bluuebunny/crossref_metadata_2025_split huggingface dataset:
Inward currents through somatic Na/K‐ATPase α1‐subunit mutants are not required for hyperaldosteronism
Authors: Meyer Dylan , Gatto Craig , Artigas Pablo 
Published: June 2021

Hyperaldosteronism (HA), the most common cause of secondary hypertension, is often initiated by an aldosterone‐producing adenoma within the zona glomerulosa of the adrenal cortex. Somatic mutations to several genes were identified in these adenomas, including to the Na/K‐ATPase (NKA) α1‐subunit. The membrane potential (Vm) of cells harboring these NKA‐α1 mutations is depolarized, which is believed to underlie the pathophysiological aspects of HA.At physiological Na+ and K+ concentrations, wild type NKA activity generates outward current due to the export of 3 Na+ and import of 2 K+ per cycle against their gradients. Under similar conditions, all HA‐associated NKA mutants evaluated thus far have dramatically reduced activity and an inward current, mediated by Na+ or H+, proposed to be a ‘gain‐of‐function’ required for Vm depolarization. The outward current of wild type NKA typically contributes minimally to Vm. Thus, for the inward current through HA‐associated NKA mutants to significantly depolarize Vm, its amplitude is expected to be significantly larger than the amplitude of wild type NKA outward current. Assuming similar expression levels, the transport rate of inward current through NKA mutant pumps should be larger than the pumping turnover rate of a single wild type NKA.To calculate the turnover rate of inward current through human NKA mutants, we utilized two‐electrode voltage clamp (TEVC) of Xenopus oocytes heterologously expressing HA‐associated mutants to measure the ouabain‐sensitive inward current amplitude in the presence of Na+, and 3H‐ouabain binding assays on the same oocyte. As ouabain binds to NKA at a 1:1 ratio, its quantity is equivalent to the number of pumps in the oocyte's plasma membrane. The ratio of current at −50 mV (a physiologically‐relevant voltage) to the moles of ouabain bound to the oocyte yielded inward current turnover rates of 22.0 ± 1.8 s−1 for L104R, 417.3 ± 29.4 s−1 for delF100‐L104, 60.7 ± 5.8 s−1 for V332G, and 19.1 ± 1.4 s−1 for EETA963S, while turnover rate of wild type K+‐induced pump current was 32.0 ± 1.5 s−1. This indicates that the inward current of several NKA mutants may not suffice to significantly depolarize Vm, and that the pathophysiological effects may simply reflect lack of function. Furthermore, oocytes expressing G99R, a mutant recently associated with HA, lacked inward current under physiological conditions, instead presenting K+‐induced outward pump current. TEVC measurements of extracellular K+‐induced current in G99R revealed a ~3‐fold reduction of K+ apparent affinity compared to wild type, while measurements of intracellular Na+‐dependent, ATP‐activated currents in giant inside‐out patches showed a ~3‐fold reduction of Na+ apparent affinity, demonstrating impaired function under physiological conditions. These results suggest that lack of function is the main mechanism leading to HA in all mutants.Support or Funding InformationSupported by NSF MCB‐1515434
Moderate resistance exercise improves the metabolic profile of adipose tissue in a model of disuse
Authors: Baer Lisa A. , Harris Johan , Sindeldecker Devin , Wade Charles E. , Stanford Kristin I. 
Published: June 2021

Treatment of trauma and several other maladies requires patient immobilization and restriction of physical activity, which can result in impaired metabolic health. In contrast to physical immobilization, physical activity and exercise improves metabolic health. Using a rodent model of disuse (hindlimb unloading) combined with resistance exercise, we determined if moderate resistance exercise could negate the effects of disuse on the metabolic profile of white adipose tissue (WAT). Male rats were assigned to four groups 1) Ambulatory No Exercise (ANE); 2) Ambulatory Exercise (AE); 3) Hindlimb Unloaded No Exercise (HUNE); 4) Hindlimb Unloaded Exercise (HUE). All rats completed a 10‐day pre‐training resistance exercise regiment prior to random group assignments. HUNE and HUE animals were placed in a tail traction system with their hindlimbs unloaded and were followed for 14 days. Animals designated to AE or HUE groups exercised for 14 days. At day 14, plasma and tissue samples were collected and analyzed. Resistance exercise resulted in a decrease in body mass in the ambulatory animals (AE vs. ANE), and hindlimb unloading significantly decreased body mass compared to ambulatory animals (HUNE and HUE vs. ANE and AE), but there was no additive effect of exercise on body mass (HUNE vs. HUE). Hindlimb unloading reduced hindlimb muscle mass (ANE and AE vs. HUNE and HUE), and resistance exercise partially restored soleus mass in the hindlimb unloaded animals (HUNE vs. HUE). Total white adipose tissue mass (WAT), perigonadal WAT (pgWAT) mass, and subcutaneous WAT (scWAT) mass and adipocyte size were significantly reduced in response to both exercise and hindlimb unloading. Expression of genes and proteins in scWAT involved in fatty acid oxidation and glucose metabolism were significantly increased with exercise (ANE vs. AE), reduced with hindlimb unloading (ANE vs. HUNE), and restored with exercise in the hindlimb unloaded animals (HUNE vs. HUE). In conclusion, these data indicate that moderate resistance exercise improves the metabolic profile of both pgWAT and scWAT in both the ambulatory state and in an animal model of disuse, providing a novel therapeutic benefit for the bed rest patients.Support or Funding InformationUS Army MRMC CDMRP W81XWH‐13‐1‐0489
Physiological Sensitivity to Salt and Uninephrectomy
Authors: Clemmer John , Pruett W. Andrew , Hester Robert 
Published: June 2021

Salt sensitivity, with or without concomitant hypertension, is associated with increased mortality. Reduced functional renal mass plays an important role in causing salt sensitive hypertension for many individuals but the mechanisms are unclear. Moreover, different derangements in human physiology can result in the same phenotype. Methodologies that could discover patterns in sensitivity to dietary change or interventions would be a revolutionary tool in personalizing medicine. We used HumMod, an integrative mathematical model of human physiology, to simulate population responses to changes in salt intake with or without reduced kidney mass (uninephrectomy). A heterogeneous population of 1000 virtual patients was created by randomly varying important physiological parameters. We examined potential physiological mechanisms responsible for the chronic blood pressure (BP) response to salt (8‐fold change in salt intake for 3 weeks) with full kidney mass and again with a single kidney in the same group of virtual patients. We also used topological data analysis (TDA), a new clustering algorithm tool, to separate patient subpopulations. In the entire population, BP increased with increased salt intake (14 ± 3 mmHg). This was associated with small changes in glomerular filtration rate (GFR) but significant increases in renal blood flow, total peripheral resistance, and distal tubular sodium reabsorption. Interestingly, TDA distinguished 5 unique clusters of salt sensitive individuals (> 15 mmHg change in BP with increased salt). While these clusters had similar BP responses to salt, they were significantly different in the mechanisms of salt sensitivity (e.g. greater reductions in GFR or impairments in the renin angiotensin system.) After kidney removal in these same individuals, the three most salt sensitive clusters were associated with a blunted increase in renal blood flow (RBF), exaggerated increases in loop and distal sodium reabsorption, and greater sodium and volume retention as compared to the salt resistant population. These data suggest that the suppression of sodium reabsorption and renin angiotensin system is key for salt resistance, while RBF in addition to GFR may be an important factor when considering criteria for potential kidney donors.Support or Funding InformationSupported by NSF EPS‐0903787, P20 GM104357, and NIH PO1 HL51971.
Comparison of Compensatory Reserve Index Before and After Analgesia
Authors: Hinojosa‐Laborde Carmen , Fernandez Jessie Renee D. , Muniz Gary W. , Nawn Corinne D. , Burns Rebecca K. , Le Thuan H. , Porter Kathy B. , Hardy John T. , Convertino Victor A. 
Published: June 2021

BACKGROUNDPrehospital assessment of cardiovascular stability during hemorrhage is unpredictable because standard vital signs do not predict cardiovascular shock until it is too late for the medic to respond with a life‐saving intervention. In an effort to improve survival from hemorrhage, a machine learning algorithm has been developed as an early predictor of cardiovascular shock. The algorithm is able to detect reductions in central blood volume during blood loss, and assess the individual's capacity to compensate for blood loss by calculating beat‐ to‐beat compensatory reserve index (CRI) from a finger photoplethysmography waveform. CRI=100 represents normovolemia and CRI=0 represents hypovolemia at the point of cardiovascular decompensation. The algorithm has been validated in blood loss studies in animals and humans; however these studies were conducted in the absence of other confounding factors associated with blood loss such as trauma and pain. The current study was designed to determine if pain, in the absence of trauma, affects CRI by comparing CRI before and after epidural anesthesia to provide analgesia during childbirth. The purpose of this study was to test the hypothesis that pain or analgesia does not affect CRI.METHODSThis study was conducted in healthy pregnant women (n=20) during the labor phase of childbirth. CRI was calculated from a finger pulse oximeter placed on the patient's index finger and connected to the DataOx™ CRI monitor. CRI was recorded for 1 hour before and 1 hour after epidural anesthesia.RESULTSCRI (mean ± SD) before analgesia was 83±11. CRI at 10, 20, 30, 40, 50, and 60 minutes after analgesia was 77±13, 80±13, 77±15, 81±16, 83±13, and 84±15, respectively. Repeated measures ANOVA revealed no statistical difference in CRI values before and after epidural anesthesia (p = 0.085). Pain scale scores before and after analgesia were 7±2 and 1±2, respectively.CONCLUSIONSThese results indicate that pain, as an independent factor (in the absence of blood loss or trauma), does not affect compensatory reserve, and provide preliminary evidence that pain associated with trauma may not affect the body's ability to compensate for blood loss. This study supports the use of pain management on the battlefield, and supports the utility of assessing compensatory reserve in the prehospital setting to improve survivability from combat trauma.Support or Funding InformationFUNDING: ­­­US Army MRMC Combat Casualty Care Research Program
GLP‐2 receptor in the brainstem is required for glucose homeostasis and insulin sensitivity
Authors: Shi Xuemei , Chacko Shaji , Brown Alexander , Li Feng , Guan Xinfu 
Published: June 2021

Background and ObjectiveGlucagon‐like peptides (GLP‐1/2) are co‐secreted from enteroendocrine L cells in the gut and preproglucagon neurons in the brainstem, and are attributed through the gut‐brain axis to improving glucose homeostasis and insulin sensitivity after gastric bypass surgery. It is unknown if GLP‐2 receptor (GLP‐2R) in the brainstem plays a physiological role in glucose homeostasis. Our objective was to determine the physiological relevance of the brainstem GLP‐2R to glycemic control.Methods and Results1) Generation of a brainstem‐specific Glp2r knockout mouse model: First, we revealed that GLP‐2R protein was localized to Phox2b neurons in the brainstem dorsal vagal complex (DVC). Second, we generated Phox2b‐specific Glp2r knockout (PKO) mice by the Cre‐LoxP system, i.e., crossing Phox2b‐cre mice with Glp2r‐loxP‐flanked (floxed) mice. Third, we validated that Glp2r was deleted specifically in the DVC Phox2b neurons. 2) Characterization of the mouse metabolic phonotype: First, we showed that the PKO mice fed regular chow (~25 g BW) had higher blood glucose baseline after overnight fast, and lower blood glucose exclusion after ip glucose tolerance challenge. Second, we quantified glucose homeostasis and insulin sensitivity in conscious mice using hyperinsulinemic euglycemic clamp (coupled with dual stable isotopic tracers 6,6‐2H2‐d‐glucose and 2H2O). The PKO mice at the basal condition had higher endogenous glucose production (5.10 ± 0.25 vs 8.02 ± 0.26 mmol/kg/h, respectively, for the WT and PKO mice). During hyperinsulinemic euglycemic clamp, the PKO mice had lower (−16%) glucose infusion rate (GIR) as resulted mainly from a decrease (−15%) in insulin‐mediated stimulation of peripheral glucose utilization (Rd).ConclusionsMice with Glp2r deletion in the brainstem DVC Phox2b neurons displayed hyperglycemia, glucose intolerance and insulin resistance, suggesting that the brainstem GLP‐2R plays an important, physiological role in the control of glucose homeostasis and insulin sensitivity.Support or Funding Information(Supported by the USDA CRIS grant 3092‐5001‐059 and the National NSFC grant 81570722).
Specificity of Compensatory Reserve vs SMO2, Lactate and ETCO2 as Early Predictors of Tolerance to Progressive Reductions in Central Blood Volume
Authors: Convertino Victor A , Howard Jeff T , Schiller Alicia M 
Published: June 2021

Circulatory shock resulting from acute uncontrolled hemorrhage remains a leading cause of death in both military and civilian trauma. Early, accurate and reliable prediction of decompensation is associated with effective intervention and improved clinical outcomes. Individual tolerance to reduced central blood volume can serve as a model to assess the sensitivity and specificity of vital sign measurements. The compensatory reserve (CR) is a measurement of this capacity. Measurements of muscle oxygen saturation (SmO2), lactate and end tidal CO2 (ETCO2) have recently gained attention as potentially superior prognostic tools for early assessment of the status of patients with progressive hemorrhage, but fail to identify individual tolerance. We hypothesized that CR would be able to better predict hemodynamic compensation and provide greater specificity and sensitivity than SmO2, lactate, or ETCO2. To test this hypothesis, we employed lower body negative pressure (LBNP) on healthy human subjects until symptoms of presyncope were evident. Receiver operating characteristic area under the curve (ROC AUC), sensitivity and specificity were used to evaluate the ability of CRI and SmO2, lactate and ETCO2 to predict hemodynamic decompensation. CR had the superior ability to predict decompensation compared to SM02, lactate and ETCO2 (ROC AUC: 0.91 vs. 0.68; 0.54; 0.70 p<0.0007), respectively. Similarly, CR also exhibited a greater sensitivity (0.87 vs. 0.65; .85, .89; p<0.05) and specificity (0.80 vs. 0.63, 0.29, 0.54, p<0.05). These findings support the notion that CR provides superior detection of hemodynamic compensation compared to measurements of tissue oxygenation, lactate and ETCO2.Support or Funding InformationUS Army MRMC Combat Casualty Care Research Program
Discovery and biosynthesis of hybrid polyketide‐nonribosomal peptides in nematodes
Authors: Butcher Rebecca A. , Feng Likui , Shou Qingyao 
Published: June 2021

Polyketides and nonribosomal peptides are well‐known for their antibiotic, antifungal, immunosuppressant, anti‐parasitic, and anti‐cancer activities. These structurally complex natural products are biosynthesized by polyketide synthases (PKSs) and nonribosomal synthetases (NRPSs), modular megasynthases that function in either an assembly‐line or iterative manner. Although polyketides and nonribosomal peptides are produced by many species of bacteria, fungi, and plants, they are extremely rare in metazoans. The genome of the nematode Caenorhabditis elegans is predicted to encode a huge, multi‐module, hybrid PKS/NRPS (PKS‐1), as well as a multi‐module NRPS (NRPS‐1), with unknown functions. Using comparative metabolomics and NMR spectroscopy, we identified and elucidated the chemical structure of nemamide, a hybrid polyketide‐nonribosomal peptide that is biosynthesized by PKS‐1/NRPS‐1 using both iterative and assembly‐line mechanisms. In recent work, we used CRISPR‐Cas9 to make specific mutations in pks‐1 and nrps‐1 in order to investigate the roles of specific enzymatic domains in the biosynthesis of nemamide. We showed that nemamide promotes survival during starvation‐induced larval arrest and facilitates recovery from arrest in response to food. Nemamide is produced in the CAN neurons under starvation conditions and downregulates insulin/ insulin‐like growth factor‐1 (IGF‐1) signaling. Our results uncover a novel mechanism by which animals respond to nutrient fluctuations to extend survival. Homologs of PKS‐1 and NRPS‐1 are present in most nematode species, including parasitic ones, and thus, nemamide likely plays an evolutionarily conserved role across nematode species.Support or Funding InformationWe acknowledge the support of the NIH/NIGMS, NSF, and Ellison Medical Foundation.
Application of Metabolomics and Fluxomics to Study Fatty Acid Synthesis in Alternative Crops
Authors: Alonso Ana Paula , Tsogtbaatar Enkhtuul , Cocuron Jean‐Christophe 
Published: June 2021

The goal of this research is to address the need for an alternative supply of jet fuel using native plants that do not compete with food crops. Pennycress (Thlaspi arvense), a member of the Brassicaceae, has been identified as a promising bioenergy crop: the chemical composition of its oil is ideally suited as a renewable source of biodiesel and jet fuel. This plant has tremendous potential for large‐scale oil production, has a short maturity time and is not a food crop. Pennycress can be commercially propagated as a winter annual, and has therefore the potential to be grown off‐season, complementing, rather than competing, the production of commodity crops, and serving as a winter cover crop. However, for this plant to become an economically viable source of specialty fuels, molecular and genetic resources need to be developed to facilitate and enhance oil production by breeding and/or genetic manipulation. This study aims to find potential biochemical step(s) that limit(s) oil synthesis, which will serve as targets for future crop improvement.To advance towards this goal, we determined the intracellular metabolite levels (metabolomics) in pennycress embryos at different stages of development. For this purpose, we have developed novel and highly sensitive methodologies using state‐of‐the‐art liquid chromatography tandem mass spectrometry (LC‐MS/MS). This metabolomics study highlighted the metabolites and pathways that were active in pennycress embryos and important for oil production. The contribution of each pathway to fatty acid synthesis in terms of carbon, reductant and energy provision is being assessed by measuring the carbon flow through the metabolic network. We are hence performing a 13C‐Metabolic Flux Analysis (fluxomics) in developing pennycress embryos to build a map of carbon flow through central metabolism. This study describes the combination of innovative tools that will pave the way for controlling seed composition in promising alternative crops.Support or Funding InformationJ.C.C was supported in part by the Agriculture and Food Research Initiative competitive grant # 2016‐67013‐24605 of the USDA National Institute of Food and Agriculture. This study is supported by the USDA‐U.S. Department of Energy Plant Feedstocks in Genomics for Bioenergy under Award Number DE‐SC0016490.
The Bioavailability and Distribution of Bovine Milk Exosomes is Distinct from that of their Cargos in Mice
Authors: Manca Sonia , Giraud David , Zempleni Janos 
Published: June 2021

BackgroundAmong extracellular vesicles, exosomes are of particular interest because of their roles in cell‐to‐cell communication, which is facilitated by the delivery of cargos such as RNAs, to recipient cells. Dietary exosomes in bovine milk are transported by intestinal and vascular endothelial cells in human and rodent cell cultures, and delivered to peripheral tissues in mice. Little is known about the fate of the cargos in milk exosomes following absorption.HypothesisRNAs in bovine milk exosomes are delivered to distinct peripheral tissues in mice.MethodsExosomes were isolated from bovine milk using ultracentrifugation. Exosome membranes were labeled using the fluorophore, DiR, whereas RNA cargos were labeled using EXO‐Glow Red in separate experiments. Identity, integrity, fine dispersion, and count of exosomes were assessed using transmission electron microscopy, western blots, and nanoparticle tracker (not shown). Exosomes were administered orally by gavage (1×1012 exosomes/g) in BALB/c mice. Absorption and distribution of exosomes and their cargos was monitored at timed intervals for up to 24 h using an iBox® Small Animal Imaging system and LiCor Odyssey CLx. At timed intervals, mice were euthanized and tissues were collected for densitometry analysis using VisionWorks®LS and Image Studio software.ResultsAt t = 12 h after administration of DiR‐labeled exosomes by gavage, only a faint DiR signal was detected in peripheral tissues (Fig. 1); labeled RNA was detected in all peripheral tissues with a preference for kidneys, brain, lungs and liver. Figure 2 depicts the densitometry analysis of labeled RNA, corrected by the signal produced by unconjugated Exo‐glow. At t = 24 h, the majority of absorbed exosomes accumulated in liver (Fig. 3), whereas the RNA cargo accumulated preferably in brain and kidneys (Figs. 4,5).ConclusionsBovine milk exosomes and their RNA cargos are bioavailable and accumulate in distinct tissues in mice. Our data suggest that bovine milk exosomes are disassembled in the intestinal mucosa, and RNA cargos are repackaged in endogenous exosomes for transfer to tissues, preferably brain and kidneys.Future studiesWe will conduct a comprehensive analysis of milk whole exosomes and RNA cargo kinetics in mice to obtain an accurate assessment of bioavailability, distribution, and elimination phenomena in health and disease.Support or Funding InformationSupport: NIFA 2015‐67017‐23181, NIFA 2016‐67001‐25301/NIH DK107264, NIH 1P20GM104320, the Gerber Foundation, and USDA Hatch Act and W3002.