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The extent to which interannual variability in Arctic sea ice influences the mid-latitude circulation has been extensively debated. While observational data support the existence of a teleconnection between November sea ice in the Barents–Kara region and the subsequent winter North Atlantic Oscillation, climate models do not consistently reproduce such a link, with only very weak inter-model consensus. We show, using the EC-Earth3 climate model, that while an ensemble of coupled EC-Earth3 simulations shows no evidence of such a teleconnection, the inclusion of stochastic parameterizations to the ocean and sea ice component results in the emergence of a robust teleconnection comparable in magnitude to that observed. While the exact mechanisms causing this remain unclear, we argue that it can be accounted for by an improved ice–ocean–atmosphere coupling due to the stochastic perturbations, which aim to represent the effect of unresolved ice and ocean variability. In particular, the weak inter-model consensus may to a large extent be due to model biases in surface coupling, with stochastic parameterizations being one possible remedy.

Abstract Adaptive optics ophthalmoscopy has enabled visualization of the in vivo human photoreceptor mosaic in health, disease and its treatment. Despite this, the clinical utility of the imaging technology has been limited by a lack of automated analysis techniques capable of accurately quantifying photoreceptor structure and a lack of an available normative image database. Here, we present a fully automated algorithm for estimating cone spacing and density over a complete adaptive optics montage along with a database of normative images and cone densities. We imaged the cone mosaics surrounding the fovea and along the horizontal and vertical meridians of fifty normal-sighted controls with a custom-built, multimodal adaptive optics scanning light ophthalmoscope. Cone spacing was automatically measured in the frequency domain and spacing measurements were converted to estimates of cone density at all locations across the montage. Consistent with previous reports, cone density measurements were highest near fovea (152,906 ± 53,209 cones/mm2) and decreased exponentially with eccentricity. A 2.5-fold variation was found in cone density estimates at 0.1 mm, this variation decreased to 1.75-fold at 1 mm. We provide all images, mosaic quantifications, and automated software open source. This database will aid investigators in translating adaptive optics ophthalmoscopy to clinical applications.

Abstract The Maintenance of Lipid Asymmetry (Mla) pathway is a multicomponent system found in all gram-negative bacteria that contributes to virulence, vesicle blebbing and preservation of the outer membrane barrier function. It acts by removing ectopic lipids from the outer leaflet of the outer membrane and returning them to the inner membrane through three proteinaceous assemblies: the MlaA-OmpC complex, situated within the outer membrane; the periplasmic phospholipid shuttle protein, MlaC; and the inner membrane ABC transporter complex, MlaFEDB, proposed to be the founding member of a structurally distinct ABC superfamily. While the function of each component is well established, how phospholipids are exchanged between components remains unknown. This stands as a major roadblock in our understanding of the function of the pathway, and in particular, the role of ATPase activity of MlaFEDB is not clear. Here, we report the structure of E. coli MlaC in complex with the MlaD hexamer in two distinct stoichiometries. Utilising in vivo complementation assays, an in vitro fluorescence-based transport assay, and molecular dynamics simulations, we confirm key residues, identifying the MlaD β6-β7 loop as essential for MlaCD function. We also provide evidence that phospholipids pass between the C-terminal helices of the MlaD hexamer to reach the central pore, providing insight into the trajectory of GPL transfer between MlaC and MlaD.

IntroductionMesenchymal stem cell-derived extracellular vesicles (MSC EVs) hold significant promise for regenerative medicine. Lyophilization of EVs significantly enhances their translational potential. While, lyophilized EVs have been studied from a morphological perspective, the functional stability of these EVs and their cargo following lyophilization need to be mechanistically investigated.MethodsIn this study, we investigated the functional and mechanistic bioactivity of fresh versus lyophilized MSC EVs, specifically focusing on functionally engineered osteoinductive EVs developed in our laboratory. We utilized dimethyl sulfoxide (DMSO) as a cryoprotectant and conducted pathway-specific in vitro and in vivo experiments to assess the stability and functionality of the EVs.ResultsOur findings show that using DMSO as a cryoprotectant before lyophilization preserves the functional stability of engineered MSC EVs. In vitro experiments demonstrated that the endocytosis, cargo integrity, and pathway-specific activity of lyophilized EVs were maintained when DMSO was used as the cryoprotectant. Additionally, in vivo bone regeneration studies revealed that the functionality of cryoprotected lyophilized EVs was comparable to that of freshly isolated EVs.DiscussionThese results provide a foundation for evaluating the functionality of lyophilized EVs and exploring the use of DMSO and other cryoprotectants in EV-based therapies. Understanding the functionality of lyophilized naïve and engineered EVs from a mechanistic perspective may enhance validation approaches for tissue regeneration strategies.

Abstract Background In recent years, stimulant use has increased among persons who use opioids in the rural U.S., leading to high rates of overdose and death. We sought to understand motivations and contexts for stimulant use among persons who use opioids in a large, geographically diverse sample of persons who use drugs (PWUD) in the rural settings. Methods We conducted semi-structured individual interviews with PWUD at 8 U.S. sites spanning 10 states and 65 counties. Content areas included general substance use, injection drug use, changes in drug use, and harm reduction practices. We used an iterative open-coding process to comprehensively itemize and categorize content shared by participants related to concurrent use. Results We interviewed 349 PWUD (64% male, mean age 36). Of those discussing current use of stimulants in the context of opioid use (n = 137, 39%), the stimulant most used was methamphetamine (78%) followed by cocaine/crack (26%). Motivations for co-use included: 1) change in drug markets and cost considerations; 2) recreational goals, e.g., seeking stronger effects after heightened opioid tolerance; 3) practical goals, such as a desire to balance or alleviate the effects of the other drug, including the use of stimulants to avoid/reverse opioid overdose, and/or control symptoms of opioid withdrawal; and 4) functional goals, such as being simultaneously energized and pain-free in order to remain productive for employment. Conclusion In a rural U.S. cohort of PWUD, use of both stimulants and opioids was highly prevalent. Reasons for dual use found in the rural context compared to urban studies included changes in drug availability, functional/productivity goals, and the use of methamphetamine to offset opioid overdose. Education efforts and harm reduction services and treatment, such as access to naloxone, fentanyl test strips, and accessible drug treatment for combined opioid and stimulant use, are urgently needed in the rural U.S. to reduce overdose and other adverse outcomes.

Abstract Choroideremia (CHM) is an X-linked retinal degeneration leading to loss of the photoreceptors, retinal pigment epithelium (RPE), and choroid. Adaptive optics optoretinography is an emerging technique for noninvasive, objective assessment of photoreceptor function. Here, we investigate parafoveal cone function in CHM using adaptive optics optoretinography and compare with cone structure and clinical assessments of vision. Parafoveal cone mosaics of 10 CHM and four normal-sighted participants were imaged with an adaptive optics scanning light ophthalmoscope. While acquiring video sequences, a 2 s 550Δ10 nm, 450 nW/deg2 stimulus was presented. Videos were registered and the intensity of each cone in each frame was extracted, normalized, standardized, and aggregated to generate the population optoretinogram (ORG) over time. A gamma-pdf was fit to the ORG and the peak was extracted as ORG amplitude. CHM ORG amplitudes were compared to normal and were correlated with bound cone density, ellipsoid zone to RPE/Bruch’s membrane (EZ-to-RPE/BrM) distance, and foveal sensitivity using Pearson correlation analysis. ORG amplitude was significantly reduced in CHM compared to normal (0.22 ± 0.15 vs. 1.34 ± 0.31). In addition, CHM ORG amplitude was positively correlated with cone density, EZ-to-RPE/BrM distance, and foveal sensitivity. Our results demonstrate promise for using ORG as a biomarker of photoreceptor function.

Background The early identification of outbreaks of both known and novel influenza-like illnesses (ILIs) is an important public health problem. Objective This study aimed to describe the design and testing of a tool that detects and tracks outbreaks of both known and novel ILIs, such as the SARS-CoV-2 worldwide pandemic, accurately and early. Methods This paper describes the ILI Tracker algorithm that first models the daily occurrence of a set of known ILIs in hospital emergency departments in a monitored region using findings extracted from patient care reports using natural language processing. We then show how the algorithm can be extended to detect and track the presence of an unmodeled disease that may represent a novel disease outbreak. Results We include results based on modeling diseases like influenza, respiratory syncytial virus, human metapneumovirus, and parainfluenza for 5 emergency departments in Allegheny County, Pennsylvania, from June 1, 2014, to May 31, 2015. We also include the results of detecting the outbreak of an unmodeled disease, which in retrospect was very likely an outbreak of the enterovirus D68 (EV-D68). Conclusions The results reported in this paper provide support that ILI Tracker was able to track well the incidence of 4 modeled influenza-like diseases over a 1-year period, relative to laboratory-confirmed cases, and it was computationally efficient in doing so. The system was also able to detect a likely novel outbreak of EV-D68 early in an outbreak that occurred in Allegheny County in 2014 as well as clinically characterize that outbreak disease accurately.

Abstract Background Genomic variants of the disease are often discovered nowadays through population-based genome-wide association studies (GWAS). Identifying genomic variations potentially underlying a phenotype, such as hypertension, in an individual is important for designing personalized treatment; however, population-level models, such as GWAS, may not capture all the important, individualized factors well. In addition, GWAS typically requires a large sample size to detect the association of low-frequency genomic variants with sufficient power. Here, we report an individualized Bayesian inference (IBI) algorithm for estimating the genomic variants that influence complex traits, such as hypertension, at the level of an individual (e.g., a patient). By modeling at the level of the individual, IBI seeks to find genomic variants observed in the individual’s genome that provide a strong explanation of the phenotype observed in this individual. Results We applied the IBI algorithm to the data from the Framingham Heart Study to explore the genomic influences of hypertension. Among the top-ranking variants identified by IBI and GWAS, there is a significant number of shared variants (intersection); the unique variants identified only by IBI tend to have relatively lower minor allele frequency than those identified by GWAS. In addition, IBI discovered more individualized and diverse variants that explain hypertension patients better than GWAS. Furthermore, IBI found several well-known low-frequency variants as well as genes related to blood pressure that GWAS missed in the same cohort. Finally, IBI identified top-ranked variants that predicted hypertension better than GWAS, according to the area under the ROC curve. Conclusions The results support IBI as a promising approach for complementing GWAS, especially in detecting low-frequency genomic variants as well as learning personalized genomic variants of clinical traits and disease, such as the complex trait of hypertension, to help advance precision medicine.

Abstract Background Overdoses have surged in rural areas in the U.S. and globally for years, but harm reduction interventions have lagged. Overdose education and naloxone distribution (OEND) programs reduce overdose mortality, but little is known about people who use drugs’ (PWUD) experience with these interventions in rural areas. Here, we analyze qualitative data with rural PWUD to learn about participants’ experiences with an OEND intervention, and about how participants’ perceptions of their rural risk environments influenced the interventions’ effects. Methods Twenty-nine one-on-one, semi-structured qualitative interviews were conducted with rural PWUD engaged in the CARE2HOPE OEND intervention in Appalachian Kentucky. Interviews were conducted via Zoom, audio-recorded, and transcribed verbatim. Thematic analysis was conducted, guided by the Rural Risk Environment Framework. Results Participants’ naloxone experiences were shaped by all domains of their rural risk environments. The OEND intervention transformed participants’ roles locally, so they became an essential component of the local rural healthcare environment. The intervention provided access to naloxone and information, thereby increasing PWUDs’ confidence in naloxone administration. Through the intervention, over half of participants gained knowledge on naloxone (access points, administration technique) and on the criminal-legal environment as it pertained to naloxone. Most participants opted to accept and carry naloxone, citing factors related to the social environment (responsibility to their community) and physical/healthcare environments (overdose prevalence, suboptimal emergency response systems). Over half of participants described recent experiences administering intervention-provided naloxone. These experiences were shaped by features of the local rural social environment (anticipated negative reaction from recipients, prior naloxone conversations). Conclusions By providing naloxone paired with non-stigmatizing health and policy information, the OEND intervention offered support that allowed participants to become a part of the healthcare environment. Findings highlight need for more OEND interventions; outreach to rural PWUD on local policy that impacts them; tailored strategies to help rural PWUD engage in productive dialogue with peers about naloxone and navigate interpersonal conflict associated with overdose reversal; and opportunities for rural PWUD to formally participate in emergency response systems as peer overdose responders. Trial registration The ClinicalTrials.gov ID for the CARE2HOPE intervention is NCT04134767. The registration date was October 19th, 2019.

Abstract Background Research conducted in urban areas has highlighted the impact of housing instability on people who inject drugs (PWID), revealing that it exacerbates vulnerability to drug-related harms and impedes syringe service program (SSP) use. However, few studies have explored the effects of houselessness on SSP use among rural PWID. This study examines the relationship between houselessness and SSP utilization among PWID in eight rural areas across 10 states. Methods PWID were recruited using respondent-driven sampling for a cross-sectional survey that queried self-reported drug use and SSP utilization in the prior 30 days, houselessness in the prior 6 months and sociodemographic characteristics. Using binomial logistic regression, we examined the relationship between experiencing houselessness and any SSP use. To assess the relationship between houselessness and the frequency of SSP use, we conducted multinomial logistic regression analyses among participants reporting any past 30-day SSP use. Results Among 2394 rural PWID, 56.5% had experienced houselessness in the prior 6 months, and 43.5% reported past 30-day SSP use. PWID who had experienced houselessness were more likely to report using an SSP compared to their housed counterparts (adjusted odds ratio [aOR] = 1.24 [95% confidence intervals [CI] 1.01, 1.52]). Among those who had used an SSP at least once (n = 972), those who experienced houselessness were just as likely to report SSP use two (aOR = 0.90 [95% CI 0.60, 1.36]) and three times (aOR = 1.18 [95% CI 0.77, 1.98]) compared to once. However, they were less likely to visit an SSP four or more times compared to once in the prior 30 days (aOR = 0.59 [95% CI 0.40, 0.85]). Conclusion This study provides evidence that rural PWID who experience houselessness utilize SSPs at similar or higher rates as their housed counterparts. However, housing instability may pose barriers to more frequent SSP use. These findings are significant as people who experience houselessness are at increased risk for drug-related harms and encounter additional challenges when attempting to access SSPs.

IntroductionClinical tools have been widely used in the diagnosis, description, and monitoring the progression of retinitis pigmentosa (RP); however, many of these methods have inherently low sensitivity and specificity, and significant photoreceptor disruption can occur before RP progression has clinically manifest. Adaptive optics scanning light ophthalmoscopy (AOSLO) has shown promise as a powerful tool for assessing photoreceptor disruption both structurally and functionally due to its increased resolution.MethodsHere we assess photoreceptor structure and function at the cellular level through AOSLO by acquiring intensity based optoretinography (iORG) in 15 individuals with no reported retinal pathology and 7 individuals with a prior clinical diagnosis of RP. Photoreceptor structure was quantified by calculating cone nearest neighbor distance (NND) across different retinal eccentricities from the AOSLO images. Cone outer segment length was measured across different retinal eccentricities using optical coherence tomography (OCT) derived longitudinal reflectivity profiles (LRPs). Finally, iORG measures of photoreceptor function were compared to retinal sensitivity as measured using the macular integrity assessment (MAIA) microperimeter.ResultsBroadly, participants with RP exhibited increasing cone nearest neighbor distances and decreasing cone outer segment length as a function of retinal eccentricity, consistent with prior reports for both controls and individuals with RP. Nearly all individuals with RP had reduced iORG amplitudes for all retinal eccentricities when compared to the control cohort, and the reduction was greater in eccentricities further from the fovea. Comparing iORG amplitudes to MAIA retinal sensitivity, we found that the iORG was more sensitive to early changes in photoreceptor function whereas MAIA was more sensitive to later stages of disease.DiscussionThis highlights the utility of iORG as a method to detect sub-clinical deficits in cone function in all stages of disease progression and supports the future use of iORG for identifying cells that are candidates for cellular based therapies.

PurposeTo characterize the effect of sampling window size on maps of foveal cone density derived from adaptive optics scanning light ophthalmoscope (AOSLO) images of the cone mosaic.MethodsForty-four AOSLO-derived montages of the foveal cone mosaic (300 x 300µm) were used for this study (from 44 individuals with normal vision). Cone photoreceptor coordinates were semi-automatically identified by one experienced grader. From these coordinates, cone density matrices across each foveal montage were derived using 10 different sampling window sizes containing 5, 10, 15, 20, 40, 60, 80, 100, 150, or 200 cones. For all 440 density matrices, we extracted the location and value of peak cone density (PCD), the cone density centroid (CDC) location, and cone density at the CDC.ResultsAcross all window sizes, PCD values were larger than those extracted at the CDC location, though the difference between these density values decreased as the sampling window size increased (p

Introduction Many rural communities bear a disproportionate share of drug-related harms. Innovative harm reduction service models, such as vending machines or kiosks, can expand access to services that reduce drug-related harms. However, few kiosks operate in the USA, and their implementation, impact and cost-effectiveness have not been adequately evaluated in rural settings. This paper describes the Kentucky Outreach Service Kiosk (KyOSK) Study protocol to test the effectiveness, implementation outcomes and cost-effectiveness of a community-tailored, harm reduction kiosk in reducing HIV, hepatitis C and overdose risk in rural Appalachia.Methods and analysis KyOSK is a community-level, controlled quasi-experimental, non-randomised trial. KyOSK involves two cohorts of people who use drugs, one in an intervention county (n=425) and one in a control county (n=325). People who are 18 years or older, are community-dwelling residents in the target counties and have used drugs to get high in the past 6 months are eligible. The trial compares the effectiveness of a fixed-site, staffed syringe service programme (standard of care) with the standard of care supplemented with a kiosk. The kiosk will contain various harm reduction supplies accessible to participants upon valid code entry, allowing dispensing data to be linked to participant survey data. The kiosk will include a call-back feature that allows participants to select needed services and receive linkage-to-care services from a peer recovery coach. The cohorts complete follow-up surveys every 6 months for 36 months (three preceding kiosk implementation and four post-implementation). The study will test the effectiveness of the kiosk on reducing risk behaviours associated with overdose, HIV and hepatitis C, as well as implementation outcomes and cost-effectiveness.Ethics and dissemination The University of Kentucky Institutional Review Board approved the protocol. Results will be disseminated in academic conferences and peer-reviewed journals, online and print media, and community meetings.Trial registration number NCT05657106.

ABSTRACT The fitness cost of an antibiotic resistance gene (ARG) can differ across host strains, creating refuges that allow the maintenance of an ARG in the absence of direct selection for its resistance phenotype. Despite the importance of such ARG-host interactions for predicting ARG dynamics, the basis of ARG fitness costs and their variability between hosts are not well understood. We determined the genetic basis of a host-dependent cost of a β-lactamase, blaTEM-116*, that conferred a significant cost in one Escherichia coli strain but was close to neutral in 11 other Escherichia spp. strains. Selection of a blaTEM-116*-encoding plasmid in the strain in which it initially had a high cost resulted in rapid and parallel compensation for that cost through mutations in a P1-like phage gene, relAP1. When the wild-type relAP1 gene was added to a strain in which it was not present and in which blaTEM-116* was neutral, it caused the ARG to become costly. Thus, relAP1 is both necessary and sufficient to explain blaTEM-116* costs in at least some host backgrounds. To our knowledge, these findings represent the first demonstrated case of the cost of an ARG being influenced by a genetic interaction with a phage gene. The interaction between a phage gene and a plasmid-borne ARG highlights the complexity of selective forces determining the maintenance and spread of ARGs and, by extension, encoding phage and plasmids in natural bacterial communities.IMPORTANCEAntibiotic resistance genes (ARGs) play a major role in the increasing problem of antibiotic resistance in clinically relevant bacteria. Selection of these genes occurs in the presence of antibiotics, but their eventual success also depends on the sometimes substantial costs they impose on host bacteria in antibiotic-free environments. We evolved an ARG that confers resistance to penicillin-type antibiotics in one host in which it did confer a cost and in one host in which it did not. We found that costs were rapidly and consistently reduced through parallel genetic changes in a gene encoded by a phage that was infecting the costly host. The unmutated version of this gene was sufficient to cause the ARG to confer a cost in a host in which it was originally neutral, demonstrating an antagonism between the two genetic elements and underlining the range and complexity of pressures determining ARG dynamics in natural populations.

Abstract Therapeutic dentin regeneration remains difficult to achieve, and a majority of the attention has been given to anabolic strategies to promote dentinogenesis directly, whereas, the available literature is insufficient to understand the role of inflammation and inflammatory complement system on dentinogenesis. The aim of this study is to determine the role of complement C5a receptor (C5aR) in regulating dental pulp stem cells (DPSCs) differentiation and in vivo dentin regeneration. Human DPSCs were subjected to odontogenic differentiation in osteogenic media treated with the C5aR agonist and C5aR antagonist. In vivo dentin formation was evaluated using the dentin injury/pulp-capping model of the C5a-deficient and wild-type mice. In vitro results demonstrate that C5aR inhibition caused a substantial reduction in odontogenic DPSCs differentiation markers such as DMP-1 and DSPP, while the C5aR activation increased these key odontogenic genes compared to control. A reparative dentin formation using the C5a-deficient mice shows that dentin regeneration is significantly reduced in the C5a-deficient mice. These data suggest a positive role of C5aR in the odontogenic DPSCs differentiation and tertiary/reparative dentin formation. This study addresses a novel regulatory pathway and a therapeutic approach for improving the efficiency of dentin regeneration in affected teeth.

Abstract Background Expanding access to sterile syringes in rural areas is vital, as injection-related epidemics expand beyond metropolitan areas globally. While pharmacies have potential to be an easily accessible source of sterile syringes, research in cities has identified moral, legal and ethical barriers that preclude over-the-counter (OTC) sales to people who inject drugs (PWID). The current study builds on prior urban-based research by elucidating (1) pharmacy OTC policies and (2) pharmacists’ rationale for, and barriers and facilitators to, OTC syringe sales in a US rural area hard hit by drug-related epidemics. Methods We conducted 14 semi-structured interviews with pharmacists recruited from two Eastern Kentucky health districts. Interview domains included experiences with, and attitudes toward, selling OTC syringes to PWID. Constructivist grounded theory methods were used to analyze verbatim transcripts. Results Most pharmacists operated “restrictive OTC” pharmacies (n = 8), where patients were required to have a prescription or proof of medical need to purchase a syringe. The remainder (n = 6) operated “open OTC” pharmacies, which allowed OTC syringe sales to most patients. Both groups believed their pharmacy policies protected their community and pharmacy from further drug-related harm, but diverging policies emerged because of stigma toward PWID, perceptions of Kentucky law, and belief OTC syringe sales were harmful rather than protective to the community. Conclusion Our results suggest that restrictive OTC pharmacy policies are rooted in stigmatizing views of PWID. Anti-stigma education about substance use disorder (SUD), human immunodeficiency virus (HIV), and Hepatitis C (HCV) is likely needed to truly shift restrictive pharmacy policy.

Abstract Beneficial mutations can become costly following an environmental change. Compensatory mutations can relieve these costs, while not affecting the selected function, so that the benefits are retained if the environment shifts back to be similar to the one in which the beneficial mutation was originally selected. Compensatory mutations have been extensively studied in the context of antibiotic resistance, responses to specific genetic perturbations, and in the determination of interacting gene network components. Few studies have focused on the role of compensatory mutations during more general adaptation, especially as the result of selection in fluctuating environments where adaptations to different environment components may often involve trade‐offs. We examine whether costs of a mutation in lacI, which deregulated the expression of the lac operon in evolving populations of Escherichia coli bacteria, were compensated. This mutation occurred in multiple replicate populations selected in environments that fluctuated between growth on lactose, where the mutation was beneficial, and on glucose, where it was deleterious. We found that compensation for the cost of the lacI mutation was rare, but, when it did occur, it did not negatively affect the selected benefit. Compensation was not more likely to occur in a particular evolution environment. Compensation has the potential to remove pleiotropic costs of adaptation, but its rarity indicates that the circumstances to bring about the phenomenon may be peculiar to each individual or impeded by other selected mutations.

Abstract Background Enrolling sufficient number of people who inject drugs (PWID) into syringe services programs (SSP) is important to curtail outbreaks of drug-related harms. Still, little is known about barriers and facilitators to SSP enrollment in rural areas with no history of such programs. This study’s purpose was to develop a grounded theory of the role of the risk environment and individual characteristics of PWID in shaping SSP enrollment in rural Kentucky. Methods We conducted one-on-one semi-structured interviews with 41 clients of 5 SSPs that were established in rural counties in Appalachian Kentucky in 2017–2018. Interviews covered PWID needs, the process of becoming aware of SSPs, and barriers and facilitators to SSP enrollment. Applying constructivist grounded theory methods and guided by the Intersectional Risk Environment Framework (IREF), we applied open, axial and selective coding to develop the grounded theory. Results Stigma, a feature of IREF’s meso-level social domain, is the main factor hampering SSP enrollment. PWID hesitated to visit SSPs because of internalized stigma and because of anticipated stigma from police, friends, family and healthcare providers. Fear of stigma was often mitigated or amplified by a constellation of meso-level environmental factors related to healthcare (e.g., SSPs) and social (PWID networks) domains and by PWID’s individual characteristics. SSPs mitigated stigma as a barrier to enrollment by providing low threshold services in a friendly atmosphere, and by offering their clients program IDs to protect them from paraphernalia charges. SSP clients spread positive information about the program within PWID networks and helped their hesitant peers to enroll by accompanying them to SSPs. Individual characteristics, including child custody, employment or high social status, made certain PWID more susceptible to drug-related stigma and hence more likely to delay SSP enrollment. Conclusions Features of the social and healthcare environments operating at the meso-level, as well as PWID’s individual characteristics, appear to enhance or mitigate the effect of stigma as a barrier to SSP enrollment. SSPs opening in locations with high stigma against PWID need to ensure low threshold and friendly services, protect their clients from police and mobilize PWID networks to promote enrollment.

Abstract Background Malaria is a major cause of death in children under five years old in low- and middle-income countries such as Malawi. Accurate diagnosis and management of malaria can help reduce the global burden of childhood morbidity and mortality. Trained healthcare workers in rural health centers manage malaria with limited supplies of malarial diagnostic tests and drugs for treatment. A clinical decision support system that integrates predictive models to provide an accurate prediction of malaria based on clinical features could aid healthcare workers in the judicious use of testing and treatment. We developed Bayesian network (BN) models to predict the probability of malaria from clinical features and an illustrative decision tree to model the decision to use or not use a malaria rapid diagnostic test (mRDT). Methods We developed two BN models to predict malaria from a dataset of outpatient encounters of children in Malawi. The first BN model was created manually with expert knowledge, and the second model was derived using an automated method. The performance of the BN models was compared to other statistical models on a range of performance metrics at multiple thresholds. We developed a decision tree that integrates predictions with the costs of mRDT and a course of recommended treatment. Results The manually created BN model achieved an area under the ROC curve (AUC) equal to 0.60 which was statistically significantly higher than the other models. At the optimal threshold for classification, the manual BN model had sensitivity and specificity of 0.74 and 0.42 respectively, and the automated BN model had sensitivity and specificity of 0.45 and 0.68 respectively. The balanced accuracy values were similar across all the models. Sensitivity analysis of the decision tree showed that for values of probability of malaria below 0.04 and above 0.40, the preferred decision that minimizes expected costs is not to perform mRDT. Conclusion In resource-constrained settings, judicious use of mRDT is important. Predictive models in combination with decision analysis can provide personalized guidance on when to use mRDT in the management of childhood malaria. BN models can be efficiently derived from data to support clinical decision making.

Abstract The differentiation of osteoblasts is under complex regulation that includes autocrine and paracrine signaling from MSCs. Exosomes are important components of the MSC secretome and their cargo contains numerous miRNAs. In this study, the importance of MSC miRNAs in modulation of osteoblastic differentiation was examined by global reduction of miRNA biosynthesis in Dicer knock down hMSCs. We additionally impaired hMSC responses to miRNAs by knockdown of Argonaute 2 expression. Knockdown of Dicer and Argonaute 2 both reduced osteoblastic differentiation of hMSCs. This was observed at the levels of hMSC culture mineralization and osteoblastic gene expression. The treatment of Dicer deficient hMSCs with wild type hMSC exosomes effectively recovered the impaired osteoblastic differentiation. Dicer knockdown reduced the quantity and diversity of miRNAs present in hMSC exosomes. miRSeq data and KEGG analysis implicated the miRNA-dependent effects on multiple osteoinductive pathways in Dicer deficient cells, including the Hippo signaling and TGF-beta signaling pathways. Treatment of hMSCs with mimics of miRNAs significantly downregulated in Dicer knockdown cells recovered functions of exosome-mediated signaling in hMSCs. These results indicate that hMSC exosomes exert miRNA-dependent control that contributes to osteoblastic differentiation.

Clinical-reasoning (CR) provides a framework for higher-order critical thinking that fosters a nurse’s ability to assess, process and remedy clinical encounters and is considered essential for the provision of quality healthcare. This study aimed to determine whether student nurses regard the inclusion of video-simulation with critical-reflection as a valuable opportunity to develop their CR skills. An existing case-based assessment was redesigned to include short video-simulations where deliberate but subtle CR flaws were included, requiring students to identify strengths and weaknesses of their own CR process. Following completion of the assessment a modified student satisfaction and self-confidence Likert scale survey with open-ended questions was conducted to identify perceptions towards the assessment task. Incorporating video-simulation and critical-reflection was perceived as a useful opportunity to develop CR skills by student nurses. Albeit, students studying in a traditional three year Bachelor of Nursing cohort were more positive of the opportunity than their peers in a two year fast-track cohort.

Abstract Background Forest dieback driven by rapid climate warming threatens ecosystems worldwide. The health of forested ecosystems depends on how tree species respond to warming during all life history stages. While it is known that seed development is temperature-sensitive, little is known about possible effects of climate warming on seed development and subsequent seedling performance. Exposure of seeds to high air temperatures may influence subsequent seedling performance negatively, though conversely, warming during seed development may aid acclimation of seedlings to subsequent thermal stress. Technical challenges associated with in-situ warming of developing tree seeds limit understanding of how tree species may respond to seed development in a warmer climate. Results We developed and validated a simple method for passively warming seeds as they develop in tree canopies to enable controlled study of climate warming on seedling performance. We quantified thermal effects of the cone-warming method across individual pine trees and stands by measuring the air temperature surrounding seed cones using thermal loggers and the temperature of seed cone tissue using thermocouples. We then investigated seedling phenotypes in relation to the warming method through a common garden study. We assessed seedling morphology, physiology, and mycorrhizal nodulation in response to experimental cone-warming in 20 seed-source-tree canopies on the San Francisco Peaks in northern Arizona, USA. The warming method increased air temperature surrounding developing seed cones by 2.1 °C, a plausible increase in mean air temperature by 2050 under current climate projections. Notable effect sizes of cone-warming were detected for seedling root length, shoot length, and diameter at root collar using Cohen’s Local f 2. Root length was affected most by cone-warming, but effect sizes of cone-warming on root length and diameter at root collar became negligible after the first year of growth. Cone-warming had small but significant effects on mycorrhizal fungal richness and seedling multispectral near-infrared indices indicative of plant health. Conclusions The method was shown to reliably elevate the temperature surrounding seed cones and thereby facilitate experimental in-situ climate warming research on forest trees. The method was furthermore shown to influence plant traits that may affect seedling performance under climate warming.

Abstract Background Protein phosphorylation networks play an important role in cell signaling. In these networks, phosphorylation of a protein kinase usually leads to its activation, which in turn will phosphorylate its downstream target proteins. A phosphorylation network is essentially a causal network, which can be learned by causal inference algorithms. Prior efforts have applied such algorithms to data measuring protein phosphorylation levels, assuming that the phosphorylation levels represent protein activity states. However, the phosphorylation status of a kinase does not always reflect its activity state, because interventions such as inhibitors or mutations can directly affect its activity state without changing its phosphorylation status. Thus, when cellular systems are subjected to extensive perturbations, the statistical relationships between phosphorylation states of proteins may be disrupted, making it difficult to reconstruct the true protein phosphorylation network. Here, we describe a novel framework to address this challenge. Results We have developed a causal discovery framework that explicitly represents the activity state of each protein kinase as an unmeasured variable and developed a novel algorithm called “InferA” to infer the protein activity states, which allows us to incorporate the protein phosphorylation level, pharmacological interventions and prior knowledge. We applied our framework to simulated datasets and to a real-world dataset. The simulation experiments demonstrated that explicit representation of activity states of protein kinases allows one to effectively represent the impact of interventions and thus enabled our framework to accurately recover the ground-truth causal network. Results from the real-world dataset showed that the explicit representation of protein activity states allowed an effective and data-driven integration of the prior knowledge by InferA, which further leads to the recovery of a phosphorylation network that is more consistent with experiment results. Conclusions Explicit representation of the protein activity states by our novel framework significantly enhances causal discovery of protein phosphorylation networks.

Lavas produced at subduction zones represent the integration of both source heterogeneity and an array of crustal processes, such as: differentiation; mixing; homogenisation; assimilation. Therefore, unravelling the relative contribution of the sub-arc mantle source versus these crustal processes is difficult when using the amalgamated end products in isolation. In contrast, plutonic xenoliths provide a complementary record of the deeper roots of the magmatic plumbing system and provide a unique record of the true chemical diversity of arc crust. Here, we present the δ56Fe record from well characterised plutonic xenoliths from two distinct volcanic centres in the Lesser Antilles volcanic arc–the islands of Martinique and Statia. The primary objective of this study is to test if the Fe isotope systematics of arc lavas are controlled by sub-arc mantle inputs or during subsequent differentiation processes during a magma’s journey through volcanic arc crust. The Fe isotopic record, coupled to petrology, trace element chemistry and radiogenic isotopes of plutonic xenoliths from the two islands reveal a hidden crustal reservoir of heavy Fe that previously hasn’t been considered. Iron isotopes are decoupled from radiogenic isotopes, suggesting that crustal and/or sediment assimilation does not control the Fe systematics of arc magmas. In contrast to arc lavas, the cumulates from both islands record MORB-like δ56Fe values. In Statia, δ56Fe decreases with major and trace element indicators of differentiation (SiO2, Na2O + K2O, Eu/Eu*, Dy/Yb), consistent with fractionating mineral assemblages along a line of liquid descent. In Martinique, δ56Fe shows no clear relationship with most indicators of differentiation (apart from Dy/Yb), suggesting that the δ56Fe signature of the plutonic xenoliths has been overprinted by later stage processes, such as percolating reactive melts. Together, these data suggest that magmatic processes within the sub-arc crust overprint any source variation of the sub-arc mantle and that a light Fe source is not a requirement to produce the light Fe isotopic compositions recorded in volcanic arc lavas. Therefore, whenever possible, the complimentary plutonic record should be considered in isotopic studies to understand the relative control of the mantle source versus magmatic processes in the crust.

Objectives COVIDTrach is a UK multicentre prospective cohort study project that aims to evaluate the outcomes of tracheostomy in patients with COVID-19 receiving mechanical ventilation and record the incidence of SARS-CoV-2 infection among healthcare workers involved in the procedure.Design Data on patient demographic, clinical history and outcomes were entered prospectively and updated over time via an online database (REDCap). Clinical variables were compared with outcomes, with logistic regression used to develop a model for mortality. Participants recorded whether any operators tested positive for SARS-CoV-2 within 2 weeks of the procedure.Setting UK National Health Service departments involved in treating patients with COVID-19 receiving mechanical ventilation.Participants The cohort comprised 1605 tracheostomy cases from 126 UK hospitals collected between 6 April and 26 August 2020.Main outcome measures Mortality following tracheostomy, successful wean from mechanical ventilation and length of time from tracheostomy to wean, discharge from hospital, complications from tracheostomy, reported SARS-CoV-2 infection among operators.Results The median time from intubation to tracheostomy was 15 days (IQR 11, 21). 285 (18%) patients died following the procedure. 1229 (93%) of the survivors had been successfully weaned from mechanical ventilation at censoring and 1049 (81%) had been discharged from hospital. Age, inspired oxygen concentration, positive end-expiratory pressure setting, fever, number of days of ventilation before tracheostomy, C reactive protein and the use of anticoagulation and inotropic support independently predicted mortality. Six reports were received of operators testing positive for SARS-CoV-2 within 2 weeks of the procedure.Conclusions Tracheostomy appears to be safe in mechanically ventilated patients with COVID-19 and to operators performing the procedure and we identified clinical parameters that are predictive of mortality.Trial registration number The study is registered with ClinicalTrials.Gov (NCT04572438).

Abstract Background High-throughput RNA sequencing (RNA-seq) has evolved as an important analytical tool in molecular biology. Although the utility and importance of this technique have grown, uncertainties regarding the proper analysis of RNA-seq data remain. Of primary concern, there is no consensus regarding which normalization and statistical methods are the most appropriate for analyzing this data. The lack of standardized analytical methods leads to uncertainties in data interpretation and study reproducibility, especially with studies reporting high false discovery rates. In this study, we compared a recently developed normalization method, UQ-pgQ2, with three of the most frequently used alternatives including RLE (relative log estimate), TMM (Trimmed-mean M values) and UQ (upper quartile normalization) in the analysis of RNA-seq data. We evaluated the performance of these methods for gene-level differential expression analysis by considering the factors, including: 1) normalization combined with the choice of a Wald test from DESeq2 and an exact test/QL (Quasi-likelihood) F-Test from edgeR; 2) sample sizes in two balanced two-group comparisons; and 3) sequencing read depths. Results Using the MAQC RNA-seq datasets with small sample replicates, we found that UQ-pgQ2 normalization combined with an exact test can achieve better performance in term of power and specificity in differential gene expression analysis. However, using an intra-group analysis of false positives from real and simulated data, we found that a Wald test performs better than an exact test when the number of sample replicates is large and that a QL F-test performs the best given sample sizes of 5, 10 and 15 for any normalization. The RLE, TMM and UQ methods performed similarly given a desired sample size. Conclusion We found the UQ-pgQ2 method combined with an exact test/QL F-test is the best choice in order to control false positives when the sample size is small. When the sample size is large, UQ-pgQ2 with a QL F-test is a better choice for the type I error control in an intra-group analysis. We observed read depths have a minimal impact for differential gene expression analysis based on the simulated data.

Abstract Aims To identify geographic “hotspots” for potential transmission of HIV and HCV and for drug overdose among persons who use heroin and cocaine in New York City and to examine historical continuities in problem drug use hotspots in the city. Methods A total of 2714 study participants were recruited among persons entering Beth Israel substance use treatment programs. A structured questionnaire was administered and blood samples for HIV and HCV testing were collected. Hotspots for potential virus transmission were defined as ZIP codes with 10+ participants, 2+ persons infected with the virus and engaging in transmission behavior, and 2+ persons not infected and engaging in acquisition behavior. ZIP codes with 3+ persons with previous overdoses were considered potential hotspots for future overdoses. Results Participants resided in 166/178 (93%) of the ZIP codes in New York City. Injecting drug use was reported in 150/178 (84%) of the ZIP codes. No zip codes were identified for injecting-related HIV transmission, 5 zip codes were identified for sexual HIV transmission, 3 for HCV transmission, and 8 for drug overdose. Many of the ZIP code potential hotspots were in neighborhoods long associated with drug use: Lower Eastside and Harlem in Manhattan, the South Bronx, and Central Brooklyn. Discussion Heroin and cocaine use requiring treatment were reported from almost all ZIP codes in New York City, indicating needs for widely dispersed harm reduction services. Identified hotspots should be targeted for reducing sexual transmission of HIV, transmission of HCV, and drug overdoses. Some of the hotspots have persisted as problem drug use areas for 40 to over 100 years. Monitoring of drug use patterns in historical hotspot neighborhoods may permit early identification of and response to emerging drug use-related health problems. Persistent historical hotspots for problem drug use present a complex problem for implementing harm reduction services that deserve additional research.

Abstract Background Healthcare access, utilization, and quality play critical roles in shaping mortality and morbidity among patients diagnosed with systemic lupus erythematosus (SLE), and yet healthcare access, utilization, and quality can be suboptimal for many people living with SLE. The aim of this qualitative study was to explore the perceived impact of a peer-led, group-based educational intervention (the Chronic Disease Self-Management Program [CDSMP]) on healthcare engagement behaviors among African American women with SLE. Methods Participants were recruited from the WELL (Women Empowered to Live with Lupus) study, a behavioral trial of the effectiveness of the CDSMP on African American women diagnosed with SLE. We conducted two waves of qualitative, one-on-one, semi-structured interviews with 24 purposively sampled WELL participants; one interview was conducted before CDSMP participation and one after. Wave 1 interviews explored health service use behaviors at baseline; Wave 2 interviews focused on changes in these behaviors post-intervention and women’s perceptions of whether and how the CDSMP shaped these changes. Transcripts were analyzed using thematic analysis methods. Results Study participants perceived the CDSMP to be a valuable resource for supporting two distinct health service use behaviors: communicating with doctors (N = 16 [88.9%]) and managing medication side effects (N = 17 [41.2%]). Women perceived that the CDSMP had the most potent and widespread effects on patients’ communication with doctors. Strategies that women believed generated improvements in patient-doctor communication included enhancing preparation for appointments and boosting patient participation during doctor’s visits. Women’s reported post-CDSMP improvements in health service use behaviors varied by disease severity and depression. Insurance coverage, while not probed directly during baseline interviews, emerged organically as a key factor affecting health service use behaviors; the CDSMP did not seem to improve participants’ ability to circumvent insurance-related barriers to accessing care. Conclusions Our findings suggest that the CDSMP may help enhance healthcare service utilization among African American women with SLE by improving doctor/patient communication and medication side effect management. If future research confirms this conclusion, African American women living with SLE should be encouraged to participate in CDSMP workshops to enhance health service use behaviors. Trial registration NCT02988661. Registered 12/07/2016

The transcriptional changes that occur in response to oxidative stress help direct the decision to maintain cell viability or enter a cell death pathway. Cyclin C-Cdk8 is a conserved kinase that associates with the RNA polymerase II Mediator complex that stimulates or represses transcription depending on the locus. In response to oxidative stress, cyclin C, but not Cdk8, displays partial translocation into the cytoplasm. These findings open the possibility that cyclin C relocalization is a regulatory mechanism governing oxidative stress-induced transcriptional changes. In the present study, the cyclin C-dependent transcriptome was determined and compared to transcriptional changes occurring in oxidatively stressed Mus musculus embryonic fibroblasts. We observed a similar number (∼2000) of genes up or downregulated in oxidatively stressed cells. Induced genes include cellular repair/survival factors while repressed loci were generally involved in proliferation or differentiation. Depleting cyclin C in unstressed cells produced an approximately equal number of genes (∼2400) that were repressed by, or whose transcription required, cyclin C. Consistent with the possibility that cyclin C nuclear release contributes to transcriptional remodeling in response to oxidative stress, we found that 37% cyclin C-dependent genes were downregulated following stress. Moreover, 20% of cyclin C- repressed genes were induced in response to stress. These findings are consistent with a model that cyclin C relocalization to the cytoplasm, and corresponding inactivation of Cdk8, represents a regulatory mechanism to repress and stimulate transcription of stress-responsive genes.

Objective This study aimed to explore the mental well-being of Syrian refugees and identify their coping mechanisms and pathways towards integration into new communities.Design Qualitative study using in-depth semi-structured interviews.Setting and participants Adult Syrian refugees (>18 years old) currently residing in South East of England.Results 12 participants (3 women and 9 men) took part in the study, all were born in Syria and the majority (n=9) were over 45 years of age. Our findings show that Syrian refugees face constant challenges as they try to integrate into a new society. Loss of and separation from loved ones as well as the nostalgia for the homeland were often cited as a source of psychological distress that created an overwhelming sense of sadness. Participants reported that they struggled for connectedness due to cultural difference and the problematic nature of rapidly formed migrant communities in their new setting. They believed in ‘being their own doctor’ and turning to faith, ritual and nature for healing and comfort. Taboo and stigma around mental health and language barriers were cited as barriers to accessing mental healthcare services.Conclusion Past experiences and present challenges frame Syrian refugees’ sense of well-being, impact use of healthcare and risk future mental health problems. It is hoped that this study will act as a catalyst for further research on this vulnerable group to promote integration, community support and culturally sensitive mental health services.