bluuebunny/crossref_metadata_2025_split huggingface dataset:
Wnt signaling promotes IL‐33 expression in the colonic epithelium
Authors: Schumacher Michael A. , Thai Megan H. , Frey Mark R. 
Published: May 2022

BackgroundInterleukin‐33 (IL‐33) released from the colonic epithelium promotes type 2 innate lymphoid cell activity as part of the colonic injury response. We have previously shown that the intracellular signaling regulator, Sprouty2, restrains colonic IL‐33 via a GSK3β‐dependent mechanism. GSK3β can regulate canonical Wnt signaling, but the role of Wnt signaling and the specific components of this pathway involved in driving IL‐33 are unknown. We tested the hypothesis that activation of discrete arms of Wnt signaling induce epithelial IL‐33 expression.MethodsBulk RNA sequencing was performed on colons from mice with an intestinal epithelial‐specific Sprouty2 deletion (Spry2IEKO) or littermate controls to determine the effect of Sprouty2 on Wnt targets. Colonoid and young adult mouse colon (YAMC) epithelial cell cultures were treated with Wnt ligands. Small molecule inhibitors were used to inhibit CBP‐ or p300‐mediated Wnt signaling arms (ICG‐001 or IQ‐1 respectively), in YAMC cells. IL‐33 levels were assayed by qPCR and ELISA.ResultsSpry2IEKO mice have increased colonic transcript levels of the Wnt target genes Mmp7 (p<0.01), Irx3 (p<0.05), Mapk10 (p<0.05), and Jag1 (p<0.05), correlating with the increased expression of IL‐33 that we have previously reported in these mice. Pathway analysis suggests specifically altered CBP/p300‐mediated Wnt signaling in Spry2IEKO colons. Treatment of wild‐type colonoids or YAMC cells with the Wnt ligands Wnt3a and R‐spondin induced IL‐33 expression by 2.1‐fold and 2.3‐fold respectively (p<0.05). Pre‐treatment of cells with ICG‐001 blocked the Wnt‐induced increase in IL‐33 at both the RNA and protein level, whereas IQ‐1 had no effect.ConclusionCBP‐mediated Wnt signaling promotes colonic epithelial expression of IL‐33 in vitro. Elevated Wnt activity in Spry2IEKO mice is associated with elevated IL‐33 levels. Dysregulated Wnt signaling may result in impairment of the injury response in inflammatory disease.
Tacrolimus induces ligand independent TGFβ receptor signaling to promote renal fibrosis
Authors: Ume Adaku , Yesomi‐Wenegieme Tara , Williams Clintoria 
Published: May 2022

BackgroundAlthough calcineurin inhibitors (CNIs) such as tacrolimus and cyclosporin have dramatically improved the quality of patient care, long‐term therapy causes irreversible damage to the kidneys in the form of renal fibrosis. These morphologic changes ultimately lead to a decline in renal function and can progress to end‐stage renal failure. These detrimental outcomes present a critical need to identify the driving mechanisms by which CNIs cause renal damage.ObjectiveIt is well established that TGFb is a major contributor to CNI‐induced renal fibrosis. However, the underlying mechanisms remain unknown. The objectives of this study are to 1) investigate whether TGFb secretion is required to stimulate TGFb receptor signaling in a model of CNI‐induced renal fibrosis and 2) investigate whether calcineurin plays a critical role in regulating TGFb receptor activity.Experimental DesignTo examine the role of calcineurin inhibition in altered TGFb receptor signaling, wild type mice were treated with either vehicle (0.01% ethanol) or 10 mg/kg tacrolimus for 7 days. To confirm in vivo findings, wild‐type mouse renal cortical fibroblasts were treated with either vehicle (0.01% ethanol) or 1nM tacrolimus for 24 hours in the presence and absence of anti‐TGFb neutralizing antibodies. TGFb receptor expression and activation, TGFb receptor downstream signaling mediators, profibrotic markers and calcineurin activity were analyzed.ResultsFindings demonstrate that tacrolimus‐induced loss of calcineurin activity is accompanied with enhanced TGFb receptor activation and signaling. Notably, increasing concentrations of anti‐TGFb neutralizing antibodies failed to abolish aberrant TGFb signaling and increased expression of profibrotic markers.ConclusionsTogether, these results demonstrate that 1) CNIs promote ligand‐independent TGFb signaling and 2) calcineurin plays a functional role in regulating TGFb receptor activity.SignificanceThis study elucidates a direct pharmacological mechanism by which CNIs promote renal fibrosis and provides a physiological role of calcineurin in regulating TGFb receptor activity. Further insight into the role of calcineurin in regulating TGFb receptor activity could improve the long‐term outcomes of patients on chronic CNI therapy.
Inhibition of Branched‐Chain Ketoacid Dehydrogenase Kinase (BDK) Attenuates BCKA Induced Suppression of Insulin‐Stimulated Glucose Uptake in Muscle Cells
Authors: Mann Gagandeep 
Published: May 2022

ObjectivePlasma levels of branched‐chain amino acids (BCAAs) and their metabolites, branched‐chain ketoacids (BCKAs) are increased in insulin resistance, a condition that can lead to type 2 diabetes mellitus (T2DM). BCAA catabolic enzymes are downregulated in diabetes and obesity in skeletal muscle. We previously showed that leucine and KIC suppressed insulin‐stimulated glucose uptake in L6 myotubes. We have also shown that knocking down branched‐chain ketoacid dehydrogenase (BCKD), an enzyme that decarboxylates BCKAs, suppressed insulin‐stimulated glucose uptake. The objective of this study was to analyze the effect of increasing BCAA catabolism on measures of insulin sensitivity. We hypothesized that upregulating BCAA catabolism increases insulin‐stimulated glucose transport and attenuates BCKA induced insulin resistance.MethodsL6 myotubes were starved of serum‐ and amino acids with vehicle (DMSO) or BDK inhibitor BT2 supplementation for 3 hours. Then myotubes were supplemented with or without BCKAs (200 uM) for 30 minutes. After, cells were incubated with or without insulin (100 nM) for 20 minutes. They were then harvested for immunoblotting or used for glucose transport assay.ResultsBDK inhibition had no effect on insulin‐stimulated glucose uptake. BCKAs suppressed insulin‐stimulated glucose uptake by 31% in control cells (p>0.05; n=3); this suppression was attenuated in cells in which BDK was inhibited. BDK inhibition also reduced KIC‐induced IRS‐1Ser612 and S6K1Thr389 phosphorylation by 28% and 60% respectively but had no effect on AktSer473 phosphorylation.ConclusionsBDK inhibition attenuated BCKA‐induced suppression of insulin‐stimulated glucose uptake, suggesting that BCAA metabolism may be implicated in the pathogenesis of insulin resistance, and is worth further investigation.
Stress‐Induced Gut Dysmotility is Associated with Altered Intestinal BDNF‐TrkB Signaling
Authors: Singh Alpana , Zhang Chengxiu , Kulkarni Subhash 
Published: May 2022

IntroductionWhile stress is associated with incidence of gut dysmotility disorders, mechanisms by which stress causes dysmotility are not well understood. In the central nervous system (CNS), stress causes behavioral deficits by increasing the levels of Glucocorticoid hormone to suppress molecular signaling between brain‐derived neurotrophic factor (BDNF) and its receptor tropomyosin receptor kinase B (TrkB). We hypothesize that stress causes a Glucocorticoid‐mediated reduction in BDNF‐TrkB signaling in the enteric nervous system (ENS), whose neurons, which modulate diverse gut functions, reside entirely within the gut wall.MethodsGut motility was assayed by analyzing the whole gut transit time (WGTT) of an orally gavaged carmine red dye in adult mice. Effect of stress was tested by comparing WGTT of Stress‐Induced Dysmotility (SID) mice, wherein age‐matched adult male mice were restrained for one hour, with that of unrestrained control mice. The effect of Glucocorticoids was tested by comparing WGTT of age‐matched adult C57/BL6 male mice dosed with Dexamethasone (DEX, a Glucocorticoid receptor agonist, intra‐peritoneal 5mg/kg), with that of saline‐dosed control mice. Effect of DEX on enteric BDNF levels (using R&D ELISA kit) was tested by using total proteins isolated from preparations of adult murine longitudinal muscle‐myenteric plexus (LM‐MP) tissues cultured with and without DEX (1 µM) for 4 hours. TrkB exists in full‐length and truncated (TrkB.T1) isoforms, and the presence of TrkB.T1 in ENS neurons was observed by immunofluorescence analyses of ENS tissue of TrkB.T1‐V5 transgenic mice. Involvement of TrkB.T1 isoform in SID was tested by comparing WGTT of male transgenic mice containing partial (T1‐Het) or complete loss (T1 KO) of TrkB.T1. Differences were compared for statistical significance using Student’s t‐test and Mann‐Whitney test.Results5 consecutive days of 1 hour restraint caused a significant delay in WGTT of adult C57/BL6 SID mice, when compared to controls (Table 1). DEX caused a significant delay in WGTT, when compared to control mice (Table 1). DEX treatment caused a significant reduction in BDNF levels (Table 1). TrkB.T1‐V5 mice show presence of TrkB.T1 on adult enteric neurons (Fig 1). A single hour of restraint caused a significant delay in WGTT in adult T1 KO mice, when compared with littermate T1 Het mice (Table 1).DiscussionRestraint stress and DEX caused similar delay in gut transit in wildtype mice, and since DEX decreased gut BDNF levels, we infer that stress‐induced increase in glucocorticoid signaling delayed gut transit by suppressing enteric BDNF levels. Increased susceptibility of T1 KO mice to SID highlights the important role of the truncated TrkB.T1 receptor in maintenance of normal gut motility during acute and moderate stress. Thus, our data implicates altered BDNF‐TrkB.T1 signaling in the etiology of Stress Induced Dysmotility.
Characterization of Hepatic Injury During the Azoxymethane Model of Acute Liver Failure
Authors: McMillin Matthew , Jhawer Ashwin , Frampton Gabriel , Bhattarai Shadikchhya M. , Troyanovskaya Eleonora 
Published: May 2022

IntroductionAcute liver failure (ALF) results from a significant loss of liver function and has few effective treatments and poor clinical outcomes. ALF is associated with neurological dysfunction, called type A hepatic encephalopathy (HE), which greatly increases mortality. One of the commonly used models of type A HE is the azoxymethane (AOM) mouse model, which is associated with hepatocyte necrosis, neuroinflammation and oxidative stress. That being said, there have been no extensive analyses into the hepatic pathology associated with AOM‐induced liver injury. Therefore, the aim of this study was to characterize the hepatic injury during the AOM model of type A HE.MethodsC57Bl/6 mice were administered 100 mg/kg of AOM and were euthanized at various stages of neurological decline and tissue was collected. These stages include normal (vehicle‐treated), prior to neurological decline (pre), minor neurological decline (minor), significant neurological decline (major), and coma. Liver injury was assessed by measuring serum ALT and AST levels as well as liver H&E staining. Serum albumin, bile acids, glutathione (GSH) and ammonia were measured to determine liver function. Various aspects of oxidative stress, inflammation and cell death were assessed by measuring CYP2E1, Nrf2, SOD1, CCL2, IL‐1β, IL‐6, LC3‐II, phosphorylated MLKL, cleaved caspase 3, p21 and TGFβ1 expression using real‐time PCR, immunoblotting, ELISA or immunohistochemistry. All data were analyzed using GraphPad Prism software with results presented as mean +/‐ SEM. Differences between two groups were compared using the Student’s t‐test and differences between three groups were determined using ANOVA.ResultsAOM‐treated mice had significant elevations of serum ALT and AST and hepatic necrosis as neurological decline progressed. CYP2E1 expression decreased throughout the stages of neurological decline, with the greatest suppression in mice at coma. Measures of oxidative stress and signaling had the largest increase when mice were at major neurological decline, which was also associated with the largest increase of inflammatory chemokines and cytokines. Measures of necroptosis, apoptosis and cellular senescence were also dysregulated, with coma mice showing the highest degrees of cell death and increased cellular senescence.ConclusionsThese data demonstrate that AOM‐treated mice have increased oxidative stress and inflammation resulting from the metabolic dysregulation from AOM toxicity. This leads to cell death through multiple different mechanisms and an impairment of regeneration due to increased cellular senescence.
Oxidative Stress Biomarkers in Patients with End Stage Renal Disease
Authors: Sharma Rishima , Sridharan Divya , Bansal Vinod , Hoppensteadt Debra , Siddiqui Fakiha , Iqbal Omer , Laddu Atul , Fareed Jawed 
Published: May 2022

IntroductionCKD5 is the final stage of chronic kidney disease in which the patient has end‐stage renal disease (ESRD) and their kidneys are about to fail. One of the larger factors that contributes to the progression of renal failure is oxidative stress. Oxidative stress occurs when there’s an imbalance between free radicals and antioxidants in the body. DNA, proteins, and lipids can become damaged as a result of this imbalance. Stable derivatives of oxidants are used as biomarkers of oxidative stress. When renal damage occurs, likely due to oxidative stress, the levels of biomarkers will likely be more elevated in ESRD patients compared to control samples. L‐FABP is a cytoplasmic protein involved in facilitating and transporting long‐chain polyunsaturated fatty acids and is very significant in many research experiments related to CKD5. The purpose of this research project is to explore the relationship between oxidative stress biomarkers in ESRD patients and how the presence of ESRD contributes to the concentration of these biomarkers in patients. Hypothesis: The levels of oxidative stress biomarkers in ESRD patients will be elevated in comparison to the concentrations of them in control patients. Materials andMethodsBlood samples were collected from CKD‐5 patients and stored in sodium citrate tubes and centrifuged at 3000 rpm to separate the plasma. Normal human plasma (NHP) was obtained from a commercial vendor. The CKD‐5 and NHP were used to profile the biomarkers through ELISA kits. The statistical and correlation analysis was done through PRISM GraphPad software and IBM SPSS. Multiple statistical analysis tests were run to determine the correlation among the biomarkers and whether the data was statistically significant. Correlation analysis, Mann‐Whitney U tests, normal distribution, skewness tests, and quartile analysis tests were done through PRISM Graphpad Software and IBM SPSS.ResultsThe results are calculated in terms of group means. L‐FABP showed statistically significantly higher values in ESRD patients compared to control patients. L‐FABP was found to be 106599.4 pg/ml in ESRD v.s. 5116.33 pg/ml in NHP. PAI‐1a was found to elevated in ESRD (17.9 ng/ml) compared to NHP (10.47 ng/ml), while NO also showed higher values in ESRD (34.74 umol/L) compared to NHP (13.79 umol/L). There was a weak correlation between the biomarkers, but ESRD patients showed highly elevated levels of L‐FABP in comparison to NO and PAI‐1.ConclusionThese results suggest that impaired renal function and kidney damage contribute to the marked increase of L‐FABP in ESRD patients. Moreover, the increase in PAI‐1 and NO suggest the upregulation of oxidative stress and hemostatic deficit.
Metal Substrate Translocation and Transport Mechanism of the Sinorhizobium melilotiP1B‐5‐type ATPase Nia Revealed by In‐vitro Transport Assays in Proteoliposomes
Authors: Abeyrathna Nisansala S. , Abeyrathna Sameera S. , Meloni Gabriele 
Published: May 2022

P1B‐type ATPases are a class of transmembrane transporter proteins responsible for metal substrate translocation, and export across cellular membranes. These ion pumps are classified into seven sub‐families (P1B‐1‐P1B‐7‐type) based on conserved amino acid motifs in their transmembrane helices that appear to control each sub‐family’s substrate selectivity. The P1B‐5‐type sub‐family is widely found in nitrogen fixing bacteria, such as Sinorhizobium meliloti,which form a unique symbiotic relationship with legumes. This symbiotic relationship is highly dependent on the nutrition trafficking between the two species, including a tight control of transition metal homeostasis. The P1B‐5‐type ATPase Nia from S. Meliloti is recognized as a putative iron and nickel transporter, and besides possessing the typical P‐type ATPase domains and topology, it features of a C‐terminal hemerythrin‐like domain containing a di‐iron binding site involved in oxygen sensing, as proposed by in‐vivo studies. However, the substrate transport across lipid bi‐layers, the overall mechanism for cargo translocation and the transport kinetic parameters remain elusive due to lack of molecular tools to study putative substrate transport across membranes in real‐time.In this study, we coupled metal‐stimulated ATPase activity assays with an experimental platform based on multiple fluorescence sensor probes, to study substrate transport mechanism and translocation kinetics in real‐time with Nia reconstituted in a native‐like environment. We expressed and purified Nia in detergent micelles and subsequently incorporated it in artificial small unilamellar lipid bi‐layer vesicles (proteoliposomes) which allow to study the substrate specificity and transport mechanism in real‐time. The proteoliposomes were used as an in‐vitrotool to determine the kinetic parameters for metal transport by encapsulating fluorescent detector probes featuring turn‐on florescence signal upon substrate ion binding and translocation.The study revealed that Fe2+, Ni2+ and Co2+are actively translocated by Nia and the kinetic parameter analysis demonstrated that Fe2+ is the preferential Nia substrate. In parallel, by encapsulating in the proteoliposome lumen pH and membrane sensitive fluorescent probes we are also revealing the chemistry underlying the mechanism of transport and potential ions involved as counter‐transported cargos in P1B‐5‐type ATPase catalytic cycle, with the aim of revealing unique molecular features for this subclass of transmembrane metal pumps.
Emerging from the Pandemic: Q‐Method Analysis of the Return to Normalcy in Undergraduate Anatomy
Authors: Metla Sai Gayathri , Akhtar‐Danesh Noori , Saini Jessica , Bayer Ilana , Wainman Bruce C. 
Published: May 2022

IntroductionAs a result of the Covid‐19 pandemic, anatomy education has had to adopt online modes of delivery. Previous research conducted on student views towards an online undergraduate anatomy and physiology course revealed areas of both strong preference (e.g., asynchronous lectures) and strong dislike (e.g., virtual specimens) within the online format. As restrictions are lessened and students transition back into in‐person learning, there exists the unique opportunity to examine the views of a consistent cohort of students towards virtual and in‐person modes of course delivery.ObjectiveThe current study seeks to compare the views of a single cohort of students experiencing both the online and in‐person anatomy and physiology course. To avoid the considerable drawbacks of course evaluations relying on Likert scales and traditional qualitative analysis, Q‐methodology is being used to measure the different perspectives of students between the two teaching modalities.MethodQ‐methodology will be used to assess opinions of students enrolled in the same undergraduate introductory anatomy and physiology course.A list of 41 opinion‐based statements regarding anatomy education was compiled. Students will sort the statements in a quasi‐normal grid based on their degree of agreement with the statements. The rankings will then undergo a by‐person factor analysis which categorizes students with shared perceptions into groups allowing educators to better understand and respond to the needs of students. Students will have the opportunity to respond to the survey at two points: at the end of the Fall 2021 semester after a primarily online mode of delivery, and at the end of the Winter 2022 semester following a primarily in person mode of delivery.ResultsData collection and analysis are anticipated to be completed in March of 2022.ConclusionWe hypothesize that Q‐methodology will enable us to discover student views specifically relevant to their experience in both the online and in‐person format of the course. We predict that this study will help identify areas of strength in both virtual and in‐person learning environments. These findings will be useful in integrating the strengths of both learning environments when developing future course offerings.
Effect of Maximal and Submaximal Muscle Activation on Corticospinal Excitability in Humans
Authors: Paish Alexander D. , Zero Alexander M. , Rice Charles L. 
Published: May 2022

IntroductionFollowing short duration (5‐10s), high intensity (≥70%MVC) muscle activation, there is an enhancement of muscle contractile properties, termed post action potentiation (PAP). During PAP induced by voluntary or electrically stimulated conditioning contractions (CC) it has been reported that the corticospinal silent period (SP), assessed by transcranial magnetic stimulation (TMS), is elongated. Although a coexistence between the two occurs, the direct effect of PAP on corticospinal excitability has not been systematically evaluated to help determine whether a direct link exists. The purpose was to assess SP duration following potentiating and non‐potentiating voluntary and electrically stimulated contractions. We hypothesized that maximal and submaximal CCs inducing PAP and those not inducing PAP enhancement, respectively, will prolong SP durations albeit to a lesser degree at submaximal intensities.MethodsTen healthy young (three females; 18‐35y) individuals free of neurological issues to date have participated in the study. Subjects were seated with their right forearm pronated and their hand was fixed to a custom finger abduction force dynamometer. Monopolar surface electromyography signals were recorded from the first dorsal interosseous muscle (FDI) with self‐adhering Ag/AgCl electrodes. Peripheral stimulated twitches and tetani (50Hz) were evoked over the ulnar nerve at the wrist via a pair of small readjustable electrodes. Maximal M‐waves (M‐max) and baseline levels of (voluntary and stimulated) PAP and force were obtained for each subject. Electrically stimulated and voluntarily induced CCs of 10s were performed at ~95% MVC and ~35% MVC for a total of four contraction conditions. Before and after these contractions SP times were obtained during the plateau of ~25% MVC held for three seconds. To obtain SP times the TMS output was set to elicit a response ~75% of M‐max. The four conditions of voluntary or stimulated, maximal and submaximal CCs were pseudo‐randomized and repeated four times per subject. The nearest three values from each subject were averaged and compared as a normalized percent change from baseline.ResultsFollowing both maximal voluntary and stimulated CCs, mean twitch torque was enhanced similarly (PAP ~180%) with no PAP at the submaximal levels. Mean change in SP duration following maximal voluntary CCs was ~14% longer than control values, while maximal tetanic CCs similarly prolonged the SP by ~13%. Both voluntary and stimulated submaximal contractions prolonged SP duration similarly by ~5% and ~6%, respectively.ConclusionThese findings indicate that corticospinal inhibition assessed as SP elongation is present not only when the muscle is enhanced under PAP but also occurs following contractions inducing no PAP. This suggests that the muscle activation required to induce PAP likely causes the observed corticospinal inhibition, not the enhanced muscle properties of PAP per se.