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An incongruence exists between the growing job opportunities in rural areas and the educational opportunities provided to rural students, especially in science, technology, engineering, and mathematics (STEM) fields. Positive change is possible, however. As one means to tackle this complex issue, this study sought to understand the perceptions and experiences of students who graduated from rural high schools and pursued STEM majors in college as exemplars of rural students who pursued postsecondary education in STEM disciplines. This phenomenological study explored how college students in STEM majors perceived their rural schools and communities as influencing their academic journeys. Rather than focusing only on deficits and inequities associated with rurality, this study employed the rural cultural wealth (RCW) framework to highlight the agency and capital rural students employed within their journeys. Qualitative data analysis revealed that participants' comments about their rural schools and communities were diverse. The findings also aligned and extended current literature and the RCW framework. These insights into these STEM students' rural backgrounds and academic journeys were then used to create recommendations for future P-20 educational research, practices, and policies.

This study investigates common features of a set of diverse schools' responses to the initial school lockdown period during the pandemic in 2020, with a focus on practices supporting learning, inclusion and wellbeing. It comprises a collective case study of four Australian schools that were selected based on their reputation for impactful support of students and teachers during the emergency remote teaching period. Methods included interviews and focus groups with school leaders, teachers and students. The schools had widely differing contexts, technology access and student needs. Despite these varied contexts, the findings provided important insights into common practices supporting effective remote teaching. Emerging principles of effective practice illuminate ways forward to mitigate the significant risks accompanying emergency remote teaching, and guide practices in a variety of school contexts.

Objectives: We tested the feasibility and efficacy of a brief mindfulness-based intervention (bMBI) for adult brain tumor (BT) survivors experiencing elevated depressive symptoms. Specifically, we examined whether there are improvements in mental health symptoms (i.e. depressive symptoms, quality of life (QOL), mental wellbeing, perceived stress) among participants at post-intervention.Methods: Nineteen BT survivors participated in a five-session, in-person, group-based bMBI intervention. Data on attendance, depressive symptoms, QOL, mental well-being, and perceived stress were collected at three timepoints. Findings: The preliminary analysis supports that this bMBI: (a) was efficacious in decreasing depressive symptoms and perceived stress, as well as increasing mental well-being and QOL, and (b) was feasible, with a retention rate greater than 70% and an attendance rate greater than 80% for adult BT survivors.Conclusions: These findings lend support for a feasible and efficacious intervention for adult BT survivors experiencing elevated depressive symptomsImplications: With the paucity of psychosocial interventions targeting adult BT survivorship, future empirically rigorous studies for this population are warranted. The uptake/adoption of this bMBI by psychosocial providers may play an important role in improving the mental health and QOL of adult BT survivors.

Abstract Global climate patterns fundamentally shape the distribution of species and ecosystems. For example, Bergmann’s rule predicts that homeothermic animals, including birds and mammals, inhabiting cooler climates are generally larger than close relatives from warmer climates. The modern world, however, lacks the comparative data needed to evaluate such macroecological rules rigorously. Here, we test for Bergmann’s rule in Mesozoic dinosaurs and mammaliaforms that radiated within relatively temperate global climate regimes. We develop a phylogenetic model that accounts for biases in the fossil record and allows for variable evolutionary dispersal rates. Our analysis also includes new fossil data from the extreme high-latitude Late Cretaceous Arctic Prince Creek Formation. We find no evidence for Bergmann’s rule in Mesozoic dinosaurs or mammaliaforms, the ancestors of extant homeothermic birds and mammals. When our model is applied to thousands of extant dinosaur (bird) and mammal species, we find that body size evolution remains independent of latitude. A modest temperature effect is found in extant, but not in Mesozoic, birds, suggesting that body size evolution in modern birds was influenced by Bergmann’s rule during Cenozoic climatic change. Our study provides a general approach for studying macroecological rules, highlighting the fossil record’s power to address longstanding ecological principles.

Abstract Background Previous research has demonstrated that early weight gain in family-based treatment (FBT) is predictive of remission for adolescents with anorexia nervosa (AN). However, no published data has addressed if early weight gain is also predictive of reaching weight restoration (i.e., 95% EBW) in patients with avoidant/restrictive food intake disorder (ARFID). Furthermore, no studies have evaluated the performance of the statistical models used to predict weight restoration at the end of treatment. This study sought to examine whether early weight gain in ARFID is predictive of weight restoration at 20 weeks using ROC analysis. Additionally, this study assessed how accurately the model classified patients and what types of misclassifications occurred. Methods Participants (n = 130, 57.7% cisgender female 70.0% white) received virtual outpatient FBT. Receiver operating characteristics (ROC) were used to predict successful weight restoration at end of treatment, using early weight gain as the predictor. Twenty weeks was considered as the end of treatment, to align with the definition of end of treatment in FBT clinical trials. ROC analyses demonstrated that gaining at least 6.2 pounds by week 5 of treatment was the strongest predictor of achieving 95% EBW at 20 weeks (AUC = 0.72 [0.63, 0.81]). ROC analyses misclassified 35% of patients; the most common misclassification was predicting that a patient would not achieve 95% EBW when they actually did (61.6%). A logistical regression model, which included the patients’ %EBW at admission in addition to early weight gain as a predictor, outperformed the ROC analyses (AUC = 0.90 [0.85, 0.95]) and provided additional context by showing the probability that a patient would succeed. Conclusion Taken together, research demonstrates that early weight gain is a useful predictor of 95% EBW at 20 weeks of treatment for patients with ARFID who require weight restoration. Furthermore, results suggest that statistical models need to take into account additional information, such as %EBW at admission, along with early weight gain in order to more accurately predict which patients will reach weight restoration at week 20.

Quantum processors based on linear arrays of trapped ions have achieved exceptional performance, but scaling to large qubit numbers requires realizing two-dimensional ion arrays as envisioned in the quantum charge-coupled device (QCCD) architecture. Here, we present a scalable method for the control of ion crystals in a grid-based surface-electrode Paul trap and characterize it in the context of transport operations that sort and reorder multispecies crystals. By combining cowiring of control electrodes at translationally symmetric locations in each grid site with the sitewise ability to exchange the voltages applied to two special electrodes gated by a binary input, site-dependent operations can be achieved using only a fixed number of analog voltage signals and a single digital input per site. In two separate experimental systems containing nominally identical grid traps, one using ^{171}Yb^{+}-^{138}Ba^{+} crystals and the other ^{137}Ba^{+}-^{88}Sr^{+}, we demonstrate this method by characterizing the conditional intrasite crystal reorder and the conditional exchange of ions between adjacent sites on the grid. Averaged across a multisite region of interest, we measure subquanta motional excitation in the axial in-phase and out-of-phase modes of the crystals following these operations at exchange rates of 2.5 kHz. In this initial demonstration, the logic controlling the voltage exchange occurs in software, but the applied signals mimic a proposed hardware implementation using crossover switches. These techniques can be further extended to implement other conditional operations in the QCCD architecture such as gates, initialization, and measurement.

Abstract Background Sars-CoV-2, the causative agent of COVID-19, has led to more than 226,000 deaths in the UK and multiple risk factors for mortality including age, sex and deprivation have been identified. This study aimed to identify which individual indicators of the Scottish Index of Multiple Deprivation (SIMD), an area-based deprivation index, were predictive of mortality. Methods This was a prospective cohort study of anonymised electronic health records of 710 consecutive patients hospitalised with Covid-19 disease between March and June 2020 in the Lothian Region of Southeast Scotland. Data sources included automatically extracted data from national electronic platforms and manually extracted data from individual admission records. Exposure variables of interest were SIMD quintiles and 12 indicators of deprivation deemed clinically relevant selected from the SIMD. Our primary outcome was mortality. Age and sex adjusted univariable and multivariable analyses were used to determine measures of association between exposures of interest and the primary outcome. Results After adjusting for age and sex, we found an increased risk of mortality in the more deprived SIMD quintiles 1 and 3 (OR 1.75, CI 0.99–3.08, p = 0.053 and OR 2.17, CI 1.22–3.86, p = 0.009, respectively), but this association was not upheld in our multivariable model containing age, sex, Performance Status and clinical parameters of severity at admission. Of the 12 pre-selected indicators of deprivation, two were associated with greater mortality in our multivariable analysis: income deprivation rate categorised by quartile (Q4 (most deprived): 2.11 (1.20–3.77) p = 0.011)) and greater than expected hospitalisations due to alcohol per SIMD data zone (1.96 (1.28–3.00) p = 0.002)). Conclusions SIMD as an aggregate measure of deprivation was not predictive of mortality in our cohort when other exposure measures were accounted for. However, we identified a two-fold increased risk of mortality in patients residing in areas with greater income-deprivation and/or number of hospitalisations due to alcohol. In areas where aggregate measures fail to capture pockets of deprivation, exploring the impact of specific SIMD indicators may be helpful in targeting resources to residents at risk of poorer outcomes from Covid-19.

Duchenne muscular dystrophy (DMD) is a progressive neuromuscular disease caused by mutations to the dystrophin gene, resulting in deficiency of dystrophin protein, loss of myofiber integrity in skeletal and cardiac muscle, and eventual cell death and replacement with fibrotic tissue. Pathologic cardiac manifestations occur in nearly every DMD patient, with the development of cardiomyopathy—the leading cause of death—inevitable by adulthood. As early cardiac abnormalities are difficult to detect, timely diagnosis and appropriate treatment modalities remain a challenge. There is no cure for DMD; treatment is aimed at delaying disease progression and alleviating symptoms. A comprehensive understanding of the pathophysiological mechanisms is crucial to the development of targeted treatments. While established hypotheses of underlying mechanisms include sarcolemmal weakening, upregulation of pro-inflammatory cytokines, and perturbed ion homeostasis, mitochondrial dysfunction is thought to be a potential key contributor. Several experimental compounds targeting the skeletal muscle pathology of DMD are in development, but the effects of such agents on cardiac function remain unclear. The synergistic integration of small molecule- and gene-target-based drugs with metabolic-, immune-, or ion balance-enhancing compounds into a combinatorial therapy offers potential for treating dystrophin deficiency-induced cardiomyopathy, making it crucial to understand the underlying mechanisms driving the disorder.

Abstract Memory impairments have been well documented in people with Duchenne muscular dystrophy (DMD). However, the underlying mechanisms are poorly understood, and there is an unmet need to develop new therapies to treat this condition. Using a novel object recognition test, we show that recognition memory impairments in D2.mdx mice are completely prevented by daily treatment with the new adiponectin receptor agonist ALY688 from day 7 to 28 of age. In comparison to age‐matched wild‐type mice, untreated D2.mdx mice demonstrated lower hippocampal mitochondrial respiration (carbohydrate substrate), greater serum interleukin‐6 cytokine content and greater hippocampal total tau and Raptor protein contents. Each of these measures was partly or fully preserved after treatment with ALY688. Collectively, these results indicate that adiponectin receptor agonism improves recognition memory in young D2.mdx mice.

Abstract Introduction Gonorrhea, caused by Neisseria gonorrhoeae (NG), is the second most common bacterial sexually transmitted infection (STI). Rates of antimicrobial resistance to standard care are increasing worldwide, with many antibiotic classes now ineffective against NG. Gepotidacin is a first-in-class, bactericidal, triazaacenaphthylene antibiotic that inhibits bacterial DNA replication by inhibition of two enzymes, where a single target-specific mutation does not significantly impact susceptibility. Gepotidacin confers activity against NG, including most strains resistant to marketed antibiotics. Here, we describe the design of a phase 3 clinical trial (EAGLE-1; NCT04010539) evaluating gepotidacin for the treatment of uncomplicated urogenital gonorrhea. Methods This phase 3, randomized, multicenter, sponsor-blinded, noninferiority study across six countries is comparing the efficacy of gepotidacin with ceftriaxone plus azithromycin in 400 patients with uncomplicated urogenital gonorrhea (microbiological intent-to-treat population) and assessing the safety of gepotidacin in approximately 600 patients (intent-to-treat population). Eligible participants 12 years of age or older with clinical suspicion of urogenital gonococcal infection and a NG-positive urogenital sample and/or purulent discharge are randomized 1:1 to receive oral gepotidacin (2 × 3000 mg 10–12 h apart) or ceftriaxone (500 mg, intramuscular) plus azithromycin (1 g, oral). The primary endpoint is culture-confirmed bacterial eradication of NG from the urogenital site at the test-of-cure (days 4–8) visit. Planned Outcomes This trial was designed in accordance with US Food and Drug Administration (2015) and European Medicines Agency (2011) guidance, particularly the primary endpoint and microbiological evaluability requirements. This study will help characterize the risk–benefit profile of gepotidacin for treating uncomplicated urogenital gonorrhea. Gepotidacin is an important potential treatment for gonorrhea to help address the urgent unmet need of multidrug resistance and the increasingly limited number of oral treatment options. Trial Registration ClinicalTrials.gov identifier, NCT04010539.

IntroductionIndividuals with type 1 diabetes (T1D) experience a complex set of alterations to skeletal muscle metabolic, neuromuscular, and vascular health; collectively referred to as diabetic myopathy. While the full scope of diabetic myopathy is still being elucidated, evidence suggests that even when individuals with T1D are physically active, indices of myopathy still exist. As such, there is a question if adherence to current physical activity guidelines elicits improvements in skeletal muscle health indices similarly between individuals with and without T1D. The objectives of this trial are to: 1) compare baseline differences in skeletal muscle health between adults with and without T1D, 2) examine the association between participation in a home-based exercise program, detraining, and retraining, with changes in skeletal muscle health, and 3) examine the roles of age and sex on these associations.Methods and analysisThis will be a prospective interventional trial. Younger (18-30 years) and older (45-65 years) males and females with T1D and matched individuals without T1D will engage in a four-phase, 18-week study sequentially consisting of a one-week lead-in period, 12-week exercise training program, one-week detraining period, and four-week retraining period. The exercise program will consist of aerobic and resistance exercise based on current guidelines set by Diabetes Canada. Metabolic, neuromuscular, and vascular outcome measures will be assessed four times: at baseline, post-exercise program, post-detraining, and post-retraining. Differences in baseline metrics between those with and without T1D will be examined with independent sample t-tests, and with two-way analyses of variance for age- and sex-stratified analyses. Changes across the duration of the study will be examined using mixed-model analyses.DisseminationFindings from this research will be shared locally and internationally with research participants, clinicians, diabetes educators, and patient advocacy organizations via in-person presentations, social media, and scientific fora.Trial registration numberNCT05740514.

Abstract Gepotidacin is a novel triazaacenaphthylene antibiotic in phase III development. Based on nonclinical in vitro characterization of gepotidacin metabolism, two phase I studies were conducted in healthy participants to investigate clinical drug–drug interactions (DDIs). We assessed gepotidacin as a DDI victim with a potent cytochrome P450 (CYP) 3A4/P‐glycoprotein (P‐gp) inhibitor (itraconazole), potent CYP3A4 inducer (rifampicin), and nonspecific organic cation transporter (OCT)/multidrug and toxic extrusion transporter (MATE) renal transport inhibitor (cimetidine) via single doses of gepotidacin before and after co‐administration with multiple doses of the modulator drugs. Gepotidacin DDI perpetrator potential for P‐gp inhibition (digoxin) and CYP3A4 inhibition (midazolam) was evaluated via single doses of the two‐drug cocktail without and with gepotidacin. The DDI magnitudes were interpreted based on area under the concentration‐time curve (AUC). A weak DDI (AUC increase 48%–50%) was observed for gepotidacin co‐administered with itraconazole. A clinically significant decrease in gepotidacin plasma AUC (52%) was observed with rifampicin coadministration, indicating a moderate DDI. There was no DDI for gepotidacin with cimetidine; a unique biomarker approach showed increased serum creatinine (24%), decreased renal clearance of creatinine (21%), and N1‐methylnicotinamide (39%), which confirmed extensive MATE inhibition and partial OCT2 inhibition. Gepotidacin was not a P‐gp DDI perpetrator, although the maximum plasma concentration of digoxin increased (53%) and is potentially clinically relevant given its narrow therapeutic index. Gepotidacin demonstrated weak CYP3A4 inhibition with midazolam (

Duchenne muscular dystrophy (DMD) is caused by genetic mutations in the cytoskeletal-sarcolemmal anchor protein dystrophin. Repeated cycles of sarcolemmal tearing and repair lead to a variety of secondary cellular and physiological stressors that are thought to contribute to weakness, atrophy, and fibrosis. Collectively, these stressors can contribute to a pro-inflammatory milieu in locomotor, cardiac, and respiratory muscles. Given the many unwanted side effects that accompany current anti-inflammatory steroid-based approaches for treating DMD (e.g., glucocorticoids), there is a need to develop new therapies that address inflammation and other cellular dysfunctions. Adiponectin receptor (AdipoR) agonists, which stimulate AdipoR1 and R2 isoforms on various cell types, have emerged as therapeutic candidates for DMD due to their anti-inflammatory, anti-fibrotic, and pro-myogenic properties in pre-clinical human and rodent DMD models. Although these molecules represent a new direction for therapeutic intervention, the mechanisms through which they elicit their beneficial effects are not yet fully understood, and DMD-specific data is limited. The overarching goal of this review is to investigate how adiponectin signaling may ameliorate pathology associated with dystrophin deficiency through inflammatory-dependent and -independent mechanisms and to determine if current data supports their future progression to clinical trials.

Abstract Biological regulation ubiquitously depends on protein allostery, but the regulatory mechanisms are incompletely understood, especially in proteins that undergo ligand-induced allostery with few structural changes. Here we used hydrogen-deuterium exchange with mass spectrometry (HDX/MS) to map allosteric effects in a paradigm ligand-responsive transcription factor, the lac repressor (LacI), in different functional states (apo, or bound to inducer, anti-inducer, and/or DNA). Although X-ray crystal structures of the LacI core domain in these states are nearly indistinguishable, HDX/MS experiments reveal widespread differences in flexibility. We integrate these results with modeling of protein-ligand-solvent interactions to propose a revised model for allostery in LacI, where ligand binding allosterically shifts the conformational ensemble as a result of distinct changes in the rigidity of secondary structures in the different states. Our model provides a mechanistic basis for the altered function of distal mutations. More generally, our approach provides a platform for characterizing and engineering protein allostery.

BackgroundAcetylcholine-induced chest pain is routinely measured during the assessment of microvascular function.AimsThe aim was to determine the relationships between acetylcholine-induced chest pain and both symptom burden and objective measures of vascular function.MethodsIn patients with angina but no obstructive coronary artery disease, invasive studies determined the presence or absence of chest pain during both acetylcholine and adenosine infusion. Thermodilution-derived coronary blood flow (CBF) and index of microvascular resistance (IMR) was determined at rest and during both acetylcholine and adenosine infusion. Patients with epicardial spasm (>90%) were excluded; vasoconstriction between 20% and 90% was considered endothelial dysfunction.ResultsEighty-seven patients met the inclusion criteria. Of these 52 patients (60%) experienced chest pain during acetylcholine while 35 (40%) did not. Those with acetylcholine-induced chest pain demonstrated: (1) Increased CBF at rest (1.6 ± 0.7 vs. 1.2 ± 0.4, p = 0.004) (2) Decreased IMR with acetylcholine (acetylcholine-IMR = 29.7 ± 16.3 vs. 40.4 ± 17.1, p = 0.004), (3) Equivalent IMR following adenosine (Adenosine-IMR: 21.1 ± 10.7 vs. 21.8 ± 8.2, p = 0.76), (4) Increased adenosine-induced chest pain (40/52 = 77% vs. 7/35 = 20%, p

Summary: Multi-cellular tumor spheroids (MCTS) have found widespread use in pre-clinical research. However, their complex three-dimensional structure makes immunofluorescent staining and imaging challenging. Here, we present a protocol for whole spheroid staining and automated imaging using laser-scanning confocal microscopy. We describe steps for cell culture, seeding of spheroids and transfer of MCTS, and adhesion to Ibidi chamber slides. We then detail fixation, immunofluorescent staining based on optimized reagent concentrations and incubation times, and confocal imaging facilitated by glycerol-based optical clearing. : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Abstract Cutaneous squamous cell carcinoma (cSCC) is a disease with globally rising incidence and poor prognosis for patients with advanced or metastatic disease. Epithelial-mesenchymal transition (EMT) is a driver of metastasis in many carcinomas, and cSCC is no exception. We aimed to provide a systematic overview of the clinical and experimental evidence for EMT in cSCC, with critical appraisal of type and quality of the methodology used. We then used this information as rationale for potential drug targets against advanced and metastatic cSCC. All primary literature encompassing clinical and cell-based or xenograft experimental studies reporting on the role of EMT markers or related signalling pathways in the progression of cSCC were considered. A screen of 3443 search results yielded 86 eligible studies comprising 44 experimental studies, 22 clinical studies, and 20 studies integrating both. From the clinical studies a timeline illustrating the alteration of EMT markers and related signalling was evident based on clinical progression of the disease. The experimental studies reveal connections of EMT with a multitude of factors such as genetic disorders, cancer-associated fibroblasts, and matrix remodelling via matrix metalloproteinases and urokinase plasminogen activator. Additionally, EMT was found to be closely tied to environmental factors as well as to stemness in cSCC via NFκB and β-catenin. We conclude that the canonical EGFR, canonical TGF-βR, PI3K/AKT and NFκB signalling are the four signalling pillars that induce EMT in cSCC and could be valuable therapeutic targets. Despite the complexity, EMT markers and pathways are desirable biomarkers and drug targets for the treatment of advanced or metastatic cSCC. Graphical Abstract

Abstract Background AMSTAR-2 is a 16-item assessment tool to check the quality of a systematic review and establish whether the most important elements are reported. ROBIS is another assessment tool which was designed to evaluate the level of bias present within a systematic review. Our objective was to compare, contrast and establish both inter-rater reliability and usability of both tools as part of two overviews of systematic reviews. Strictly speaking, one tool assesses methodological quality (AMSTAR-2) and the other assesses risk of bias (ROBIS), but there is considerable overlap between the tools in terms of the signalling questions. Methods Three reviewers independently assessed 31 systematic reviews using both tools. The inter-rater reliability of all sub-sections using each instrument (AMSTAR-2 and ROBIS) was calculated using Gwet’s agreement coefficient (AC1 for unweighted analysis and AC2 for weighted analysis). Results Thirty-one systematic reviews were included. For AMSTAR-2, the median agreement for all questions was 0.61. Eight of the 16 AMSTAR-2 questions had substantial agreement or higher (> 0.61). For ROBIS, the median agreement for all questions was also 0.61. Eleven of the 24 ROBIS questions had substantial agreement or higher. Conclusion ROBIS is an effective tool for assessing risk of bias in systematic reviews and AMSTAR-2 is an effective tool at assessing quality. The median agreement between raters for both tools was identical (0.61). Reviews that included a meta-analysis were easier to rate with ROBIS; however, further developmental work could improve its use in reviews without a formal synthesis. AMSTAR-2 was more straightforward to use; however, more response options would be beneficial.

In recent years, there has been an increase in programs designed to promote involved and responsible fatherhood. While the literature provides insight into how existing organizations serving fathers can improve the quality of their service delivery, little is known about starting a fatherhood program from the ground up. This article contributes to the needed discussion on such programs by exploring the development of the Man Up fatherhood program. Featured in this discussion is Man Up’s program development model, which combines parent education and community engagement events and activities and engages fathers at a level that transcends their involvement as program participants or research subjects. Engaging and promoting responsible fatherhood through community events is one of the ways that distinguishes Man Up from other fatherhood programs.

Metastatic cutaneous squamous cell carcinoma (CSCC) is a highly morbid disease requiring radical surgery and adjuvant therapy, which is associated with a poor prognosis. Yet, compared to other advanced malignancies, relatively little is known of the genomic landscape of metastatic CSCC. We have previously reported the mutational signatures and mutational patterns of CCCTC-binding factor (CTCF) regions in metastatic CSCC. However, many other genomic components (indel signatures, non-coding drivers, and structural variants) of metastatic CSCC have not been reported. To this end, we performed whole genome sequencing on lymph node metastases and blood DNA from 25 CSCC patients with regional metastases of the head and neck. We designed a multifaceted computational analysis at the whole genome level to provide a more comprehensive perspective of the genomic landscape of metastatic CSCC. In the non-coding genome, 3′ untranslated region (3′UTR) regions of EVC (48% of specimens), PPP1R1A (48% of specimens), and ABCA4 (20% of specimens) along with the tumor-suppressing long non-coding RNA (lncRNA) LINC01003 (64% of specimens) were significantly functionally altered (Q-value < 0.05) and represent potential non-coding biomarkers of CSCC. Recurrent copy number loss in the tumor suppressor gene PTPRD was observed. Gene amplification was much less frequent, and few genes were recurrently amplified. Single nucleotide variants driver analyses from three tools confirmed TP53 and CDKN2A as recurrently mutated genes but also identified C9 as a potential novel driver in this disease. Furthermore, indel signature analysis highlighted the dominance of ID signature 13 (ID13) followed by ID8 and ID9. ID9 has previously been shown to have no association with skin melanoma, unlike ID13 and ID8, suggesting a novel pattern of indel variation in metastatic CSCC. The enrichment analysis of various genetically altered candidates shows enrichment of “TGF-beta regulation of extracellular matrix” and “cell cycle G1 to S check points.” These enriched terms are associated with genetic instability, cell proliferation, and migration as mechanisms of genomic drivers of metastatic CSCC.

Abstract Background With increasing concerns about the impact of frequent antibiotic usage on the human microbiome, it is important to characterize the potential for such effects in early antibiotic drug development clinical trials. In a randomised Phase 2a clinical trial study that evaluated the pharmacokinetics of repeated oral doses of gepotidacin, a first-in-chemical-class triazaacenaphthylene antibiotic with a distinct mechanism of action, in adult females with uncomplicated urinary tract infections for gepotidacin (GSK2140944) we evaluated the potential changes in microbiome composition across multiple time points and body-sites ( ClinicalTrials.gov : NCT03568942). Results Samples of gastrointestinal tract (GIT), pharyngeal cavity and vaginal microbiota were collected with consent from 22 patients at three time points relative to the gepotidacin dosing regimen; Day 1 (pre-dose), Day 5 (end of dosing) and Follow-up (Day 28 ± 3 days). Microbiota composition was determined by DNA sequencing of 16S rRNA gene variable region 4 amplicons. By Day 5, significant changes were observed in the microbiome diversity relative to pre-dose across the tested body-sites. However, by the Follow-up visit, microbiome diversity changes were reverted to compositions comparable to Day 1. The greatest range of microbiome changes by body-site were GIT followed by the pharyngeal cavity then vagina. In Follow-up visit samples we found no statistically significant occurrences of pathogenic taxa. Conclusion Our findings suggest that gepotidacin alteration of the human microbiome after 5 days of dosing is temporary and rebound to pre-dosing states is evident within the first month post-treatment. We recommend that future antibiotic drug trials include similar exploratory investigations into the duration and context of microbiome modification and recovery. Trial registration NCT03568942 . Registered 26 June 2018.

Abstract Chronic kidney disease (CKD) leads to musculoskeletal impairments that are impacted by muscle metabolism. We tested the hypothesis that 10-weeks of voluntary wheel running can improve skeletal muscle mitochondria activity and function in a rat model of CKD. Groups included (n = 12–14/group): (1) normal littermates (NL); (2) CKD, and; (3) CKD-10 weeks of voluntary wheel running (CKD-W). At 35-weeks old the following assays were performed in the soleus and extensor digitorum longus (EDL): targeted metabolomics, mitochondrial respiration, and protein expression. Amino acid-related compounds were reduced in CKD muscle and not restored by physical activity. Mitochondrial respiration in the CKD soleus was increased compared to NL, but not impacted by physical activity. The EDL respiration was not different between NL and CKD, but increased in CKD-wheel rats compared to CKD and NL groups. Our results demonstrate that the soleus may be more susceptible to CKD-induced changes of mitochondrial complex content and respiration, while in the EDL, these alterations were in response the physiological load induced by mild physical activity. Future studies should focus on therapies to improve mitochondrial function in both types of muscle to determine if such treatments can improve the ability to adapt to physical activity in CKD.

Abstract Background Accurate diagnosis in patients with suspected coronavirus disease 2019 (COVID-19) is essential to guide treatment and limit spread of the virus. The combined nasal and throat swab is used widely, but its diagnostic performance is uncertain. Methods In a prospective, multi-centre, cohort study conducted in secondary and tertiary care hospitals in Scotland, we evaluated the combined nasal and throat swab with reverse transcriptase-polymerase chain reaction (RT-PCR) for severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) in consecutive patients admitted to hospital with suspected COVID-19. Diagnostic performance of the index and serial tests was evaluated for a primary outcome of confirmed or probable COVID-19, and a secondary outcome of confirmed COVID-19 on serial testing. The diagnosis was adjudicated by a panel, who recorded clinical, laboratory and radiological features blinded to the test results. Results We enrolled 1368 consecutive patients (median age 68 [interquartile range, IQR 53–80] years, 47% women) who underwent a total of 3822 tests (median 2 [IQR 1–3] tests per patient). The primary outcome occurred in 36% (496/1368), of whom 65% (323/496) and 35% (173/496) had confirmed and probable COVID-19, respectively. The index test was positive in 255/496 (51%) patients with the primary outcome, giving a sensitivity and specificity of 51.4% (95% confidence interval [CI] 48.8 to 54.1%) and 99.5% (95% CI 99.0 to 99.8%). Sensitivity increased in those undergoing 2, 3 or 4 tests to 60.1% (95% CI 56.7 to 63.4%), 68.3% (95% CI 64.0 to 72.3%) and 77.6% (95% CI 72.7 to 81.9%), respectively. The sensitivity of the index test was 78.9% (95% CI 74.4 to 83.2%) for the secondary outcome of confirmed COVID-19 on serial testing. Conclusions In patients admitted to hospital, a single combined nasal and throat swab with RT-PCR for SARS-CoV-2 has excellent specificity, but limited diagnostic sensitivity for COVID-19. Diagnostic performance is significantly improved by repeated testing.

Introduction Patients with major depressive disorder (MDD) with acute suicidal ideation or behavior (MDSI) require immediate intervention. Though oral antidepressants can be effective at reducing depressive symptoms, they can take 4–6 weeks to reach full effect. Objectives This study aimed to identify unmet needs in the treatment of patients with MDSI, specifically exploring the potential clinical benefits of rapid reduction of depressive symptoms. Methods A Delphi panel consisting of practicing psychiatrists (n=12) from the US, Canada and EU was conducted between December 2020–June 2021. Panelists were screened to ensure they had sufficient experience with managing patients with MDD and MDSI. Panelists completed two survey rounds, and a virtual consensus meeting. Results This research confirmed current unmet needs in the treatment of patients with MDSI. Hopelessness, functional impairment, worsening of MDD symptoms, recurrent hospitalization and higher risk of suicide attempt were considered as key consequences of the slow onset of action of oral antidepressants. Treatment with rapid acting antidepressant was anticipated by panelists to provide short-term benefit such as rapid reduction of core MDD symptoms which may contribute to shorter hospital stays and improved patient engagement/compliance, allowing for earlier interventions and improved patient outcomes. For long-term benefits, panelists agreed that improved daily functioning and increased trust/confidence in treatment options, constitute key benefits of rapid-acting treatments Conclusions There is need for rapid-acting treatments which may help address key unmet needs and provide clinically meaningful benefits driven by the rapid relief of depressive symptoms particularly in patients with MDSI. Disclosure SB, ED, KJ, MO’H, QZ, MM, MH, SR, JA and DZ are employees of Janssen and hold stock in Johnson & Johnson Inc. AN is currently employed by Neurocrine Biosciences Inc. RP is an employee of Adelphi Values PROVE hired by Janssen.

Cutaneous squamous cell carcinoma (cSCC) of the head and neck region is the second most prevalent skin cancer, with metastases to regional lymph nodes occurring in 2%–5% of cases. To further our understanding of the molecular events characterizing cSCC invasion and metastasis, we conducted targeted cancer progression gene expression and pathway analysis in non-metastasizing (PRI-) and metastasizing primary (PRI+) cSCC tumors of the head and neck region, cognate lymph node metastases (MET), and matched sun-exposed skin (SES). The highest differentially expressed genes in metastatic (MET and PRI+) versus non-metastatic tumors (PRI-) and SES included PLAU, PLAUR, MMP1, MMP10, MMP13, ITGA5, VEGFA, and various inflammatory cytokine genes. Pathway enrichment analyses implicated these genes in cellular pathways and functions promoting matrix remodeling, cell survival and migration, and epithelial to mesenchymal transition, which were all significantly activated in metastatic compared to non-metastatic tumors (PRI-) and SES. We validated the overexpression of urokinase plasminogen activator receptor (uPAR, encoded by PLAUR) in an extended patient cohort by demonstrating higher uPAR staining intensity in metastasizing tumors. As pathway analyses identified epidermal growth factor (EGF) as a potential upstream regulator of PLAUR, the effect of EGF on uPAR expression levels and cell motility was functionally validated in human metastatic cSCC cells. In conclusion, we propose that uPAR is an important driver of metastasis in cSCC and represents a potential therapeutic target in this disease.

Abstract Attitudes, which can be thought of as the sum of individuals' thoughts, feelings, and beliefs concerning an attitude object, inform how people interact with the world around them. An understanding of attitudes may play an important role in promoting desirable human behavior, and attitudes studies should be incorporated into any behavior‐change intervention. One framework for understanding attitudes is the ABC, or “tripartite” model, which says that affect (“A,” i.e., emotional response); previous behavior (“B”); and cognition (“C,” i.e., beliefs) are the basis for an individual's attitude. Although this framework is widely used in social psychology, few conservation studies break down the “attitude” monolith into these more usable components. In this study, we sought apply the ABC framework to understand how affect and cognitive beliefs relate to overall attitude toward predators across southern Kenya and north‐west Zimbabwe. We used a factor analysis approach to identify latent affective (n = 3) and cognitive (n = 3) factors relating to human–predator interactions. These factors were then used to construct a regression model, which examined the power of the psychological factors to explain overall attitudes toward predators. We found that the model explained 27% of variation in attitudes, with four independently significant factors: location; perceived harms of living with predators; perceived benefits of killing predators; and positive affect. Although effect sizes were relatively small, these results suggest that cognitive beliefs may substantially influence attitudes, and therefore that interventions which seek to (a) remove the factual basis for negative beliefs and (b) improve perceptions or dispel unfounded beliefs may play a significant role in changing overall attitudes toward predators.

Respiratory system damage is the primary cause of mortality in individuals who are exposed to vesicating agents including sulfur mustard (SM). Despite these devastating health complications, there are no fielded therapeutics that are specific for such injuries. Previous studies reported that SM inhalation depleted the tracheobronchial airway epithelial stem cell (TSC) pool and supported the hypothesis, TSC replacement will restore airway epithelial integrity and improve health outcomes for SM-exposed individuals. TSC express Major Histocompatibility Complex (MHC-I) transplantation antigens which increases the chance that allogeneic TSC will be rejected by the patient’s immune system. However, previous studies reported that Beta-2 microglobulin (B2M) knockout cells lacked cell surface MHC-I and suggested that B2M knockout TSC would be tolerated as an allogeneic graft. This study used a Cas9 ribonucleoprotein (RNP) to generate B2M-knockout TSC, which are termed Universal Donor Stem Cells (UDSC). Whole genome sequencing identified few off-target modifications and demonstrated the specificity of the RNP approach. Functional assays demonstrated that UDSC retained their ability to self-renew and undergo multilineage differentiation. A preclinical model of SM inhalation was used to test UDSC efficacy and identify any treatment-associated adverse events. Adult male Sprague-Dawley rats were administered an inhaled dose of 0.8 mg/kg SM vapor which is the inhaled LD50 on day 28 post-challenge. On recovery day 2, vehicle or allogeneic Fisher rat UDSC were delivered intravenously (n = 30/group). Clinical parameters were recorded daily, and planned euthanasia occurred on post-challenge days 7, 14, and 28. The vehicle and UDSC treatment groups exhibited similar outcomes including survival and a lack of adverse events. These studies establish a baseline which can be used to further develop UDSC as a treatment for SM-induced airway disease.

Background: Hospital wastewater is a major source of antimicrobial resistance (AMR) outflow into the environment. This study uses metagenomics to study how hospital clinical activity impacts antimicrobial resistance genes (ARGs) abundances in hospital wastewater.Methods: Sewage was collected over a 24-h period from multiple wastewater collection points (CPs) representing different specialties within a tertiary hospital site and simultaneously from community sewage works. High throughput shotgun sequencing was performed using Illumina HiSeq4000. ARG abundances were correlated to hospital antimicrobial usage (AMU), data on clinical activity and resistance prevalence in clinical isolates.Results: Microbiota and ARG composition varied between CPs and overall ARG abundance was higher in hospital wastewater than in community influent. ARG and microbiota compositions were correlated (Procrustes analysis, p=0.014). Total antimicrobial usage was not associated with higher ARG abundance in wastewater. However, there was a small positive association between resistance genes and antimicrobial usage matched to ARG phenotype (IRR 1.11, CI 1.06–1.16, p

Measurements of atmospheric O2 have been used to quantify large-scale fluxes of carbon between the oceans, atmosphere and land since 1992 (Keeling and Shertz, 1992). With time, datasets have grown and estimates of fluxes have become more precise, but a key uncertainty in these calculations is the exchange ratio of O2 and CO2 associated with the net land carbon sink (αB). We present measurements of atmospheric O2 and CO2 collected over a 6-year period from a mixed deciduous forest in central Massachusetts, USA (42.537∘ N, 72.171∘ W). Using a differential fuel-cell-based instrument for O2 and a nondispersive infrared analyzer for CO2, we analyzed airstreams collected within and ∼5 m above the forest canopy. Averaged over the entire period of record, we find these two species covary with a slope of -1.081±0.007 mol of O2 per mole of CO2 (the mean and standard error of 6 h periods). If we limit the data to values collected on summer days within the canopy, the slope is -1.03±0.01. These are the conditions in which biotic influences are most likely to dominate. This result is significantly different from the value of −1.1 widely used in O2-based calculations of the global carbon budget, suggesting the need for a deeper understanding of the exchange ratios of the various fluxes and pools comprising the net sink.

Background: SARS-CoV-2 antibody tests are used for population surveillance and might have a future role in individual risk assessment. Lateral flow immunoassays (LFIAs) can deliver results rapidly and at scale, but have widely varying accuracy. Methods: In a laboratory setting, we performed head-to-head comparisons of four LFIAs: the Rapid Test Consortium's AbC-19TM Rapid Test, OrientGene COVID IgG/IgM Rapid Test Cassette, SureScreen COVID-19 Rapid Test Cassette, and Biomerica COVID-19 IgG/IgM Rapid Test. We analysed blood samples from 2,847 key workers and 1,995 pre-pandemic blood donors with all four devices. Findings: We observed a clear trade-off between sensitivity and specificity: the IgG band of the SureScreen device and the AbC-19TM device had higher specificities but OrientGene and Biomerica higher sensitivities. Based on analysis of pre-pandemic samples, SureScreen IgG band had the highest specificity (98.9%, 95% confidence interval 98.3 to 99.3%), which translated to the highest positive predictive value across any pre-test probability: for example, 95.1% (95% uncertainty interval 92.6, 96.8%) at 20% pre-test probability. All four devices showed higher sensitivity at higher antibody concentrations (“spectrum effects”), but the extent of this varied by device. Interpretation: The estimates of sensitivity and specificity can be used to adjust for test error rates when using these devices to estimate the prevalence of antibody. If tests were used to determine whether an individual has SARS-CoV-2 antibodies, in an example scenario in which 20% of individuals have antibodies we estimate around 5% of positive results on the most specific device would be false positives. Funding: Public Health England.

Background Biological sex is an important modifier of cardiovascular disease and women generally have better outcomes compared with men. However, the contribution of cardiac fibroblasts (CFs) to this sexual dimorphism is relatively unexplored. Methods and Results Isoproterenol (ISO) was administered to rats as a model for chronic β‐adrenergic receptor (β‐AR)‐mediated cardiovascular disease. ISO‐treated males had higher mortality than females and also developed fibrosis whereas females did not. Gonadectomy did not abrogate this sex difference. To determine the cellular contribution to this phenotype, CFs were studied. CFs from both sexes had increased proliferation in vivo in response to ISO, but CFs from female hearts proliferated more than male cells. In addition, male CFs were significantly more activated to myofibroblasts by ISO. To investigate potential regulatory mechanisms for the sexually dimorphic fibrotic response, β‐AR mRNA and PKA (protein kinase A) activity were measured. In response to ISO treatment, male CFs increased expression of β1‐ and β2‐ARs, whereas expression of both receptors decreased in female CFs. Moreover, ISO‐treated male CFs had higher PKA activity relative to vehicle controls, whereas ISO did not activate PKA in female CFs. Conclusions Chronic in vivo β‐AR stimulation causes fibrosis in male but not female rat hearts. Male CFs are more activated than female CFs, consistent with elevated fibrosis in male rat hearts and may be caused by higher β‐AR expression and PKA activation in male CFs. Taken together, our data suggest that CFs play a substantial role in mediating sex differences observed after cardiac injury.

Introduction: Older adults, who already have higher levels of social isolation, loneliness, and sedentary behavior, are particularly susceptible to negative impacts from social distancing mandates meant to control the spread of COVID-19. We sought to explore the physical, mental, and social health impacts of the pandemic on older adults and their coping techniques.Materials and Methods: We conducted 25 semi-structured interviews with a sub-sample of participants in an ongoing sedentary behavior reduction intervention. Interviews were recorded and transcribed, and iterative coding was used to extract key themes.Results: Most participants reported an increase in sedentary behavior due to limitations on leaving their home and increased free time to pursue seated hobbies (e.g., reading, knitting, tv). However, many participants also reported increased levels of intentional physical activity and exercise, particularly outdoors or online. Participants also reported high levels of stress and a large decrease in in-person social connection. Virtual connection with others through phone and video was commonly used to stay connected with friends and family, engage in community groups and activities, and cope with stress and social isolation. Maintenance of a positive attitude and perspective gained from past hardships was also an important coping strategy for many participants.Discussion: The COVID-19 pandemic and associated social distancing measures have impacted older adults' perceived levels of activity, stress, and social isolation, but many leveraged technology and prior life experiences to cope. These themes could inform future interventions for older adults dealing with chronic stress and isolation.

Abstract Type 1 diabetes (T1D) has been reported to negatively affect the health of skeletal muscle, though the underlying mechanisms are unknown. Myostatin, a myokine whose increased expression is associated with muscle‐wasting diseases, has not been reported in humans with T1D but has been demonstrated to be elevated in preclinical diabetes models. Thus, the purpose of this study was to determine if there is an elevated expression of myostatin in the serum and skeletal muscle of persons with T1D compared to controls. Secondarily, we aimed to explore relationships between myostatin expression and clinically important metrics (e.g., HbA1c, strength, lean mass) in women and men with (N = 31)/without T1D (N = 24) between 18 and 72 years old. Body composition, baseline strength, blood sample and vastus lateralis muscle biopsy were evaluated. Serum, but not muscle, myostatin expression was significantly elevated in those with T1D versus controls, and to a greater degree in T1D women than T1D men. Serum myostatin levels were not significantly associated with HbA1c nor disease duration. A significant correlation between serum myostatin expression and maximal voluntary contraction (MVC) and body fat mass was demonstrated in control subjects, but these correlations did not reach significance in those with T1D (MVC: R = 0.64 controls vs. R = 0.37 T1D; Body fat: R = −0.52 controls/R = −0.02 T1D). Collectively, serum myostatin was correlated with lean mass (R = 0.45), and while this trend was noted in both groups separately, neither reached statistical significance (R = 0.47 controls/R = 0.33 T1D). Overall, while those with T1D exhibited elevated serum myostatin levels (particularly females) myostatin expression was not correlated with clinically relevant metrics despite some of these relationships existing in controls (e.g., lean/fat mass). Future studies will be needed to fully understand the mechanisms underlying increased myostatin in T1D, with relationships to insulin dosing being particularly important to elucidate.

In Duchenne muscular dystrophy, a lack of dystrophin leads to extensive muscle weakness and atrophy that is linked to cellular metabolic dysfunction and oxidative stress. This dystrophinopathy results in a loss of tethering between microtubules and the sarcolemma. Microtubules are also believed to regulate mitochondrial bioenergetics potentially by binding the outer mitochondrial membrane voltage dependent anion channel (VDAC) and influencing permeability to ADP/ATP cycling. The objective of this investigation was to determine if a lack of dystrophin causes microtubule disorganization concurrent with mitochondrial dysfunction in skeletal muscle, and whether this relationship is linked to altered binding of tubulin to VDAC. In extensor digitorum longus (EDL) muscle from 4-week old D2.mdx mice, microtubule disorganization was observed when probing for α-tubulin. This cytoskeletal disorder was associated with a reduced ability of ADP to stimulate respiration and attenuate H2O2 emission relative to wildtype controls. However, this was not associated with altered α-tubulin-VDAC2 interactions. These findings reveal that microtubule disorganization in dystrophin-deficient EDL is associated with impaired ADP control of mitochondrial bioenergetics, and suggests that mechanisms alternative to α-tubulin's regulation of VDAC2 should be examined to understand how cytoskeletal disruption in the absence of dystrophin may cause metabolic dysfunctions in skeletal muscle.