bluuebunny/crossref_metadata_2025_split huggingface dataset:
Functional roles of neuromedin B and gastrin‐releasing peptide in regulating itch and pain in the spinal cord of primates
Authors: Ding Huiping , Kiguchi Norikazu , Park Sun Hee , Kishioka Shiroh , Shiozawa Yusuke , Peters Christopher M , Ko Mei-Chuan 
Published: April 2020

Neuromedin B (NMB) has been demonstrated to elicit itch and pain in rodents. However, the functional role of NMB and its cognate receptor (NMBR) in regulating itch and pain in the primate spinal cord is unknown. The aim of this study was to determine the functional profile of spinal NMB and compare its behavioral effects with those elicited by spinal gastrin‐releasing peptide (GRP) in rhesus monkeys (Macaca mulatta). Following intrathecal administration, GRP (3–10 nmol) dose‐dependently elicited robust scratching responses. In contrast, intrathecal NMB (30–200 nmol) only elicited mild scratching responses. Intrathecal GRP‐ and NMB‐elicited scratching activities were attenuated by a GRP receptor (GRPR) antagonist (RC‐3095) and a NMBR antagonist (PD168368), respectively. Unlike intrathecal substance P (100 nmol) producing thermal allodynia manifested as a reduced tail‐withdrawal latency in 46C water, intrathecal administration of GRP or NMB (1–100 nmol) did not change the thermal nociceptive threshold of primates. In addition, intrathecal administration of a mu opioid receptor agonist, DAMGO (3 nmol), inhibited capsaicin‐induced thermal allodynia. Unlike DAMGO, both RC‐3095 and PD16838 (100–300 nmol) intrathecally did not produce antiallodynic effects. Taken together, these pharmacological findings indicate that spinal NMB‐NMBR and GRP‐GRPR systems have a minimum role in the neurotransmission of pain and both spinal GRPR and NMBR function independently to elicit itch/scratching responses in primates.Support or Funding InformationThe US‐PHS grants AR064456 and AR069861
Halotolerance in the Southeastern Michigan Soil Microbiome
Authors: Bowen John , Zwiesler-Vollick Julie , Siddiq Fauzia 
Published: April 2020

Many communities in the Midwest and Northeastern United States use salt to treat icy roads in the winter. Studies have shown that the many years of salt application have had harmful effects on freshwater ecosystems, roadside flora and fauna, and overall wetland biomes. We were interested in investigating the impact that road salt use has had on the soil microbiome. Specifically, we sought to determine if microbes that reside near salted roads have become more tolerant of salt, or more halotolerant, than microbes that reside far from roads. We accomplished this by first collecting soil samples from near roads (exposed to road salt) and far from roads (not exposed to road salt) from various parks across Southeastern Michigan. Salinity measurements were taken by mixing 1 g soil per 1 mL distilled water. We then
used these soil samples to compare the number of colony forming units on 5% NaCl nutrient agar plates and standard nutrient agar plates, investigate growth rate differences between these microbes in 5% NaCl nutrient broth, and sequence various salt intolerant, halotolerant, and halophilic microbes. This allowed us to uncover relationships between the soil salt concentrations, the microbiome distances from roads, and the halotolerance of the microbiomes, as well as to gain some insight into the microbiome population makeup. Preliminary data suggests that there may be a correlation between the salt concentration in soil (ppm) and the overall soil microbiome halotolerance. This indicates that microbiomes closer to roads (which we have shown to have greater soil salt concentration) are likely more halotolerant than microbiomes further from roads (which we have shown to have lesser soil salt concentration). We will investigate the growth rate differences between halotolerant and nonhalotolerant strains as well as determine the genus of the microbes to provide further insight into the composition of the microbiome. This study suggests that the application of road salt is not only affecting wetland biomes but may also be having an effect on the soil microbiome as well.Support or Funding InformationSpecial thanks to Lawrence Technological University for laboratory space, equipment, and funding to carry out this study.
Subcellular Localization of Prohibitin 1 is Mediated by Akt Phosphorylation at Thr258
Authors: Thompson Austin , Martin Cole , Dong Christopher , Snyder Zachary , McClung J. Keith 
Published: April 2020

The overexpression of intratumoral Prohibitin 1 (PHB1), an evolutionarily conserved gene, has been associated with a 5‐year survival rate of less than 35% in muscle‐invasive bladder cancer patients. When localized to the mitochondria, PHB1 promotes tumor cell proliferation by supporting mitochondrial integrity, whereas nuclear PHB1 assists in inducing cell cycle arrest. As the subcellular localization of PHB1 dictates its function, the mechanisms governing the change in PHB1’s subcellular localization could be exploited to develop novel cancer therapeutics. PHB1 has a mitochondrial signal sequence, but previous work suggested that phosphorylation of PHB1 by Akt at Thr258 is required for mitochondrial localization. Our study confirms and expands this work by using immunofluorescence microscopy to visualize the subcellular location of PHB1 in a muscle‐invasive bladder cancer cell line (5637) after the use of Akt inhibitors. Akt inhibition liberated PHB1 from the mitochondria causing a significant increase in nuclear localization. However, blocking Akt disrupts multiple cellular processes, including those involving the function of PHB1, presenting a potentially confounding explanation for these results. To negate the off‐target effects of Akt inhibition, site‐directed mutagenesis was used to create two mutant PHB1 cDNA plasmids. In the first plasmid, Thr258 was mutated into an aspartate, T258D, which mimics Akt phosphorylation. The second mutation created an alanine, T258A, non‐phosphorylatable. Transfection of T258D showed elevated mitochondrial localization of PHB1, while non‐phosphorylatable T258A pooled in the cytoplasm and failed to enter the mitochondria. Therefore, we concluded that phosphorylation of Thr258 by Akt increases the mitochondrial localization of PHB1.Support or Funding InformationR.J. McElroy Trust Student/Faculty Research FundWartburg College Undergraduate Research FundWartburg College Biology DepartmentMany generous donors to the Group Alpha Research Fund
Metabolic Regulation of Stem Cell Fate and Function
Authors: Shapira Suzanne N. , Christofk Heather R. 
Published: April 2020

The overarching goal of this project is to understand how diet‐derived nutrients regulate stem cell metabolism and downstream fate decisions in the setting of normal development and disease. Quiescent adult stem cells typically favor glycolytic metabolism over the oxidative metabolism utilized by differentiating cells. Importantly, manipulating stem cell metabolism is sufficient to alter cell fate decisions. While the serum concentrations of endogenous metabolites such as glucose and lactate are under homeostatic constraints, diet can cause vast fluctuations in circulating levels of certain metabolites that serve as co‐factors for chromatin and DNA modifying enzymes. As such, exogenous nutrients can control the epigenome and gene expression programs, thus altering differentiation and cell fate. However, little is known about how changing nutrient levels will affect the metabolism and chromatin status in the adult stem cell populations. To address this question, we have adapted a recently described mouse liver organoid system. The liver coordinates whole‐body metabolism and detoxification, and therefore is acutely tuned to respond to dietary changes. Liver organoids generate 3D assemblies that mirror the native physiology of the tissue and self‐renew in vitro due to the activity of Wnt‐responsive stem cells that reside in the hepatic ductal compartment. We have found that treatment with physiologically attainable concentrations of ascorbic acid inhibits hepatic organoid expansion in a dose‐dependent manner. Ascorbic acid, commonly known as Vitamin C, is a water‐soluble antioxidant that is obtained exclusively through the diet in humans. Ascorbic acid is an essential co‐factor for the Fe‐ and α‐ketoglutarate dependent dioxygenase (α‐KGDDs) class of enzymes, which includes collagen hydroxylases, prolyl hydroxylases, and epigenetic erasers such as DNA and histone demethylases. To determine if activation of α‐KGDDs enzymes suppresses organoid expansion, we treated hepatic organoids with a cell permeable form of α‐KG and observed growth inhibition identical to that observed with ascorbic acid treatment. α‐KG/succinate ratios and expression of the ascorbate transporters increases in differentiated hepatic organoids compared to cultures maintained in expansion media, suggesting that ascorbic acid may preferentially accumulate and function during terminal differentiation. Taken together, we propose that ascorbic acid acts as a cofactor for α‐KGDD enzymes in order to restrain stem cell activity and may play a role in promoting differentiation to the hepatic lineage.Support or Funding InformationS.N.S is supported by a postdoctoral grant from the Eli and Edythe Broad Stem Cell Research Center at UCLA. H.R.C is supported by a CTSI seed grant from the David Geffen School of Medicine at UCLA.
Peripheral Blood Mononuclear Cell‐Derived Superoxide Production, Spontaneous Baroreflex Sensitivity, and Vascular Function in Chronic Kidney Disease Patients
Authors: Kaur Jasdeep , Sabnani Manoj K. , Stephens Brandi Y. , Nandadeva Damsara , Weidanz Jon A. , Fadel Paul J. 
Published: April 2020

Patients with chronic kidney disease (CKD) have an increased risk of cardiovascular morbidity and mortality. Previous studies have shown that CKD patients have high oxidative stress, which may be a potential underlying mechanism contributing to vascular dysfunction and impaired baroreflex sensitivity in this patient group. However, the exact mechanism(s) leading to elevated oxidative stress in CKD remains unclear. Peripheral blood mononuclear cells (PBMCs) have been identified as a primary source of NADPH oxidase‐derived superoxide production, leading to elevated systemic oxidative stress. PBMCs can cross the blood brain barrier and increase oxidative stress centrally, which could potentially reduce arterial baroreflex sensitivity (BRS) in these patients. Indeed, there is an emerging body of evidence indicating that elevated oxidative stress impairs BRS. Herein, we tested the hypothesis that CKD patients have greater superoxide production in PBMCs that is associated with impaired vascular function and attenuated BRS. To test this hypothesis, we studied 7 CKD patients stage 3 and 4 (age = 62 ± 3 years, BMI = 35 ± 2 kg/m2, estimated glomerular filtration rate (eGFR) = 43 ± 7 ml/min/1.73 m2) and 5 age‐matched controls (age = 57 ± 3 years, BMI = 25 ± 1 kg/m2, eGFR = 80 ± 5 ml/min/1.73 m2). PBMCs were isolated from whole blood and intracellular superoxide production in PBMCs was measured using dihydroethidium fluorescence. Resting heart rate (ECG) and arterial blood pressure (finger photoplethysmography) were measured for 10 minutes and spontaneous cardiac BRS was determined using the sequence technique. In addition, central artery stiffness (pulse wave velocity, SphygmoCor) and brachial artery flow‐mediated dilation (duplex Doppler ultrasound) were measured to assess vascular function. CKD patients had significantly elevated PBMC‐derived superoxide production compared to controls (CKD = 32769 ± 4067 vs. controls = 18421 ± 3515 relative fluorescence units; p = 0.03). However, cardiac BRS was not different between the groups (18.2 ± 3.5 in CKD vs 15.4 ± 1.5 ms/mmHg in controls; p = 0.35). Central artery stiffness appeared greater (CKD = 7.8 ± 0.6 vs. controls = 6.3 ± 0.2 m/s; p = 0.07), and endothelial function lower in CKD patients (CKD = 3.5 ± 0.6% vs. controls = 5.8 ± 1.4%; p = 0.19) but neither reached statistical significance in the cohort studied thus far. These preliminary findings suggest that CKD patients exhibit elevated PBMC‐derived superoxide production, which may be one of the mechanisms contributing to impaired vascular function in this population. However, this elevated oxidative stress was not associated with impaired spontaneous cardiac BRS in CKD patients.Support or Funding InformationSupported by NIH R01 HL127071
Nuclear Pregnane X Receptor Transduces Glucose Signal to Suppress Estrogen Sulfotransferase Gene in Human Liver Cells
Authors: Hu Hao , Negishi Masahiko 
Published: April 2020

Estrogen sulfotransferase (SULT1E1) metabolically inactivates estrogen and SULT1E1 expression is tightly regulated by multiple nuclear receptors. Previously, it was found that nuclear receptors HNF4a and RORa form as complexes binding to the SULT1E1 enhancer (−943/−922 bp) to activate gene expression in high glucose (450 mg/dL) treated human liver cells. However, the mechanism by which low glucose signal (40 mg/dL) mediated SULT1E1 suppression in human liver cells remain mysterious. Here, we have demonstrated that low glucose signal stimulates PXR phosphorylation at Ser350 on its ligand binding domain, preventing its heterodimerization with RXR and enabling it to interact with the HNF4a/RORa binding complexes on the enhancer, thereby suppressing SULT1E1 gene transcription. SULT1E1 was significantly repressed at both mRNA and protein levels in low glucose cultured human liver cells and this suppression was found to be PXR dependent. Vaccinia‐related kinase 1 (VRK1) was found to phosphorylate PXR at Ser350 in low glucose treated cells. Transient transfection analysis of SULT1E1 promoter‐luciferase reporter genes revealed that phosphorylated PXR significantly suppressed SULT1E1 promoter activity via the enhancer. Chromatin immunoprecipitation and gel shift assays elucidated that phosphorylated PXR bound to the SULT1E1 enhancer and disrupted the formation of the HNF4a/RORa binding complexes on it at low glucose condition. The physical interactions among these nuclear receptors were subsequently demonstrated by a series of co‐immunoprecipitation assays. Our study has shown that xenobiotic sensing nuclear receptor PXR transduces low glucose signal by its conserved phosphorylation to suppress SULT1E1 expression in human liver cells.Support or Funding InformationThis work was supported by National Institutes of Health intramural research program Z01ES1005‐01.
High Altitude Hypoxia Induces Cellular Immaturity of Pulmonary Arteries in the Fetal Lamb: Assessment of Protein Biomarkers
Authors: Jones Breanna , Leslie Eric , Alvarez Rafael , Dasgupta Chiranjib , Zhang Lubo , Wilson Sean M. 
Published: April 2020

Pulmonary Arterial Hypertension (PAH) in newborns can be mediated by intrauterine hypoxia and which can cause significant aberrations on vascular structure and function. Previous evidence suggests that long term hypoxic stress can induce phenotypic modifications to various vascular beds, including those of the lung. We are now seeking to interrogate the influence of intrauterine hypoxia on pulmonary vascular phenotypes and to test the hypothesis that intrauterine hypoxia causes the vasculature to shift from a mature to an immature phenotype. This was evaluated by examining protein expression using proteomic analyses on pulmonary arteries isolated from near term fetal lambs that were exposed to intrauterine high altitude hypoxia (3801 m) for the latter 110+ days of pregnancy or those gestated near sea level. Protein networks were created via Ingenuity Pathway Analysis (IPA) and Search Tool for the Retrieval of Interacting Genes/Proteins (STRING). Intrauterine hypoxia caused a number of significant changes including reducing the expression of smooth muscle myosin, fibronectin, and collagen along with upregulation of proteins involved in nucleotide synthesis, mitosis/cell proliferation, as well as cell adhesion and migration. The observed changes are consistent with a hypoxia‐induced phenotypic shift towards cells being more synthetic, proliferative, and migratory. Such findings provide insight into the mechanistic underpinnings related to the development of pulmonary hypertension due to gestational hypoxia. This work opens new avenues of investigation to determine common signaling pathways affected by intrauterine hypoxia with the aim of discovering the fundamental mechanisms driving hypoxia‐induced remodeling and senescence.Support or Funding InformationSupported in part by NIH grants HD083132 (LZ), HD098477 (SMW), RA was a Apprenticeship Bridge to College summer research fellow through the LLU Center for Health Disparities Research with funding from P20MD006988 (M. DeLeon) from the National Institute of Health Disparities and Minority Health of the National Institutes of Health.
Control of Initiation and Aggressiveness of DMBA‐Induced Breast Cancer in Rats by Dietary Consumption of Hinute‐AM.
Authors: Das Salil K. , Sharan Chakradhari , Maebuchi Motohiro , Mukherjee Shyamali K. 
Published: April 2020

We reported earlier that the consumption of soy protein controlled not only the proliferation of dimethylbenz [a] anthracene (DMBA)‐induced breast cancer in rats but also its aggressiveness. Soy protein is hydrolyzed in the gut to small peptides which are easily absorbed and are involved in various cellular functions. Fuji Oil Company, Osaka, Japan, has commercially marketed a soy protein hydrolysate, termed Hinute‐AM, composed of primarily di‐ and tripeptides, to be used as a health food, and it has anti‐inflammatory property. The purpose of the present study was to investigate whether dietary intake of Hinute‐AM can delay the development of DMBA‐induced breast cancer and its aggressiveness in rats. For this study, we obtained 22‐days old female Sprague Dawley rats from Charles River (Wilmington, MA). Animals were divided into 5 groups, each containing 5 animals. Group 1 received standard AIN‐76A diet (Envigo – Teklad Diets, Madison, WI) containing 20% casein and 0% Hinute‐AM; Group 2 received the same diet containing 15% casein + 5% Hinute‐AM; Group 3 received the same diet, but containing 10% casein + 10% Hinute‐AM; Group 4 received the same diet but containing 5% casein + 15% Hinute‐AM; and Group 5 received the same diet but containing 0% casein + 20% Hinute‐AM. For induction of mammary tumors, all groups received a single intragastric administration of DMBA (Sigma Chemical Co., St. Louis) dissolved in sesame oil (80 mg/kg b.wt.) at 50 days of age. At 120 days post DMBA injection, the animals were euthanized by CO2 asphyxiation. All tumors were weighed and measured for volume, and a section of tumor was subjected to histological grading by a certified pathologist. Our preliminary data revealed that replacement of casein by Hinute‐AM as a source of dietary protein caused a dose‐dependent delay in the development of breast tumor, decrease in breast cancer incidence, a decrease in tumor volume, and tumor grade.Support or Funding InformationThis work was supported by grants from Fuji Oil Company, Osaka, Japan and by the Meharry Translational Research Center grant NIH 5U54MD007593
Involvement of PDZ‐SAP97 Interactions in Regulating AQP2 Translocation in Response to Vasopressin in LLC‐PK1 cells
Authors: Bahouth Suleiman W. , Nooh Mohammed M. 
Published: April 2020

Arginine‐vasopressin‐ (AVP) mediated translocation of aquaporin‐2 (AQP2) protein forming water channels from storage vesicles to the membrane of renal collecting ducts is critical for renal conservation of water. The type‐1 PDZ‐binding motif (PBM) in AQP2 “GTKA” is a critical barcode for its translocation, but its precise role and that of its interacting protein partners in this process remains obscure. We determined that SAP97, a MAGUK protein involved in establishing epithelial cell polarity was an avid binding partner to the PBM of AQP2. The role of the PBM and SAP97 on AQP2 redistribution in response to AVP was assessed in LLC‐PK1 renal collecting cells by confocal microscopy and by cell surface biotinylation techniques. These studies indicated that the distribution of AQP2 and SAP97 overlapped in the kidneys and in LLC‐PK1 and that knockdown of SAP97 inhibited the translocation of AQP2 in response to AVP. The binding between AQP2 and SAP97 was mediated by specific interactions between the second PDZ of SAP97 and the PBM of AQP2. Mechanistically, inactivation of the PBM of AQP2, global delocalization of protein kinase A (PKA) or knockdown of SAP97, inhibited AQP2 translocation as well as AVP‐ and forskolin‐mediated phosphorylation of serine256 in AQP2, which serves as the major translocation barcode of AQP2. These results suggest that targeting of PKA to the AQP2 microdomain via SAP97‐AQP2 interactions in association with cross‐talk between two barcodes in AQP2, namely the PBM and phospho‐Ser256, play an important role in the translocation of AQP2 forming channels in the kidney.Support or Funding InformationThis work was supported by the National Institutes of Health grant: HL‐085848 and by Bridge Funding support by the University of Tennessee‐HSC.
Fragment‐Based Screening to Identify Thioesterase Superfamily Member 2 (Them2) Inhibitors for the Management of Obesity‐Related Disorders
Authors: Krumm Christopher S. , Adura Carolina , Liu Xu , Li Yingxia , Bracken W. Clay , Ginn John D. , Glickman J. Fraser , Ortlund Eric A. , Cohen David E. 
Published: April 2020

BackgroundThe prevalence of obesity‐related disorders continues to increase, but current management options remained limited. In the setting of chronic overnutrition, excessive tissue concentrations of free fatty acid (FFA) contribute to the pathogenesis of obesity‐related disorders. Thioesterase superfamily member 2 (Them2) is an acyl‐CoA thioesterase (Acot) that hydrolyzes long‐chain fatty acyl‐CoAs into free fatty acids and CoASH. Them2 is enriched in oxidative tissues, and Them2−/− mice are protected from diet‐induced obesity, diabetes, and non‐alcoholic fatty liver disease. These findings suggest that chemical inhibition of Them2 could be leveraged in the management of obesity‐related disorders.AimThis study was designed to develop a small molecule inhibitor that targets the enzymatic activity of Them2.MethodsRecombinant His‐tagged human Them2 was expressed in E. coli, and purified by affinity chromatography and size exclusion chromatography. Two fragment‐based screens (FBS) were performed: 1) A 1056 fragment library was screened by differential scanning fluorimetry (DSF), and 2) a 500 19F‐labeled fragment library was screened by 19F‐nuclear magnetic resonance (19F‐NMR). Secondary orthogonal biochemical (microscale thermophoresis; MST) assays confirmed binding interactions between fragment compound hits and Them2. A microplate‐based fluorometric Acot activity assay was used to measure the Acot activity of Them2.Results30 DSF‐derived fragment compounds (2.8% hit rate) that decreased the melting temperature of Them2 by greater than 4°C (i.e. Them2 de‐stabilizers) were identified. Deconvoluted NMR chemical shifts revealed binding of 15 19F‐labeled fragment compounds to Them2 (3% hit rate). The most promising fragment compound hits exhibited Kd values in the μM range by MST. The most potent fragment compound hits were those that suppressed Acot activity.ConclusionsComplementary biochemical approaches identify fragment compounds capable of both binding and inhibiting Them2. Following structural characterization of ligand binding by x‐ray crystallography, medicinal chemistry strategies can be leveraged to improve potency. Lead compounds that inhibit Them2 should prove attractive in the management of obesity‐related disorders.Support or Funding InformationThis work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grants R01 DK056626, R37 DK048873, and T32 DK116970 (to D.E.C).