The role of Gag in HIV-1 DNA synthesis and sensitivity to reverse transcriptase inhibitor drugs

We hypothesise that HIV-1 DNA synthesis occurs inside intact viral capsid (CA) cores. We propose that dNTPs are transported into the CA via an electrostatic channel, formed by six positively charged arginines in the centre of CA hexamers. Here, we consider whether reverse transcriptase inhibitor (RTI) sensitivity is altered when the nature of the channel is changed, either by Gag mutation or exchange with Gag from a non-pandemic HIV isolate with a different structure. There are two classes of RTIs: nucleoside/nucleotide based (NRTI) and non-nucleoside inhibitors (NNRTI). We hypothesised that negatively charged NRTIs would recruit to CA hexamers, to be transported into cores. However, NNRTIs are uncharged, yet potently inhibit DNA synthesis, suggesting that NNRTIs enter cores by diffusion or inhibit after uncoating. We tested HIV-1 vector sensitivity to RTIs, either bearing lab adapted M-group, transmitted founder, O-group or mutant Gag sequences. Viral inhibition was measured by comparing IC50 and IC90 values in a range of cell lines. Our data shows that some differences in Gag demonstrate a cell type-dependent effect on viral sensitivity to RTIs. We also tested the stage of RTI inhibition, measuring early and late–reverse transcription (RT) products of HIV-1 (M) and HIV-1 (O) virus in the presence of inhibitors. Our data show that both HIV-1 (M) and (O) vectors are inhibited after 2nd DNA strand transfer. We determined that a small number of vDNA strands are required to infect a U87 cell, which increases in the presence of RTIs or on R18G mutation. We conclude that differences in Gag have some small cell type-dependent effects on RTI sensitivity. We hypothesise this may be due to differences in the timing of CA uncoating between cell types, supported by our finding that all RTIs tested inhibit RT predominantly after 2nd strand transfer

Open Access and the REF: Issues and Potential Solutions Workshop

This report provides a summary of the discussion and findings of the Open Access and the REF: Issues and Potential Solutions workshop held as part of the End-to-End Project. The workshop was highly interactive and feedback received indicated it was considered an excellent event, and that it was vital and useful to bring together various key stakeholders to discuss problems and procedures and develop ideas

Pregabalin Dispensing Patterns in Amman-Jordan: an Observational Study from Community Pharmacies

Objectives Pregabalin is currently approved for the treatment of epilepsy, generalized anxiety disorder, neuropathic pain and fibromyalgia. Rising attention to the abuse liability of pregabalin causing addictive behaviors is partially based on case reports and published literature of pregabalin used in dosages that override the approved therapeutic range. This study was conducted to provide background data regarding the abuse/misuse of pregabalin from community pharmacy in Jordan. Methods A prospective cross-sectional observational study design was used, which was conducted at different community pharmacies in Amman-Jordan. During the study period (November 2016-January 2017), a total 77 requests for pregabalin were observed from 14 pharmacies. A structured interview was conducted with all customers to gather information regarding their demographic and their request of pregabalin. Results A total of 77 pregabalin requests form 77 customers in a community pharmacy setting were observed in this study. Spinal disc herniation was the most common complaint for which the customer asked for the medication (n= 27, 35.1%). Self-medication was the most frequent method of requesting pregabalin (n= 44, 57.1%), while a total of 33 customers (42.9%) asked for the product using a prescription. During the observation period the number of customers suspected of abusing pregabalin for non-medical reason was 35 (45.5%). A total of 33 out of the 35 suspected customers (94.3%) asked for the product without a prescription, and 19/35 weren‘t sold due to suspicion of abuse (54.3%). Conclusion The study underscores the need for regulatory efforts to manage pregabalin abuse, through the addition of pregabalin containing products to the controlled drug list which can’t be purchased without a prescription. Also, pharmacists and customers must be educated at a community pharmacy level regarding potential hazards of pregabalin abuse

H1N1 Antibody Persistence 1 Year After Immunization With an Adjuvanted or Whole-Virion Pandemic Vaccine and Immunogenicity and Reactogenicity of Subsequent Seasonal Influenza Vaccine: A Multicenter Follow-on Study

Background. We investigated antibody persistence in children 1 year after 2 doses of either an AS03B-adjuvanted split-virion or nonadjuvanted whole-virion monovalent pandemic influenza vaccine and assessed the immunogenicity and reactogenicity of a subsequent dose of trivalent influenza vaccine (TIV). Methods. Children previously immunized at age 6 months to 12 years in the original study were invited to participate. After a blood sample was obtained to assess persistence of antibody against swine influenza A/H1N1(2009) pandemic influenza, children received 1 dose of 2010/2011 TIV, reactogenicity data were collected for 7 days, and another blood sample was obtained 21 days after vaccination. Results. Of 323 children recruited, 302 received TIV. Antibody persistence (defined as microneutralization [MN] titer ≥1:40) 1 year after initial vaccination was significantly higher in the AS03B-adjuvanted compared with the whole-virion vaccine group, 100% (95% confidence interval [CI], 94.1%–100%) vs 32.4% (95% CI, 21.5%–44.8%) in children immunized <3 years old and 96.9% (95% CI, 91.3%–99.4%) vs 65.9% (95% CI, 55.3%–75.5%) in those 3–12 years old at immunization, respectively (P < .001 for both groups). All children receiving TIV had post-vaccination MN titers ≥1:40. Although TIV was well tolerated in all groups, reactogenicity in children <5 years old was slightly greater in those who originally received AS03B-adjuvanted vaccine. Conclusions. This study provides serological evidence that 2 doses of AS03B-adjuvanted pandemic influenza vaccine may be sufficient to maintain protection across 2 influenza seasons. Administration of TIV to children who previously received 2 doses of either pandemic influenza vaccine is safe and is immunogenic for the H1N1 strain

Regulation of CHK1 inhibitor resistance by a c-Rel and USP1 dependent pathway

Previously, we discovered that deletion of c-Rel in the Em-Myc mouse model of lymphoma results in earlier onset of disease, a finding that contrasted with the expected function of this NF-κB subunit in B-cell malignancies. Here we report that Em-Myc/cRel−/− cells have an unexpected and major defect in the CHK1 pathway. Total and phospho proteomic analysis revealed that Em-Myc/cRel−/− lymphomas highly resemble wild-type (WT) Em-Myc lymphomas treated with an acute dose of the CHK1 inhibitor (CHK1i) CCT244747. Further analysis demonstrated that this is a consequence of Em-Myc/cRel−/ − lymphomas having lost expression of CHK1 protein itself, an effect that also results in resistance to CCT244747 treatment in vivo. Similar down-regulation of CHK1 protein levels was also seen in CHK1i resistant U2OS osteosarcoma and Huh7 hepatocellular carcinoma cells. Further investigation revealed that the deubiquitinase USP1 regulates CHK1 proteolytic degradation and that its down-regulation in our model systems is responsible, at least in part, for these effects. We demonstrate that treating WT Em-Myc lymphoma cells with the USP1 inhibitor ML323 was highly effective at reducing tumour burden in vivo. Targeting USP1 activity may thus be an alternative therapeutic strategy in MYC-driven tumours

Evaluation of the Clinical and Microbiological Response to Salmonella Paratyphi A Infection in the First Paratyphoid Human Challenge Model.

BACKGROUND: To expedite the evaluation of vaccines against paratyphoid fever, we aimed to develop the first human challenge model of Salmonella enterica serovar Paratyphi A infection. METHODS: Two groups of 20 participants underwent oral challenge with S. Paratyphi A following sodium bicarbonate pretreatment at 1 of 2 dose levels (group 1: 1-5 × 103 colony-forming units [CFU] and group 2: 0.5-1 × 103 CFU). Participants were monitored in an outpatient setting with daily clinical review and collection of blood and stool cultures. Antibiotic treatment was started when prespecified diagnostic criteria were met (temperature ≥38°C for ≥12 hours and/or bacteremia) or at day 14 postchallenge. RESULTS: The primary study objective was achieved following challenge with 1-5 × 103 CFU (group 1), which resulted in an attack rate of 12 of 20 (60%). Compared with typhoid challenge, paratyphoid was notable for high rates of subclinical bacteremia (at this dose, 11/20 [55%]). Despite limited symptoms, bacteremia persisted for up to 96 hours after antibiotic treatment (median duration of bacteremia, 53 hours [interquartile range, 24-85 hours]). Shedding of S. Paratyphi A in stool typically preceded onset of bacteremia. CONCLUSIONS: Challenge with S. Paratyphi A at a dose of 1-5 × 103 CFU was well tolerated and associated with an acceptable safety profile. The frequency and persistence of bacteremia in the absence of clinical symptoms was notable, and markedly different from that seen in previous typhoid challenge studies. We conclude that the paratyphoid challenge model is suitable for the assessment of vaccine efficacy using endpoints that include bacteremia and/or symptomatology. CLINICAL TRIALS REGISTRATION: NCT02100397

Longitudinal observation and decline of neutralizing antibody responses in the three months following SARS-CoV-2 infection in humans

Antibody responses to SARS-CoV-2 can be detected in most infected individuals 10–15 d after the onset of COVID-19 symptoms. However, due to the recent emergence of SARS-CoV-2 in the human population, it is not known how long antibody responses will be maintained or whether they will provide protection from reinfection. Using sequential serum samples collected up to 94 d post onset of symptoms (POS) from 65 individuals with real-time quantitative PCR-confirmed SARS-CoV-2 infection, we show seroconversion (immunoglobulin (Ig)M, IgA, IgG) in >95% of cases and neutralizing antibody responses when sampled beyond 8 d POS. We show that the kinetics of the neutralizing antibody response is typical of an acute viral infection, with declining neutralizing antibody titres observed after an initial peak, and that the magnitude of this peak is dependent on disease severity. Although some individuals with high peak infective dose (ID50 > 10,000) maintained neutralizing antibody titres >1,000 at >60 d POS, some with lower peak ID50 had neutralizing antibody titres approaching baseline within the follow-up period. A similar decline in neutralizing antibody titres was observed in a cohort of 31 seropositive healthcare workers. The present study has important implications when considering widespread serological testing and antibody protection against reinfection with SARS-CoV-2, and may suggest that vaccine boosters are required to provide long-lasting protection

Regulation of CHK1 inhibitor resistance by a c-Rel and USP1 dependent pathway

AbstractWe previously discovered that deletion of c-Rel in the Eμ-Myc mouse model of lymphoma results in earlier onset of disease, a finding that contrasted with the expected function of this NF-κB subunit in B-cell malignancies. Here we report that c-rel -/- Eµ-Myc cells have an unexpected and major defect in the CHK1 pathway, with almost undetectable levels of CHK1 and CLSPN protein leading to therapeutic resistance to the highly specific CHK1 inhibitor (CHK1i) CCT244747. Similar downregulation of CHK1 levels was also seen in CCT244747 resistant U20S osteosarcoma cells. Further investigation revealed that downregulation of the deubiquitinase USP1 is responsible, at least in part, for these effects. Importantly, we demonstrate that c-rel -/- Eµ-Myc lymphoma cells survive though upregulation of compensatory PI3K/AKT pathway activity. Moreover, targeting this pathway with Pictilisib (GDC-0941) effectively killed c-rel -/- Eµ-Myc in vivo, while having no effect on wild type Eμ-Myc cells. This data reveals an NF-κB regulated pathway controlling CHK1 activity in cancer cells and identifies a potential mechanism for both acquiring and overcoming CHK1i resistance in cancer patients.</jats:p

The evaluation of the North Atlantic climate system in UKESM1 historical simulations for CMIP6

Earth System models enable a broad range of climate interactions that physical climate models are unable to simulate. However, the extent to which adding Earth System components changes or improves the simulation of the physical climate is not well understood. Here we present a broad multi-variate evaluation of the North Atlantic climate system in historical simulations of the UK Earth System Model (UKESM1) performed for CMIP6. In particular, we focus on the mean state and the decadal timescale evolution of important variables that span the North Atlantic Climate system. In general, UKESM1 performs well and realistically simulates many aspects of the North Atlantic climate system. Like the physical version of the model, we find that changes in external forcing, and particularly aerosol forcing, are an important driver of multi-decadal change in UKESM1, especially for Atlantic Multi-decadal Variability and the Atlantic Meridional Overturning Circulation. However, many of the shortcomings identified are similar to common biases found in physical climate models, including the physical climate model that underpins UKESM1. For example, the summer jet is too weak and too far poleward; decadal variability in the winter jet is underestimated; intra-seasonal stratospheric polar vortex variability is poorly represented; and Arctic sea ice is too thick. Forced shortwave changes may be also too strong in UKESM1, which, given the important role of historical aerosol forcing in shaping the evolution of the North Atlantic in UKESM1, motivates further investigation. Therefore, physical model development, alongside Earth System development, remains crucial in order to improve climate simulations

Abstracts from the NIHR INVOLVE Conference 2017

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