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Background: HIV-1 infections initiated by multiple founder variants are characterised by a higher viral load and a worse clinical prognosis than those initiated with single founder variants, yet little is known about the routes of exposure through which transmission of multiple founder variants is most probable. Here we used individual patient data to calculate the probability of multiple founders stratified by route of HIV exposure and study methodology. Methods: We conducted a systematic review and meta-analysis of studies that estimated founder variant multiplicity in HIV-1 infection, searching MEDLINE, Embase, and Global Health databases for papers published between Jan 1, 1990, and Sept 14, 2020. Eligible studies must have reported original estimates of founder variant multiplicity in people with acute or early HIV-1 infections, have clearly detailed the methods used, and reported the route of exposure. Studies were excluded if they reported data concerning people living with HIV-1 who had known or suspected superinfection, who were documented as having received pre-exposure prophylaxis, or if the transmitting partner was known to be receiving antiretroviral treatment. Individual patient data were collated from all studies, with authors contacted if these data were not publicly available. We applied logistic meta-regression to these data to estimate the probability that an HIV infection is initiated by multiple founder variants. We calculated a pooled estimate using a random effects model, subsequently stratifying this estimate across exposure routes in a univariable analysis. We then extended our model to adjust for different study methods in a multivariable analysis, recalculating estimates across the exposure routes. This study is registered with PROSPERO, CRD42020202672. Findings: We included 70 publications in our analysis, comprising 1657 individual patients. Our pooled estimate of the probability that an infection is initiated by multiple founder variants was 0·25 (95% CI 0·21–0·29), with moderate heterogeneity (Q=132·3, p2=64·2%). Our multivariable analysis uncovered differences in the probability of multiple variant infection by exposure route. Relative to a baseline of male-to-female transmission, the predicted probability for female-to-male multiple variant transmission was significantly lower at 0·13 (95% CI 0·08–0·20), and the probabilities were significantly higher for transmissions in people who inject drugs (0·37 [0·24–0·53]) and men who have sex with men (0·30 [0·33–0·40]). There was no significant difference in the probability of multiple variant transmission between male-to-female transmission (0·21 [0·14–0·31]), post-partum transmission (0·18 [0·03–0·57]), pre-partum transmission (0·17 [0·08–0·33]), and intra-partum transmission (0·27 [0·14–0·45]). Interpretation: We identified that transmissions in people who inject drugs and men who have sex with men are significantly more likely to result in an infection initiated by multiple founder variants, and female-to-male infections are significantly less probable. Quantifying how the routes of HIV infection affect the transmission of multiple variants allows us to better understand how the evolution and epidemiology of HIV-1 determine clinical outcomes. Funding: Medical Research Council Precision Medicine Doctoral Training Programme and a European Research Council Starting Grant.

PurposeUnlike protests against police brutality in the past (2014 and earlier), police officers responding to First Amendment-protected demonstrations in summer 2020 likely were wearing body-worn cameras(BWCs). This study seeks to understand police perceptions of the effects of BWCs when used in the George Floyd protests.Design/methodology/approachThe authors use survey data from 100 agencies with federally-funded BWCs to assess the prevalence of BWC deployment to George Floyd protests and perceived benefits and limitations of the technology within this unique context.FindingsAbout three-quarters of agencies encountered some level of demonstration/protest related to the killing of George Floyd, and the majority of those deployed BWCs during these demonstrations. Respondents indicated evidentiary value of footage was a key reason for doing so, and at least three preconditions for a civilizing effect were present.Originality/valueResearch has documented numerous benefits associated with BWCs, from reductions in use of force and citizen complaints to evidentiary value. However, the extent to which BWC benefits extend to public protests is unclear. The George Floyd protests represent an opportunity to understand the prevalence and usefulness of BWCs in policing public protests.

TDP-43 is an RNA-binding protein with a crucial nuclear role in splicing, and mislocalises from the nucleus to the cytoplasm in a range of neurodegenerative disorders. TDP-43 proteinopathy spans a spectrum of incurable, heterogeneous, and increasingly prevalent neurodegenerative diseases, including the amyotrophic lateral sclerosis and frontotemporal dementia disease spectrum and a significant fraction of Alzheimer’s disease. There are currently no directed disease-modifying therapies for TDP-43 proteinopathies, and no way to distinguish who is affected before death. It is now clear that TDP-43 proteinopathy leads to a number of molecular changes, including the de-repression and inclusion of cryptic exons. Importantly, some of these cryptic exons lead to the loss of crucial neuronal proteins and have been shown to be key pathogenic players in disease pathogenesis (e.g., STMN2), as well as being able to modify disease progression (e.g., UNC13A). Thus, these aberrant splicing events make promising novel therapeutic targets to restore functional gene expression. Moreover, presence of these cryptic exons is highly specific to patients and areas of the brain affected by TDP-43 proteinopathy, offering the potential to develop biomarkers for early detection and stratification of patients. In summary, the discovery of cryptic exons gives hope for novel diagnostics and therapeutics on the horizon for TDP-43 proteinopathies.

Amyotrophic lateral sclerosis and frontotemporal dementia are devastating neurological diseases. Yet, there is no effective treatment for them. We uncovered a genetic cause of these diseases leading to a cryptic process in neurons caused by a malfunctioning protein. Our finding might help provide a potential new therapy for such neurological diseases.

We present 109 near full-length HIV genomes amplified from blood serum samples obtained during early 1986 from across Uganda, which to our knowledge is the earliest and largest population sample from the initial phase of the HIV epidemic in Africa. Consensus sequences were made from paired-end Illumina reads with a target-capture approach to amplify HIV material following poor success with standard approaches. In comparisons with a smaller ‘intermediate’ genome dataset from 1998 to 1999 and a ‘modern’ genome dataset from 2007 to 2016, the proportion of subtype D was significantly higher initially, dropping from 67% (73/109), to 57% (26/46) to 17% (82/465) respectively (p env sequences were predicted to be X4 tropic compared with none of the 24 subtype A1. We also analysed the frequency of subtype in the envelope region of inter-subtype recombinants, and found that subtype A1 is over-represented in env, suggesting recombination and selection have acted to remove subtype D env from circulation. The reduction of subtype D frequency over three decades therefore appears to be a result of selective pressure against X4 tropism and its higher virulence. Lastly, we find a subtype D specific codon deletion at position 24 of the V3 loop, which may explain the higher propensity for subtype D to utilise X4 tropism.

The Sustainable East Africa Research in Community Health (SEARCH) trial was a universal test-and-treat (UTT) trial in rural Uganda and Kenya, aiming to lower regional HIV-1 incidence. Here, we quantify breakthrough HIV-1 transmissions occurring during the trial from population-based, dried blood spot samples. Between 2013 and 2017, we obtained 549 gag and 488 pol HIV-1 consensus sequences from 745 participants: 469 participants infected prior to trial commencement and 276 SEARCH-incident infections. Putative transmission clusters, with a 1.5% pairwise genetic distance threshold, were inferred from maximum likelihood phylogenies; clusters arising after the start of SEARCH were identified with Bayesian time-calibrated phylogenies. Our phylodynamic approach identified nine clusters arising after the SEARCH start date: eight pairs and one triplet, representing mostly opposite-gender linked (6/9), within-community transmissions (7/9). Two clusters contained individuals with non-nucleoside reverse transcriptase inhibitor (NNRTI) resistance, both linked to intervention communities. The identification of SEARCH-incident, within-community transmissions reveals the role of unsuppressed individuals in sustaining the epidemic in both arms of a UTT trial setting. The presence of transmitted NNRTI resistance, implying treatment failure to the efavirenz-based antiretroviral therapy (ART) used during SEARCH, highlights the need to improve delivery and adherence to up-to-date ART recommendations, to halt HIV-1 transmission.

Spatial selective listening and auditory choice underlie important processes including attending to a speaker at a cocktail party and knowing how (or whether) to respond. To examine task encoding and the relative timing of potential neural substrates underlying these behaviors, we developed a spatial selective detection paradigm for monkeys, and recorded activity in primary auditory cortex (AC), dorsolateral prefrontal cortex (dlPFC), and the basolateral amygdala (BLA). A comparison of neural responses among these three areas showed that, as expected, AC encoded the side of the cue and target characteristics before dlPFC and BLA. Interestingly, AC also encoded the choice of the monkey before dlPFC and around the time of BLA. Generally, BLA showed weak responses to all task features except the choice. Decoding analyses suggested that errors followed from a failure to encode the target stimulus in both AC and dlPFC, but again, these differences arose earlier in AC. The similarities between AC and dlPFC responses were abolished during passive sensory stimulation with identical trial conditions, suggesting that the robust sensory encoding in dlPFC is contextually gated. Thus, counter to a strictly PFC-driven decision process, in this spatial selective listening task AC neural activity represents the sensory and decision information before dlPFC. Unlike in the visual domain, in this auditory task, the BLA does not appear to be robustly involved in selective spatial processing.SIGNIFICANCE STATEMENTWe examined neural correlates of an auditory spatial selective listening task by recording single-neuron activity in behaving monkeys from the amygdala, dorsolateral prefrontal cortex, and auditory cortex. We found that auditory cortex coded spatial cues and choice-related activity before dorsolateral prefrontal cortex or the amygdala. Auditory cortex also had robust delay period activity. Therefore, we found that auditory cortex could support the neural computations that underlie the behavioral processes in the task.

No treatment for frontotemporal dementia (FTD), the second most common type of early-onset dementia, is available, but therapeutics are being investigated to target the 2 main proteins associated with FTD pathological subtypes: TDP-43 (FTLD-TDP) and tau (FTLD-tau). Testing potential therapies in clinical trials is hampered by our inability to distinguish between patients with FTLD-TDP and FTLD-tau. Therefore, we evaluated truncated stathmin-2 (STMN2) as a proxy of TDP-43 pathology, given the reports that TDP-43 dysfunction causes truncated STMN2 accumulation. Truncated STMN2 accumulated in human induced pluripotent stem cell-derived neurons depleted of TDP-43, but not in those with pathogenic TARDBP mutations in the absence of TDP-43 aggregation or loss of nuclear protein. In RNA-Seq analyses of human brain samples from the NYGC ALS cohort, truncated STMN2 RNA was confined to tissues and disease subtypes marked by TDP-43 inclusions. Last, we validated that truncated STMN2 RNA was elevated in the frontal cortex of a cohort of patients with FTLD-TDP but not in controls or patients with progressive supranuclear palsy, a type of FTLD-tau. Further, in patients with FTLD-TDP, we observed significant associations of truncated STMN2 RNA with phosphorylated TDP-43 levels and an earlier age of disease onset. Overall, our data uncovered truncated STMN2 as a marker for TDP-43 dysfunction in FTD.

SummaryBackgroundHIV-1 infections initiated by multiple founder variants are characterised by a higher viral load and a worse clinical prognosis, yet little is known about the routes of exposure through which transmission of multiple founder variants is most likely.MethodsWe conducted a systematic review of studies that estimated founder variant multiplicity in HIV-1 infection, searching MEDLINE, EMBASE and Global Health databases for papers published between 1st January 1990 and 14th September 2020 (PROSPERO study CRD42020202672). Leveraging individual patient estimates from these studies, we performed a logistic meta-regression to estimate the probability that an HIV infection is initiated by multiple founder variants. We calculated a pooled estimate using a random effects model, subsequently stratifying this estimate across nine transmission routes in a univariable analysis. We then extended our model to adjust for different study methods in a multivariable analysis, recalculating estimates across the nine transmission routes.FindingsWe included 70 publications in our analysis, comprising 1657 individual patients. Our pooled estimate of the probability that an infection is initiated by multiple founder variants was 0·25 (95% CI: 0·21-0·29), with moderate heterogeneity (Q = 132 · 3, p < 0 · 001, I2 = 64 · 2%). Our multivariable analysis uncovered differences in the probability of multiple variant infection by transmission route. Relative to a baseline of male-to-female transmission, the predicted probability for female-to-male multiple variant transmission was significantly lower at 0·13 (95% CI: 0·08-0·20), while the probabilities for people-who-inject-drugs (PWID) and men-who-have-sex-with-men (MSM) transmissions were significantly higher at 0·37 (0·24-0·53) and 0·30 (0·33-0·40), respectively. There was no significant difference in the probability of multiple variant transmission between male-to-female transmission (0·21 (0·14-0·31)), post-partum mother-to-child (0·18 (0·03-0·57)), pre-partum mother-to-child (0·17 (0·08-0·33)), intrapartum mother-to-child (0·27 (0·14-0·40)).InterpretationWe identified PWID and MSM transmissions are significantly more likely to result in an infection initiated by multiple founder variants, whilst female-to-male infections are significantly less likely. Quantifying how the routes of HIV infection impact the transmission of multiple variants allows us to better understand how the evolution and epidemiology of HIV-1 determine clinical outcomes.FundingThis study was supported by the MRC Precision Medicine Doctoral Training Programme (ref: 2259239) and an ERC Starting Grant awarded to KEA (award number 757688). The funding sources played no role in study design, data collection, data analysis, data interpretation, or writing of the report.Panel: Research in contextEvidence before this studyMost HIV-1 infections are initiated by a single, genetically homogeneous founder variant. Infections initiated by multiple founders, however, are associated with a significantly faster decline of CD4+ T cells in untreated individuals, ultimately leading to an earlier onset of AIDS. Through our systematic search of MEDLINE, EMBASE and Global Health databases, we identified 82 studies that classify the founder variant multiplicity of early HIV infections. As these studies vary in the methodology used to calculate the number of founder variants, it is difficult to evaluate the multiplicity of founder variants across routes of exposure.Added value of this studyWe estimated the probability that an HIV infection is initiated by multiple founder variants across exposure routes, leveraging individual patient data from 70 of the identified studies. Our multivariable meta-regression adjusted for heterogeneity across study methodology and uncovered differences in the probability that an infection is initiated by multiple founder variants by exposure route. While overall, we estimated that 25% of infections are initiated by multiple founder variants, our analysis found that this probability for female-to-male transmission is significantly lower than for male-to-female transmission. By contrast, this probability was significantly higher among people-who-inject-drugs (PWID) and men-who-have-sex-with-men (MSM). There was no difference in the probability of multiple founder variant transmission for mother-to-child transmission when compared with male-to-female sexual transmission.Implications of all the available evidenceBecause HIV-1 infections initiated by multiple founders are associated with a poorer prognosis, determining whether the route of exposure affects the probability with which infections are initiated by multiple variants facilitates an improved understanding of how the evolution and epidemiology of HIV-1 determine clinical progression. Our results identify that PWID and MSM transmissions are significantly more likely to result in an infection initiated by multiple founder variants compared to male-to-female. This reiterates the need for focussed public health programmes that reduce the burden of HIV-1 in these risk groups.

Cell and tissue functions rely on an elaborate intracellular transport system responsible for distributing bioactive molecules with high spatiotemporal accuracy. The tubular network of the Endoplasmic Reticulum (ER) constitutes a system for the delivery of luminal solutes it stores, including Ca2+, across the cell periphery. The physical nature and factors underlying the ER’s functioning as a fluidics system are unclear. Using an improved ER transport visualisation methodology combined with optogenetic Ca2+ dynamics imaging, we observed that ER luminal transport is modulated by natural ER tubule narrowing and dilation, directly proportional to the amount of an ER membrane morphogen, Reticulon 4 (RTN4). Consequently, the ER morphoregulatory effect of RTN4 defines ER’s capacity for peripheral Ca2+ delivery and thus controls axonogenesis. Excess RTN4 limited ER luminal transport, Ca2+ release and iPSC-derived cortical neurons’ axonal extension, while RTN4 elimination reversed the effects. Summary Intracellular transport through the lumen of the ER network is modulated through narrowing/dilation of ER tubules by a membrane morphogen – RTN4, a process controlling axonogenesis by limiting the delivery of ER-stored Ca2+.

TDP-43 aggregation and redistribution have been recognised as a hallmark of amyotrophic lateral sclerosis, frontotemporal dementia and other neurological disorders. While TDP-43 has been studied extensively in neuronal tissues, TDP-43 inclusions have also been described in the muscle of inclusion body myositis patients, highlighting the need to understand the role of TDP-43 beyond the central nervous system. Using RNA-seq we performed the first direct comparison of TDP-43-mediated transcription and alternative splicing in muscle (C2C12) and neuronal (NSC34) mouse cells. Our results clearly show that TDP-43 displays a tissue-characteristic behaviour targeting unique transcripts in each cell type. This is not due to variable transcript abundance but rather due to cell-specific expression of RNA-binding proteins, which influences TDP-43 performance. Among splicing events commonly dysregulated in both cell lines, we identified some that are TDP-43-dependent also in human cells and show that inclusion levels of these alternative exons appear to be differentially altered in affected tissues of FTLD and IBM patients. We therefore propose that TDP-43 dysfunction, reflected in aberrant splicing, contributes to disease development but it does so in a tissue- and disease-specific manner.

Variants within the UNC13A gene have long been known to increase risk of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), two related neurodegenerative diseases defined by mislocalization of the RNA-binding protein TDP-43. Here, we show that TDP-43 depletion induces robust inclusion of a cryptic exon (CE) within UNC13A, a critical synaptic gene, resulting in nonsense-mediated decay and protein loss. Strikingly, two common polymorphisms strongly associated with ALS/FTD risk directly alter TDP-43 binding within the CE or downstream intron, increasing CE inclusion in cultured cells and in patient brains. Our findings, which are the first to demonstrate a genetic link specifically between loss of TDP-43 nuclear function and disease, reveal both the mechanism by which UNC13A variants exacerbate the effects of decreased nuclear TDP-43 function, and provide a promising therapeutic target for TDP-43 proteinopathies. One-Sentence Summary Shared ALS/FTD risk variants increase the sensitivity of a cryptic exon in the synaptic gene UNC13A to TDP-43 depletion.

Variants within the UNC13A gene have long been known to increase risk of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), two related neurodegenerative diseases defined by mislocalization of the RNA-binding protein TDP-43. Here, we show that TDP-43 depletion induces robust inclusion of a cryptic exon (CE) within UNC13A, a critical synaptic gene, resulting in nonsense-mediated decay and protein loss. Strikingly, two common polymorphisms strongly associated with ALS/FTD risk directly alter TDP-43 binding within the CE or downstream intron, increasing CE inclusion in cultured cells and in patient brains. Our findings, which are the first to demonstrate a genetic link specifically between loss of TDP-43 nuclear function and disease, reveal both the mechanism by which UNC13A variants exacerbate the effects of decreased nuclear TDP-43 function, and provide a promising therapeutic target for TDP-43 proteinopathies.One-Sentence SummaryShared ALS/FTD risk variants increase the sensitivity of a cryptic exon in the synaptic gene UNC13A to TDP-43 depletion.

Auditory selective listening and decision making underlies important processes, including attending to a single speaker in a crowded room, often referred to as the cocktail party problem. To examine the neural mechanisms underlying these behaviors, we developed a novel auditory selective listening paradigm for monkeys. In this task, monkeys had to detect a difficult to discriminate target embedded in noise when presented in a pre-cued location (either left or right) and ignore it if it was in the opposite location. While the animals carried out the task we recorded neural activity in primary auditory cortex (AC), dorsal lateral prefrontal cortex (dlPFC) and the basal lateral amygdala (BLA), given that these areas have been implicated in auditory decision making, selective listing, and/or reward-guided decision making. There were two main findings in the neural data. First, primary AC encoded the side of the cue and target, and the monkey’s choice, before either dlPFC or the amygdala. The BLA encoded cue and target variables negligibly, but was engaged at the time of the monkey’s choice. Second, decoding analyses suggested that errors followed primarily from a failure to encode the target stimulus in both AC and PFC, but earlier in AC. Thus, AC neural activity is poised to represent the sensory volley and decision making during selective listening before dlPFC, and they both precede activity in BLA.

Recombination is an important feature of HIV evolution, occurring both within and between the major branches of diversity (subtypes). The Ugandan epidemic is primarily composed of two subtypes, A1 and D, that have been co-circulating for 50 years, frequently recombining in dually infected patients. Here, we investigate the frequency of recombinants in this population and the location of breakpoints along the genome. As part of the PANGEA-HIV consortium, 1,472 consensus genome sequences over 5 kb have been obtained from 1,857 samples collected by the MRC/UVRI & LSHTM Research unit in Uganda, 465 (31.6 per cent) of which were near full-length sequences (>8 kb). Using the subtyping tool SCUEAL, we find that of the near full-length dataset, 233 (50.1 per cent) genomes contained only one subtype, 30.8 per cent A1 (n = 143), 17.6 per cent D (n = 82), and 1.7 per cent C (n = 8), while 49.9 per cent (n = 232) contained more than one subtype (including A1/D (n = 164), A1/C (n = 13), C/D (n = 9); A1/C/D (n = 13), and 33 complex types). K-means clustering of the recombinant A1/D genomes revealed a section of envelope (C2gp120-TMgp41) is often inherited intact, whilst a generalized linear model was used to demonstrate significantly fewer breakpoints in the gag–pol and envelope C2-TM regions compared with accessory gene regions. Despite similar recombination patterns in many recombinants, no clearly supported circulating recombinant form (CRF) was found, there was limited evidence of the transmission of breakpoints, and the vast majority (153/164; 93 per cent) of the A1/D recombinants appear to be unique recombinant forms. Thus, recombination is pervasive with clear biases in breakpoint location, but CRFs are not a significant feature, characteristic of a complex, and diverse epidemic. 1. Introduction 2. Methods 2.1 Sample collection 2.2 Sequencing and alignment 2.3 Subtyping 2.4 Identification of transmitted breakpoints 2.5 Recombination pattern classification 2.6 Breakpoint and genome location model framework 3. Results 3.1 Subtype distribution 3.2 Identification of CRFs and transmitted breakpoints 3.3 Recombinant groupings 3.4 Breakpoint distribution 4. Discussion

The neuromuscular junction (NMJ) is the highly specialised peripheral synapse formed between lower motor neuron terminals and muscle fibres. Post-synaptic acetylcholine receptors (AChRs), which are found in high density in the muscle membrane, bind to acetylcholine released into the synaptic cleft of the NMJ, ultimately facilitating the conversion of motor action potentials to muscle contractions. NMJs have been studied for many years as a general model for synapse formation, development and function, and are known to be early sites of pathological changes in many neuromuscular diseases. However, information is limited on the diversity of NMJs in different muscles, whether muscle fibre type impacts NMJ morphology and growth, and the relevance of these parameters to neuropathology. Here, this crucial gap was addressed using a robust and standardised semi-automated workflow called NMJ-morph to quantify features of pre- and post-synaptic NMJ architecture in an unbiased manner. Five wholemount muscles from wild-type mice were dissected and compared at immature (post-natal day, P7) and early adult (P31-32) timepoints. Post-synaptic AChR morphology was found to be more variable between muscles than that of the motor neuron terminal and there were greater differences in the developing NMJ than at the mature synapse. Post-synaptic architecture, but not neuronal morphology or post-natal synapse growth, correlates with fibre type and is largely independent of muscle fibre diameter. Counter to previous observations, this study indicates that smaller NMJs tend to innervate muscles with higher proportions of fast twitch fibres and that NMJ growth rate is not conserved across all muscles. Furthermore, healthy pre- and post-synaptic NMJ morphological parameters were collected for five anatomically and functionally distinct mouse muscles, generating reference data that will be useful for the future assessment of neuromuscular disease models.Graphical Abstract

Background: As of June 18, 2020, Uganda had registered 741 COVID-19 cases, and several intervention measures have been implemented to control the spread of the SARS-CoV2. With increasing risk of community transmission, it is important to forecast the extent on the COVID-19 epidemic in Uganda. This study aimed to design a predictive model to provide reliable estimates for COVID-19 in Uganda. Methods: A stochastic model of a modified SEAIR (Susceptible, Exposed, Infected Asymptomatic, Infected Symptomatic and Removed) type, which made assumptions on the local SARS-CoV2 transmission, was used to forecast the impact of COVID-19 in relation to the varying levels of compliance to mitigation measures. Results: Our results predict between 2,000 to 4,000 cases within 100 days from lifting the lockdown if no or weak interventions are in place. However, if social distancing was implemented at 40% effectiveness, to prevent interaction between susceptible and infected populations with no use of face-masks, we would expect 200 to 400 cases. The number of infections decreases with increase in the level of effectiveness of social distancing measures and the curve flattens at ≥60% level of social distancing. Wearing face-masks at ≥40% consistency and social distancing at 50% reduces the number of infections and flattens the curve. Interpretation: The prompt public health response in Uganda has so far limited COVID-19 to imported cases, comprising mainly of truck drivers. Although lifting the lockdown measures is feasible, social distancing strategies, as the most effective measure, should be maintained at high levels and supplemented with wearing face-masks. Funding Statement: None to declare Declaration of Interests: All authors have no reported conflicts of interest. Ethics Approval Statement: Not required

Mutations in the RNA-binding protein FUS cause amyotrophic lateral sclerosis (ALS), a devastating neurodegenerative disease. FUS plays a role in numerous aspects of RNA metabolism, including mRNA splicing. However, the impact of ALS-causative mutations on splicing has not been fully characterized, as most disease models have been based on overexpressing mutant FUS, which will alter RNA processing due to FUS autoregulation. We and others have recently created knockin models that overcome the overexpression problem, and have generated high depth RNA-sequencing on FUS mutants in parallel to FUS knockout, allowing us to compare mutation-induced changes to genuine loss of function. We find that FUS-ALS mutations induce a widespread loss of function on expression and splicing. Specifically, we find that mutant FUS directly alters intron retention levels in RNA-binding proteins. Moreover, we identify an intron retention event in FUS itself that is associated with its autoregulation. Altered FUS levels have been linked to disease, and we show here that this novel autoregulation mechanism is altered by FUS mutations. Crucially, we also observe this phenomenon in other genetic forms of ALS, including those caused by TDP-43, VCP and SOD1 mutations, supporting the concept that multiple ALS genes interact in a regulatory network.

Background: Harm reduction has dramatically reduced HIV incidence among people who inject drugs (PWID). In Glasgow, Scotland, Methods: We collected Scottish pol sequences and identified closely related sequences from public databases. Genetic linkage was ascertained among 228 Scottish, 1820 UK, and 524 global sequences. The outbreak cluster was extracted to estimate epidemic parameters.Results: All 104 outbreak sequences originated from Scotland and contained E138A and V179E. Mean genetic distance was Conclusions: In contrast to other recent HIV outbreaks among PWID, harm reduction services were not clearly reduced in Scotland. Nonetheless, the high proportion of individuals with a history of homelessness (45%) suggests that services were inadequate for those in precarious living situations. The high prevalence of hepatitis C (>90%) is indicative of sharing of injecting equipment. Monitoring the epidemic phylogenetically in real time may accelerate public health action.

The current study examined the effects of an intervention aimed at blocking the transfer of frustration from a previous experience (i.e. recall task) to a subsequent and unrelated task (i.e. ultimatum bargaining task). Participants who went through the intervention were more likely to accept unfair offers in the ultimatum bargaining task than those who did not go through the intervention. These results show that participants who were blocked from transferring their feelings of frustration from the recall task to the subsequent bargaining task (no-transfer condition) more likely accepted unfair offers than those who inadvertently transferred their feelings of frustration (transfer condition). The effect of conditions on accept-reject decisions in the ultimatum bargaining was mediated by reported feelings of frustration.

Background Phylogenetic mapping of HIV-1 lineages circulating across defined geographical locations is promising for better understanding HIV transmission networks to design optimal prevention interventions. Methods We obtained near full-length HIV-1 genome sequences from people living with HIV (PLWH), including participants on antiretroviral treatment in the Botswana Combination Prevention Project, conducted in 30 Botswana communities in 2013–2018. Phylogenetic relationships among viral sequences were estimated by maximum likelihood. Results We obtained 6078 near full-length HIV-1C genome sequences from 6075 PLWH. We identified 984 phylogenetically distinct HIV-1 lineages (molecular HIV clusters) circulating in Botswana by mid-2018, with 2–27 members per cluster. Of these, dyads accounted for 62%, approximately 32% (n = 316) were found in single communities, and 68% (n = 668) were spread across multiple communities. Men in clusters were approximately 3 years older than women (median age 42 years, vs 39 years; P Conclusions A large number of circulating phylogenetically distinct HIV-1C lineages (molecular HIV clusters) suggests highly diversified HIV transmission networks across Botswana communities by 2018.

Although fishing communities (FCs) in Uganda are disproportionately affected by HIV-1 relative to the general population (GP), the transmission dynamics are not completely understood. We earlier found most HIV-1 transmissions to occur within FCs of Lake Victoria. Here, we test the hypothesis that HIV-1 transmission in FCs is isolated from networks in the GP. We used phylogeography to reconstruct the geospatial viral migration patterns in 8 FCs and 2 GP cohorts and a Bayesian phylogenetic inference in BEAST v1.8.4 to analyse the temporal dynamics of HIV-1 transmission. Subtype A1 (pol region) was most prevalent in the FCs (115, 45.1%) and GP (177, 50.4%). More recent HIV transmission pairs from FCs were found at a genetic distance (GD)

Research in historical archaeology has often focused on analyzing the degree to which African diaspora material culture has been shaped by the beliefs and practices of particular African cultures impacted by the trans-Atlantic slave trade. Landscapes, architecture, and objects can, at times, be analyzed to evaluate whether their design, construction, or use has been influenced by such cultural traditions. Through the concepts of innovation and creolization, material culture such as this can be reinterpreted in new ways. This article reexamines English manufactured smoking pipes recovered from Newton Plantation cemetery on Barbados. I argue that within an African diasporic burial context, unmodified European manufactured objects, such as English manufactured smoking pipes, could carry cultural significance shaped by African cultural beliefs and practices. Artifacts such as these are embedded in both diversity and situational complexity.

OBJECTIVES Transmission of drug-resistant HIV-1 has decreased in the UK since the early 2000s. This analysis reports recent trends and characteristics of transmitted drug resistance (TDR) in the UK from 2010 to 2013. METHODS Resistance tests conducted in antiretroviral treatment (ART)-naive individuals between 2010 and 2013 were analysed for the presence of transmitted drug resistance mutations (TDRMs), defined as any mutations from a modified 2009 World Health Organization surveillance list, or a modified 2013 International Antiviral Society-USA list for integrase tests. Logistic regression was used to examine associations between demographics and the prevalence of TDRMs. RESULTS TDRMs were observed in 1223 (7.5%) of 16 425 individuals; prevalence declined from 8.1% in 2010 to 6.6% in 2013 (P = 0.02). The prevalence of TDRMs was higher among men who have sex with men (MSM) compared with heterosexual men and women (8.7% versus 6.4%, respectively) with a trend for decreasing TDRMs among MSM (P = 0.008) driven by a reduction in nucleoside reverse transcriptase inhibitor (NRTI)-related mutations. The most frequently detected TDRMs were K103N (2.2%), T215 revertants (1.6%), M41L (0.9%) and L90M (0.7%). Predicted phenotypic resistance to first-line ART was highest to the nonnucleoside reverse transcriptase inhibitors (NNRTIs) rilpivirine and efavirenz (6.2% and 3.4%, respectively) but minimal to NRTIs, including tenofovir, and protease inhibitors (PIs). No major integrase TDRMs were detected among 101 individuals tested while ART-naive. CONCLUSIONS We observed a decrease in TDRMs in recent years. However, this was confined to the MSM population and rates remained stable in those with heterosexually acquired HIV infection. Resistance to currently recommended first-line ART, including integrase inhibitors, remained reassuringly low.

SUMMARY Clonal evolution of osimertinib-resistance mechanisms in EGFR mutant lung adenocarcinoma is poorly understood. Using multi-region whole-exome and RNA sequencing of prospectively collected pre- and post-osimertinib-resistant tumors, including at rapid autopsies, we identify a likely mechanism driving osimertinib resistance in all patients analyzed. The majority of patients acquire two or more resistance mechanisms either concurrently or in temporal sequence. Focal copy-number amplifications occur subclonally and are spatially and temporally separated from common resistance mutations such as EGFR C797S. MET amplification occurs in 66% (n = 6/9) of first-line osimertinib-treated patients, albeit spatially heterogeneous, often co-occurs with additional acquired focal copy-number amplifications and is associated with early progression. Noteworthy osimertinib-resistance mechanisms discovered include neuroendocrine differentiation without histologic transformation, PD-L1, KRAS amplification, and ESR1-AKAP12, MKRN1-BRAF fusions. The subclonal co-occurrence of acquired genomic alterations upon osimertinib resistance will likely require targeting multiple resistance mechanisms by combination therapies., In Brief Roper et al. perform multi-region whole-exome and RNA sequencing of pre- and post-resistant tumors from EGFR mutant lung cancer patients treated with osimertinib. They uncover the subclonal co-occurrence of acquired genomic alterations upon osimertinib resistance suggesting combination first-line therapies may prevent or delay key resistance mechanisms., Graphical Abstract

Background: Memory CD4+ T cells (mCD4s) containing integrated HIV DNA are considered the main barrier to a cure for HIV infection. Here we analysed HIV DNA reservoirs in antigen-specific subsets of mCD4s to delineate the mechanisms by which HIV reservoirs persist during ART.Methods/Results: HIV Gag, Cytomegalovirus (CMV) and Tetanus Toxoid (TT) specific mCD4s were isolated from peripheral blood samples obtained from 11 individual subjects, 2-11 years after commencing ART. Antigen-specific mCD4s were identified by the sensitive OX40 assay and purified by cell sorting. Total HIV DNA levels were quantified by real-time PCR, and clonal viral sequences generated from mCD4 subsets and pre-ART plasma samples. Quantitative results and sequence analysis were restricted to 5 and 3 study participants respectively, likely due to the low frequency of the antigen-specific mCD4s and relatively low HIV DNA proviral loads. Median HIV Gag-, CMV-, and TT-specific mCD4s were 0.61%, 2.46% and 0.78% of total mCD4s, and contained a median of 2.50, 2.38, and 2.55 log10 copies of HIV DNA per 106 cells respectively. HIV DNA sequences derived from antigen-specific mCD4s clustered with sequences derived from pre-ART plasma samples. There was a trend towards increased viral diversity in clonal viral sequences derived from CMV-specific relative to TT-specific mCD4s.Discussion/Conclusions: Despite limitations, this study provides direct evidence that HIV reservoirs persist in memory CD4+ T cell subsets maintained by homeostatic proliferation (TT) and adds to growing evidence against viral evolution during ART. Similar future studies require techniques that sample diverse HIV reservoirs and with improved sensitivity.

Viral phylogenetic methods contribute to understanding how HIV spreads in populations, and thereby help guide the design of prevention interventions. So far, most analyses have been applied to well-sampled concentrated HIV-1 epidemics in wealthy countries. To direct the use of phylogenetic tools to where the impact of HIV-1 is greatest, the Phylogenetics And Networks for Generalized HIV Epidemics in Africa (PANGEA-HIV) consortium generates full-genome viral sequences from across sub-Saharan Africa. Analyzing these data presents new challenges, since epidemics are principally driven by heterosexual transmission and a smaller fraction of cases is sampled. Here, we show that viral phylogenetic tools can be adapted and used to estimate epidemiological quantities of central importance to HIV-1 prevention in sub-Saharan Africa. We used a community-wide methods comparison exercise on simulated data, where participants were blinded to the true dynamics they were inferring. Two distinct simulations captured generalized HIV-1 epidemics, before and after a large community-level intervention that reduced infection levels. Five research groups participated. Structured coalescent modelling approaches were most successful: phylogenetic estimates of HIV-1 incidence, incidence reductions, and the proportion of transmissions from individuals in their first three months of infection correlated with the true values (Pearson correlation > 90%), with small bias. However, on some simulations, true values were markedly outside reported confidence or credibility intervals. The blinded comparison revealed current limits and strengths in using HIV phylogenetics in challenging settings, provided benchmarks for future methods’ development, and supports using the latest generation of phylogenetic tools to advance HIV surveillance and prevention.

This article reviews existing clinical practices and sensor research undertaken to monitor fetal well-being during labour. Current clinical practices that include fetal heart rate monitoring and fetal scalp blood sampling are shown to be either inadequate or time-consuming. Monitoring of lactate in blood is identified as a potential alternative for intrapartum fetal monitoring due to its ability to distinguish between different types of acidosis. A literature review from a medical and technical perspective is presented to identify the current advancements in the field of lactate sensors for this application. It is concluded that a less invasive and a more continuous monitoring device is required to fulfill the clinical needs of intrapartum fetal monitoring. Potential specifications for such a system are also presented in this paper.

SUMMARY Intratumor mutational heterogeneity has been documented in primary non-small-cell lung cancer. Here, we elucidate mechanisms of tumor evolution and heterogeneity in metastatic thoracic tumors (lung adenocarcinoma and thymic carcinoma) using whole-exome and transcriptome sequencing, SNP array for copy-number alterations (CNAs), and mass-spectrometry-based quantitative proteomics of metastases obtained by rapid autopsy. APOBEC mutagenesis, promoted by increased expression of APOBEC3 region transcripts and associated with a high-risk APOBEC3 germline variant, correlated with mutational tumor heterogeneity. TP53 mutation status was associated with APOBEC hypermutator status. Interferon pathways were enriched in tumors with high APOBEC mutagenesis and IFN-γ-induced expression of APOBEC3B in lung adenocarcinoma cells, suggesting that the immune microenvironment may promote mutational heterogeneity. CNAs occurring late in tumor evolution correlated with downstream transcriptomic and proteomic heterogeneity, although global proteomic heterogeneity was significantly greater than transcriptomic and CNA heterogeneity. These results illustrate key mechanisms underlying multi-dimensional heterogeneity in metastatic thoracic tumors., In Brief Roper et al. perform integrated exome, transcriptome, and quantitative proteomics analyses of metastases obtained through rapid autopsy of patients with thoracic malignancies. They identify APOBEC mutagenesis, driven by germline variants, mutant TP53, and the tumor microenvironment, and late copy-number alterations as key mechanisms underlying proteogenomic evolution and heterogeneity., Graphical Abstract

Background & methods The ICONIC project has developed an automated high-throughput pipeline to generate HIV nearly full-length genomes (NFLG, i.e. from gag to nef) from next-generation sequencing (NGS) data. The pipeline was applied to 420 HIV samples collected at University College London Hospitals NHS Trust and Barts Health NHS Trust (London) and sequenced using an Illumina MiSeq at the Wellcome Trust Sanger Institute (Cambridge). Consensus genomes were generated and subtyped using COMET, and unique recombinants were studied with jpHMM and SimPlot. Maximum-likelihood phylogenetic trees were constructed using RAxML to identify transmission networks using the Cluster Picker. Results The pipeline generated sequences of at least 1Kb of length (median = 7.46Kb, IQR = 4.01Kb) for 375 out of the 420 samples (89%), with 174 (46.4%) being NFLG. A total of 365 sequences (169 of them NFLG) corresponded to unique subjects and were included in the down-stream analyses. The most frequent HIV subtypes were B (n = 149, 40.8%) and C (n = 77, 21.1%) and the circulating recombinant form CRF02_AG (n = 32, 8.8%). We found 14 different CRFs (n = 66, 18.1%) and multiple URFs (n = 32, 8.8%) that involved recombination between 12 different subtypes/CRFs. The most frequent URFs were B/CRF01_AE (4 cases) and A1/D, B/C, and B/CRF02_AG (3 cases each). Most URFs (19/26, 73%) lacked breakpoints in the PR+RT pol region, rendering them undetectable if only that was sequenced. Twelve (37.5%) of the URFs could have emerged within the UK, whereas the rest were probably imported from sub-Saharan Africa, South East Asia and South America. For 2 URFs we found highly similar pol sequences circulating in the UK. We detected 31 phylogenetic clusters using the full dataset: 25 pairs (mostly subtypes B and C), 4 triplets and 2 quadruplets. Some of these were not consistent across different genes due to inter- and intra-subtype recombination. Clusters involved 70 sequences, 19.2% of the dataset. Conclusions The initial analysis of genome sequences detected substantial hidden variability in the London HIV epidemic. Analysing full genome sequences, as opposed to only PR+RT, identified previously undetected recombinants. It provided a more reliable description of CRFs (that would be otherwise misclassified) and transmission clusters.

Background & Methods The ICONIC project has developed an automated high-throughput pipeline to generate HIV nearly full-length genomes (NFLG, i.e. from gag to nef ) from next-generation sequencing (NGS) data. The pipeline was applied to 420 HIV samples collected at University College London Hospital and Barts Health NHS Trust (London) and sequenced using an Illumina MiSeq at the Wellcome Trust Sanger Institute (Cambridge). Consensus genomes were generated and subtyped using COMET, and unique recombinants were studied with jpHMM and SimPlot. Maximum-likelihood phylogenetic trees were constructed using RAxML to identify transmission networks using the Cluster Picker. Results The pipeline generated sequences of at least 1Kb of length (median=7.4Kb) for 375 out of the 420 samples (89%), with 174 (46.4%) being NFLG. A total of 365 sequences (169 of them NFLG) corresponded to unique subjects and were included in the down-stream analyses. The most frequent HIV subtypes were B (n=149, 40.8%) and C (n=77, 21.1%) and the circulating recombinant form CRF02_AG (n=32, 8.8%). We found 14 different CRFs (n=66, 18.1%) and multiple URFs (n=32, 8.8%) that involved recombination between 12 different subtypes/CRFs. The most frequent URFs were B/CRF01_AE (4 cases) and A1/D, B/C, and B/CRF02_AG (3 cases each). Most URFs (19/26, 73%) lacked breakpoints in the PR+RT pol region, rendering them undetectable if only that was sequenced. Twelve (37.5%) of the URFs could have emerged within the UK, whereas the rest were probably imported from sub-Saharan Africa, South East Asia and South America. For 2 URFs we found highly similar pol sequences circulating in the UK. We detected 31 phylogenetic clusters using the full dataset: 25 pairs (mostly subtypes B and C), 4 triplets and 2 quadruplets. Some of these were not consistent across different genes due to inter- and intra-subtype recombination. Clusters involved 70 sequences, 19.2% of the dataset. Conclusions The initial analysis of genome sequences detected substantial hidden variability in the London HIV epidemic. Analysing full genome sequences, as opposed to only PR+RT, identified previously undetected recombinants. It provided a more reliable description of CRFs (that would be otherwise misclassified) and transmission clusters.

Patients who do not disclose their sexuality, including men who do not disclose same-sex behaviour, are difficult to characterise through traditional epidemiological approaches such as interviews. Using a recently developed method to detect large networks of viral sequences from time-resolved trees, we localised non-disclosed men who have sex with men (MSM) in UK transmission networks, gaining crucial insight into the behaviour of this group.For this phylogenetic analysis, we obtained HIV pol sequences from the UK HIV Drug Resistance Database (UKRDB), a central repository for resistance tests done as part of routine clinical care throughout the UK. Sequence data are linked to demographic and clinical data held by the UK Collaborative HIV Cohort study and the national HIV/AIDS reporting system database. Initially, we reconstructed maximum likelihood phylogenies from these sequences, then sequences were selected for time-resolved analysis in BEAST if they were clustered with at least one other sequence at a genetic distance of 4·5% or less with support of at least 90%. We used time-resolved phylogenies to create networks by linking together nodes if sequences shared a common ancestor within the previous 5 years. We identified potential non-disclosed MSM (pnMSM), defined as self-reported heterosexual men who clustered only with men. We measured the network position of pnMSM, including betweenness (a measure of connectedness and importance) and assortativity (the propensity for nodes sharing attributes to link).14 405 individuals were in the network, including 8452 MSM, 1743 heterosexual women and 1341 heterosexual men. 249 pnMSM were identified (18·6% of all clustered heterosexual men) in the network. pnMSM were more likely to be black African (p0·0001), less likely to be infected with subtype B (p=0·006), and were slightly older (p=0·002) than the MSM they clustered with. Mean betweenness centrality was lower for pnMSM than for MSM (1·31, 95% CI 0·48-2·15 in pnMSM vs 2·24, 0·98-3·51 in MSM; p=0·002), indicating that pnMSM were in peripheral positions in MSM clusters. Assortativity by risk group was higher than expected (0·037 vs -0·037, p=0·01) signifying that pnMSM were linked to each other. We found that self-reported heterosexual men were more likely to link MSM and heterosexual women than heterosexual women were to link MSM and heterosexual men (Fisher's exact test p=0·0004; OR 2·24) but the number of such transmission chains was small (only 54 in total vs 32 in women).pnMSM are a subgroup distinct from both MSM and from heterosexual men. They are more likely to choose sexual partners who are also pnMSM and might exhibit lower-risk sexual behaviour than MSM (eg, choosing low-risk partners or consistently using condoms). Heterosexual men are the group most likely to be diagnosed with late-stage disease (ie, low CD4 counts) and non-disclosed MSM might put female partners at higher risk than heterosexual men because non-disclosed MSM have male partners. Hence, pnMSM require specific consideration to ensure they are included in public health interventions.National Institutes of Health.

Introduction Phylogenetic information provides new horizons for clinical case finding in HIV, but raises issues of acceptability, privacy and even criminalisation. We reviewed studies describing use of phylogenetics to directly inform case finding in community acquired stigmatised infectious diseases. Methods A search in MEDLINE, Embase, CINAHL and PsychINFO for articles where phylogenetics have been used to facilitate case finding in sexually transmitted infections, TB, HBV or HCV, published until July 2016 in English. Results 26 of 6042 papers screened met the inclusion criteria; 17 TB, 9 HIV. 19 studies reported using phylogenetics to identify and investigate HIV outbreaks but did not report its role in case finding. Case finding strategies included confirming the source of an outbreak to prompt wider investigation (HIV); investigation of phylogenetically clustered cases (TB, HIV); combined cluster and geographical information to target screening (TB); screening informed by discrepancies between genotypic and epidemiological data (TB); phylogenetic characterisation to inform a screening intervention (HIV); epidemiological data to identify of a source (HIV); and contact tracing with genotype matching to a phenotype (HIV). Facilitators included sharing molecular surveillance data to establish community support in targeted TB screening. Barriers included delayed results, time lapse between cases and refusal of access to premises for screening. However patient barriers were rarely reported. Ethical issues included media coverage of an HIV sources identity. Conclusion Phylogenetics-informed approaches to case finding are feasible in stigmatised infections. However studies reporting their use in clinical and public health practice provide limited information on patient related barriers, acceptability, or on ethical challenges such as identification of “core” transmitters or criminalisation. Research into patient views on acceptability, risks and preferred approaches to using phylogenetic information for case finding in HIV is needed to inform interventions.

To characterize HIV-1 transmission dynamics in regions where the burden of HIV-1 is greatest, the “Phylogenetics and Networks for Generalised HIV Epidemics in Africa” consortium (PANGEA-HIV) is sequencing full-genome viral isolates from across sub-Saharan Africa. We report the first 3,985 PANGEA-HIV consensus sequences from four cohort sites (Rakai Community Cohort Study, n = 2,833; MRC/UVRI Uganda, n = 701; Mochudi Prevention Project, n = 359; Africa Health Research Institute Resistance Cohort, n = 92). Next-generation sequencing success rates varied: more than 80% of the viral genome from the gag to the nef genes could be determined for all sequences from South Africa, 75% of sequences from Mochudi, 60% of sequences from MRC/UVRI Uganda, and 22% of sequences from Rakai. Partial sequencing failure was primarily associated with low viral load, increased for amplicons closer to the 3′ end of the genome, was not associated with subtype diversity except HIV-1 subtype D, and remained significantly associated with sampling location after controlling for other factors. We assessed the impact of the missing data patterns in PANGEA-HIV sequences on phylogeny reconstruction in simulations. We found a threshold in terms of taxon sampling below which the patchy distribution of missing characters in next-generation sequences (NGS) has an excess negative impact on the accuracy of HIV-1 phylogeny reconstruction, which is attributable to tree reconstruction artifacts that accumulate when branches in viral trees are long. The large number of PANGEA-HIV sequences provides unprecedented opportunities for evaluating HIV-1 transmission dynamics across sub-Saharan Africa and identifying prevention opportunities. Molecular epidemiological analyses of these data must proceed cautiously because sequence sampling remains below the identified threshold and a considerable negative impact of missing characters on phylogeny reconstruction is expected.

Summary Background Therapeutic options for the management of hepatitis C virus (HCV) infection have evolved rapidly over the past two decades, with a consequent improvement in cure rates. Novel therapeutic agents are an area of great interest in the research community, with a number of these agents showing promise in the clinical setting. Aims To assess and present the available evidence for the use of novel therapeutic agents for the treatment of HCV, updating previous guidelines. Methods All Phase 2 and 3 studies, as well as abstract presentations from international Hepatology meetings were identified and reviewed for suitable inclusion, based on studies of new therapies in HCV. Treatment-naive and experienced individuals, as well as cirrhotic and co-infected individuals were included. Results Sofosbuvir, simeprevir and faldaprevir, along with pegylated interferon and ribavirin, have a role in the treatment of chronic HCV infection. The precise regimens are largely dependent on the patient characteristics, patient and physician preferences, and cost implication. Conclusions Therapies for chronic HCV have evolved dramatically in recent years. Interferon-free regimens are now possible without compromise in the rate of sustained viral response. The decision as to which regimen is most appropriate is multifactorial, and based on efficacy, safety and cost.

9049 Background: Osimertinib, a 3rd generation EGFR TKI, has been approved for treatment naïve patients (pts) with metastatic EGFR mutant NSCLC. Mechanisms of resistance to osimertinib are emerging from limited studies using targeted sequencing platforms. Methods: We performed whole exome (WES) and RNA-sequencing of osimertinib resistant tumors of EGFR-mutant NSCLC pts treated in a prospective clinical trial (NCT02759835). Treatment naïve EGFR mutant pts or pts with T790M-positive NSCLC after EGFR-TKI treatment receive osimertinib. Upon progression, pts with ≤5 progressing sites undergo local ablative therapy (LAT; surgery, radiation, RFA) and resume osimertinib. We analyzed paired pre-treatment and post-progression tumors in 10 patients and post-progression tumors in 3 additional patients and investigated intra- and inter-metastatic tumor heterogeneity using tumors procured from LAT surgeries and autopsies. Results: Acquired, focal copy number amplifications (CNA) of oncogenes occurred in the majority of patients (54%, n = 7/13) whereas acquired osimertinib resistance mutation EGFR C797S was less common (15%, n = 2/13). Early progression on osimertinib ( < 12 months PFS) in treatment naïve pts was associated with acquired, focal CNA. Despite pre-existing EGFR amplification, further amplification of the mutant allele of EGFR was the most common focal CNA (33%, n = 3/9). Other oncogenes amplified in resistant tumors include MET, KRAS, ERBB2 and YES1. CD274 (PD-L1) amplification occurred as the only putative mechanism of resistance in a pt without prior EGFR-TKI treatment. Using RNA-seq, we identified a pt who retained NSCLC histology, but upregulated genes associated with neuroendocrine differentiation upon osimertinib resistance. Clonal evolutionary analysis using WES of prospectively collected sensitive and resistant tumor tissue, including at autopsy is underway. Conclusions: Unbiased WES and transcriptome sequencing revealed heterogeneity, clonal evolution and novel osimertinib resistance mechanisms. Majority of pts had two or more resistance mechanisms suggesting the requirement of combination therapies to overcome resistance. Clinical trial information: NCT02759835.

Human immunodeficiency virus type 1 (HIV-1) is pandemic, but its contemporary global transmission network has not been characterized. A better understanding of the properties and dynamics of this network is essential for surveillance, prevention, and eventual eradication of HIV. Here, we apply a simple and computationally efficient network-based approach to all publicly available HIV polymerase sequences in the global database, revealing a contemporary picture of the spread of HIV-1 within and between countries. This approach automatically recovered well-characterized transmission clusters and extended other clusters thought to be contained within a single country across international borders. In addition, previously undescribed transmission clusters were discovered. Together, these clusters represent all known modes of HIV transmission. The extent of international linkage revealed by our comprehensive approach demonstrates the need to consider the global diversity of HIV, even when describing local epidemics. Finally, the speed of this method allows for near-real-time surveillance of the pandemic's progression.

West Central Africa has been implicated as the epicenter of the HIV-1 epidemic, and almost all group M subtypes can be found there. Previous analysis of early HIV-1 group M sequences from Kinshasa in the Democratic Republic of Congo, formerly Zaire, revealed that isolates from a number of individuals fall in different positions in phylogenetic trees constructed from sequences from opposite ends of the genome as a result of recombination between viruses of different subtypes. Here, we use discrete ancestral trait mapping to develop a procedure for quantifying HIV-1 group M intersubtype recombination across phylogenies, using individuals' gag (p17) and env (gp41) subtypes. The method was applied to previously described HIV-1 group M sequences from samples obtained in Kinshasa early in the global radiation of HIV. Nine different p17 and gp41 intersubtype recombinant combinations were present in the data set. The mean number of excess ancestral subtype transitions (NEST) required to map individuals' p17 subtypes onto the gp14 phylogeny samples, compared to the number required to map them onto the p17 phylogenies, and vice versa, indicated that excess subtype transitions occurred at a rate of approximately 7 × 10 −3 to 8 × 10 −3 per lineage per year as a result of intersubtype recombination. Our results imply that intersubtype recombination may have occurred in approximately 20% of lineages evolving over a period of 30 years and confirm intersubtype recombination as a substantial force in generating HIV-1 group M diversity.

Viral phylogenetic methods contribute to understanding how HIV spreads in populations, and thereby help guide the design of prevention interventions. So far, most analyses have been applied to well-sampled concentrated HIV-1 epidemics in wealthy countries. To direct the use of phylogenetic tools to where the impact of HIV-1 is greatest, the Phylogenetics And Networks for Generalized HIV Epidemics in Africa (PANGEA-HIV) consortium generates full-genome viral sequences from across sub-Saharan Africa. Analyzing these data presents new challenges, since epidemics are principally driven by heterosexual transmission and a smaller fraction of cases is sampled. Here, we show that viral phylogenetic tools can be adapted and used to estimate epidemiological quantities of central importance to HIV-1 prevention in sub-Saharan Africa. We used a community-wide methods comparison exercise on simulated data, where participants were blinded to the true dynamics they were inferring. Two distinct simulations captured generalized HIV-1 epidemics, before and after a large community-level intervention that reduced infection levels. Five research groups participated. Structured coalescent modeling approaches were most successful: phylogenetic estimates of HIV-1 incidence, incidence reductions, and the proportion of transmissions from individuals in their first 3 months of infection correlated with the true values (Pearson correlation > 90%), with small bias. However, on some simulations, true values were markedly outside reported confidence or credibility intervals. The blinded comparison revealed current limits and strengths in using HIV phylogenetics in challenging settings, provided benchmarks for future methods’ development, and supports using the latest generation of phylogenetic tools to advance HIV surveillance and prevention., Molecular Biology and Evolution, 34 (1), ISSN:0737-4038, ISSN:1537-1719

Summary. By the H-phosphonate approach on a solid support we have synthesised a 28-mer homopolymer of 2′-deoxycytidine (SdC28), a 28-mer phosphorothioate oligodeoxynucleotide (Srev) complementary to the HIV-1 regulatory gene and a number of its base modified derivatives. Anti HIV-1 activity of S-dC28 and Srev in chronically infected T-cells was studied. Melting temperatures of rev-comp rev and Srev-comp rev were also measured.

Swine have often been considered as a mixing vessel for different influenza strains. In order to assess their role in more detail, we undertook a retrospective sequencing study to detect and characterize the reassortants present in European swine and to estimate the rate of reassortment between H1N1, H1N2 and H3N2 subtypes with Eurasian (avian-like) internal protein-coding segments. We analysed 69 newly obtained whole genome sequences of subtypes H1N1–H3N2 from swine influenza viruses sampled between 1982 and 2008, using Illumina and 454 platforms. Analyses of these genomes, together with previously published genomes, revealed a large monophyletic clade of Eurasian swine-lineage polymerase segments containing H1N1, H1N2 and H3N2 subtypes. We subsequently examined reassortments between the haemagglutinin and neuraminidase segments and estimated the reassortment rates between lineages using a recently developed evolutionary analysis method. High rates of reassortment between H1N2 and H1N1 Eurasian swine lineages were detected in European strains, with an average of one reassortment every 2–3 years. This rapid reassortment results from co-circulating lineages in swine, and in consequence we should expect further reassortments between currently circulating swine strains and the recent swine-origin H1N1v pandemic strain.

Although cemented total hip replacement has long been recognized as a situation that can lead to wear, the wear generated on the femoral stem has not been well documented, especially with regard to how this wear is initiated and propagated. This present work aimed to further investigate this issue based on a comprehensive study on surface morphology of the femoral stem and the bone cement, which were collected from seven in vitro wear simulations. It was shown that the wear locations on the stem surface compared well with the results of retrieval studies, and the boundaries of the worn areas matched well the edges of the micropores present in the bone cement surface. This indicated that the micropores could potentially contribute to the generation of femoral stem wear. In addition, metallic debris was detected around the micropores from the simulation with increased loading cycles. However, no evidence of macro-cracks was observed across the cement mantle in spite of the presence of micro-cracks initiated at the edge of the micropores. This study demonstrated a possible cause for progression of femoral stem wear and it may have an important bearing on the long term durability of cemented hip prosthesis.

The optimisation of unbiased deep-sequencing methods for full-length HBV genomes will inform detailed insights into the evolution and diversity of this pathogen. Nanopore-based sequencing technologies offer the potential for real-time detection and sequencing of blood-borne viruses, useful for clinical diagnosis, drug-resistance calling and high-resolution molecular epidemiology. To date, sequencing accuracy has limited the full potential of Oxford Nanopore’s long-read capabilities. We here describe use of an isothermal rolling-circle amplification (RCA) method, (i) to amplify HBV DNA and (ii) to derive concatemers of whole HBV genomes thus providing a mechanism to correct sequencing errors.