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Providencia alcalifaciens is a Gram-negative bacterium found in various water and land environments and organisms, including insects and mammals. Some P. alcalifaciens strains encode gene homologs of virulence factors found in pathogenic Enterobacterales members, such as Salmonella enterica serovar Typhimurium and Shigella flexneri. Whether these genes are pathogenic determinants in P. alcalifaciens is not known. In this study, we investigated P. alcalifaciens-host interactions at the cellular level, focusing on the role of two type III secretion systems (T3SS) belonging to the Inv-Mxi/Spa family. T3SS1b is widespread in Providencia spp. and encoded on the chromosome. A large plasmid that is present in a subset of P. alcalifaciens strains, primarily isolated from diarrheal patients, encodes for T3SS1a. We show that P. alcalifaciens 205/92 is internalized into eukaryotic cells, lyses its internalization vacuole, and proliferates in the cytosol. This triggers caspase-4-dependent inflammasome responses in gut epithelial cells. The requirement for the T3SS1a in entry, vacuole lysis, and cytosolic proliferation is host cell type-specific, playing a more prominent role in intestinal epithelial cells than in macrophages or insect cells. In a bovine ligated intestinal loop model, P. alcalifaciens colonizes the intestinal mucosa and induces mild epithelial damage with negligible fluid accumulation in a T3SS1a- and T3SS1b-independent manner. However, T3SS1b was required for the rapid killing of Drosophila melanogaster. We propose that the acquisition of two T3SS has allowed P. alcalifaciens to diversify its host range, from a highly virulent pathogen of insects to an opportunistic gastrointestinal pathogen of animals.

Successful integration of point-of-care testing (POCT) into clinical settings requires improved assay sensitivity and precision to match laboratory standards. Here, we show how innovations in amplified biosensing, imaging, and data processing, coupled with deep learning, can help improve POCT. To demonstrate the performance of our approach, we present a rapid and cost-effective paper-based high-sensitivity vertical flow assay (hs-VFA) for quantitative measurement of cardiac troponin I (cTnI), a biomarker widely used for measuring acute cardiac damage and assessing cardiovascular risk. The hs-VFA includes a colorimetric paper-based sensor, a portable reader with time-lapse imaging, and computational algorithms for digital assay validation and outlier detection. Operating at the level of a rapid at-home test, the hs-VFA enabled the accurate quantification of cTnI using 50 μL of serum within 15 min per test and achieved a detection limit of 0.2 pg/mL, enabled by gold ion amplification chemistry and time-lapse imaging. It also achieved high precision with a coefficient of variation of

Biallelic loss of cyclin-dependent kinase 12 (CDK12) defines a metastatic castration-resistant prostate cancer (mCRPC) subtype. It remains unclear, however, whether CDK12 loss drives prostate cancer (PCa) development or uncovers pharmacologic vulnerabilities. Here, we show Cdk12 ablation in murine prostate epithelium is sufficient to induce preneoplastic lesions with lymphocytic infiltration. In allograft-based CRISPR screening, Cdk12 loss associates positively with Trp53 inactivation but negatively with Pten inactivation. Moreover, concurrent Cdk12/Trp53 ablation promotes proliferation of prostate-derived organoids, while Cdk12 knockout in Pten-null mice abrogates prostate tumor growth. In syngeneic systems, Cdk12/Trp53-null allografts exhibit luminal morphology and immune checkpoint blockade sensitivity. Mechanistically, Cdk12 inactivation mediates genomic instability by inducing transcription-replication conflicts. Strikingly, CDK12-mutant organoids and patient-derived xenografts are sensitive to inhibition or degradation of the paralog kinase, CDK13. We therein establish CDK12 as a bona fide tumor suppressor, mechanistically define how CDK12 inactivation causes genomic instability, and advance a therapeutic strategy for CDK12-mutant mCRPC.

Early stages of Alzheimer's disease (AD) are associated with volume reductions in specific subregions of the medial temporal lobe (MTL). Using a manual segmentation method-the Olsen-Amaral-Palombo (OAP) protocol-previous work in healthy older adults showed that reductions in grey matter volumes in MTL subregions were associated with lower scores on the Montreal Cognitive Assessment (MoCA), suggesting atrophy may occur prior to diagnosis of mild cognitive impairment, a condition that often progresses to AD. However, current "gold standard" manual segmentation methods are labour intensive and time consuming. Here, we examined the utility of Automatic Segmentation of Hippocampal Subfields (ASHS) to detect volumetric differences in MTL subregions of healthy older adults who varied in cognitive status as determined by the MoCA. We trained ASHS on the OAP protocol to create the ASHS-OAP atlas and then examined how well automated segmentation replicated manual segmentation. Volumetric measures obtained from the ASHS-OAP atlas were also contrasted against those from the ASHS-PMC atlas, a widely used atlas provided by the ASHS team. The pattern of volumetric results was similar between the ASHS-OAP atlas and manual segmentation for anterolateral entorhinal cortex and perirhinal cortex, suggesting that ASHS-OAP is a viable alternative to current manual segmentation methods for detecting group differences based on cognitive status. Although ASHS-OAP and ASHS-PMC produced varying volumes for most regions of interest, they both identified early signs of neurodegeneration in CA2/CA3/DG and identified marginal differences in entorhinal cortex. Our findings highlight the utility of automated segmentation methods but still underscore the need for a unified and harmonized MTL segmentation atlas.

Profound functional switch of key regulatory factors may play a major role in homeostasis and disease. Dysregulation of circadian rhythm (CR) is strongly implicated in cancer with mechanisms poorly understood. We report here that the function of REV-ERBα, a major CR regulator of the orphan nuclear receptor subfamily, is dramatically altered in tumors in both its genome binding and functional mode. Loss of CR is linked to a functional inversion of REV-ERBα from a repressor in control of CR and metabolic gene programs in normal tissues to a strong activator in different cancers. Through changing its association from NCoR/HDAC3 corepressor complex to BRD4/p300 coactivators, REV-ERBα directly activates thousands of genes including tumorigenic programs such as MAPK and PI3K-Akt signaling. Functioning as a master transcriptional activator, REV-ERBα partners with pioneer factor FOXA1 and directly stimulates a large number of signaling genes, including multiple growth factors, receptor tyrosine kinases, RASs, AKTs, and MAPKs. Moreover, elevated REV-ERBα reprograms FOXA1 to bind new targets through a BRD4-mediated increase in local chromatin accessibility. Pharmacological targeting with SR8278 diminishes the function of both REV-ERBα and FOXA1 and synergizes with BRD4 inhibitor in effective suppression of tumorigenic programs and tumor growth. Thus, our study revealed a functional inversion by a CR regulator in driving gene reprogramming as an unexpected paradigm of tumorigenesis mechanism and demonstrated a high effectiveness of therapeutic targeting such switch.

Inquiries into properties of brain structure and function have progressed due to developments in magnetic resonance imaging (MRI). To sustain progress in investigating and quantifying neuroanatomical details in vivo, the reliability and validity of brain measurements are paramount. Quality control (QC) is a set of procedures for mitigating errors and ensuring the validity and reliability of brain measurements. Despite its importance, there is little guidance on best QC practices and reporting procedures. The study of hippocampal subfields in vivo is a critical case for QC because of their small size, inter-dependent boundary definitions, and common artifacts in the MRI data used for subfield measurements. We addressed this gap by surveying the broader scientific community studying hippocampal subfields on their views and approaches to QC. We received responses from 37 investigators spanning 10 countries, covering different career stages, and studying both healthy and pathological development and aging. In this sample, 81% of researchers considered QC to be very important or important, and 19% viewed it as fairly important. Despite this, only 46% of researchers reported on their QC processes in prior publications. In many instances, lack of reporting appeared due to ambiguous guidance on relevant details and guidance for reporting, rather than absence of QC. Here, we provide recommendations for correcting errors to maximize reliability and minimize bias. We also summarize threats to segmentation accuracy, review common QC methods, and make recommendations for best practices and reporting in publications. Implementing the recommended QC practices will collectively improve inferences to the larger population, as well as have implications for clinical practice and public health.

The peripheral endoplasmic reticulum (ER) forms a dense, interconnected, and constantly evolving network of membrane-bound tubules in eukaryotic cells. While individual structural elements and the morphogens that stabilize them have been described, a quantitative understanding of the dynamic large-scale network topology remains elusive. We develop a physical model of the ER as an active liquid network, governed by a balance of tension-driven shrinking and new tubule growth. This minimalist model gives rise to steady-state network structures with density and rearrangement timescales predicted from the junction mobility and tubule spawning rate. Several parameter-independent geometric features of the liquid network model are shown to be representative of ER architecture in live mammalian cells. The liquid network model connects the timescales of distinct dynamic features such as ring closure and new tubule growth in the ER. Furthermore, it demonstrates how the steady-state network morphology on a cellular scale arises from the balance of microscopic dynamic rearrangements.

BACKGROUND: The research community has historically failed to enroll diverse groups of participants in dementia clinical trials. A unique aspect of dementia care research is the requirement of a study partner, who can attest to the care recipients clinical and functional capacity. The aim of this study is to assess racial and ethnic differences and the importance of various trial considerations among dementia caregivers, in their decision to participate in clinical research as study partners. METHOD: We embedded a vignette about a hypothetical dementia clinical trial in a nationally representative survey of U.S. dementia caregivers, oversampling non-Hispanic Black and Hispanic caregivers. Dementia caregivers were asked about their willingness to participate in the trial with their care recipient and rated the importance of nine considerations in hypothetical decisions to participate. Caregiver demographic characteristics were analyzed as predictors of trial participation in a base demographic model. In a second reasons model caregiver demographic characteristics and the rated importance of the nine considerations were separately analyzed as predictors; both models used survey-weighted logistic regression. RESULT: The sample consisted of 610 dementia caregivers, including 156 non-Hispanic Black and 122 Hispanic caregiver participants. In the base demographic model, hypothetical trial participation was negatively associated with older caregiver age (OR (odds ratio) = 0.72, p =

Study designRetrospective cohort study of prospectively accrued data.ObjectiveTo evaluate a large, prospective, multicentre dataset of surgically treated degenerative cervical myelopathy (DCM) cases on the contemporary risk of C5 palsy with surgical approach.Summary of background dataThe influence of surgical technique on postoperative C5 palsy after decompression for DCM is intensely debated. Comprehensive, covariate-adjusted analyses are needed using contemporary data.MethodsPatients with moderate to severe DCM were prospectively enrolled in the multicenter, randomized, Phase III CSM-Protect clinical trial and underwent either anterior or posterior decompression between Jan 31, 2012 and May 16, 2017. The primary outcome was the incidence of postoperative C5 palsy, defined as the onset of muscle weakness by at least one grade in manual muscle test at the C5 myotome with slight or absent sensory disruption after cervical surgery. Two comparative cohorts were made based on the anterior or posterior surgical approach. Multivariate hierarchical mixed-effects logistic regression was used to estimate odds ratios (OR) with 95% confidence intervals (CI) for C5 palsy.ResultsA total of 283 patients were included, and 53.4% underwent posterior decompression. The total incidence of postoperative C5 palsy was 7.4% and was significantly higher in patients who underwent posterior decompression compared with anterior decompression (11.26% vs. 3.03%, P =0.008). After multivariable regression, the posterior approach was independently associated with greater than four times the likelihood of postoperative C5 palsy ( P =0.017). Rates of C5 palsy recovery were comparable between the two surgical approaches.ConclusionThe odds of postoperative C5 palsy are significantly higher after posterior decompression compared to anterior decompression for DCM. This may influence surgical decision-making when there is equipoise in deciding between anterior and posterior treatment options for DCM.Level of evidenceTherapeutic Level-II.

Tumors can induce systemic disturbances in distant organs, leading to physiological changes that enhance host morbidity. In Drosophila cancer models, tumors have been known for decades to cause hypervolemic "bloating" of the abdominal cavity. Here we use allograft and transgenic tumors to show that hosts display fluid retention associated with autonomously defective secretory capacity of fly renal tubules, which function analogous to those of the human kidney. Excretion from these organs is blocked by abnormal cells that originate from inappropriate activation of normally quiescent renal stem cells (RSCs). Blockage is initiated by IL-6-like oncokines that perturb renal water-transporting cells and trigger a damage response in RSCs that proceeds pathologically. Thus, a chronic inflammatory state produced by the tumor causes paraneoplastic fluid dysregulation by altering cellular homeostasis of host renal units.

BACKGROUND: Overdose is a leading cause of maternal mortality; in response, maternal mortality review committees have recommended expanding substance use disorder (SUD) screening, improving collaboration between obstetric and SUD treatment providers, and reducing fragmentation in systems of care. We undertook an analysis of the perinatal SUD treatment landscape in Baltimore, Maryland in order to identify barriers to treatment engagement during pregnancy and the postpartum period and guide system improvement efforts. METHODS: We conducted a survey of seven birthing hospitals, 31 prenatal care practices, and 108 SUD treatment providers in Baltimore from April-June 2023. Organizations were asked to quantify care for perinatal patients with opioid use disorder (OUD) as well as about screening, service availability, referral practices, and support needed to improve care. RESULTS: 61% of the 145 contacted organizations responded. Birthing hospitals reported caring for pregnant persons with OUD with greater frequency than prenatal care practices or SUD treatment programs. Most birthing hospitals and prenatal care practices reported screening for OUD at intake, but the minority reported using validated tools. Service availability varied by type of organization and type of service. In general, prenatal care practices offered the fewest number of SUD-related services. Most SUD treatment programs that offered buprenorphine or methadone to the general population also offered these medications to pregnant patients. Withdrawal management for comorbid alcohol/benzodiazepine use disorders during pregnancy was more limited. The majority of birthing hospitals and prenatal care practices reported offering neither direct naloxone distribution nor prescriptions. Few SUD treatment programs offered tailored services for perinatal patients or for parents of young children, and many programs do not permit children onsite. Respondents reported high levels of interest in education and consultative support on SUD treatment in pregnancy within obstetric settings and on pregnancy-related medical concerns within SUD programs. CONCLUSIONS: This project provides a comprehensive picture of services available for treatment of perinatal OUD in a major US city. Results have served as a guide for ongoing citywide system improvement efforts by our project team and offer a model for other jurisdictions hoping to strengthen services for perinatal OUD and reduce maternal mortality.

Mozambique is making progress towards elimination of trachoma as a public health problem, but in some districts trachomatous inflammation-follicular (TF) prevalence remains above the 5% elimination threshold despite years of various interventions, including antibiotic mass drug administration. To characterize transmission in four districts, we incorporated testing of ocular infection and serology into routine trachoma impact surveys (TIS) in August 2022. We examined residents aged ≥ 1 year for trachoma and collected information on household water, sanitation, and hygiene. Among children aged 1-9 years, we tested conjunctival swabs for Chlamydia trachomatis nucleic acid and dried blood spots for C. trachomatis antibodies. We modeled age-dependent seroprevalence to estimate seroconversion rate (SCR). We examined 4841 children aged 1-9 years. TF prevalence ranged between 1.1 and 6.0% with three districts below the 5% threshold. PCR-confirmed infection prevalence ranged between 1.1 and 4.8%, and Pgp3 seroprevalence ranged between 8.8 and 24.3%. Pgp3 SCR was 1.9 per 100 children per year in the district with the lowest TF prevalence. Two other districts with TF

Wildlife biodiversity is essential for healthy, resilient and sustainable ecosystems. For biologists, this diversity also represents a treasure trove of genetic, molecular and developmental mechanisms that deepen our understanding of the origins and rules of life. However, the rapid decline in biodiversity reported recently foreshadows a potentially catastrophic collapse of many important ecosystems and the associated irreversible loss of many forms of life on our planet. Immediate action by conservationists of all stripes is required to avert this disaster. In this Spotlight, we draw together insights and proposals discussed at a recent workshop hosted by Revive & Restore, which gathered experts to discuss how stem cell technologies can support traditional conservation techniques and help protect animal biodiversity. We discuss reprogramming, in vitro gametogenesis, disease modelling and embryo modelling, and we highlight the prospects for leveraging stem cell technologies beyond mammalian species.

Enhancing the efficacy of immunotherapy in brain metastases (BrM) requires an improved understanding of the immune composition of BrM and how this is affected by radiation and dexamethasone. Our two-arm pilot study (NCT04895592) allocated 26 patients with BrM to either low (Arm A) or high (Arm B) dose peri-operative dexamethasone followed by pre-operative stereotactic radiosurgery (pSRS) and resection (n= 13 per arm). The primary endpoint, a safety analysis at 4 months, was met. The secondary clinical endpoints of overall survival, distant brain failure, leptomeningeal disease and local recurrence at 12-months were 66%, 37.3%, 6%, and 0% respectively and were not significantly different between arms (p= 0.7739, p= 0.3884, p= 0.3469). Immunological data from two large retrospective BrM datasets and confirmed by correlates from both arms of this pSRS prospective trial revealed that BrM CD8 T cells were composed of predominantly PD1+ TCF1+ stem-like and PD1+ TCF1-TIM3+ effector-like cells. Clustering of TCF1+ CD8 T cells with antigen presenting cells in immune niches was prognostic for local control, even without pSRS. Following pSRS, CD8 T cell and immune niche density were transiently reduced compared to untreated BrM, followed by a rebound 6+ days post pSRS with an increased frequency of TCF1- effector-like cells. In sum, pSRS is safe and therapeutically beneficial, and these data provide a framework for how pSRS may be leveraged to maximize intracranial CD8 T cell responses.

The effects of the COVID-19 pandemic on comprehensive maternal deaths in Brazil have not been fully explored. Using publicly available data from the Brazilian Mortality Information (SIM) and Information System on Live Births (SINASC) databases, we used two complementary forecasting models to predict estimates of maternal mortality ratios using maternal deaths (MMR) and comprehensive maternal deaths (MMRc) in the years 2020 and 2021 based on data from 2008 to 2019. We calculated national and regional standardized mortality ratio estimates for maternal deaths (SMR) and comprehensive maternal deaths (SMRc) for 2020 and 2021. The observed MMRc in 2021 was more than double the predicted MMRc based on the Holt-Winters and autoregressive integrated moving average models (127.12 versus 60.89 and 59.12 per 100,000 live births, respectively). We found persisting sub-national variation in comprehensive maternal mortality: SMRc ranged from 1.74 (95% confidence interval [CI] 1.64, 1.86) in the Northeast to 2.70 (95% CI 2.45, 2.96) in the South in 2021. The observed national estimates for comprehensive maternal deaths in 2021 were the highest in Brazil in the past three decades. Increased resources for prenatal care, maternal health, and postpartum care may be needed to reverse the national trend in comprehensive maternal deaths.

BACKGROUND: The Neuroform Atlas Stent System is an established treatment modality for unruptured anterior and posterior circulation intracranial aneurysms. Location-specific results are needed to guide treatment decision-making. However, it is unclear whether there are differences in safety and efficacy outcomes between carotid and more distal anterior circulation aneurysms. METHODS: The ATLAS IDE trial was a prospective, multicenter, single-arm, open-label interventional study that evaluated the safety and efficacy of the Neuroform Atlas Stent System. We compared differences in efficacy and safety outcomes of proximal internal carotid artery (ICA) versus distal and bifurcation anterior circulation aneurysms. RESULTS: Of 182 cases, there were 70 aneurysms in the ICA and 112 in the distal anterior circulation (including ICA terminus/bifurcation). There were no significant differences in the primary efficacy endpoint (85.5% vs 83.9%, p=0.78) and complete aneurysm occlusion rates (88.7% vs 87.9%, p=0.78) between proximal ICA aneurysms and distal aneurysms, respectively. Complications were more often encountered in distal and bifurcation aneurysms, but the overall rate of major safety events was low and comparable between the two groups (1.4% vs 6.3%, p=0.14). Recanalization and retreatment rates were also similar between the groups. CONCLUSION: The results of this study suggest that the Neuroform Atlas Stent System is a safe and efficacious treatment modality for unruptured anterior circulation intracranial aneurysms, regardless of aneurysm location. TRIAL REGISTRATION NUMBER: NCT02340585.

OBJECTIVE: This study aimed to validate claims-based algorithms for identifying SLE and lupus nephritis (LN) in Medicare data, enhancing the use of the Lupus Index for geospatial research on SLE prevalence and outcomes. METHODS: We retrospectively evaluated the performance of rule-based algorithms using the International Classification of Diseases, 10th Revision (ICD-10) codes to identify SLE and LN in a well-defined prospective longitudinal cohort of patients with and without SLE from a South Carolina registry and rheumatology outpatient clinics. The analysis included comparison of algorithms based on Medicare fee-for-service claims data to these rigorously phenotyped populations. The primary classification for SLE cases was based on the American College of Rheumatology and Systemic Lupus Erythematosus International Collaborating Clinics criteria for SLE and LN. Algorithms were based on the number of ICD-10 codes with and without a 30-day separation in the observation period, including all of 2016-2018. RESULTS: The algorithm using two ICD-10 codes for SLE, with or without a 30-day separation, showed the best overall performance. For LN, specific ICD-10 codes outperformed combinations of SLE and renal/proteinuria codes that were found in ICD-9. CONCLUSIONS: The findings of this study highlight the performance of specific ICD-10 code algorithms in identifying SLE and LN cases within Medicare data, providing a valuable tool for informing use of the Lupus Index. This index allows for improved geographical targeting of clinical resources, health disparity studies and clinical trial site selection. The study underscores the importance of algorithm selection based on research objectives, recommending more specific algorithms for precise tasks like clinical trial site identification and less specific ones for broader applications such as health disparities research.

BACKGROUND: The Tigertriever device offers a unique feature that enables gradual control of the radial expansion. We sought to evaluate the safety and efficacy of the Tigertriever device in patients with large vessel occlusion (LVO) and underlying intracranial atherosclerotic disease (ICAD). The patients were part of the TIGER trial. METHODS: The presence of underlying ICAD was determined by a core imaging laboratory using CT angiography and digital subtraction angiography. The primary outcomes included successful reperfusion, puncture to reperfusion time, and complications associated with the use of the Tigertriever device. Patients underwent mechanical thrombectomy with the Tigertriever device for up to three passes, and alternative devices were employed for subsequent passes. RESULTS: A total of 160 patients were enrolled in the TIGER trial, and 32 patients had ICAD. Among the patients with ICAD, 78% achieved successful reperfusion within three passes of the Tigertriever device, without requiring rescue therapy. Additionally, a first pass effect was observed in 46.8%. The median time from puncture to reperfusion was 22 minutes. There were no device-related complications. The National Institutes of Health Stroke Scale (NIHSS) score at 24 hours was significantly reduced, from an average of 17 at baseline to 8. At the 3 month follow-up, 50% of patients achieved a modified Rankin Scale score of ≤2. CONCLUSION: Endovascular therapy (EVT) with the Tigertriever device for LVO in patients with underlying ICAD is effective and safe. When compared with historical data from other devices employed in similar cases, we observed a high rate of successful reperfusion, along with a shorter puncture to reperfusion time.

OBJECTIVES: This study aimed to evaluate pulmonary diffusing capacity for nitric oxide (DLNO) and pulmonary diffusing capacity for carbon monoxide (DLCO) in Mexican Hispanics born and raised at 2240 m altitude (midlanders) compared with those born and raised at sea level (lowlanders). It also aimed to assess the effectiveness of race-specific reference equations for pulmonary diffusing capacity (white people vs Mexican Hispanics) in minimising root mean square errors (RMSE) compared with race-neutral equations. METHODS: DLNO, DLCO, alveolar volume (VA) and gas transfer coefficients (KNO and KCO) were measured in 392 Mexican Hispanics (5 to 78 years) and compared with 1056 white subjects (5 to 95 years). Reference equations were developed using segmented linear regression (DLNO, DLCO and VA) and multiple linear regression (KNO and KCO) and validated with Least Absolute Shrinkage and Selection Operator. RMSE comparisons between race-specific and race-neutral models were conducted using repeated k-fold cross-validation and random forests. RESULTS: Midlanders exhibited higher DLCO (mean difference: +4 mL/min/mm Hg), DLNO (mean difference: +7 mL/min/mm Hg) and VA (mean difference: +0.17 L) compared with lowlanders. The Bayesian information criterion favoured race-specific models and excluding race as a covariate increased RMSE by 61% (DLNO), 18% (DLCO) and 4% (KNO). RMSE values for VA and KCO were comparable between race-specific and race-neutral models. For DLCO and DLNO, race-neutral equations resulted in 3% to 6% false positive rates (FPRs) in Mexican Hispanics and 20% to 49% false negative rates (FNRs) in white subjects compared with race-specific equations. CONCLUSIONS: Mexican Hispanics born and raised at 2240 m exhibit higher DLCO and DLNO compared with lowlanders. Including race as a covariate in reference equations lowers the RMSE for DLNO, DLCO and KNO and reduces FPR and FNR compared with race-neutral models. This study highlights the need for altitude-specific and race-specific reference equations to improve pulmonary function assessments across diverse populations.

Ex vivo human skin models are valuable tools in skin research due to their physiological relevance. Traditionally, standard cultivation is performed in a cell culture incubator with a defined temperature of 37 °C and a specific atmosphere enriched with CO2 to ensure media stability. Maintaining the model under these specific conditions limits its flexibility in assessing exposures to which the skin is exposed to in daily life, for example changes in atmospheric compositions. In this study we demonstrated that the foreskin-derived skin model can be successfully cultured at room temperature outside a CO2 incubator using a CO2-independent, serum-free media. Over a cultivation period of three days, the integrity of the tissue and the preservation of immune cells is well maintained, indicating the models stability and resilience under the given conditions. Exposing our Medical University of Graz - human Organotypic Skin Explant Culture (MUG-hOSEC) model to cytotoxic and inflammatory stimuli results in responses analyzable within the supernatant. Besides the common analysis of released proteins upon treatment, such as cytokines and enzymes, we have included extracellular vesicle to obtain a more comprehensive picture of cell communication.

To investigate how clinicians correct patient misconceptions, we analyzed 23 video recordings of primary care visits. Analysis focused on operationalizing, identifying, and characterizing clinician corrections, integrating two inductive approaches: microanalysis of clinical interaction and conversation analysis. According to our definition, patient misconception-clinician correction episodes met three essential criteria: (1) the clinician refuted something the patient had said, (2) which the patient had presented without uncertainty, and (3) which contained a proposition that was factually incorrect. We identified 59 such episodes; the patient misconceptions most commonly related to medication issues; fewer than half had foreseeable implications for patients' future actions. We identified seven clinician correction practices: Three direct practices (displaying surprise, marking disagreement, contradicting the patient) and four indirect practices (presenting the correct proposition, providing explanations, invoking an outside authority, demonstrating with evidence). We found an almost equal distribution of these direct and indirect practices.

We recently completed the Fund Consciousness Science! Project: a workshop and subawards program aimed to align United States federal funding mechanisms and consciousness research. Here we describe the project’s motivation, execution, and outcomes to motivate similar efforts both locally and globally.

BACKGROUND: Outcome measures are crucial to support a treat-to-target approach to rheumatoid arthritis (RA) care, yet their integration into clinical practice remains inconsistent. We developed an Electronic Heath Record-integrated, patient-facing side-car application to display RA outcomes (disease activity, functional status, pain scores), medications, and lab results during clinical visits (RA PRO Dashboard). The study aimed to evaluate patient perceptions and attitudes towards the implementation of a novel patient-facing dashboard during clinical visits using a mixed-methods approach. METHODS: RA patients whose clinicians used the dashboard at least once during their clinical visit were invited to complete a survey regarding its usefulness in care. We also conducted semi-structured interviews with a subset of patients to assess their perceptions of the dashboard. The interviews were transcribed verbatim and analyzed thematically using deductive and inductive techniques. Emerging themes and subthemes were organized into four domains of the Ecological Model of Health. RESULTS: Out of 173 survey respondents, 79% were interested in seeing the dashboard again at a future visit, 71% felt it improved their understanding of their disease, and 65% believed it helped with decision-making about their RA care. Many patients reported that the dashboard helped them discuss their RA symptoms (76%) and medications (72%) with their clinician. Interviews with 29 RA patients revealed 10 key themes: the dashboard was perceived as a valuable visual tool that improved patients understanding of RA outcome measures, enhanced their involvement in care, and increased their trust in clinicians and the clinic. Common reported limitations included concerns about reliability of RA outcome questionnaires for some RA patients and inconsistent collection and explanation of these measures by clinicians. CONCLUSIONS: In both the quantitative and qualitative components of the study, patients reported that the dashboard improved their understanding of their RA, enhanced patient-clinician communication, supported shared decision-making, and increased patient engagement in care. These findings support the use of dashboards or similar data visualization tools in RA care and can be used in future interventions to address challenges in data collection and patient education.

Many central nervous system (CNS) disorders lack approved treatment options. Previous research demonstrated that peptide CAQK can bind to chondroitin sulfate proteoglycans (CSPGs) in the extracellular matrix of the CNS. In vivo studies have investigated CAQK conjugated to nanoparticles containing therapeutic agents with varying methodologies/outcomes. This paper presents the first systematic review assessing its properties, applications, and outcomes secondary to its use. Following PRISMA guidelines, a comprehensive search was performed across multiple databases. Studies utilizing CAQK as a therapeutic agent/homing molecule in animal/human models were selected. Sixteen studies met the inclusion criteria. Mice and rats were the predominant animal models. All studies except one used CAQK to deliver a therapeutic agent. The reviewed studies mostly included models of brain and spinal cord injuries. Most studies had intravenous administration of CAQK. All studies demonstrated various benefits and that CAQK conjugation facilitated localization to target tissues. No studies directly evaluated the effects of CAQK alone. The data are limited by the heterogeneity in study methodologies and the lack of direct comparison between CAQK and conjugated agents. Overall, these findings present CAQK utilization to deliver a therapeutic agent as a promising targeting strategy in the management of disorders where CSPGs are upregulated.

BACKGROUND: The prevalence of hypertensive disorders of pregnancy (HDPs) in India is 11%, which is one of the highest rates globally. Existing research on HDPs in India primarily focuses on biological risk factors, with minimal research on how socioecological factors combine to increase risk of HDPs. We conducted a rapid review using Bronfenbrenners Ecological Model to understand the social and cultural factors associated with HDPs among Indian pregnant women to identify possible intervention targets that may uniquely improve health in this population. Bronfenbrenners Ecological Model is a framework that can be used to understand the complex relationship between multiple influences on health. METHODS: We reviewed studies published between January 2010 and January 2024 using PubMed, Science Direct, and Scopus databases. Search terms included variants of hypertension, pregnancy, and India. Inclusion criteria were: (1) peer-reviewed journal article; (2) published between January 2010 to January 2024; (3) participants consisted of Indian women living in India; (4) studies evaluated socioecological risk factors associated with HDPs. One independent reviewer performed searches, screening, data extraction, and quality assessment. Each included study was then organized within Bronfenbrenners Ecological Model. RESULTS: A total of 921 studies were generated from the initial search, with 157 exclusions due to duplicates. Following screening for inclusion and exclusion criteria at the title/abstract and full text levels, 17 studies remained in the final review. Socioecological risk factors of HDPs were identified at each level, with the most commonly identified influences including: low socioeconomic status (SES), lacking community education and knowledge on HDP management and prevention, and lacking prenatal HDP screening. CONCLUSION: This study determined that the high risk for HDPs in India is influenced by many intertwined socioecological factors. Women in rural and low SES areas need more health education on HDP management and prevention. There also needs to be more adequate prenatal HDP screening, with at least 4 and ideally 8 prenatal visits. Prenatal screenings should be accompanied with culturally appropriate patient education, especially for low SES women who have limited literacy, so that they can effectively make individual and microsystemic lifestyle decisions aimed at either managing or preventing HDPs.

BACKGROUND: The University of Californias Diabetes Prevention Program (UC DPP) Initiative was implemented systemwide to address diabetes and obesity risk on all 10 campuses. As little is known about implementing lifestyle change programs in university settings, we examined implementation partners (i.e., UC DPP leaders and campus leads) perceptions of factors influencing program success on UC campuses. METHODS: We conducted qualitative interviews with UC DPP leaders and campus leads to examine challenges and opportunities with university-based DPP delivery models. Interviews were recorded, professionally transcribed, and reviewed in detail by the research team. Transcripts were analyzed using rapid qualitative analysis (RQA). The study was approved by the UCLA Institutional Review Board. All implementation partners provided verbal informed consent. RESULTS: Twenty-six implementation partners (8 UC DPP leaders and 18 campus leads) completed interviews in 2021. Seven themes were identified as critical for implementation, including (1) marketing and recruitment (i.e., market and recruit broadly through established channels as well as target at-risk populations); (2) enrollment (i.e., offer the program during convenient times and let participants know what to expect); (3) use an adaptable, evidence-based program; (4) secure funding for the program, participants, lifestyle coaches, and space; (5) hire experienced and dedicated staff and lifestyle coaches; (6) ensure leadership support; and (7) utilize campus linkages and resources. Perceptions of challenges faced with respect to these themes are also described. CONCLUSIONS: This is one of the first studies to examine the challenges and opportunities of delivering an intensive lifestyle change program across 10 university sites. Understanding factors that enhance success of university-based diabetes prevention programs can facilitate UC DPP efforts and help inform delivery strategies of health and wellness programs across other university settings more broadly.

Study objectivesInsomnia symptoms are prevalent along the trajectory of Alzheimer's disease (AD), but the neurobiological underpinning of their interaction is poorly understood. Here, we assessed structural and functional brain measures within and between the default mode network (DMN), salience network, and central executive network (CEN).MethodsWe selected 320 participants from the ADNI database and divided them by their diagnosis: cognitively normal (CN), Mild Cognitive Impairment (MCI), and AD, with and without self-reported insomnia symptoms. We measured the gray matter volume (GMV), structural covariance (SC), degrees centrality (DC), and functional connectivity (FC), testing the effect and interaction of insomnia symptoms and diagnosis on each index. Subsequently, we performed a within-group linear regression across each network and ROI. Finally, we correlated observed abnormalities with changes in cognitive and affective scores.ResultsInsomnia symptoms were associated with FC alterations across all groups. The AD group also demonstrated an interaction between insomnia and diagnosis. Within-group analyses revealed that in CN and MCI, insomnia symptoms were characterized by within-network hyperconnectivity, while in AD, within- and between-network hypoconnectivity was ubiquitous. SC and GMV alterations were nonsignificant in the presence of insomnia symptoms, and DC indices only showed network-level alterations in the CEN of AD individuals. Abnormal FC within and between DMN and CEN hubs was additionally associated with reduced cognitive function across all groups, and increased depressive symptoms in AD.ConclusionsWe conclude that patients with clinical AD present with a unique pattern of insomnia-related functional alterations, highlighting the profound interaction between both conditions.

Behavioral speech tasks have been widely used to understand the mechanisms of speech motor control in typical speakers as well as in various clinical populations. However, determining which neural functions differ between typical speakers and clinical populations based on behavioral data alone is difficult because multiple mechanisms may lead to the same behavioral differences. For example, individuals with cerebellar ataxia (CA) produce atypically large compensatory responses to pitch perturbations in their auditory feedback, compared to typical speakers, but this pattern could have many explanations. Here, computational modeling techniques were used to address this challenge. Bayesian inference was used to fit a state feedback control (SFC) model of voice fundamental frequency (fo) control to the behavioral pitch perturbation responses of speakers with CA and typical speakers. This fitting process resulted in estimates of posterior likelihood distributions for five model parameters (sensory feedback delays, absolute and relative levels of auditory and somatosensory feedback noise, and controller gain), which were compared between the two groups. Results suggest that the speakers with CA may proportionally weight auditory and somatosensory feedback differently from typical speakers. Specifically, the CA group showed a greater relative sensitivity to auditory feedback than the control group. There were also large group differences in the controller gain parameter, suggesting increased motor output responses to target errors in the CA group. These modeling results generate hypotheses about how CA may affect the speech motor system, which could help guide future empirical investigations in CA. This study also demonstrates the overall proof-of-principle of using this Bayesian inference approach to understand behavioral speech data in terms of interpretable parameters of speech motor control models.

Our knowledge of the contribution of genetic interactions (epistasis) to variation in human complex traits remains limited, partly due to the lack of efficient, powerful, and interpretable algorithms to detect interactions. Recently proposed approaches for set-based association tests show promise in improving the power to detect epistasis by examining the aggregated effects of multiple variants. Nevertheless, these methods either do not scale to large Biobank data sets or lack interpretability. We propose QuadKAST, a scalable algorithm focused on testing pairwise interaction effects (quadratic effects) within small to medium-sized sets of genetic variants (window size ≤100) on a trait and provide quantified interpretation of these effects. Comprehensive simulations show that QuadKAST is well-calibrated. Additionally, QuadKAST is highly sensitive in detecting loci with epistatic signals and accurate in its estimation of quadratic effects. We applied QuadKAST to 52 quantitative phenotypes measured in ≈300,000 unrelated white British individuals in the UK Biobank to test for quadratic effects within each of 9515 protein-coding genes. We detect 32 trait-gene pairs across 17 traits and 29 genes that demonstrate statistically significant signals of quadratic effects (accounting for the number of genes and traits tested). Across these trait-gene pairs, the proportion of trait variance explained by quadratic effects is comparable to additive effects, with five pairs having a ratio >1. Our method enables the detailed investigation of epistasis on a large scale, offering new insights into its role and importance.

SNP heritability, the proportion of phenotypic variation explained by genotyped SNPs, is an important parameter in understanding the genetic architecture underlying various diseases and traits. Methods that aim to estimate SNP heritability from individual genotype and phenotype data are limited by their ability to scale to Biobank-scale data sets and by the restrictions in access to individual-level data. These limitations have motivated the development of methods that only require summary statistics. Although the availability of publicly accessible summary statistics makes them widely applicable, these methods lack the accuracy of methods that utilize individual genotypes. Here we present a SUMmary-statistics-based Randomized Haseman-Elston regression (SUM-RHE), a method that can estimate the SNP heritability of complex phenotypes with accuracies comparable to approaches that require individual genotypes, while exclusively relying on summary statistics. SUM-RHE employs Genome-Wide Association Study (GWAS) summary statistics and statistics obtained on a reference population, which can be efficiently estimated and readily shared for public use. Our results demonstrate that SUM-RHE obtains estimates of SNP heritability that are substantially more accurate compared with other summary statistic methods and on par with methods that rely on individual-level data.

Recent research highlights the significant impact of the gut microbiota on health and disease. Thalassemia, a hereditary blood disorder, requires regular blood transfusions, leading to an accumulation of iron in the body. Such changes could potentially alter the intestinal microbiota, thereby increasing the susceptibility of thalassemic patients to infection. In this study, we analyzed the fecal microbiota of 70 non-transfusion-dependent (NTDT) β-thalassemia/HbE patients and 30 healthy controls. Our findings indicate that iron chelation intervention had no detectable effect on the microbiome profile of thalassemic patients. However, the cross-sectional analysis revealed that the bacterial diversity and community structure in patients were significantly less diverse and distinct compared to those of healthy subjects. Using reference frames, we were also able to demonstrate that bacterial taxa that are known to produce short chain fatty acids, from the genera Alistipes, Coprococcus, and Oscillospira, and those from the family Ruminococcaceae, were less prevalent in the patients. In contrast, bacterial taxa associated with an unhealthy gut, including the genus Clostridium and those from the families Fusobacteriaceae, Enterobacteriaceae, and Peptostrptococcaceae, were more prevalent in patients and found to be correlated with higher levels of ferritin. Collectively, these changes in the microbiota could be regarded as markers of raised ferritin levels, and therefore, awareness should be exercised as they could interfere, albeit indirectly, with the treatment of the co-morbidities of thalassemia.

BACKGROUND: Pre-diagnostic disturbances in the microbiome-derived metabolome have been associated with an increased risk of diabetes in non-pregnant populations. However, the roles of microbiome-derived metabolites, the end-products of microbial metabolism, in gestational diabetes (GDM) remain understudied. We examined the prospective association of microbiome-derived metabolites in early to mid-pregnancy with GDM risk in a diverse population. METHODS: We conducted a prospective discovery and validation study, including a case-control sample of 91 GDM and 180 non-GDM individuals within the multi-racial/ethnic The Pregnancy Environment and Lifestyle Study (PETALS) as the discovery set, a random sample from the PETALS (42 GDM, 372 non-GDM) as validation set 1, and a case-control sample (35 GDM, 70 non-GDM) from the Gestational Weight Gain and Optimal Wellness randomized controlled trial as validation set 2. We measured untargeted fasting serum metabolomics at gestational weeks (GW) 10-13 and 16-19 by gas chromatography/time-of-flight mass spectrometry (TOF-MS), liquid chromatography (LC)/quadrupole TOF-MS, and hydrophilic interaction LC/quadrupole TOF-MS. GDM was diagnosed using the 3-h, 100-g oral glucose tolerance test according to the Carpenter-Coustan criteria around GW 24-28. RESULTS: Among 1362 annotated compounds, we identified 140 of gut microbiome metabolism origin. Multivariate enrichment analysis illustrated that carbocyclic acids and branched-chain amino acid clusters at GW 10-13 and the unsaturated fatty acids cluster at GW 16-19 were positively associated with GDM risk (FDR

Pancreatic ductal adenocarcinoma (PDAC) is a leading cause of cancer-related deaths, with adenosquamous carcinoma of the pancreas (ASCP), a rare variant, representing 1-10% of cases. Standard chemotherapy trials for pancreatic cancer exclude ASCP, leaving its optimal treatment uncertain. This report describes a 68-year-old male with metastatic ASCP and a KRAS G12C mutation, progressing through multiple lines of systemic therapy, including targeted inhibition of KRAS G12C. Notably, the patient exhibited a robust response to single-agent immune checkpoint inhibition (ICI) with pembrolizumab, despite intact mismatch repair proteins. The limited success of traditional therapies in pancreatic cancer, coupled with the rarity of ASCP, presents a challenge in establishing effective treatment strategies. While KRAS G12C inhibitors demonstrated modest benefits, this case highlights the remarkable response to ICI in a patient with squamous histology. The distinct tumor microenvironment of ASCP, characterized by squamous differentiation, may contribute to this exceptional response. This underscores the need for further research and clinical trials focused on ICI in ASCP, with an ongoing multi-center phase 2 trial investigating outcomes in this specific subset.

We investigate the emergence, mutation profile, and dissemination of SARS-CoV-2 lineage B.1.214.2, first identified in Belgium in January 2021. This variant, featuring a 3-amino acid insertion in the spike protein similar to the Omicron variant, was speculated to enhance transmissibility or immune evasion. Initially detected in international travelers, it substantially transmitted in Central Africa, Belgium, Switzerland, and France, peaking in April 2021. Our travel-aware phylogeographic analysis, incorporating travel history, estimated the origin to the Republic of the Congo, with primary European entry through France and Belgium, and multiple smaller introductions during the epidemic. We correlate its spread with human travel patterns and air passenger data. Further, upon reviewing national reports of SARS-CoV-2 outbreaks in Belgian nursing homes, we found this strain caused moderately severe outcomes (8.7% case fatality ratio). A distinct nasopharyngeal immune response was observed in elderly patients, characterized by 80% unique signatures, higher B- and T-cell activation, increased type I IFN signaling, and reduced NK, Th17, and complement system activation, compared to similar outbreaks. This unique immune response may explain the variants epidemiological behavior and underscores the need for nasal vaccine strategies against emerging variants.

INTRODUCTION: The Fear of Pain Questionnaire (FOPQ) is a self-report tool designed to measure an individuals fear of pain (FOP). While the Persian version of the FOPQ (FOPQ-P) has been developed, its validity and reliability have not yet been assessed in the Iranian context. This study aims to evaluate the psychometric properties of the FOPQ-P among Iranian patients after surgery. METHODS: A methodological study was conducted in 2023 involving 400 post-surgery patients selected with a convenience sampling. The FOPQ was translated into Persian, and its psychometric properties were analyzed using network analysis, exploratory factor analysis (EFA), confirmatory factor analysis (CFA), as well as assessments of convergent and discriminant validity. Internal consistency was measured using Cronbachs alpha, McDonalds Omega, average inter-item correlation coefficient, Composite Reliability, and Maximal Reliability. RESULTS: The EFA results with Promax and Kaiser Normalization rotation identified two factors that explained 54.32% of the variance, comprising seven items. The CFA confirmed the models validity. Both convergent and discriminant validity were established. The reliability analyses showed that Cronbachs alpha, McDonalds omega, composite reliability, and MaxR for all constructs were above 0.7. Additionally, the average inter-item correlation coefficient was greater than 0.5, indicating strong internal consistency and construct reliability. CONCLUSION: The findings suggest that the FOPQ-P possesses a valid structure and was acceptable reliability in patients cultural context of Iran post-surgery, making it a suitable instrument for measuring fear of pain in this population.

BACKGROUND: Varicoceles affect up to 30% of postpubertal adolescent males. Studying this population remains difficult due to this topics sensitive nature. Using the popularity of social media in this cohort and natural language processing (NLP) techniques, our aim was to identify perceptions of adolescent males on an internet varicocele forum to inform how physicians may better evaluate and counsel this pediatric population. OBJECTIVE: We aimed to characterize themes of discussion and specific concerns expressed by adolescents using a mixed methods approach involving quantitative NLP and qualitative annotation of an online varicocele community. METHODS: We extracted posts from the Reddit community r/varicocele (5100 members) with criteria of discussant age ≤21 years and word count >20. We used qualitative thematic analysis and the validated constant comparative method, as well as an NLP technique called the meaning extraction method with principal component analysis (MEM/PCA), to identify discussion themes. Two investigators independently interrogated 150 randomly selected posts to further characterize content based on NLP-identified themes and calculated the Kaiser-Meyer-Olkin (KMO) statistic and the Bartlett test. Both quantitative and qualitative approaches were then compared to identify key themes of discussion. RESULTS: A total of 1103 posts met eligibility criteria from July 2015 to June 2022. Among the 150 randomly selected posts, MEM/PCA and qualitative thematic analysis separately revealed key themes: an overview of varicocele (40/150, 27%), management (29/150, 19%), postprocedural experience (28/150, 19%), seeking community (26/150, 17%) and second opinions after visiting a physician (27/150, 18%). Quantitative analysis also identified hypogonadism and semen analysis as concerns when discussing their condition. The KMO statistic was >0.60 and the Bartlett test was

Autism spectrum disorder (ASD) is a neurodevelopmental disorder (NDD) that affects approximately 4% of males and 1% of females in the United States. While causes of ASD are multi-factorial, single rare genetic variants contribute to around 20% of cases. Here, we report a case series of seven unrelated probands (6 males, 1 female) with ASD or another variable NDD phenotype attributed to de novo heterozygous loss of function or missense variants in the gene LARP1 (La ribonucleoprotein 1). LARP1 encodes an RNA-binding protein that post-transcriptionally regulates the stability and translation of thousands of mRNAs, including those regulating cellular metabolism and metabolic plasticity. Using lymphocytes collected and immortalized from an index proband who carries a truncating variant in one allele of LARP1, we demonstrated that lower cellular levels of LARP1 protein cause reduced rates of aerobic respiration and glycolysis. As expression of LARP1 increases during neurodevelopment, with higher levels in neurons and astrocytes, we propose that LARP1 haploinsufficiency contributes to ASD or related NDDs through attenuated metabolic activity in the developing fetal brain.

PurposeTo evaluate functional and structural assessments as endpoints for clinical trials for USH2A-related retinal degeneration.MethodsPeople with biallelic disease-causing variants in USH2A, visual acuity ≥ 20/80, and visual field ≥ 10° diameter were enrolled in a 4-year, natural history study. Participants underwent static perimetry, microperimetry, visual acuity, fullfield stimulus testing (FST), and optical coherence tomography annually. Rates of change estimated from mixed-effects linear models and percentages of eyes with changes exceeding the coefficient of repeatability (CoR) or thresholds conforming with U.S. Food and Drug Administration (FDA) guidelines were evaluated.ResultsRates of change were generally more sensitive to change than proportions of eyes exceeding a threshold such as the CoR. Baseline ellipsoid zone area ≥ 3 mm2 was necessary to detect change. Mean sensitivity and volumetric hill of vision measures on static perimetry had similar properties and were the most sensitive to changes of the continuous measures. The highest 4-year proportions of eyes exceeding the CoR were from FST testing (47%) and microperimetry (32%). Specification of loci as functional transition points (FTPs) resulted in 45% (static perimetry) and 46% (microperimetry) at 4 years, meeting FDA guidelines for progression.ConclusionsRate of change of mean sensitivity on static perimetry was a sensitive perimetric continuous measure. Percentages of within-eye change were largest with FST testing and microperimetry. FTPs appear to be particularly sensitive to change. These results affect clinical trial design for USH2A-related retinal degeneration.Translational relevanceAnalyses of natural history data from the Rate of Progression in USH2A-Related Retinal Degeneration (RUSH2A) study can inform eligibility criteria and endpoints for clinical trials.

Whole genome sequences (WGS) enable discovery of rare variants which may contribute to missing heritability of coronary artery disease (CAD). To measure their contribution, we apply the GREML-LDMS-I approach to WGS of 4949 cases and 17,494 controls of European ancestry from the NHLBI TOPMed program. We estimate CAD heritability at 34.3% assuming a prevalence of 8.2%. Ultra-rare (minor allele frequency ≤ 0.1%) variants with low linkage disequilibrium (LD) score contribute ~50% of the heritability. We also investigate CAD heritability enrichment using a diverse set of functional annotations: i) constraint; ii) predicted protein-altering impact; iii) cis-regulatory elements from a cell-specific chromatin atlas of the human coronary; and iv) annotation principal components representing a wide range of functional processes. We observe marked enrichment of CAD heritability for most functional annotations. These results reveal the predominant role of ultra-rare variants in low LD on the heritability of CAD. Moreover, they highlight several functional processes including cell type-specific regulatory mechanisms as key drivers of CAD genetic risk.

The μ-opioid receptor (μOR), a prototypical G protein-coupled receptor (GPCR), is the target of opioid analgesics such as morphine and fentanyl. Due to the severe side effects of current opioid drugs, there is considerable interest in developing novel modulators of μOR function. Most GPCR ligands today are small molecules, however biologics, including antibodies and nanobodies, represent alternative therapeutics with clear advantages such as affinity and target selectivity. Here, we describe the nanobody NbE, which selectively binds to the μOR and acts as an antagonist. We functionally characterize NbE as an extracellular and genetically encoded μOR ligand and uncover the molecular basis for μOR antagonism by determining the cryo-EM structure of the NbE-μOR complex. NbE displays a unique ligand binding mode and achieves μOR selectivity by interactions with the orthosteric pocket and extracellular receptor loops. Based on a β-hairpin loop formed by NbE that deeply protrudes into the μOR, we design linear and cyclic peptide analogs that recapitulate NbEs antagonism. The work illustrates the potential of nanobodies to uniquely engage with GPCRs and describes lower molecular weight μOR ligands that can serve as a basis for therapeutic developments.

Rosacea is a chronic inflammatory skin disorder, whose underlying cellular and molecular mechanisms remain obscure. Here, we generate a single-cell atlas of facial skin from female rosacea patients and healthy individuals. Among keratinocytes, a subpopulation characterized by IFNγ-mediated barrier function damage is found to be unique to rosacea lesions. Blocking IFNγ signaling alleviates rosacea-like phenotypes and skin barrier damage in mice. The papulopustular rosacea is featured by expansion of pro-inflammatory fibroblasts, Schwann, endothelial and macrophage/dendritic cells. The frequencies of type 1/17 and skin-resident memory T cells are increased, and vascular mural cells are characterized by activation of inflammatory pathways and impaired muscle contraction function in rosacea. Most importantly, fibroblasts are identified as the leading cell type producing pro-inflammatory and vasodilative signals in rosacea. Depletion of fibroblasts or knockdown of PTGDS, a gene specifically upregulated in fibroblasts, blocks rosacea development in mice. Our study provides a comprehensive understanding of the aberrant alterations of skin-resident cell populations and identifies fibroblasts as a key determinant in rosacea development.

Persistent racial disparities in health outcomes have catalyzed legislative reforms and heightened scientific focus recently. However, despite the well-documented properties of skin pigments in binding drug compounds, their impact on therapeutic efficacy and adverse drug responses remains insufficiently explored. This perspective examines the intricate relationships between variation in melanin-based skin pigmentation and pharmacokinetics and -dynamics, highlighting the need for considering diversity in skin pigmentation as a variable to advance the equitability of pharmacological interventions. The article provides guidelines on the selection of New Approach Methods (NAMs) to foster inclusive study designs in preclinical drug development pipelines, leading to an improved level of translatability to the clinic.

The ultrafast photochemical reaction mechanism, transient spectra, and transition kinetics of the human blue cone visual pigment have been recorded at room temperature. Ultrafast time-resolved absorption spectroscopy revealed the progressive formation and decay of several metastable photo-intermediates, corresponding to the Batho to Meta-II photo-intermediates previously observed with bovine rhodopsin and human green cone opsin, on the picosecond to millisecond timescales following pulsed excitation. The experimental data reveal several interesting similarities and differences between the photobleaching sequences of bovine rhodopsin, human green cone opsin, and human blue cone opsin. While Meta-II formation kinetics are comparable between bovine rhodopsin and blue cone opsin, the transition kinetics of earlier photo-intermediates and qualitative characteristics of the Meta-I to Meta-II transition are more similar for blue cone opsin and green cone opsin. Additionally, the blue cone photo-intermediate spectra exhibit a high degree of overlap with uniquely small spectral shifts. The observed variation in Meta-II formation kinetics between rod and cone visual pigments is explained based on key structural differences.

The release of GPT-4 and other large language models (LLMs) has the potential to transform healthcare. However, existing research evaluating LLM performance on real-world clinical notes is limited. Here, we conduct a highly-powered study to determine whether LLMs can provide clinical recommendations for three tasks (admission status, radiological investigation(s) request status, and antibiotic prescription status) using clinical notes from the Emergency Department. We randomly selected 10,000 Emergency Department visits to evaluate the accuracy of zero-shot, GPT-3.5-turbo- and GPT-4-turbo-generated clinical recommendations across four different prompting strategies. We found that both GPT-4-turbo and GPT-3.5-turbo performed poorly compared to a resident physician, with accuracy scores 8% and 24%, respectively, lower than physician on average. Both LLMs tended to be overly cautious in its recommendations, with high sensitivity at the cost of specificity. Our findings demonstrate that, while early evaluations of the clinical use of LLMs are promising, LLM performance must be significantly improved before their deployment as decision support systems for clinical recommendations and other complex tasks.

BACKGROUND: People living with HIV experience HIV stigma alongside a spectrum of aging-related health conditions that accelerate their vulnerability to the ill effects of loneliness and social isolation. Group-singing interventions are efficacious in improving psychosocial well-being among older people in the general population; however, the social curative effects of group singing have not been explored in relation to HIV stigma. By promoting group identification, bonding, and pride, group singing may reduce loneliness, social isolation, and other negative impacts of HIV stigma among older people living with HIV. Access to group-singing programs may be enhanced by technology. OBJECTIVE: While group singing has been extensively studied in older adults, group-singing interventions have not been adapted for older people living with HIV to target loneliness and social isolation in the context of HIV stigma. The objective of this study was to describe the systematic development of a group-singing intervention to reduce loneliness and social isolation among older people living with HIV. METHODS: In the San Francisco Bay Area between February 2019 and October 2019, we engaged older people living with HIV in a rigorous, 8-stage, community-engaged intervention adaptation process using the Assessment, Decision, Adaptation, Production, Topical Experts, Integration, Training, and Testing (ADAPT-ITT) framework. On the basis of a formative assessment of the needs and preferences of older people living with HIV, we selected an evidence-based group-singing intervention for older adults and systematically adapted the intervention components by administering them to a community advisory council (n=13). RESULTS: The result was United Voices, a 12-week hybrid (web-based and in-person) group-singing intervention for older people living with HIV. United Voices comprises 12 web-based (ie, via Zoom [Zoom Video Communications]) rehearsals, web-based and in-person drop-in helpdesk sessions, and a professionally produced final concert recording. CONCLUSIONS: Through an iterative process and in consultation with stakeholders and topic experts, we refined and manualized United Voices and finalized the design of a pilot randomized controlled trial to evaluate the feasibility and acceptability of the intervention protocol and procedures. The findings provide insights into the barriers and facilitators involved in culturally tailoring interventions for older people living with HIV, implementing intervention adaptations within web-based environments, and the promise of developing hybrid music-based interventions for older adults with HIV.

Pulmonary veno-occlusive disease (PVOD) is a rare but severe form of pulmonary hypertension characterized by the obstruction of pulmonary arteries and veins, causing increased pulmonary artery pressure and leading to right ventricular (RV) heart failure. PVOD is often resistant to conventional pulmonary arterial hypertension (PAH) treatments and has a poor prognosis, with a median survival time of 2-3 years after diagnosis. We previously showed that the administration of a chemotherapy agent mitomycin C (MMC) in rats mediates PVOD through the activation of the eukaryotic initiation factor 2 (eIF2) kinase protein kinase R (PKR) and the integrated stress response (ISR), resulting in the impairment of vascular endothelial junctional structure and barrier function. Here, we demonstrate that aged rats over 1 year exhibit more severe vascular remodeling and RV hypertrophy than young adult rats following MMC treatment. This is attributed to an age-associated elevation of basal ISR activity and depletion of protein phosphatase 1, leading to prolonged eIF2 phosphorylation and sustained ISR activation. Pharmacological blockade of PKR or ISR mitigates PVOD phenotypes in both age groups, suggesting that targeting the PKR/ISR axis could be a potential therapeutic strategy for PVOD.

Increasingly, scRNA-Seq studies explore cell populations across different samples and the effect of sample heterogeneity on organisms phenotype. However, relatively few bioinformatic methods have been developed which adequately address the variation between samples for such population-level analyses. We propose a framework for representing the entire single-cell profile of a sample, which we call a GloScope representation. We implement GloScope on scRNA-Seq datasets from study designs ranging from 12 to over 300 samples and demonstrate how GloScope allows researchers to perform essential bioinformatic tasks at the sample-level, in particular visualization and quality control assessment.

Oral broad-spectrum antivirals are urgently needed for the treatment of many emerging and contemporary RNA viruses. We previously synthesized 1-O-octadecyl-2-O-benzyl-sn-glyceryl-P-RVn (ODBG-P-RVn, V2043), a phospholipid prodrug of GS-441524 (remdesivir nucleoside, RVn), and demonstrated its in vivo efficacy in a SARS-CoV-2 mouse model. Structure-activity relationship studies focusing on the prodrug scaffold identified two modifications, 3-fluoro-4-methoxy-benzyl (V2053) and 4-cyano-benzyl (V2067), that significantly enhanced the in vitro broad-spectrum antiviral activity against multiple RNA viruses when compared to V2043. Here, we demonstrate that V2043, V2053, and V2067 are all orally bioavailable, well-tolerated, and achieve high sustained plasma levels after single oral daily dosing. All three phospholipid prodrugs are significantly more active than RVn in vitro and significantly reduce SARS-CoV-2 lung titers in prophylaxis and treatment mouse models of SARS-CoV-2 B.1.351 infection. On a molar basis, V2043 and V2067 are substantially more active than obeldesivir/GS-5245 and molnupiravir in vivo. Together, these data support the continued development of phospholipid RVn prodrugs for the treatment of SARS-CoV-2 and other RNA viruses of clinical concern.

Despite known treatments, tuberculosis (TB) remains the worlds top infectious killer, highlighting the pressing need for new drug regimens. To prioritize the most efficacious drugs for clinical testing, we previously developed a PK-PD translational platform with bacterial dynamics that reliably predicted short-term monotherapy outcomes in Phase IIa trials from preclinical mouse studies. In this study, we extended our platform to include PK-PD models that account for drug-drug interactions in combination regimens and bacterial regrowth in our bacterial dynamics model to predict cure at the end of treatment and relapse 6 months post-treatment. The Phase III STAND trial testing a new regimen comprised of pretomanid (Pa), moxifloxacin (M), and pyrazinamide (Z) (PaMZ) was suspended after a separate ongoing trial (NC-005) suggested that adding bedaquiline (B) to the PaMZ regimen would improve efficacy. To forecast if the addition of B would, indeed, benefit the PaMZ regimen, we applied an extended translational platform to both regimens. We predicted currently available short- and long-term clinical data well for drug combinations related to BPaMZ. We predicted the addition of B to PaMZ to shorten treatment duration by 2 months and to have similar bacteriological success to standard HRZE treatment (considering only treatment success but not withdrawal from side effects and other adverse events), both at the end of treatment for treatment efficacy and 6 months after treatment has ended in relapse prevention. Using BPaMZ as a case study, we have demonstrated our translational platform can predict Phase II and III outcomes prior to actual trials, allowing us to better prioritize the regimens most likely to succeed.

Casein kinase 1δ (CK1δ) controls essential biological processes including circadian rhythms and wingless-related integration site (Wnt) signaling, but how its activity is regulated is not well understood. CK1δ is inhibited by autophosphorylation of its intrinsically disordered C-terminal tail. Two CK1 splice variants, δ1 and δ2, are known to have very different effects on circadian rhythms. These variants differ only in the last 16 residues of the tail, referred to as the extreme C termini (XCT), but with marked changes in potential phosphorylation sites. Here, we test whether the XCT of these variants have different effects in autoinhibition of the kinase. Using NMR and hydrogen/deuterium exchange mass spectrometry, we show that the δ1 XCT is preferentially phosphorylated by the kinase and the δ1 tail makes more extensive interactions across the kinase domain. Mutation of δ1-specific XCT phosphorylation sites increases kinase activity both in vitro and in cells and leads to changes in the circadian period, similar to what is reported in vivo. Mechanistically, loss of the phosphorylation sites in XCT disrupts tail interaction with the kinase domain. δ1 autoinhibition relies on conserved anion-binding sites around the CK1 active site, demonstrating a common mode of product inhibition of CK1δ. These findings demonstrate how a phosphorylation cycle controls the activity of this essential kinase.