Ultrafast Two-Photon Imaging of a High-Gain Voltage Indicator in Awake Behaving Mice.

Optical interrogation of voltage in deep brain locations with cellular resolution would be immensely useful for understanding how neuronal circuits process information. Here, we report ASAP3, a genetically encoded voltage indicator with 51% fluorescence modulation by physiological voltages, submillisecond activation kinetics, and full responsivity under two-photon excitation. We also introduce an ultrafast local volume excitation (ULoVE) method for kilohertz-rate two-photon sampling in vivo with increased stability and sensitivity. Combining a soma-targeted ASAP3 variant and ULoVE, we show single-trial tracking of spikes and subthreshold events for minutes in deep locations, with subcellular resolution and with repeated sampling over days. In the visual cortex, we use soma-targeted ASAP3 to illustrate cell-type-dependent subthreshold modulation by locomotion. Thus, ASAP3 and ULoVE enable high-speed optical recording of electrical activity in genetically defined neurons at deep locations during awake behavior.

Receptor tyrosine kinases regulate signal transduction through a liquid-liquid phase separated state.

The recruitment of signaling proteins into activated receptor tyrosine kinases (RTKs) to produce rapid, high-fidelity downstream response is exposed to the ambiguity of random diffusion to the target site. Liquid-liquid phase separation (LLPS) overcomes this by providing elevated, localized concentrations of the required proteins while impeding competitor ligands. Here, we show a subset of phosphorylation-dependent RTK-mediated LLPS states. We then investigate the formation of phase-separated droplets comprising a ternary complex including the RTK, (FGFR2); the phosphatase, SHP2; and the phospholipase, PLCγ1, which assembles in response to receptor phosphorylation. SHP2 and activated PLCγ1 interact through their tandem SH2 domains via a previously undescribed interface. The complex of FGFR2 and SHP2 combines kinase and phosphatase activities to control the phosphorylation state of the assembly while providing a scaffold for active PLCγ1 to facilitate access to its plasma membrane substrate. Thus, LLPS modulates RTK signaling, with potential consequences for therapeutic intervention.

Factors associated with COVID-19-related death using OpenSAFELY.

Coronavirus disease 2019 (COVID-19) has rapidly affected mortality worldwide1. There is unprecedented urgency to understand who is most at risk of severe outcomes, and this requires new approaches for the timely analysis of large datasets. Working on behalf of NHS England, we created OpenSAFELY-a secure health analytics platform that covers 40% of all patients in England and holds patient data within the existing data centre of a major vendor of primary care electronic health records. Here we used OpenSAFELY to examine factors associated with COVID-19-related death. Primary care records of 17,278,392 adults were pseudonymously linked to 10,926 COVID-19-related deaths. COVID-19-related death was associated with: being male (hazard ratio (HR) 1.59 (95% confidence interval 1.53-1.65)); greater age and deprivation (both with a strong gradient); diabetes; severe asthma; and various other medical conditions. Compared with people of white ethnicity, Black and South Asian people were at higher risk, even after adjustment for other factors (HR 1.48 (1.29-1.69) and 1.45 (1.32-1.58), respectively). We have quantified a range of clinical factors associated with COVID-19-related death in one of the largest cohort studies on this topic so far. More patient records are rapidly being added to OpenSAFELY, we will update and extend our results regularly.

Adapting the in vitro micronucleus assay (OECD Test Guideline No. 487) for testing of manufactured nanomaterials: recommendations for best practices.

The current Organisation for Economic Co-Operation and Development test guideline number 487 (OECD TG No. 487) provides instruction on how to conduct the in vitro micronucleus assay. This assay is one of the gold standard approaches for measuring the mutagenicity of test items; however, it is directed at testing low molecular weight molecules and may not be appropriate for particulate materials (e.g. engineered nanoparticles [ENPs]). This study aimed to adapt the in vitro micronucleus assay for ENP testing and underpins the development of an OECD guidance document. A harmonized, nano-specific protocol was generated and evaluated by two independent laboratories. Cell lines utilized were human lymphoblastoid (TK6) cells, human liver hepatocytes (HepG2) cells, Chinese hamster lung fibroblast (V79) cells, whole blood, and buffy coat cells from healthy human volunteers. These cells were exposed to reference ENPs from the Joint Research Council (JRC): SiO2 (RLS-0102), Au5nm and Au30nm (RLS-03, RLS-010), CeO2 (NM212), and BaSO4 (NM220). Tungsten carbide-cobalt (WC/Co) was used as a trial particulate positive control. The chemical controls were positive in all cell cultures, but WC/Co was only positive in TK6 and buffy coat cells. In TK6 cells, mutagenicity was observed for SiO2- and both Au types. In HepG2 cells, Au5nm and SiO2 showed sub-two-fold increases in micronuclei. In V79 cells, whole blood, and buffy coat cells, no genotoxicity was detected with the test materials. The data confirmed that ENPs could be tested with the harmonized protocol, additionally, concordant data were observed across the two laboratories with V79 cells. WC/Co may be a suitable particulate positive control in the in vitro micronucleus assay when using TK6 and buffy coat cells. Detailed recommendations are therefore provided to adapt OECD TG No. 487 for testing ENP.

Selenium speciation studies in cancer patients to evaluate the responses of biomarkers of selenium status to different selenium compounds.

This work presents the first systematic comparison of selenium (Se) speciation in plasma from cancer patients treated orally with three Se compounds (sodium selenite, SS; L-selenomethionine, SeMet; or Se-methylselenocysteine, MSC) at 400 µg/day for 28 days. The primary goal was to investigate how these chemical forms of Se affect the plasma Se distribution, aiming to identify the most effective Se compound for optimal selenoprotein expression. This was achieved using methodology based on HPLC-ICP-MS after sample preparation/fractionation approaches. Measurements of total Se in plasma samples collected before and after 4 weeks of treatment showed that median total Se levels increased significantly from 89.6 to 126.4 µg kg-1 Se (p < 0.001), particularly when SeMet was administered (190.4 µg kg-1 Se). Speciation studies showed that the most critical differences between treated and baseline samples were seen for selenoprotein P (SELENOP) and selenoalbumin after administration with MSC (p = 5.8 × 10-4) and SeMet (p = 6.8 × 10-5), respectively. Notably, selenosugar-1 was detected in all low-molecular-weight plasma fractions following treatment, particularly with MSC. Two different chromatographic approaches and spiking experiments demonstrated that about 45% of that increase in SELENOP levels (to ~ 8.8 mg L-1) with SeMet is likely due to the non-specific incorporation of SeMet into the SELENOP affinity fraction. To the authors' knowledge, this has not been reported to date. Therefore, SELENOP is probably part of both the regulated (55%) and non-regulated (45%) Se pools after SeMet administration, whereas SS and MSC mainly contribute to the regulated one.

Determining the toxicological effects of indoor air pollution on both a healthy and an inflammatory-comprised model of the alveolar epithelial barrier in vitro.

Exposure to indoor air pollutants (IAP) has increased recently, with people spending more time indoors (i.e. homes, offices, schools and transportation). Increased exposures of IAP on a healthy population are poorly understood, and those with allergic respiratory conditions even less so. The objective of this study, therefore, was to implement a well-characterised in vitro model of the human alveolar epithelial barrier (A549 + PMA differentiated THP-1 incubated with and without IL-13, IL-5 and IL-4) to determine the effects of a standardised indoor particulate (NIST 2583) on both a healthy lung model and one modelling a type-II (stimulated with IL-13, IL-5 and IL-4) inflammatory response (such as asthma).Using concentrations from the literature, and an environmentally appropriate exposure we investigated 232, 464 and 608ng/cm2 of NIST 2583 respectively. Membrane integrity (blue dextran), viability (trypan blue), genotoxicity (micronucleus (Mn) assay) and (pro-)/(anti-)inflammatory effects (IL-6, IL-8, IL-33, IL-10) were then assessed 24 h post exposure to both models. Models were exposed using a physiologically relevant aerosolisation method (VitroCell Cloud 12 exposure system).No changes in Mn frequency or membrane integrity in either model were noted when exposed to any of the tested concentrations of NIST 2583. A significant decrease (p < 0.05) in cell viability at the highest concentration was observed in the healthy model. Whilst cell viability in the "inflamed" model was decreased at the lower concentrations (significantly (p < 0.05) after 464ng/cm2). A significant reduction (p < 0.05) in IL-10 and a significant increase in IL-33 was seen after 24 h exposure to NIST 2583 (464, 608ng/cm2) in the "inflamed" model.Collectively, the results indicate the potential for IAP to cause the onset of a type II response as well as exacerbating pre-existing allergic conditions. Furthermore, the data imposes the importance of considering unhealthy individuals when investigating the potential health effects of IAP. It also highlights that even in a healthy population these particles have the potential to induce this type II response and initiate an immune response following exposure to IAP.

Challenges in Estimating the Effectiveness of COVID-19 Vaccination Using Observational Data.

The COVID-19 vaccines were developed and rigorously evaluated in randomized trials during 2020. However, important questions, such as the magnitude and duration of protection, their effectiveness against new virus variants, and the effectiveness of booster vaccination, could not be answered by randomized trials and have therefore been addressed in observational studies. Analyses of observational data can be biased because of confounding and because of inadequate design that does not consider the evolution of the pandemic over time and the rapid uptake of vaccination. Emulating a hypothetical "target trial" using observational data assembled during vaccine rollouts can help manage such potential sources of bias. This article describes 2 approaches to target trial emulation. In the sequential approach, on each day, eligible persons who have not yet been vaccinated are matched to a vaccinated person. The single-trial approach sets a single baseline at the start of the rollout and considers vaccination as a time-varying variable. The nature of the confounding depends on the analysis strategy: Estimating "per-protocol" effects (accounting for vaccination of initially unvaccinated persons after baseline) may require adjustment for both baseline and "time-varying" confounders. These issues are illustrated by using observational data from 2 780 931 persons in the United Kingdom aged 70 years or older to estimate the effect of a first dose of a COVID-19 vaccine. Addressing the issues discussed in this article should help authors of observational studies provide robust evidence to guide clinical and policy decisions.

Analysis of Uncertainty and Sensitivity in Tailings Dam Breach-Runout Numerical Modelling.

Tailings dam breaches (TDBs) and subsequent flows can pose significant risk to public safety, the environment, and the economy. Numerical runout models are used to simulate potential tailings flows and understand their downstream impacts. Due to the complex nature of the breach-runout processes, the mobility and downstream impacts of these types of failures are highly uncertain. We applied the first-order second-moment (FOSM) methodology to a database of 11 back-analyzed historical tailings flows to evaluate uncertainties in TDB runout modelling and conducted a sensitivity analysis to identify key factors contributing to the variability of the HEC-RAS model output, including at different locations along the runout path. The results indicate that prioritizing resources toward advancements in estimating the values of primary contributors to the sensitivity of the selected model outputs is necessary for more reliable model results. We found that the total released volume is among the top contributors to the sensitivity of modelled inundation area and maximum flow depth, while surface roughness is among the top contributors to the sensitivity of modelled maximum flow velocity and flow front arrival time. However, the primary contributors to the sensitivity of the model outputs varied depending on the case study; therefore, the selection of appropriate rheological models and consideration of site-specific conditions are crucial for accurate predictions. The study proposes and demonstrates the FOSM methodology as an approximate probabilistic approach to model-based tailings flow runout prediction, which can help improve the accuracy of risk assessments and emergency response plans. Supplementary Information: The online version contains supplementary material available at 10.1007/s10230-024-00970-w.

Las roturas de presas de relaves (TDBs) y los flujos subsiguientes pueden suponer un riesgo significativo para la seguridad pública, el medio ambiente y la economía. Los modelos numéricos de desbordamiento se utilizan para simular posibles flujos de relaves y comprender su impacto aguas abajo. Debido a la naturaleza compleja de los procesos de rotura-desbordamiento, la movilidad y los impactos aguas abajo de este tipo de fallos tienen mucha incertidumbre. Se aplicó la metodología del segundo-momento de primer-orden (FOSM) a una base de datos de 11 flujos históricos de relaves analizados retrospectivamente para evaluar las incertidumbres en la modelización del desbordamiento de TDB y se realizó un análisis de sensibilidad para identificar los factores clave que contribuyen a la variabilidad de los resultados del modelo HEC-RAS, incluso en diferentes ubicaciones a lo largo de la trayectoria de fuga. Los resultados indican que es necesario priorizar los recursos hacia avances en la estimación de los valores de los principales contribuyentes a la sensibilidad de los resultados del modelo seleccionado para obtener resultados más fiables del modelo. El volumen total liberado se encuentra entre los principales contribuyentes a la sensibilidad del área de inundación modelizada y la profundidad máxima del flujo, mientras que la rugosidad de la superficie se encuentra entre los principales contribuyentes a la sensibilidad de la velocidad máxima del flujo modelizado y el tiempo de llegada del frente de flujo. Sin embargo, los principales factores que contribuyen a la sensibilidad de los resultados del modelo varían dependiendo del caso de estudio; por lo tanto, la selección de modelos reológicos apropiados y la consideración de las condiciones específicas del emplazamiento son cruciales para obtener predicciones precisas. El estudio propone y muestra la metodología FOSM como un enfoque probabilístico aproximado para la predicción de la extensión de flujos de relaves basada en modelos, que puede ayudar a mejorar la precisión de las evaluaciones de riesgos y los planes de respuesta a emergencias.

Antigen binding by conformational selection in near-germline antibodies.

Conformational flexibility in antibody-combining sites has been hypothesized to facilitate polyspecificity toward multiple unique epitopes and enable the limited germline repertoire to match an overwhelming diversity of potential antigens; however, elucidating the mechanisms of antigen recognition by flexible antibodies has been understandably challenging. Here, multiple liganded and unliganded crystal structures of the near-germline anticarbohydrate antibodies S25-2 and S25-39 are reported, which reveal an unprecedented diversity of complementarity-determining region H3 conformations in apparent equilibrium. These structures demonstrate that at least some germline or near-germline antibodies are flexible entities sensitive to their chemical environments, with conformational selection available as an evolved mechanism that preserves the inherited ability to recognize common pathogens while remaining adaptable to new threats.

The S-layer homology domains of Paenibacillus alvei surface protein SpaA bind to cell wall polysaccharide through the terminal monosaccharide residue.

Self-assembling (glyco)protein surface layers (S-layers) are ubiquitous prokaryotic cell-surface structures involved in structural maintenance, nutrient diffusion, host adhesion, virulence, and other processes, which makes them appealing targets for therapeutics and biotechnological applications as biosensors or drug delivery systems. However, unlocking this potential requires expanding our understanding of S-layer properties, especially the details of surface-attachment. S-layers of Gram-positive bacteria often are attached through the interaction of S-layer homology (SLH) domain trimers with peptidoglycan-linked secondary cell wall polymers (SCWPs). Cocrystal structures of the SLH domain trimer from the Paenibacillus alvei S-layer protein SpaA (SpaASLH) with synthetic, terminal SCWP disaccharide and trisaccharide analogs, together with isothermal titration calorimetry binding analyses, reveal that while SpaASLH accommodates longer biologically relevant SCWP ligands within both its primary (G2) and secondary (G1) binding sites, the terminal pyruvylated ManNAc moiety serves as the nearly exclusive SCWP anchoring point. Binding is accompanied by displacement of a flexible loop adjacent to the receptor site that enhances the complementarity between protein and ligand, including electrostatic complementarity with the terminal pyruvate moiety. Remarkably, binding of the pyruvylated monosaccharide SCWP fragment alone is sufficient to cause rearrangement of the receptor-binding sites in a manner necessary to accommodate longer SCWP fragments. The observation of multiple conformations in longer oligosaccharides bound to the protein, together with the demonstrated functionality of two of the three SCWP receptor-binding sites, reveals how the SpaASLH-SCWP interaction has evolved to accommodate longer SCWP ligands and alleviate the strain inherent to bacterial S-layer adhesion during growth and division.

Kilohertz two-photon fluorescence microscopy imaging of neural activity in vivo.

Understanding information processing in the brain requires monitoring neuronal activity at high spatiotemporal resolution. Using an ultrafast two-photon fluorescence microscope empowered by all-optical laser scanning, we imaged neuronal activity in vivo at up to 3,000 frames per second and submicrometer spatial resolution. This imaging method enabled monitoring of both supra- and subthreshold electrical activity down to 345 µm below the brain surface in head-fixed awake mice.

Severity of Severe Acute Respiratory System Coronavirus 2 (SARS-CoV-2) Alpha Variant (B.1.1.7) in England.

BACKGROUND: The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) alpha variant (B.1.1.7) is associated with higher transmissibility than wild-type virus, becoming the dominant variant in England by January 2021. We aimed to describe the severity of the alpha variant in terms of the pathway of disease from testing positive to hospital admission and death. METHODS: With the approval of NHS England, we linked individual-level data from primary care with SARS-CoV-2 community testing, hospital admission, and Office for National Statistics all-cause death data. We used testing data with S-gene target failure as a proxy for distinguishing alpha and wild-type cases, and stratified Cox proportional hazards regression to compare the relative severity of alpha cases with wild-type diagnosed from 16 November 2020 to 11 January 2021. RESULTS: Using data from 185 234 people who tested positive for SARS-CoV-2 in the community (alpha = 93 153; wild-type = 92 081), in fully adjusted analysis accounting for individual-level demographics and comorbidities as well as regional variation in infection incidence, we found alpha associated with 73% higher hazards of all-cause death (adjusted hazard ratio [aHR]: 1.73; 95% confidence interval [CI]: 1.41-2.13; P < .0001) and 62% higher hazards of hospital admission (1.62; 1.48-1.78; P < .0001) compared with wild-type virus. Among patients already admitted to the intensive care unit, the association between alpha and increased all-cause mortality was smaller and the CI included the null (aHR: 1.20; 95% CI: .74-1.95; P = .45). CONCLUSIONS: The SARS-CoV-2 alpha variant is associated with an increased risk of both hospitalization and mortality than wild-type virus.

Overall and cause-specific hospitalisation and death after COVID-19 hospitalisation in England: A cohort study using linked primary care, secondary care, and death registration data in the OpenSAFELY platform.

BACKGROUND: There is concern about medium to long-term adverse outcomes following acute Coronavirus Disease 2019 (COVID-19), but little relevant evidence exists. We aimed to investigate whether risks of hospital admission and death, overall and by specific cause, are raised following discharge from a COVID-19 hospitalisation. METHODS AND FINDINGS: With the approval of NHS-England, we conducted a cohort study, using linked primary care and hospital data in OpenSAFELY to compare risks of hospital admission and death, overall and by specific cause, between people discharged from COVID-19 hospitalisation (February to December 2020) and surviving at least 1 week, and (i) demographically matched controls from the 2019 general population; and (ii) people discharged from influenza hospitalisation in 2017 to 2019. We used Cox regression adjusted for age, sex, ethnicity, obesity, smoking status, deprivation, and comorbidities considered potential risk factors for severe COVID-19 outcomes. We included 24,673 postdischarge COVID-19 patients, 123,362 general population controls, and 16,058 influenza controls, followed for ≤315 days. COVID-19 patients had median age of 66 years, 13,733 (56%) were male, and 19,061 (77%) were of white ethnicity. Overall risk of hospitalisation or death (30,968 events) was higher in the COVID-19 group than general population controls (fully adjusted hazard ratio [aHR] 2.22, 2.14 to 2.30, p < 0.001) but slightly lower than the influenza group (aHR 0.95, 0.91 to 0.98, p = 0.004). All-cause mortality (7,439 events) was highest in the COVID-19 group (aHR 4.82, 4.48 to 5.19 versus general population controls [p < 0.001] and 1.74, 1.61 to 1.88 versus influenza controls [p < 0.001]). Risks for cause-specific outcomes were higher in COVID-19 survivors than in general population controls and largely similar or lower in COVID-19 compared with influenza patients. However, COVID-19 patients were more likely than influenza patients to be readmitted or die due to their initial infection or other lower respiratory tract infection (aHR 1.37, 1.22 to 1.54, p < 0.001) and to experience mental health or cognitive-related admission or death (aHR 1.37, 1.02 to 1.84, p = 0.039); in particular, COVID-19 survivors with preexisting dementia had higher risk of dementia hospitalisation or death (age- and sex-adjusted HR 2.47, 1.37 to 4.44, p = 0.002). Limitations of our study were that reasons for hospitalisation or death may have been misclassified in some cases due to inconsistent use of codes, and we did not have data to distinguish COVID-19 variants. CONCLUSIONS: In this study, we observed that people discharged from a COVID-19 hospital admission had markedly higher risks for rehospitalisation and death than the general population, suggesting a substantial extra burden on healthcare. Most risks were similar to those observed after influenza hospitalisations, but COVID-19 patients had higher risks of all-cause mortality, readmission or death due to the initial infection, and dementia death, highlighting the importance of postdischarge monitoring.

Ethnic differences in SARS-CoV-2 infection and COVID-19-related hospitalisation, intensive care unit admission, and death in 17 million adults in England: an observational cohort study using the OpenSAFELY platform.

BACKGROUND: COVID-19 has disproportionately affected minority ethnic populations in the UK. Our aim was to quantify ethnic differences in SARS-CoV-2 infection and COVID-19 outcomes during the first and second waves of the COVID-19 pandemic in England. METHODS: We conducted an observational cohort study of adults (aged ≥18 years) registered with primary care practices in England for whom electronic health records were available through the OpenSAFELY platform, and who had at least 1 year of continuous registration at the start of each study period (Feb 1 to Aug 3, 2020 [wave 1], and Sept 1 to Dec 31, 2020 [wave 2]). Individual-level primary care data were linked to data from other sources on the outcomes of interest: SARS-CoV-2 testing and positive test results and COVID-19-related hospital admissions, intensive care unit (ICU) admissions, and death. The exposure was self-reported ethnicity as captured on the primary care record, grouped into five high-level census categories (White, South Asian, Black, other, and mixed) and 16 subcategories across these five categories, as well as an unknown ethnicity category. We used multivariable Cox regression to examine ethnic differences in the outcomes of interest. Models were adjusted for age, sex, deprivation, clinical factors and comorbidities, and household size, with stratification by geographical region. FINDINGS: Of 17 288 532 adults included in the study (excluding care home residents), 10 877 978 (62·9%) were White, 1 025 319 (5·9%) were South Asian, 340 912 (2·0%) were Black, 170 484 (1·0%) were of mixed ethnicity, 320 788 (1·9%) were of other ethnicity, and 4 553 051 (26·3%) were of unknown ethnicity. In wave 1, the likelihood of being tested for SARS-CoV-2 infection was slightly higher in the South Asian group (adjusted hazard ratio 1·08 [95% CI 1·07-1·09]), Black group (1·08 [1·06-1·09]), and mixed ethnicity group (1·04 [1·02-1·05]) and was decreased in the other ethnicity group (0·77 [0·76-0·78]) relative to the White group. The risk of testing positive for SARS-CoV-2 infection was higher in the South Asian group (1·99 [1·94-2·04]), Black group (1·69 [1·62-1·77]), mixed ethnicity group (1·49 [1·39-1·59]), and other ethnicity group (1·20 [1·14-1·28]). Compared with the White group, the four remaining high-level ethnic groups had an increased risk of COVID-19-related hospitalisation (South Asian group 1·48 [1·41-1·55], Black group 1·78 [1·67-1·90], mixed ethnicity group 1·63 [1·45-1·83], other ethnicity group 1·54 [1·41-1·69]), COVID-19-related ICU admission (2·18 [1·92-2·48], 3·12 [2·65-3·67], 2·96 [2·26-3·87], 3·18 [2·58-3·93]), and death (1·26 [1·15-1·37], 1·51 [1·31-1·71], 1·41 [1·11-1·81], 1·22 [1·00-1·48]). In wave 2, the risks of hospitalisation, ICU admission, and death relative to the White group were increased in the South Asian group but attenuated for the Black group compared with these risks in wave 1. Disaggregation into 16 ethnicity groups showed important heterogeneity within the five broader categories. INTERPRETATION: Some minority ethnic populations in England have excess risks of testing positive for SARS-CoV-2 and of adverse COVID-19 outcomes compared with the White population, even after accounting for differences in sociodemographic, clinical, and household characteristics. Causes are likely to be multifactorial, and delineating the exact mechanisms is crucial. Tackling ethnic inequalities will require action across many fronts, including reducing structural inequalities, addressing barriers to equitable care, and improving uptake of testing and vaccination. FUNDING: Medical Research Council.

Describing the population experiencing COVID-19 vaccine breakthrough following second vaccination in England: a cohort study from OpenSAFELY.

BACKGROUND: While the vaccines against COVID-19 are highly effective, COVID-19 vaccine breakthrough is possible despite being fully vaccinated. With SARS-CoV-2 variants still circulating, describing the characteristics of individuals who have experienced COVID-19 vaccine breakthroughs could be hugely important in helping to determine who may be at greatest risk. METHODS: With the approval of NHS England, we conducted a retrospective cohort study using routine clinical data from the OpenSAFELY-TPP database of fully vaccinated individuals, linked to secondary care and death registry data and described the characteristics of those experiencing COVID-19 vaccine breakthroughs. RESULTS: As of 1st November 2021, a total of 15,501,550 individuals were identified as being fully vaccinated against COVID-19, with a median follow-up time of 149 days (IQR: ​107-179). From within this population, a total of 579,780 (<4%) individuals reported a positive SARS-CoV-2 test. For every 1000 years of patient follow-up time, the corresponding incidence rate (IR) was 98.06 (95% CI 97.93-98.19). There were 28,580 COVID-19-related hospital admissions, 1980 COVID-19-related critical care admissions and 6435 COVID-19-related deaths; corresponding IRs 4.77 (95% CI 4.74-4.80), 0.33 (95% CI 0.32-0.34) and 1.07 (95% CI 1.06-1.09), respectively. The highest rates of breakthrough COVID-19 were seen in those in care homes and in patients with chronic kidney disease, dialysis, transplant, haematological malignancy or who were immunocompromised. CONCLUSIONS: While the majority of COVID-19 vaccine breakthrough cases in England were mild, some differences in rates of breakthrough cases have been identified in several clinical groups. While it is important to note that these findings are simply descriptive and cannot be used to answer why certain groups have higher rates of COVID-19 breakthrough than others, the emergence of the Omicron variant of COVID-19 coupled with the number of positive SARS-CoV-2 tests still occurring is concerning and as numbers of fully vaccinated (and boosted) individuals increases and as follow-up time lengthens, so too will the number of COVID-19 breakthrough cases. Additional analyses, to assess vaccine waning and rates of breakthrough COVID-19 between different variants, aimed at identifying individuals at higher risk, are needed.

Site-directed M2 proton channel inhibitors enable synergistic combination therapy for rimantadine-resistant pandemic influenza.

Pandemic influenza A virus (IAV) remains a significant threat to global health. Preparedness relies primarily upon a single class of neuraminidase (NA) targeted antivirals, against which resistance is steadily growing. The M2 proton channel is an alternative clinically proven antiviral target, yet a near-ubiquitous S31N polymorphism in M2 evokes resistance to licensed adamantane drugs. Hence, inhibitors capable of targeting N31 containing M2 (M2-N31) are highly desirable. Rational in silico design and in vitro screens delineated compounds favouring either lumenal or peripheral M2 binding, yielding effective M2-N31 inhibitors in both cases. Hits included adamantanes as well as novel compounds, with some showing low micromolar potency versus pandemic "swine" H1N1 influenza (Eng195) in culture. Interestingly, a published adamantane-based M2-N31 inhibitor rapidly selected a resistant V27A polymorphism (M2-A27/N31), whereas this was not the case for non-adamantane compounds. Nevertheless, combinations of adamantanes and novel compounds achieved synergistic antiviral effects, and the latter synergised with the neuraminidase inhibitor (NAi), Zanamivir. Thus, site-directed drug combinations show potential to rejuvenate M2 as an antiviral target whilst reducing the risk of drug resistance.

A de novo ACTB gene pathogenic variant in identical twins with phenotypic variation for hydrops and jejunal atresia.

The beta-actin gene (ACTB) encodes a ubiquitous cytoskeletal protein, essential for embryonic development in humans. De novo heterozygous missense variants in the ACTB are implicated in causing Baraitser-Winter cerebrofrontofacial syndrome (BWCFFS; MIM#243310). ACTB pathogenic variants are rarely associated with intestinal malformations. We report on a rare case of monozygotic twins presenting with proximal small bowel atresia and hydrops in one, and apple-peel bowel atresia and laryngeal dysgenesis in the other. The twin with hydrops could not be resuscitated. Intensive and surgical care was provided to the surviving twin. Rapid trio genome sequencing identified a de novo missense variant in ACTB (NM_00101.3:c.1043C>T; p.(Ser348Leu)) that guided the care plan. The identical variant subsequently was identified in the demised twin. To characterize the functional effect, the variant was recreated as a pseudoheterozygote in a haploid wild-type S. cerevisiae strain. There was an obvious growth defect of the yACT1S348L/WT pseudoheterozygote compared to a yACT1WT/WT strain when grown at 22°C but not when grown at 30°C, consistent with the yACT1 S348L variant having a functional defect that is dominant over the wild-type allele. The functional results provide supporting evidence that the Ser348Leu variant is likely to be a pathogenic variant, including being associated with intestinal malformations in BWCFFS, and can demonstrate variable expressivity within monozygotic twins.

The Influence of Nanobubble Size and Stability on Ultrasound Enhanced Drug Delivery.

Lipid-shelled nanobubbles (NBs) are emerging as potential dual diagnostic and therapeutic agents. Similar to their micron-scale counterparts, microbubbles (1-10 µm), they can act as ultrasound contrast agents as well as locally enhance therapeutic uptake. Recently, it has been shown that the reduced size of NBs (<1 µm) promotes increased uptake and accumulation in tumor interstitial space, which can enhance their diagnostic and therapeutic performance. However, accurate characterization of NB size and concentration is challenging and may limit their translation into clinical use. Their submicron nature limits accuracy of conventional microscopy techniques, while common light scattering techniques fail to distinguish between subpopulations present in NB samples (i.e., bubbles and liposomes). Due to the difficulty in the characterization of NBs, relatively little is known about the influence of size on their therapeutic performance. In this study, we describe a novel method of using a commercially available nanoparticle tracking analysis system, to distinguish between NBs and liposomes based on their differing optical properties. We used this technique to characterize three NB populations of varying size, isolated via centrifugation, and subsequently used this to assess their potential for enhancing localized delivery. Confocal fluorescence microscopy and image analysis were used to quantify the ultrasound enhanced uptake of fluorescent dextran into live colorectal cancer cells. Our results showed that the amount of localized uptake did not follow the expected trends, in which larger NB populations out-perform smaller NBs, at matched concentration. To understand this observed behavior, the stability of each NB population was assessed. It was found that dilution of the NB samples from their stock concentration influences their stability, and it is hypothesized that both the total free lipid and interbubble distance play a role in NB lifetime, in agreement with previously proposed theories and models.

Transparency of high-dimensional propensity score analyses: Guidance for diagnostics and reporting.

PURPOSE: The high-dimensional propensity score (HDPS) is a semi-automated procedure for confounder identification, prioritisation and adjustment in large healthcare databases that requires investigators to specify data dimensions, prioritisation strategy and tuning parameters. In practice, reporting of these decisions is inconsistent and this can undermine the transparency, and reproducibility of results obtained. We illustrate reporting tools, graphical displays and sensitivity analyses to increase transparency and facilitate evaluation of the robustness of analyses involving HDPS. METHODS: Using a study from the UK Clinical Practice Research Datalink that implemented HDPS we demonstrate the application of the proposed recommendations. RESULTS: We identify seven considerations surrounding the implementation of HDPS, such as the identification of data dimensions, method for code prioritisation and number of variables selected. Graphical diagnostic tools include assessing the balance of key confounders before and after adjusting for empirically selected HDPS covariates and the identification of potentially influential covariates. Sensitivity analyses include varying the number of covariates selected and assessing the impact of covariates behaving empirically as instrumental variables. In our example, results were robust to both the number of covariates selected and the inclusion of potentially influential covariates. Furthermore, our HDPS models achieved good balance in key confounders. CONCLUSIONS: The data-adaptive approach of HDPS and the resulting benefits have led to its popularity as a method for confounder adjustment in pharmacoepidemiological studies. Reporting of HDPS analyses in practice may be improved by the considerations and tools proposed here to increase the transparency and reproducibility of study results.

Recording of 'COVID-19 vaccine declined': a cohort study on 57.9 million National Health Service patients' records in situ using OpenSAFELY, England, 8 December 2020 to 25 May 2021.

BackgroundPriority patients in England were offered COVID-19 vaccination by mid-April 2021. Codes in clinical record systems can denote the vaccine being declined.AimWe describe records of COVID-19 vaccines being declined, according to clinical and demographic factors.MethodsWith the approval of NHS England, we conducted a retrospective cohort study between 8 December 2020 and 25 May 2021 with primary care records for 57.9 million patients using OpenSAFELY, a secure health analytics platform. COVID-19 vaccination priority patients were those aged ≥ 50 years or ≥ 16 years clinically extremely vulnerable (CEV) or 'at risk'. We describe the proportion recorded as declining vaccination for each group and stratified by clinical and demographic subgroups, subsequent vaccination and distribution of clinical code usage across general practices.ResultsOf 24.5 million priority patients, 663,033 (2.7%) had a decline recorded, while 2,155,076 (8.8%) had neither a vaccine nor decline recorded. Those recorded as declining, who were subsequently vaccinated (n = 125,587; 18.9%) were overrepresented in the South Asian population (32.3% vs 22.8% for other ethnicities aged ≥ 65 years). The proportion of declining unvaccinated patients was highest in CEV (3.3%), varied strongly with ethnicity (black 15.3%, South Asian 5.6%, white 1.5% for ≥ 80 years) and correlated positively with increasing deprivation.ConclusionsClinical codes indicative of COVID-19 vaccinations being declined are commonly used in England, but substantially more common among black and South Asian people, and in more deprived areas. Qualitative research is needed to determine typical reasons for recorded declines, including to what extent they reflect patients actively declining.