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Regina Sala,1 Lorene Amet,2 Natasa Blagojevic-Stokic,3 Paul Shattock,4 Paul Whiteley5 1Centre for Psychiatry, Wolfson Institute, Barts & The London School of Medicine & Dentistry Queen Mary University of London, London, UK; 2Autism Treatment Plus, Edinburgh, UK; 3Thinking Autism, London, UK; 4Education & Services for People with Autism, Sunderland, UK; 5Education & Services for People with Autism Research, Sunderland, UKCorrespondence: Dr Regina SalaCentre for Psychiatry, Wolfson Institute, Barts & The London School of Medicine & Dentistry Queen Mary University of London, London, UKEmail regina.sala@nhs.netAbstract: Autism spectrum disorder (ASD) is a highly complex and heterogeneous developmental disorder that affects how individuals communicate with other people and relate to the world around them. Research and clinical focus on the behavioural and cognitive manifestations of ASD, whilst important, have obscured the recognition that ASD is also commonly associated with a range of physical and mental health conditions. Many physical conditions appear with greater frequency in individuals with ASD compared to non-ASD populations. These can contribute to a worsening of social communication and behaviour, lower quality of life, higher morbidity and premature mortality. We highlight some of the key physical comorbidities affecting the immune and the gastrointestinal systems, metabolism and brain function in ASD. We discuss how healthcare professionals working with individuals with ASD and parents/carers have a duty to recognise their needs in order to improve their overall health and wellbeing, deliver equality in their healthcare experiences and reduce the likelihood of morbidity and early mortality associated with the condition.Keywords: autism spectrum disorder, ASD, physical health, comorbidity, inequality

Paul Whiteley, Kevin Carr, Paul Shattock ESPA Research, Sunderland, UKCorrespondence: Paul WhiteleyThe Robert Luff Laboratory, Unit 133i Business Innovation Centre (BIC), ESPA Research, Wearfield, Sunderland SR5 2TA, UKTel +44 191 516 6366Email paul.whiteley@espa-research.org.ukAbstract: Autism or autism spectrum disorder (ASD) is described as a lifelong condition with core behavioural symptoms appearing during infancy or early childhood. Genetic and other effects occurring during the earliest times of life are thought to play a significant contributory role to the presentation of autism, denoting that autism is typically seen as an innate or inborn condition. Such descriptions have, and continue to, define autism research and clinical practice. Inspection of the existing research literature, however, suggests that within the vast heterogeneity of autism, not everyone experiences autism in such a prescribed way. Various reports have observed the presentation of “acquired autism” following a period of typical development. Other findings have documented an abatement of clinically relevant autistic features and related comorbid pathology for some. Such reports offer important insights into the heterogeneity and complexity of autism.Keywords: autism, regression, innate, lifelong, acquired, heterogeneity

Abstract Background The Mini-Mental State Examination (MMSE) and the Montreal Cognitive Assessment (MoCA) questionnaires are commonly used to measure global cognition in clinical trials. Because these scales are discrete and bounded with ceiling and floor effects and highly skewed, their analysis as continuous outcomes presents challenges. Normality assumptions of linear regression models are usually violated, which may result in failure to detect associations with variables of interest. Methods Alternative approaches to analyzing the results of these cognitive batteries include transformations (standardization, square root, or log transformation) of the scores in the multivariate linear regression (MLR) model, the use of nonlinear beta-binomial regression (which is not dependent on the assumption of normality), or Tobit regression, which adds a latent variable to account for bounded data. We aim to empirically compare the model performance of all proposed approaches using four large randomized controlled trials (ORIGIN, TRANSCEND, COMPASS, and NAVIGATE-ESUS), and using as metrics the Akaike information criterion (AIC). We also compared the treatment effects for the methods that have the same unit of measure (i.e., untransformed MLR, beta-binomial, and Tobit). Results The beta-binomial consistently demonstrated superior model performance, with the lowest AIC values among nearly all the approaches considered, followed by the MLR with square root and log transformations across all four studies. Notably, in ORIGIN, a substantial AIC reduction was observed when comparing the untransformed MLR to the beta-binomial, whereas other studies had relatively small AIC reductions. The beta-binomial model also resulted in a significant treatment effect in ORIGIN, while the untransformed MLR and Tobit regression showed no significance. The other three studies had similar and insignificant treatment effects among the three approaches. Conclusion When analyzing discrete and bounded outcomes, such as cognitive scores, as continuous variables, a beta-binomial regression model improves model performance, avoids spurious significance, and allows for a direct interpretation of the actual cognitive measure. Trials registration ORIGIN (NCT00069784). Registered on October 1, 2003; TRANSCEND (NCT00153101). Registered on September 9, 2005; COMPASS (NCT01776424). Registered on January 24, 2013; NAVIGATE-ESUS (NCT02313909). Registered on December 8, 2014.

Abstract Advances in omics technologies have enabled the in-depth study of microbial communities and their metabolic profiles from all environments. Here metagenomes were sampled from piranha (Serrasalmus rhombeus) and from river water from the Rio São Benedito (Amazon Basin). Shotgun metagenome sequencing was used to explore diversity and to test whether fish microbiomes are a good proxy for river microbiome studies. The results showed that the fish microbiomes were not significantly different from the river water microbiomes at higher taxonomic ranks. However, at the genus level, fish microbiome alpha diversity decreased, and beta diversity increased. This result repeated for functional gene abundances associated with specific metabolic categories (SEED level 3). A clear delineation between water and fish was seen for beta diversity. The piranha microbiome provides a good and representative subset of its river water microbiome. Variations seen in beta biodiversity were expected and can be explained by temporal variations in the fish microbiome in response to stronger selective forces on its biodiversity. Metagenome assembled genomes construction was better from the fish samples. This study has revealed that the microbiome of a piranha tells us a lot about its river water microbiome and function.

ABSTRACT Grapes have been cultivated for wine production for millennia. Wine production involves a complex biochemical process where sugars in grape must are converted into alcohol and other compounds by microbial fermentation, primarily by the yeast Saccharomyces cerevisiae. Commercially available S. cerevisiae strains are often used in winemaking, but indigenous (native) strains are gaining attention for their potential to contribute unique flavors. Recent advancements in high-throughput DNA sequencing have revolutionized our understanding of microbial communities during wine fermentation. Indeed, transcriptomic analysis of S. cerevisiae during wine fermentation has revealed a core gene expression program and provided insights into how this yeast adapts to fermentation conditions. Here, we assessed how the age of vines impacts the grape fungal microbiome and used transcriptomics to characterize microbial functions in grape must fermented with commercial and native S. cerevisiae. We discovered that ~130-year-old Zinfandel vines harbor higher fungal loads on their grapes compared to 20-year-old Zinfandel vines, but fungal diversity is similar. Additionally, a comparison of inoculated and uninoculated fermentations showed distinct fungal dynamics, with uninoculated fermentations harboring the yeasts Metschnikowia and Pichia. Transcriptomic analysis revealed significant differences in gene expression between fermentations inoculated and not inoculated with a commercial S. cerevisiae strain. Genes related to metabolism, stress response, and cell adhesion were differentially expressed, indicating varied functionality of S. cerevisiae in these fermentations. These findings provide insights into S. cerevisiae function during fermentation and highlight the potential for indigenous yeast to contribute to wine diversity.IMPORTANCEUnderstanding microbial functions during wine fermentation, particularly the role of Saccharomyces cerevisiae, is crucial for enhancing wine quality. While commercially available S. cerevisiae strains are commonly used, indigenous strains can offer unique flavors, potentially reflecting vineyard terroir. By leveraging high-throughput DNA sequencing and transcriptomic analysis, we explored the impact of vine age on the grape mycobiome and characterized microbial functions during grape fermentation. Our findings revealed that older vines harbor higher fungal loads, but fungal diversity remains similar across vine ages. Additionally, uninoculated fermentations exhibited diverse fungal dynamics, including the beneficial wine yeasts Metschnikowia and Pichia. Transcriptomic analysis uncovered significant differences in S. cerevisiae gene expression between inoculated and uninoculated fermentations, highlighting the potential of indigenous yeast to enhance wine diversity and inform winemaking practices.

Background: The rapid development of less invasive and traumatic medical procedures has resulted in a mixture of terms used to describe them, without any agreed definition for each. This is confusing to both medical professionals and patients and can lead to unrealistic patient expectations. The aim of this article is to show the current confused nomenclature and to suggest a new, simple classification based on access and invasiveness (AI) that can be applied to any medical procedure. Methods: We performed an online search for definitions for ‘non-invasive’, ‘non-surgical’, ‘minimally invasive’, ‘minimal access’, ‘pinhole’ and ‘keyhole’. We then searched peer-reviewed medical papers (PRMPs) and patient facing websites (PFWs) for the following index procedures, to see which of the original 6 terms were used to describe them: transvaginal ultrasound, sclerotherapy for leg veins, botulinum toxin injections, dermal fillers, endovenous thermal ablation and laparoscopic gall bladder removal. Results: We found a wide variety of definitions for each of the initial terms. In both PRMPs and PFWs, there were a variety of terms used for each index procedure (i.e.: transvaginal ultrasound, injections of sclerotherapy, botulinum toxin or dermal fillers being both ‘non-invasive’ and ‘minimally invasive’) showing confusion in the classification of procedures. We suggested the ‘AI classification’ based on access (A - none, B - natural orifice or C - penetrating an epithelial surface) and invasiveness (1 - none, 2 - surface damage, 3 - needle = 21G but not a surgical trocar, 5 - surgical trocars or small incisions and 6 - incisions). Conclusion: The current confusion of terms used for procedures that are less invasive than the open surgical alternatives leads to confusion and possible false patient expectations. We have proposed an AI classification that can be applied easily to any procedure, giving a uniform classification for medical professionals and patients to understand.

Abstract Background Mississippi (MS) experiences disproportionally high rates of new HIV infections and limited availability of pre-exposure prophylaxis (PrEP). Federally Qualified Health Centers (FQHCs) are poised to increase access to PrEP. However, little is known about the implementation strategies needed to successfully integrate PrEP services into FQHCs in MS. Purpose The study had two objectives: identify barriers and facilitators to PrEP use and to develop tailored implementation strategies for FQHCs. Methods Semi-structured interviews were conducted with 19 staff and 17 PrEP-eligible patients in MS FQHCs between April 2021 and March 2022. The interview was guided by the integrated-Promoting Action on Research Implementation in Health Services (i-PARIHS) framework which covered PrEP facilitators and barriers. Interviews were coded according to the i-PARIHS domains of context, innovation, and recipients, followed by thematic analysis of these codes. Identified implementation strategies were presented to 9 FQHC staff for feedback. Results Data suggested that PrEP use at FQHCs is influenced by patient and clinic staff knowledge with higher levels of knowledge reflecting more PrEP use. Perceived side effects are the most significant barrier to PrEP use for patients, but participants also identified several other barriers including low HIV risk perception and untrained providers. Despite these barriers, patients also expressed a strong motivation to protect themselves, their partners, and their communities from HIV. Implementation strategies included education and provider training which were perceived as acceptable and appropriate. Conclusions Though patients are motivated to increase protection against HIV, multiple barriers threaten uptake of PrEP within FQHCs in MS. Educating patients and providers, as well as training providers, are promising implementation strategies to overcome these barriers.

Abstract The first dose of COVID-19 vaccines led to an overall reduction in cardiovascular events, and in rare cases, cardiovascular complications. There is less information about the effect of second and booster doses on cardiovascular diseases. Using longitudinal health records from 45.7 million adults in England between December 2020 and January 2022, our study compared the incidence of thrombotic and cardiovascular complications up to 26 weeks after first, second and booster doses of brands and combinations of COVID-19 vaccines used during the UK vaccination program with the incidence before or without the corresponding vaccination. The incidence of common arterial thrombotic events (mainly acute myocardial infarction and ischaemic stroke) was generally lower after each vaccine dose, brand and combination. Similarly, the incidence of common venous thrombotic events, (mainly pulmonary embolism and lower limb deep venous thrombosis) was lower after vaccination. There was a higher incidence of previously reported rare harms after vaccination: vaccine-induced thrombotic thrombocytopenia after first ChAdOx1 vaccination, and myocarditis and pericarditis after first, second and transiently after booster mRNA vaccination (BNT-162b2 and mRNA-1273). These findings support the wide uptake of future COVID-19 vaccination programs.

David John Mott,1 Jake Hitch,1,2 Samantha Nier,3 Zack Pemberton-Whiteley,3,4 Chris Skedgel1 1Office of Health Economics, London, UK; 2Health Economics Research Centre, Nuffield Department of Population Health, University of Oxford, Oxford, UK; 3Acute Leukemia Advocates Network, Bern, Switzerland; 4Leukemia Care, Worcester, UKCorrespondence: David John Mott, Email dmott@ohe.orgBackground: Acute leukemia is a cancer of the white blood cells which progresses rapidly and aggressively. There are two types: acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML). The latter has a rare subtype: acute promyelocytic leukemia (APL). For some patients, following first-line treatment, remission is not achieved (“refractory disease”), and for others the leukemia returns after achieving remission (“relapse”). For these individuals, outcomes are typically poor. It is, therefore, important to understand patients’ treatment priorities in this context.Methods: Building upon formative qualitative research, an online survey containing a discrete choice experiment (DCE) was designed to explore patients’ treatment preferences in the relapsed/refractory setting. The DCE attributes were mode of administration; quality of life during treatment; chance of response; duration of response; and quality of life during response. Each respondent completed twelve scenarios containing two hypothetical treatments. Participants were eligible if they lived in the United Kingdom and had a diagnosis of acute leukemia. The data were analysed using a latent class model.Results: A total of 95 patients completed the survey. The latent class analysis identified two classes. For both, chance of response was the most important attribute. For class 1, every attribute was important, whereas for class 2, the only important attributes were quality of life (during treatment and response) and chance of response. A greater proportion of respondents would fall into class 1 overall, and those with ALL or APL and those more recently diagnosed were more likely to be in class 2.Conclusion: Our results indicate that patients are strongly concerned about the chance of response, as well as quality of life (to a lesser extent), when faced with different treatment options in the relapsed/refractory setting. However, there is significant preference heterogeneity within the patient population, and other treatment characteristics also matter to many.Keywords: acute leukemia, hematology, oncology, patient preferences, discrete choice experiment

ABSTRACT Mycobacteroides abscessus (MAB) causes lung infections in people with cystic fibrosis (pwCF), and infecting strains show significant genetic variability both between and within individuals. MAB isolates can be divided into dominant clonal clusters (DCCs) or non-clustering groups and can present as smooth or rough colonies on agar plates. Both DCCs and the rough colony morphology have been linked to increased pathogenicity, but the mechanisms are unclear. This study explored the genomes of MAB isolates collected from individuals within the CF@LANTA CF center along with publicly available genomes to identify genes associated with more pathogenic MAB DCCs. Sixty-eight isolates from 26 CF individuals colonized by MAB were morphotyped and sequenced, with almost half of these isolates being members of DCC group 1 (DCC1). While lung function was not significantly impacted by colonization with DCC1 or rough isolates, 102 genes were specifically associated with DCC1 isolates. These genes were enriched for functions in sulfur-based DNA modification, DNA integration, and phenylacetic acid (PAA) catabolism. PAA is produced by the human gut microbiota and found throughout the human body. We show that strains containing PAA metabolic genes allow MAB to use PAA as a sole carbon and energy source. Although the benefits of PAA metabolic genes and other enriched pathways remain unclear, these findings highlight genes associated with emerging MAB CF strains.IMPORTANCEA primary challenge in treating bacterial infections is the wide spectrum of disease and genetic variability across bacterial strains. This is particularly evident in Mycobacteroides abscessus (MAB), an emerging pathogen affecting people with cystic fibrosis (pwCF). MAB exhibits significant genetic diversity both within and between individuals. However, seven dominant circulating clones (DCCs) have emerged as the major cause of human infections, demonstrating increased pathogenicity. Understanding the mechanisms underlying this increased pathogenicity and the associated genetic factors is crucial for developing novel treatment strategies. Our findings reveal that specific genes are associated with the DCC1 isolate of MAB, many of which are implicated in antimicrobial susceptibility or virulence.

ABSTRACT Brook Trout (Salvelinus fontinalis) populations have experienced marked declines throughout their native range and are presently threatened due to isolation in small habitat fragments, land use changes, and climate change. The existence of numerous, spatially distinct populations poses substantial challenges for monitoring population status (e.g., abundance, recruitment, or occupancy). Genetic monitoring with estimates of effective number of breeders (Nb) provides a potentially powerful metric to complement existing population monitoring, assessment, and prioritization. We estimated Nb for 71 Brook Trout habitat units in mid‐Atlantic region of the United States and obtained a mean Nb of 73.2 (range 6.90–493). Our modeling approach tested whether Nb estimates were sensitive to differences in habitat size, presence of non‐native salmonids, base flow index, temperature, acidic precipitation, and indices of anthropogenic disturbance. We found significant support for three of our hypotheses including the positive influences of available habitat and base flow index and negative effect of temperature. Our results are consistent with presently observed and predicted future impacts of climate change on populations of this cold‐water fish. Importantly, these findings support the use of Nb in population assessments as an index of relative population status.

Cannabinoid hyperemesis syndrome presents as a complex of symptoms and signs encompassing nausea, vomiting, abdominal pain, and hot water bathing behavior, most typically in a heavy cannabis user. Its presentation is frequently associated with hypothalamic-pituitary-adrenal axis activation with stress and weight loss. Recent investigation has identified five statistically significant mutations in patients distinct from those of frequent cannabis users who lack the symptoms, affecting the TRPV1 receptor, two dopamine genes, the cytochrome P450 2C9 enzyme that metabolizes tetrahydrocannabinol, and the adenosine triphosphate-binding cassette transporter. The syndrome is associated with escalating intake of high potency cannabis, or alternatively, other agonists of the cannabinoid-1 receptor including synthetic cannabinoids. Some patients develop environmental triggers in scents or foods that suggest classical conditioned responses. Various alternative “causes” are addressed and refuted in the text, including exposure to pesticides, neem oil or azadirachtin. Nosological confusion of cannabinoid hyperemesis syndrome has arisen with cyclic vomiting syndrome, whose presentation and pathophysiology are clearly distinct. The possible utilization of non-intoxicating antiemetic cannabis components in cannabis for treatment of cannabinoid hyperemesis syndrome is addressed, along with future research suggestions in relation to its genetic foundation and possible metabolomic signatures.

ABSTRACT Understanding the molecular mechanisms governing microbial interactions is crucial for unraveling the complexities of microbial communities and their ecological impacts. Here, we employed a two-species model system comprising the oral bacteria Aggregatibacter actinomycetemcomitans and Streptococcus gordonii to investigate how synergistic and antagonistic interactions between microbes impact their resilience to environmental change and invasion by other microbes. We used an in vitro colony biofilm model and focused on two S. gordonii-produced extracellular molecules, L-lactate and H2O2, which are known to impact fitness of this dual-species community. While the ability of A. actinomycetemcomitans to cross-feed on S. gordonii-produced L-lactate enhanced its fitness during co-culture, this function showed little impact on the ability of co-cultures to resist environmental change. In fact, the ability of A. actinomycetemcomitans to catabolize L-lactate may be detrimental in the presence of tetracycline, highlighting the complexity of interactions under antimicrobial stress. Furthermore, H2O2, known for its antimicrobial properties, had negative impacts on both species in our model system. However, H2O2 production by S. gordonii enhanced A. actinomycetemcomitans tolerance to tetracycline, suggesting a protective role under antibiotic pressure. Finally, S. gordonii significantly inhibited the bacterium Serratia marcescens from invading in vitro biofilms, but this inhibition was lost during co-culture with A. actinomycetemcomitans and in a murine abscess model, where S. gordonii actually promoted S. marcescens invasion. These data indicate that microbial interactions can impact fitness of a bacterial community upon exposure to stresses, but these impacts are highly environment dependent.IMPORTANCEMicrobial interactions are critical modulators of the emergence of microbial communities and their functions. However, how these interactions impact the fitness of microbes in established communities upon exposure to environmental stresses is poorly understood. Here, we utilized a two-species community consisting of Aggregatibacter actinomycetemcomitans and Streptococcus gordonii to examine the impact of synergistic and antagonistic interactions on microbial resilience to environmental fluctuations and susceptibility to microbial invasion. We focused on the S. gordonii-produced extracellular molecules, L-lactate and H2O2, which have been shown to mediate interactions between these two microbes. We discovered that seemingly beneficial functions, such as A. actinomycetemcomitans cross-feeding on S. gordonii-produced L-Lactate, can paradoxically exacerbate vulnerabilities, such as susceptibility to antibiotics. Moreover, our data highlight the context-dependent nature of microbial interactions, emphasizing that a seemingly potent antimicrobial, such as H2O2, can have both synergistic and antagonistic effects on a microbial community dependent on the environment.

Summary: Despite extensive global research into genetic predisposition for severe COVID-19, knowledge on the role of rare host genetic variants and their relation to other risk factors remains limited. Here, 52 genes with prior etiological evidence were sequenced in 1,772 severe COVID-19 cases and 5,347 population-based controls from Spain/Italy. Rare deleterious TLR7 variants were present in 2.4% of young (

Abstract The preservation of genetic variation is fundamental in biodiversity conservation, yet its importance for population viability remains contentious. Mixed‐source reintroductions, where individuals are translocated into a single vacant habitat from multiple genetically divergent and often depauperate populations, provide an opportunity to evaluate how genetic variation and hybridization influence individual and relative population fitness. Population genetic theory predicts that individuals with higher genetic variation and hybrids among populations should have higher fitness. We tested these two hypotheses by analyzing individual and population‐scale data for westslope cutthroat trout (Oncorhynchus clarkii lewisi) in four mixed‐source reintroductions. We observed more hybrid and fewer nonhybrid offspring than expected across four independent mixed‐source reintroductions. We also found clear evidence that heterozygosity influenced individual reproductive and relative population fitness. Overall, we found a strong, positive relationship between genetic variation, hybridization, and transplant fitness, emphasizing the importance of genetic variation and population mixing in conservation.

BackgroundTrust is a foundation of the therapeutic relationship and is associated with important patient outcomes. Building trust between parents of children with medical complexity (CMC) and physicians during inpatient care is complicated by lack of relational continuity, cumulative (sometimes negative) parent experiences and the need to adjust roles and expectations to accommodate parental expertise. This study's objective was to describe how parents of CMC conceptualize trust with physicians within the pediatric inpatient setting and to provide recommendations for building trust in these relationships.MethodsInterviews with 16 parents of CMC were completed and analyzed using interpretive description methodology.ResultsThe research team identified one overarching meta theme regarding factors that influence trust development: situational awareness is needed to inform personalized care of children and families. There were also six major themes: (1) ensuring that the focus is on the child and family, (2) respecting both parent and physician expertise, (3) collaborating effectively, (4) maintaining a flow of communication, (5) acknowledging the impact of personal attributes, and (6) recognizing issues related to the healthcare system.DiscussionMany elements that facilitated trust development were also components of patient- and family-centered care. Parents in this study approached trust with inpatient physicians as something that needs to be earned and reciprocated. To gain the trust of parents of CMC, inpatient physicians should personalize medical care to address the needs of each child and should explore the perceptions, expertise, and previous experiences of their parents.

Abstract Decline in cognitive function is the most feared aspect of ageing. Poorer midlife cognitive function is associated with increased dementia and stroke risk. The mechanisms underlying variation in cognitive function are uncertain. Here, we assessed associations between 1160 proteins’ plasma levels and two measures of cognitive function, the digit symbol substitution test (DSST) and the Montreal Cognitive Assessment in 1198 PURE-MIND participants. We identified five DSST performance-associated proteins (NCAN, BCAN, CA14, MOG, CDCP1), with NCAN and CDCP1 showing replicated association in an independent cohort, GS (N = 1053). MRI-assessed structural brain phenotypes partially mediated (8–19%) associations between NCAN, BCAN, and MOG, and DSST performance. Mendelian randomisation analyses suggested higher CA14 levels might cause larger hippocampal volume and increased stroke risk, whilst higher CDCP1 levels might increase intracranial aneurysm risk. Our findings highlight candidates for further study and the potential for drug repurposing to reduce the risk of stroke and cognitive decline.

Recently, laparoscopic surgeries (LPSs) are becoming increasingly prevalent. Incorporating these methods into clinical practice necessitates an in-depth understanding of the surgical techniques and multifaceted instrumentation that are specific to a slightly invasive operation. Within this systematic review, attention is directed to the technique for pneumoperitoneum creation, like the open and closed technique, to select optimal procedures along with appropriate utilization, with prominence on complication avoidance. This systematic literature review (SLR) examined significant findings from 2018 to 2023. This SLR complies with the quality standards suggested by the PRISMA document. Web of Science, SCOPUS, ProQuest, ScienceDirect, and SpringerLink are the resources. The exclusion criteria included case reports, abstracts, and letters, as well as inclusion criteria included randomized, quasi-randomized, non-randomized, and cohort investigations on human patients, provided that they liked access safety methods and presented values. According to the findings, it can be concluded that the LP inspection of the abdominal cavity (AC), which necessitates cannula implantation, is safe and effective. Closed (Veress needle technique) and open (Hasson technique) pneumoperitoneum induction techniques are frequently utilized. Most laparoscopy (LP) surgeons prefer closed LP utilizing a Veress needle and the first trocar’s masked insertion. Critics of the Veress needle, however, assert that this technique increases the risk of vascular injury. According to proponents of the open Hasson technique, vascular injury could be entirely avoided. The use of an open approach utilizing a blunt-tipped trocar for the formation of pneumoperitoneum may provide a potentially safer alternative during LPSs, depending on safety considerations.

Abstract Infection with SARS-CoV-2 is associated with an increased risk of arterial and venous thrombotic events, but the implications of vaccination for this increased risk are uncertain. With the approval of NHS England, we quantified associations between COVID-19 diagnosis and cardiovascular diseases in different vaccination and variant eras using linked electronic health records for ~40% of the English population. We defined a ‘pre-vaccination’ cohort (18,210,937 people) in the wild-type/Alpha variant eras (January 2020-June 2021), and ‘vaccinated’ and ‘unvaccinated’ cohorts (13,572,399 and 3,161,485 people respectively) in the Delta variant era (June-December 2021). We showed that the incidence of each arterial thrombotic, venous thrombotic and other cardiovascular outcomes was substantially elevated during weeks 1-4 after COVID-19, compared with before or without COVID-19, but less markedly elevated in time periods beyond week 4. Hazard ratios were higher after hospitalised than non-hospitalised COVID-19 and higher in the pre-vaccination and unvaccinated cohorts than the vaccinated cohort. COVID-19 vaccination reduces the risk of cardiovascular events after COVID-19 infection. People who had COVID-19 before or without being vaccinated are at higher risk of cardiovascular events for at least two years.

Abstract Alzheimer’s disease (AD) risk is increased in carriers of the apolipoprotein E (APOE) ε4 allele and decreased in ε2 allele carriers compared with the ε3ε3 genotype. The aim of this study was to determine whether: the APOE genotype affects brain grey (GM) or white matter (WM) structure; and if differences exist, the age when they become apparent and whether there are differential effects by sex. We used cross-sectional magnetic resonance imaging data from ~43,000 (28,494 after pre-processing) white British cognitively healthy participants (7,446 APOE ε4 carriers) aged 45–80 years from the UK Biobank cohort and investigated image-derived phenotypes (IDPs). We observed no statistically significant effects of APOE genotype on GM structure volumes or median T2* in subcortical structures, a measure related to iron content. The volume of white matter hyperintensities differed significantly between APOE genotype groups with higher volumes in APOE ε4ε4 (effect size 0.14 standard deviations [SD]) and ε3ε4 carriers (effect size 0.04 SD) but no differences in ε2 carriers compared with ε3ε3 carriers. WM integrity measures in the dorsal (mean diffusivity [MD]) and ventral cingulum (MD and intracellular volume fraction), posterior thalamic radiation (MD and isotropic volume fraction) and sagittal stratum (MD) indicated lower integrity in APOE ε4ε4 carriers (effect sizes around 0.2–0.3 SD) and ε3ε4 (effect sizes around 0.05 SD) carriers but no differences in ε2 carriers compared with the APOE ε3ε3 genotype. Effects did not differ between men and women. APOE ε4 homozygotes had lower WM integrity specifically at older ages with a steeper decline of WM integrity from the age of 60 that corresponds to around 5 years greater “brain age”. APOE genotype affects various white matters measures, which might be indicative of preclinical AD processes. This hypothesis can be assessed in future when clinical outcomes become available.

Clinical trials for Duchenne muscular dystrophy (DMD) are assessing the therapeutic efficacy of systemically delivered adeno-associated virus (AAV) carrying a modified DMD transgene. High vector doses (>1E14 vg/kg) are needed to globally transduce skeletal muscles; however, such doses trigger immune-related adverse events. Mitigating these immune responses is crucial for widespread application of AAV-based therapies. We used single-cell RNA sequencing and T cell receptor (TCR) sequencing on peripheral blood mononuclear cells from five participants prior to, and after, dosing. One subject in the high-dose cohort experienced thrombotic microangiopathy (TMA). Few changes in cell frequencies occurred after treatment; however, differential gene expression demonstrated induction of interferon response genes in most T cell types. T cell clonotype and clumping analysis showed the expansion or appearance of groups of related TCR sequences in the post-treatment samples. Three of these expanded clumps could be assigned to prior human herpesvirus infections, two of which were present in the participant that exhibited TMA. These data provide insight on the mechanistic basis of human immune-AAV interactions and lay a foundation for improved understanding of why TMA arises in some patients and not others.

The move toward early detection and treatment of cancer presents challenges for value assessment using traditional endpoints. Current cancer management rarely considers the full economic and societal benefits of therapies. Our study used a modified Delphi process to develop principles for defining and assessing value of cancer therapies that aligns with the current trajectory of oncology research and reflects broader notions of value. 24 experts participated in consensus-building activities across 5 months (16 took part in structured interactions, including a survey, plenary sessions, interviews, and off-line discussions, while 8 participated in interviews). Discussion focused on: 1) which oncology-relevant endpoints should be used for assessing treatments for early-stage cancer and access decisions for early-stage treatments, and 2) the importance of additional value components and how these can be integrated in value assessments. The expert group reached consensus on 4 principles in relation to the first area (consider oncology-relevant endpoints other than overall survival; build evidence for endpoints that provide earlier indication of efficacy; develop evidence for the next generation of predictive measures; use managed entry agreements supported by ongoing evidence collection to address decision-maker evidence needs) and 3 principles in relation to the second (routinely use patient reported outcomes in value assessments; assess broad economic impact of new medicines; consider other value aspects of relevance to patients and society).

Copy number variation in large gene families is well characterized for plant resistance genes, but similar studies are rare in animals. The zebrafish (Danio rerio) has hundreds of NLR immune genes, making this species ideal for studying this phenomenon. By sequencing 93 zebrafish from multiple wild and laboratory populations, we identified a total of 1513 NLRs, many more than the previously known 400. Approximately half of those are present in all wild populations, but only 4% were found in 80% or more of the individual fish. Wild fish have up to two times as many NLRs per individual and up to four times as many NLRs per population than laboratory strains. In contrast to the massive variability of gene copies, nucleotide diversity in zebrafish NLR genes is very low: around half of the copies are monomorphic and the remaining ones have very few polymorphisms, likely a signature of purifying selection.

Background: COVID-19 vaccination rates are lower in the Southern United States compared to other regions. This study investigated COVID-19 vaccination hesitancy in Mississippi (MS) to identify preferences that may boost MS vaccination strategies in areas with poor vaccine uptake. Methods: Qualitative interviews were completed between April 2021 and January 2022 with staff and patients at four Federally Qualified Health Centers in MS. Interviews included the following COVID-19 vaccine topics: willingness to be vaccinated, barriers and facilitators, and methods for providing vaccine information. Data were organized with NVivo software and analyzed using reflexive thematic analysis. Results: Fifteen clinic staff and 49 patients were interviewed. Barriers to vaccine uptake included a lack of knowledge and understanding of how the vaccine worked, distrust of the government, fear of side effects, and social pressure to stay unvaccinated. Vaccination facilitators included its widespread accessibility, a desire to protect themselves and vulnerable populations, and a previous unpleasant COVID-19 illness experience. Participants stated that vaccine information should be provided by health organizations and familiar, respected community members. Conclusions: Results identified barriers to vaccination, such as mistrust of the government and healthcare system, and facilitators like vaccination advocacy originating from congregations and religious leaders. These findings can inform future COVID-19 vaccination efforts to increase overall immunization rates in MS. Future research in other locations could further assess commonalities and differences in the barriers and facilitators to vaccination.

ABSTRACTThe ESKAPEE pathogen Pseudomonas aeruginosa is a common cause of chronic wound and cystic fibrosis lung infections, as well as acute burn and nosocomial infections. Many of these infections are recalcitrant to conventional antibiotic therapies due to both traditional antibiotic resistance mechanisms and antimicrobial tolerance. Recent successes with bacteriophage (phage) therapy to treat chronic human P. aeruginosa infections have led to a renewed interest in isolating and characterizing new P. aeruginosa phages. Here, we isolated and characterized a new lytic phage (termed PIP, pili-infecting phage) capable of infecting P. aeruginosa PA14. PIP is a tailed phage with an icosahedral head and flexible tail containing a genome that is 57,462 bp in length. Phylogenetic analysis reveals that PIP belongs to the subfamily Queuovirinae and genus Nipunavirus but is highly divergent in gene content from known Nipunaviruses. By isolating and characterizing a P. aeruginosa strain that spontaneously evolved resistance to PIP, we show that the receptor for PIP is Type IV pili. In summary, we isolated a new P. aeruginosa phage species with a unique genome, thus increasing the diversity of phages known to infect this important human pathogen.IMPORTANCEThe opportunistic pathogen Pseudomonas aeruginosa causes both acute and chronic human infections. These infections are notoriously difficult to treat due to both antibiotic resistance and antibiotic tolerance. The increasing frequency of antibiotic failure in P. aeruginosa infections has led scientists to explore other treatment options, including bacteriophage (phage) therapy. To this end, there has been a significant effort to identify new Pseudomonas phages. Here, we isolated and characterized a bacteriophage (termed PIP, pili-infecting phage) that infects P. aeruginosa PA14. Examination of the PIP genome revealed that this phage represents a new species in the subclass Queuovirinae. The isolation and characterization of spontaneous PA14 mutants that are resistant to PIP infection revealed Type IV pili as the PIP receptor. Ultimately, this study characterizes a new species of Pseudomonas phage, thus enhancing the known diversity of phages that infect this important pathogen.

# Background After Anterior Cruciate Ligament Reconstruction (ACLR) athletes face the challenge of regaining their previous competitive level while avoiding re-injury and early knee joint cartilage degeneration. Quadriceps and hamstrings strength reductions and neuromuscular alterations potentially related to risk of re-injury are present after ACLR and relate to deficits in muscle activation. # Design Cross-sectional laboratory study # Purpose To examine quadriceps and hamstrings muscle activation during repeated hops in healthy pivoting-sport athletes and those who had undergone ACLR (bone-tendon-bone and semitendinosus graft) who had met functional criteria allowing return to training. # Methods Surface electromyography (SEMG) was recorded from vastus medialis and lateralis and medial and lateral hamstrings bilaterally during 30 seconds' repeated hopping in male athletes on average eight months after ACLR surgery (5-12 months). All patients underwent hamstring (HS) (n=24) or bone-tendon-bone (BTB) reconstruction (n=20) and were compared to healthy controls (n=31). The SEMG signals were normalized to those obtained during maximal voluntary isometric contraction. # Results A significant time shift in peak muscle activation (earlier) was seen for: vastus medialis and vastus lateralis activation in the control group, in the BTB group's healthy (but not injured) leg and both legs of the HS group. A significant time shift in peak muscle activation was seen for lateral hamstrings (earlier) in all but the BTB group's injured leg and the medial hamstrings in the control group only. Lower peak activation levels of the vastus lateralis (p\

Abstract Background Role models have been identified as a potential means to tackle the persisting low levels of physical activity among young girls. The aim of this research was to explore the involvement of community- and peer role models within the CHARMING (CHoosing Active Role Models to INspire Girls) intervention, an intervention which aims to increase and sustain physical activity among 9–10-year-old girls. The research questions were, is it feasible and acceptable to recruit role models? and what are the perceived barriers and facilitators to the inclusion of peer role models within the intervention? Methods A mixed methods process evaluation was embedded within a larger feasibility study, involving three secondary schools and four adjoining primary schools in South Wales, United Kingdom. One-to-one interviews were conducted with teachers (N = 10) across the seven schools and community role models (N = 10). Focus groups were conducted with 18 peer role models (older girls from adjoining secondary schools) and 18 girls aged 9–10-years who had participated in the intervention. Primary school teachers kept observation logs of each intervention session. A researcher completed observation logs of two random sessions per school. Qualitative data were analysed using thematic analysis with a combined deductive and inductive coding approach. Observation data were analysed using descriptive statistics. Data were triangulated and comparative analyses conducted across schools. Results Twenty-three peer role models (aged 12–16-years) and 16 community role models participated in intervention delivery. Overall, the inclusion of both types of role models was shown as acceptable and feasible within the CHARMING intervention. Observation data highlighted key areas (i.e., intervention components delivered inconsistently) for further qualitative exploration. Six themes were identified during analyses; reach and access, communication, logistics, existing systems, interpersonal relationships, and perceived impacts. Themes were intertwined across the barriers and facilitators of recruitment and implementation. Areas for future improvement were highlighted. Conclusions Findings can be used to optimise the CHARMING intervention and inform wider interventions or policies employing several role models across settings to promote physical activity among children.

Melissa J Kiely, Ashleigh Poulsen, Simon D Muschamp, Chloe Sallis, Mark S Whiteley Research Department, the Whiteley Clinic, Guildford, Surrey, UKCorrespondence: Mark S Whiteley, The Whiteley Clinic, 1 Stirling House, Stirling Road, Guildford, Surrey, GU2 7RF, UK, Email mark@thewhiteleyclinic.co.ukBackground: Cellulite affects 80– 90% of post-pubertal females and, although cosmetic, has proven adverse psychological effects. A new compression garment with patented “vari-pad” technology has been developed, aiming to stimulate lymphatic return from the buttocks and thighs. The primary aim of this small pilot study was to assess participant satisfaction after using this garment in the short term and secondarily, to look for any objective improvements.Methods: Eighteen female participants (mean 47.5y, range 26– 60) completed the study. There were 2 weeks of “stabilisation”, 4 weeks of wearing the garment, and 2 weeks of further assessment. With the initial assessment, there were 9 visits for each participant. At each visit, weight, participant satisfaction (0– 10), 3D images (Canfield, USA) and transcutaneous water content (LymphScanner, Delfin Technologies, Finland) and comfort (0– 10) were recorded.Results: There was no significant weight change in any of the participants during the trial period. After 4 weeks of wearing the garment, there was a significant improvement in participant satisfaction of their cellulite (n = 15, p = 0.0036) with a mean improvement of 18.5%. 3D image analysis of dimple volume showed no significant change in buttock cellulite, a significant decrease in left thigh cellulite (n = 17, p = 0.0123), but not on the right (n = 14, p = 0.6890). There was a significant decrease of localised water (n = 36, p = 0.0041). Comfort was 86% (range 40– 100%) and there were no adverse reactions to the garment.Conclusion: The vari-padded compression garment produced a significant improvement in participant satisfaction after just 4 weeks of treatment. Objective measurements failed to show a significant difference in cellulite on the buttocks but showed a significant improvement in the thighs by analysis of both dimple volumes and reduced transcutaneous water measurements. Further study is suggested to demonstrate if longer timeframes for use of the apparel will lead to further significant improvement in the appearance of cellulite.NCT Number: NCT05748678.Keywords: cellulite, compression, garment, hosiery

This pilot study investigated the feasibility of using magnetic resonance elastography (MRE) for the non-invasive detection and quantification of liver fibrosis in the Oncopig cancer model. Seven 8-week-old Oncopigs underwent alcoholic liver fibrosis induction and serial MRE imaging and liver biopsy at 1, 2, and 3 months post procedure. MRE was utilized to quantify liver stiffness, and liver fibrosis was histologically graded using the METAVIR system. The primary outcome measure was the capability to detect and quantify liver fibrosis using MRE with radiologic–pathologic correlation. Liver fibrosis induction, MRE imaging, and liver biopsy were successfully performed. MRE liver fibrosis was evident in 57% (4/7), 50% (3/6), and 40% (2/5) animal subjects 1, 2, and 3 months after fibrosis induction, with mean liver stiffness of 2.94, 3.25, and 2.91 kPa, respectively. Histological liver fibrosis was noted in 71% (5/7), 100% (5/5), and 100% (5/5) of animal subjects with available tissue samples. There was no significant statistical correlation between the MRE-measured liver stiffness and the METAVIR fibrosis scores. In conclusion, quantifiable liver fibrosis may be induced in the Oncopig. MRE has potential utility in non-invasively detecting liver stiffness in this large-animal preclinical model, though tissue biopsy was more sensitive in demonstrating disease.

# Background Jump testing performance and limb symmetry measures are important metrics for clinicians to monitor during rehabilitation after Anterior Cruciate Ligament (ACL) reconstruction, however they require hardware and software which is not commonly available in clinical practice. Video-based solutions may present a feasible alternative, but their veracity in classifying patients using limb-symmetry of 90% has not been established, nor have the clinimetric values for the performance measures been reported in this population. # Purpose To describe the diagnostic accuracy (pass/fail using 90% LSI) and clinimetrics of an iPad-based app ("MyJump") compared to reference force plate analyses for limb symmetry, jump/hop height, contact time, flight time, and reactive strength index # Study design Prospective cohort, diagnostic accuracy # Methods Fifty-one consecutive patients recovering from ACL reconstruction undertaking routine independent clinical evaluation of their hop and jump performance were concurrently and independently examined using force plates and the MyJump app. Diagnostic accuracy of MyJump was compared to reference force plate analyses using a criterion of 90% limb symmetry. # Results Diagnostic accuracy of the MyJump app was very good: positive predictive value for jump height was 0.83 and 1.0 for reactive strength index, and negative predictive value was 0.95 and 1.0 for the same metrics, respectively. Of the 131 classifications made using the MyJump app, there were five false positives and three false negatives -- all of these were in classification of jump height with no misclassifications of RSI. Irrespective of jump type, the MyJump app displayed excellent reliability (ICC\>0.95) for both height and reactive strength index. Minimum detectable changes were approximately 1cm for height, 0.1 for reactive strength index, 0.02s for contact time, and 0.3s for flight time. # Conclusion Where force plates are unavailable, the MyJump app is a valid and reliable substitute for criteria assessment of jump/hop height and reactive strength index in those recovering from ACL surgery using a 90% limb symmetry threshold. The minimum detectable changes vary by metric but are likely sufficiently accurate to detect clinical changes. # Level Of Evidence Level 3

Abstract Background Drugs targeting the spindle assembly checkpoint (SAC), such as inhibitors of Aurora kinase B (AURKB) and dual specific protein kinase TTK, are in different stages of clinical development. However, cell response to SAC abrogation is poorly understood and there are no markers for patient selection. Methods A panel of 53 tumor cell lines of different origins was used. The effects of drugs were analyzed by MTT and flow cytometry. Copy number status was determined by FISH and Q-PCR; mRNA expression by nCounter and RT-Q-PCR and protein expression by Western blotting. CRISPR-Cas9 technology was used for gene knock-out (KO) and a doxycycline-inducible pTRIPZ vector for ectopic expression. Finally, in vivo experiments were performed by implanting cultured cells or fragments of tumors into immunodeficient mice. Results Tumor cells and patient-derived xenografts (PDXs) sensitive to AURKB and TTK inhibitors consistently showed high expression levels of BH3-interacting domain death agonist (BID), while cell lines and PDXs with low BID were uniformly resistant. Gene silencing rendered BID-overexpressing cells insensitive to SAC abrogation while ectopic BID expression in BID-low cells significantly increased sensitivity. SAC abrogation induced activation of CASP-2, leading to cleavage of CASP-3 and extensive cell death only in presence of high levels of BID. Finally, a prevalence study revealed high BID mRNA in 6% of human solid tumors. Conclusions The fate of tumor cells after SAC abrogation is driven by an AURKB/ CASP-2 signaling mechanism, regulated by BID levels. Our results pave the way to clinically explore SAC-targeting drugs in tumors with high BID expression.

Objective: To develop an electronic surveillance system that provides prompt in-depth situational infectious disease risk and linkage analysis for inpatients in a tertiary hospital. Patients and Methods: All patients admitted to Singapore General Hospital (SGH), a 1900-bedded tertiary care hospital, are included in routine surveillance. The 3-Dimensional Disease Outbreak Surveillance System (3D-DOSS) was developed to spatiotemporally represent inpatient surveillance data on a “digital twin” of SGH and evaluated for performance in surveillance, contact tracing, and outbreak investigations. This study was conducted over a 12 month period (October 1, 2020 to September 30, 2021). Results: The 3D-DOSS surveillance module identified an influenza cluster of 10 inpatients in November 2018, mapping retrospective data between September 2018 and December 2018. Seventy-six clusters of 2 or more linked patients with health care–associated Klebsiella pneumoniae carbapenemase–type carbapenemase-producing Enterobacteriaceae were detected in SGH in 2 years (2018 and 2019). The 3D-DOSS contact tracing module promptly identified 44 primary and 162 secondary inpatient contacts, after exposure to a health care worker with coronavirus disease 2019 in April 2021. For outbreak mapping, 24 patients with OXA-48 were mapped on October 22, 2020, using 3D-DOSS to determine their spatiotemporal distribution. Conclusion: The integration of health care data and representation on a virtual hospital digital twin is a useful tool in an outbreak alert and response framework. Infectious disease surveillance systems, which are syndrome-based, that can access real-time data, and can incorporate movement networks, can potentially enhance health care–associated infection prevention and preparedness for disease X.

Many current gene therapy targets use recombinant adeno-associated virus (AAV). The majority of delivered AAV therapeutics persist as episomes, separate from host DNA, yet some viral DNA can integrate into host DNA in different proportions and at genomic locations. The potential for viral integration leading to oncogenic transformation has led regulatory agencies to require investigation into AAV integration events following gene therapy in preclinical species. In the present study, tissues were collected from cynomolgus monkeys and mice 6 and 8 weeks, respectively, following administration of an AAV vector delivering transgene cargo. We compared three different next-generation sequencing approaches (shearing extension primer tag selection ligation-mediated PCR, targeted enrichment sequencing [TES], and whole-genome sequencing) to contrast the specificity, scope, and frequency of integration detected by each method. All three methods detected dose-dependent insertions with a limited number of hotspots and expanded clones. While the functional outcome was similar for all three methods, TES was the most cost-effective and comprehensive method of detecting viral integration. Our findings aim to inform the direction of molecular efforts to ensure a thorough hazard assessment of AAV viral integration in our preclinical gene therapy studies.

Introduction Endovenous therapy is the first choice management for symptomatic varicose veins in NICE guidelines, with 56–70 000 procedures performed annually in the UK. Venous thromboembolism (VTE), including deep vein thrombosis (DVT) and pulmonary embolism (PE), is a known complication of endovenous therapy, occurring at a rate of up to 3.4%. Despite 73% of UK practitioners administering pharmacological thromboprophylaxis to reduce VTE, no high-quality evidence supporting this practice exists. Pharmacological thromboprophylaxis may have clinical and cost benefit in preventing VTE; however, further evidence is needed. This study aims to establish whether when endovenous therapy is undertaken: a single dose or course of pharmacological thromboprophylaxis alters the risk of VTE; pharmacological thromboprophylaxis is associated with an increased rate of bleeding events; pharmacological prophylaxis is cost effective.Methods and analysis A multi-centre, assessor-blind, randomised controlled trial (RCT) will recruit 6660 participants from 40 NHS and private sites across the UK. Participants will be randomised to intervention (single dose or extended course of pharmacological thromboprophylaxis plus compression) or control (compression alone). Participants will undergo a lower limb venous duplex ultrasound scan at 21–28 days post-procedure to identify asymptomatic DVT. The duplex scan will be conducted locally by blinded assessors. Participants will be contacted remotely for follow-up at 7 days and 90 days post-procedure. The primary outcome is imaging-confirmed lower limb DVT with or without symptoms or PE with symptoms within 90 days of treatment. The main analysis will be according to the intention-to-treat principle and will compare the rates of VTE at 90 days, using a repeated measures analysis of variance, adjusting for any pre-specified strongly prognostic baseline covariates using a mixed effects logistic regression.Ethics and dissemination Ethical approval was granted by Brent Research Ethics Committee (22/LO/0261). Results will be disseminated in a peer-reviewed journal and presented at national and international conferences.Trial registration number ISRCTN18501431.

Summary: Long interspersed nuclear element-1 (L1 or LINE-1) is a highly abundant mobile genetic element in both humans and mice, comprising almost 20% of each genome. L1s are silenced by several mechanisms, as their uncontrolled expression has the potential to induce genomic instability. However, L1s are paradoxically expressed at high levels in differentiating neural progenitor cells. Using in vitro and in vivo techniques to modulate L1 expression, we report that L1s play a critical role in both human and mouse brain development by regulating the rate of neural differentiation in a reverse-transcription-independent manner.

Background: Visit-to-visit variability in single biological measurements has been associated with cognitive decline and an elevated risk of cardiovascular diseases (CVD). However, the effect of visit-to-visit variability in multiple biological measures is underexplored. We investigated the effect of visit-to-visit variability in blood pressure (BP), heart rate (HR), weight, fasting plasma glucose, cholesterol, and triglycerides on cognitive performance and CVD. Methods: Data on BP, HR, weight, glucose, cholesterol, and triglycerides from study visits in the Outcome Reduction with Initial Glargine Intervention (ORIGIN) trial were used to estimate the association between visit-to-visit variability, cognitive performance (Mini Mental State Examination (MMSE) score) and CVD (non-fatal stroke, non-fatal myocardial infarction, or cardiovascular death). Visit-to-visit variation for each measurement was estimated by calculating each individuals visit-to-visit standard deviation for that measurement. Participants whose standard deviation was in the highest quarter were classified as having high variation. Participants were grouped into those having 0, 1, 2, 3, or ≥ 4 high variation measurements. Regression and survival models were used to estimate the association between biological measures with MMSE and CVD with adjustment for confounders and mean measurement value. Results: After adjustment for covariates, higher visit-to-visit variability in BP, HR, weight, and FPG were associated with poorer MMSE and a higher risk of CVD. Effect sizes did not vary greatly by measurement. The effects of high visit-to-visit variability were additive; compared to participants who had no measurements with high visit-to-visit variability, those who had high visit-to-visit variability in ≥4 measurements had poorer MMSE scores (-0.63 (95 % CI -0.96 to -0·31). Participants with ≥4 measurements with high visit-to-visit variability compared to participants with none had higher risk of CVD (hazard ratio 2.46 (95 % CI 1.63 to 3.70). Conclusion: Visit-to-visit variability in several measurements were associated with cumulatively poorer cognitive performance and a greater risk of CVD.

ABSTRACT Bacteria rely on extracellular chemical cues to sense their environment, interact with one another, and shape their chemical landscape. Understanding this biochemistry can help us elucidate the mechanisms by which bacterial interactions impact community function. By applying mass spectrometry to mono- and co-cultures of the oral pathogen Aggregatibacter actinomycetemcomitans and the oral commensal Streptococcus gordonii, we identified hundreds of extracellular small molecules produced by these bacteria. We discovered that A. actinomycetemcomitans secretes millimolar amounts of glutathione and identified a five-gene operon (called gttABCDE) that is important for maximum secretion. The metabolomics data set generated in this study provides a valuable data set for unraveling the importance of individual molecules for mediating polymicrobial interactions in the oral cavity. IMPORTANCE Microbes produce a large array of extracellular molecules, which serve as signals and cues to promote polymicrobial interactions and alter the function of microbial communities. This has been particularly well studied in the human oral microbiome, where key metabolites have been shown to impact both health and disease. Here, we used an untargeted mass spectrometry approach to comprehensively assess the extracellular metabolome of the pathogen Aggregatibacter actinomycetemcomitans and the commensal Streptococcus gordonii during mono- and co-culture. We generated and made publicly available a metabolomic data set that includes hundreds of potential metabolites and leveraged this data set to identify an operon important for glutathione secretion in A. actinomycetemcomitans.

A 42-year-old man presented with very large lower limb varicose veins bilaterally and an 8-month history of fainting when standing up from a squatting position. He had been investigated for syncope by a cardiologist with no abnormality found on electrocardiogram, echocardiogram, 24-h electrocardiogram or 48-h blood pressure monitoring. Venous duplex ultrasonography showed bilaterally great saphenous vein reflux, with each vein having a diameter of 23.5 and 24.0 mm at the groin, and a similar dilation along the whole vein to ankle. These veins were successfully ablated with endovenous laser ablation, immediately following which the syncope disappeared and has not returned. We calculate that before treatment, the patient had approximately 500 ml of blood refluxing passively with gravity from his deep venous system on standing into his incompetent great saphenous veins and associated varicosities, which would account for his postural syncope.

ABSTRACT Hard-wired within the genome of a pathogen is the information regarding factors responsible for its pathogenicity. Advances in functional genomics, particularly gene expression analysis, have made possible genome-wide interrogation of a pathogen to decipher pathogenicity-associated genes. Standard protocols assess differential expression of genes during pathogenesis; however, often a conservative approach is taken, which requires expression fold change above an arbitrarily defined threshold and a statistical significance test to infer a gene as differentially expressing. This renders a high-confidence set of differentially expressed genes in pathogenesis, however, at the cost of numerous false negatives. To circumvent this problem and comprehensively catalog pathogenicity-associated genes, we have developed a novel pipeline that uses standard protocol in combination with gene co-expression network of a pathogen constructed using publicly available RNA-Seq data sets. We assessed the efficacy of this pipeline on Pseudomonas aeruginosa PAO1, a model bacterial pathogen, highlighting the power of our network-based approach in discovering novel genes or pathways associated with the pathogenesis, or antibiotic resistance of this strain. Complementing standard protocol with a gene network-based method thus elevated the ability to identify pathogenicity-associated genes in P. aeruginosa PAO1.IMPORTANCEWe present here a new systems-level approach to decipher genetic factors and biological pathways associated with virulence and/or antibiotic treatment of bacterial pathogens. The power of this approach was demonstrated by application to a well-studied pathogen Pseudomonas aeruginosa PAO1. Our gene co-expression network-based approach unraveled known and unknown genes and their networks associated with pathogenicity in P. aeruginosa PAO1. The systems-level investigation of P. aeruginosa PAO1 helped identify putative pathogenicity and resistance-associated genetic factors that could not otherwise be detected by conventional approaches of differential gene expression analysis. The network-based analysis uncovered modules that harbor genes not previously reported by several original studies on P. aeruginosa virulence and resistance. These could potentially act as molecular determinants of P. aeruginosa PAO1 pathogenicity and responses to antibiotics.

Abstract Introduction The diagnosis of cardiac contusion, caused by blunt chest trauma, remains a challenge due to the non-specific symptoms it causes and the lack of ideal tests to diagnose myocardial damage. A cardiac contusion can be life-threatening if not diagnosed and treated promptly. Several diagnostic tests have been used to evaluate the risk of cardiac complications, but the challenge of identifying patients with contusions nevertheless remains. Aim of the study To evaluate the accuracy of diagnostic tests for detecting blunt cardiac injury (BCI) and its complications, in patients with severe chest injuries, who are assessed in an emergency department or by any front-line emergency physician. Methods A targeted search strategy was performed using Ovid MEDLINE and Embase databases from 1993 up to October 2022. Data on at least one of the following diagnostic tests: electrocardiogram (ECG), serum creatinine phosphokinase-MB level (CPK-MB), echocardiography (Echo), Cardiac troponin I (cTnI) or Cardiac troponin T (cTnT). Diagnostic tests for cardiac contusion were evaluated for their accuracy in meta-analysis. Heterogeneity was assessed using the I 2 and the QUADAS-2 tool was used to assess bias of the studies. Results This systematic review yielded 51 studies (n = 5,359). The weighted mean incidence of myocardial injuries after sustaining a blunt force trauma stood at 18.3% of cases. Overall weighted mean mortality among patients with blunt cardiac injury was 7.6% (1.4–36.4%). Initial ECG, cTnI, cTnT and transthoracic echocardiography TTE all showed high specificity (> 80%), but lower sensitivity (

Abstract Background Sex determination is the process whereby the bipotential embryonic gonads become committed to differentiate into testes or ovaries. In genetic sex determination (GSD), the sex determining trigger is encoded by a gene on the sex chromosomes, which activates a network of downstream genes; in mammals these include SOX9, AMH and DMRT1 in the male pathway, and FOXL2 in the female pathway. Although mammalian and avian GSD systems have been well studied, few data are available for reptilian GSD systems. Results We conducted an unbiased transcriptome-wide analysis of gonad development throughout differentiation in central bearded dragon (Pogona vitticeps) embryos with GSD. We found that sex differentiation of transcriptomic profiles occurs at a very early stage, before the gonad consolidates as a body distinct from the gonad-kidney complex. The male pathway genes dmrt1 and amh and the female pathway gene foxl2 play a key role in early sex differentiation in P. vitticeps, but the central player of the mammalian male trajectory, sox9, is not differentially expressed in P. vitticeps at the bipotential stage. The most striking difference from GSD systems of other amniotes is the high expression of the male pathway genes amh and sox9 in female gonads during development. We propose that a default male trajectory progresses if not repressed by a W-linked dominant gene that tips the balance of gene expression towards the female trajectory. Further, weighted gene expression correlation network analysis revealed novel candidates for male and female sex differentiation. Conclusion Our data reveal that interpretation of putative mechanisms of GSD in reptiles cannot solely depend on lessons drawn from mammals.

Abstract Background Computational text phenotyping is the practice of identifying patients with certain disorders and traits from clinical notes. Rare diseases are challenging to be identified due to few cases available for machine learning and the need for data annotation from domain experts. Methods We propose a method using ontologies and weak supervision, with recent pre-trained contextual representations from Bi-directional Transformers (e.g. BERT). The ontology-driven framework includes two steps: (i) Text-to-UMLS, extracting phenotypes by contextually linking mentions to concepts in Unified Medical Language System (UMLS), with a Named Entity Recognition and Linking (NER+L) tool, SemEHR, and weak supervision with customised rules and contextual mention representation; (ii) UMLS-to-ORDO, matching UMLS concepts to rare diseases in Orphanet Rare Disease Ontology (ORDO). The weakly supervised approach is proposed to learn a phenotype confirmation model to improve Text-to-UMLS linking, without annotated data from domain experts. We evaluated the approach on three clinical datasets, MIMIC-III discharge summaries, MIMIC-III radiology reports, and NHS Tayside brain imaging reports from two institutions in the US and the UK, with annotations. Results The improvements in the precision were pronounced (by over 30% to 50% absolute score for Text-to-UMLS linking), with almost no loss of recall compared to the existing NER+L tool, SemEHR. Results on radiology reports from MIMIC-III and NHS Tayside were consistent with the discharge summaries. The overall pipeline processing clinical notes can extract rare disease cases, mostly uncaptured in structured data (manually assigned ICD codes). Conclusion The study provides empirical evidence for the task by applying a weakly supervised NLP pipeline on clinical notes. The proposed weak supervised deep learning approach requires no human annotation except for validation and testing, by leveraging ontologies, NER+L tools, and contextual representations. The study also demonstrates that Natural Language Processing (NLP) can complement traditional ICD-based approaches to better estimate rare diseases in clinical notes. We discuss the usefulness and limitations of the weak supervision approach and propose directions for future studies.

Despite its widespread use in adults, the Nordic hamstring exercise remains underexplored in athletic youth populations. Further, given the dynamic nature of growth and maturation, comparisons with elite adult populations may be inaccurate. Here we describe absolute and body mass-normalised eccentric hamstring normative values for football, athletics and multi-sport youth populations. 676 routine standardised tests were conducted, including 244 U12–U18 student-athletes (football, athletics, multi-sport) and 346 Qatar Stars League (QSL) football players using the NordBord. The average maximum values for the left and right leg from 3 repetitions were recorded. Significant increases in absolute strength were seen across chronological (e.g., 150 N±15 for U12 to 330 N±40 for U18) and skeletal (142.9 N±13.9 for skeletal age of 12 compared to 336.2 N±71.2 for skeletal age of 18) age groups. The differences in values normalised to body mass were smaller at 3.6 N/kg±0.25 for the U-13 group, but similar for the U14 to U18 groups (4.5 N/kg±0.16, 4.6 N/kg±0.11, 4.6 N/kg±0.27, 4.7 N/kg±0.14, 4.5 N/kg±0.18). Students displayed lower absolute strength than the professional football players (272.1 N compared to 297.3 N, p < 0.0001) but higher relative strength (4.7 N/kg compared to 4.2 N/kg, p < 0.0001). Comparing Nordic hamstring strength values between athletes, and between skeletal and chronological age groups can be done when values are normalised to the athlete’s body mass. For the U14s and onwards age categories, body mass normalised values are comparable to professional football players.

Abstract Background Management of adults with atrial fibrillation (AF) or atrial flutter in the emergency department (ED) includes rate reduction, cardioversion, and stroke prevention. Different approaches to these components of care may lead to variation in frequency of hospitalization and stroke prevention actions, with significant implications for patient experience, cost of care, and risk of complications. Standardization using evidence-based recommendations could reduce variation in management, preventable hospitalizations, and stroke risk. Methods We describe the rationale for our ED-based AF treatment recommendations. We also describe the development of an electronic clinical decision support system (CDSS) to deliver these recommendations to emergency physicians at the point of care. We implemented the CDSS at three pilot sites to assess feasibility and solicit user feedback. We will evaluate the impact of the CDSS on hospitalization and stroke prevention actions using a stepped-wedge cluster randomized pragmatic clinical trial across 13 community EDs in Northern California. Discussion We hypothesize that the CDSS intervention will reduce hospitalization of adults with isolated AF or atrial flutter presenting to the ED and increase anticoagulation prescription in eligible patients at the time of ED discharge and within 30 days. If our hypotheses are confirmed, the treatment protocol and CDSS could be recommended to other EDs to improve management of adults with AF or atrial flutter. Trial registration ClinicalTrials.gov NCT05009225 . Registered on 17 August 2021.

Madalitso M Mlozen,1,2 Jacqueline Van Marwijk,1 Brendan Shane Wilhelmi,1 Chris Whiteley1 1Department of Biochemistry and Microbiology, Rhodes University, Makhanda (Grahamstown), South Africa; 2Malawi Adventist University, Malamulo Campus, Department of Biomedical Sciences, Makwasa, MalawiCorrespondence: Madalitso M Mlozen, Malawi Adventist University, Malamulo campus, Department of Biomedical Sciences, P.O.Box 55, Makwasa, Tel +265 884628334, Email mlozenim@mchs.advenist.orgBackground: Regardless of the efforts to ease cases of human African trypanosomiasis (HAT), an increased number of cases get reported annually. This is because of drug resistant Trypanosoma brucei (Tb), the causative agent of the illness. This has renewed the need for creative methods to find new anti-trypanosomal drugs. The blood stream form (BSF) of the parasite depends exclusively on the glycolytic pathway for energy production while it is in the human host. Interruptions in this pathway efficiently kills the parasite. Trypanosoma brucei hexokinase (TbHK) is the first enzyme in glycolysis, and any effectors or inhibitors of TbHK would have potential as anti-trypanosomal agents.Methods: TbHK and human glucokinase (hGCK) were over-expressed with a 6 histidine-tag in E. coli BL21(DE3) cells having the pRARE2 plasmid.Results: TbHK had thermal and pH stability between 30°C and 55°C and 7.5 and 8.5, respectively, while hGCK exhibited thermal and pH stability between 30°C and 40°C and 7.0 and 8.0, respectively. Kinetically, TbHK had a Km of 39.3 μM, Vmax of 0.066 μmol.min− 1.mL− 1, kcat of 2.05 min− 1 and kcat/Km of 0.0526 min− 1.μmol− 1. hGCK exhibited a Km of 4.5 μM, Vmax of 0.032 μnmol.min− 1.mL− 1, kcat of 11.25 min− 1, and kcat/Km of 2.5 min− 1.μmol− 1. Interaction kinetic studies of silver nanoparticles (AgNPs) (0.1 μM) of average size of 6 nm with TbHK and hGCK were conducted. AgNPs selectively inhibited TbHK over hGCK. TbHK showed a non-competitive inhibition with a 50% and 28% decrease in Vmax, and kcat/km, respectively. HsGCK showed a 33% increase in affinity, 9% decrease in Vmax, and a 50% increase in enzyme efficiency.Conclusion: The observed pattern of hGCK and AgNPs falls under the uncompetitive inhibition. The observed highly selective inhibitory effects of AgNPs between TbHK and hGCK may be used in development of new anti-trypanosomal drugs.Keywords: nanoparticles, hexokinase, trypanosomiasis, nanomedicine

Background Type I interferons are cytokines involved in innate immunity against viruses. Genetic disorders of type I interferon regulation are associated with a range of autoimmune and cerebrovascular phenotypes. Carriers of pathogenic variants involved in genetic disorders of type I interferons are generally considered asymptomatic. Preliminary data suggests, however, that genetically determined dysregulation of type I interferon responses is associated with autoimmunity, and may also be relevant to sporadic cerebrovascular disease and dementia. We aim to determine whether functional variants in genes involved in type I interferon regulation and signalling are associated with the risk of autoimmunity, stroke, and dementia in a population cohort. Methods We will perform a hypothesis-driven candidate pathway association study of type I interferon-related genes using rare variants in the UK Biobank (UKB). We will manually curate type I interferon regulation and signalling genes from a literature review and Gene Ontology, followed by clinical and functional filtering. Variants of interest will be included based on pre-defined clinical relevance and functional annotations (using LOFTEE, M-CAP and a minor allele frequency

Protein stability is fundamental to maintain pharmaceutical efficacy in the nascent field of biologics. One particular property that is essential for therapeutic effect is retention of the folded 3-dimensional conformation, i.e. once unfolding has occurred the biologic is often rendered inactive. In this work we propose a modified form of a recently published UV spectroscopic method that identifies protein unfolding. In this study we determine concentration limits to avoid protein unfolding of two model surfactants, namely polysorbate 20 and polysorbate 80, by correlating surfactant concentration with percentage ‘unfolded’ for three model proteins. For each scenario two distinct regions were observed, firstly surfactant concentrations at which no unfolding had occurred, followed by a second region whereby unfolding steadily increased with surfactant concentration. In general for the combinations analysed in this study, this second region began to appear around ten times below the critical micellar concentration of each surfactant, regardless of the protein or polysorbate chosen. It is therefore proposed that this adapted method could be used by researchers in the early stages of formulation development as a convenient and simple screening tool to confirm the ‘onset of unfolding’ concentration for protein-surfactant formulations, thus helping to optimise surfactant concentration selection in pharmaceutical formulations to maintain the benefits of surfactants yet avoid inadvertent unfolding.

IntroductionProteus mirabilis is a key pathobiont in catheter-associated urinary tract infections (CA-UTIs), which is well known to form crystalline biofilms that occlude catheters. Urease activity alkylates urine through the release of ammonia, consequentially resulting in higher levels of Mg2+ and Ca2+ and formation of crystals. In this study, we showed that N-acetyl cysteine (NAC), a thiol antioxidant, is a potent urease inhibitor that prevents crystalline biofilm formation.MethodsTo quantify urease activity, Berthelot’s method was done on bacterial extracts treated with NAC. We also used an in vitro catheterised glass bladder model to study the effect of NAC treatment on catheter occlusion and biofilm encrustation in P. mirabilis infections. Inductively-coupled plasma mass spectrometry (ICP-MS) was performed on catheter samples to decipher elemental profiles.ResultsNAC inhibits urease activity of clinical P. mirabilis isolates at concentrations as low as 1 mM, independent of bacterial killing. The study also showed that NAC is bacteriostatic on P. mirabilis, and inhibited biofilm formation and catheter occlusion in an in vitro. A significant 4-8log10 reduction in viable bacteria was observed in catheters infected in this model. Additionally, biofilms in NAC treated catheters displayed a depletion of calcium, magnesium, or phosphates (>10 fold reduction), thus confirming the absence of any urease activity in the presence of NAC. Interestingly, we also showed that not only is NAC anti-inflammatory in bladder epithelial cells (BECs), but that it mutes its inflammatory response to urease and P. mirabilis infection by reducing the production of IL-6, IL-8 and IL-1b.DiscussionUsing biochemical, microbiological and immunological techniques, this study displays the functionality of NAC in preventing catheter occlusion by inhibiting urease activity. The study also highlights NAC as a strong anti-inflammatory antibiofilm agent that can target both bacterial and host factors in the treatment of CA-UTIs.

IntroductionAnterior cruciate ligament (ACL) injuries cause knee instability, knee pain, weight-bearing adjustments, and functional deficits but their association to patellar tendon quality is unknown. Our purpose was to investigate quadriceps strength, patellar tendon quality, relative load exposure, perceived knee stability, knee pain, extension angle, and time from ACL injury; in addition to examining their relative associations.MethodsInjured and uninjured legs of 81 male athletes of different sports with a unilateral ACL injury (18–45 years) were examined. Participants reported location and intensity of knee pain and their perceived stability using a numerical rating scale (NRS 0–10). Strength was tested with an isokinetic device. Tendon quality was measured using ultrasound tissue characterization. Means ± standard deviation (SD) of perceived knee stability, knee extension angle, knee pain, isokinetic quadriceps strength in relation to body mass, proportion of echo-types (I–IV), tendon volume, and number of days from ACL injury to assessment are reported. Values of effect sizes (ES) and correlations (rs) were calculated.ResultsACL injured leg demonstrated reduced reported knee stability (6.3 ± 2.5), decreased knee extension angle (−0.7 ± 3.1° vs. −2.7 ± 2.2°; ES = 0.7; P

BackgroundNatural language processing (NLP) has the potential to automate the reading of radiology reports, but there is a need to demonstrate that NLP methods are adaptable and reliable for use in real-world clinical applications.MethodsWe tested the F1 score, precision, and recall to compare NLP tools on a cohort from a study on delirium using images and radiology reports from NHS Fife and a population-based cohort (Generation Scotland) that spans multiple National Health Service health boards. We compared four off-the-shelf rule-based and neural NLP tools (namely, EdIE-R, ALARM+, ESPRESSO, and Sem-EHR) and reported on their performance for three cerebrovascular phenotypes, namely, ischaemic stroke, small vessel disease (SVD), and atrophy. Clinical experts from the EdIE-R team defined phenotypes using labelling techniques developed in the development of EdIE-R, in conjunction with an expert researcher who read underlying images.ResultsEdIE-R obtained the highest F1 score in both cohorts for ischaemic stroke, ≥93%, followed by ALARM+, ≥87%. The F1 score of ESPRESSO was ≥74%, whilst that of Sem-EHR is ≥66%, although ESPRESSO had the highest precision in both cohorts, 90% and 98%. For F1 scores for SVD, EdIE-R scored ≥98% and ALARM+ ≥90%. ESPRESSO scored lowest with ≥77% and Sem-EHR ≥81%. In NHS Fife, F1 scores for atrophy by EdIE-R and ALARM+ were 99%, dropping in Generation Scotland to 96% for EdIE-R and 91% for ALARM+. Sem-EHR performed lowest for atrophy at 89% in NHS Fife and 73% in Generation Scotland. When comparing NLP tool output with brain image reads using F1 scores, ALARM+ scored 80%, outperforming EdIE-R at 66% in ischaemic stroke. For SVD, EdIE-R performed best, scoring 84%, with Sem-EHR 82%. For atrophy, EdIE-R and both ALARM+ versions were comparable at 80%.ConclusionsThe four NLP tools show varying F1 (and precision/recall) scores across all three phenotypes, although more apparent for ischaemic stroke. If NLP tools are to be used in clinical settings, this cannot be performed “out of the box.” It is essential to understand the context of their development to assess whether they are suitable for the task at hand or whether further training, re-training, or modification is required to adapt tools to the target task.