Site-specific silencing of regulatory elements as a mechanism of X inactivation.

The inactive X chromosome's (Xi) physical territory is microscopically devoid of transcriptional hallmarks and enriched in silencing-associated modifications. How these microscopic signatures relate to specific Xi sequences is unknown. Therefore, we profiled Xi gene expression and chromatin states at high resolution via allele-specific sequencing in mouse trophoblast stem cells. Most notably, X-inactivated transcription start sites harbored distinct epigenetic signatures relative to surrounding Xi DNA. These sites displayed H3-lysine27-trimethylation enrichment and DNaseI hypersensitivity, similar to autosomal Polycomb targets, yet excluded Pol II and other transcriptional hallmarks, similar to nontranscribed genes. CTCF bound X-inactivated and escaping genes, irrespective of measured chromatin boundaries. Escape from X inactivation occurred within, and X inactivation was maintained exterior to, the area encompassed by Xist in cells subject to imprinted and random X inactivation. The data support a model whereby inactivation of specific regulatory elements, rather than a simple chromosome-wide separation from transcription machinery, governs gene silencing over the Xi.

Widespread transcriptional pausing and elongation control at enhancers.

Regulation by gene-distal enhancers is critical for cell type-specific and condition-specific patterns of gene expression. Thus, to understand the basis of gene activity in a given cell type or tissue, we must identify the precise locations of enhancers and functionally characterize their behaviors. Here, we demonstrate that transcription is a nearly universal feature of enhancers in Drosophila and mammalian cells and that nascent RNA sequencing strategies are optimal for identification of both enhancers and superenhancers. We dissect the mechanisms governing enhancer transcription and discover remarkable similarities to transcription at protein-coding genes. We show that RNA polymerase II (RNAPII) undergoes regulated pausing and release at enhancers. However, as compared with mRNA genes, RNAPII at enhancers is less stable and more prone to early termination. Furthermore, we found that the level of histone H3 Lys4 (H3K4) methylation at enhancers corresponds to transcriptional activity such that highly active enhancers display H3K4 trimethylation rather than the H3K4 monomethylation considered a hallmark of enhancers. Finally, our work provides insights into the unique characteristics of superenhancers, which stimulate high-level gene expression through rapid pause release; interestingly, this property renders associated genes resistant to the loss of factors that stabilize paused RNAPII.

Gastric Alimetry® Testing and Healthcare Economic Analysis in Nausea and Vomiting Syndromes.

BACKGROUND: Chronic nausea and vomiting syndromes (CNVS), gastroparesis and functional dyspepsia (FD) are complex disorders. Body Surface Gastric Mapping (BSGM), a new test of gastric function, using Gastric Alimetry® (Alimetry, New Zealand) may be useful for de-escalating healthcare utilisation. This study aimed to define healthcare costs and estimate health economic impacts of implementing this test in patients with chronic gastroduodenal symptoms. METHODS: Consecutive patients at a tertiary referral centre evaluated with Gastric Alimetry were included. Frequency and cost data relating to medical investigations, hospital and outpatient presentations were evaluated. Costs of healthcare utilisation were calculated, and the potential cost savings of implementing Gastric Alimetry within a diagnostic decision-tree model were estimated. RESULTS: Overall, 31 consecutive patients (mean age 36.1 years; 83.9% female; predominant symptoms: nausea [83.9%], pain [61.3%], vomiting [67.7%] and bloating [35.5%]) completed Gastric Alimetry testing. Repeat gastroscopy and abdominal CT rates were 29% (8/28) and 85% (11/13), respectively. Gastric Alimetry testing identified spectral abnormalities in 45.2% of patients, and symptom profiling classified a further 29.1% of patients. Median annualised cost difference after test introduction was NZ$-12,032. Estimated reductions in investigation-related costs when incorporating Gastric Alimetry into the diagnostic workflow model were approximately NZ$1,300 per patient. CONCLUSIONS: Healthcare utilisation and confirmatory testing rates remain high in nausea and vomiting syndromes. This study presents real-world data, together with a decision-tree analysis, showing Gastric Alimetry can streamline clinical care pathways, resulting in reduced healthcare utilisation and cost.

Pausing of RNA polymerase II regulates mammalian developmental potential through control of signaling networks.

The remarkable capacity for pluripotency and self-renewal in embryonic stem cells (ESCs) requires a finely tuned transcriptional circuitry wherein the pathways and genes that initiate differentiation are suppressed, but poised to respond rapidly to developmental signals. To elucidate transcriptional control in mouse ESCs in the naive, ground state, we defined the distribution of engaged RNA polymerase II (Pol II) at high resolution. We find that promoter-proximal pausing of Pol II is most enriched at genes regulating cell cycle and signal transduction and not, as expected, at developmental or bivalent genes. Accordingly, ablation of the primary pause-inducing factor NELF does not increase expression of lineage markers, but instead causes proliferation defects, embryonic lethality, and dysregulation of ESC signaling pathways. Indeed, ESCs lacking NELF have dramatically attenuated FGF/ERK activity, rendering them resistant to differentiation. This work thus uncovers a key role for NELF-mediated pausing in establishing the responsiveness of stem cells to developmental cues.

Measuring Vaccine Efficacy Against Infection and Disease in Clinical Trials: Sources and Magnitude of Bias in Coronavirus Disease 2019 (COVID-19) Vaccine Efficacy Estimates.

BACKGROUND: Phase III trials have estimated coronavirus disease 2019 (COVID-19) vaccine efficacy (VE) against symptomatic and asymptomatic infection. We explore the direction and magnitude of potential biases in these estimates and their implications for vaccine protection against infection and against disease in breakthrough infections. METHODS: We developed a mathematical model that accounts for natural and vaccine-induced immunity, changes in serostatus, and imperfect sensitivity and specificity of tests for infection and antibodies. We estimated expected biases in VE against symptomatic, asymptomatic, and any severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections and against disease following infection for a range of vaccine characteristics and measurement approaches, and the likely overall biases for published trial results that included asymptomatic infections. RESULTS: VE against asymptomatic infection measured by polymerase chain reaction (PCR) or serology is expected to be low or negative for vaccines that prevent disease but not infection. VE against any infection is overestimated when asymptomatic infections are less likely to be detected than symptomatic infections and the vaccine protects against symptom development. A competing bias toward underestimation arises for estimates based on tests with imperfect specificity, especially when testing is performed frequently. Our model indicates considerable uncertainty in Oxford-AstraZeneca ChAdOx1 and Janssen Ad26.COV2.S VE against any infection, with slightly higher than published, bias-adjusted values of 59.0% (95% uncertainty interval [UI] 38.4-77.1) and 70.9% (95% UI 49.8-80.7), respectively. CONCLUSIONS: Multiple biases are likely to influence COVID-19 VE estimates, potentially explaining the observed difference between ChAdOx1 and Ad26.COV2.S vaccines. These biases should be considered when interpreting both efficacy and effectiveness study results.

Does a multidisciplinary menopausal symptoms after cancer clinic reduce symptoms?

PURPOSE: This study aimed to measure the prevalence of menopausal symptoms in patients attending a multidisciplinary model of care clinic at their initial clinic visit and their subsequent follow-up consultation using a validated patient-reported outcome measure to assess whether menopausal symptoms after cancer had improved. METHODS: A retrospective review was conducted of patients attending the clinic in a 12-month period in 2017 (n = 189). Recorded variables included patient demographics, details of index cancer, previous treatments, and menopausal symptom management strategies. Severity of menopausal symptoms was evaluated using the Greene Climacteric Scale. The extent to which patients were bothered by symptoms was combined into two categories and dichotomized (present/absent). Differences in symptom prevalence between the initial consultation and first follow-up visit were examined using McNemar's test. RESULTS: The majority of patients attending the clinic had a history of breast cancer (72%). Fifty-five percent of patients were prescribed a non-hormonal therapy at their initial visit, most commonly gabapentin. Significantly fewer patients reported being bothered by hot flushes, fatigue, sleep difficulties, and loss of interest in sex, anxiety, or troubles concentrating at the first follow-up visit compared to their initial consultation (p < 0.01). CONCLUSION: In this study, there was an improvement in self-reported menopausal symptoms in a significant proportion of cancer survivors attending a multidisciplinary menopause clinic between their initial and first subsequent follow-up consultations.

Arabidopsis thaliana G3BP Ortholog Rescues Mammalian Stress Granule Phenotype across Kingdoms.

Stress granules (SGs) are dynamic RNA-protein complexes localized in the cytoplasm that rapidly form under stress conditions and disperse when normal conditions are restored. The formation of SGs depends on the Ras-GAP SH3 domain-binding protein (G3BP). Formations, interactions and functions of plant and human SGs are strikingly similar, suggesting a conserved mechanism. However, functional analyses of plant G3BPs are missing. Thus, members of the Arabidopsis thaliana G3BP (AtG3BP) protein family were investigated in a complementation assay in a human G3BP knock-out cell line. It was shown that two out of seven AtG3BPs were able to complement the function of their human homolog. GFP-AtG3BP fusion proteins co-localized with human SG marker proteins Caprin-1 and eIF4G1 and restored SG formation in G3BP double KO cells. Interaction between AtG3BP-1 and -7 and known human G3BP interaction partners such as Caprin-1 and USP10 was also demonstrated by co-immunoprecipitation. In addition, an RG/RGG domain exchange from Arabidopsis G3BP into the human G3BP background showed the ability for complementation. In summary, our results support a conserved mechanism of SG function over the kingdoms, which will help to further elucidate the biological function of the Arabidopsis G3BP protein family.

Collaborative Cross mice reveal extreme epilepsy phenotypes and genetic loci for seizure susceptibility.

OBJECTIVE: Animal studies remain essential for understanding mechanisms of epilepsy and identifying new therapeutic targets. However, existing animal models of epilepsy do not reflect the high level of genetic diversity found in the human population. The Collaborative Cross (CC) population is a genetically diverse recombinant inbred panel of mice. The CC offers large genotypic and phenotypic diversity, inbred strains with stable genomes that allow for repeated phenotypic measurements, and genomic tools including whole genome sequence to identify candidate genes and candidate variants. METHODS: We evaluated multiple complex epileptic traits in a sampling of 35 CC inbred strains using the flurothyl-induced seizure and kindling paradigm. We created an F2 population of 297 mice with extreme seizure susceptibility and performed quantitative trait loci (QTL) mapping to identify genomic regions associated with seizure sensitivity. We used quantitative RNA sequencing from CC hippocampal tissue to identify candidate genes and whole genome sequence to identify genetic variants likely affecting gene expression. RESULTS: We identified new mouse models with extreme seizure susceptibility, seizure propagation, epileptogenesis, and SUDEP (sudden unexpected death in epilepsy). We performed QTL mapping and identified one known and seven novel loci associated with seizure sensitivity. We combined whole genome sequencing and hippocampal gene expression to pinpoint biologically plausible candidate genes (eg, Gabra2) and variants associated with seizure sensitivity. SIGNIFICANCE: New mouse models of epilepsy are needed to better understand the complex genetic architecture of seizures and to identify therapeutics. We performed a phenotypic screen utilizing a novel genetic reference population of CC mice. The data we provide enable the identification of protective/risk genes and novel molecular mechanisms linked to complex seizure traits that are currently challenging to study and treat.

The auto-inhibitory domain and ATP-independent microtubule-binding region of Kinesin heavy chain are major functional domains for transport in the Drosophila germline.

The major motor Kinesin-1 provides a key pathway for cell polarization through intracellular transport. Little is known about how Kinesin works in complex cellular surroundings. Several cargos associate with Kinesin via Kinesin light chain (KLC). However, KLC is not required for all Kinesin transport. A putative cargo-binding domain was identified in the C-terminal tail of fungal Kinesin heavy chain (KHC). The tail is conserved in animal KHCs and might therefore represent an alternative KLC-independent cargo-interacting region. By comprehensive functional analysis of the tail during Drosophila oogenesis we have gained an understanding of how KHC achieves specificity in its transport and how it is regulated. This is, to our knowledge, the first in vivo structural/functional analysis of the tail in animal Kinesins. We show that the tail is essential for all functions of KHC except Dynein transport, which is KLC dependent. These tail-dependent KHC activities can be functionally separated from one another by further characterizing domains within the tail. In particular, our data show the following. First, KHC is temporally regulated during oogenesis. Second, the IAK domain has an essential role distinct from its auto-inhibitory function. Third, lack of auto-inhibition in itself is not necessarily detrimental to KHC function. Finally, the ATP-independent microtubule-binding motif is required for cargo localization. These results stress that two unexpected highly conserved domains, namely the auto-inhibitory IAK and the auxiliary microtubule-binding motifs, are crucial for transport by Kinesin-1 and that, although not all cargos are conserved, their transport involves the most conserved domains of animal KHCs.

Cytoplasmic streaming in Drosophila oocytes varies with kinesin activity and correlates with the microtubule cytoskeleton architecture.

Cells can localize molecules asymmetrically through the combined action of cytoplasmic streaming, which circulates their fluid contents, and specific anchoring mechanisms. Streaming also contributes to the distribution of nutrients and organelles such as chloroplasts in plants, the asymmetric position of the meiotic spindle in mammalian embryos, and the developmental potential of the zygote, yet little is known quantitatively about the relationship between streaming and the motor activity which drives it. Here we use Particle Image Velocimetry to quantify the statistical properties of Kinesin-dependent streaming during mid-oogenesis in Drosophila. We find that streaming can be used to detect subtle changes in Kinesin activity and that the flows reflect the architecture of the microtubule cytoskeleton. Furthermore, based on characterization of the rheology of the cytoplasm in vivo, we establish estimates of the number of Kinesins required to drive the observed streaming. Using this in vivo data as the basis of a model for transport, we suggest that the disordered character of transport at mid-oogenesis, as revealed by streaming, is an important component of the localization dynamics of the body plan determinant oskar mRNA.

Fertility biomarkers to estimate metabolic risks in women with polycystic ovary syndrome.

PURPOSE: We sought to evaluate the relationship between the polycystic ovary syndrome (PCOS)-defining characteristics and the risk of developing metabolic complications in women presenting with complaints of infertility and/or menstrual irregularities and subsequently diagnosed with PCOS. METHODS: This was a cross-sectional study. Women presenting with complaints of infertility and/or irregular menses and diagnosed with PCOS by the Rotterdam criteria, underwent endocrine, metabolic, and ultrasound assessment in the early follicular phase. Reproductive and metabolic parameters were included in regression analysis models with the PCOS-defining characteristics; ROC curves were calculated for the significant predictors. RESULTS: Three hundred and seventy-four women with PCOS were included in our study. Oligo-anovulation, menstrual irregularities, and hirsutism were not predictive of any of the variables. Ovarian volume, follicle count, and biochemical hyperandrogenism were predictors for hormonal, metabolic, and endometrial complications. The relationships were independent of age and body mass index. ROC curves identified lower cut-off values of the PCOS-defining characteristics to predict patients' risks of hyperinsulinemia, dyslipidemia, and glucose intolerance. CONCLUSIONS: Adverse metabolic effects of PCOS are already present in women at the time they present complaining of infertility and/or irregular menses. Hyperandrogenism and ultrasound can assist in predicting the patients' concomitant metabolic abnormalities and can aid physicians in tailoring counseling for effective preventive strategies.

What to do when under scrutiny.

IN AUGUST, the Nursing and Midwifery Council (NMC) concluded an 81-day hearing into care failings at Brithdir Care Home in Caerphilly, Wales. The hearing ended the UK's biggest inquiry into allegations of neglect to date. It led to three nurses being struck off, a fourth being suspended for one year and a fifth being given a caution order.

Transcription precedes loss of Xist coating and depletion of H3K27me3 during X-chromosome reprogramming in the mouse inner cell mass.

Repression of Xist RNA expression is considered a prerequisite to reversal of X-chromosome inactivation (XCI) in the mouse inner cell mass (ICM), and reactivation of X-linked genes is thought to follow loss of Xist RNA coating and heterochromatic markers of inactivation, such as methylation of histone H3. We analyzed X-chromosome activity in developing ICMs and show that reactivation of gene expression from the inactive-X initiates in the presence of Xist coating and H3K27me3. Furthermore, depletion of Xist RNA coating through forced upregulation of NANOG does not result in altered reactivation kinetics. Taken together, our observations suggest that in the ICM, X-linked gene transcription and Xist coating are uncoupled. These data fundamentally alter our perception of the reactivation process and support the existence of a mechanism to reactivate Xp-linked genes in the ICM that operates independently of loss of Xist RNA and H3K27me3 from the imprinted inactive-X.

Implementation and adherence to regular asymptomatic testing in a COVID-19 vaccine trial.

BACKGROUND: For pathogens which cause infections that present asymptomatically, evaluating vaccine efficacy (VE) against asymptomatic infection is important for understanding a vaccine's potential epidemiological impact. Regular testing for subclinical infections is a potentially valuable strategy but its success hinges on participant adherence and minimising false positives. This paper describes the implementation and adherence to weekly testing in a COVID-19 vaccine trial. METHODS: COV002 was a phase 2/3 trial assessing the ChAdOx1 nCoV-19 vaccine against SARS-CoV-2. Asymptomatic infections were detected using weekly self-administered swabs for RT-PCR testing. We analysed adherence using mixed-effects regression models and estimated the probability of true and false positive asymptomatic infections using estimates of adherence and testing characteristics. FINDINGS: 356,551 tests were self-administered by 10,811 participants during the 13-month follow-up. Median adherence was 75.0% (IQR 42·6-90·9), which translated to a 74·5% (IQR 50·9-78·8) probability of detecting a positive asymptomatic infection during the swabbing period, and between 21 and 96 false positives during VE evaluation. The odds of returning a swab declined by 8% per week and further after testing positive and unblinding. Adherence was higher in older age groups, females and non-healthcare workers. INTERPRETATION: The COV002 trial demonstrated the feasibility of running a long-term regular asymptomatic testing strategy. This information could be valuable for designing future phase III vaccine trials in which infection is an outcome. FUNDING: UK Research and Innovation, National Institutes for Health Research (NIHR), Coalition for Epidemic Preparedness Innovations, NIHR Oxford Biomedical Research Centre, Thames Valley and South Midland's NIHR Clinical Research Network, AstraZeneca.

Developmental RNA processing of 3'UTRs in Hox mRNAs as a context-dependent mechanism modulating visibility to microRNAs.

The Drosophila Hox gene Ultrabithorax (Ubx) controls the development of thoracic and abdominal segments, allocating segment-specific features to different cell lineages. Recent studies have shown that Ubx expression is post-transcriptionally regulated by two microRNAs (miRNAs), miR-iab4 and miR-iab8, acting on target sites located in the 3' untranslated regions (UTRs) of Ubx mRNAs. Here, we show that during embryonic development Ubx produces mRNAs with variable 3'UTRs in different regions of the embryo. Analysis of the resulting remodelled 3'UTRs shows that each species harbours different sets of miRNA target sites, converting each class of Ubx mRNA into a considerably different substrate for miRNA regulation. Furthermore, we show that the distinct developmental distributions of Ubx 3'UTRs are established by a mechanism that is independent of miRNA regulation and therefore are not the consequence of miR-iab4/8-mediated RNA degradation acting on those sensitive mRNA species; instead, we propose that this is a hard-wired 3'UTR processing system that is able to regulate target mRNA visibility to miRNAs according to developmental context. We show that reporter constructs that include Ubx short and long 3'UTR sequences display differential expression within the embryonic central nervous system, and also demonstrate that mRNAs of three other Hox genes suffer similar and synchronous developmental 3'UTR processing events during embryogenesis. Our work thus reveals that developmental RNA processing of 3'UTR sequences is a general molecular strategy used by a key family of developmental regulators so that their transcripts can display different levels of visibility to miRNA regulation according to developmental cues.

Drosophila PAT1 is required for Kinesin-1 to transport cargo and to maximize its motility.

Kinesin heavy chain (KHC), the force-generating component of Kinesin-1, is required for the localization of oskar mRNA and the anchoring of the nucleus in the Drosophila oocyte. These events are crucial for the establishment of the anterior-posterior and dorsal-ventral axes. KHC is also essential for the localization of Dynein and for all ooplasmic flows. Interestingly, oocytes without Kinesin light chain show no major defects in these KHC-dependent processes, suggesting that KHC binds its cargoes and is activated by a novel mechanism. Here, we shed new light on the molecular mechanism of Kinesin function in the germline. Using a combination of genetic, biochemical and motor-tracking studies, we show that PAT1, an APP-binding protein, interacts with Kinesin-1, functions in the transport of oskar mRNA and Dynein and is required for the efficient motility of KHC along microtubules. This work suggests that the role of PAT1 in cargo transport in the cell is linked to PAT1 function as a positive regulator of Kinesin motility.

Serum markers of ovarian reserve and ovarian histology in adult mice treated with cyclophosphamide in pre-pubertal age.

PURPOSE: AMH is used to quantify the extent of follicular pool in postpubertal women, but its value after chemotherapy is unclear. We tested AMH as a marker of follicular reserve in adult mice treated with cyclophosphamide (CTX) in prepubertal age. METHODS: Mice received placebo or CTX at age 18 days. AMH and FSH were assessed on day 43, 56, and 95 of life. Ovaries were fixed in formalin, embedded in paraffin, and stained with H&E and TUNEL. Follicular apoptosis was graded. RESULTS: All mice exposed to CTX had a decreased number of follicles/mm(2) and significantly decreased AMH, but only 48 % of pubertal and 81 % of adult mice had increased FSH. Over time, there was an increase in FSH (p < 0.05), but not a concurrent decrease in AMH, while in controls, FSH remained stable and AMH decreased. There was no correlation between histological and serological markers. CONCLUSIONS: CTX administration to pre-pubertal mice caused various degrees of residual function, which were reflected by FSH, but not by AMH or by the number of ovarian follicles. AMH served as a marker of quantitative, and FSH of qualitative, residual ovarian function.

Impact of poor air quality while deployed on respiratory health: a systematic review.

INTRODUCTION: British military personnel deploy internationally to areas with high levels of ambient air pollution. Air pollution can cause acute respiratory symptoms which lead to concern about potential long-lasting health effects. There is a requirement for evidence-based policy on chronic respiratory disease associated with military deployments to areas with poor air quality (AQ). This literature review examines the published evidence relating to the development of chronic respiratory disease in military personnel after exposure to poor AQ while deployed. METHODS: A literature search was conducted in accordance with the Preferred Reporting Items for Systematic Reviews and Meta-Analysis guidelines. Embase, MEDLINE and Global health databases were searched for English language studies published since 2014 examining the respiratory health of military personnel deployed to Southwest Asia since 2001. A quality appraisal of selected articles was conducted using the Critical Appraisals Skills Programme checklist and a descriptive review completed. RESULTS: Eleven studies were found, eight of which had objective outcome measures. Two prospective cohort studies were included; the remainder of the data were retrospective. CONCLUSION: High rates of respiratory symptoms are reported by personnel who deploy to areas of poor AQ, giving rise to high levels of concern. Spirometry testing has found mild deficits, mostly of an obstructive nature, in a third of those with ongoing symptoms post deployment. These have not been consistently linked with deployment length. An increased risk of asthma appears to be multi-factorial in aetiology and there is no evidence for an increased risk of chronic obstructive pulmonary disease or histological pathology post deployment. At present, there is no definitive evidence of chronic respiratory disease due to exposure to poor AQ while deployed. Further objective longitudinal studies are required to continue to investigate the association, diagnosis and management of those with ongoing symptoms.