A), displays high levels of promoter-proximally stalled pol II despite being constitutively active. Consistent with pol II stalling, we detect considerable pausing complex (NELF/DSIF) association with Cp. Significantly, we observe substantial Cp-specific pTEFb recruitment that stimulates high-level pol II CTD serine 2 phosphorylation at distal regions (up to +75 kb), promoting elongation. We reveal that Cp-specific pol II accumulation is directed by DNA sequences unfavourable for nucleosome assembly that increase TBP access and pol II recruitment. Stalled pol II then maintains Cp nucleosome depletion. Our data indicate that pTEFb is recruited to Cp by the bromodomain protein Brd4, with polymerase stalling facilitating stable association of pTEFb. The Brd4 inhibitor JQ1 and the pTEFb inhibitors DRB and Flavopiridol significantly reduce Cp, but not LMP1 transcript production indicating that Brd4 and pTEFb are required for Cp transcription. Taken together our data indicate that pol II stalling at Cp promotes transcription of essential immortalizing genes during EBV infection by (i) preventing promoter-proximal nucleosome assembly and ii) necessitating the recruitment of pTEFb thereby maintaining serine 2 CTD phosphorylation at distal regions

A Novel Cre Recombinase Imaging System for Tracking Lymphotropic Virus Infection In Vivo

BACKGROUND:Detection, isolation, and identification of individual virus infected cells during long term infection are critical to advance our understanding of mechanisms of pathogenesis for latent/persistent viruses. However, current approaches to study these viruses in vivo have been hampered by low sensitivity and effects of cell-type on expression of viral encoded reporter genes. We have designed a novel Cre recombinase (Cre)-based murine system to overcome these problems, and thereby enable tracking and isolation of individual in vivo infected cells. METHODOLOGY/PRINCIPAL FINDINGS:Murine gammaherpesvirus 68 (MHV-68) was used as a prototypic persistent model virus. A Cre expressing recombinant virus was constructed and characterised. The virus is attenuated both in lytic virus replication, producing ten-fold lower lung virus titres than wild type virus, and in the establishment of latency. However, despite this limitation, when the sEGFP7 mouse line containing a Cre-activated enhanced green fluorescent protein (EGFP) was infected with the Cre expressing virus, sites of latent and persistent virus infection could be identified within B cells and macrophages of the lymphoid system on the basis of EGFP expression. Importantly, the use of the sEGFP7 mouse line which expresses high levels of EGFP allowed individual virus positive cells to be purified by FACSorting. Virus gene expression could be detected in these cells. Low numbers of EGFP positive cells could also be detected in the bone marrow. CONCLUSIONS/SIGNIFICANCE:The use of this novel Cre-based virus/mouse system allowed identification of individual latently infected cells in vivo and may be useful for the study and long-term monitoring of other latent/persistent virus infections

ADVANCE integrated group intervention to address both substance use and intimate partner abuse perpetration by men in substance use treatment: a feasibility randomised controlled trial

Background: Substance use is a risk factor for intimate partner abuse (IPA) perpetration. Delivering perpetrator interventions concurrently with substance use treatment shows promise. Methods: The feasibility of conducting an efficacy and cost-effectiveness trial of the ADVANCE 16-week intervention to reduce IPA by men in substance use treatment was explored. A multicentre, parallel group individually randomised controlled feasibility trial and formative evaluation was conducted. Over three temporal cycles, 104 men who had perpetrated IPA towards a female (ex) partner in the past year were randomly allocated to receive the ADVANCE intervention + substance use treatment as usual (TAU) (n = 54) or TAU only (n = 50) and assessed 16-weeks post-randomisation. Participants’ (ex) partners were offered support and 27 provided outcome data. Thirty-one staff and 12 men who attended the intervention participated in focus groups or interviews that were analysed using the framework approach. Pre-specified criteria assessed the feasibility of progression to a definitive trial: 1) ≥ 60% of eligible male participants recruited; 2) intervention acceptable to staff and male participants; 3) ≥ 70% of participants followed-up and 4) levels of substance use and 5) IPA perpetrated by men in the intervention arm did not increase from average baseline level at 16-weeks post-randomisation. Results: 70.7% (104/147) of eligible men were recruited. The formative evaluation confirmed the intervention’s acceptability. Therapeutic alliance and session satisfaction were rated highly. The overall median rate of intervention session attendance (of 14 compulsory sessions) was 28.6% (range 14.3–64.3% by the third cycle). 49.0% (51/104) of men and 63.0% (17/27) of their (ex) partners were followed-up 16-weeks post-randomisation. This increased to 100% of men and women by cycle three. At follow-up, neither substance use nor IPA perpetration had worsened for men in the intervention arm. Conclusions: It was feasible to deliver the ADVANCE intervention in substance use treatment services, although it proved difficult to collect data from female (ex)partners. While some progression criteria were met, others were not, although improvements were demonstrated by the third cycle. Lessons learned will be implemented into the study design for a definitive trial of the ADVANCE intervention

Strategies to facilitate integrated care for people with alcohol and other drug problems: a systematic review

Background: There is a growing body of research highlighting the potential benefits of integrated care as a way of addressing the needs of people with alcohol and other drug (AOD) problems, given the broad range of other issues clients often experience. However, there has been little academic attention on the strategies that treatment systems, agencies and clinicians could implement to facilitate integrated care. Methods: We synthesised the existing evidence on strategies to improve integrated care in an AOD treatment context by conducting a systematic review of the literature. We searched major academic databases for peer-reviewed articles that evaluated strategies that contribute to integrated care in an AOD context between 1990 and 2014. Over 2600 articles were identified, of which 14 met the study inclusion criteria of reporting on an empirical study to evaluate the implementation of integrated care strategies. The types of strategies utilised in included articles were then synthesised. Results: We identified a number of interconnected strategies at the funding, organisational, service delivery and clinical levels. Ensuring that integrated care is included within service specifications of commissioning bodies and is adequately funded was found to be critical in effective integration. Cultivating positive inter-agency relationships underpinned and enabled the implementation of most strategies identified. Staff training in identifying and responding to needs beyond clinicians' primary area of expertise was considered important at a service level. However, some studies highlight the need to move beyond discrete training events and towards longer term coaching-type activities focussed on implementation and capacity building. Sharing of client information (subject to informed consent) was critical for most integrated care strategies. Case-management was found to be a particularly good approach to responding to the needs of clients with multiple and complex needs. At the clinical level, screening in areas beyond a clinician's primary area of practice was a common strategy for facilitating referral and integrated care, as was joint care planning. Conclusion: Despite considerable limitations and gaps in the literature in terms of the evaluation of integrated care strategies, particularly between AOD services, our review highlights several strategies that could be useful at multiple levels. Given the interconnectedness of integrated care strategies identified, implementation of multi-level strategies rather than single strategies is likely to be preferable

TILLING - a shortcut in functional genomics

Recent advances in large-scale genome sequencing projects have opened up new possibilities for the application of conventional mutation techniques in not only forward but also reverse genetics strategies. TILLING (Targeting Induced Local Lesions IN Genomes) was developed a decade ago as an alternative to insertional mutagenesis. It takes advantage of classical mutagenesis, sequence availability and high-throughput screening for nucleotide polymorphisms in a targeted sequence. The main advantage of TILLING as a reverse genetics strategy is that it can be applied to any species, regardless of its genome size and ploidy level. The TILLING protocol provides a high frequency of point mutations distributed randomly in the genome. The great mutagenic potential of chemical agents to generate a high rate of nucleotide substitutions has been proven by the high density of mutations reported for TILLING populations in various plant species. For most of them, the analysis of several genes revealed 1 mutation/200–500 kb screened and much higher densities were observed for polyploid species, such as wheat. High-throughput TILLING permits the rapid and low-cost discovery of new alleles that are induced in plants. Several research centres have established a TILLING public service for various plant species. The recent trends in TILLING procedures rely on the diversification of bioinformatic tools, new methods of mutation detection, including mismatch-specific and sensitive endonucleases, but also various alternatives for LI-COR screening and single nucleotide polymorphism (SNP) discovery using next-generation sequencing technologies. The TILLING strategy has found numerous applications in functional genomics. Additionally, wide applications of this throughput method in basic and applied research have already been implemented through modifications of the original TILLING strategy, such as Ecotilling or Deletion TILLING

Quantifying Age-Related Differences in Information Processing Behaviors When Viewing Prescription Drug Labels

Adverse drug events (ADEs) are a significant problem in health care. While effective warnings have the potential to reduce the prevalence of ADEs, little is known about how patients access and use prescription labeling. We investigated the effectiveness of prescription warning labels (PWLs, small, colorful stickers applied at the pharmacy) in conveying warning information to two groups of patients (young adults and those 50+). We evaluated the early stages of information processing by tracking eye movements while participants interacted with prescription vials that had PWLs affixed to them. We later tested participants’ recognition memory for the PWLs. During viewing, participants often failed to attend to the PWLs; this effect was more pronounced for older than younger participants. Older participants also performed worse on the subsequent memory test. However, when memory performance was conditionalized on whether or not the participant had fixated the PWL, these age-related differences in memory were no longer significant, suggesting that the difference in memory performance between groups was attributable to differences in attention rather than differences in memory encoding or recall. This is important because older adults are recognized to be at greater risk for ADEs. These data provide a compelling case that understanding consumers’ attentive behavior is crucial to developing an effective labeling standard for prescription drugs

Human Amniotic Epithelial Cell Transplantation Induces Markers of Alternative Macrophage Activation and Reduces Established Hepatic Fibrosis

Chronic hepatic inflammation from multiple etiologies leads to a fibrogenic response that can progress to cirrhosis and liver failure. Transplantation of human amniotic epithelial cells (hAEC) from term delivered placenta has been shown to decrease mild to moderate hepatic fibrosis in a murine model. To model advanced human liver disease and assess the efficacy of hAEC therapy, we transplanted hAEC in mice with advanced hepatic fibrosis. Immunocompetent C57BL/6 mice were administered carbon tetrachloride (CCl4) twice weekly resulting in bridging fibrosis by 12 weeks. hAEC (2×106) were infused via the tail vein at week 8 or weeks 8 and 10 (single and double dose, respectively). Human cells were detected in mouse liver four weeks after transplantation showing hAEC engraftment. CCl4 treated mice receiving single or double hAEC doses showed a significant but similar decrease in liver fibrosis area associated with decreased activation of collagen-producing hepatic stellate cells and decreased hepatic protein levels of the pro-fibrogenic cytokine, transforming growth factor-beta1. CCl4 administration caused hepatic T cell infiltration that decreased significantly following hAEC transplantation. Hepatic macrophages play a crucial role in both fibrogenesis and fibrosis resolution. Mice exposed to CCl4 demonstrated increased numbers of hepatic macrophages compared to normal mice; the number of macrophages decreased significantly in CCl4 treated mice given hAEC. These mice had significantly lower hepatic protein levels of the chemokine monocyte chemoattractant protein-1 than mice given CCl4 alone. Alternatively activated M2 macrophages are associated with fibrosis resolution. CCl4 treated mice given hAEC showed increased expression of genes associated with M2 macrophages including YM-1, IL-10 and CD206. We provide novel data showing that hAEC transplantation induces a wound healing M2 macrophage phenotype associated with reduction of established hepatic fibrosis that justifies further investigation of this potential cell-based therapy for advanced hepatic fibrosis

The Mechanism of Release of P-TEFb and HEXIM1 from the 7SK snRNP by Viral and Cellular Activators Includes a Conformational Change in 7SK

The positive transcription elongation factor, P-TEFb, is required for the production of mRNAs, however the majority of the factor is present in the 7SK snRNP where it is inactivated by HEXIM1. Expression of HIV-1 Tat leads to release of P-TEFb and HEXIM1 from the 7SK snRNP in vivo, but the release mechanisms are unclear.We developed an in vitro P-TEFb release assay in which the 7SK snRNP immunoprecipitated from HeLa cell lysates using antibodies to LARP7 was incubated with potential release factors. We found that P-TEFb was directly released from the 7SK snRNP by HIV-1 Tat or the P-TEFb binding region of the cellular activator Brd4. Glycerol gradient sedimentation analysis was used to demonstrate that the same Brd4 protein transfected into HeLa cells caused the release of P-TEFb and HEXIM1 from the 7SK snRNP in vivo. Although HEXIM1 binds tightly to 7SK RNA in vitro, release of P-TEFb from the 7SK snRNP is accompanied by the loss of HEXIM1. Using a chemical modification method, we determined that concomitant with the release of HEXIM1, 7SK underwent a major conformational change that blocks re-association of HEXIM1.Given that promoter proximally paused polymerases are present on most human genes, understanding how activators recruit P-TEFb to those genes is critical. Our findings reveal that the two tested activators can extract P-TEFb from the 7SK snRNP. Importantly, we found that after P-TEFb is extracted a dramatic conformational change occurred in 7SK concomitant with the ejection of HEXIM1. Based on our findings, we hypothesize that reincorporation of HEXIM1 into the 7SK snRNP is likely the regulated step of reassembly of the 7SK snRNP containing P-TEFb

Evidence That Gene Activation and Silencing during Stem Cell Differentiation Requires a Transcriptionally Paused Intermediate State

A surprising portion of both mammalian and Drosophila genomes are transcriptionally paused, undergoing initiation without elongation. We tested the hypothesis that transcriptional pausing is an obligate transition state between definitive activation and silencing as human embryonic stem cells (hESCs) change state from pluripotency to mesoderm. Chromatin immunoprecipitation for trimethyl lysine 4 on histone H3 (ChIP-Chip) was used to analyze transcriptional initiation, and 3′ transcript arrays were used to determine transcript elongation. Pluripotent and mesodermal cells had equivalent fractions of the genome in active and paused transcriptional states (∼48% each), with ∼4% definitively silenced (neither initiation nor elongation). Differentiation to mesoderm changed the transcriptional state of 12% of the genome, with roughly equal numbers of genes moving toward activation or silencing. Interestingly, almost all loci (98–99%) changing transcriptional state do so either by entering or exiting the paused state. A majority of these transitions involve either loss of initiation, as genes specifying alternate lineages are archived, or gain of initiation, in anticipation of future full-length expression. The addition of chromatin dynamics permitted much earlier predictions of final cell fate compared to sole use of conventional transcript arrays. These findings indicate that the paused state may be the major transition state for genes changing expression during differentiation, and implicate control of transcriptional elongation as a key checkpoint in lineage specification

The effect of parental rearing conditions on offspring life history in Anopheles stephensi

Background The environmental conditions experienced by parents are increasingly recognized to impact the success of offspring. Little is known on the presence of such parental effects in Anopheles. If present, parental effects could influence mosquito breeding programmes, some malaria control measures and have epidemiological and evolutionary consequences. Methods The presence of parental effects on offspring emergence time, size, survival, blood meal size and fecundity in laboratory reared An. stephensi were tested. Results Parental rearing conditions did not influence the time taken for offspring to emerge, or their size or survival as adults. However, parental effects were influential in determining the fecundity of daughters. Counter-intuitively, daughters of parents reared in low food conditions produced larger egg clutches than daughters of parents reared in high food conditions. Offspring reared in low food conditions took larger blood meals if their parents had also experienced a low food environment. Conclusion So far as we are aware, this is the first evidence of parental effects on progeny in Anophele