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Abstract Self-replicating RNA (srRNA) technology, in comparison to mRNA vaccines, has shown dose-sparing by approximately 10-fold and more durable immune responses. However, no improvements are observed in the adverse events profile. Here, we develop an srRNA vaccine platform with optimized non-coding regions and demonstrate immunogenicity and safety in preclinical and clinical development. Optimized srRNA vaccines generate protective immunity (according to the WHO defined thresholds) at doses up to 1,000,000-fold lower than mRNA in female mouse models of influenza and rabies. Clinically, safety and immunogenicity of RBI-4000, an srRNA vector encoding the rabies glycoprotein, was evaluated in a Phase I study (NCT06048770). RBI-4000 was able to elicit de novo protective immunity in the majority of healthy participants when administered at a dose of 0.1, 1, or 10 microgram (71%, 94%, 100%, respectively) in a prime-boost schedule. Similarly, we observe immunity above the WHO benchmark of protection following a single administration in most participants at both 1 and 10 microgram doses. There are no serious adverse events reported across all cohorts. These data establish the high therapeutic index of optimized srRNA vectors, demonstrating feasibility of both low dose and single dose approaches for vaccine applications.

Abstract Filoviruses, including Ebola, Marburg, Sudan, and Taï Forest viruses, are zoonotic pathogens that can cause severe viral hemorrhagic fever and death. Developing vaccines that provide durable, broad immunity against multiple filoviruses is a high global health priority. In this Phase 1 trial, we enrolled 60 healthy U.S. adults and evaluated the safety, reactogenicity and immunogenicity of homologous and heterologous MVA-BN®-Filo and Ad26.ZEBOV prime-boost schedules followed in select arms by MVA-BN®-Filo boost at 1 year (NCT02891980). We found that all vaccine regimens had acceptable safety and reactogenicity. The heterologous prime-boost strategy elicited superior Ebola binding and neutralizing antibody, antibody-dependent cellular cytotoxicity (ADCC), and cellular responses compared to homologous prime-boost. The MVA-BN®-Filo boost administered at 1 year resulted in robust humoral and cellular responses that persisted through 6-month follow-up. Overall, our data demonstrated that a heterologous Ad26.ZEBOV/MVA-BN®-Filo prime-boost was safe and immunogenic and established immunologic memory primed to respond after re-exposure. Clinicaltrials.gov, NCT02891980, registered September 1, 2016.

Introduction COVID-19 vaccines may be administered with other vaccines during the same healthcare visit. COVID-19 monovalent (Fall 2021) and bivalent (Fall 2022) vaccine recommendations coincided with annual seasonal influenza vaccination. Data describing the frequency of the co-administration of COVID-19 vaccines with other vaccines are limited. Methods We used V-safe, a voluntary smartphone-based U.S. safety surveillance system established by the CDC, to describe trends in the administration of COVID-19 vaccines with other vaccines reported to V-safe during December 14, 2020 – May 19, 2023. Results Of the 21 million COVID-19 vaccinations reported to V-safe, 2.2% (459,817) were administered with at least 1 other vaccine. Co-administration most frequently occurred during the first week of October 2023 (27,092; 44.1%). Most reports of co-administration included influenza vaccine (393,003; 85.5%). Co-administration was most frequently reported for registrants aged 6 months-6 years (4,872; 4.4%). Conclusion Reports of co-administration to V-safe peaked during October 2023, when influenza vaccination most often occurs, possibly reflecting increased opportunities for multiple vaccinations and greater acceptability of the co-administration of COVID-19 vaccine with other vaccines, especially influenza vaccine.

Abstract NLRP3 inflammasome has been implicated in neutrophil polarization and extrusion of neutrophil extracellular traps (NETs) in vitro and facilitates secretion of Il1-beta (IL-1β). Permanent ligation of the left anterior descending artery was used to induce MI in WT and NLRP3−/− mice as well as in NLRP3−/− recipient mice transfused with either WT or NLRP3−/− neutrophils. NLRP3 deficiency reduced infarct size to roughly a third of WT heart injury and preserved left ventricular (LV) function at 12 h after MI as assessed by echocardiography and triphenyltetrazolium chloride staining of live tissue. Transfusion of WT but not NLRP3−/− neutrophils after MI increased infarct size in NLRP3−/− mice and significantly reduced LV function. The key features of myocardial tissue in WT neutrophil transfused recipients were increased H3Cit-positive deposits with NET-like morphology and increased tissue levels of IL-1β and plasma levels of von Willebrand Factor (VWF). Flow cytometry analysis also revealed that neutrophil NLRP3 increased the number of labeled and transfused neutrophils in the bone marrow of recipient mice following MI. Our data suggest a key role for neutrophil NLRP3 in the production of IL-1β and deposition of NETs in cardiac tissue exacerbating injury following MI. We provide evidence for a link between neutrophil NLRP3 and VWF release likely enhancing thromboinflammation in the heart. Neutrophil NLRP3 deficiency conferred similar cardioprotective effects to general NLRP3 deletion in MI rendering anti-neutrophil NLRP3 therapy a promising target for early cardioprotective treatment.

ABSTRACT Diffuse panbronchiolitis (DPB) is a rare, idiopathic inflammatory disease primarily diagnosed in East Asian populations. DPB is characterized by diffuse pulmonary lesions, inflammation of the respiratory bronchioles, and bacterial infections of the airway. Historically, sputum cultures reveal Pseudomonas aeruginosa in 22% of DPB patients, increasing to 60% after 4 years from disease onset. Although DPB patients have a known susceptibility to respiratory P. aeruginosa infections, as is observed in other chronic lung diseases such as cystic fibrosis (CF), the characterization of DPB P. aeruginosa strains is limited. In this study, we characterized 24 strains obtained from a cohort of DPB patients for traits previously associated with virulence, including growth, motility, antibiotic susceptibility, lipopolysaccharide structure, and genomic diversity. Our cohort of DPB P. aeruginosa strains exhibits considerable genomic variability when compared with isolates from people with cystic fibrosis chronically colonized with P. aeruginosa and acute P. aeruginosa infection isolates. Similar to CF, DPB P. aeruginosa strains produce a diverse array of modified lipid A structures. Antibiotic susceptibility testing revealed increased resistance to erythromycin, a representative agent of the macrolide antibiotics used to manage DPB patients. Differences in the O-antigen type among P. aeruginosa strains collected from these different backgrounds were also observed. Ultimately, the characterization of DPB P. aeruginosa strains highlights several unique qualities of P. aeruginosa strains collected from chronically diseased airways, underscoring the challenges in treating DPB, CF, and other obstructive respiratory disease patients with P. aeruginosa infections.IMPORTANCEDiffuse panbronchiolitis (DPB), a chronic lung disease characterized by persistent P. aeruginosa infection, serves as an informative comparator to more common chronic lung diseases, such as cystic fibrosis (CF). This study aimed to better address the interplay between P. aeruginosa and chronically compromised airway environments through the examination of DPB P. aeruginosa strains, as existing literature regarding DPB is limited to case reports, case series, and clinical treatment guidelines. The evaluation of these features in the context of DPB, in tandem with prevailing knowledge of P. aeruginosa strains collected from more common chronic lung diseases (e.g., CF), can aid in the development of more effective strategies to combat respiratory P. aeruginosa infections in patients with chronic lung diseases.

Objective: To estimate the potential contributions of reported changes in frequency of penile-vaginal sex (PVS), condom use and STI screening to changes in gonorrhea and chlamydial diagnoses from 2012 to 2019. Methods: An agent-based model of the heterosexual population in the U.S. simulated the STI epidemics. Baseline was calibrated to 2012 diagnosis rates, testing, condom use, and frequency of PVS. Counterfactuals used behaviors from the 2017-2019 NSFG, and we evaluated changes in diagnosis and incidence rates in 2019. Results: Higher testing rates increased gonorrhea and chlamydia diagnosis by 14% and 13%, respectively, but did not reduce incidence. Declining frequency of PVS reduced the diagnosis rate for gonorrhea and chlamydia 6% and 3% respectively while reducing incidence by 10% and 9% respectively. Declining condom use had negligible impact on diagnosis and incidence. Conclusion: Understanding how changing behavior drives STI incidence is essential to addressing the growing epidemics. Changes in testing and frequency of PVS likely contributed to some, but not all, of the changes in diagnoses. More research is needed to understand the context within which changing sexual behavior and testing are occurring.

Extremity trauma, including ischemia (e.g., prolonged tourniquet application or crush), is common among battlefield injuries. Injured muscle releases toxins leading to rhabdomyolysis and, potentially, acute kidney injury (AKI). The goal of this study was to characterize sequelae of ischemic extremity injury over 72 h, focusing on time courses of rhabdomyolysis and AKI. Male Sprague Dawley rats were placed into two groups. Ischemic injury was produced in anesthetized rats using bilateral tourniquets (TK; n = 10) for 5 h; control (CON; n = 9) rats were treated identically without TK application. Indicators of rhabdomyolysis and renal function were measured in conscious rats 1 day preinjury (baseline, BL) and then at 1.5, 24, 48, and 72 h post-TK release. Prolonged TK application produced necrosis in both muscle and bone marrow but not in kidney. The wet/dry weights indicated edema in injured limbs at 72 h (4.1 (0.5) (TK) vs. 2.9 (0.1) (CON); p < 0.001). TK rats exhibited a 100-fold increase in creatine kinase activity compared to CON at 1.5 h (20,040 (7265) U/L vs. 195 (86) U/L (mean (SD); p < 0.0001). TK decreased the mean glomerular filtration rate (GFR; p < 0.001) at 1.5 h, but these values recovered by 24 h in concert with elevated urinary flow and alkalinization. Prolonged ischemic extremity injury therefore produced severe rhabdomyolysis without irreversible renal damage.

Heterogeneity in endothelial cell (EC) sub-phenotypes is becoming increasingly appreciated in atherosclerosis progression. Still, studies quantifying EC heterogeneity across whole transcriptomes and epigenomes in both in vitro and in vivo models are lacking. Multiomic profiling concurrently measuring transcriptomes and accessible chromatin in the same single cells was performed on six distinct primary cultures of human aortic ECs (HAECs) exposed to activating environments characteristic of the atherosclerotic microenvironment in vitro. Meta-analysis of single-cell transcriptomes across 17 human ex vivo arterial specimens was performed and two computational approaches quantitatively evaluated the similarity in molecular profiles between heterogeneous in vitro and ex vivo cell profiles. HAEC cultures were reproducibly populated by four major clusters with distinct pathway enrichment profiles and modest heterogeneous responses: EC1-angiogenic, EC2-proliferative, EC3-activated/mesenchymal-like, and EC4-mesenchymal. Quantitative comparisons between in vitro and ex vivo transcriptomes confirmed EC1 and EC2 as most canonically EC-like, and EC4 as most mesenchymal with minimal effects elicited by siERG and IL1B. Lastly, accessible chromatin regions unique to EC2 and EC4 were most enriched for coronary artery disease (CAD)-associated single-nucleotide polymorphisms from Genome Wide Association Studies (GWAS), suggesting that these cell phenotypes harbor CAD-modulating mechanisms. Primary EC cultures contain markedly heterogeneous cell subtypes defined by their molecular profiles. Surprisingly, the perturbations used here only modestly shifted cells between subpopulations, suggesting relatively stable molecular phenotypes in culture. Identifying consistently heterogeneous EC subpopulations between in vitro and ex vivo models should pave the way for improving in vitro systems while enabling the mechanisms governing heterogeneous cell state decisions.

ABSTRACT Pseudomonas aeruginosa can survive in a myriad of environments, partially due to modifications of its lipid A, the membrane anchor of lipopolysaccharide. We previously demonstrated that divergent late acyltransferase paralogs, HtrB1 and HtrB2, add acyloxyacyl laurate to lipid A 2- and 2′-acyl chains, respectively. The genome of P. aeruginosa also has genes which encode two dioxygenase enzymes, LpxO1 and LpxO2, that individually hydroxylate a specific secondary laurate. LpxO1 acts on the 2′-acyloxyacyl laurate (added by HtrB2), whereas LpxO2 acts on the 2-acyloxyacyl laurate (added by HtrB1) in a site-specific manner. Furthermore, while both enzyme pairs are evolutionarily linked, phylogenomic analysis suggests the LpxO1/HtrB2 enzyme pair as being of ancestral origin, present throughout the Pseudomonas lineage, whereas the LpxO2/HtrB1 enzyme pair likely arose via horizontal gene transfer and has been retained in P. aeruginosa over time. Using a murine pulmonary infection model, we showed that both LpxO1 and LpxO2 enzymes are functional in vivo, as direct analysis of in vivo lipid A structure from bronchoalveolar lavage fluid revealed 2-hydroxylated lipid A. Gene expression analysis reveals increased lpxO2 but unchanged lpxO1 expression in vivo, suggesting differential regulation of these enzymes during infection. We also demonstrate that loss-of-function mutations arise in lpxO1 and lpxO2 during chronic lung infection in people with cystic fibrosis (CF), indicating a potential role for pathogenesis and airway adaptation. Collectively, our study characterizes lipid A 2-hydroxylation during P. aeruginosa airway infection that is regulated by two distinct lipid A dioxygenase enzymes.IMPORTANCEPseudomonas aeruginosa is an opportunistic pathogen that causes severe infection in hospitalized and chronically ill individuals. During infection, P. aeruginosa undergoes adaptive changes to evade host defenses and therapeutic interventions, increasing mortality and morbidity. Lipid A structural alteration is one such change that P. aeruginosa isolates undergo during chronic lung infection in CF. Investigating genetic drivers of this lipid A structural variation is crucial in understanding P. aeruginosa adaptation during infection. Here, we describe two lipid A dioxygenases with acyl-chain site specificity, each with different evolutionary origins. Further, we show that loss of function in these enzymes occurs in CF clinical isolates, suggesting a potential pathoadaptive phenotype. Studying these bacterial adaptations provides insight into selection pressures of the CF airway on P. aeruginosa phenotypes that persist during chronic infection. Understanding these adaptive changes may ultimately provide clinicians better control over bacterial populations during chronic infection.

Mitochondrial membrane potential directly powers many critical functions of mitochondria, including ATP production, mitochondrial protein import, and metabolite transport. Its loss is a cardinal feature of aging and mitochondrial diseases, and cells closely monitor membrane potential as an indicator of mitochondrial health. Given its central importance, it is logical that cells would modulate mitochondrial membrane potential in response to demand and environmental cues, but there has been little exploration of this question. We report that loss of the Sit4 protein phosphatase in yeast increases mitochondrial membrane potential, both by inducing the electron transport chain and the phosphate starvation response. Indeed, a similarly elevated mitochondrial membrane potential is also elicited simply by phosphate starvation or by abrogation of the Pho85-dependent phosphate sensing pathway. This enhanced membrane potential is primarily driven by an unexpected activity of the ADP/ATP carrier. We also demonstrate that this connection between phosphate limitation and enhancement of mitochondrial membrane potential is observed in primary and immortalized mammalian cells as well as in Drosophila. These data suggest that mitochondrial membrane potential is subject to environmental stimuli and intracellular signaling regulation and raise the possibility for therapeutic enhancement of mitochondrial function even in defective mitochondria.

Background Citrulline may amplify the effects of L-arginine and nitric oxide concentration, which may augment vasodilation and blood flow, thereby enhancing aerobic exercise performance. The purpose of this randomized, double-blind, placebo-controlled crossover study was to investigate effects of L-citrulline + Glutathione on aerobic exercise performance and blood flow in well-trained men. Methods Twenty-five males (Mean ± SD; Age: 22.2 ± 2.4 yrs; Height: 177.0 ± 4.8 cm; Weight: 75.3 ± 6.9 kg) were randomly assigned to the L-citrulline + Glutathione (Setria Performance Blend: SPB; L-citrulline [2 g] + glutathione [200 mg], 6 capsules) or placebo (PL; 3.1 g cellulose, 6 capsules) group. Participants performed a maximal oxygen consumption treadmill test to determine peak velocity (PV) and returned after eight days of ingesting either PL or SPB. Three timed treadmill runs to exhaustion (TTE) were performed at 90%, 100%, and 110% PV. Brachial artery blood flow and vessel diameter were assessed using ultrasound at 1-hr prior to exercise (1hrPrEX), after each exercise bout, immediately post-exercise (immediate PEX), and 30 minutes post exercise (30minPEX) at visits 2 and 4. Blood analytes were assessed via venous blood draws at visit 1, visit 3, and 1hrPEX, immediate PEX, and 30minPEX at visits 2 and 4. After a 14-day washout, participants repeated the same procedures, ingesting the opposite treatment. Separate repeated measures ANOVAs were performed for TTE, vessel diameter, blood flow, and blood analytes. Results Blood flow was significantly augmented 30minPEX (p = 0.04) with SPB in comparison with PL. L-citrulline and L-arginine plasma concentrations were significantly elevated immediately PEX (p = 0.001) and 30-minPEX (p = 0.001) following SPB in comparison to PL. Conclusion Acute ingestion of SPB after eight days may enhance blood flow, L-citrulline, and L-arginine plasma concentrations after high-intensity exercise, which may enhance performance. Clinical Trial Registration [https://clinicaltrials.gov/ct2/show/nct04090138], identifier [NCT04090138].

Pre-sleep nutrition habits in elite female athletes have yet to be evaluated. A retrospective analysis was performed with 14 NCAA Division I female soccer players who wore a WHOOP, Inc. band – a wearable device that quantifies recovery by measuring sleep, activity, and heart rate metrics through actigraphy and photoplethysmography, respectively – 24 h a day for an entire competitive season to measure sleep and recovery. Pre-sleep food consumption data were collected via surveys every 3 days. Average pre-sleep nutritional intake (mean ± sd: kcals 330 ± 284; cho 46.2 ± 40.5 g; pro 7.6 ± 7.3 g; fat 12 ± 10.5 g) was recorded. Macronutrients and kcals were grouped into high and low categories based upon the 50th percentile of the mean to compare the impact of a high versus low pre-sleep intake on sleep and recovery variables. Sleep duration (p = 0.10, 0.69, 0.16, 0.17) and sleep disturbances (p = 0.42, 0.65, 0.81, 0.81) were not affected by high versus low kcal, PRO, fat, CHO intake, respectively. Recovery (p = 0.81, 0.06, 0.81, 0.92), RHR (p = 0.84, 0.64, 0.26, 0.66), or HRV (p = 0.84, 0.70, 0.76, 0.93) were also not affected by high versus low kcal, PRO, fat, or CHO consumption, respectively. Consuming a small meal before bed may have no impact on sleep or recovery.

Abstract Protein arginine deiminases (PAD) 4 is an enzyme that catalyzes citrullination of protein and its role in autoimmune diseases has been established through clinical genetics and gene knock out studies in mice. Further, studies with PAD4 – deficient mice have shown that PAD4 deficiency does not lead to increased infection or immune suppression, which makes PAD4 an attractive therapeutic target for auto-immune and inflammatory diseases. PAD4 has critical enzymatic role of promoting chromatin decondensation and neutrophil extracellular traps (NETs) formation that is associated with a number of immune-mediated pathological conditions. Here, we present a non-covalent PAD4 inhibitor JBI-589 with high PAD4 isoform selectivity and delineated its binding mode at 2.88 Å resolution by X-ray crystallography. We confirmed its effectiveness in inhibiting NET formation in vitro. Additionally, by using two mouse arthritis models for human rheumatoid arthritis (RA), the well-known disease associated with PAD4 clinically, we established its efficacy in vivo. These results suggest that JBI-589 would be beneficial for both PAD4 and NET-associated pathological conditions.

Abstract NLR family pyrin domain containing 3 (NLRP3) inflammasome mediates caspase-1-dependent processing of inflammatory cytokines such as IL-1β, an essential endothelial activator, and contributes to the pathology of inflammatory diseases. To evaluate the role of NLRP3 in neutrophils in endothelial activation, which is still elusive, we used the thioglycollate-induced peritonitis model characterized by an early neutrophil influx, on Nlrp3 −/− and Nlrp3 +/+ mice. Nlrp3 −/− mice recruited fewer neutrophils than Nlrp3 +/+ into the peritoneum and showed lower IL-1β in peritoneal lavage fluid. The higher production of IL-1β in Nlrp3 +/+ was neutrophil-dependent as neutrophil depletion prevented the IL-1β production. The Nlrp3 +/+ neutrophils collected from the peritoneal fluid formed significantly more filaments (specks) than Nlrp3 −/− neutrophils of ASC (apoptosis-associated speck-like protein containing a caspase activating and recruitment domain), a readout for inflammasome activation. Intravital microscopy revealed that leukocytes rolled significantly slower in Nlrp3 +/+ venules than in Nlrp3 −/− . Nlrp3 −/− endothelial cells isolated from mesenteric vessels demonstrated a lower percentage of P-selectin-positive cells with lower intensity of surface P-selectin expression than the Nlrp3 +/+ endothelial cells evaluated by flow cytometry. We conclude that neutrophils orchestrate acute thioglycollate-induced peritonitis by producing IL-1β in an NLRP3-dependent manner. This increases endothelial P-selectin expression and leukocyte transmigration.

Summary: Pseudomonas aeruginosa (Pa) is a pathogen causing chronic pulmonary infections in patients with cystic fibrosis (CF). Manipulation of lipids is an important feature of Pa infection and on a tissue-level scale is poorly understood. Using a mouse model of acute Pa pulmonary infection, we explored the whole-lung phospholipid response using mass spectrometry imaging (MSI) and spatial lipidomics. Using a histology-driven analysis, we isolated airways and parenchyma from both mock- and Pa-infected lungs and used systems biology tools to identify enriched metabolic pathways from the differential phospholipid identities. Infection was associated with a set of 26 ions, with 11 unique to parenchyma and 6 unique to airways. Acyl remodeling was differentially enriched in infected parenchyma as the predominant biological function. These functions correlated with markers of polymorphonuclear (PMN) cell influx, a defining feature of the lung response to Pa infection, implicating enzymes active in phospholipid remodeling.

In animal models, maternal obesity is associated with development of neurodevelopmental disorder like phenotypes. Here the authors show in a macaque model that in obese dams, cell-free fetal DNA methylation, inflammatory cytokines, and metabolites correlated with infant brain DNA methylation, lipids, and metabolites.

ABSTRACT Individuals with cystic fibrosis (CF) suffer from frequent and recurring microbial airway infections. The Gram-negative bacterium Pseudomonas aeruginosa is one of the most common organisms isolated from CF patient airways. P. aeruginosa establishes chronic infections that persist throughout a patient’s lifetime and is a major cause of morbidity and mortality. Throughout the course of infection, P. aeruginosa must evolve and adapt from an initial state of early, transient colonization to chronic colonization of the airways. Here, we examined isolates of P. aeruginosa from children under the age of 3 years old with CF to determine genetic adaptations the bacterium undergoes during this early stage of colonization and infection. These isolates were collected when early aggressive antimicrobial therapy was not the standard of care and therefore highlight strain evolution under limited antibiotic pressure. Examination of specific phenotypic adaptations, such as lipid A palmitoylation, antibiotic resistance, and loss of quorum sensing, did not reveal a clear genetic basis for such changes. Additionally, we demonstrate that the geography of patient origin, within the United States or among other countries, does not appear to significantly influence genetic adaptation. In summary, our results support the long-standing model that patients acquire individual isolates of P. aeruginosa that subsequently become hyperadapted to the patient-specific airway environment. This study provides a multipatient genomic analysis of isolates from young CF patients in the United States and contributes data regarding early colonization and adaptation to the growing body of research about P. aeruginosa evolution in the context of CF airway disease. IMPORTANCE Chronic lung infection with Pseudomonas aeruginosa is of major concern for patients with cystic fibrosis (CF). During infection, P. aeruginosa undergoes genomic and functional adaptation to the hyperinflammatory CF airway, resulting in worsening lung function and pulmonary decline. All studies that describe these adaptations use P. aeruginosa obtained from older children or adults during late chronic lung infection; however, children with CF can be infected with P. aeruginosa as early as 3 months of age. Therefore, it is unclear when these genomic and functional adaptations occur over the course of CF lung infection, as access to P. aeruginosa isolates in children during early infection is limited. Here, we present a unique cohort of CF patients who were identified as being infected with P. aeruginosa at an early age prior to aggressive antibiotic therapy. Furthermore, we performed genomic and functional characterization of these isolates to address whether chronic CF P. aeruginosa phenotypes are present during early infection.

The transcription factor SREBP2 is the main regulator of cholesterol homeostasis and is central to the mechanism of action of lipid-lowering drugs, such as statins, which are responsible for the largest overall reduction in cardiovascular risk and mortality in humans with atherosclerotic disease. Recently, SREBP2 has been implicated in leukocyte innate and adaptive immune responses by upregulation of cholesterol flux or direct transcriptional activation of pro-inflammatory genes. Here, we investigate the role of SREBP2 in endothelial cells (ECs), since ECs are at the interface of circulating lipids with tissues and crucial to the pathogenesis of cardiovascular disease. Loss of SREBF2 inhibits the production of pro-inflammatory chemokines but amplifies type I interferon response genes in response to inflammatory stimulus. Furthermore, SREBP2 regulates chemokine expression not through enhancement of endogenous cholesterol synthesis or lipoprotein uptake but partially through direct transcriptional activation. Chromatin immunoprecipitation sequencing of endogenous SREBP2 reveals that SREBP2 bound to the promoter regions of two nonclassical sterol responsive genes involved in immune modulation, BHLHE40 and KLF6. SREBP2 upregulation of KLF6 was responsible for the downstream amplification of chemokine expression, highlighting a novel relationship between cholesterol homeostasis and inflammatory phenotypes in ECs.

Abstract Carbon is often used as a conductive additive in catalyst layers to increase conductivity and catalytic activity. However, the effect of carbon addition to perovskites on the oxygen reduction (ORR) and oxygen evolution (OER) reactions is convoluted. In this work, composites of perovskite Ba0.5Sr0.5Co0.8Fe0.2O3‐δ (BSCF) and conductive additives, carbon and indium doped tin oxide are compared. It is found that the conductive additives have differing effects on the ORR and OER activities and cobalt redox behavior, with carbon having a much more significant effect. In order to elucidate further these differences between BSCF and BSCF/carbon, operando X‐ray absorption spectroscopy (XAS) is measured simultaneously with cyclic voltammetry into the ORR and OER regions and the continuous changes in the Co oxidation state are observed with high time resolution. We theorize that carbon is enhancing the Co redox activity and as a result, the ORR and OER activities are likewise improved.

Abstract Current seasonal and pre-pandemic influenza vaccines induce short-lived predominantly strain-specific and limited heterosubtypic responses. To better understand how vaccine adjuvants AS03 and MF59 may provide improved antibody responses to vaccination, we interrogated serum from subjects who received 2 doses of inactivated monovalent influenza A/Indonesia/05/2005 vaccine with or without AS03 or MF59 using hemagglutinin (HA) microarrays (NCT01317758 and NCT01317745). The arrays were designed to reflect both full-length and globular head HA derived from 17 influenza A subtypes (H1 to H16 and H18) and influenza B strains. We observed significantly increased strain-specific and broad homo- and heterosubtypic antibody responses with both AS03 and MF59 adjuvanted vaccination with AS03 achieving a higher titer and breadth of IgG responses relative to MF59. The adjuvanted vaccine was also associated with the elicitation of stalk-directed antibody. We established good correlation of the array antibody responses to H5 antigens with standard HA inhibition and microneutralization titers.

As COVID-19 vaccine coverage increases, screening programs that focus on the unvaccinated population test fewer individuals. This modeling study identifies vaccine coverage and effectiveness scenarios where such programs are (and are not) impactful.

Rework is a common practice used in the dairy industry as a strategy to help minimize waste from processing steps or errors that might otherwise render the product unsaleable. Dairy processors may rework their high-temperature, short-time (HTST) fluid milk products up to code date (21 d) at a typical dilution rate of ≤20% rework into ≥80% fresh raw milk. Bacterial spores present in raw milk that can survive pasteurization and grow at refrigeration temperatures are often responsible for milk spoilage. However, the potential impact of growth and thermal resistance of organisms in reworked product has not been investigated. Our objective was to characterize growth, sporulation, and thermal resistance of Paenibacillus odorifer under conditions representative of extreme storage conditions (time and temperature) of reduced fat (2%) and chocolate milk to evaluate whether product containing rework would have a reduced shelf life. Commercial UHT-pasteurized 2% milk and chocolate milk were independently inoculated with 4 strains of P. odorifer at 1 to 2 log cfu/mL and stored at 4°C and 7°C for 30 d. Changes in P. odorifer cell densities were determined by standard serial dilution with spread plating on tryptic soy agar with yeast extract and incubation at 25°C for 48 h. Spore counts were determined following thermal treatment at 80°C for 12 min. Thermal resistance of a cocktail of P. odorifer in milk was determined after treatments at 63°C for 30 min and 72°C for 15 s. Strains of P. odorifer grew rapidly at 7°C and reached a maximum cell density of ~8 log cfu/g in both 2% and chocolate milk within 12 d. All strains grew more slowly at 4°C and had not reached maximum cell density by 21 d. With extreme temperature abuse (25°C, 24 h), P. odorifer will sporulate in milk; however, thermally resistant subpopulations, including spores, did not develop in milk at 4°C until after stationary phase was achieved (>24 d). Vegetative cells of P. odorifer were verified to be sensitive to pasteurization (>7 log reduction); therefore, P. odorifer would not be expected to contribute to reduced shelf life of fluid milk products containing rework, even with extended storage before rework.

BackgroundMaternal obesity has been associated with a higher risk of pregnancy-related complications in mothers and offspring; however, effective interventions have not yet been developed. We tested two interventions, calorie restriction and pravastatin administration, during pregnancy in a rhesus macaque model with the hypothesis that these interventions would normalize metabolic dysregulation in pregnant mothers leading to an improvement in infant metabolic and cognitive/social development.MethodsA total of 19 obese mothers were assigned to either one of the two intervention groups (n = 5 for calorie restriction; n = 7 for pravastatin) or an obese control group (n = 7) with no intervention, and maternal gestational samples and postnatal infant samples were compared with lean control mothers (n = 6) using metabolomics methods.ResultsGestational calorie restriction normalized one-carbon metabolism dysregulation in obese mothers, but altered energy metabolism in her offspring. Although administration of pravastatin during pregnancy tended to normalize blood cholesterol in the mothers, it potentially impacted the gut microbiome and kidney function of their offspring. In the offspring, both calorie restriction and pravastatin administration during pregnancy tended to normalize the activity of AMPK in the brain at 6 months, and while results of the Visual Paired-Comparison test, which measures infant recognition memory, was not significantly impacted by either of the interventions, gestational pravastatin administration, but not calorie restriction, tended to normalize anxiety assessed by the Human Intruder test.ConclusionsAlthough the two interventions tested in a non-human primate model led to some improvements in metabolism and/or infant brain development, negative impacts were also found in both mothers and infants. Our study emphasizes the importance of assessing gestational interventions for maternal obesity on both maternal and offspring long-term outcomes.

Fieldwork education is a vital component of occupational therapy education. Academic fieldwork coordinators face a shortage of qualified occupational therapists who are prepared to be fieldwork educators. This pilot study aimed to evaluate the effects of an online learning module developed to prepare occupational therapists to become fieldwork educators. A pre- and post-survey were used to measure changes in perceived preparedness following completion of an online learning module. A 39-item electronic survey measured perceived preparedness of the following fieldwork educator competencies: education, supervision, evaluation, and administration. Sixteen participants completed all three components of the study: pre-survey, the learning module, and post-survey. Significant findings indicate completion of the online learning module led to participants feeling more prepared to: (a) implement a professional development plan; (b) use a variety of instructional strategies; (c) use current supervision models and theories; (d) initiate interaction to resolve conflict; (e) communicate and collaborate with academic programs to integrate the academic curriculum; (f) complete and provide the academic program with required paperwork; (g) use fieldwork evaluation tools to accurately measure student performance and provide feedback; (h) design and implement a fieldwork program in collaboration with the academic fieldwork coordinator in accordance with Accreditation Council for Occupational Therapy Education (ACOTE) Standards; (i) document an organized, systematic, fieldwork program; (j) identify the legal and health care policies that directly influence fieldwork; and (k) complete an orientation for the student. Implementing an online fieldwork educator learning module had a positive impact on occupational therapists preparing for the role of Level II fieldwork educator.

Despite numerous clinically available vaccines and therapeutics, aged patients remain at increased risk for COVID-19 morbidity. Furthermore, various patient populations, including the aged can have suboptimal responses to SARS-CoV-2 vaccine antigens. Here, we characterized vaccine-induced responses to SARS-CoV-2 synthetic DNA vaccine antigens in aged mice. Aged mice exhibited altered cellular responses, including decreased IFNγ secretion and increased TNFα and IL-4 secretion suggestive of TH2-skewed responses. Aged mice exhibited decreased total binding and neutralizing antibodies in their serum but significantly increased TH2-type antigen-specific IgG1 antibody compared to their young counterparts. Strategies to enhance vaccine-induced immune responses are important, especially in aged patient populations. We observed that co-immunization with plasmid-encoded adenosine deaminase (pADA)enhanced immune responses in young animals. Ageing is associated with decreases in ADA function and expression. Here, we report that co-immunization with pADA enhanced IFNγ secretion while decreasing TNFα and IL-4 secretion. pADA expanded the breadth and affinity SARS-CoV-2 spike-specific antibodies while supporting TH1-type humoral responses in aged mice. scRNAseq analysis of aged lymph nodes revealed that pADA co-immunization supported a TH1 gene profile and decreased FoxP3 gene expression. Upon challenge, pADA co-immunization decreased viral loads in aged mice. These data support the use of mice as a model for age-associated decreased vaccine immunogenicity and infection-mediated morbidity and mortality in the context of SARS-CoV-2 vaccines and provide support for the use of adenosine deaminase as a molecular adjuvant in immune-challenged populations.

Environmental cues, such as physical forces and heterotypic cell interactions play a critical role in cell function, yet their collective contributions to transcriptional changes are unclear. Focusing on human endothelial cells, we performed broad individual sample analysis to identify transcriptional drifts associated with environmental changes that were independent of genetic background. Global gene expression profiling by RNA sequencing and protein expression by liquid chromatography–mass spectrometry directed proteomics distinguished endothelial cells in vivo from genetically matched culture (in vitro) samples. Over 43% of the transcriptome was significantly changed by the in vitro environment. Subjecting cultured cells to long-term shear stress significantly rescued the expression of approximately 17% of genes. Inclusion of heterotypic interactions by co-culture of endothelial cells with smooth muscle cells normalized approximately 9% of the original in vivo signature. We also identified novel flow dependent genes, as well as genes that necessitate heterotypic cell interactions to mimic the in vivo transcriptome. Our findings highlight specific genes and pathways that rely on contextual information for adequate expression from those that are agnostic of such environmental cues.

Idiopathic pulmonary arterial hypertension is a rare and fatal disease with a heterogeneous treatment response. Here the authors show that unsupervised machine learning of whole blood transcriptomes from 359 patients with idiopathic pulmonary arterial hypertension identifies 3 subgroups (endophenotypes) that improve risk stratification and provide new molecular insights.

IntroductionOver the last decade, the field of systems vaccinology has emerged, in which high throughput transcriptomics and other omics assays are used to probe changes of the innate and adaptive immune system in response to vaccination. The goal of this study was to benchmark key technical and analytical parameters of RNA sequencing (RNA-seq) in the context of a multi-site, double-blind randomized vaccine clinical trial.MethodsWe collected longitudinal peripheral blood mononuclear cell (PBMC) samples from 10 subjects before and after vaccination with a live attenuated Francisella tularensis vaccine and performed RNA-Seq at two different sites using aliquots from the same sample to generate two replicate datasets (5 time points for 50 samples each). We evaluated the impact of (i) filtering lowly-expressed genes, (ii) using external RNA controls, (iii) fold change and false discovery rate (FDR) filtering, (iv) read length, and (v) sequencing depth on differential expressed genes (DEGs) concordance between replicate datasets. Using synthetic mRNA spike-ins, we developed a method for empirically establishing minimal read-count thresholds for maintaining fold change accuracy on a per-experiment basis. We defined a reference PBMC transcriptome by pooling sequence data and established the impact of sequencing depth and gene filtering on transcriptome representation. Lastly, we modeled statistical power to detect DEGs for a range of sample sizes, effect sizes, and sequencing depths.Results and DiscussionOur results showed that (i) filtering lowly-expressed genes is recommended to improve fold-change accuracy and inter-site agreement, if possible guided by mRNA spike-ins (ii) read length did not have a major impact on DEG detection, (iii) applying fold-change cutoffs for DEG detection reduced inter-set agreement and should be used with caution, if at all, (iv) reduction in sequencing depth had a minimal impact on statistical power but reduced the identifiable fraction of the PBMC transcriptome, (v) after sample size, effect size (i.e. the magnitude of fold change) was the most important driver of statistical power to detect DEG. The results from this study provide RNA sequencing benchmarks and guidelines for planning future similar vaccine studies.

Asymptomatic esophageal eosinophilia (aEE) is a rare presentation, where patients have increased eosinophils in esophageal mucosa but lack any esophagus-related symptoms. Cases of aEE have only been documented in adults, and little is known about its clinical significance and whether treatment is warranted. We report a case of an 11-year-old patient with uncontrolled severe persistent asthma who underwent flexible bronchoscopy and upper endoscopy as a part of complete aerodigestive evaluation. Elevated intraepithelial eosinophils in the esophageal mucosa were noted, suggesting an aEE-like presentation. This case documents a pediatric patient with aEE and highlights the importance of combined aerodigestive assessment with pulmonology and gastroenterology teams for the evaluation of severe asthma.

BackgroundIn the United States, the rates of primary and secondary syphilis have increased more rapidly among men who have sex with men (MSM) than among any other subpopulation. Rising syphilis rates among MSM reflect changes in both individual behaviors and the role of sexual networks (eg, persons linked directly or indirectly by sexual contact) in the spread of the infection. Decades of research examined how sexual networks influence sexually transmitted infections (STIs) among MSM; however, few longitudinal data sources focusing on syphilis have collected network characteristics. The Centers for Disease Control and Prevention, in collaboration with 3 sites, enrolled a prospective cohort of MSM in 3 US cities to longitudinally study sexual behaviors and STIs, including HIV, for up to 24 months. ObjectiveThe Network Epidemiology of Syphilis Transmission (NEST) study aimed to collect data on the factors related to syphilis transmission and acquisition among MSM. MethodsThe NEST study was a prospective cohort study that enrolled 748 MSM in Baltimore, Maryland; Chicago, Illinois; and Columbus, Ohio. NEST recruitment used a combination of convenience sampling, venue-based recruitment, and respondent-driven sampling approaches. At quarterly visits, participants completed a behavioral questionnaire and were tested for syphilis, HIV, gonorrhea, and chlamydia. The participants also provided a list of their sexual partners and described their 3 most recent partners in greater detail. ResultsThe NEST participants were enrolled in the study from July 2018 to December 2021. At baseline, the mean age of the participants was 31.5 (SD 9.1) years. More than half (396/727. 54.5%) of the participants were non-Hispanic Black, 29.8% (217/727) were non-Hispanic White, and 8.8% (64/727) were Hispanic or Latino. Multiple recruitment strategies across the 3 study locations, including respondent-driven sampling, clinic referrals, flyers, and social media advertisements, strengthened NEST participation. Upon the completion of follow-up visits in March 2022, the mean number of visits per participant was 5.1 (SD 3.2; range 1-9) in Baltimore, 2.2 (SD 1.6; range 1-8) in Chicago, and 7.2 (SD 2.9; range 1-9) in Columbus. Using a community-based participatory research approach, site-specific staff were able to draw upon collaborations with local communities to address stigma concerning STIs, particularly syphilis, among potential NEST participants. Community-led efforts also provided a forum for staff to describe the NEST study objectives and plans for research dissemination to the target audience. Strategies to bolster data collection during the COVID-19 pandemic included telehealth visits (all sites) and adaptation to self-collection of STI specimens (Baltimore only). ConclusionsData from NEST will be used to address important questions regarding individual and partnership-based sexual risk behaviors among MSM, with the goal of informing interventions to prevent syphilis in high-burden areas. International Registered Report Identifier (IRRID)RR1-10.2196/40095

Henry Devlin,1,* Stephanie Fulaz,1,* Dishon Wayne Hiebner,1 James P O’Gara,2 Eoin Casey1 1UCD School of Chemical and Bioprocess Engineering, University College Dublin, Dublin, Ireland; 2Department of Microbiology, School of Natural Sciences, National University of Ireland, Galway, Ireland*These authors contributed equally to this workCorrespondence: Eoin CaseyUCD School of Chemical and Bioprocess Engineering, University College Dublin, Belfield, Dublin, IrelandEmail eoin.casey@ucd.ieBackground: Staphylococcus aureus biofilms pose a unique challenge in healthcare due to their tolerance to a wide range of antimicrobial agents. The high cost and lengthy timeline to develop novel therapeutic agents have pushed researchers to investigate the use of nanomaterials to deliver antibiofilm agents and target biofilm infections more efficiently. Previous studies have concentrated on improving the efficacy of antibiotics by deploying nanoparticles as nanocarriers. However, the dispersal of the extracellular polymeric substance (EPS) matrix in biofilm-associated infections is also critical to the development of novel nanoparticle-based therapies.Methods: This study evaluated the efficacy of enzyme-functionalized mesoporous silica nanoparticles (MSNs) against methicillin-resistant S. aureus (MRSA) and methicillin-sensitive S. aureus (MSSA) biofilms. MSNs were functionalized with the enzyme lysostaphin, which causes cell lysis of S. aureus bacteria. This was combined with two other enzyme functionalized MSNs, serrapeptase and DNase I which will degrade protein and eDNA in the EPS matrix, to enhance eradication of the biofilm. Cell viability after treatment with enzyme-functionalized MSNs was assessed using a MTT assay and CLSM, while crystal violet staining was used to assess EPS removal.Results: The efficacy of all three enzymes against S. aureus cells and biofilms was significantly improved when they were immobilized onto MSNs. Treatment efficacy was further enhanced when the three enzymes were used in combination against both MRSA and MSSA. Regardless of biofilm maturity (24 or 48 h), near-complete dispersal and killing of MRSA biofilms were observed after treatment with the enzyme-functionalized MSNs. Disruption of mature MSSA biofilms with a polysaccharide EPS was less efficient, but cell viability was significantly reduced.Conclusion: The combination of these three enzymes and their functionalization onto nanoparticles might extend the therapeutic options for the treatment of S. aureus infections, particularly those with a biofilm component.Keywords: MRSA, lysostaphin, antimicrobial, antibiofilm, EPS matrix

Bone non-unions resulting from severe traumatic injuries pose significant clinical challenges, and the biological factors that drive progression towards and healing from these injuries are still not well understood. Recently, a dysregulated systemic immune response following musculoskeletal trauma has been identified as a contributing factor for poor outcomes and complications such as infections. In particular, myeloid-derived suppressor cells (MDSCs), immunosuppressive myeloid-lineage cells that expand in response to traumatic injury, have been highlighted as a potential therapeutic target to restore systemic immune homeostasis and ultimately improve functional bone regeneration. Previously, we have developed a novel immunomodulatory therapeutic strategy to deplete MDSCs using Janus gold nanoparticles that mimic the structure and function of antibodies. Here, in a preclinical delayed treatment composite injury model of bone and muscle trauma, we investigate the effects of these nanoparticles on circulating MDSCs, systemic immune profiles, and functional bone regeneration. Unexpectedly, treatment with the nanoparticles resulted in depletion of the high side scatter subset of MDSCs and an increase in the low side scatter subset of MDSCs, resulting in an overall increase in total MDSCs. This overall increase correlated with a decrease in bone volume (P = 0.057) at 6 weeks post-treatment and a significant decrease in mechanical strength at 12 weeks post-treatment compared to untreated rats. Furthermore, MDSCs correlated negatively with endpoint bone healing at multiple timepoints. Single cell RNA sequencing of circulating immune cells revealed differing gene expression of the SNAb target molecule S100A8/A9 in MDSC sub-populations, highlighting a potential need for more targeted approaches to MDSC immunomodulatory treatment following trauma. These results provide further insights on the role of systemic immune dysregulation for severe trauma outcomes in the case of non-unions and composite injuries and suggest the need for additional studies on targeted immunomodulatory interventions to enhance healing.

Summary: Pseudomonas aeruginosa infections can be difficult to treat and new therapeutics are needed. Bacteriophage therapy is a promising alternative to traditional antibiotics, but large numbers of isolated and characterized phages are lacking. We collected 23 diverse P. aeruginosa isolates from people with cystic fibrosis (CF) and clinical infections, and used them to screen and isolate over a dozen P. aeruginosa-targeting phages from hospital wastewater. Phages were characterized with genome sequencing, comparative genomics, and lytic activity screening against all 23 bacterial host isolates. We evolved bacterial mutants that were resistant to phage infection for four different phages, and used genome sequencing and functional analysis to study them further. We also tested phages for their ability to kill P. aeruginosa grown in biofilms in vitro and ex vivo on CF airway epithelial cells. Overall, this study demonstrates how systematic genomic and phenotypic characterization can be deployed to develop bacteriophages as precision antibiotics.

Abstract Prognosis communication in heart failure is often narrowly defined as a discussion of life expectancy, but as clinical guidelines and research suggest, these discussions should provide a broader understanding of the disease, including information about disease trajectory, the experiences of living with heart failure, potential burden on patients and families, and mortality. Furthermore, despite clinical guidelines recommending early discussions, evidence suggests that these discussions occur infrequently or late in the disease trajectory. We review the literature concerning patient, caregiver, and clinician perspectives on discussions of this type, including the frequency, timing, desire for, effects of, and barriers to their occurrence. We propose an alternate view of prognosis communication, in which the patient and family/caregiver are educated about the nature of the disease at the time of diagnosis, and a process of engagement is undertaken so that the patient's full participation in their care is marshalled, and the care team engages the patient in the informed decision making that will guide care throughout the disease trajectory. We also identify and discuss evidence gaps concerning (i) patient preferences and readiness for prognosis information along the trajectory; (ii) best practices for communicating prognosis information; and (iii) effects of prognosis communication on patient's quality of life, mental health, engagement in critical self‐care, and clinical outcomes. Research is needed to determine best practices for engaging patients in prognosis communication and for evaluating the effects of this communication on patient engagement and clinical outcomes.

Stephanie Fulaz,1,* Henry Devlin,1,* Stefania Vitale,1 Laura Quinn,1 James P O’Gara,2 Eoin Casey1 1UCD School of Chemical and Bioprocess Engineering, University College Dublin, Dublin, Ireland; 2Department of Microbiology, School of Natural Sciences, National University of Ireland, Galway, Ireland*These authors contributed equally to this workCorrespondence: Eoin CaseyUCD School of Chemical and Bioprocess Engineering, University College Dublin, Belfield, Dublin 4, Dublin, IrelandEmail eoin.casey@ucd.ieBackground: Considering the timeline required for the development of novel antimicrobial drugs, increased attention should be given to repurposing old drugs and improving antimicrobial efficacy, particularly for chronic infections associated with biofilms. Methicillin-susceptible Staphylococcus aureus (MSSA) and methicillin-resistant S. aureus (MRSA) are common causes of biofilm-associated infections but produce different biofilm matrices. MSSA biofilm cells are typically embedded in an extracellular polysaccharide matrix, whereas MRSA biofilms comprise predominantly of surface proteins and extracellular DNA (eDNA). Nanoparticles (NPs) have the potential to enhance the delivery of antimicrobial agents into biofilms. However, the mechanisms which influence the interactions between NPs and the biofilm matrix are not yet fully understood.Methods: To investigate the influence of NPs surface chemistry on vancomycin (VAN) encapsulation and NP entrapment in MRSA and MSSA biofilms, mesoporous silica nanoparticles (MSNs) with different surface functionalization (bare-B, amine-D, carboxyl-C, aromatic-A) were synthesised using an adapted Stöber method. The antibacterial efficacy of VAN-loaded MSNs was assessed against MRSA and MSSA biofilms.Results: The two negatively charged MSNs (MSN-B and MSN-C) showed a higher VAN loading in comparison to the positively charged MSNs (MSN-D and MSN-A). Cellular binding with MSN suspensions (0.25 mg mL− 1) correlated with the reduced viability of both MSSA and MRSA biofilm cells. This allowed the administration of low MSNs concentrations while maintaining a high local concentration of the antibiotic surrounding the bacterial cells.Conclusion: Our data suggest that by tailoring the surface functionalization of MSNs, enhanced bacterial cell targeting can be achieved, leading to a novel treatment strategy for biofilm infections.Keywords: Staphylococcus aureus, mesoporous silica nanoparticles, EPS matrix, antimicrobial, vancomycin, nanoparticle-biofilm interactions

Background Associations between depression, incident heart failure (HF), and mortality are well documented in predominately White samples. Yet, there are sparse data from racial minorities, including those who are women, and depression is underrecognized and undertreated in the Black population. Thus, we examined associations between baseline depressive symptoms, incident HF, and all‐cause mortality across 10 years. Methods and Results We included Jackson Heart Study (JHS) participants with no history of HF at baseline (n=2651; 63.9% women; median age, 53 years). Cox proportional hazards models tested if the risk of incident HF or mortality differed by clinically significant depressive symptoms at baseline (Center for Epidemiological Studies–Depression scores ≥16 versus

Regulation of gene expression requires the combinatorial binding of sequence-specific transcription factors (TFs) at promoters and enhancers. Prior studies showed that alterations in the spacing between TF binding sites can influence promoter and enhancer activity. However, the relative importance of TF spacing alterations resulting from naturally occurring insertions and deletions (InDels) has not been systematically analyzed. To address this question, we first characterized the genome-wide spacing relationships of 73 TFs in human K562 cells as determined by ChIP-seq (chromatin immunoprecipitation sequencing). We found a dominant pattern of a relaxed range of spacing between collaborative factors, including 45 TFs exclusively exhibiting relaxed spacing with their binding partners. Next, we exploited millions of InDels provided by genetically diverse mouse strains and human individuals to investigate the effects of altered spacing on TF binding and local histone acetylation. These analyses suggested that spacing alterations resulting from naturally occurring InDels are generally tolerated in comparison to genetic variants directly affecting TF binding sites. To experimentally validate this prediction, we introduced synthetic spacing alterations between PU.1 and C/EBPβ binding sites at six endogenous genomic loci in a macrophage cell line. Remarkably, collaborative binding of PU.1 and C/EBPβ at these locations tolerated changes in spacing ranging from 5 bp increase to >30 bp decrease. Collectively, these findings have implications for understanding mechanisms underlying enhancer selection and for the interpretation of non-coding genetic variation.

Background Damage control laparotomy (DCL) is a life-saving procedure in patients with abdominal hemorrhage. After DCL, patients are sometimes left with an open abdomen (OA) so they may undergo multiple exploratory laparotomies (EXLAP), or re-explorations. Patients with OA are at increased risk of infectious complications (ICs). The association between number of re-explorations after DCL and the number of ICs is not clear. We hypothesized that each additional re-exploration increases the risk of developing IC.Methods This 6-year retrospective cohort study included patients aged ≥16 years from the NTDB who had DCL defined as EXLAP within 2 hours of arrival (ICD-9: 54.11, 54.12, 54.19) with at least one re-exploration. The primary outcome was IC (ie, superficial surgical site infection (SSI), organ space SSI, deep SSI, sepsis, pneumonia, or catheter-related bloodstream infection), examined dichotomously (present/absent) and ordinally as the number of ICs. Multivariate Poisson regression was used to assess the association between number of re-explorations and number of ICs. Significance was assigned at p

Glycosylphosphatidylinositol-anchored high density lipoprotein–binding protein 1 (GPIHBP1), the protein that shuttles LPL to the capillary lumen, is essential for plasma triglyceride metabolism. When GPIHBP1 is absent, LPL remains stranded within the interstitial spaces and plasma triglyceride hydrolysis is impaired, resulting in severe hypertriglyceridemia. While the functions of GPIHBP1 in intravascular lipolysis are reasonably well understood, no one has yet identified DNA sequences regulating GPIHBP1 expression. In the current studies, we identified an enhancer element located ∼3.6 kb upstream from exon 1 of mouse Gpihbp1. To examine the importance of the enhancer, we used CRISPR/Cas9 genome editing to create mice lacking the enhancer (Gpihbp1 Enh/Enh). Removing the enhancer reduced Gpihbp1 expression by >90% in the liver and by ∼50% in heart and brown adipose tissue. The reduced expression of GPIHBP1 was insufficient to prevent LPL from reaching the capillary lumen, and it did not lead to hypertriglyceridemia–even when mice were fed a high-fat diet. Compound heterozygotes (Gpihbp1 Enh/− mice) displayed further reductions in Gpihbp1 expression and exhibited partial mislocalization of LPL (increased amounts of LPL within the interstitial spaces of the heart), but the plasma triglyceride levels were not perturbed. The enhancer element that we identified represents the first insight into DNA sequences controlling Gpihbp1 expression.

Background: Diet quality, exercise and sleep are dynamic modifiable factors that impact mental health. However, taking into consideration gender and age-groups as risk factors may be necessary to customize therapies. The purpose of the study was to assess the dynamic interactions between diet quality, and lifestyle factors in relation to mental distress in these sub-groups. Method: A total of 52 adult participants provided 4 week-daily records using the Food-Mood questionnaire. Data were collected for 2 years during the summer and fall seasons. Spearman's correlation as well as multivariate and multilevel regression analyses were used to identify correlations and model the relationships, respectively, between the variables of interest. Results: There was a mild negative correlation between AHEI and K-6 scores;rs= -0.08 (95% confidence interval = [-0.14, -0.02]). Men and participants aged 30 years or older had lower K-6 scores than women (0.8 vs 1.8, p < 0.001), and younger adults (0.6 vs 1.9, p < 0.001), respectively. Lower K-6 scores associated with 20 min or more exercise compared to days without exercise or exercised less than 20 min (1.2 versus 1.9, p < 0.001). Seasonal changes were associated with alterations in diet quality and mental wellbeing (P < 0.001). Limitations: A convenience sample was used. Conclusion: Our results suggest that adjusting one modifiable factor may lead to improvement in others. In addition, these factors are age and gender dependent. Therefore, customization of dietary and lifestyle factors based on gender and age-groups is recommended to optimize mental wellbeing.

Summary: While the immediate and transitory response of breast cancer cells to pathological stiffness in their native microenvironment has been well explored, it remains unclear how stiffness-induced phenotypes are maintained over time after cancer cell dissemination in vivo. Here, we show that fibrotic-like matrix stiffness promotes distinct metastatic phenotypes in cancer cells, which are preserved after transition to softer microenvironments, such as bone marrow. Using differential gene expression analysis of stiffness-responsive breast cancer cells, we establish a multigenic score of mechanical conditioning (MeCo) and find that it is associated with bone metastasis in patients with breast cancer. The maintenance of mechanical conditioning is regulated by RUNX2, an osteogenic transcription factor, established driver of bone metastasis, and mitotic bookmarker that preserves chromatin accessibility at target gene loci. Using genetic and functional approaches, we demonstrate that mechanical conditioning maintenance can be simulated, repressed, or extended, with corresponding changes in bone metastatic potential.

The John Day region of central Oregon, United States contains ∼50 million years of near-continuous, fossiliferous sedimentation, representing one of the world’s richest archives of Cenozoic terrestrial ecosystems and climate. Stable isotope proxy data from this region are commonly used to infer the elevation history of the Cascades, which intercept westerly moisture in transit to the John Day region. However, the Blue Mountains, which accreted in the Mesozoic, create a region of local high topography that can confound signals of Cascades uplift. John Day deposits, including the John Day Formation, are divided into an eastern facies located within the Blue Mountains and a western facies in the adjacent plains. As a result, the Blue Mountains may have supported gradients in climate and ecology between the eastern and western facies, and constraining these gradients is necessary for reconstructing past topography and ecosystem change. In order to define the Cenozoic extent and magnitude of Blue Mountains topography we use oxygen isotopes in authigenic clay minerals to construct a spatially resolved map of local elevation. We find that the oxygen isotope composition of clay minerals within the Blue Mountains is ∼3‰ lower than in the adjacent high plains, and this offset is mostly constant throughout our record (spanning ∼50 – 5 Ma). We attribute this offset to Blue Mountains topography, either directly from upslope rainout or indirectly through the effect of elevation on local variations in precipitation seasonality. Our results highlight the importance of local topographic features in regional paleotopography reconstructions and provide important biogeographical context for the rich paleo-floral and -faunal records preserved in John Day sediments.

The purpose of this study was to establish the test/re-test reliability of two interprofessional education (IPE) instruments, the Readiness for Interprofessional Learning Scale (RIPLS) and the Interdisciplinary Education Perception Scale (IEPS) among occupational therapy (OT) graduate students. The intent was to compare results based on previous IPE experience and year in the program. The RIPLS and IEPS were distributed to 111 OT students at one university. Both instruments were distributed a second time 10-14 days later. Cronbach’s alpha, weighted Kappas, intraclass correlation coefficients (ICC), standard error of measurement, and minimal detectable change were calculated for each instrument. Assessments occurred for all subjects, between students with and without previous IPE experience, and first and second-year students in the program. Overall and between group composite score reliability for the RIPLS and IEPS were fair to excellent (ICC≥0.72). RIPLS subscale ICC’s were variable per previous IPE experience and year in program, ranging from fair-excellent (ICC=0.45-0.93). IEPS subscale ICC’s were excellent for second-year students (ICC≥0.79), and fair-excellent for students with or without previous experience and first-year students (ICC=0.50-0.84). There were no differences for the RIPLS within or between sessions or groups. First-year students had significantly higher scores compared to second-year students within sessions for the IEPS composite score, Competency and Autonomy subscale, and Perception of Actual Cooperation subscale (p≤0.035). Both instruments have acceptable test-re-test reliability; however, previous IPE experience and year in program should be accounted for when distributing the instruments and interpreting the results.

ObjectiveNumerous adverse prenatal exposures have been individually associated with risk for psychiatric illness in the offspring. However, such exposures frequently co-occur, raising questions about their cumulative impact. We evaluated effects of cumulative adverse prenatal exposure burden on psychopathology risk in school-aged children.MethodsUsing baseline surveys from the U.S.-based Adolescent Brain and Cognitive Development (ABCD) Study (7,898 non-adopted, unrelated children from 21 sites, age 9-10, and their primary caregivers), we examined 8 retrospectively-reported adverse prenatal exposures in relation to caregiver-reported total and subscale Child Behavior Checklist (CBCL) scores. We also assessed cumulative effects of these factors on CBCL total as a continuous measure, as well as on odds of clinically significant psychopathology (CBCL total ≥60), in both the initial set and a separate ABCD sample comprising an additional 696 sibling pairs. Analyses were conducted before and after adjustment for 14 demographic and environmental covariates.ResultsIn minimally and fully adjusted models, 6 exposures (unplanned pregnancy; maternal alcohol, marijuana, and tobacco use early in pregnancy; pregnancy complications; and birth complications) independently associated with significant but small increases in CBCL total score. Among these 6, none increased the odds of crossing the threshold for clinically significant symptoms by itself. However, odds of exceeding this threshold became significant with 2 exposures (OR = 1.86, 95% CI 1.47-2.36), and increased linearly with each level of exposure (OR = 1.39, 95% CI 1.31-1.47), up to 3.53-fold for ≥4 exposures versus none. Similar effects were observed in confirmatory analysis among siblings. Within sibling pairs, greater discordance for exposure load associated with greater CBCL total differences, suggesting that results were not confounded by unmeasured family-level effects.ConclusionChildren exposed to multiple common, adverse prenatal events showed dose-dependent increases in broad, clinically significant psychopathology at age 9-10. Fully prospective studies are needed to confirm and elaborate upon this pattern.

Using RNAseq, we identified a 61 gene-based circulating transcriptomic profile most correlated with four indices of pulmonary arterial hypertension severity. In an independent dataset, 13/61 (21%) genes were differentially expressed in lung tissues of pulmonary arterial hypertension cases versus controls, highlighting potentially novel candidate genes involved in pulmonary arterial hypertension development.

ABSTRACT Toxoplasma gondii’s tropism for and persistence in the central nervous system (CNS) underlies the symptomatic disease that T. gondii causes in humans. Our recent work has shown that neurons are the primary CNS cell with which Toxoplasma interacts and which it infects in vivo. This predilection for neurons suggests that T. gondii’s persistence in the CNS depends specifically upon parasite manipulation of the host neurons. Yet, most work on T. gondii-host cell interactions has been done in vitro and in nonneuronal cells. We address this gap by utilizing our T. gondii-Cre system that allows permanent marking and tracking of neurons injected with parasite effector proteins in vivo. Using laser capture microdissection (LCM) and RNA sequencing using RNA-seq, we isolated and transcriptionally profiled T. gondii-injected neurons (TINs), Bystander neurons (nearby non-T. gondii-injected neurons), and neurons from uninfected mice (controls). These profiles show that TIN transcriptomes significantly differ from the transcriptomes of Bystander and control neurons and that much of this difference is driven by increased levels of transcripts from immune cells, especially CD8+ T cells and monocytes. These data suggest that when we used LCM to isolate neurons from infected mice, we also picked up fragments of CD8+ T cells and monocytes clustering in extreme proximity around TINs and, to a lesser extent, Bystander neurons. In addition, we found that T. gondii transcripts were primarily found in the TIN transcriptome, not in the Bystander transcriptome. Collectively, these data suggest that, contrary to common perception, neurons that directly interact with or harbor parasites can be recognized by CD8+ T cells. IMPORTANCE Like other persistent intracellular pathogens, Toxoplasma gondii, a protozoan parasite, has evolved to evade the immune system and establish a chronic infection in specific cells and organs, including neurons in the CNS. Understanding T. gondii’s persistence in neurons holds the potential to identify novel, curative drug targets. The work presented here offers new insights into the neuron-T. gondii interaction in vivo. By transcriptionally profiling neurons manipulated by T. gondii, we unexpectedly revealed that immune cells, and specifically CD8+ T cells, appear to cluster around these neurons, suggesting that CD8+ T cells specifically recognize parasite-manipulated neurons. Such a possibility supports evidence from other labs that questions the long-standing dogma that neurons are often persistently infected because they are not directly recognized by immune cells such as CD8+ T cells. Collectively, these data suggest we reconsider the broader role of neurons in the context of infection and neuroinflammation.