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Abstract A reduction in the thickness and volume of the brain entorhinal cortex (EC), together with changes in blood arachidonic acid (AA) and docosahexaenoic acid (DHA), are associated with Alzheimer’s disease (AD) among apolipoprotein E ε4 carriers. Magnetic Resonance Imaging (n = 631) and plasma lipidomics (n = 181) were performed using the LIMBIC/CENC cohort to examine the influence of ε4 on AA- and DHA-lipids and EC thickness and volume in relation to mild traumatic brain injury (mTBI). Results showed that left EC thickness was higher among ε4 carriers with mTBI. Repeated mTBI (r-mTBI) was associated with reduced right EC thickness after controlling for ε4, age and sex. Age, plus mTBI chronicity were linked to increased EC White Matter Volume (WMV). After controlling for age and sex, the advancing age of ε4 carriers with blast mTBI was associated with reduced right EC Grey Matter Volume (GMV) and thickness. Among ε4 carriers, plasma tau and Aβ40 were associated with mTBI and blast mTBI, respectively. Chronic mTBI, ε4 and AA to DHA ratios in phosphatidylcholine, ethanolamides, and phosphatidylethanolamine were associated with decreased left EC GMV and WMV. Further research is needed to explore these as biomarkers for detecting AD pathology following mTBI.

Abstract Facioscapulohumeral muscular dystrophy (FSHD) affects roughly 1 in 7500 individuals. While at the population level there is a general pattern of affected muscles, there is substantial heterogeneity in muscle expression across- and within-patients. There can also be substantial variation in the pattern of fat and water signal intensity within a single muscle. While quantifying individual muscles across their full length using magnetic resonance imaging (MRI) represents the optimal approach to follow disease progression and evaluate therapeutic response, the ability to automate this process has been limited. The goal of this work was to develop and optimize an artificial intelligence-based image segmentation approach to comprehensively measure muscle volume, fat fraction, fat fraction distribution, and elevated short-tau inversion recovery signal in the musculature of patients with FSHD. Intra-rater, inter-rater, and scan-rescan analyses demonstrated that the developed methods are robust and precise. Representative cases and derived metrics of volume, cross-sectional area, and 3D pixel-maps demonstrate unique intramuscular patterns of disease. Future work focuses on leveraging these AI methods to include upper body output and aggregating individual muscle data across studies to determine best-fit models for characterizing progression and monitoring therapeutic modulation of MRI biomarkers.

Abstract Single amino acid (AA) supplementations in foods are increasing, however their potential nutritional and physiological impacts are not fully understood. This study examined the effects of l-lysine (Lys) supplementation on protein quality of diets, serum AA concentrations and associations between the ratio of supplemental Lys to dietary protein (X) with body weight gain (BWG) in Sprague–Dawley male rats. Rats were fed one of 10 diets containing either 7% or 20% casein and supplemented with 0% (Control), 1.5%, 3%, 6% Lys or 6% Lys + 3% l-arginine (Arg) (8 rats/diet group) for 1 week. Lys supplementation reduced the protein quality of the casein-based diets (p 0.18. Free Lys supplementation dose-dependently increased serum Lys levels (p 0.05). In the 7% casein diets, ≥ 1.5% supplemental Lys reduced serum alanine, asparagine, glycine, isoleucine, leucine, serine, tyrosine, valine, carnitine, ornithine, and increased urea. Supplementation of ≥ 3% Lys additionally reduced tryptophan and increased histidine, methionine and α-aminoadipic acid (α-AAA) compared to the Control (p

Abstract The development of high intensity petawatt lasers has created new possibilities for ion acceleration and nuclear fusion using solid targets. In such laser-matter interaction, multiple ion species are accelerated with broad spectra up to hundreds of MeV. To measure ion yields and for species identification, CR-39 solid-state nuclear track detectors are frequently used. However, these detectors are limited in their applicability for multi-ion spectra differentiation as standard image recognition algorithms can lead to a misinterpretation of data, there is no unique relation between track diameter and particle energy, and there are overlapping pit diameter relationships for multiple particle species. In this report, we address these issues by first developing an algorithm to overcome user bias during image processing. Second, we use calibration of the detector response for protons, carbon and helium ions (alpha particles) from 0.1 to above 10 MeV and measurements of statistical energy loss fluctuations in a forward-fitting procedure utilizing multiple, differently filtered CR-39, altogether enabling high-sensitivity, multi-species particle spectroscopy. To validate this capability, we show that inferred CR-39 spectra match Thomson parabola ion spectrometer data from the same experiment. Filtered CR-39 spectrometers were used to detect, within a background of ~ 2 × 1011 sr−1 J−1 protons and carbons, (1.3 ± 0.7) × 108 sr−1 J−1 alpha particles from laser-driven proton-boron fusion reactions.

BackgroundDevelopment of reliable disease activity biomarkers is critical for diagnostics, prognostics, and novel drug development. Although computed tomography (CT) is the gold-standard for quantification of bone erosions, there are no consensus approaches or rationales for utilization of specific outcome measures of erosive arthritis in complex joints. In the case of preclinical models, such as sexually dimorphic tumor necrosis factor transgenic (TNF-Tg) mice, disease severity is routinely quantified in the ankle through manual segmentation of the talus or small regions of adjacent bones primarily due to the ease in measurement. Herein, we sought to determine the particular hindpaw bones that represent reliable biomarkers of sex-dependent disease progression to guide future investigation and analysis.MethodsHindpaw micro-CT was performed on wild-type (n = 4 male, n = 4 female) and TNF-Tg (n = 4 male, n = 7 female) mice at monthly intervals from 2-5 (females) and 2-8-months (males) of age, since female TNF-Tg mice exhibit early mortality from cardiopulmonary disease at approximately 5-6-months. Further, 8-month-old WT (n = 4) and TNF-Tg males treated with anti-TNF monoclonal antibodies (n = 5) or IgG placebo isotype controls (n = 6) for 6-weeks were imaged with micro-CT every 3-weeks. For image analysis, we utilized our recently developed high-throughput and semi-automated segmentation strategy in Amira software. Synovial and osteoclast histology of ankle joints was quantified using Visiopharm.ResultsFirst, we demonstrated that the accuracy of automated segmentation, determined through analysis of ~9000 individual bones by a single user, was comparable in wild-type and TNF-Tg hindpaws before correction (79.2±8.9% vs 80.1±5.1%, p = 0.52). Compared to other bone compartments, the tarsal region demonstrated a sudden, specific, and significant bone volume reduction in female TNF-Tg mice, but not in males, by 5-months (4-months 4.3± 0.22 vs 5-months 3.4± 0.62 mm3, pConclusionsWe demonstrated that sexually dimorphic changes in arthritic hindpaws of TNF-Tg mice are bone-specific, where the cuboid serves as a reliable early biomarker of erosive arthritis in female mice. Adoption of automated segmentation approaches in pre-clinical or clinical models has potential to translate quantitative biomarkers to monitor bone erosions in disease and evaluate therapeutic efficacy.

Aging is associated with a wide range of physiological and behavioral changes in many species. Zebrafish, like humans, rodents, and birds, exhibits gradual senescence, and thus may be a useful model organism for identifying evolutionarily conserved mechanisms related to aging. Here, we compared behavior in the novel tank test of young (6-month-old) and middle aged (12-month-old) zebrafish from two strains (TL and TU) and both sexes. We find that this modest age difference results in a reduction in locomotor activity in male fish. We also found that background strain modulated the effects of age on predator avoidance behaviors related to anxiety: older female TL fish increased bottom dwelling whereas older male TU fish decreased thigmotaxis. Although there were no consistent effects of age on either short-term (within session) or long-term (next day) habituation to the novel tank, strain affected the habituation response. TL fish tended to increase their distance from the bottom of the tank whereas TU fish had no changes in bottom distance but instead tended to increase thigmotaxis. Our findings support the use of zebrafish for the study of how age affects locomotion and how genetics interacts with age and sex to alter exploratory and emotional behaviors in response to novelty.

Bacteriophage therapy is a rapidly growing field of study. Narrow host ranges, bacterial resistance, and limited antibiotic availability make lytic phages a feasible therapeutic potential. Phage discovery, a critical step in developing phage therapy, is a pathway to accessible treatment. This has always been a laborious, time-consuming and resource-intensive process. In this paper, we describe a 96-well plate low-volume bacteriophage enrichment method with concentrated environmental sources to rapidly discover and isolate phages targeting multiple organisms simultaneously. Samples from natural water sources, wastewater influent, and activated sludge were tested in large volume enrichment cultures and low-volume 96-well plate format. Each plate has the capacity to run as many as 48 different combinations with multiple bacterial hosts. The time to identify the presence of phage in a sample was 5 to 10 hours in the low-volume format versus a minimum of 2 days in the traditional enrichment method. The labor and expense involved also favor the 96-well plate format. There was some loss of discovered phages using this technique, primarily targeting bacterial species less prevalent in the environment. This is an easily modifiable method that is amenable to automation and a variety of potential phage sources.

IntroductionThe Delft Self-Grasping Hand (SGH) is an adjustable passive hand prosthesis that relies on wrist flexion to adjust the aperture of its grasp. The mechanism requires engagement of the contralateral hand meaning that hand is not available for other tasks. A commercialised version of this prosthesis, known as the mHand Adapt, includes a new release mechanism, which avoids the need to press a release button, and changes to the hand shape. This study is the first of its kind to compare two passive adjustable hand prostheses on the basis of quantitative scoring and contralateral hand involvement.Methods10 anatomically intact participants were asked to perform the Southampton Hand Assessment Procedure (SHAP) with the mHand. Functionality and contralateral hand involvement were recorded and compared against SGH data originating from a previous trial involving a nearly identical testing regime.ResultsmHand exhibited higher functionality scores and less contralateral hand interaction time, especially during release-aiding interactions. Additionally, a wider range of tasks could be completed using the mHand than the SGH.DiscussionGeometric changes make the mHand more capable of manipulating smaller objects. The altered locking mechanism means some tasks can be performed without any contralateral hand involvement and a higher number of tasks do not require contralateral involvement when releasing. Some participants struggled with achieving a good initial grip due to the inability to tighten the grasp once already formed.ConclusionThe mHand offers the user higher functionality scores with less contralateral hand interaction time and the ability to perform a wider range of tasks. However, there are some design trade-offs which may make it slightly harder to learn to use.

Abstract Y-box binding protein 1 (YBX1 or YB1) is a therapeutically relevant oncoprotein capable of RNA and DNA binding and mediating protein–protein interactions that drive proliferation, stemness, and resistance to platinum-based therapies. Given our previously published findings, the potential for YB1-driven cisplatin resistance in medulloblastoma (MB), and the limited studies exploring YB1-DNA repair protein interactions, we chose to investigate the role of YB1 in mediating radiation resistance in MB. MB, the most common pediatric malignant brain tumor, is treated with surgical resection, cranio-spinal radiation, and platinum-based chemotherapy, and could potentially benefit from YB1 inhibition. The role of YB1 in the response of MB to ionizing radiation (IR) has not yet been studied but remains relevant for determining potential anti-tumor synergy of YB1 inhibition with standard radiation therapy. We have previously shown that YB1 drives proliferation of cerebellar granular neural precursor cells (CGNPs) and murine Sonic Hedgehog (SHH) group MB cells. While others have demonstrated a link between YB1 and homologous recombination protein binding, functional and therapeutic implications remain unclear, particularly following IR-induced damage. Here we show that depleting YB1 in both SHH and Group 3 MB results not only in reduced proliferation but also synergizes with radiation due to differential response dynamics. YB1 silencing through shRNA followed by IR drives a predominantly NHEJ-dependent repair mechanism, leading to faster γH2AX resolution, premature cell cycle re-entry, checkpoint bypass, reduced proliferation, and increased senescence. These findings show that depleting YB1 in combination with radiation sensitizes SHH and Group 3 MB cells to radiation.

Much of the chronic disease burden in the U.S. population can be traced to poor diet. There has been a sustained focus on influencing children's diets and encouraging healthier eating habits by changing policies for what foods and beverages can be served to children through large federally-funded nutrition assistance programs. Yet without attention to how nutrition policies are implemented, and the surrounding context for these policies, these policy changes may not have the intended results. In this perspective, we used Bullock et al.'s (2021) Process Model of Implementation from a Policy Perspective to analyze how the complexities of the implementation process of large-scale nutrition policies can dilute potential health outcomes. We examine the Child and Adult Care Food Program (CACFP), a federal program focused on supporting the provision of nutritious meals to over 4 million children attending childcare, as a case study. We examine how the larger societal contexts of food insecurity, attitudes towards the social safety net, and a fragmented childcare system interact with CACFP. We review the “policy package” of CACFP itself, in terms of its regulatory requirements, and the various federal, state, and local implementation agencies that shape CACFP's on-the-ground implementation. We then review the evidence for how each component of the CACFP policy implementation process impacts uptake, costs, feasibility, equity, and effectiveness at improving children's nutrition. Our case study demonstrates how public health researchers and practitioners must consider the complexities of policy implementation processes to ensure effective implementation of nutrition policies intended to improve population health.

YiiP from Shewanella oneidensis is a prokaryotic Zn2+/H+ antiporter that serves as a model for the Cation Diffusion Facilitator (CDF) superfamily, members of which are generally responsible for homeostasis of transition metal ions. Previous studies of YiiP as well as related CDF transporters have established a homodimeric architecture and the presence of three distinct Zn2+ binding sites named A, B, and C. In this study, we use cryo-EM, microscale thermophoresis and molecular dynamics simulations to address the structural and functional roles of individual sites as well as the interplay between Zn2+ binding and protonation. Structural studies indicate that site C in the cytoplasmic domain is primarily responsible for stabilizing the dimer and that site B at the cytoplasmic membrane surface controls the structural transition from an inward facing conformation to an occluded conformation. Binding data show that intramembrane site A, which is directly responsible for transport, has a dramatic pH dependence consistent with coupling to the proton motive force. A comprehensive thermodynamic model encompassing Zn2+ binding and protonation states of individual residues indicates a transport stoichiometry of 1 Zn2+ to 2–3 H+ depending on the external pH. This stoichiometry would be favorable in a physiological context, allowing the cell to use the proton gradient as well as the membrane potential to drive the export of Zn2+.

Abstract Diabetic retinopathy (DR) is the most common diabetic microvascular complication and cause of blindness in adults under the age of 65. Our results suggest that, when comparing transcriptomes of cultures grown in hypoxic conditions versus room-air, cybrids containing mitochondria from African and Asian diabetic subjects ([Afr + Asi]/DM) have some uniquely different transcriptome profiles compared to European/diabetic (Euro/DM) cybrids (e.g., fatty acid metabolism: EnrichR rank 10 in [Afr + Asi]/DM, rank 85 in Euro/DM; Endocytosis: rank 25 in [Afr + Asi]/DM, rank 5 in Euro/DM; Ubiquitin Mediated Proteolysis: rank 34 in [Afr + Asi]/DM, rank 7 in Euro/DM). As determined by both RNA-seq and qRT-PCR results, transcription of the gene encoding oleoyl-ACP hydrolase (OLAH) was significantly increased in [Afr + Asi]/DM cybrids compared to Euro/DM cybrids in hypoxic conditions. Additionally, our results show that in hypoxic conditions, Euro/DM cybrids and [Afr + Asi]/DM cybrids show similar decreases in ROS production. All cybrids showed decreased ZO1-minus protein levels, but their phagocytic functions were not significantly altered in hypoxic conditions. In conclusion, our findings suggest that the "molecular memory" imparted by [Afr + Asi]/DM mtDNA may act through one of the molecular pathways seen in transcriptome analysis, such as fatty acid metabolism, without significantly changing essential RPE functions.

Meeting the needs of persons with upper limb loss in Uganda requires an understanding of the needs and desires of the local population. The limitations of resources and accessibility for the individual gave rise to a focused design methodology for delivering a culturally acceptable solution using 3D Printing technology. A series of co-design activities were held in Uganda and provided direct feedback to drive the design of two prototypes based on acceptable aesthetics and priority Activities of Daily Living. Two terminal device prototypes were 3D printed in the UK. These can be directly attached to a standard proximal socket thread. The passive hand was printed in a flexible filament and the prehensor was printed in a durable impact resistant material. Local researchers in Uganda have similar 3D printers, filaments, and assembly hardware, which allowed for concurrent development and refinement of the prototypes. Local participation provides a rich user feedback environment to understand which elements of prosthetic device design are integral to delivering acceptable prosthetics solutions for fabrication in Uganda. 3D printing can provide a viable route to addressing the needs of the user. The proposed terminal devices are now in the process of being printed locally for field testing.

The transcriptional regulator SsrB acts as a switch between virulent and biofilm lifestyles of non-typhoidal Salmonella enterica serovar Typhimurium. During infection, phosphorylated SsrB activates genes on Salmonella Pathogenicity Island-2 (SPI-2) essential for survival and replication within the macrophage. Low pH inside the vacuole is a key inducer of expression and SsrB activation. Previous studies demonstrated an increase in SsrB protein levels and DNA-binding affinity at low pH; the molecular basis was unknown (Liew et al., 2019). This study elucidates its underlying mechanism and in vivo significance. Employing single-molecule and transcriptional assays, we report that the SsrB DNA-binding domain alone (SsrBc) is insufficient to induce acid pH-sensitivity. Instead, His12, a conserved residue in the receiver domain confers pH sensitivity to SsrB allosterically. Acid-dependent DNA binding was highly cooperative, suggesting a new configuration of SsrB oligomers at SPI-2-dependent promoters. His12 also plays a role in SsrB phosphorylation; substituting His12 reduced phosphorylation at neutral pH and abolished pH-dependent differences. Failure to flip the switch in SsrB renders Salmonella avirulent and represents a potential means of controlling virulence.

Abstract Coronaviruses are important viral pathogens across a range of animal species including humans. They have a high potential for cross-species transmission as evidenced by the emergence of COVID-19 and may be the origin of future pandemics. There is therefore an urgent need to study coronaviruses in depth and to identify new therapeutic targets. This study shows that distant coronaviruses such as Alpha-, Beta-, and Deltacoronaviruses can share common host immune associated pathways and genes. Differentially expressed genes (DEGs) in the transcription profile of epithelial cell lines infected with swine acute diarrhea syndrome, severe acute respiratory syndrome coronavirus 2, or porcine deltacoronavirus, showed that DEGs within 10 common immune associated pathways were upregulated upon infection. Twenty Three pathways and 21 DEGs across 10 immune response associated pathways were shared by these viruses. These 21 DEGs can serve as focused targets for therapeutics against newly emerging coronaviruses. We were able to show that even though there is a positive correlation between PDCoV and SARS-CoV-2 infections, these viruses could be using different strategies for efficient replication in their cells from their natural hosts. To the best of our knowledge, this is the first report of comparative host transcriptome analysis across distant coronavirus genres.

(1) Background: This study investigated the feasibility of conducting a two-week “real-world” trial of the Self Grasping Hand (SGH), a novel 3D printed passive adjustable prosthesis for hand absence; (2) Methods: Single-group pilot study of nine adults with trans-radial limb absence; five used body-powered split-hooks, and four had passive cosmetic hands as their usual prosthesis. Data from activity monitors were used to measure wear time and bilateral activity. At the end of the two-week trial, function and satisfaction were measured using the Orthotics and Prosthetics Users’ Survey Function Scale (OPUS) and the prosthesis satisfaction sub-scales of the Trinity Amputations and Prosthesis Experience Scale (TAPES). Semi-structured interviews captured consumer feedback and suggestions for improvement; (3) Results: Average SGH wear time over 2 weeks was 17.5 h (10% of total prosthesis wear time) for split-hook users and 83.5 h (63% of total prosthesis wear time) for cosmetic hand users. Mean satisfaction was 5.2/10, and mean function score was 47.9/100; (4) Two-week real-world consumer testing of the SGH is feasible using the methods described. Future SGH designs need to be more robust with easier grasp lock/unlock.

Climate change and climate variability are affecting marine mammal species and these impacts are projected to continue in the coming decades. Vulnerability assessments provide a framework for evaluating climate impacts over a broad range of species using currently available information. We conducted a trait-based climate vulnerability assessment using expert elicitation for 108 marine mammal stocks and stock groups in the western North Atlantic, Gulf of Mexico, and Caribbean Sea. Our approach combined the exposure (projected change in environmental conditions) and sensitivity (ability to tolerate and adapt to changing conditions) of marine mammal stocks to estimate vulnerability to climate change, and categorize stocks with a vulnerability index. The climate vulnerability score was very high for 44% (n = 47) of these stocks, high for 29% (n = 31), moderate for 20% (n = 22), and low for 7% (n = 8). The majority of stocks (n = 78; 72%) scored very high exposure, whereas 24% (n = 26) scored high, and 4% (n = 4) scored moderate. The sensitivity score was very high for 33% (n = 36) of these stocks, high for 18% (n = 19), moderate for 34% (n = 37), and low for 15% (n = 16). Vulnerability results were summarized for stocks in five taxonomic groups: pinnipeds (n = 4; 25% high, 75% moderate), mysticetes (n = 7; 29% very high, 57% high, 14% moderate), ziphiids (n = 8; 13% very high, 50% high, 38% moderate), delphinids (n = 84; 52% very high, 23% high, 15% moderate, 10% low), and other odontocetes (n = 5; 60% high, 40% moderate). Factors including temperature, ocean pH, and dissolved oxygen were the primary drivers of high climate exposure, with effects mediated through prey and habitat parameters. We quantified sources of uncertainty by bootstrapping vulnerability scores, conducting leave-one-out analyses of individual attributes and individual scorers, and through scoring data quality for each attribute. These results provide information for researchers, managers, and the public on marine mammal responses to climate change to enhance the development of more effective marine mammal management, restoration, and conservation activities that address current and future environmental variation and biological responses due to climate change.

IntroductionWe are conducting a multicenter study to identify classifiers predictive of disease-specific survival in patients with primary melanomas. Here we delineate the unique aspects, challenges, and best practices for optimizing a study of generally small-sized pigmented tumor samples including primary melanomas of at least 1.05mm from AJTCC TNM stage IIA-IIID patients. We also evaluated tissue-derived predictors of extracted nucleic acids' quality and success in downstream testing. This ongoing study will target 1,000 melanomas within the international InterMEL consortium.MethodsFollowing a pre-established protocol, participating centers ship formalin-fixed paraffin embedded (FFPE) tissue sections to Memorial Sloan Kettering Cancer Center for the centralized handling, dermatopathology review and histology-guided coextraction of RNA and DNA. Samples are distributed for evaluation of somatic mutations using next gen sequencing (NGS) with the MSK-IMPACTTM assay, methylation-profiling (Infinium MethylationEPIC arrays), and miRNA expression (Nanostring nCounter Human v3 miRNA Expression Assay).ResultsSufficient material was obtained for screening of miRNA expression in 683/685 (99%) eligible melanomas, methylation in 467 (68%), and somatic mutations in 560 (82%). In 446/685 (65%) cases, aliquots of RNA/DNA were sufficient for testing with all three platforms. Among samples evaluated by the time of this analysis, the mean NGS coverage was 249x, 59 (18.6%) samples had coverage below 100x, and 41/414 (10%) failed methylation QC due to low intensity probes or insufficient Meta-Mixed Interquartile (BMIQ)- and single sample (ss)- Noob normalizations. Six of 683 RNAs (1%) failed Nanostring QC due to the low proportion of probes above the minimum threshold. Age of the FFPE tissue blocks (pConclusionOur experience with many archival tissues demonstrates that with careful management of tissue processing and quality control it is possible to conduct multi-omic studies in a complex multi-institutional setting for investigations involving minute quantities of FFPE tumors, as in studies of early-stage melanoma. The study describes, for the first time, the optimal strategy for obtaining archival and limited tumor tissue, the characteristics of the nucleic acids co-extracted from a unique cell lysate, and success rate in downstream applications. In addition, our findings provide an estimate of the anticipated attrition that will guide other large multicenter research and consortia.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection in immunocompromised individuals is associated with prolonged virus shedding and evolution of viral variants. Rapamycin and its analogs (rapalogs, including everolimus, temsirolimus, and ridaforolimus) are FDA approved as mTOR inhibitors for the treatment of human diseases, including cancer and autoimmunity. Rapalog use is commonly associated with an increased susceptibility to infection, which has been traditionally explained by impaired adaptive immunity. Here, we show that exposure to rapalogs increased susceptibility to SARS-CoV-2 infection in tissue culture and in immunologically naive rodents by antagonizing the cell-intrinsic immune response. We identified 1 rapalog (ridaforolimus) that was less potent in this regard and demonstrated that rapalogs promote spike-mediated entry into cells, by triggering the degradation of the antiviral proteins IFITM2 and IFITM3 via an endolysosomal remodeling program called microautophagy. Rapalogs that increased virus entry inhibited mTOR-mediated phosphorylation of the transcription factor TFEB, which facilitated its nuclear translocation and triggered microautophagy. In rodent models of infection, injection of rapamycin prior to and after virus exposure resulted in elevated SARS-CoV-2 replication and exacerbated viral disease, while ridaforolimus had milder effects. Overall, our findings indicate that preexisting use of certain rapalogs may elevate host susceptibility to SARS-CoV-2 infection and disease by activating lysosome-mediated suppression of intrinsic immunity.

Dysregulation in neutrophil extracellular trap (NET) formation and degradation may play a role in the pathogenesis and severity of COVID-19; however, its role in the pediatric manifestations of this disease, including multisystem inflammatory syndrome in children (MIS-C) and chilblain-like lesions (CLLs), otherwise known as “COVID toes,” remains unclear. Studying multinational cohorts, we found that, in CLLs, NETs were significantly increased in serum and skin. There was geographic variability in the prevalence of increased NETs in MIS-C, in association with disease severity. MIS-C and CLL serum samples displayed decreased NET degradation ability, in association with C1q and G-actin or anti-NET antibodies, respectively, but not with genetic variants of DNases. In adult COVID-19, persistent elevations in NETs after disease diagnosis were detected but did not occur in asymptomatic infection. COVID-19–affected adults displayed significant prevalence of impaired NET degradation, in association with anti-DNase1L3, G-actin, and specific disease manifestations, but not with genetic variants of DNases. NETs were detected in many organs of adult patients who died from COVID-19 complications. Infection with the Omicron variant was associated with decreased NET levels when compared with other SARS-CoV-2 strains. These data support a role for NETs in the pathogenesis and severity of COVID-19 in pediatric and adult patients.

Objective Integrated community case management (iCCM) of childhood illness in Uganda involves protocol-based care of malaria, pneumonia and diarrhoea for children under 5 years old. This study assessed volunteer village health workers’ (VHW) ability to provide correct iCCM care according to the national protocol and change in their performance over time since initial training.Setting VHWs affiliated with the Ugandan national programme provide community-based care in eight villages in Bugoye Subcounty, a rural area in Kasese District. The first cohort of VHWs began providing iCCM care in March 2013, the second cohort in July 2016.Participants All children receiving iCCM care in 18 430 clinical encounters occurring between April 2014 and December 2018.Primary and secondary outcome measures The descriptive primary outcome measure was the proportion of patients receiving overall correct care, defined as adherence to the iCCM protocol for the presenting condition (hereafter quality of care). The analytic primary outcome was change in the odds of receiving correct care over time, assessed using logistic regression models with generalised estimating equations. Secondary outcome measures included a set of binary measures of adherence to specific elements of the iCCM protocol. Preplanned and final measures were the same.Results Overall, VHWs provided correct care in 74% of clinical encounters. For the first cohort of VHWs, regression modelling demonstrated a modest increase in quality of care until approximately 3 years after their initial iCCM training (OR 1.022 per month elapsed, 95% CI 1.005 to 1.038), followed by a modest decrease thereafter (OR 0.978 per month, 95% CI 0.970 to 0.986). For the second cohort, quality of care was essentially constant over time (OR 1.007 per month, 95% CI 0.989 to 1.025).Conclusion Quality of care was relatively constant over time, though the trend towards decreasing quality of care after 3 years of providing iCCM care requires further monitoring.

In 2016, four clusters of local mosquitoborne Zika virus transmission were identified in Miami-Dade County, Florida, USA, generating “red zones” (areas into which pregnant women were advised against traveling). The Miami-Dade County Mosquito Control Division initiated intensive control activities, including property inspections, community education, and handheld sprayer applications of larvicides and adulticides. For the first time, the Mosquito Control Division used a combination of areawide ultralow-volume adulticide and low-volume larvicide spraying to effectively control Aedes aegypti mosquitoes, the primary Zika virus vector within the county. The number of mosquitoes rapidly decreased, and Zika virus transmission was interrupted within the red zones immediately after the combination of adulticide and larvicide spraying.

Coronaviruses cause respiratory and gastrointestinal diseases in diverse host species. Deltacoronaviruses (DCoVs) have been identified in various songbird species and in leopard cats in China. In 2009, porcine deltacoronavirus (PDCoV) was detected in fecal samples from pigs in Asia, but its etiologic role was not identified until 2014, when it caused major diarrhea outbreaks in swine in the United States. Studies have shown that PDCoV uses a conserved region of the aminopeptidase N protein to infect cell lines derived from multiple species, including humans, pigs, and chickens. Because PDCoV is a potential zoonotic pathogen, investigations of its prevalence in humans and its contribution to human disease continue. We report experimental PDCoV infection and subsequent transmission among poultry. In PDCoV-inoculated chicks and turkey poults, we observed diarrhea, persistent viral RNA titers from cloacal and tracheal samples, PDCoV-specific serum IgY antibody responses, and antigen-positive cells from intestines.

BackgroundThe emotional health of adolescent and young adult (AYA) cancer survivors is compromised both during and after cancer treatment. Targeted programs designed to support AYAs’ ability to cope with stress in the years following treatment completion are lacking. Mind-body programs may ameliorate the negative psychological and emotional effects of stress and assist AYAs with managing the psychosocial challenges of early survivorship. ObjectiveOur randomized waitlist-control trial aims to assess the feasibility, acceptability, and preliminary efficacy of a virtual group program (Bounce Back) to promote stress management and resiliency among posttreatment AYAs. MethodsBounce Back is a stress management and resiliency program delivered via videoconference by a trained mental health clinician. Sessions were adapted from an evidence-based mind-body program (Stress Management and Resiliency Training - Relaxation Response Resiliency Program [SMART-3RP]) grounded in relaxation response elicitation, mindfulness, cognitive behavioral therapy, and positive psychology. Seventy-two AYAs (diagnosed with cancer between ages 14 years and 29 years and had completed cancer treatment within the last 5 years) were randomly assigned to the Bounce Back program or waitlist-control group and completed assessments at baseline, 3 months postbaseline, and 6 months postbaseline. The primary aim of the study is to determine the feasibility and acceptability of the Bounce Back program. Descriptive statistics, including means, frequencies, and ranges supplemented by qualitative exit interview feedback will be used to characterize the sample and to summarize feasibility and acceptability. The exploratory aims are to evaluate the preliminary effects of the program on stress coping and psychosocial outcome measures (ie, anxiety, depression) collected across the 3 time points. ResultsThis study was funded by the National Cancer Institute in July 2017. Study procedures were approved by the Dana-Farber Harvard Cancer Center Institutional Review Board in October 2018 (Protocol 18-428). The randomized trial was conducted from July 2019 to March 2021. Quantitative data collection is complete, and qualitative exit interview data collection is ongoing. Results are expected to be published in peer-reviewed journals and presented at local, national, or international meetings in the coming years. ConclusionsFew evidence-based programs exist that tackle the key transitional issues faced by AYA cancer survivors. Future analyses will help us determine the feasibility and acceptability of the Bounce Back program and its impact on AYA stress coping and psychological well-being. Trial RegistrationClinicalTrials.gov NCT03768336; https://clinicaltrials.gov/ct2/show/NCT03768336 International Registered Report Identifier (IRRID)DERR1-10.2196/34033

Zebrafish have made significant contributions to our understanding of the vertebrate brain and the neural basis of behavior, earning a place as one of the most widely used model organisms in neuroscience. Their appeal arises from the marriage of low cost, early life transparency, and ease of genetic manipulation with a behavioral repertoire that becomes more sophisticated as animals transition from larvae to adults. To further enhance the use of adult zebrafish, we created the first fully segmented three-dimensional digital adult zebrafish brain atlas (AZBA). AZBA was built by combining tissue clearing, light-sheet fluorescence microscopy, and three-dimensional image registration of nuclear and antibody stains. These images were used to guide segmentation of the atlas into over 200 neuroanatomical regions comprising the entirety of the adult zebrafish brain. As an open source, online (azba.wayne.edu), updatable digital resource, AZBA will significantly enhance the use of adult zebrafish in furthering our understanding of vertebrate brain function in both health and disease.

Abstract This case discusses a patient who presented with immune thrombocytopenia purpura (ITP) one week after COVID‐19 vaccination, introducing ITP as a consideration for a potential, but rare, adverse reaction to COVID‐19 vaccination.

Prosocial behavior, in particular helping others in need, occurs preferentially in response to distress of one’s own group members. In order to explore the neural mechanisms promoting mammalian helping behavior, a discovery-based approach was used here to identify brain-wide activity correlated with helping behavior in rats. Demonstrating social selectivity, rats helped others of their strain (‘ingroup’), but not rats of an unfamiliar strain (‘outgroup’), by releasing them from a restrainer. Analysis of brain-wide neural activity via quantification of the early-immediate gene c-Fos identified a shared network, including frontal and insular cortices, that was active in the helping test irrespective of group membership. In contrast, the striatum was selectively active for ingroup members, and activity in the nucleus accumbens, a central network hub, correlated with helping. In vivo calcium imaging showed accumbens activity when rats approached a trapped ingroup member, and retrograde tracing identified a subpopulation of accumbens-projecting cells that was correlated with helping. These findings demonstrate that motivation and reward networks are associated with helping an ingroup member and provide the first description of neural correlates of ingroup bias in rodents.

Type three secretion systems enable bacterial pathogens to inject effectors into the cytosol of eukaryotic hosts to reprogram cellular functions. It is technically challenging to label effectors and the secretion machinery without disrupting their structure/function. Herein, we present a new approach for labeling and visualization of previously intractable targets. Using genetic code expansion, we site-specifically labeled SsaP, the substrate specificity switch, and SifA, a here-to-fore unlabeled secreted effector. SsaP was secreted at later infection times; SsaP labeling demonstrated the stochasticity of injectisome and effector expression. SifA was labeled after secretion into host cells via fluorescent unnatural amino acids or non-fluorescent labels and a subsequent click reaction. We demonstrate the superiority of imaging after genetic code expansion compared to small molecule tags. It provides an alternative for labeling proteins that do not tolerate N- or C-terminal tags or fluorophores and thus is widely applicable to other secreted effectors and small proteins.

Many enveloped viruses induce multinucleated cells (syncytia), reflective of membrane fusion events caused by the same machinery that underlies viral entry. These syncytia are thought to facilitate replication and evasion of the host immune response. Here, we report that co-culture of human cells expressing the receptor ACE2 with cells expressing SARS-CoV-2 spike, results in synapse-like intercellular contacts that initiate cell-cell fusion, producing syncytia resembling those we identify in lungs of COVID-19 patients. To assess the mechanism of spike/ACE2-driven membrane fusion, we developed a microscopy-based, cell-cell fusion assay to screen ~6000 drugs and >30 spike variants. Together with quantitative cell biology approaches, the screen reveals an essential role for biophysical aspects of the membrane, particularly cholesterol-rich regions, in spike-mediated fusion, which extends to replication-competent SARS-CoV-2 isolates. Our findings potentially provide a molecular basis for positive outcomes reported in COVID-19 patients taking statins and suggest new strategies for therapeutics targeting the membrane of SARS-CoV-2 and other fusogenic viruses.

Abstract Purpose: Research demonstrates that children receive twice as much medical radiation from Computed Tomography (CT) scans performed at non-pediatric facilities as equivalent CTs performed at pediatric trauma centers (PTCs). In 2014, AFMC outreach staff educated Emergency Department (ED) staff on appropriate CT imaging utilization to reduce unnecessary medical radiation exposure. We set out to determine the educational campaign’s impact on injured children received radiation dose. Methods: All injured children who underwent CT imaging and were transferred to a Level I PTC during 2010 to 2013 (pre-campaign) and 2015 (post-campaign) were reviewed. Patient demographics, mode of transportation, ED length of stay, scanned body region, injury severity score, and trauma center level were analyzed. Median effective radiation dose (ERD) controlled for each variable, pre-campaign and post-campaign, was compared using Wilcoxon rank sum test. Results: Three hundred eighty-five children under 17 years were transferred from 45 and 48 hospitals, pre- and post-campaign. Most (43%) transferring hospitals were urban or critical access hospitals (30%). Pre- and post-campaign patient demographics were similar. We analyzed 482 and 398 CT scans pre- and post-campaign. Overall, median ERD significantly decreased from 3.80 to 2.80. Abdominal CT scan ERD declined significantly from 7.2 to 4.13 (P-value 0.03). Head CT scan ERD declined from 3.27 to 2.45 (P-value < 0.0001). Conclusion: A statewide, CT scan educational campaign contributed to ERD decline (lower dose scans and fewer repeat scans) among transferred injured children seen at PTCs. State-level interventions are feasible and can be effective in changing radiology provider practices.

The neuroinvasive disease caused by Jamestown Canyon virus (JCV) infection is rare. However, increasing incidence and widespread occurrence of the infection make JCV a growing public health concern. Presently, clinical diagnosis is achieved through serological testing, and mosquito pool surveillance requires virus isolation and identification. A rapid molecular detection test, such as real-time RT-PCR, for diagnosis and surveillance of JCV has not been widely utilized. To enhance testing and surveillance, here, we describe the development and validation of a real-time RT-PCR test for the detection of JCV RNA. Three primer and probe sets were evaluated for analytical sensitivity and specificity. One probe set, JCV132FAM, was found to be the most sensitive test detecting 7.2 genomic equivalents/µL. While less sensitive, a second probe set JCV231cFAM was the most specific test with limited detection of Keystone virus at high RNA loads. Taken together, these data indicate both probe sets can be utilized for a primary sensitive screening assay and a secondary specific confirmatory assay. While both primer and probe sets detected high viral loads of Keystone virus, these assays did not detect any virus in the California encephalitis virus clade, including negative detection of the medically important La Crosse virus (LACV) and snowshoe hare virus (SSHV). The real-time RT-PCR assay described herein could be utilized in diagnosis and surveillance in regions with co-circulation of JCV and LACV or SSHV to inform public health action.

Background Drug therapy yields different results depending on its recipient population. Cisplatin, a commonly used chemotherapeutic agent, causes different levels of resistance and side effects for different patients, but the mechanism(s) are presently unknown. It has been assumed that this variation is a consequence of differences in nuclear (n) DNA, epigenetics, or some external factor(s). There is accumulating evidence that an individual’s mitochondrial (mt) DNA may play a role in their response to medications. Variations within mtDNA can be observed, and an individual’s mtDNA can be categorized into haplogroups that are defined by accumulations of single nucleotide polymorphisms (SNPs) representing different ethnic populations. Methods The present study was conducted on transmitochondrial cytoplasmic hybrids (cybrids) that possess different maternal-origin haplogroup mtDNA from African (L), Hispanic [A+B], or Asian (D) backgrounds. Cybrids were created by fusing Rho0 ARPE-19 cells (lacking mtDNA) with platelets, which contain numerous mitochondria but no nuclei. These cybrid cells were cultured to passage five, treated with cisplatin, incubated for 48 h, then analyzed for cell metabolic activity (tetrazolium dye (MTT) assay), mitochondrial membrane potential (JC-1 assay), cytotoxicity (lactate dehydrogenase (LDH) assay), and gene expression levels for ALK, BRCA1, EGFR, and ERBB2/HER2. Results Results indicated that untreated cybrids with varying mtDNA haplogroups had similar relative metabolic activity before cisplatin treatment. When treated with cisplatin, (1) the decline in metabolic activity was greatest in L (27.4%, p [A+B], p = 0.0270). (4) The expression levels decreased for ALK in L (p

To gain a comprehensive view of the changes in host gene expression underlying Zika virus (ZIKV) pathogenesis, we performed whole-genome RNA sequencing (RNA-seq) of ZIKV-infected Drosophila adult flies. RNA-seq analysis revealed that ZIKV infection alters several and diverse biological processes, including stress, locomotion, lipid metabolism, imaginal disc morphogenesis and regulation of JAK/STAT signaling. To explore the interaction between ZIKV infection and JAK/STAT signaling regulation, we generated genetic constructs overexpressing ZIKV-specific non-structural proteins NS2A, NS2B, NS4A and NS4B. We found that ectopic expression of non-structural proteins in the developing Drosophila eye significantly restricts growth of the larval and adult eye and correlates with considerable repression of the in vivo JAK/STAT reporter, 10XStat92E-GFP. At the cellular level, eye growth defects are associated with reduced rate of proliferation without affecting the overall rate of apoptosis. In addition, ZIKV NS4A genetically interacts with the JAK/STAT signaling components; co-expression of NS4A along with the dominant-negative form of domeless or StatRNAi results in aggravated reduction in eye size, while co-expression of NS4A in HopTuml (also known as hopTum) mutant background partially rescues the hop-induced eye overgrowth phenotype. The function of ZIKV NS4A in regulating growth is maintained in the wing, where ZIKV NS4A overexpression in the pouch domain results in reduced growth linked with diminished expression of Notch targets, Wingless (Wg) and Cut, and the Notch reporter, NRE-GFP. Thus, our study provides evidence that ZIKV infection in Drosophila results in restricted growth of the developing eye and wing, wherein eye phenotype is induced through regulation of JAK/STAT signaling, whereas restricted wing growth is induced through regulation of Notch signaling. The interaction of ZIKV non-structural proteins with the conserved host signaling pathways further advance our understanding of ZIKV-induced pathogenesis. This article has an associated First Person interview with the first author of the paper.

Porcine deltacoronavirus (PDCoV) is an emerging enteropathogenic coronavirus of swine that causes acute diarrhoea, vomiting, dehydration and mortality in seronegative neonatal piglets. PDCoV was first reported in Hong Kong in 2012 and its etiological features were first characterized in the United States in 2014. Currently, PDCoV is a concern due to its broad host range, including humans. Chickens, turkey poults, and gnotobiotic calves can be experimentally infected by PDCoV. Therefore, as discussed in this review, a comprehensive understanding of the origin, evolution, cross-species transmission and zoonotic potential of epidemic PDCoV strains is urgently needed.

In response to the 2016 Zika outbreak, Aedes aegypti mosquitoes from 38 locations across Puerto Rico were screened using Centers for Disease Control and Prevention bottle bioassays for sensitivity to insecticides used for mosquito control. All populations were resistant to pyrethroids. Naled, an organophosphate, was the most effective insecticide, killing all mosquitoes tested.

ABSTRACT IMPACT: This is the first examination of risk factors for prolonged opioid use after an ICU stay and will inform efforts to strengthen prescribing guidelines and care transition models for patients after critical illness. OBJECTIVES/GOALS: The majority of patients in intensive care units (ICU) receive opioids during admission, and up to 25% receive a prescription at discharge. However, transitions of care and prolonged use after discharge remain unknown. We aim to characterize risk factors for prolonged opioid use after an ICU stay. METHODS/STUDY POPULATION: A retrospective study using insurance claims from Optum Clinformatics ®Data Mart was conducted for opioid-naive adult patients (18-64 years) with an ICU admission from 2010 to 2019. The primary outcome was new persistent opioid use, defined as a continued prescription fill 91-180 days after discharge, in addition to a fill in the first 90 days. The primary exposure was an opioid fill at discharge. The ICU admission was characterized using the Clinical Classification System from the Agency of Healthcare Research and Quality, based on patients’primary diagnosis code. Diagnoses were combined into 11 groups highlighting the affected organ system/mechanism of injury. Logistic regression evaluated the associations of patient demographic and clinical characteristics with the probability of persistent opioid use. RESULTS/ANTICIPATED RESULTS: In this cohort of 90,721 patients discharged from the ICU, 3.3% continued to fill opioids at 6 months. An opioid prescription fill (OR 3.1; 95% CI 28 - 3.3) and benzodiazepine prescription fill (OR 1.6; 95% CI 1.4 - 1.8) within 3 days of ICU discharge were each significantly associated with the development of new persistent opioid use. Patient diagnosis groups of Musculoskeletal/Trauma (OR 2.3; 95% CI 2.0 - 2.6), Neoplasms (OR 1.6; 95% CI 1.5 - 1.9), and GI/Hepatobiliary (OR 1.5; 95% CI 1.3 - 1.8) were significantly more likely to develop new persistent use when compared to the Cardiovascular diagnosis group. DISCUSSION/SIGNIFICANCE OF FINDINGS: Opioid prescriptions at discharge after an ICU stay increase the odds of prolonged opioid use. These results will inform efforts to strengthen prescribing guidelines and care models after a critical illness. Further work will characterize the trajectory of prescribing and patient exposure to high-risk prescribing after ICU discharge.

Hepatitis E virus is an important emerging pathogen producing a lethal impact on the pregnant population and immunocompromised patients. Starting in 1983, it has been described as the cause for acute hepatitis transmitted via the fecal–oral route. However, zoonotic and blood transfusion transmission of HEV have been reported in the past few decades, leading to the detailed research of HEV pathogenesis. The reason behind HEV being highly virulent to the pregnant population particularly during the third trimester, leading to maternal and fetal death, remains unknown. Various host factors (immunological, nutritional, hormonal) and viral factors have been studied to define the key determinants assisting HEV to be virulent in pregnant and immunocompromised patients. Similarly, chronic hepatitis is seen particularly in solid organ transplant patients, resulting in fatal conditions. This review describes recent advances in the immunopathophysiology of HEV infections in general, pregnant, and immunocompromised populations, and further elucidates the in vitro and in vivo models utilized to understand HEV pathogenesis.

We isolated a strain of Zika virus, MB16-23, from Aedes aegypti mosquitoes collected in Miami Beach, Florida, USA, on September 2, 2016. Phylogenetic analysis suggests that MB16-23 most likely originated from the Caribbean region.

After Salmonella is phagocytosed, it resides in an acidic vacuole. Its cytoplasm acidifies to pH 5.6; acidification activates pathogenicity island 2 (SPI-2). SPI-2 encodes a type three secretion system whose effectors modify the vacuole, driving endosomal tubulation. Using super-resolution imaging in single bacterial cells, we show that low pH induces expression of the SPI-2 SsrA/B signaling system. Single particle tracking, atomic force microscopy, and single molecule unzipping assays identified pH-dependent stimulation of DNA binding by SsrB. A so-called phosphomimetic form (D56E) was unable to bind to DNA in live cells. Acid-dependent DNA binding was not intrinsic to regulators, as PhoP and OmpR binding was not pH-sensitive. The low level of SPI-2 injectisomes observed in single cells is not due to fluctuating SsrB levels. This work highlights the surprising role that acid pH plays in virulence and intracellular lifestyles of Salmonella; modifying acid survival pathways represents a target for inhibiting Salmonella.

[This corrects the article DOI: 10.1371/journal.pone.0191073.].

Immune priming in insects involves an initial challenge with a non-pathogenic microbe or exposure to a low dose of pathogenic microorganisms, which provides a certain degree of protection against a subsequent pathogenic infection. The protective effect of insect immune priming has been linked to the activation of humoral or cellular features of the innate immune response during the preliminary challenge, and these effects might last long enough to promote the survival of the infected animal. The fruit fly Drosophila melanogaster is a superb model to dissect immune priming processes in insects due to the availability of molecular and genetic tools, and the comprehensive understanding of the innate immune response in this organism. Previous investigations have indicated that the D. melanogaster immune system can be primed efficiently. Here we have extended these studies by examining the result of immune priming against two potent entomopathogenic bacteria, Photorhabdus luminescens and P. asymbiotica. We have found that rearing D. melanogaster on diet containing a non-pathogenic strain of Escherichia coli alone or in combination with Micrococcus luteus upregulates the antibacterial peptide immune response in young adult flies, but it does not prolong fly life span. Also, subsequent intrathoracic injection with P. luminescens or P. asymbiotica triggers the Immune deficiency and Toll signaling pathways in flies previously exposed to a live or heat-killed mix of the non-pathogenic bacteria, but the immune activation fails to promote fly survival against the pathogens. These findings suggest that immune priming in D. melanogaster, and probably in other insects, is determined by the type of microbes involved as well as the mode of microbial exposure, and possibly requires a comprehensive and precise alteration of immune signaling and function to provide efficient protection against pathogenic infection.