Lrp1 is a host entry factor for Rift Valley fever virus.

Rift Valley fever virus (RVFV) is a zoonotic pathogen with pandemic potential. RVFV entry is mediated by the viral glycoprotein (Gn), but host entry factors remain poorly defined. Our genome-wide CRISPR screen identified low-density lipoprotein receptor-related protein 1 (mouse Lrp1/human LRP1), heat shock protein (Grp94), and receptor-associated protein (RAP) as critical host factors for RVFV infection. RVFV Gn directly binds to specific Lrp1 clusters and is glycosylation independent. Exogenous addition of murine RAP domain 3 (mRAPD3) and anti-Lrp1 antibodies neutralizes RVFV infection in taxonomically diverse cell lines. Mice treated with mRAPD3 and infected with pathogenic RVFV are protected from disease and death. A mutant mRAPD3 that binds Lrp1 weakly failed to protect from RVFV infection. Together, these data support Lrp1 as a host entry factor for RVFV infection and define a new target to limit RVFV infections.

Discovering selective antiferroptotic inhibitors of the 15LOX/PEBP1 complex noninterfering with biosynthesis of lipid mediators.

Programmed ferroptotic death eliminates cells in all major organs and tissues with imbalanced redox metabolism due to overwhelming iron-catalyzed lipid peroxidation under insufficient control by thiols (Glutathione (GSH)). Ferroptosis has been associated with the pathogenesis of major chronic degenerative diseases and acute injuries of the brain, cardiovascular system, liver, kidneys, and other organs, and its manipulation offers a promising new strategy for anticancer therapy. This explains the high interest in designing new small-molecule-specific inhibitors against ferroptosis. Given the role of 15-lipoxygenase (15LOX) association with phosphatidylethanolamine (PE)-binding protein 1 (PEBP1) in initiating ferroptosis-specific peroxidation of polyunsaturated PE, we propose a strategy of discovering antiferroptotic agents as inhibitors of the 15LOX/PEBP1 catalytic complex rather than 15LOX alone. Here we designed, synthesized, and tested a customized library of 26 compounds using biochemical, molecular, and cell biology models along with redox lipidomic and computational analyses. We selected two lead compounds, FerroLOXIN-1 and 2, which effectively suppressed ferroptosis in vitro and in vivo without affecting the biosynthesis of pro-/anti-inflammatory lipid mediators in vivo. The effectiveness of these lead compounds is not due to radical scavenging or iron-chelation but results from their specific mechanisms of interaction with the 15LOX-2/PEBP1 complex, which either alters the binding pose of the substrate [eicosatetraenoyl-PE (ETE-PE)] in a nonproductive way or blocks the predominant oxygen channel thus preventing the catalysis of ETE-PE peroxidation. Our successful strategy may be adapted to the design of additional chemical libraries to reveal new ferroptosis-targeting therapeutic modalities.

Chiropterans Are a Hotspot for Horizontal Transfer of DNA Transposons in Mammalia.

Horizontal transfer of transposable elements (TEs) is an important mechanism contributing to genetic diversity and innovation. Bats (order Chiroptera) have repeatedly been shown to experience horizontal transfer of TEs at what appears to be a high rate compared with other mammals. We investigated the occurrence of horizontally transferred (HT) DNA transposons involving bats. We found over 200 putative HT elements within bats; 16 transposons were shared across distantly related mammalian clades, and 2 other elements were shared with a fish and two lizard species. Our results indicate that bats are a hotspot for horizontal transfer of DNA transposons. These events broadly coincide with the diversification of several bat clades, supporting the hypothesis that DNA transposon invasions have contributed to genetic diversification of bats.

Disparities in Access to Outpatient Surgery Related to Removal of Procedures from Medicare's Inpatient Only List.

OBJECTIVE: We assessed the shift from inpatient to outpatient surgical care related to changes to the Inpatient Only List in 2020 and 2021 compared to 2019. SUMMARY BACKGROUND DATA: The extent to which procedures shift from the inpatient to outpatient setting following removal from Medicare's Inpatient Only List is unknown. Many health systems also encouraged a shift from inpatient to outpatient surgery during the COVID-19 pandemic. Assessing the relative change in outpatient surgical utilization for procedures removed from the Inpatient Only List during COVID-19 would provide empirical data on whether reimbursement policy changes or inpatient capacity needs during the pandemic were more likely to shift care from the inpatient to outpatient setting. METHODS: We used administrative data from the PINC AI Healthcare Database across 723 hospitals to determine the within-facility relative change in outpatient vs inpatient procedural volume in 2020 and 2021 compared to 2019 using a multivariable conditional fixed-effects Poisson regression model. We also assessed whether outpatient surgical utilization varied by race and ethnicity. Using a multivariable linear probability model, we assessed the absolute change in risk-adjusted 30-day complication, readmission, and mortality rates for inpatient and outpatient surgical procedures. RESULTS: In 2020 and 2021 compared to 2019 respectively, there was a 5.3% (95% CI, 1.4% to 9.5%) and 41.3% (95% CI 33.1% to 50.0%) relative increase in outpatient elective procedural volume. Outpatient procedural volume increased most significantly for hip replacement which was removed from the Inpatient Only List in 2020 (increase in outpatient surgical utilization of 589.3% (95% CI, 524.9% to 660.3%)). The shift to outpatient hip replacement procedures was concentrated among White patients; in 2021, hip replacement procedural volume increased by 271.1% (95% CI, 241.2% and 303.7%) for White patients and 29.5% (95% CI, 24.4% and 34.9%) for Black patients compared to 2019 levels. There were no consistent or large changes in 30-day complication, readmission, or mortality risk in 2020 and 2021 compared to 2019. CONCLUSION: There was a modest increase in elective outpatient surgeries and a pronounced increase in outpatient orthopedic surgeries which were removed from the Inpatient Only List during the COVID-19 pandemic. Utilization of outpatient surgical procedures was concentrated among White patients.

Evaluating Chemical Footprinting-Induced Perturbation of Protein Higher Order Structure.

Specific amino acid footprinting mass spectrometry (MS) is an increasingly utilized method for elucidating protein higher order structure (HOS). It does this by adding to certain amino acid residues a mass tag, whose reaction extent depends on solvent accessibility and microenvironment of the protein. Unlike reactive free radicals and carbenes, these specific footprinters react slower than protein unfolding. Thus, their footprinting, under certain conditions, provokes structural changes to the protein, leading to labeling on non-native structures. It is critical to establish conditions (i.e., reagent concentrations, time of reaction) to ensure that the structure of the protein following footprinting remains native. Here, we compare the efficacy of five methods in assessing protein HOS following footprinting at the intact protein level and then further localize the perturbation at the peptide level. Three are MS-based methods that provide dose-response plot analysis, evaluation of Poisson distributions of precursor and products, and determination of the average number of modifications. These MS-based methods reliably and effectively indicate HOS perturbation at the intact protein level, whereas spectroscopic methods (circular dichroism (CD) and dynamic light scattering (DLS)) are less sensitive in monitoring subtle HOS perturbation caused by footprinting. Evaluation of HOS at the peptide level indicates regions that are sensitive to localized perturbations. Peptide-level analysis also provides higher resolution of the HOS perturbation, and we recommend using it for future footprinting studies. Overall, this work shows conclusive evidence for HOS perturbation caused by footprinting. Implementation of quality control workflows can identify conditions to avoid the perturbation, for footprinting, allowing accurate and reliable identification of protein structural changes that accompany, for example, ligand interactions, mutations, and changes in solution environment.

Thyroid hormones mediate the impact of early-life stress on ventral tegmental area gene expression and behavior.

Proper thyroid function is essential to the developing brain, including dopamine neuron differentiation, growth, and maintenance. Stress across the lifespan impacts thyroid hormone signaling and anxiety disorders and depression have been associated with thyroid dysfunction (both hypo- and hyper-active). However, less is known about how stress during postnatal development impacts thyroid function and related brain development. Our previous work in mice demonstrated that early-life stress (ELS) transiently impinged on expression of a transcription factor in dopamine neurons, Otx2, shown to be regulated by thyroid hormones. We hypothesized that thyroid hormone signaling may link experience of ELS with transcriptional dysregulation within the dopaminergic midbrain, and ultimately behavior. Here, we find that ELS transiently increases thyroid-stimulating hormone levels (inversely related to thyroid signaling) in both male and female mice at P21, an effect which recovers by adolescence. We next tested whether transient treatment of ELS mice with synthetic thyroid hormone (levothyroxine, LT4) could ameliorate the impact of ELS on sensitivity to future stress, and on expression of genes related to dopamine neuron development and maintenance, thyroid signaling, and plasticity within the ventral tegmental area. Among male mice, but not females, juvenile LT4 treatment prevented hypersensitivity to adult stress. We also found that rescuing developmental deficits in thyroid hormone signaling after ELS restored levels of some genes altered directly by ELS, and prevented alterations in expression of other genes sensitive to the second hit of adult stress. These findings suggest that thyroid signaling mediates the deleterious impact of ELS on VTA development, and that temporary treatment of hypothyroidism after ELS may be sufficient to prevent future stress hypersensitivity.

Clinical effectiveness and cost-impact after 2 years of a ketogenic diet and virtual coaching intervention for patients with diabetes.

AIM: We previously evaluated the impacts at 5 months of a digitally delivered coaching intervention in which participants are instructed to adhere to a very low carbohydrate, ketogenic diet. With extended follow-up (24 months), we assessed the longer-term effects of this intervention on changes in clinical outcomes, health care utilization and costs associated with outpatient, inpatient and emergency department use in the Veterans Health Administration. MATERIALS AND METHODS: We employed a difference-in-differences model with a waiting list control group to estimate the 24-month change in glycated haemoglobin, body mass index, blood pressure, prescription medication use, health care utilization rates and associated costs. The analysis included 550 people with type 2 diabetes who were overweight or obese and enrolled in the Veterans Health Administration for health care. Data were obtained from electronic health records from 2018 to 2021. RESULTS: The virtual coaching and ketogenic diet intervention was associated with significant reductions in body mass index [-1.56 (SE 0.390)] and total monthly diabetes medication usage [-0.35 (SE 0.054)]. No statistically significant differences in glycated haemoglobin, blood pressure, outpatient visits, inpatient visits, or emergency department visits were observed. The intervention was associated with reductions in per-patient, per-month outpatient spending [-USD286.80 (SE 97.175)] and prescription drug costs (-USD105.40 (SE 30.332)]. CONCLUSIONS: A virtual coaching intervention with a ketogenic diet component offered modest effects on clinical and cost parameters in people with type 2 diabetes and with obesity or overweight. Health care systems should develop methods to assess participant progress and engagement over time if they adopt such interventions, to ensure continued patient engagement and goal achievement.

Differences in adverse outcomes across race and ethnicity among Veterans with similar predicted risks of an overdose or suicide-related event.

OBJECTIVE: To evaluate the degree to which differences in incidence of mortality and serious adverse events exist across patient race and ethnicity among Veterans Health Administration (VHA) patients receiving outpatient opioid prescriptions and who have similar predicted risks of adverse outcomes. Patients were assigned scores via the VHA Stratification Tool for Opioid Risk Mitigation (STORM), a model used to predict the risk of experiencing overdose- or suicide-related health care events or death. Individuals with the highest STORM risk scores are targeted for case review. DESIGN: Retrospective cohort study of high-risk veterans who received an outpatient prescription opioid between 4/2018-3/2019. SETTING: All VHA medical centers. PARTICIPANTS: In total, 84 473 patients whose estimated risk scores were between 0.0420 and 0.0609, the risk scores associated with the top 5%-10% of risk in the STORM development sample. METHODS: We examined the expected probability of mortality and serious adverse events (SAEs; overdose or suicide-related events) given a patient's risk score and race. RESULTS: Given a similar risk score, Black patients were less likely than White patients to have a recorded SAE within 6 months of risk score calculation. Black, Hispanic, and Asian patients were less likely than White patients with similar risk scores to die within 6 months of risk score calculation. Some of the mortality differences were driven by age differences in the composition of racial and ethnic groups in our sample. CONCLUSIONS: Our results suggest that relying on the STORM model to identify patients who may benefit from an interdisciplinary case review may identify patients with clinically meaningful differences in outcome risk across race and ethnicity.

Sexual dimorphism in the dorsal root ganglia of neonatal mice identified by protein expression profiling with single-cell mass cytometry.

Development of neuronal and glial populations in the dorsal root ganglia (DRG) is required for detection of touch, body position, temperature, and noxious stimuli. While female-male differences in somatosensory perception have been previously reported, no study has examined global sex differences in the abundance of DRG cell types, and the developmental origin of these differences has not been characterized. To investigate whether sex-specific differences in neuronal and glial cell types arise in the DRG during development, we performed single-cell mass cytometry analysis on sex-separated DRGs from 4 separate litter replicates of postnatal day 0 (P0) C57/BL6 mouse pups. In this analysis, we observed that females had a higher abundance of total neurons (p = 0.0266), as well as an increased abundance of TrkB+ (p = 0.031) and TrkC+ (p = 0.04) neurons for mechanoreception and proprioception, while males had a higher abundance of TrkA+ (p = 0.025) neurons for thermoreception and nociception. Pseudotime comparison of the female and male datasets indicates that male neurons are more mature and differentiated than female neurons at P0. These findings warrant further studies to determine whether these differences are maintained across development, and their impact on somatosensory perception.

Strikingly High Activity of 15-Lipoxygenase Towards Di-Polyunsaturated Arachidonoyl/Adrenoyl-Phosphatidylethanolamines Generates Peroxidation Signals of Ferroptotic Cell Death.

The vast majority of membrane phospholipids (PLs) include two asymmetrically positioned fatty acyls: oxidizable polyunsaturated fatty acids (PUFA) attached predominantly at the sn2 position, and non-oxidizable saturated/monounsaturated acids (SFA/MUFA) localized at the sn1 position. The peroxidation of PUFA-PLs, particularly sn2-arachidonoyl(AA)- and sn2-adrenoyl(AdA)-containing phosphatidylethanolamines (PE), has been associated with the execution of ferroptosis, a program of regulated cell death. There is a minor subpopulation (≈1-2 mol %) of doubly PUFA-acylated phospholipids (di-PUFA-PLs) whose role in ferroptosis remains enigmatic. Here we report that 15-lipoxygenase (15LOX) exhibits unexpectedly high pro-ferroptotic peroxidation activity towards di-PUFA-PEs. We revealed that peroxidation of several molecular species of di-PUFA-PEs occurred early in ferroptosis. Ferrostatin-1, a typical ferroptosis inhibitor, effectively prevented peroxidation of di-PUFA-PEs. Furthermore, co-incubation of cells with di-AA-PE and 15LOX produced PUFA-PE peroxidation and induced ferroptotic death. The decreased contents of di-PUFA-PEs in ACSL4 KO A375 cells was associated with lower levels of di-PUFA-PE peroxidation and enhanced resistance to ferroptosis. Thus, di-PUFA-PE species are newly identified phospholipid peroxidation substrates and regulators of ferroptosis, representing a promising therapeutic target for many diseases related to ferroptotic death.

Stereospecific and Regioselective Synthesis of E-Allylic Alcohols through Reductive Cross Coupling of Terminal Alkynes.

We have developed a convergent method for the synthesis of allylic alcohols that involves a reductive coupling of terminal alkynes with α-chloro boronic esters. The new method affords allylic alcohols with excellent regioselectivity (anti-Markovnikov) and an E/Z ratio greater than 200:1. The reaction can be performed in the presence of a wide range of functional groups and has a substrate scope that complements the stoichiometric alkenylation of α-chloro boronic esters performed using alkenyl lithium and Grignard reagents. The transformation is stereospecific and allows for the robust and highly selective synthesis of chiral allylic alcohols. Our studies support a mechanism that involves hydrocupration of the alkyne and cross-coupling of the alkenyl copper intermediate with α-chloro boronic esters. Experimental evidence excludes a radical mechanism of the cross-coupling step and is consistent with the formation of a boron-ate intermediate and a 1,2-metalate shift.

Workflow for Validating Specific Amino Acid Footprinting Reagents for Protein Higher Order Structure Elucidation.

Protein footprinting mass spectrometry probes protein higher order structure and dynamics by labeling amino acid side-chains or backbone amides as a function of solvent accessibility. One category of footprinting uses residue-specific, irreversible covalent modifications, affording flexibility of sample processing for bottom-up analysis. Although several specific amino acid footprinting technologies are becoming established in structural proteomics, there remains a need to assess fundamental properties of new reagents before their application. Often, footprinting reagents are applied to complex or novel protein systems soon after their discovery and sometimes without a thorough investigation of potential downsides of the reagent. In this work, we assemble and test a validation workflow that utilizes cyclic peptides and a model protein to characterize benzoyl fluoride, a recently published, next-generation nucleophile footprinter. The workflow includes the characterization of potential side-chain reactive groups, reaction "quench" efficacies, reagent considerations and caveats (e.g., buffer pH), residue-specific kinetics compared to those of established reagents, and protein-wide characterization of modification sites with considerations for proteolysis. The proposed workflow serves as a starting point for improved footprinting reagent discovery, validation, and introduction, the aspects of which we recommend before applying to unknown protein systems.

Characterization of Higher Order Structural Changes of a Thermally Stressed Monoclonal Antibody via Mass Spectrometry Footprinting and Other Biophysical Approaches.

Characterizing changes in the higher order structure (HOS) of monoclonal antibodies upon stressed conditions is critical to gaining a better understanding of the product and process. One single biophysical approach may not be best suited to assess HOS comprehensively; thus, the synergy from multiple, complementary approaches improves characterization accuracy and resolution. In this study, we employed two mass spectrometry (MS )-based footprinting techniques, namely, fast photochemical oxidation of proteins (FPOP)-MS and hydrogen-deuterium exchange (HDX)-MS, supported by dynamic light scattering (DLS), differential scanning calorimetry (DSC), circular dichroism (CD), and nuclear magnetic resonance (NMR) to study changes to the HOS of a mAb upon thermal stress. The biophysical techniques report a nuanced characterization of the HOS in which CD detects no changes to the secondary or tertiary structure, yet DLS measurements show an increase in the hydrodynamic radius. DSC indicates that the stability decreases, and chemical or conformational changes accumulate with incubation time according to NMR. Furthermore, whereas HDX-MS does not indicate HOS changes, FPOP-MS footprinting reveals conformational changes at residue resolution for some amino acids. The local phenomena observed with FPOP-MS indicate that several residues show various patterns of degradation during thermal stress: no change, an increase in solvent exposure, and a biphasic response to solvent exposure. All evidences show that FPOP-MS efficiently resolves subtle structural changes and novel degradation pathways upon thermal stress treatment at residue-level resolution.

Effect of a Predictive Analytics-Targeted Program in Patients on Opioids: a Stepped-Wedge Cluster Randomized Controlled Trial.

BACKGROUND: Risk of overdose, suicide, and other adverse outcomes are elevated among sub-populations prescribed opioid analgesics. To address this, the Veterans Health Administration (VHA) developed the Stratification Tool for Opioid Risk Mitigation (STORM)-a provider-facing dashboard that utilizes predictive analytics to stratify patients prescribed opioids based on risk for overdose/suicide. OBJECTIVE: To evaluate the impact of the case review mandate on serious adverse events (SAEs) and all-cause mortality among high-risk Veterans. DESIGN: A 23-month stepped-wedge cluster randomized controlled trial in all 140 VHA medical centers between 2018 and 2020. PARTICIPANTS: A total of 44,042 patients actively prescribed opioid analgesics with high STORM risk scores (i.e., percentiles 1% to 5%) for an overdose or suicide-related event. INTERVENTION: A mandate requiring providers to perform case reviews on opioid analgesic-prescribed patients at high risk of overdose/suicide. MAIN MEASURES: Nine serious adverse events (SAEs), case review completion, number of risk mitigation strategies, and all-cause mortality. KEY RESULTS: Mandated review inclusion was associated with a significant decrease in all-cause mortality within 4 months of inclusion (OR: 0.78; 95% CI: 0.65-0.94). There was no detectable effect on SAEs. Stepped-wedge analyses found that mandated review patients were five times more likely to receive a case review than non-mandated patients with similar risk (OR: 5.1; 95% CI: 3.64-7.23) and received more risk mitigation strategies than non-mandated patients (0.498; CI: 0.39-0.61). CONCLUSIONS: Among VHA patients prescribed opioid analgesics, identifying high risk patients and mandating they receive an interdisciplinary case review was associated with a decrease in all-cause mortality. Results suggest that providers can leverage predictive analytic-targeted population health approaches and interdisciplinary collaboration to improve patient outcomes. TRIAL REGISTRATION: ISRCTN16012111.

Sedation Weaning Initiative Targeting Methadone Exposure: Single Center Improvements in Withdrawal Symptoms and Hospital Length of Stay for Pediatric Cardiac Critical Care.

OBJECTIVES: Sedation and pain medications are necessary in the management of postoperative pediatric cardiac patients. Prolonged exposure to these medications can lead to negative side effects including withdrawal. We hypothesized that standardized weaning guidelines would decrease exposure to sedation medications and decrease withdrawal symptoms. The primary aim was to decrease average days of methadone exposure to within goal for moderate- and high-risk patients within 6 months. DESIGN: Quality improvement methods were used to standardize sedation medication weaning in a pediatric cardiac ICU. SETTING: This study took place at Duke Children's Hospital Pediatric Cardiac ICU in Durham, North Carolina from January 1, 2020, to December 31, 2021. PATIENTS: Children less than 12 months old admitted to the pediatric cardiac ICU who underwent cardiac surgery. INTERVENTIONS: Sedation weaning guidelines were implemented over the course of 12 months. Data were tracked every 6 months and compared with the 12 months pre-intervention. Patients were stratified into low, moderate, and high risk withdrawal categories based on duration of opioid infusion exposure. MEASUREMENTS AND MAIN RESULTS: Total sample size was 94 patients in the moderate and high risk categories. Process measures included documentation of Withdrawal Assessment Tool scores and appropriate methadone prescription in patients which increased to 100% post-intervention. For outcome measures, we observed decreased dexmedetomidine infusion duration, decreased methadone wean duration, decreased frequency of elevated Withdrawal Assessment Tool scores, and decreased hospital length of stay post-intervention. For the primary aim, methadone wean duration consistently decreased after each study period. Our intervention did not adversely impact balancing measures. CONCLUSIONS: A quality improvement initiative to standardize sedation weaning in a Pediatric Cardiac ICU was successfully implemented and was correlated with decreased duration of sedation medications, decreased withdrawal scores, and decreased length of stay.

Impact of Mandated Case Review Policy on Opioid Discontinuation and Mortality Among High-Risk Long-Term Opioid Therapy Patients: The STORM Stepped-Wedge Cluster Randomized Controlled Trial.

BACKGROUND: Although long-term opioid therapy (LTOT) has its own risks, opioid discontinuation could pose harm for high-risk Veterans Health Administration (VHA) patients receiving LTOT. There is limited information on the impact of a mandate requiring providers to perform case reviews on high-risk patients with an active opioid prescription (ie, mandated case review policy) on opioid discontinuation and mortality. METHODS: Our study is a secondary data analysis of a 23-month stepped-wedge cluster randomized controlled trial between April 2018 and March 2020. The study included 10 685 LTOT patients with a predicted risk of a serious adverse event between the top 1% to 5% nationally who entered the risk range between 4/18/2018 and 11/9/2019. We examined whether the mandated case review policy had an impact on opioid discontinuation and mortality for the patients. RESULTS: Among 10 685 LTOT patients (88.2% male; mean [SD] age, 61.1 [11.7] years), 29.1% experienced discontinuation and the mortality rate was 9.5%. Patients under mandated case review had a decreased risk of opioid discontinuation (average marginal effect [AME], -11.16 [95% CI, -15.30 to -7.01] percentage points) and all-cause mortality (AME, -3.31 [95% CI, -5.63 to -1.00] percentage points), relative to patients who were not under the mandate. CONCLUSIONS: The VHA mandated case review policy was associated with lower probability of discontinuation and all-cause mortality for high-risk patients receiving LTOT. Interventions that maintain care engagement while optimizing pain management for high-risk patients may be beneficial for minimizing mortality and other risks associated with discontinuation.

Perception versus reality: analysis of time spent on bedside rounds in an academic ICU (Intensive Care Unit).

BACKGROUND: Despite its importance, teaching at the bedside is declining over time. This purported decline has not been quantified. Quantifying bedside teaching is challenging, and we found only one study quantifying bedside teaching on a hospitalist service. OBJECTIVE: We conducted a study to understand the prevalence of bedside teaching in our medical intensive care unit. METHODS: We conducted a single-center single-unit study in the medical intensive care unit of an academic tertiary care institution. We used a survey tool to assess perceived time spent on bedside teaching, quality of teaching, and total rounding time. In parallel, independent observers objectively measured time spent on rounds and on bedside teaching. Residents were asked to complete the survey once a week. Independent observers collected data daily and weekly averages were obtained. RESULTS: 43 responses were collected over a 4-month period. Most respondents (73%) reported a total rounding time of either 90-120 min or greater than 120 min. Median reported bedside teaching time was 16-20 min with 16 respondents (37%) reporting less than 15 min and 27 respondents (63%) reporting 16 min or more. The amount of time spent on bedside teaching was reported as adequate or more than adequate by 77% (33) of respondents with 58% (25) reporting that bedside teaching was very or extremely effective in helping them learn. Mean census reported by the independent observers was 12.75 patients per team. Bedside teaching represented an average of 12% of total rounding time, 16.85 min per day. While total rounding time increased with increasing census, there was no decline in bedside teaching time. CONCLUSION: It is reported that bedside teaching has decreased over time. Our study has demonstrated that bedside teaching occurs in our Medical ICU, and though it represents a minority of the time spent on rounds, residents still reported teaching in the ICU to be adequate.

Harnessing changes in open chromatin determined by ATAC-seq to generate insulin-responsive reporter constructs.

BACKGROUND: Gene regulation is critical for proper cellular function. Next-generation sequencing technology has revealed the presence of regulatory networks that regulate gene expression and essential cellular functions. Studies investigating the epigenome have begun to uncover the complex mechanisms regulating transcription. Assay for transposase-accessible chromatin by sequencing (ATAC-seq) is quickly becoming the assay of choice for many epigenomic investigations. However, whether intervention-mediated changes in accessible chromatin determined by ATAC-seq can be harnessed to generate intervention-inducible reporter constructs has not been systematically assayed. RESULTS: We used the insulin signaling pathway as a model to investigate chromatin regions and gene expression changes using ATAC- and RNA-seq in insulin-treated Drosophila S2 cells. We found correlations between ATAC- and RNA-seq data, especially when stratifying differentially-accessible chromatin regions by annotated feature type. In particular, our data demonstrated a weak but significant correlation between chromatin regions annotated to enhancers (1-2 kb from the transcription start site) and downstream gene expression. We cloned candidate enhancer regions upstream of luciferase and demonstrate insulin-inducibility of several of these reporters. CONCLUSIONS: Insulin-induced chromatin accessibility determined by ATAC-seq reveals enhancer regions that drive insulin-inducible reporter gene expression.

Benzoyl Transfer for Footprinting Alcohol-Containing Residues in Higher Order Structural Applications of Mass-Spectrometry-Based Proteomics.

Protein footprinting mass spectrometry (MS), an emerging approach to elucidate higher-order structure (HOS) and binding, benefits from the iterative development of reaction strategies to expand the covalent labeling toolbox. Herein, we introduce a footprinting reagent for nucleophiles and demonstrate its efficacy for differential covalent labeling MS analysis. Benzoyl fluoride (BF), although reactive with water, is more practical for modifying nucleophilic functional groups than other acid halides and serves as an acyl-transfer reagent for proteins. BF is 10 times more reactive with phenolic Tyr than the current generation nucleophile footprinter. BF modifies, in addition to Tyr, Lys, His, and the N-terminus, weak nucleophiles Ser and Thr, for which few footprinters exist, imparting broad applicability with a range of nucleophiles. We applied benzoylation to a model Ser- and Thr-rich protein-ligand binding system without perturbing the protein HOS. This efficacious footprinting method expands the toolbox of reagents and provides promise for future reaction strategies including possibly membrane proteins.

Automated Specific Amino Acid Footprinting Mass Spectrometry: Repurposing an HDX Platform for Determining Reagent Feasibility.

Protein footprinting is a mass spectrometry (MS)-based approach to measure protein conformational changes. One approach, specific amino acid labeling, imparts often an irreversible modification to protein side chains but requires careful selection of the reactive reagent and often time-consuming optimization of experimental parameters prior to submission to bottom-up MS analysis. In this work, we repurpose a hydrogen-deuterium exchange MS (HDX-MS) LEAP HDX system for automated specific amino acid footprinting MS, demonstrating its efficacy in reaction optimization and monitoring applicability to specific ligand binding systems. We screened reagent conditions for two model ligand-binding systems and demonstrate the method's efficacy for measuring differences induced by ligand binding. Our proof-of-concept experiments provide a platform for rapidly screening specific amino acid reagents and reaction conditions for protein systems to be studied by footprinting.