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Abstract Increasing biological nitrogen (N) fixation (BNF) in maize production could reduce the environmental impacts of N fertilizer use, but reactive N in the rhizosphere of maize limits the BNF process. Using non-transgenic methods, we developed gene-edited strains of Klebsiella variicola (Kv137-2253) and Kosakonia sacchari (Ks6-5687) bacteria optimized for root-associated BNF and ammonium excretion in N-rich conditions. The aim of this research was to elucidate the mechanism of action of these strains. We present evidence from in vitro, in planta and field experiments that confirms that our genetic remodeling strategy derepresses BNF activity in N-rich systems and increases ammonium excretion by orders of magnitude above the respective wildtype strains. BNF is demonstrated in controlled environments by the transfer of labeled 15N2 gas from the rhizosphere to the chlorophyll of inoculated maize plants. This was corroborated in several 15N isotope tracer field experiments where inoculation with the formulated, commercial-grade product derived from the gene-edited strains (PIVOT BIO PROVEN® 40) provided on average 21 kg N ha-1 to the plant by the VT-R1 growth stages. Data from small-plot and on-farm trials suggest that this technology can improve crop N status pre-flowering and has potential to mitigate the risk of yield loss associated with a reduction in synthetic N fertilizer inputs.

Abstract Background Total elbow arthroplasty (TEA) is a mainstay treatment for elbow arthropathy and elbow fracture that can relieve pain and restore functional motion. The purpose of this study was to determine the role of surgical indication in the incidence of short-term adverse events and risk factors influencing complications in TEA to treat arthropathy compared to TEA for fracture. Matched cohorts were created to better isolate the impact of the surgical indication. Methods The study identified patients within the American College of Surgeons National Surgical Quality Improvement Program (ACS-NSQIP) database that underwent TEA to treat arthropathy or fracture from 2010 to 2020. Matched cohorts were created to analyze the incidence of 30 day adverse events and risk factors for complications. Results The study included 599 patients for analysis. The overall incidence of any adverse event was 9.7%. In matched cohorts of 218 patients, the fracture group experienced a higher incidence of overall adverse events (13.8% vs. 6.0% in the arthropathy group, p = 0.009) and postoperative transfusions (9.6% vs. 1.8% in the arthropathy group, p

Abstract Precipitation channelled down tree stems (stemflow) or into drip points of ‘throughfall’ beneath trees results in spatially concentrated inputs of water and chemicals to the ground. Currently, these flows are poorly characterised due to uncertainties about which branches redirect rainfall to stemflow or throughfall drip points. We introduce a graph theoretic algorithm that ‘prunes’ quantitative structural models of trees (derived from terrestrial LiDAR) to identify branches contributing to stemflow and those contributing to throughfall drip points. To demonstrate the method's utility, we analysed two trees with similar canopy sizes but contrasting canopy architecture and rainfall partitioning behaviours. For both trees, the branch ‘watershed’ area contributing to stemflow (under conditions assumed to represent moderate precipitation intensity) was found to be only half of the total ground area covered by the canopy. The study also revealed significant variations between trees in the number and median contribution areas of modelled throughfall drip points (69 vs. 94 drip points tree−1, with contributing projected areas of 28.6 vs. 7.8 m2 tree−1, respectively). Branch diameter, surface area, volumes and woody area index of components contributing to stemflow and throughfall drip points may play a role in the trees' differing rainfall partitioning behaviours. Our pruning algorithm, enabled by the proliferation of LiDAR observations of canopy structure, promises to enhance studies of canopy hydrology. It offers a novel approach to refine our understanding of how trees interact with rainfall, thereby broadening the utility of existing LiDAR data in environmental research.

The increased use of immune checkpoint inhibitors (ICIs) across cancer programs has created the need for standardized monitoring and management of immune-related adverse events (irAEs). Delayed recognition without appropriate treatment can have serious and life-threatening consequences. The management of irAEs presents a unique set of challenges that must be addressed at a multidisciplinary level. Although various national and international guidelines and working groups provide high-level recommendations for the management of irAEs, practical guidance is lacking. Furthermore, timely collaboration between specialists requires institutional protocols that enable the early recognition, assessment, and treatment of irAEs. Such protocols should be developed by institution specialists and include algorithms for all healthcare providers involved in the care of patients treated with ICIs. At William Osler Health System in Brampton, Ontario, practical step-by-step multidisciplinary treatment approaches with recommendations for the management of irAEs were developed in collaboration with experts across Canada. Here, we provide an in-depth description of the approaches, outlining baseline investigations prior to the initiation of ICIs, as well as the monitoring and management of irAEs based on symptoms, severity, and involved organ systems. We encourage other centres to adapt and modify our approaches according to their specific needs and requirements.

Abstract The extremely high levels of genetic polymorphism within the human major histocompatibility complex (MHC) limit the usefulness of reference-based alignment methods for sequence assembly. We incorporate a short-read, de novo assembly algorithm into a workflow for novel application to the MHC. MHConstructor is a containerized pipeline designed for high-throughput, haplotype-informed, reproducible assembly of both whole genome sequencing and target capture short-read data in large, population cohorts. To-date, no other self-contained tool exists for the generation of de novo MHC assemblies from short-read data. MHConstructor facilitates wide-spread access to high-quality, alignment-free MHC sequence analysis.

Abstract Esophageal squamous cell carcinoma (ESCC) is highly heterogeneous. Our understanding of full molecular and immune landscape of ESCC remains limited, hindering the development of personalised therapeutic strategies. To address this, we perform genomic-transcriptomic characterizations and AI-aided histopathological image analysis of 120 Chinese ESCC patients. Here we show that ESCC can be categorized into differentiated, metabolic, immunogenic and stemness subtypes based on bulk and single-cell RNA-seq, each exhibiting specific molecular and histopathological features based on an amalgamated deep-learning model. The stemness subgroup with signature genes, such as WFDC2, SFRP1, LGR6 and VWA2, has the poorest prognosis and is associated with downregulated immune activities, a high frequency of EP300 mutation/activation, functional mutation enrichment in Wnt signalling and the highest level of intratumoural heterogeneity. The immune profiling by transcriptomics and immunohistochemistry reveals ESCC cells overexpress natural killer cell markers XCL1 and CD160 as immune evasion. Strikingly, XCL1 expression also affects the sensitivity of ESCC cells to common chemotherapy drugs. This study opens avenues for ESCC treatment and provides a valuable public resource to better understand ESCC.

Abstract Background Falls are the leading cause of injury related morbidity and mortality in older adults. Primary and secondary prevention strategies that address modifiable risk factors are critically important to reduce the number of falls and fall related injuries. A number of evidence-based fall prevention programs are available, but few offer potential for broad dissemination and public health impact due to implementation barriers, such as a need for trained program leaders and clinicians. Methods The study will use a randomized controlled trial design to evaluate incorporating physical therapy exercises (primary prevention strategy) within an existing intervention called Walk with Ease. While Walk with Ease has an established evidence-base related to the management of arthritis pain and symptoms, the present study will determine the potential to also reduce falls and fall risk in community-dwelling older adults. The integrated process and outcome evaluation will determine the relative effectiveness of individually-prescribed exercises (compared to standardized exercises) as well as the potential of ‘habit training’ resources (relative to generic behavior prompts) to improve compliance with exercises in this population. Discussion The study, conducted through a local clinical-community partnership will advance both the science and practice of community-based fall prevention programming, while also informing implementation strategies needed to promote broader dissemination. Trial registration ClinicalTrials.gov, NCT05693025, Registered January 20, 2023, Updated March 1, 2023.

During early development of biopharmaceuticals, suboptimal producing clones and production conditions can result in limited quantities of high-purity products. Here we describe a systematic approach, which requires minimal amounts of protein (~10 mg) to assess critical quality attributes of a monoclonal antibody (mAb). A commercial anti-PCSK9 IgG2 (evolocumab, Repatha®) and an early-stage biosimilar candidate were compared head-to-head using a range of high-throughput physicochemical and in-vitro binding analytical methods. Overall, both mAbs were shown to be highly pure and primarily monomeric, to share an identical primary structure, and to have similar higher-order structural integrity, apparent solubility, aggregation propensity, and physical stability profiles under temperature and pH stress conditions. Low levels of dimers were detected for the innovator (1.2%) and the biosimilar candidate mAb (0.3%), which also presented fragments (1.2%). Regarding charge heterogeneity, the amount of the main charge isoform was 53.6% for the innovator and 61.6% for the biosimilar candidate mAb. Acidic species were 38% for the innovator and 30% for the biosimilar candidate. Variations in the relative content of a few N-glycan species were found. The in-vitro binding affinity to PCSK9 was monitored, and no differences were detected. The mathematical approach called “error spectral difference” (ESD), proposed herein, enabled a quantitative comparison of the biophysical datasets. The workflow used in the present work to characterize CQAs at early stages is helpful in supporting the development of biosimilar mAb candidates.

Submarine groundwater discharge (SGD) is a recognized contributor to the hydrological and biogeochemical functioning of coral reef ecosystems located along coastlines. However, the distribution, size, and thermal properties of SGD remain poorly understood at most land–reef margins. We developed, deployed, and demonstrated an operational method for airborne detection and mapping of SGD using the 200 km coastline of western Hawai‘i Island as a testing and analysis environment. Airborne high spatial resolution (1 m) thermal imaging produced relative sea surface temperature (SST) maps that aligned geospatially with boat-based transects of SGD presence–absence. Boat-based SST anomaly measurements were highly correlated with airborne SST anomaly measurements (R2 = 0.85; RMSE = 0.04 °C). Resulting maps of the relative difference in SST inside and outside of SGD plumes, called delta-SST, revealed 749 SGD plumes in 200 km of coastline, with nearly half of the SGD plumes smaller than 0.1 ha in size. Only 9% of SGD plumes were ≥1 ha in size, and just 1% were larger than 10 ha. Our findings indicate that small SGD is omnipresent in the nearshore environment. Furthermore, we found that the infrequent, large SGD plumes (>10 ha) displayed the weakest delta-SST values, suggesting that large discharge plumes are not likely to provide cooling refugia to warming coral reefs. Our operational approach can be applied frequently over time to generate SGD information relative to terrestrial substrate, topography, and pollutants. This operational approach will yield new insights into the role that land-to-reef interactions have on the composition and condition of coral reefs along coastlines.

Introduction: In this narrative review, we identified concepts related to the process of transforming a psychiatry curriculum for New Zealand medical students. Method: A literature search was performed on four databases (Embase, Medline, PsycInfo, and Scopus) for articles related to curriculum development in psychiatry, including relevant aspects of culture and technology. Results: Ninety-three articles met the inclusion criteria. Three main themes were identified: the needs of learners; curriculum frameworks that optimise learning; and the role of technology. The key features of an effective psychiatry curriculum are the extent to which it integrates with other disciplines, develops key competencies, supports authentic learning, and promotes cultural safety. Conclusions: Transforming curricula is an iterative process that prioritises learners’ needs, establishes psychiatry within the teaching context, integrates learning evidence, and responds to the changing demands of society. The findings from this review apply to medical curricula more generally: a well-integrated specialist curriculum, in this case psychiatry, enables medical students to build essential competencies and depends upon effective collaboration with stakeholders, attention to cultural safety, and incorporating technology into the teaching context.

Abstract AI image classification algorithms have shown promising results when applied to skin cancer detection. Most public skin cancer image datasets are comprised of dermoscopic photos and are limited by selection bias, lack of standardization, and lend themselves to development of algorithms that can only be used by skilled clinicians. The SLICE-3D (“Skin Lesion Image Crops Extracted from 3D TBP”) dataset described here addresses those concerns and contains images of over 400,000 distinct skin lesions from seven dermatologic centers from around the world. De-identified images were systematically extracted from sensitive 3D Total Body Photographs and are comparable in optical resolution to smartphone images. Algorithms trained on lower quality images could improve clinical workflows and detect skin cancers earlier if deployed in primary care or non-clinical settings, where photos are captured by non-expert physicians or patients. Such a tool could prompt individuals to visit a specialized dermatologist. This dataset circumvents many inherent limitations of prior datasets and may be used to build upon previous applications of skin imaging for cancer detection.

Abstract Growing restrictions and bans on animal testing for chemical safety assessment under different regulations have led to an increasing use of alternative methods. Read-across is one of the major approaches used for this purpose, which relies on the identification of toxicological hazards of a data-poor or untested (target) chemical from data on other already-tested (source) similar chemicals. This requires the target substance to be first assigned to a group or category of ‘similar’ chemicals. The ‘similarity’ may be in terms of structural features alone, or in combination with certain rules that are based on mechanistic and/or toxicological aspects. In this regard, the OECD QSAR toolbox—a major free-access in silico platform—is widely used to derive toxicity predictions for a range of (eco) toxicological endpoints. The Toolbox allows the user to identify a set of similar chemicals (analogues) by computational ‘profilers’ that incorporate different structural alerts, or a combination of structural alerts and physicochemical and/or toxicokinetic rules relevant to a specific toxicological endpoint. The overall aim of this study was to assess the performance of the in silico profilers provided in the OECD QSAR Toolbox for reliability for identifying chemical analogues for category formation in a number of high-quality databases on mutagenicity, carcinogenicity, and skin sensitisation. The study also aimed to identify the reasons for any limitations in the performance of the profilers, and propose ways to improve their overall accuracy. The results showed that whilst some structural alerts are fit-for-purpose as such within the acceptable limits, others need refinement or a consideration for their possible exclusion from the profiler. Such refinements are imperative for a reliable use of the profilers in read-across and grouping/categorisation for classification, labelling and risk assessment of chemicals.

Abstract Two-dimensional (2D) semiconductors are promising candidates for optoelectronic application and quantum information processes due to their inherent out-of-plane 2D confinement. In addition, they offer the possibility of achieving low-dimensional in-plane exciton confinement, similar to zero-dimensional quantum dots, with intriguing optical and electronic properties via strain or composition engineering. However, realizing such laterally confined 2D monolayers and systematically controlling size-dependent optical properties remain significant challenges. Here, we report the observation of lateral confinement of excitons in epitaxially grown in-plane MoSe2 quantum dots (~15-60 nm wide) inside a continuous matrix of WSe2 monolayer film via a sequential epitaxial growth process. Various optical spectroscopy techniques reveal the size-dependent exciton confinement in the MoSe2 monolayer quantum dots with exciton blue shift (12-40 meV) at a low temperature as compared to continuous monolayer MoSe2. Finally, single-photon emission (g2(0) ~ 0.4) was also observed from the smallest dots at 1.6 K. Our study opens the door to compositionally engineered, tunable, in-plane quantum light sources in 2D semiconductors.

Abstract Activation-induced cytidine deaminase (AID) is a B cell-specific mutator required for antibody diversification. However, it is also implicated in the etiology of several B cell malignancies. Evaluating the AID-induced mutation load in patients at-risk for certain blood cancers is critical in assessing disease severity and treatment options. We have developed a digital PCR (dPCR) assay that allows us to quantify mutations resulting from AID modification or DNA double-strand break (DSB) formation and repair at sites known to be prone to DSBs. Implementation of this assay shows that increased AID levels in immature B cells increase genome instability at loci linked to chromosomal translocation formation. This includes the CRLF2 locus that is often involved in translocations associated with a subtype of acute lymphoblastic leukemia (ALL) that disproportionately affects Hispanics, particularly those with Latin American ancestry. Using dPCR, we characterize the CRLF2 locus in B cell-derived genomic DNA from both Hispanic ALL patients and healthy Hispanic donors and found increased mutations in both, suggesting that vulnerability to DNA damage at CRLF2 may be driving this health disparity. Our ability to detect and quantify these mutations will potentiate future risk identification, early detection of cancers, and reduction of associated cancer health disparities.

Abstract Background Legislative bodies across the country have increasingly allowed off-road vehicles (ORVs) including all-terrain vehicles (ATVs) and utility task vehicles (UTVs) on public roads, an environment for which they are not designed. In 2004, Iowa gave individual counties the discretion to pass ordinances allowing ORVs on public roadways. The objective of this study was to evaluate the relationship between the passage of ORV ordinances and ORV crash rates, especially on public roads. Methods An Iowa ORV roadway ordinance database and an Iowa ORV crash database (2002–2018) for all 99 counties were compiled. Crashes for which county location could not be determined were excluded. Utilizing a zero-inflated Poisson model, correcting for background crash frequency trends and population, investigators compared the relative rates of crashes after ordinance passage to time points before ordinance implementation and to counties without such ordinances. Sub-analyses, including that focused on more recent years (2008–2018), were also performed. Results Forty-five county ORV roadway ordinances went into effect between 2011 and 2018 and 2,347 crashes (69%) met inclusion criteria. Adjusted for year, there was a 58% greater ORV crash rate in counties after passing an ORV roadway ordinance (incidence rate ratio (IRR) 1.58, 95% CI 1.32–1.90). Roadway crashes (n = 834) increased 48% after ordinance passage (IRR 1.48, 95% CI 1.14–1.94). This roadway crash association remained statistically significant when analysis was limited to the years 2008–2018 (IRR 1.39, CI 1.06–1.83, n = 544); to ATV crashes only (IRR 1.70, CI 1.20–2.40, n = 683); and to ATV crashes excluding counties with UTV-only ordinances (IRR 1.74, CI 1.40–2.15, n = 2,011). Conclusions ORV roadway and total crashes increased significantly after implementation of county ordinances allowing ORVs on public roadways and when compared to counties without such ordinances. It is likely that these increased crashes have resulted in more injuries and possibly deaths. Results from this study may help inform policymakers as they consider legislation regarding ORV usage on public roads.

Abstract We propose to use atoms and molecules as quantum sensors of axion-mediated monopole-dipole forces. We show that electron spin precession experiments using atomic and molecular beams are well-suited for axion searches thanks to the presence of co-magnetometer states and single-shot temporal resolution. Experimental strategies to detect axion gradients from localised sources and the earth are presented, taking ACME III as a prototype example. Other possibilities including atomic beams, and laser-cooled atoms and molecules are discussed.

Abstract Melioidosis is an often-fatal neglected tropical disease caused by an environmental bacterium Burkholderia pseudomallei. However, our understanding of the disease-causing bacterial lineages, their dissemination, and adaptive mechanisms remains limited. To address this, we conduct a comprehensive genomic analysis of 1,391 B. pseudomallei isolates collected from nine hospitals in northeast Thailand between 2015 and 2018, and contemporaneous isolates from neighbouring countries, representing the most densely sampled collection to date. Our study identifies three dominant lineages, each with unique gene sets potentially enhancing bacterial fitness in the environment. We find that recombination drives lineage-specific gene flow. Transcriptome analyses of representative clinical isolates from each dominant lineage reveal increased expression of lineage-specific genes under environmental conditions in two out of three lineages. This underscores the potential importance of environmental persistence for these dominant lineages. The study also highlights the influence of environmental factors such as terrain slope, altitude, and river direction on the geographical dispersal of B. pseudomallei. Collectively, our findings suggest that environmental persistence may play a role in facilitating the spread of B. pseudomallei, and as a prerequisite for exposure and infection, thereby providing useful insights for informing melioidosis prevention and control strategies.

Abstract With the success of immunotherapy in cancer, understanding the tumor immune microenvironment (TIME) has become increasingly important; however in pediatric brain tumors this remains poorly characterized. Accordingly, we developed a clinical immune-oncology gene expression assay and used it to profile a diverse range of 1382 samples with detailed clinical and molecular annotation. In low-grade gliomas we identify distinct patterns of immune activation with prognostic significance in BRAF V600E-mutant tumors. In high-grade gliomas, we observe immune activation and T-cell infiltrates in tumors that have historically been considered immune cold, as well as genomic correlates of inflammation levels. In mismatch repair deficient high-grade gliomas, we find that high tumor inflammation signature is a significant predictor of response to immune checkpoint inhibition, and demonstrate the potential for multimodal biomarkers to improve treatment stratification. Importantly, while overall patterns of immune activation are observed for histologically and genetically defined tumor types, there is significant variability within each entity, indicating that the TIME must be evaluated as an independent feature from diagnosis. In sum, in addition to the histology and molecular profile, this work underscores the importance of reporting on the TIME as an essential axis of cancer diagnosis in the era of personalized medicine.

Older adults face unique barriers and challenges related to physical activity (PA) participation. Motivational interviewing (MI) is a commonly used health coaching strategy to support behavior change that holds potential for older adults. Previous research on MI strategies has focused primarily on face-to-face delivery, limiting insights regarding virtual programs. Objectives: The purpose of this study was to determine if MI could be delivered telephonically with high fidelity and high acceptability in older adult participants. The study is designed to inform future trials evaluating its effectiveness in supporting virtual PA programs. Methods: This study evaluated the feasibility and acceptability of telephonic MI among older adults that enrolled in an online version of the Walk with Ease program. Results: Of 39 participants referred, 29 enrolled and 27 provided feedback. Participants were highly accepting and adherent, with 74 % of patients attending at least five of six sessions, and 96 % of participants indicating satisfaction with the MI provided. Coaches improved program enjoyment by helping set effective goals and providing ongoing accountability. Conclusions: Evaluations documented adequate fidelity and high acceptability of telephonic delivery, and motivation results revealed large, significant increases in autonomous regulation (quality of motivation). Innovation: Telephonic MI using non-healthcare professional coaches is feasibly delivered with high fidelity, and is acceptable to older adult participants. The innovative delivery format offers novel opportunities supporting virtual and telehealth interventions to reduce chronic disease risk among older adults.

Purpose: To assess radiomics and deep learning (DL) methods in identifying symptomatic Carotid Artery Disease (CAD) from carotid CT angiography (CTA) images. We further compare the performance of these novel methods to the conventional calcium score. Methods: Carotid CT angiography (CTA) images from symptomatic patients (ischaemic stroke/transient ischaemic attack within the last 3 months) and asymptomatic patients were analysed. Carotid arteries were classified into culprit, non-culprit and asymptomatic. The calcium score was assessed using the Agatston method. 93 radiomic features were extracted from regions-of-interest drawn on 14 consecutive CTA slices. For DL, convolutional neural networks (CNNs) with and without transfer learning were trained directly on CTA slices. Predictive performance was assessed over 5-fold cross validated AUC scores. SHAP and GRAD-CAM algorithms were used for explainability. Results: 132 carotid arteries were analysed (41 culprit, 41 non-culprit, and 50 asymptomatic). For asymptomatic vs symptomatic arteries, radiomics attained a mean AUC of 0.96(± 0.02), followed by DL 0.86(± 0.06) and then calcium 0.79(± 0.08). For culprit vs non-culprit arteries, radiomics achieved a mean AUC of 0.75(± 0.09), followed by DL 0.67(± 0.10) and then calcium 0.60(± 0.02). For multi-class classification, the mean AUCs were 0.95(± 0.07), 0.79(± 0.05), and 0.71(± 0.07) for radiomics, DL and calcium, respectively. Explainability revealed consistent patterns in the most important radiomic features. Conclusions: Our study highlights the potential of novel image analysis techniques in extracting quantitative information beyond calcification in the identification of CAD. Though further work is required, the transition of these novel techniques into clinical practice may eventually facilitate better stroke risk stratification.

Salmonella was isolated from 23/79 (29.1%) pooled gecko stool samples from households in southern Malawi. Whole genome sequencing of 47 individual isolates within this collection revealed 27 Salmonella serovars spanning two subspecies. Our results demonstrate that geckos play an important role in the carriage of Salmonella within households.

ABSTRACT Photorhabdus is a bacterial genus containing both insect and emerging human pathogens. Most insect-restricted species display temperature restriction, unable to grow above 34°C, while Photorhabdus asymbiotica can grow at 37°C to infect mammalian hosts and cause Photorhabdosis. Metabolic adaptations have been proposed to facilitate the survival of this pathogen at higher temperatures, yet the biological mechanisms underlying these are poorly understood. We have reconstructed an extensively manually curated genome-scale metabolic model of P. asymbiotica (iEC1073, BioModels ID MODEL2309110001), validated through in silico gene knockout and nutrient utilization experiments with an excellent agreement between experimental data and model predictions. Integration of iEC1073 with transcriptomics data obtained for P. asymbiotica at temperatures of 28°C and 37°C allowed the development of temperature-specific reconstructions representing metabolic adaptations the pathogen undergoes when shifting to a higher temperature in a mammalian compared to insect host. Analysis of these temperature-specific reconstructions reveals that nucleotide metabolism is enriched with predicted upregulated and downregulated reactions. iEC1073 could be used as a powerful tool to study the metabolism of P. asymbiotica, in different genetic or environmental conditions.IMPORTANCEPhotorhabdus bacterial species contain both human and insect pathogens, and most of these species cannot grow in higher temperatures. However, Photorhabdus asymbiotica, which infects both humans and insects, can grow in higher temperatures and undergoes metabolic adaptations at a temperature of 37°C compared to that of insect body temperature. Therefore, it is important to examine how this bacterial species can metabolically adapt to survive in higher temperatures. In this work, using a mathematical model, we have examined the metabolic shift that takes place when the bacteria switch from growth conditions in 28°C to 37°C. We show that P. asymbiotica potentially experiences predicted temperature-induced metabolic adaptations at 37°C predominantly clustered within the nucleotide metabolism pathway.

Bacillus thuringiensis crystal (Cry) proteins have been expressed in commercial transgenic crops for nearly 30 years, providing safe and effective control of insect pests and significantly reducing the application of hazardous chemical pesticides. B. thuringiensis crystal proteins have also been shown to target parasitic nematodes, including plant parasitic nematodes. Recently, transgenic soybean crops expressing Cry14Ab have been shown to provide control against the soybean cyst nematode Heterodera glycines, marking the first time a crystal protein is being commercialized in transgenic crops for control of a nematode pest. However, apart from H. glycines and the free-living nematode, Caenorhabditis elegans, the breadth of nematode activity of Cry14Ab, e.g., against gastrointestinal parasitic nematodes (GINs), has not been reported. Here we study the efficacy of Cry14Ab against a wide range of gastrointestinal nematode parasites (GINs) in vitro and in vivo. We find that Cry14Ab is effective in vitro against the barber's pole worm Haemonchus contortus larvae, small strongyles cyathostomin larvae, the hookworm Ancylostoma ceylanicum adults, the roundworm Ascaris suum L4 larvae, and the whipworm Trichuris muris adults. In rodents infected with GIN parasites, Cry14Ab is effective as an in vivo anthelmintic against the hookworms A. ceylanicum and N. americanus, against the mouse parasite Heligmosomoides polygyrus bakeri, and against the roundworm A. suum. Cry14Ab also variably reduces the reproduction of the whipworm T. muris in vivo. Using optimized profile Markov Models, we looked for other putative anthelmintic Cry proteins and, within this list, identified a Bt crystal protein, GenBank accession no. MF893203, that we produced and demonstrated intoxicated GINs. This protein, with 90% amino acid identity to Cry14Ab, is active against C. elegans, A. ceylanicum adults, and A. suum L4 larvae in vitro. MF893203 was given the official designation of Cry14Ac. Cry14Ac is also an effective in vivo anthelmintic against A. ceylanicum hookworms in hamsters and intestinal A. suum in mice. Taken together, our results demonstrate that Cry14Ab and Cry14Ac have wide therapeutic utility against GINs.

This article describes a novel approach of integrating writing center professionals into online EdD dissertation committees to enhance student success and writing proficiency. Departing from the traditional “master-apprentice” model of doctoral studies, the study explores the rationale behind restructuring dissertation committees to better align with evolving programmatic outcomes and the diverse career trajectories of doctoral students. Drawing on existing scholarship and theoretical foundations, it clarifies the ambiguity surrounding the roles of committee members beyond the dissertation advisor and advocates for a coalition of experts approach to dissertation committees. We describe the establishment and functions of a Research and Writing Development Center (RWDC), highlighting its role in supporting students throughout the dissertation process. Emphasizing close collaboration between faculty advisors and RWDC professionals, the article shares the successful relationship forged to provide comprehensive feedback and guidance to scholarly practitioners writing dissertations of practice. Programmatic overviews and outcomes underscore the efficacy of this collaborative advising model in enhancing student completion rates and addressing the evolving landscape of EdD programs. This study offers valuable insight into reimagining dissertation committees to foster the holistic development of scholarly practitioners in EdD programs.

Recent and projected changes in climate over this century pose an unprecedented threat to the health, diversity, and productivity of forest ecosystems. Forests have migrated and adapted to long-term changes in climate over thousands to millions of years; however, natural migration rates and adaptive responses of tree populations cannot match the rapid pace of current climate change. Consequently, more climate-informed approaches to reforestation are needed as current reforestation strategies using local seed sources may no longer be adequate to meet forest management objectives. Assisted migration is a climate change adaptation technique that can help maintain the ecosystem services and economic value that forests provide. Forestry assisted migration (FAM) focusses on the movement of populations of widespread, commercially, or ecologically important forest tree species within or just beyond their current ranges as a way to maintain forest productivity and health in the face of climate change. Although the forestry community recognizes FAM as a reforestation tool, guidance for planning and implementation of FAM is lacking and a framework that provides this guidance can prove useful to land managers with limited time and resources available who want to undertake FAM. We developed a practical framework (the FAM Framework) to provide a structured approach to ensure the most important considerations and best available science are utilized by land managers wanting to implement FAM on their land base. The FAM Framework incorporates multiple factors for the application of FAM in four sequential phases: assessment and analysis, climate-based plant material selection, seed procurement and deployment, and documentation and monitoring. The FAM Framework was tested by developing an assisted migration plan for the Superior National Forest, Minnesota (MN), and lessons learned from the development of this specific plan were used to revise and improve the FAM Framework for suitability across all lands. While originally designed to meet the needs of National Forest System land managers, it is relevant and applicable across the spectrum of land ownership because it incorporates consideration of critical elements in planning and implementing FAM on any landscape while facilitating adaptive management for active learning and future implementation.

The Photorhabdus virulence cassette (PVC) is an extracellular contractile injection system. In the producing bacterium, N-terminal signal peptides enable effector ‘payloads’ to be loaded into the PVC’s hollow tube—facilitated by the ‘ATPases associated with diverse cellular activities’ (AAA) ATPase, Pvc15—ready for injection of the toxin or virulence factor into eukaryotic cytosols. Pvc15’s function and its interaction with the signal peptide were unclear. This study describes the signal peptide diversity in extracellular contractile injection system clades and interrogates the Pvc15–signal peptide interaction using ATPase assays, cell respiratory assays and western blot quantification of Escherichia coli lysates and co-purifications of PVCs with their payloads. This study found that extracellular contractile injection system signal peptides can be grouped according to sequence alignment, owing to potentially homologous loading mechanisms. Pvc15 contains three domains, including tandem AAA domains D1 and D2. By constructing Pvc15 mutants, we found that while each domain is necessary for PVC-payload loading, domain D2 is the sole bioactive ATPase domain and rescues unstable payloads via the signal peptide. Finally, truncating the signal peptide abolishes Pvc15-dependent PVC loading and has varying effects on payload stability. This study provides crucial insights into extracellular contractile injection system effector loading mechanisms and their ATPase chaperones, and suggests that these devices could be bioengineered for injection of therapeutic proteins into human cells.

ABSTRACT The Enterobacteriaceae are a scientifically and medically important clade of bacteria, containing the model organism Escherichia coli, as well as major human pathogens including Salmonella enterica and Klebsiella pneumoniae. Essential gene sets have been determined for several members of the Enterobacteriaceae, with the Keio E. coli single-gene deletion library often regarded as a gold standard. However, it remains unclear how gene essentiality varies between related strains and species. To investigate this, we have assembled a collection of 13 sequenced high-density transposon mutant libraries from five genera within the Enterobacteriaceae. We first assess several gene essentiality prediction approaches, investigate the effects of transposon density on essentiality prediction, and identify biases in transposon insertion sequencing data. Based on these investigations, we develop a new classifier for gene essentiality. Using this new classifier, we define a core essential genome in the Enterobacteriaceae of 201 universally essential genes. Despite the presence of a large cohort of variably essential genes, we find an absence of evidence for genus-specific essential genes. A clear example of this sporadic essentiality is given by the set of genes regulating the σE extracytoplasmic stress response, which appears to have independently acquired essentiality multiple times in the Enterobacteriaceae. Finally, we compare our essential gene sets to the natural experiment of gene loss in obligate insect endosymbionts that have emerged from within the Enterobacteriaceae. This isolates a remarkably small set of genes absolutely required for survival and identifies several instances of essential stress responses masked by redundancy in free-living bacteria.IMPORTANCEThe essential genome, that is the set of genes absolutely required to sustain life, is a core concept in genetics. Essential genes in bacteria serve as drug targets, put constraints on the engineering of biological chassis for technological or industrial purposes, and are key to constructing synthetic life. Despite decades of study, relatively little is known about how gene essentiality varies across related bacteria. In this study, we have collected gene essentiality data for 13 bacteria related to the model organism Escherichia coli, including several human pathogens, and investigated the conservation of essentiality. We find that approximately a third of the genes essential in any particular strain are non-essential in another related strain. Surprisingly, we do not find evidence for essential genes unique to specific genera; rather it appears a substantial fraction of the essential genome rapidly gains or loses essentiality during evolution. This suggests that essentiality is not an immutable characteristic but depends crucially on the genomic context. We illustrate this through a comparison of our essential genes in free-living bacteria to genes conserved in 34 insect endosymbionts with naturally reduced genomes, finding several cases where genes generally regarded as being important for specific stress responses appear to have become essential in endosymbionts due to a loss of functional redundancy in the genome.

Background Heart failure (HF) is not included in atrial fibrillation (AF) bleeding risk prediction scores, reflecting uncertainty regarding its importance as a risk factor for major haemorrhage. We aimed to report the relative risk of first major haemorrhage in people with HF and AF compared with people with AF without HF (‘AF only’).Methods English primary care cohort study of 2 178 162 people aged ≥45 years in the Clinical Practice Research Datalink from January 2000 to December 2018, linked to secondary care and mortality databases. We used traditional survival analysis and competing risks methods, accounting for all-cause mortality and anticoagulation.Results Over 7.56 years median follow-up, 60 270 people were diagnosed with HF and AF of whom 4996 (8.3%) had a major haemorrhage and 36 170 died (60.0%), compared with 8256 (6.4%) and 34 375 (27.2%), respectively, among 126 251 people with AF only. Less than half those with AF were prescribed an anticoagulant (45.6% from 2014 onwards), although 75.7% were prescribed an antiplatelet or anticoagulant. In a fully adjusted Cox model, the HR for major haemorrhage was higher among people with HF and AF (2.52, 95% CI 2.44 to 2.61) than AF only (1.87, 95% CI 1.82 to 1.92), even in a subgroup analysis of people prescribed anticoagulation. However, in a Fine and Gray competing risk model, the HR of major haemorrhage was similar for people with AF only (1.82, 95% CI 1.77 to 1.87) or HF and AF (1.71, 95% CI 1.66 to 1.78).Conclusions People with HF and AF are at increased risk of major haemorrhage compared with those with AF only and current prediction scores may underestimate the risk of haemorrhage in HF and AF. However, people with HF and AF are more likely to die than have a major haemorrhage and therefore an individual’s expected prognosis should be carefully considered when predicting future bleeding risk.

Abstract Antimicrobial resistance (AMR) is a growing public health crisis that requires innovative solutions. Current susceptibility testing approaches limit our ability to rapidly distinguish between antimicrobial-susceptible and -resistant organisms. Salmonella Typhimurium (S. Typhimurium) is an enteric pathogen responsible for severe gastrointestinal illness and invasive disease. Despite widespread resistance, ciprofloxacin remains a common treatment for Salmonella infections, particularly in lower-resource settings, where the drug is given empirically. Here, we exploit high-content imaging to generate deep phenotyping of S. Typhimurium isolates longitudinally exposed to increasing concentrations of ciprofloxacin. We apply machine learning algorithms to the imaging data and demonstrate that individual isolates display distinct growth and morphological characteristics that cluster by time point and susceptibility to ciprofloxacin, which occur independently of ciprofloxacin exposure. Using a further set of S. Typhimurium clinical isolates, we find that machine learning classifiers can accurately predict ciprofloxacin susceptibility without exposure to it or any prior knowledge of resistance phenotype. These results demonstrate the principle of using high-content imaging with machine learning algorithms to predict drug susceptibility of clinical bacterial isolates. This technique may be an important tool in understanding the morphological impact of antimicrobials on the bacterial cell to identify drugs with new modes of action.

Abstract Background Hypertension (HTN) involves genetic variability in the renin‐angiotensin system and influences antihypertensive response. We previously reported that angiotensinogen (AGT) messenger RNA (mRNA) is endogenously bound by miR‐122‐5p and rs699 A > G decreases reporter mRNA in the microRNA functional‐assay PASSPORT‐seq. The AGT promoter variant rs5051 C > T is in linkage disequilibrium (LD) with rs699 A > G and increases AGT transcription. The independent effect of these variants is understudied due to their LD therefore we aimed to test the hypothesis that increased AGT by rs5051 C > T counterbalances AGT decreased by rs699 A > G, and when these variants occur independently, it translates to HTN‐related phenotypes. Methods We used in silico, in vitro, in vivo, and retrospective models to test this hypothesis. Results In silico, rs699 A > G is predicted to increase miR‐122‐5p binding affinity by 3%. Mir‐eCLIP results show rs699 is 40–45 nucleotides from the strongest microRNA‐binding site in the AGT mRNA. Unexpectedly, rs699 A > G increases AGT mRNA in an AGT‐plasmid‐cDNA HepG2 expression model. Genotype‐Tissue Expression (GTEx) and UK Biobank analyses demonstrate liver AGT expression and HTN phenotypes are not different when rs699 A > G occurs independently from rs5051 C > T. However, GTEx and the in vitro experiments suggest rs699 A > G confers cell‐type‐specific effects on AGT mRNA abundance, and suggest paracrine renal renin‐angiotensin‐system perturbations could mediate the rs699 A > G associations with HTN. Conclusions We found that rs5051 C > T and rs699 A > G significantly associate with systolic blood pressure in Black participants in the UK Biobank, demonstrating a fourfold larger effect than in White participants. Further studies are warranted to determine if altered antihypertensive response in Black individuals might be due to rs5051 C > T or rs699 A > G. Studies like this will help clinicians move beyond the use of race as a surrogate for genotype.

Abstract Background Although elective procedures have life-changing potential, all surgeries come with an inherent risk of reoperation. There is a gap in knowledge investigating the risk of reoperation across orthopaedics. We aimed to identify the elective orthopaedic procedures with the highest rate of unplanned reoperation and the reasons for these procedures having such high reoperation rates. Methods Patients in the NSQIP database were identified using CPT and ICD-10 codes. We isolated 612,815 orthopaedics procedures from 2018 to 2020 and identified the 10 CPT codes with the greatest rate of unplanned return to the operating room. For each index procedure, we identified the ICD-10 codes for the reoperation procedure and categorized them into infection, mechanical failure, fracture, wound disruption, hematoma or seroma, nerve pathology, other, and unspecified. Results Below knee amputation (BKA) (CPT 27880) had the highest reoperation rate of 6.92% (37 of 535 patients). Posterior-approach thoracic (5.86%) or cervical (4.14%) arthrodesis and cervical laminectomy (3.85%), revision total hip arthroplasty (5.23%), conversion to total hip arthroplasty (4.33%), and revision shoulder arthroplasty (4.22%) were among the remaining highest reoperation rates. The overall leading causes of reoperation were infection (30.1%), mechanical failure (21.1%), and hematoma or seroma (9.4%) for the 10 procedures with the highest reoperation rates. Conclusions This study successfully identified the elective orthopaedic procedures with the highest 30-day return to OR rates. These include BKA, posterior thoracic and cervical spinal arthrodesis, revision hip arthroplasty, revision total shoulder arthroplasty, and cervical laminectomy. With this data, we can identify areas across orthopaedics in which revising protocols may improve patient outcomes and limit the burden of reoperations on patients and the healthcare system. Future studies should focus on the long-term physical and financial impact that these reoperations may have on patients and hospital systems. Level of clinical evidence IV.

Abstract The master DNA damage repair histone protein, H2AX, is essential for orchestrating the recruitment of downstream mediator and effector proteins at damaged chromatin. The phosphorylation of H2AX at S139, γH2AX, is well-studied for its DNA repair function. However, the extended C-terminal tail is not characterized. Here, we define the minimal motif on H2AX for the canonical function in activating the MDC1-RNF8-RNF168 phosphorylation-ubiquitination pathway that is important for recruiting repair proteins, such as 53BP1 and BRCA1. Interestingly, H2AX recruits 53BP1 independently from the MDC1-RNF8-RNF168 pathway through its evolved C-terminal linker region with S139 phosphorylation. Mechanistically, 53BP1 recruitment to damaged chromatin is mediated by the interaction between the H2AX C-terminal tail and the 53BP1 Oligomerization-Tudor domains. Moreover, γH2AX-linker mediated 53BP1 recruitment leads to camptothecin resistance in H2AX knockout cells. Overall, our study uncovers an evolved mechanism within the H2AX C-terminal tail for regulating DNA repair proteins at damaged chromatin.

Abstract Background Infections caused by multidrug-resistant gram-negative bacteria present a severe threat to global public health. The WHO defines drug-resistant Klebsiella pneumoniae as a priority pathogen for which alternative treatments are needed given the limited treatment options and the rapid acquisition of novel resistance mechanisms by this species. Longitudinal descriptions of genomic epidemiology of Klebsiella pneumoniae can inform management strategies but data from sub-Saharan Africa are lacking. Methods We present a longitudinal analysis of all invasive K. pneumoniae isolates from a single hospital in Blantyre, Malawi, southern Africa, from 1998 to 2020, combining clinical data with genome sequence analysis of the isolates. Results We show that after a dramatic increase in the number of infections from 2016 K. pneumoniae becomes hyperendemic, driven by an increase in neonatal infections. Genomic data show repeated waves of clonal expansion of different, often ward-restricted, lineages, suggestive of hospital-associated transmission. We describe temporal trends in resistance and surface antigens, of relevance for vaccine development. Conclusions Our data highlight a clear need for new interventions to prevent rather than treat K. pneumoniae infections in our setting. Whilst one option may be a vaccine, the majority of cases could be avoided by an increased focus on and investment in infection prevention and control measures, which would reduce all healthcare-associated infections and not just one.

Abstract Recent geologic and modeled evidence suggests that the grounding line of the Siple Coast of the West Antarctic Ice Sheet (WAIS) retreated hundreds of kilometers beyond its present position in the middle to late Holocene and readvanced within the past 1.7 ka. This grounding line reversal has been attributed to both changing rates of isostatic rebound and regional climate change. Here, we test these two hypotheses using a proxy-informed ensemble of ice sheet model simulations with varying ocean thermal forcing, global glacioisostatic adjustment (GIA) model simulations, and coupled ice sheet-GIA simulations that consider the interactions between these processes. Our results indicate that a warm to cold ocean cavity regime shift is the most likely cause of this grounding line reversal, but that GIA influences the rate of ice sheet response to oceanic changes. This implies that the grounding line here is sensitive to future changes in sub-ice shelf ocean circulation.

Abstract Global mean sea level has risen at an accelerating rate in the last decade and will continue to rise for centuries. The Amundsen Sea Embayment in West Antarctica is a critical region for present and future ice loss, however most studies consider only a worst-case future for the region. Here we use ice sheet model sensitivity experiments to investigate the centennial scale implications of short-term periods of enhanced ocean driven sub-ice shelf melting on ice loss and assess what future reduction in melting is necessary to mitigate ice stream retreat and offset global sea level rise. Our findings reveal that restoring elevated melt rates to present-day levels within 100 years causes rates of ice discharge to immediately decline, thereby limiting the overall sea level contribution from the region. However, while ice stream re-advance and slowed ice discharge is possible with reduced basal melting, a centennial scale increase in accumulation must occur to offset the extensive ice loss.

In September 2021, a total of 25 patients diagnosed with COVID-19 developed acute melioidosis after (median 7 days) admission to a COVID-19 field hospital in Thailand. Eight nonpotable tap water samples and 6 soil samples were culture-positive for Burkholderia pseudomallei. Genomic analysis suggested contaminated tap water as the likely cause of illness.

Introduction: Falls present a significant public health concern in the United States as a primary cause of unintentional injury-related deaths among older adults. A fall risk assessment toolkit STEADI developed by the CDC has been shown to predict future falls. However, STEADI has issues with accurate evaluations due to the disagreement on cut-off scores in functional assessments and history-taking questionnaires. Wearable sensor technology offers a practical and quantifiable alternative for assessing an individual's movement performance in real-world environments. The use of Inertial Measurement Units (IMUs) offers considerable potential to enhance fall risk screening. Purpose: The primary aim of this study is to test the agreement of STEADI functional assessment performance measured by the IMUs in comparison to human-based measurements. Method: 27 participants (Age: 74.37 ± 7.21) performed STEADI, including the Four-Stage Balance Test (4SBT), Timed Up & Go Test (TUG), 30-second Chair Stand (30sCS) with IMU placed at the fifth lumbar vertebra which is the proxy location of whole-body Center of Mass. By adopting an equivalent test, the STEADI agreement was tested between the human rater and IMU measurements, giving α = 0.05. Result: Between the results from evaluators and IMU, the difference in TUG is -0.23 s, and the difference in 30sCS is 0.37, which is equivalent to within 4 % and 8 % for TUG and 30sCS, respectively. The difference in single-leg stance during the 4SBT is 0.59 s; however, the calculated equivalence zone is larger (22.7 %). Conclusion: This study demonstrates the feasibility of using IMU sensors to enhance fall risk screening protocols based on the STEADI. Future refinement may still need to enable broader application and effective screening practices on a larger scale of the population.

Abstract Networks of marine protected areas (MPAs) are being increasingly implemented worldwide as conservation management tools. We report here on MPA effectiveness using data from a 17‐year long remotely operated vehicle (ROV) monitoring program spanning mesophotic (~ 20–130 m) depths in 26 MPAs across California's MPA network. We utilize a spatial modeling approach that includes important environmental covariates as well as spatial dependence in the data, and allows the separation of statewide and regional trends in the abundance of focal species from additional trends specific to MPAs. We demonstrate that there have been statewide and/or regional recoveries in abundance for the majority of our 10 focal demersal fish species, with all four statewide species assessments and 18 out of 22 species‐region combinations assessed displaying positive trends. We also demonstrate that MPA protection has had an additional positive effect on the abundance of the majority of these focal species inside MPAs compared with reference areas, with positive effects for all four statewide species, and 18 out of 22 statewide/regions assessed showing positive effects, four showing no statistically detectable differences, and no negative MPA effects found. Comparisons with theoretical expectations of MPA recovery for our focal species showed that 2 out of 4 statewide, and 11 out of 17 species‐region combinations assessed displayed higher mean MPA effects than expected. Our results highlight strong trajectories of increasing abundance and additional MPA effects for many of our focal species, demonstrating that MPAs are having positive effects in mesophotic depths across the network as well as at previously reported shallower depths, and that image‐based platforms such as ROVs provide an important tool to support timely reporting on the effectiveness of MPA networks.

Key Clinical Message Morel‐Lavallee lesions are uncommon injuries and there is a lack of data to support what the most effective management is. In the case of a young athlete with a small lesion, we propose that conservative treatment with rest and close follow up is appropriate.

Abstract Rapid and accurate maps of floods across large domains, with high temporal resolution capturing event peaks, have applications for flood forecasting and resilience, damage assessment, and parametric insurance. Satellite imagery produces incomplete observations spatially and temporally, and hydrodynamic models require tradeoffs between computational efficiency and accuracy. We address these challenges with a novel flood model which predicts surface water area from the U.S. National Water Model using a convolutional neural network (NWM‐CNN). We trained NWM‐CNN on 780 flood events, at a 250 m resolution with an RMSE of 4.58% on held out validation geographies. We demonstrate NWM‐CNN across California during the 2023 atmospheric rivers, comparing predictions against Sentinel‐1 mapped flood observations. We compared historical predictions from 1979 to 2023 to flood damage reports in Sacramento County, California. Results show that NWM‐CNN captures inundation extent better than the Height Above Nearest Drainage (HAND) approach (25%–36% RMSE, respectively).

This study uses secondary data analysis to assess the relationship between social isolation (SI) and population density in the US, as well as any moderating influence that race/ethnicity may have on that relationship. A recent consensus on the measure of SI is that it focuses on the objective absence of social connections, whereas loneliness refers to subjective assessments of one’s social connections. Therefore, while the original study measured both objective and subjective SI may be innovative, it may also be overly ambitious. SI puts older people at risk for health problems, including an increased chance of dying. The AARP Foundation gathered the initial convenience sample, which included 8149 senior citizens. The study determined population density using self-reported zip codes, measured as persons per square mile, and divided the results into tertiles. Linear mixed models were used to investigate the moderating role of race/ethnicity between population density and SI. The findings revealed that greater population density was associated with less SI for individuals residing in zip codes with a higher percentage of the same race/ethnicity, but more SI for those in zip codes with a lower percentage of the same race/ethnicity. These results suggest that race/ethnicity should be considered in future studies or when developing policies and interventions to address SI among older adults in high-population-density areas. For example, when policymakers aim to address SI in a community, they may want to collect data based on zip codes and create targeted interventions for specific racial/ethnic groups within those zip code areas.

Organophosphorus nerve agents (OPNA) are hazardous environmental exposures to the civilian population and have been historically weaponized as chemical warfare agents (CWA). OPNA exposure can lead to several neurological, sensory, and motor symptoms that can manifest into chronic neurological illnesses later in life. There is still a large need for technological advancement to better understand changes in brain function following OPNA exposure. The human-relevant in vitro multi-electrode array (MEA) system, which combines the MEA technology with human stem cell technology, has the potential to monitor the acute, sub-chronic, and chronic consequences of OPNA exposure on brain activity. However, the application of this system to assess OPNA hazards and risks to human brain function remains to be investigated. In a concentration-response study, we have employed a human-relevant MEA system to monitor and detect changes in the electrical activity of engineered neural networks to increasing concentrations of the sarin surrogate 4-nitrophenyl isopropyl methylphosphonate (NIMP). We report a biphasic response in the spiking (but not bursting) activity of neurons exposed to low (i.e., 0.4 and 4 μM) versus high concentrations (i.e., 40 and 100 μM) of NIMP, which was monitored during the exposure period and up to 6 days post-exposure. Regardless of the NIMP concentration, at a network level, communication or coordination of neuronal activity decreased as early as 60 min and persisted at 24 h of NIMP exposure. Once NIMP was removed, coordinated activity was no different than control (0 μM of NIMP). Interestingly, only in the high concentration of NIMP did coordination of activity at a network level begin to decrease again at 2 days post-exposure and persisted on day 6 post-exposure. Notably, cell viability was not affected during or after NIMP exposure. Also, while the catalytic activity of AChE decreased during NIMP exposure, its activity recovered once NIMP was removed. Gene expression analysis suggests that human iPSC-derived neurons and primary human astrocytes resulted in altered genes related to the cell’s interaction with the extracellular environment, its intracellular calcium signaling pathways, and inflammation, which could have contributed to how neurons communicated at a network level.

The bone-cartilage interface is defined by a unique arrangement of cells and tissue matrix. Injury to the interface can contribute to the development of arthritic joint disease. Attempts to repair osteochondral damage through clinical trials have generated mixed outcomes. Tissue engineering offers the potential of integrated scaffold design with multiregional architecture to assist in tissue regeneration, such as the bone-cartilage interface. Challenges remain in joining distinct materials in a single scaffold mass while maintaining integrity and avoiding delamination. The aim of the current work is to examine the possibility of joining two closely related acrylamide derivatives such as, poly n-isopropyl acrylamide (pNIPAM) and poly n‑tert‑butyl acrylamide (pNTBAM). The target is to produce a single scaffold unit with distinct architectural regions in the favour of regenerating the osteochondral interface. Longitudinal phosphate glass fibres (PGFs) with the formula 50P2O5.30CaO.20Na2O were incorporated to provide additional bioactivity by degradation to release ions such as calcium and phosphate which are considered valuable to assist the mineralization process. Polymers were prepared via atom transfer radical polymerization (ATRP) and solutions cast to ensure the integration of polymers chains. Scaffold was characterized using scanning electron microscope (SEM) and Fourier transform infra-red (FTIR) techniques. The PGF mass degradation pattern was inspected using micro computed tomography (µCT). Biological assessment of primary human osteoblasts (hOBs) and primary human chondrocytes (hCHs) upon scaffolds was performed using alizarin red and colorimetric calcium assay for mineralization assessment; alcian blue staining and dimethyl-methylene blue (DMMB) assay for glycosaminoglycans (GAGs); immunostaining and enzyme-linked immunosorbent assay (ELISA) to detect functional proteins expression by cells such as collagen I, II, and annexin A2. FTIR analysis revealed an intact unit with gradual transformation from pNIPAM to pNTBAM. SEM images showed three distinct architectural regions with mean pore diameter of 54.5 µm (pNIPAM), 16.5 µm (pNTBAM) and 118 µm at the mixed interface. Osteogenic and mineralization potential by cells was observed upon the entire scaffold's regions. Chondrogenic activity was relevant on the pNTBAM side of the scaffold only with minimal evidence in the pNIPAM region. PGFs increased mineralization potential of both hOBs and hCHs, evidenced by elevated collagens I, X, and annexin A2 with reduction of collagen II in PGFs scaffolds. In conclusion, pNIPAM and pNTBAM integration created a multiregional scaffold with distinct architectural regions. Differential chondrogenic, osteogenic, and mineralized cell performance, in addition to the impact of PGF, suggests a potential role for phosphate glass-incorporated, acrylamide-derivative scaffolds in osteochondral interface regeneration.

Background: Institutional academic productivity varies on an individual level. This study aims to analyze the research output of adult reconstruction and arthroplasty fellowship programs in the United States. Methods: The American Association of Hip and Knee Surgeons Fellowship Directory was used to evaluate 112 adult reconstruction and arthroplasty fellowships in the United States. Publication data and Hirsch index (h-index) were collected from the Scopus Database. All of each author’s total publications were analyzed with their current institution, regardless of their affiliation at the time of publication. Multivariate logistic regressions were performed to determine the effect of program size on research productivity. Results: The total number of publications per institution ranged from 2 to 3743, with a mean of 289 and a median of 135. The h-index of individual faculty members ranged from 0 to 103, with a mean of 16 and a median of 11. The number of faculty (P < .001) and number of fellows (P = .003) per program had a significant effect on the total number of publications. The number of faculty did not have a significant effect on the median number of publications (P = .12) or the median h-index (P = .31). The number of fellows had a significant effect on the median number of publications (P < .001) and the median h-index (P < .001). Conclusions: Academic productivity in adult reconstruction and arthroplasty fellowships within the United States varies widely, with the top few institutions responsible for a majority of the overall output.

Background: Physical activity (PA) is essential following an acute cardiac event. Cardiac rehabilitation (CR) is commonly prescribed, and PA after CR is recommended. Because of age-related changes in functional ability and multi-comorbidity, many older cardiac patients struggle to continue performing PA at home after CR. Depressive symptoms and anxiety are prevalent in cardiac patients and associated with poor self-care, including lack of daily PA. Yoga has been demonstrated to improve psychological and physical health outcomes in cardiac patients, but it is unknown whether yoga, modified for older CR patients – Gentle Yoga – is beneficial in managing psychological distress and maintaining PA following phase II CR. Our specific aims are to:1) determine the feasibility and acceptability of a modified gentle yoga intervention delivered via video conferencing for older cardiac patients; 2) compare, at 3-month follow-up, the effects and determine effect sizes of a gentle yoga intervention versus control on psychological health and physical health. Methods: We are conducting a 2-group (intervention versus control) randomized controlled pilot study. The intervention is a 12-week gentle yoga program delivered via video conference. Short-term effects will be evaluated at 3-month. Conclusion: This study is designed to be suited for older cardiac patients who would not have access to supervised PA opportunities after facility-based CR to enhance PA. This study will provide data about the feasibility and acceptability of the protocol for older cardiac patients and will offer effect sizes to determine sample size for a fully powered randomized controlled trial.

A quasi-steady, CFD-based modeling approach is employed to investigate the heat loading within a small package of twenty-five 18650 Li-ion cells. The quasi-steady approach allows for computationally efficient simulations to capture the compressible and turbulent flow field through the safety vent structure and out into the space surrounding a failing cell. Combustion of vent gases leads to high heat loading on neighboring cells and nearby surfaces. Heat transfer mechanisms within the enclosure include convection from hot gases, radiation from the participating medium, and radiation exchange between surfaces. Simulations provide insight into the magnitude of each heat transfer mechanism, and the spatial distribution of heat flux on nearby cells and surfaces within the pack. The complex geometry of the safety vent geometry resulted in an asymmetric jet flow pattern, which induces highly localized impingement heat transfer on specific cells within the enclosure. Radiation from hot surfaces was more significant than radiation from hot gases and soot to neighboring cells. The quasi-steady simulations may be used in the future to develop reduced-order heat transfer models that include the effects of venting and combustion on propagating failure.

Abstract INTRODUCTION Neuropsychiatric symptoms (NPS) are highly prevalent in Alzheimer's disease (AD). There are no effective treatments targeting these symptoms. METHODS To facilitate identification of causative mechanistic pathways, we initiated an effort (NIH: U01AG079850) to collate, harmonize, and analyze all available NPS data (≈ 100,000 samples) of diverse ancestries with whole‐genome sequencing data from the Alzheimer's Disease Sequencing Project (ADSP). RESULTS This study will generate a genomic resource for Alzheimer's disease with both harmonized whole‐genome sequencing and NPS phenotype data that will be publicly available through NIAGADS. Primary analyses will (1) identify novel genetic risk factors associated with NPS in AD, (2) characterize the shared genetic architecture of NPS in AD and primary psychiatric disorders, and (3) assess the role of ancestry effects in the etiology of NPS in AD. DISCUSSION Expansion of the ADSP to harmonize and refine NPS phenotypes coupled with the proposed core analyses will lay the foundation to disentangle the molecular mechanisms underlying these detrimental symptoms in AD in diverse populations. Highlights Neuropsychiatric symptoms (NPS) are highly prevalent in Alzheimer's disease (AD). There are no effective treatments targeting NPS in AD. The current effort aims to collate, harmonize, and analyze all NPS data from the Alzheimer's Disease Sequencing Project. Core analyses will identify underlying genetic factors and mechanistic pathways. The harmonized genomic and phenotypic data from this initiative will be available through National Institute on Aging Genetics of Alzheimer's Disease Data Storage Site.

IntroductionGenetic predisposition to autoimmune encephalitis with antibodies against N-methyl-D-aspartate receptor (NMDAR) is poorly understood. Given the diversity of associated environmental factors (tumors, infections), we hypothesized that human leukocyte antigen (HLA) and killer-cell immunoglobulin-like receptors (KIR), two extremely polymorphic gene complexes key to the immune system, might be relevant for the genetic predisposition to anti-NMDAR encephalitis. Notably, KIR are chiefly expressed by Natural Killer (NK) cells, recognize distinct HLA class I allotypes and play a major role in anti-tumor and anti-infection responses.MethodsWe conducted a Genome Wide Association Study (GWAS) with subsequent control-matching using Principal Component Analysis (PCA) and HLA imputation, in a multi-ethnic cohort of anti-NMDAR encephalitis (n=479); KIR and HLA were further sequenced in a large subsample (n=323). PCA-controlled logistic regression was then conducted for carrier frequencies (HLA and KIR) and copy number variation (KIR). HLA-KIR interaction associations were also modeled. Additionally, single cell sequencing was conducted in peripheral blood mononuclear cells from 16 cases and 16 controls, NK cells were sorted and phenotyped.ResultsAnti-NMDAR encephalitis showed a weak HLA association with DRB1*01:01~DQA1*01:01~DQB1*05:01 (OR=1.57, 1.51, 1.45; respectively), and DRB1*11:01 (OR=1.60); these effects were stronger in European descendants and in patients without an underlying ovarian teratoma. More interestingly, we found increased copy number variation of KIR2DL5B (OR=1.72), principally due to an overrepresentation of KIR2DL5B*00201. Further, we identified two allele associations in framework genes, KIR2DL4*00103 (25.4% vs. 12.5% in controls, OR=1.98) and KIR3DL3*00302 (5.3% vs. 1.3%, OR=4.44). Notably, the ligands of these KIR2DL4 and KIR3DL3, respectively, HLA-G and HHLA2, are known to act as immune checkpoint with immunosuppressive functions. However, we did not find differences in specific KIR-HLA ligand interactions or HLA-G polymorphisms between cases and controls. Similarly, gene expression of CD56dim or CD56bright NK cells did not differ between cases and controls.DiscussionOur observations for the first time suggest that the HLA-KIR axis might be involved in anti-NMDAR encephalitis. While the genetic risk conferred by the identified polymorphisms appears small, a role of this axis in the pathophysiology of this disease appears highly plausible and should be analyzed in future studies.

ABSTRACT Coccidioidomycosis, also known as Valley fever, is a disease caused by the fungal pathogen Coccidioides. Unfortunately, patients are often misdiagnosed with bacterial pneumonia, leading to inappropriate antibiotic treatment. The soil Bacillus subtilis-like species exhibits antagonistic properties against Coccidioides in vitro; however, the antagonistic capabilities of host microbiota against Coccidioides are unexplored. We sought to examine the potential of the tracheal and intestinal microbiomes to inhibit the growth of Coccidioides in vitro. We hypothesized that an uninterrupted lawn of microbiota obtained from antibiotic-free mice would inhibit the growth of Coccidioides, while partial in vitro depletion through antibiotic disk diffusion assays would allow a niche for fungal growth. We observed that the microbiota grown on 2×GYE (GYE) and Columbia colistin and nalidixic acid with 5% sheep’s blood agar inhibited the growth of Coccidioides, but microbiota grown on chocolate agar did not. Partial depletion of the microbiota through antibiotic disk diffusion revealed diminished inhibition and comparable growth of Coccidioides to controls. To characterize the bacteria grown and identify potential candidates contributing to the inhibition of Coccidioides, 16S rRNA sequencing was performed on tracheal and intestinal agar cultures and murine lung extracts. We found that the host bacteria likely responsible for this inhibition primarily included Lactobacillus and Staphylococcus. The results of this study demonstrate the potential of the host microbiota to inhibit the growth of Coccidioides in vitro and suggest that an altered microbiome through antibiotic treatment could negatively impact effective fungal clearance and allow a niche for fungal growth in vivo.IMPORTANCECoccidioidomycosis is caused by a fungal pathogen that invades the host lungs, causing respiratory distress. In 2019, 20,003 cases of Valley fever were reported to the CDC. However, this number likely vastly underrepresents the true number of Valley fever cases, as many go undetected due to poor testing strategies and a lack of diagnostic models. Valley fever is also often misdiagnosed as bacterial pneumonia, resulting in 60%–80% of patients being treated with antibiotics prior to an accurate diagnosis. Misdiagnosis contributes to a growing problem of antibiotic resistance and antibiotic-induced microbiome dysbiosis; the implications for disease outcomes are currently unknown. About 5%–10% of symptomatic Valley fever patients develop chronic pulmonary disease. Valley fever causes a significant financial burden and a reduced quality of life. Little is known regarding what factors contribute to the development of chronic infections and treatments for the disease are limited.