Optimizing the production of dsRNA biocontrols in microbial systems using multiple transcriptional terminators.

Crop pests and pathogens annually cause over $220 billion in global crop damage, with insects consuming 5%-20% of major grain crops. Current crop pest and disease control strategies rely on insecticidal and fungicidal sprays, plant genetic resistance, transgenes, and agricultural practices. Double-stranded RNA (dsRNA) is emerging as a novel sustainable method of plant protection as an alternative to traditional chemical pesticides. Successful commercialization of dsRNA-based biocontrols requires the economical production of large quantities of dsRNA combined with suitable delivery methods to ensure RNAi efficacy against the target pest. In this study, we have optimized the design of plasmid DNA constructs to produce dsRNA biocontrols in Escherichia coli, by employing a wide range of alternative synthetic transcriptional terminators before measurement of dsRNA yield. We demonstrate that a 7.8-fold increase of dsRNA was achieved using triple synthetic transcriptional terminators within a dual T7 dsRNA production system compared to the absence of transcriptional terminators. Moreover, our data demonstrate that batch fermentation production dsRNA using multiple transcriptional terminators is scalable and generates significantly higher yields of dsRNA generated in the absence of transcriptional terminators at both small-scale batch culture and large-scale fermentation. In addition, we show that application of these dsRNA biocontrols expressed in E. coli cells results in increased insect mortality. Finally, novel mass spectrometry analysis was performed to determine the precise sites of transcriptional termination at the different transcriptional terminators providing important further mechanistic insight.

Analysis of Silver Alert Reporting System Activations for Missing Adults With Dementia in Texas, 2017 to 2022.

This cross-sectional study analyzes data from Silver Alert activations in Texas from 2017 to 2022 to identify temporal, geographic, and wandering characteristics of missing adults with dementia.

Homo- and Heteronuclear Group 12 Metallothionein Type B Cluster Analogs: Synthesis, Structure, 1H NMR and ESI-MS.

Metallothioneins (MTs) are a ubiquitous class of small cysteine-rich metal-binding proteins involved in metal homeostasis and detoxification with highly versatile metal binding properties. Despite the long-standing association of MT with M3S3 and M4S5 metal clusters, synthetic complexes with these core architectures are exceptionally rare. Here, we demonstrate an approach to synthesizing and characterizing aggregates of group 12 metal ions with monocyclic M3S3 cores in acetonitrile solution without the protection of a protein. Multidentate monothiol ligand N,N-bis(2-pyridylmethyl)-2-aminoethanethiol (L1H) provided [Cd3(L1)3](ClO4)3 (1), the first structurally characterized nonproteinaceous aggregate with a metallothionein-like monocyclic Cd3S3 core. In addition, [Zn3(L1)3](ClO4)3·4CH3CN (2·4CH3CN) was characterized by X-ray crystallography. The complex cations of 1 and 2 had comparable structures despite being nonisomorphic. Variable temperature and concentration 1H NMR were used to investigate aggregation equilibria of 1, 2, and a precipitate with composition "Hg(L1)(ClO4)" (3). Cryogenic 1H NMR studies of 3 revealed a J(199Hg1H) coupling constant pattern consistent with an aggregate possessing a cyclic core. ESI-MS was used for gas-phase characterization of 1-3, as well as mixed-metal [M2M'(L1)3(ClO4)2]+ ions prepared in situ by pairwise acetonitrile solution combinations of the group 12 complexes of L1. Access to synthetic variants of metallothionein-like group 12 aggregates provides an additional approach to understanding their behavior.

Analysis of Emergency Department Patient-Visit Volumes in an Academic Health System During a COVID-19 Pandemic Statewide 'Safer at Home' Order.

BACKGROUND: We describe patient-visit volumes, patient acuity, and demographics in our 4 academic health system emergency departments (ED) before, during, and after implementation of a COVID-19 pandemic safer-at-home order. METHODS: Data were collected from the electronic health record, including patient-visit volumes, chief complaint, Emergency Severity Index (ESI), and patient demographics. Descriptive statistics were performed. RESULTS: There was a 37% decrease in combined ED patient-visit volume during the safer-at-home order period (42% at the academic medical center). ED patient-visit volumes increased after the safer-at-home order concluded. During the safer-at-home order period, there was an increase in the proportion of ESI-2 visits and admission rates from EDs across the system. CONCLUSIONS: Significant differences in ED patient-visit volumes and patient acuity were associated with a safer-at-home order in our academic health system. These differences are similar to experiences of other hospital systems across the country.

Benchtop Electrochemical Growth and Controlled Alloying of Polycrystalline In x Ga1-x As Thin Films.

Compared to Si, GaAs offers unique material advantages such as high carrier mobility and energy conversion efficiency, making GaAs a leading competitor to replace Si on several technological fronts related to optoelectronics and solar energy conversion. Alloying the GaAs lattice with elemental In allows the direct bandgap of the resulting ternary alloy to be tuned across the near-infrared (NIR) region of the electromagnetic spectrum from ∼0.9 to 3.5 µm. However, methods of fabricating high-quality crystalline GaAs are currently limited by their high cost and low throughput relative to Si growth methods, suggesting the need for alternative low-cost routes to GaAs growth and alloying. This research documents the first instance in the literature of the electrodeposition and controlled alloying of polycrystalline In x Ga1-x As films at ambient pressure and near-room temperature using the electrochemical liquid-liquid-solid (ec-LLS) process. X-ray diffraction and Raman spectroscopy support the polycrystalline growth of (111)-oriented In x Ga1-x As films. Consistent redshifts of the GaAs-like TO peaks were observed in the Raman data as the In composition of the liquid metal electrode was increased. Optical bandgaps, determined via diffuse reflectance measurements, displayed a consistent decrease with the increase in the In composition of In x Ga1-x As films. While Raman, diffuse reflectance, and energy-dispersive X-ray spectroscopy data support controlled alloying efforts, all techniques suggest an overall decrease of the In/Ga ratios present in deposited films relative to those of the liquid metal electrodes. These results lend support for the continued development of ec-LLS as a viable method of achieving crystalline growth and alloying of binary and ternary semiconductor material systems using a benchtop setup under ambient pressure and near-room temperature.

Prioritization of autoimmune disease-associated genetic variants that perturb regulatory element activity in T cells.

Genome-wide association studies (GWASs) have uncovered hundreds of autoimmune disease-associated loci; however, the causal genetic variants within each locus are mostly unknown. Here, we perform high-throughput allele-specific reporter assays to prioritize disease-associated variants for five autoimmune diseases. By examining variants that both promote allele-specific reporter expression and are located in accessible chromatin, we identify 60 putatively causal variants that enrich for statistically fine-mapped variants by up to 57.8-fold. We introduced the risk allele of a prioritized variant (rs72928038) into a human T cell line and deleted the orthologous sequence in mice, both resulting in reduced BACH2 expression. Naive CD8 T cells from mice containing the deletion had reduced expression of genes that suppress activation and maintain stemness and, upon acute viral infection, displayed greater propensity to become effector T cells. Our results represent an example of an effective approach for prioritizing variants and studying their physiologically relevant effects.

Systematic identification of genomic elements that regulate FCGR2A expression and harbor variants linked with autoimmune disease.

BACKGROUND: FCGR2A binds antibody-antigen complexes to regulate the abundance of circulating and deposited complexes along with downstream immune and autoimmune responses. Although the abundance of FCRG2A may be critical in immune-mediated diseases, little is known about whether its surface expression is regulated through cis genomic elements and non-coding variants. In the current study, we aimed to characterize the regulation of FCGR2A expression, the impact of genetic variation and its association with autoimmune disease. METHODS: We applied CRISPR-based interference and editing to scrutinize 1.7 Mb of open chromatin surrounding the FCGR2A gene to identify regulatory elements. Relevant transcription factors (TFs) binding to these regions were defined through public databases. Genetic variants affecting regulation were identified using luciferase reporter assays and were verified in a cohort of 1996 genotyped healthy individuals using flow cytometry. RESULTS: We identified a complex proximal region and five distal enhancers regulating FCGR2A. The proximal region split into subregions upstream and downstream of the transcription start site, was enriched in binding of inflammation-regulated TFs, and harbored a variant associated with FCGR2A expression in primary myeloid cells. One distal enhancer region was occupied by CCCTC-binding factor (CTCF) whose binding site was disrupted by a rare genetic variant, altering gene expression. CONCLUSIONS: The FCGR2A gene is regulated by multiple proximal and distal genomic regions, with links to autoimmune disease. These findings may open up novel therapeutic avenues where fine-tuning of FCGR2A levels may constitute a part of treatment strategies for immune-mediated diseases.

Genome-wide enhancer maps link risk variants to disease genes.

Genome-wide association studies (GWAS) have identified thousands of noncoding loci that are associated with human diseases and complex traits, each of which could reveal insights into the mechanisms of disease1. Many of the underlying causal variants may affect enhancers2,3, but we lack accurate maps of enhancers and their target genes to interpret such variants. We recently developed the activity-by-contact (ABC) model to predict which enhancers regulate which genes and validated the model using CRISPR perturbations in several cell types4. Here we apply this ABC model to create enhancer-gene maps in 131 human cell types and tissues, and use these maps to interpret the functions of GWAS variants. Across 72 diseases and complex traits, ABC links 5,036 GWAS signals to 2,249 unique genes, including a class of 577 genes that appear to influence multiple phenotypes through variants in enhancers that act in different cell types. In inflammatory bowel disease (IBD), causal variants are enriched in predicted enhancers by more than 20-fold in particular cell types such as dendritic cells, and ABC achieves higher precision than other regulatory methods at connecting noncoding variants to target genes. These variant-to-function maps reveal an enhancer that contains an IBD risk variant and that regulates the expression of PPIF to alter the membrane potential of mitochondria in macrophages. Our study reveals principles of genome regulation, identifies genes that affect IBD and provides a resource and generalizable strategy to connect risk variants of common diseases to their molecular and cellular functions.

Global discovery of lupus genetic risk variant allelic enhancer activity.

Genome-wide association studies of Systemic Lupus Erythematosus (SLE) nominate 3073 genetic variants at 91 risk loci. To systematically screen these variants for allelic transcriptional enhancer activity, we construct a massively parallel reporter assay (MPRA) library comprising 12,396 DNA oligonucleotides containing the genomic context around every allele of each SLE variant. Transfection into the Epstein-Barr virus-transformed B cell line GM12878 reveals 482 variants with enhancer activity, with 51 variants showing genotype-dependent (allelic) enhancer activity at 27 risk loci. Comparison of MPRA results in GM12878 and Jurkat T cell lines highlights shared and unique allelic transcriptional regulatory mechanisms at SLE risk loci. In-depth analysis of allelic transcription factor (TF) binding at and around allelic variants identifies one class of TFs whose DNA-binding motif tends to be directly altered by the risk variant and a second class of TFs that bind allelically without direct alteration of their motif by the variant. Collectively, our approach provides a blueprint for the discovery of allelic gene regulation at risk loci for any disease and offers insight into the transcriptional regulatory mechanisms underlying SLE.

MAUDE: inferring expression changes in sorting-based CRISPR screens.

Improved methods are needed to model CRISPR screen data for interrogation of genetic elements that alter reporter gene expression readout. We create MAUDE (Mean Alterations Using Discrete Expression) for quantifying the impact of guide RNAs on a target gene's expression in a pooled, sorting-based expression screen. MAUDE quantifies guide-level effects by modeling the distribution of cells across sorting expression bins. It then combines guides to estimate the statistical significance and effect size of targeted genetic elements. We demonstrate that MAUDE outperforms previous approaches and provide experimental design guidelines to best leverage MAUDE, which is available on https://github.com/Carldeboer/MAUDE.

Prioritizing disease and trait causal variants at the TNFAIP3 locus using functional and genomic features.

Genome-wide association studies have associated thousands of genetic variants with complex traits and diseases, but pinpointing the causal variant(s) among those in tight linkage disequilibrium with each associated variant remains a major challenge. Here, we use seven experimental assays to characterize all common variants at the multiple disease-associated TNFAIP3 locus in five disease-relevant immune cell lines, based on a set of features related to regulatory potential. Trait/disease-associated variants are enriched among SNPs prioritized based on either: (1) residing within CRISPRi-sensitive regulatory regions, or (2) localizing in a chromatin accessible region while displaying allele-specific reporter activity. Of the 15 trait/disease-associated haplotypes at TNFAIP3, 9 have at least one variant meeting one or both of these criteria, 5 of which are further supported by genetic fine-mapping. Our work provides a comprehensive strategy to characterize genetic variation at important disease-associated loci, and aids in the effort to identify trait causal genetic variants.

Comparison of the Bleeding Cricothyrotomy Model to SimMan for Training Students and Residents Emergency Cricothyrotomy.

INTRODUCTION: A cricothyroidotomy is an emergency procedure that few emergency medicine residents see or perform during their training. Therefore, there is a need for low cost, high fidelity models for training. In this study, we explore a new training model for cricothyroidotomies (the bleeding CRIC [cost-effective realistic interactive cricothyroidotomy]) to determine if this new task-trainer is non-inferior compared to the current standard of training. METHODS: Authors conducted a randomized control non-inferiority study. There were seventeen residents and medical students enrolled by convenience sample to partake in the study. The participants were randomized by block randomization to be taught how to perform a cricothyroidotomy on either the new task trainer or the current standard task trainer and then were asked to perform the procedure on a pig trachea model. Primary outcome measures were scores on a previously validated objective assessment tool and secondary outcomes were comfort levels and realism scores based on pre and post survey results which were analyzed with ANOVA. RESULTS: There was found to be no statistically significant difference between the groups in assessment scores, time to completion, or comfort levels pre- and post-intervention. There was a statistically significant difference in that the participants gave higher realism scores in post-test analysis to the Bleeding CRIC compared to the SimMan. Both groups demonstrated that they had significantly improved comfort levels from baseline post-intervention. CONCLUSION: Overall, the new task trainer was rated by learners to feel more realistic than the current standard. This study demonstrates non-inferiority of the new task trainer and further studies with larger sample sizes should be conducted to determine its true efficacy.

Defining T Cell States Associated with Response to Checkpoint Immunotherapy in Melanoma.

Treatment of cancer has been revolutionized by immune checkpoint blockade therapies. Despite the high rate of response in advanced melanoma, the majority of patients succumb to disease. To identify factors associated with success or failure of checkpoint therapy, we profiled transcriptomes of 16,291 individual immune cells from 48 tumor samples of melanoma patients treated with checkpoint inhibitors. Two distinct states of CD8+ T cells were defined by clustering and associated with patient tumor regression or progression. A single transcription factor, TCF7, was visualized within CD8+ T cells in fixed tumor samples and predicted positive clinical outcome in an independent cohort of checkpoint-treated patients. We delineated the epigenetic landscape and clonality of these T cell states and demonstrated enhanced antitumor immunity by targeting novel combinations of factors in exhausted cells. Our study of immune cell transcriptomes from tumors demonstrates a strategy for identifying predictors, mechanisms, and targets for enhancing checkpoint immunotherapy.

Positional specificity of different transcription factor classes within enhancers.

Gene expression is controlled by sequence-specific transcription factors (TFs), which bind to regulatory sequences in DNA. TF binding occurs in nucleosome-depleted regions of DNA (NDRs), which generally encompass regions with lengths similar to those protected by nucleosomes. However, less is known about where within these regions specific TFs tend to be found. Here, we characterize the positional bias of inferred binding sites for 103 TFs within ∼500,000 NDRs across 47 cell types. We find that distinct classes of TFs display different binding preferences: Some tend to have binding sites toward the edges, some toward the center, and some at other positions within the NDR. These patterns are highly consistent across cell types, suggesting that they may reflect TF-specific intrinsic structural or functional characteristics. In particular, TF classes with binding sites at NDR edges are enriched for those known to interact with histones and chromatin remodelers, whereas TFs with central enrichment interact with other TFs and cofactors such as p300. Our results suggest distinct regiospecific binding patterns and functions of TF classes within enhancers.

Choosing emergency medicine: Influences on medical students' choice of emergency medicine.

BACKGROUND: Relatively little is understood about which factors influence students' choice of specialty and when learners ultimately make this decision. OBJECTIVE: The objective is to understand how experiences of medical students relate to the timing of selection of Emergency Medicine (EM) as a specialty. Of specific interest were factors such as how earlier and more positive specialty exposure may impact the decision-making process of medical students. METHODS: A cross-sectional survey study of EM bound 4th year US medical students (MD and DO) was performed exploring when and why students choose EM as their specialty. An electronic survey was distributed in March 2015 to all medical students who applied to an EM residency at 4 programs representing different geographical regions. Descriptive analyses and multinomial logistic regressions were performed. RESULTS: 793/1372 (58%) responded. Over half had EM experience prior to medical school. When students selected EM varied: 13.9% prior to, 50.4% during, and 35.7% after their M3 year. Early exposure, presence of an EM residency program, previous employment in the ED, experience as a pre-hospital provider, and completion of an M3 EM clerkship were associated with earlier selection. Delayed exposure to EM was associated with later selection of EM. CONCLUSIONS: Early exposure and prior life experiences were associated with choosing EM earlier in medical school. The third year was identified as the most common time for definitively choosing the specialty.

A Comparison of Collaborative Care Outcomes in Two Health Care Systems: VA Clinics and Federally Qualified Health Centers.

OBJECTIVE: Collaborative care for depression results in symptom reduction when compared with usual care. No studies have systematically compared collaborative care outcomes between veterans treated at Veterans Affairs (VA) clinics and civilians treated at publicly funded federally qualified health centers (FQHCs) after controlling for demographic and clinical characteristics. METHODS: Data from two randomized controlled trials that used a similar collaborative care intervention for depression were combined to conduct post hoc analyses (N=759). The Telemedicine-Enhanced Antidepressant Management intervention was delivered in VA community-based outpatient clinics (CBOCs), and the Outreach Using Telemedicine for Rural Enhanced Access in Community Health intervention was delivered in FQHCs. Multivariate logistic regression was used to determine whether veteran status moderated the effect of the intervention on treatment response (>50% reduction in symptoms). RESULTS: There was a significant main effect for intervention (odds ratio [OR]=5.23, p<.001) and a moderating effect for veteran status, with lower response rates among veterans compared with civilians (OR=.21, p=.01). The addition of variables representing medication dosage and number of mental health and general health appointments did not influence the moderating effect. A sensitivity analysis stratified by gender found a significant moderating effect of veteran status for men but not women. CONCLUSIONS: Veteran status was a significant moderator of collaborative care effectiveness for depression, indicating that veterans receiving collaborative care at a CBOC are at risk of nonresponse. Unmeasured patient- or system-level characteristics may contribute to poorer response among veterans.

Resistance to checkpoint blockade therapy through inactivation of antigen presentation.

Treatment with immune checkpoint blockade (CPB) therapies often leads to prolonged responses in patients with metastatic melanoma, but the common mechanisms of primary and acquired resistance to these agents remain incompletely characterized and have yet to be validated in large cohorts. By analyzing longitudinal tumor biopsies from 17 metastatic melanoma patients treated with CPB therapies, we observed point mutations, deletions or loss of heterozygosity (LOH) in beta-2-microglobulin (B2M), an essential component of MHC class I antigen presentation, in 29.4% of patients with progressing disease. In two independent cohorts of melanoma patients treated with anti-CTLA4 and anti-PD1, respectively, we find that B2M LOH is enriched threefold in non-responders (~30%) compared to responders (~10%) and associated with poorer overall survival. Loss of both copies of B2M is found only in non-responders. B2M loss is likely a common mechanism of resistance to therapies targeting CTLA4 or PD1.

Alcohol Use Disorder Moderates the Effect of Age on Heart Rate Variability in Veterans With Posttraumatic Stress Disorder.

Research on heart rate variability (HRV) in posttraumatic stress disorder (PTSD) and comorbid alcohol use disorder (AUD) is limited despite its use as a biomarker of both disorders. This study examined whether AUD comorbidity contributes an additive effect on HRV for veterans with PTSD. HRV was assessed in 70 male Operation Enduring Freedom/Operation Iraqi Freedom veterans with PTSD, including 32 with co-occurring AUD. Mean HRV values for both groups were below the mean for healthy adults, but additive effects of PTSD and AUD on HRV were not observed. Consistent with prior studies, hierarchical regressions showed that HRV decreased with age in the PTSD-only group. However, HRV increased slightly with age among veterans with both PTSD and AUD. This interaction remained significant after controlling for common HRV covariates. These findings support HRV as a biomarker of PTSD and extend research by demonstrating the complex relationship between PTSD and HRV in the context of co-occurring AUD.