bluuebunny/crossref_metadata_2025_split huggingface dataset:
Stability‐function trade‐offs during the evolution of antimicrobial resistance
Authors: Matange Nishad 
Published: April 2020

Structural stability is a major constraint on the evolution of protein sequences. However, under strong directional selection, mutations that confer novel phenotypes but compromise structural stability of proteins may be permissible. During the evolution of antibiotic resistance, mutations that confer drug resistance often have pleiotropic effects on the structure and function of antibiotic‐target proteins. In the present study, I use trimethoprim (TMP)‐resistant alleles of dihydrofolate reductase from Escherichia coli (EcDHFR) to exemplify how drug‐resistance can often be associated with protein misfolding and aggregation. I show that misfolding of TMP‐resistant DHFR is associated with its reduced steady state levels, resulting in a fitness cost. Importantly, the effect of destabilizing mutations on drug‐resistance is contingent on the folding environment of the cell. In particular, the presence or absence of the lon‐protease, a quality control protease in E. coli, determines the genotype‐phenotype relationship between DHFR mutations and their level of trimethoprim‐resistance. The status of lon‐protease also determines the mutational repertoire accessible to EcDHFR to evolve drug resistance. This study demonstrates that trade‐offs between structural stability and function can influence innate drug resistance as well as the potential for mutationally acquired drug resistance of an enzyme.Support or Funding InformationDST‐INSPIRE Faculty Fellowship, Department of Science and Technology, Govt. Of IndiaFigure 1Figure 2
Molecular and structural characterization of a TEAD mutation at the origin of Sveinsson’s chorioretinal atrophy
Authors: Bokhovchuk Fedir , Mesrouze Yannick , Izaac Aude , Meyerhofer Marco , Zimmermann Catherine , Fontana Patrizia , Schmelzle Tobias , Erdmann Dirk , Furet Pascal , Kallen Joerg , Chène Patrick 
Published: April 2020

Four TEAD transcription factors (TEAD1–4) mediate the signalling output of the Hippo pathway that controls organ size in humans. TEAD transcriptional activity is regulated via interactions with the YAP, TAZ and VGLL proteins. A mutation in the TEAD1 gene, Tyr421His, has been identified in patients suffering from Sveinsson’s chorioretinal atrophy (SCA), an autosomal dominant eye disease. This mutation prevents the YAP/TAZ:TEAD1 interaction. In this study, we measure the affinity of YAP, TAZ and VGLL1 for the four human TEADs and find that they have a similar affinity for all TEADs. We quantitate the effect of the mutation found in SCA patients and show that it destabilizes the YAP/TAZ:TEAD interaction by about 3 kcal·mol–1. We determine the structure of YAP in complex with this mutant form of TEAD and show that the histidine residue adopts different conformations at the binding interface. The presence of this flexible residue induces the destabilization of several H‐bonds and the loss of van der Waals contacts, which explains why the Tyr421HisTEAD1 mutation has such a large destabilizing effect on the formation of the YAP:TEAD complex.Close view of the region surrounding key Tyr/His429 in the superimosed wtYAP:wtTEAD4 and wtYAP:Tyr429HisTEAD4 complexesFigure 1
Vascular Expression of Toll‐Like Receptor 4 and Myeloid Differentiation Factor‐88 Mediate Endothelial Dysfunction in Response to Lipopolysaccharide Independent of Circulating Immune Cells
Authors: Coate Ian D. , Didion Sean P. 
Published: April 2020

The goal of this study was to test the hypothesis that vascular expression of toll‐like receptor 4 (TLR4) and its primary downstream signaling pathway, myeloid‐differentiation factor‐88 (MyD88), mediate endothelial dysfunction in response to lipopolysaccharide (LPS). Carotid arteries from wild‐type (C57Bl/6), Tlr4‐Lps‐d (Tlr4 mutant mice), and Myd88‐deficient (Myd88−/−) mice were incubated with either vehicle (saline) or LPS (0.05–0.50 μg/ml) for 22 hrs. LPS, but not vehicle, treatment produced concentration‐dependent impairment (P<0.05) of endothelium‐dependent relaxation to acetylcholine (ACh) in wild‐type mice. In contrast, in carotid arteries from Tlr4‐Lps‐d or Myd88−/− mice endothelial responses to ACh following LPS incubation were found to be similar (P>0.05) to responses in wild‐type mice incubated with vehicle. LPS has no effect (P>0.05) on vascular response to sodium nitroprusside in any of the six experimental groups. Both basal and NADPH‐stimulated superoxide levels were selectively increased (P<0.05) in vessels from wild‐type, but not Tlr4‐Lps‐d or Myd88−/−, mice treated with LPS. RNA expression revealed that both TLR4 and MyD88 are indeed expressed in the vascular wall under basal conditions and that LPS induces differential expression of a number of inflammatory gene products, including Tlr4, Myd88, and Nfkb1. Pathway analysis revealed a linkage between TLR4 and MyD88 in the vessels wall with IL‐6 and downstream targets such as Nox2 and Nox4. These findings serve to link activation of TLR4 and downstream signaling through MyD88 and NFkB1 with increases in vascular superoxide and functional impairment of endothelial function in response to LPS. Taken together, these findings demonstrate that both Tlr4 and Myd88 expression and activity within the vascular wall contributes to LPS‐induced impairment of endothelial function. The fact that experiments were performed ex vivo, demonstrate that the effects of LPS on endothelial function occur directly within the vascular wall independently of circulating immune cell activation.Support or Funding InformationSupported in part by NIH/NIBIB R01‐EB2006.
Breathing Pattern Differs Between Male and Female Rats in Escherichia coli Induced Septicemia
Authors: Campanaro Cara , Clifford Caitlyn , Tackett Shiloh , Horton Kofi-Kermit , Nethery David , Hsieh Yee-Hsee , Dick Thomas , Jacono Frank 
Published: April 2020

Septicemia is a potentially fatal infection marked by bacteria in the bloodstream coupled with a dysregulated inflammatory response. Escherichia coli (E. coli) is a common cause of septicemia. We hypothesized that in E. coli induced septicemia, respiratory frequency (fR) and the ventilatory pattern variability (VPV) would depend on sex. Male (n=6) and female (n=6) Sprague Dawley rats were inoculated with E. coli (100×106 colony forming units) contained within a clot formed of thrombin, fibrinogen, and phosphate buffered saline. We measured ventilation in unanesthetized rats with whole‐body plethysmography before (0h) and 12h after inoculation. Before inoculation, the fR male rats was greater than that of females (fR: 78.4 ± 11.9 vs. 58.7±6.98 breaths per minute (brthspm); p=0.007). In contrast, VPV did not differ between males and female (autocorrelation coefficient (r) at 1 cycle length 0.58±0.17 vs 0.56±0.13 and sample entropy 1.24±0.04 vs 1.34±0.11 Nats). However, 12h after inoculation, fR in male rats was much greater than female (142.73±22.03 vs. 64.59±6.61 brthspm; p= 0.0001) and analysis of VPV found differences in autocorrelation (0.814±0.084 vs. 0.578±0.160; p=0.01) and sample entropy (0.98±0.10 vs. 1.19±0.13 Nats; p=0.01). Further, in both male and female rats, fR increased 12h after inoculation rats (males, p=0.001; females p=0.02). These preliminary data indicate that male rats have a higher resting fR and greater ventilatory response to septicemia than female rats. Pending analysis of pro‐inflammatory cytokines will indicate whether the magnitude of these ventilatory response correlates to the magnitude of the inflammatory response.Support or Funding InformationFunded by: VA Research Service (I01BX004197); NIH (U01 EB021960)Figure 1
Connectivity Enhanced Structure Activity Relationship (ceSAR): A Novel Approach to Increase Accuracy and Speed of Virtual Drug Discovery
Authors: Thorman Alexander William , Reigle James , Chutipongtanate Somchai , Shamsaei Behrouz , Pilarczyk Marcin , Fazel-Najafabadi Mehdi , Adamczak Rafal , Kouril Michal , Medvedovic Mario , Meller Jarek 
Published: April 2020

Experimental drug discovery and repurposing is a costly and time‐consuming process. Here, a novel approach, dubbed connectivity enhanced structure activity relationship (ceSAR) is introduced. ceSAR combines traditional virtual screening methodologies with cheminformatics and Library of Integrated Network‐Based Cellular Signatures (LINCS) gene expression signatures of genetic and chemical perturbations. This methodology combines structural similarity of small molecules with transcriptional connectivity and virtual screening‐predicted specific interacting molecules for the enhancement of structure activity relationship (SAR) and lead compound discovery. The transcriptional connectivity aspect of ceSAR is implemented as an R Shiny package, titled Sig2Lead, that employs connectivity between chemical and genetic signatures included in the LINCS library to identify small molecule inhibitors of user‐targeted pathways. Chemical libraries are reduced with ceSAR by first measuring concordance of transcriptomic signatures between genetic and chemical perturbation, identifying putative pathway inhibitors, followed by traditional docking approaches to re‐score the remaining library to identify compounds likely to interact with the target of interest. This combination identifies compounds that are both pathway inhibitors and have shape complementarity to the target of interest, making the most likely scenario that the molecules are directly interacting with the target protein of interest to modulate the pathway. Through integration of these orthogonal approaches, both an increased accuracy and speed can be observed, with the speed increase being several orders of magnitude.Support or Funding InformationThis work was supported in part by the National Institutes of Health grants U54 HL127624ceSAR combines transcriptional connectivity and virtual screening to identify novel inhibitors from a screened chemical library. The library is first reduced using transcriptional connectivity to a knockdown of the target gene to restrict the search to putative pathway inhibitors. These pathway inhibitors are then reduced further using traditional virtual screening approaches to find compounds with shape complementarity to the protein of interest.Figure 1Comparison of results of an early drug screen using a combination of docking with AutoDock4.2.6 and Sig2Lead shows a dramatic reduction in compounds without losing the best‐performing compounds. If used in this case, the ceSAR method would have excluded 51 of the 147 tested compounds, of which 45 had no observable IC50 via a Fluorescence Polarization assay, thus driving library enrichment. This reduction would have saved time and reagents through preventing testing of as many spurious compounds.Figure 2
Human Lung Microvascular Endothelial Cell Death in Response to Cell‐free Hemoglobin
Authors: Tomasek Toria , Meegan Jamie E. , Ware Lorraine B. , Bastarache Julie A. 
Published: April 2020

During sepsis, circulating levels of cell‐free hemoglobin (CFH) are often elevated due to the lysis of red blood cells, and higher concentrations are associated with worse clinical outcomes. Sepsis is caused by a widespread inflammation response, which leads to the release of pro‐inflammatory molecules and cytokines into the circulation. These compounds can prime and activate the endothelium rendering it more susceptible to damage from other circulating molecules. Sepsis is a leading cause of acute respiratory distress syndrome (ARDS), which is characterized by vascular leak and fluid accumulation in the lungs. In our previous studies in murine models of sepsis, lung endothelial cell death was observed, which may contribute to the alveolar capillary barrier dysfunction that characterizes ARDS. Here we investigate whether CFH can initiate cell death in vitro and whether this is potentiated by exposure to pro‐inflammatory signals. Primary human lung microvascular endothelial cells (HLMVECs) were cultured to confluence and exposed to various clinically relevant concentrations of CFH diluted in complete media for up to 24 hours or pre‐treated for 4 hours with 5 ng/ml Cytomix (equal parts IL‐1β, TNF‐α, and INFγ), followed by treatment with 1.0 mg/ml CFH for 24 hours. Cell viability was determined using Draq7, a cell membrane impermeable nuclear stain. Cell death was measured by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining, which detects DNA fragmentation from late apoptosis or necrosis. While there was a time‐ and concentration‐dependent trend, no significant differences between CFH treated and untreated cells were seen in the absence of cytomix, even at the highest concentration of 1.0 mg/ml CFH at 24 hours for the TUNEL assay (57 TUNEL+
cells per FOV in CFH‐treated vs. 17 in control, p=0.8245). However, there was a significant increase in Draq7 staining with 1.0 mg/ml CFH treatment after 24 hours (7.9% Draq7+ cells in CFH‐treated vs. 2.3% in control, p=0.0240). Exposure to cytomix augmented the decrease in viability from CFH as measured by Draq7 assay (32.4% Draq7+ cells in Cyto + CFH‐treated cells vs.
14.5% in CFH‐treated cells, p<0.0001). These findings indicate that CFH alone has modest effects on lung endothelial cell viability. However, during sepsis when circulating cytokine levels are increased, the effect of CFH on endothelial cell death is enhanced, which may contribute to the microvascular leak and alveolar fluid accumulation that are characteristic of acute lung injury.Support or Funding InformationThis work is funded by NIH HL135849, HL103836, and HL094296.Figure 1
Effect of URAT1 inhibition with verinurad on proximal tubule intracellular lactate: A mathematical modeling analysis and hypothesis for antiproteinuric effect
Authors: Nieves Emily , Hallow Melissa , Greasley Peter , Helmlinger Gabriel , Parkinson Joanna 
Published: April 2020

Verinurad (RDEA3170) lowers serum uric acid by inhibiting URAT1, a urate/carboxylate co‐transporter in the proximal tubule (PT) of the kidney. In a recent clinical trial, verinurad also reduced proteinuria, but the mechanism remains unknown. One proposed hypothesis is that the inhibition of URAT1 augments protein reabsorption by increasing intracellular lactate in proximal tubule cells. The increased availability of lactate may lead to a restoration of the reabsorption of protein by increasing ATP levels and/ or improving endocytosis of protein by lowering endosomal pH. The goal of this study was to mathematically evaluate the effect of URAT1 inhibition with verinurad on intracellular lactate concentration.A mathematical model of urate and lactate transport in the rat proximal tubule was adapted to simulate transport in humans. The transporter model was further adjusted to represent states of hyperuricemia and altered expression of uric acid transporters that likely occur in hyperuricemia. The transporter model was linked to a systems description of plasma uric acid and the pharmacokinetics and pharmacodynamics of verinurad were incorporated into the model.The model was able to reproduce observed verinurad pharmacokinetics, as well as the observed changes in plasma and urinary uric acid. The model predicted that URAT1 inhibition with verinurad increases intracellular lactate concentration in the human PT by 26% in normouricemic and 20% in hyperuricemic conditions.Model simulations indicate that verinurad may substantially increase the availability of lactate inside PT epithelial cells. Since lactate is used as a fuel by the PT, increased availability of lactate may potentially help restore protein reabsorption capacity in fuel‐deficient PT cells by increasing ATP levels. In addition, increased intracellular lactate could help maintain the low endosomal pH necessary for protein reabsorption. This hypothesis remains to be experimentally proven; further non‐clinical/clinical data would be valuable to support model predictions.Urate and lactate proximal tubule transport model.Figure 1
Essential Anatomy for Primary Care vs. Non‐Primary Care Clerkships and Electives
Authors: Hankin Mark , Harmon Derek , Martindale Jim , Keim Sarah , Topping Daniel , Farias Anna , Harrell Kelley , Marzban Hassan , Cotter Meghan , Royer Danielle , Davies David , Lopez Lisa , Carpenetti Tiffany , Downs Mary Beth , McNary Thomas , Latacha Kimberly , Zumwalt Ann , Mork Amy , Wineski Lawrence , Sanky Charles , Laitman Jeffrey , Reidenberg Joy S. , Brooks William , Kirera Francis , Lewis Steven , Granite Guinevere , McWhorter David , Pratt Rebecca , Notebaert Andrew , Miriyala Sumitra , Gest Thomas , Doroudi Majid , Giannaris Eustathia Lela , Kar Rekha , Sakaguchi Alan , Lackey-Cornelison Wendy , Hisley Kenneth 
Published: April 2020

IntroductionTo prepare medical students for clinical training and practice, it is critical to understand the anatomical knowledge considered most important for different clinical specialties.AimTo address this issue, a consortium of anatomists in the US and Canada is collecting data from clinical educators in clerkships and electives to identify the anatomy they consider essential for their specialty.MethodsAn IRB‐approved, online survey (Qualtrics, Seattle, WA) was used to assess the importance of 98 anatomical topics in seven body regions. The study first examined the nominal relationship between groups of primary care (Family Medicine, General Internal Medicine, General Pediatrics) and non‐primary care specialties, and whether they considered a given anatomical region important to their clerkship/elective. Second, the study compared the rank assigned by each specialty to each anatomical topic using an ordinal scale from 1 (not important) to 4 (essential).ResultsAt the time of abstract submission, data had been collected from 598 clinicians (clerkship/elective directors and attending physicians) in 19 clerkships/electives at 33 medical schools (allopathic n=28; osteopathic n=5). Relative to Non‐Primary care physicians, Primary Care physicians showed a statistically significant, higher percentage of “Yes” responses for all seven anatomical regions (Table
1). Further data analysis has identified the highest ranking anatomical topics within each region for Primary Care and Non‐Primary Care Specialties.Discussion and ConclusionThis database provides detailed information regarding the most clinically relevant anatomical topics as identified by clinical educators. This information can aid in focusing preclinical learning to best prepare medical students for success in their undergraduate and graduate clinical experiences.Responses to the question: Is the anatomy of this body region important to your clinical specialty?Table 1
Using Breath Metabolic Profiling to Identify Genetic Variants in Alcohol Metabolism
Authors: Kang Sunwoo , White Joseph Robert , Gross Eric Richard 
Published: April 2020

Aldehyde dehydrogenase 2 (ALDH2) metabolizes acetaldehyde to acetic acid. An ALDH2 genetic variant, ALDH2*2, limits acetaldehyde metabolism. As a result, after consuming alcohol, the ALDH2*2 genetic variant causes acetaldehyde accumulation, facial flushing, and tachycardia; commonly referred to as Asian glow. Here we describe how we developed a machine learning algorithm to distinguish between genetic variants of ALDH2 using data collected from the human breath mass spectrum.To develop this machine learning algorithm, we recruited human volunteers to perform an alcohol challenge after obtaining IRB approval. For these volunteers, we performed genotyping in addition to subjecting volunteers to a 0.25g/kg alcohol challenge. During the alcohol challenge, we measured the human breath mass spectra for 16 people (8 ALDH2*1*1 and 8 ALDH2*1*2) using selective ion flow mass spectrometry. This created a metabolic profile for each person of the full mass spectrum which consisted of 1,155 ion measurements complemented with 14 metabolite panel measurements.To develop a machine learning algorithm to predict ALDH2 genotype, standard scaler (SS) and principal component analysis (PCA) were applied to normalize the variance in ion counts and maximize high dimensional input. The visualization of metabolomics data showed two distinct clusters that are dependent on the ethanol intake (Figure
1A) and on ALDH2 genotype (Figure
1B). Based on the cluster distinction, we were able to verify that our data could characterize ethanol metabolism. Next, we applied ~20 classifier algorithms such as Support Vector Machine, Perceptron, Gaussian Naïve Bayes, Stochastic Gradient Descent, and Decision trees. The prediction through Random Forest yielded three novel features from the raw mass spectra that are not included in metabolite panel. These algorithms were able to achieve a training accuracy above 90% threshold for identifying between ALDH2 genotypes. These finding suggests a non‐invasive method to determine ALDH2 genotype that may be used to complement existing methods to determine the response a person has to alcohol.Support or Funding InformationThis research received grant funding from Stanford Chem‐H and NIH NIGMS GM119522.Visualization of Temporal HEB Metabolomics Profiling. Figure 1A: 3D PCA on Ethanol Catabolism with SVM distinction kernel. Blue=baseline, red=after ethanol exposure / Figure 1B: 3D Visualization of Partial Gradient of Acetaldehyde and Ethanol. Blue=ALDH2 cohort, red=ALDH2*2 cohortFigure 1
A Larval Zebrafish Model for Assessing Hypoxic‐Induced In Vivo Cardiomyocyte Damage: Time Course for Induction and Cardiac Output Recovery
Authors: Burggren Warren , Abramova Regina , Bautista Naim , Danielson Regina Fritsche , Gupta Avi , Hansson Kenny , Iyer Neha , Jagadeeswaran Pudur , Jennbacken Karin , Patel Vishal , Raman Revathi , Trivedi Hersh , Wang Qing-Dong 
Published: April 2020

Larval zebrafish are used to study regenerative properties of cardiac muscle and treatments that may enhance this process. Cardiac injury models in zebrafish larva currently include cryoinjury, laser ablation, pharmacological treatment and cardiac dysfunction mutations. Effective in damaging cardiomyocytes, these models lack the critical element of tissue hypoxia, which induces molecular cascades within cardiac muscle. We have developed a potentially more realistic model for studying cardiac arrest and recovery in larval zebrafish (Danio rerio) by using acute, severe hypoxic exposure to briefly induce cardiac arrest. Exposure to PO2s of 6–9 mmHg induces loss of mobility followed by cardiac arrest within 120 min in 5 days post fertilization (dpf) and within 40 min at 10 dpf. Approximately 90% of 5dpf larvae survived acute, severe hypoxic exposure, but survival fell to 30% by 10 dpf. Heart beat resumed in surviving larvae and returned to air‐saturated water at 4 min in 5 dpf and 6–8 min at 8–10 dpf. Heart rate, stroke volume and cardiac output in control 6–7 dpf larvae before hypoxic exposure were 188±5 bpm, 0.20±0.001nl and 35.5.3±2.2 nl/min (n=35), respectively. In larvae with hypoxia‐induced cardiac arrest, heart rate returned to control values by 24 h of recovery, but stroke volume and cardiac output remained ~50% depressed from control values at 24 h of recovery. Full restoration of cardiac performance was evident at 72h, indicating that regenerative properties of the zebrafish heart extend to critical physiological measures. Molecular markers for apoptosis (Caspase‐3) were elevated >5‐fold immediately after resumption of heart beat, peaking after 4 hours into the recovery period. A cell regeneration marker (PCNA) increased >4‐fold beginning after a lag of 2 hours, and was still elevated ~6 hours after resumption of heart beat, presumably contributing to recovery of heart function. The highly reproducible cardiac effects of acute hypoxia‐induced cardiac arrest in the larval zebrafish presents an alternative, realistic model for studying cardiac tissue hypoxia injury and recovery of function.Support or Funding InformationAstraZenecaTime course of changes in heart rate, stroke volume, and cardiac output in 5–7 day larval zebrafish following induction of cardiac arrest by acute hypoxic exposure (PO2 ~ 7–9 mmHg).Figure 1