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ABSTRACTRozanolixizumab is a humanized anti-neonatal Fc receptor (FcRn) monoclonal antibody (mAb) of the immunoglobulin G4 (IgG4) sub-class, currently in clinical development for the treatment of IgG autoantibody-driven diseases. This format is frequently used for therapeutic mAbs due to its intrinsic lower affinity for Fc gamma receptors (FcγR) and lack of C1q engagement. However, with growing evidence suggesting that no Fc-containing agent is truly “silent” in this respect, we explored the engagement of FcγRs and potential functional consequences with rozanolixizumab. In the study presented here, rozanolixizumab was shown to bind to FcγRs in both protein-protein and cell-based assays, and the kinetic data were broadly as expected based on published data for an IgG4 mAb. Rozanolixizumab was also able to mediate antibody bipolar bridging (ABB), a phenomenon that led to a reduction of labeled FcγRI from the surface of human macrophages in an FcRn-dependent manner. However, the presence of exogenous human IgG, even at low concentrations, was able to prevent both binding and ABB events. Furthermore, data from in vitro experiments using relevant human cell types that express both FcRn and FcγRI indicated no evidence for functional sequelae in relation to cellular activation events (e.g., intracellular signaling, cytokine production) upon either FcRn or FcγR binding of rozanolixizumab. These data raise important questions about whether therapeutic antagonistic mAbs like rozanolixizumab would necessarily engage FcγRs at doses typically administered to patients in the clinic, and hence challenge the relevance and interpretation of in vitro assays performed in the absence of competing IgG.

Abstract Despite the high prevalence of heart failure in the western world, there are few effective treatments. Fibulin-3 is a protein involved in extracellular matrix (ECM) structural integrity, however its role in the heart is unknown. We have demonstrated, using single cell RNA-seq, that fibulin-3 was highly expressed in quiescent murine cardiac fibroblasts, with expression highest prior to injury and late post-infarct (from ~ day-28 to week-8). In humans, fibulin-3 was upregulated in left ventricular tissue and plasma of heart failure patients. Fibulin-3 knockout (Efemp1 −/−) and wildtype mice were subjected to experimental myocardial infarction. Fibulin-3 deletion resulted in significantly higher rate of cardiac rupture days 3–6 post-infarct, indicating a weak and poorly formed scar, with severe ventricular remodelling in surviving mice at day-28 post-infarct. Fibulin-3 knockout mice demonstrated less collagen deposition at day-3 post-infarct, with abnormal collagen fibre-alignment. RNA-seq on day-3 infarct tissue revealed upregulation of ECM degradation and inflammatory genes, but downregulation of ECM assembly/structure/organisation genes in fibulin-3 knockout mice. GSEA pathway analysis showed enrichment of inflammatory pathways and a depletion of ECM organisation pathways. Fibulin-3 originates from cardiac fibroblasts, is upregulated in human heart failure, and is necessary for correct ECM organisation/structural integrity of fibrotic tissue to prevent cardiac rupture post-infarct.

Abstract RNA-binding proteins (RBPs) are key players regulating RNA processing and are associated with disorders ranging from cancer to neurodegeneration. Here, we present a proteomics workflow for large-scale identification of RBPs and their RNA-binding regions in the mammalian brain identifying 526 RBPs. Analysing brain tissue from males of the Huntington’s disease (HD) R6/2 mouse model uncovered differential RNA-binding of the alternative splicing regulator RBM5. Combining several omics workflows, we show that RBM5 binds differentially to transcripts enriched in pathways of neurodegeneration in R6/2 brain tissue. We further find these transcripts to undergo changes in splicing and demonstrate that RBM5 directly regulates these changes in human neurons derived from embryonic stem cells. Finally, we reveal that RBM5 interacts differently with several known huntingtin interactors and components of huntingtin aggregates. Collectively, we demonstrate the applicability of our method for capturing RNA interactor dynamics in the contexts of tissue and disease.

ABSTRACTTrYbe® is an Fc-free therapeutic antibody format, capable of engaging up to three targets simultaneously, with long in vivo half-life conferred by albumin binding. This format is shown by small-angle X-ray scattering to be conformationally flexible with favorable ‘reach’ properties. We demonstrate the format’s broad functionality by co-targeting of soluble and cell surface antigens. The benefit of monovalent target binding is illustrated by the lack of formation of large immune complexes when co-targeting multivalent antigens. TrYbes® are manufactured using standard mammalian cell culture and protein A affinity capture processes. TrYbes® have been formulated at high concentrations and have favorable drug-like properties, including stability, solubility, and low viscosity. The unique functionality and inherent developability of the TrYbe® makes it a promising multi-specific antibody fragment format for antibody therapy.

School-based interventions for the prevention of dating and relationship violence (DRV) and gender-based violence (GBV) take advantage of universal opportunities for intervention. Information on differential effectiveness of interventions is important to assess if they ameliorate or worsen social gradients in specific outcomes. This is especially important in DRV and GBV prevention given the gendered context of these behaviours and their common aetiologies in patriarchal gender norms, and social acceptance in school contexts of sexual harassment, such as catcalling or unwanted groping. We undertook a systematic review of moderation analyses in randomised trials of school-based interventions for DRV and GBV prevention. We searched 21 databases and used supplementary search methods without regard to publication type, language or year of publication, and synthesised moderation tests relating to equity-relevant characteristics (principally sex and prior history of the outcome) for DRV and GBV perpetration and victimisation. Across 23 included outcome evaluations, programme effects on DRV victimisation were not moderated by gender or prior experience of DRV victimisation, but DRV perpetration outcomes were greater for boys, particularly for emotional and physical DRV perpetration. Findings for GBV outcomes were counterintuitive. Our findings suggest that practitioners should carefully monitor local intervention effectiveness and equity to ensure that interventions are working as intended. However, one of the most surprising findings from our analysis—with clear relevance for uncertainties in practice—was that differential impacts by sexuality or sexual minority status were not frequently evaluated.

Unlike single-gene mutations leading to Mendelian conditions, common human diseases are likely to be emergent phenomena arising from multilayer, multiscale, and highly interconnected interactions. Atrial and ventricular septal defects are the most common forms of cardiac congenital anomalies in humans. Atrial septal defects (ASD) show an open communication between the left and right atria postnatally, potentially resulting in serious hemodynamic consequences if untreated. A milder form of atrial septal defect, patent foramen ovale (PFO), exists in about one-quarter of the human population, strongly associated with ischaemic stroke and migraine. The anatomic liabilities and genetic and molecular basis of atrial septal defects remain unclear. Here, we advance our previous analysis of atrial septal variation through quantitative trait locus (QTL) mapping of an advanced intercross line (AIL) established between the inbred QSi5 and 129T2/SvEms mouse strains, that show extremes of septal phenotypes. Analysis resolved 37 unique septal QTL with high overlap between QTL for distinct septal traits and PFO as a binary trait. Whole genome sequencing of parental strains and filtering identified predicted functional variants, including in known human congenital heart disease genes. Transcriptome analysis of developing septa revealed downregulation of networks involving ribosome, nucleosome, mitochondrial, and extracellular matrix biosynthesis in the 129T2/SvEms strain, potentially reflecting an essential role for growth and cellular maturation in septal development. Analysis of variant architecture across different gene features, including enhancers and promoters, provided evidence for the involvement of non-coding as well as protein-coding variants. Our study provides the first high-resolution picture of genetic complexity and network liability underlying common congenital heart disease, with relevance to human ASD and PFO.

Sopp et al describe an approach, which exploits the tailpiece of the naturally multimeric IgM to augment hexamerisation of IgG. Their approach provides a newly engineered format of antibodies for promoting hexamerisation and enhanced complement-dependent cytotoxicity only when cell surface bound.

Abstract High-throughput single-cell RNA-seq (scRNA-seq) is a powerful tool for studying gene expression in single cells. Most current scRNA-seq bioinformatics tools focus on analysing overall expression levels, largely ignoring alternative mRNA isoform expression. We present a computational pipeline, Sierra, that readily detects differential transcript usage from data generated by commonly used polyA-captured scRNA-seq technology. We validate Sierra by comparing cardiac scRNA-seq cell types to bulk RNA-seq of matched populations, finding significant overlap in differential transcripts. Sierra detects differential transcript usage across human peripheral blood mononuclear cells and the Tabula Muris, and 3 ′UTR shortening in cardiac fibroblasts. Sierra is available at https://github.com/VCCRI/Sierra .

Introduction Sexual violence among higher education students is a public health concern, threatening the general safety of students, often with significant physical and mental health implications for victims. Establishing the prevalence estimates of sexual violence at higher education institutions (HEIs) is essential for designing and resourcing responses to sexual violence, including monitoring the effectiveness of prevention initiatives and institutional programmes. Yet, to date, there have been no rigorous studies assessing prevalence of sexual violence at HEIs in the UK.Methods and analysis Informed by guidance from Universities UK, the University of Oxford administration and the related student advocacy groups working within the University, Oxford Understanding Relationships, Sex, Power, Abuse and Consent Experiences is a cross-sectional survey of all undergraduate and graduate students over the age of 18 enrolled at the University of Oxford, UK. The survey design uses a complete sampling approach and measures adapted from previous campus climate surveys in the USA as well as the Sexual Experiences Survey (USA). The analysis will estimate the prevalence of sexual harassment and sexual violence perpetration and victimisation, and will examine whether ethnicity, gender identity, and sexual orientation are associated with these primary outcomes.Ethics and dissemination Ethical approval was obtained by the Social Sciences and Humanities Interdivisional Research Ethics Committee at the University of Oxford which is a subcommittee of the Central University Research Ethics Committee (ref no.: R73805/RE001). The research team will disseminate findings through peer-reviewed journal articles and conference presentations. A report cowritten by authors and stakeholders will be shared with Oxford University students.

BackgroundTo strengthen the impact of cash transfers, these interventions have begun to be packaged as cash-plus programmes, combining cash with additional transfers, interventions, or services. The intervention's complementary ("plus") components aim to improve cash transfer effectiveness by targeting mediating outcomes or the availability of supplies or services. This study examined whether cash-plus interventions for infants and children Methods and findingsForty-two databases, donor agencies, grey literature sources, and trial registries were systematically searched, yielding 5,097 unique articles (as of 06 April 2021). Randomised and quasi-experimental studies were eligible for inclusion if the intervention package aimed to improve outcomes for children ConclusionsIn this study, we observed that few cash-plus combinations were more effective than cash transfers alone. Cash combined with food transfers and primary healthcare show the greatest signs of added effectiveness. More research is needed on when and how cash-plus combinations are more effective than cash alone, and work in this field must ensure that these interventions improve outcomes among the most vulnerable children.

Fractionation in Gram-negative bacteria is used to identify the subcellular localization of proteins, in particular the localization of exported recombinant proteins. The process of cell fractionation can be fraught with cross-contamination issues and often lacks supporting data for fraction purity. Here, we compare three periplasm extraction and two cell disruption techniques in different combinations to investigate which process gives uncontaminated compartments from Escherichia coli. From these data, a robust method named PureFrac was compiled that gives pure periplasmic fractions and a superior recovery of soluble cytoplasmic proteins. The process extracts periplasm using cold osmotic shock with magnesium, prior to sonication and ultracentrifugation to separate the cytoplasm from insoluble material. This method handles cells cultivated in various conditions and allows preparation of active proteins in their respective compartments.

Objective While mobile health-based human immunodeficiency virus (HIV) interventions are often designed to promote health equity, systematic differences in the use of and access to mobile technologies may counteract that and widen treatment gaps. This systematic review applies an equity lens to investigate whether existing research provides adequate evidence on the ethical implications of mHealth technologies in HIV treatment and prevention. Methods This study included a two-stage methodology, consisting of (a) a systematic review of systematic reviews and (b) an evidence synthesis of primary studies. For the review of reviews we searched eight electronic databases, eight electronic journals and Google Scholar. We also screened reference lists and consulted authors of included studies. Primary studies were extracted from eligible reviews. We based our data extraction and analysis on the Place of residence, Race, Occupation, Gender/Sex, Religion, Education, Socioeconomic status, Social capital and other disadvantage related characteristics (PROGRESS-Plus) framework and the use of harvest plots, focusing on the socio-demographic distribution of mHealth effects. Results A total of 8786 citations resulted in 19 eligible reviews and 39 eligible primary studies. Existing reviews did not provide any analyses of the equity impacts of mobile health-based HIV initiatives. Information availability was higher in primary studies, predominantly suggesting no social gradient of mobile health-based HIV interventions. Overall, evidence remains weak and not sufficient to allow for confident equity statements. Conclusions Despite the negative force of socio-demographic inequities and the emerging nature of mobile health technologies, evidence on the equity implications of mobile health interventions for HIV care remains scarce. Not knowing how the effects of mobile health technologies differ across population subgroups inevitably limits our capacities to equitably adopt, adjust and integrate mobile health interventions towards reaching those disproportionally affected by the epidemic.

BackgroundTheories of health outcomes often hypothesize that living in more socially and economically disadvantaged neighbourhoods will lead to worse health. Multiple measures of neighbourhood disadvantage are available to researchers, which may serve as better or worse proxies for each other across time. To inform longitudinal study design and interpretation we investigated how perceived and objective measures of neighbourhood disadvantage vary over time and the factors underlying this variation.MethodsData were from 8,918 mothers with at least three time-points of neighbourhood data in the Avon Longitudinal Study of Parents and Children in the UK. We analyzed measures of objective (Indices of Multiple Deprivation) and perceived (neighbourhood quality, social cohesion, and stress) exposure to neighbourhood disadvantage at 10 time-points over 18 years. We used group-based trajectory modelling to determine the overlap in participants' trajectories on the different measures and evaluated the baseline factors associated with different perceived trajectories over time.ResultsThere was evidence of heterogeneity in both perceived and objective measures of neighbourhood disadvantage over time (e.g., on the objective measure, 5% of participants moved to more deprived neighbourhoods, 11% moved to less deprived neighbourhoods, 20% consistently lived in deprived neighbourhoods, and 64% consistently lived in non-deprived neighbourhoods). Perceived social cohesion showed the weakest relationship with exposure to objective neighbourhood deprivation: most participants in each trajectory group of objective neighbourhood deprivation followed non-corresponding trajectories of perceived social cohesion (61-80%). Accounting for objective deprivation exposure, poorer socioeconomic and psychosocial indicators at baseline were associated with following more negative perceived neighbourhood trajectories (e.g., high neighbourhood stress) over time.ConclusionTrajectories of perceived and objective measures of neighbourhood disadvantage varied over time, with the extent of variation depending on the time point of measurement and individual-level social factors. Researchers should be mindful of this variation when choosing and determining the timing of measures of neighbourhood disadvantage in longitudinal studies and when inferring effect mechanisms.

Background Child maltreatment is a major public health problem affecting one quarter of children in England and Wales. Good epidemiological data are needed to establish how many and which children are most at risk, and to evaluate the impact of policies and interventions. However, a comprehensive data source on child maltreatment is currently lacking. Aim We aimed to create a rich data source on the incidence of Child maltreatment over Time (iCoverT) in England and Wales. Methods We developed systematic methods to search and identify administrative data sources that regularly measured child maltreatment. Data sources were investigated and assessed against pre-specified eligibility criteria and a bespoke quality assessment tool. Relevant data were extracted, digitalised, and harmonised over time. All data and their accompanying documentation were prepared to form an open access data source: the iCoverT (osf.io/cf7mv). Results We identified 13 unique sources of administrative data, six of which met our eligibility criteria: Child protection statistics, Children in care, Criminal statistics, Homicide index, Mortality statistics and NSPCC statistics. Data and documentation were prepared and combined to form the iCoverT, including 272 variables, over 43,500 data points, and spanning over 150 years. A subsequent time series analysis demonstrated the utility of the iCoverT; identifying large overall decreases in child maltreatment from 1858 to 2016 (e.g. 90% decrease in child homicides (2.7 per fewer per 100,000 children)) but worrying recent increases from 2000 to 2016. Conclusion We systematically developed a rich data source on child maltreatment in England and Wales. Our methodology overcomes practical obstacles and offers a new approach for harnessing administrative data for research. Our resulting data source is a valuable public health surveillance tool, which can be used to monitor national levels of child maltreatment and to evaluate the effectiveness of child protection initiatives.

Abstract Background Over 100 mammalian G protein-coupled receptors are yet to be matched with endogenous ligands; these so-called orphans are prospective drug targets for the treatment of disease. GPR37L1 is one such orphan, abundant in the brain and detectable as mRNA in the heart and kidney. GPR37L1 ablation was reported to cause hypertension and left ventricular hypertrophy, and thus, we sought to further define the role of GPR37L1 in blood pressure homeostasis. Methods We investigated the cardiovascular effects of GPR37L1 using wild-type (GPR37L1wt/wt) and null (GPR37L1KO/KO) mice established on a C57BL/6J background, both under baseline conditions and during AngII infusion. We profiled GPR37L1 tissue expression, examining the endogenous receptor by immunoblotting and a β-galactosidase reporter mouse by immunohistochemistry. Results GPR37L1 protein was abundant in the brain but not detectable in the heart and kidney. We measured blood pressure in GPR37L1wt/wt and GPR37L1KO/KO mice and found that deletion of GPR37L1 causes a female-specific increase in systolic, diastolic, and mean arterial pressures. When challenged with short-term AngII infusion, only male GPR37L1KO/KO mice developed exacerbated left ventricular hypertrophy and evidence of heart failure, while the female GPR37L1KO/KO mice were protected from cardiac fibrosis. Conclusions Despite its absence in the heart and kidney, GPR37L1 regulates baseline blood pressure in female mice and is crucial for cardiovascular compensatory responses in males. The expression of GPR37L1 in the brain, yet absence from peripheral cardiovascular tissues, suggests this orphan receptor is a hitherto unknown contributor to central cardiovascular control.

Abstract We recently read the article by Benton et al. which reviewed risk of bias in natural experimental studies investigating the impact of the built environment on physical activity (Benton et al., 2016; Int J Behav Nutr Phys Act 13:107). As a technical exercise in assessing risk of bias to understand study quality, we found the results of this study both interesting and potentially useful. However, it prompted a number of concerns with the use of risk of bias tools for assessing the quality of evidence from studies exploiting natural experiments. As we discuss in this commentary, the rigid application of such tools could have adverse effects on the uptake and use of natural experiments in population health research and practice.

Summary: Background: It is unclear whether child maltreatment is increasing or decreasing in England and Wales. More evidence is needed, from multiple sources and over longer periods of time, to explore trends in child maltreatment. We investigated whether the annual incidence of child maltreatment has changed over time, using official record data and time-series methods to establish long-term trends. Methods: In this observational time-series analysis, we used six data sources (Government records for child mortality, police-recorded child homicides, crimes against children, child protection, and children in care; and NSPCC data) to estimate the incidence of child maltreatment in England and Wales and examine long-term trends. We included nationally representative data that could estimate the incidence of child maltreatment for more than 25 years. Our primary outcomes were the number of victims (age 16 years) of child maltreatment per 12-month period in England, including or excluding Wales. We fitted Poisson regression models with year as the exposure and the number of victims or perpetrators of child maltreatment as the outcome (adjusted for population age-structure and size). When a linear trend was not appropriate, we fitted generalised additive models with penalised splines to visualise trends. Findings: The incidence of child mortality by homicide or assault decreased by 90% (2·7 per 100 000 children) between 1858 and 2016 and the incidence of people guilty of child cruelty or neglect decreased by 83% (6·7 per 100 000 adults) between 1893 and 2016, whereas child protection registrations increased by 182% (328·7 per 100 000 children) between 1988 and 2016. Crimes against children and children entering care increased between 2000 and 2016. In 2016, 40 children died by homicide, with twice as many adolescent (15–19 years) deaths than infant (age

Monoclonal antibody (mAb) immunotherapy has transformed the treatment of allergy, autoimmunity, and cancer. The interaction of mAb with Fc gamma receptors (FcγR) is often critical for efficacy. The genes encoding the low-affinity FcγR have single nucleotide polymorphisms (SNPs) and copy number variation that can impact IgG Fc:FcγR interactions. Leukocyte-based in vitro assays remain one of the industry standards for determining mAb efficacy and predicting adverse responses in patients. Here we addressed the impact of FcγR genetics on immune cell responses in these assays and investigated the importance of assay format. FcγR genotyping of 271 healthy donors was performed using a Multiplex Ligation-Dependent Probe Amplification assay. Freeze-thawed/pre-cultured peripheral blood mononuclear cells (PBMCs) and whole blood samples from donors were stimulated with reagents spanning different mAb functional classes to evaluate the association of FcγR genotypes with T-cell proliferation and cytokine release. Using freeze-thawed/pre-cultured PBMCs, agonistic T-cell-targeting mAb induced T-cell proliferation and the highest levels of cytokine release, with lower but measurable responses from mAb which directly require FcγR-mediated cellular effects for function. Effects were consistent for individual donors over time, however, no significant associations with FcγR genotypes were observed using this assay format. In contrast, significantly elevated IFN-γ release was associated with the FCGR2A-131H/H genotype compared to FCGR2A-131R/R in whole blood stimulated with Campath (p ≤ 0.01) and IgG1 Fc hexamer (p ≤ 0.05). Donors homozygous for both the high affinity FCGR2A-131H and FCGR3A-158V alleles mounted stronger IFN-γ responses to Campath (p ≤ 0.05) and IgG1 Fc Hexamer (p ≤ 0.05) compared to donors homozygous for the low affinity alleles. Analysis revealed significant reductions in the proportion of CD14hi monocytes, CD56dim NK cells (p ≤ 0.05) and FcγRIIIa expression (p ≤ 0.05), in donor-matched freeze-thawed PBMC compared to whole blood samples, likely explaining the difference in association between FcγR genotype and mAb-mediated cytokine release in the different assay formats. These findings highlight the significant impact of FCGR2A and FCGR3A SNPs on mAb function and the importance of using fresh whole blood assays when evaluating their association with mAb-mediated cytokine release in vitro. This knowledge can better inform on the utility of in vitro assays for the prediction of mAb therapy outcome in patients.

Objective: Parental obesity can induce metabolic phenotypes in offspring independent of the inherited DNA sequence. Here we asked whether such non-genetic acquired metabolic traits can be passed on to a second generation that has never been exposed to obesity, even as germ cells. Methods: We examined the F1, F2, and F3 a/a offspring derived from F0 matings of obese prediabetic Avy/a sires and lean a/a dams. After F0, only lean a/a mice were used for breeding. Results: We found that F1 sons of obese founder males exhibited defects in glucose and lipid metabolism, but only upon a post-weaning dietary challenge. F1 males transmitted these defects to their own male progeny (F2) in the absence of the dietary challenge, but the phenotype was largely attenuated by F3. The sperm of F1 males exhibited changes in the abundance of several small RNA species, including the recently reported diet-responsive tRNA-derived fragments. Conclusions: These data indicate that induced metabolic phenotypes may be propagated for a generation beyond any direct exposure to an inducing factor. This non-genetic inheritance likely occurs via the actions of sperm noncoding RNA. Keywords: Paternal effects, Epigenetic inheritance, Noncoding RNA, Sperm RNA

Child maltreatment is a major public health problem, which is plagued with research challenges. Good epidemiological data can help to establish the nature and scope of past and present child maltreatment, and monitor its progress going forward. However, high quality data sources are currently lacking for England and Wales. We employed systematic methodology to harness pre-existing datasets (including non-digitalised datasets) and develop a rich data source on the incidence of Child maltreatment over Time (iCoverT) in England and Wales. The iCoverT consists of six databases and accompanying data documentation: Child Protection Statistics, Children In Care Statistics, Criminal Statistics, Homicide Index, Mortality Statistics and NSPCC Statistics. Each database is a unique indicator of child maltreatment incidence with 272 data variables in total. The databases span from 1858 to 2016 and therefore extends current data sources by over 80 years. We present a proof-of-principle analysis of a subset of the data to show how time series methods may be used to address key research challenges. This example demonstrates the utility of iCoverT and indicates that it will prove to be a valuable data source for researchers, clinicians and policy-makers concerned with child maltreatment. The iCoverT is freely available at the Open Science Framework (osf.io/cf7mv).