bluuebunny/crossref_metadata_2025_split huggingface dataset:
In vitro Metabolism studies of Catechol‐Containing Natural Products and Analogues
Authors: Hermanson Jake , Bohannan Amanda , Schwartz Zachary , Jemal Mauricio , Kerwin Sean M. 
Published: April 2020

Many biologically active natural products contain phenol groups that are prone to rapid phase‐II metabolism. In order to better understand the structural basis for these metabolic transformations, we have investigated the in vitro metabolism of rooperol, a bis‐catechol with anti‐cancer activity, and caffeic acid phenethyl amide (CAPA), a synthetic analog of a natural product with antioxidant and cytoprotective activities. In the presence of pig liver microsomes supplemented with UDP‐glucuronic acid, both of these catechols undergo rapid phase‐II metabolism to form mono‐ (CAPA and rooperol) and bis‐ (rooperol) glucuronide metabolites. The identity of these metabolites was determined by HPLC‐MS. We followed the kinetics of these in vitro metabolism reactions using HPLC. In both cases, the disappearance of the compound over time corresponded to a first‐order process with half‐life of 4.0 ± 0.5 min for rooperol and 5.1 ± 0.8 min for CAPA. In the case of rooperol, this data is in agreement with previously published pharmacokinetic (PK) studies in humans, in which only phase‐II metabolites were detected after oral administration, as well as PK studies in baboons which also report rapid phase‐II metabolism following IV administration. While human or non‐human primate PK data for CAPA have not been reported, our data suggest that this compound will also suffer from rapid phase‐II metabolism, and thus more metabolically stable analogs of both rooperol and CAPA are required.Support or Funding InformationThis research was supported by the U.S. Department of Education HSI STEM program (841.031c), Award #P021C160036The Robert and Ella Owens Medical Research Foundation
Rapamycin‐Dependent Gln3 Transcription Factor Dephosphorylation and Nuclear Localization Depend On Distinct Regions of the Gln3 Protein
Authors: Tate Jennifer J. , Cooper Terrance G 
Published: April 2020

Yeast cell survival in nitrogen‐depleted environments depends on Gln3 entering the nucleus and activating: (i) Nitrogen Catabolite Repression‐sensitive genes whose products scavenge poor nitrogen sources from its extra‐cellular surroundings and (ii) the ATG14 gene required for autophagic recycling nitrogen from intra‐cellular constituents. The TorC1 global serine/threonine kinase complex is one of multiple regulators that controls intracellular Gln3 localization and function. When TorC1 is specifically inhibited by rapamycin, Gln3 is dephosphorylated in a Sit4 phosphatase‐dependent manner, enters the nucleus and activates transcription. However, rapamycin‐elicited Gln3 dephosphorylation itself is insufficient to achieve this outcome, as the hypo‐phosphorylated Gln3 fails to enter the nucleus in the absence of a second phosphatase, PP2A. These observations led to the hypothesis that rapamycin‐elicited outcomes involve distinct target sequences within the Gln3 protein. Supporting this hypothesis, our experiments demonstrate that abolishing the Gln3‐Tor1 interaction, rapamycin‐response or Gln3 cytoplasmic sequestration targets abrogates nuclear Gln3 localization but without effect on rapamycin‐elicited Gln3 dephosphorylation. Truncated Gln3 proteins further show that summary loss of all three regulatory targets also has no effect on Gln3 dephosphorylation. Finally, we have identified multiple specific serine/threonine residues in the N‐terminal region of Gln3 that are dephosphorylated in rapamycin treated cells and when substituted with phosphomimetic aspartate residues abolish Gln3 localization in an otherwise full‐length wild type protein.Support or Funding InformationThis work supported by NIH GM35642‐27 and LTAUSA18162.
Oxidative Stress‐Induced Senescence Alters Glutamate Transporter Expression in Human Brainstem Astrocytes
Authors: Sivasubramanian Mahesh Kumar , Monteiro Raisa , Balasubramanian Priya , Subramanian Madhan 
Published: April 2020

Oxidative stress in the rostral ventrolateral medulla (RVLM), a key brainstem region for sympathetic cardiovascular control, has been implicated in the pathogenesis of sympathetic nervous system overactivity in obesity. Previous studies from our lab have shown that increased oxidative stress is associated with DNA damage and induction of cellular senescence in the brainstem of obese mice. Neurons are post‐mitotic and do not undergo senescence. On the other hand, glial cells especially astrocytes have been reported to undergo senescence in aging and neurodegenerative diseases. In the brainstem, astrocytes play a critical role in regulating excitatory neurotransmission through extracellular glutamate reuptake mediated by astrocytic glutamate transporters (EAAT1 and EAAT2). We hypothesized that oxidative stress induces senescence and affects glutamate transporter expression in brainstem astrocytes. To address this hypothesis, we established a cell culture system to investigate the molecular changes associated with oxidative stress‐induced senescence in human brainstem astrocytes. To mimic oxidative stress observed in high fat diet‐induced obesity, we treated cultured human brainstem astrocytes with hydrogen peroxide (H2O2) at a dose of 300μM for 2 hrs. Senescence was assessed in the cells after 5 days by SA‐beta gal staining and using gene expression analysis of senescence markers at the end of 7 days. Data was analyzed by unpaired student's t‐test and a p‐value less than 0.05 was considered statistically significant. H2O2 treatment reduced cell count and increased SA‐beta gal activity in human brainstem astrocytes. Further, we also observed increased gene expression levels of cell cycle inhibitor p21 and senescence‐associated secretory phenotype (SASP) factors including MCP1, CXCL1, IL8, CXCL10 and MMP3, suggesting oxidative stress induces senescence in human brainstem astrocytes. More importantly, senescent brainstem astrocytes had significant downregulation of glutamate reuptake transporters, EAAT1 and EAAT2. This suggests that senescent astrocytes could mediate sympathoexcitation through enhanced glutamate neurotransmission in the brainstem in obesity. Future in‐vivo studies will investigate the mechanism behind senescence‐induced alterations in glia‐neuron interaction in obesity mediated sympathoexcitation.Support or Funding InformationNIH‐HL148844 and RED account funds
Countermeasures to the impact of cosmic radiation exposure on myocardial mitochondrial function.
Authors: Edwards John , Weiss Madison , Tefft Kristy , Nikisher Bridgette , Katseff Adiya 
Published: April 2020

Space travel increases solar particle radiation exposure to astronauts and this is significantly elevated once travel moves beyond low Earth orbit. This includes γ radiation, as well as a combinations of high energy protons and heavy ions such as 56Fe, 28Si, and 16O.There are distinct differences in the biological response to low‐energy transfer (x‐rays) or high‐energy transfer (High‐LET) (56Fe) events. Airline flight crews are chronically exposed to High‐LET and higher incidents of cancer are reported for these individuals. Much of what we know about the consequences of high‐LET exposure comes from studies of nuclear workers and atom bomb survivors. For flight crews (astronauts), most estimates of galactic radiation exposure have a low fluence rate that is much lower that patients undergoing radiotherapy or the acute exposure of those that survived the A‐bomb. However, the cumulative radiation dose is likely to have long‐term deleterious effects on the health of astronauts. And this risk increases as flight duration is increased.The underlying mechanisms for these elevated risks are unknown, although direct DNA damage and elevated oxidant stress are highly suspected. Given the relatively low fluence rate of exposure it might be possible to manage these deleterious effects using small molecules currently available. To that end we have utilized the FDA approved drug library from MedChem Express (723 drugs) to perform a high throughput screen of cultured cells following exposure to cosmic radiation. The H9c2 cell and RBL‐2H3 mast cell lines were exposed to a total of 75 cGy using a simplified 5‐ion GCR protocol developed by NASA at the NSRL facility at the Brookhaven National Laboratory. Following GCR exposure cells were split onto 96 well test plates and treated for 7 days with vehicle or 10 μM drug. Media and drugs were changed on alternate days. At harvest cells were tested for viability (MTT), oxidant stress (DHE), cellular senescence (ONPG) and mitochondrial function (ATP). Using the non‐drug treated cells as controls, Z‐scores were calculated and a composite score was developed for each drug. The top 160 composite scores were retested following a similar protocol using1 μM of each drug. Within the 160 drugs, 33 are considered to have an anti‐inflammatory capacity. 5 of the eight 5‐HT antagonists considered useful were 5‐HT3 specific. 12 of 25 angiotensin converting enzyme inhibitors or AT1 antagonists were observed to be effective while no AT2 antagonists appeared useful. 10 of 46 drugs indentified as interacting with histamine receptors were deemed effective. 7 of 54 COX inhibitors deemed effective were COX2 but not COX1 inhibitors. Collectively these findings suggest that drugs interacting with GPCR/G coupled proteins that modulate inflammation and anti‐oxidant pathways may be useful in the management of cosmic radiation exposure of long duration flight crews.Support or Funding InformationSupported in part by NASA 80NSSC19K0436
Role of anti‐angiogenic protein TSP‐1 in MAGE‐B2 driven cell proliferation
Authors: Bagsby Clarke , Colemon Ashley , Ramanathan Saumya 
Published: April 2020

Melanoma Antigen Genes (MAGEs) are defined as cancer‐testis antigens due to their unique expression pattern. There are two families of MAGEs, Type I and Type II. Type I MAGEs are expressed in the testis and in no other normal somatic tissue and then re‐expressed in many cancers. Type II MAGEs on the other hand, are ubiquitous in their expression, meaning they are expressed in various somatic tissues. MAGE‐B2 is a member of the Type I subfamily. Expression of MAGE‐B2 causes increases in cell proliferation by as yet unknown mechanisms. Furthermore, depletion of MAGE‐B2 from cancer cells decreases cell proliferation. Our data indicate that the levels of anti‐angiogenic protein Thrombospondin‐1 (TSP‐1) is lowered in MAGE‐B2 expressing cells. TSP‐1 is also known as a regulator of TGFb signaling, because it activates TGFb. We will first determine whether TGFb signaling effectors such as TGFb, TGFb‐receptor, or Smad proteins are altered in expression and/or activity because of aberrant levels of MAGE‐B2. Then, we will determine whether TSP‐1 and TGFb signaling pathway effectors are needed for MAGE‐B2 driven proliferation, using gain and loss of function assays. In summary, these studies will shed light on the mechanisms that MAGE‐B2 uses to drive cell proliferation.Support or Funding InformationNSF HBCU UP: Research Initiation Award HRD1764201 to S.R.
The Toxic Effect of Environmental Cadmium on Visceral Adipose Tissue
Authors: Prabhu Ramya , Ribeiro Marta , Kajdacsy-Balla Andre 
Published: April 2020

Cadmium is an environmental toxin strongly associated with the development of cancer, kidney and liver disease, and bone demineralization. Whether cadmium causes obesity in humans remains a subject of controversy in the literature. An NHANES study that examined the effect of various toxic metals on central obesity demonstrated a decrease in the BMI of subjects exposed to cadmium. The purpose of this study is to examine the effect of cadmium on visceral adipose tissue (VAT), the potential for chelation to reverse cadmium‐induced changes, and to investigate the mechanism by which these changes occur. VAT is the subset of adipose tissue most heavily implicated in the pathogenesis of obesity, insulin resistance, and metabolic syndrome. ICR mice were placed into one of three groups: control, cadmium followed by washout, and cadmium followed by chelation. Mice in the two experimental groups were treated with 100ppm CdCl2 drinking water for 8 weeks followed by either 8 weeks of washout or 8 weeks of chelation with DMSA. Digital analysis of H&E images was performed using ImageJ and morphometric parameters of approximately 400 adipocytes per group were obtained. A 14.6% reduction in adipocyte size was observed in the group treated with cadmium followed by washout relative to the control group (p < 0.0001). In the group that underwent chelation after cadmium exposure, there was an increase in adipocyte size relative to the cadmium‐washout group (p<0.002). Additionally, animals in the cadmium‐washout group had a 5% reduction in body weight relative to control (p<0.0001). There was no significant difference in weight between the control group and the cadmium‐chelation group. In conclusion, the mouse model we have developed demonstrates that chronic low dose cadmium exposure causes weight loss and that chelation has the potential to reverse this change. Although the effect of cadmium on body weight remains controversial, this mouse model may serve to clarify this relationship and address the mechanism of cadmium’s action.Support or Funding InformationNIEHS Grant # P30 ES027792‐02
Bile Acids Modulate Colonic MAdCAM‐1 Expression in a Murine Model of PSC‐IBD
Authors: Gao Rachel Y. , Shearn Colin , Orlicky David , Battista Kayla , Alexeev Erica , Cartwright Ian , Kostelecky Rachael , Colgan Sean , Fennimore Blair 
Published: April 2020

Background & ObjectivePrimary Sclerosing Cholangitis (PSC) is a progressive cholestatic liver disease of unknown etiology that carries a strong association with inflammatory bowel disease (IBD). PSC‐associated IBD (PSC‐IBD) displays a unique colitis phenotype characterized by right side predominant inflammation and increased risk of colorectal cancer compared to non‐PSC‐IBD. Although the initiation/progression PSC‐IBD remains unclear, some evidence supports an integral role for the gut‐specific mucosal vascular addressin cell adhesion molecule (MAdCAM‐1) in modulating both hepatic and intestinal inflammation. Multidrug resistance protein 2 (Mdr2) knockout mice develop spontaneous cholestatic liver injury and fibrosis mirroring human PSC. To further elucidate disease mechanisms of PSC‐IBD, we combined Mdr2−/− mice with the chemically inducible model of DSS colitis (Mdr2−/−/DSS). We hypothesize that alterations in bile acids promote mucosal inflammation in this murine model of combined cholestasis/colitis. There are currently no good models for PSC‐IBD, so our objective is to develop a murine model mimicking PSC‐IBD for studying the gut‐liver axis and proinflammatory crosstalk.Methods & ResultsMice were fed 1.5% DSS for 5 days and recovered for 4 days before being euthanized. Transformed sinusoidal endothelial cells (TSECs) were treated with 5 ng/ml of TNF‐αand/or 100μM of ursodeoxycholic acid (UDCA). UDCA treated mice were fed 0.5% UDCA fortified chow for 2 weeks and throughout the previously described DSS course.In this model, Mdr2−/− mice displayed marked alterations in intestinal bile acid composition and increased susceptibility to DSS colitis based on weight loss, disease activity index, histology, and inflammatory cytokines, etc. Mdr2−/− mice also displayed an upregulation of colonic MAdCAM‐1 expression as compared to C57BL/6 colitis controls. In vitro work utilizing transformed sinusoidal endothelial cells (TSECs) and bEnd.3 cells (brain endothelial cells) revealed that MAdCAM‐1 was potently induced by TNF‐α and that this induction was significantly attenuated by co‐treatment with ursodeoxycholic acid (UDCA). In vivo, DSS‐treated Mdr2−/− mice fed with UCDA fortified chow demonstrated significant attenuation of colitis susceptibility and decreased colonic MAdCAM‐1 expression as compared to standard chow controls.ConclusionsThese findings suggest a unique interplay between alterations in bile acid homeostasis and intestinal MAdCAM‐1 expression in modulating pro‐inflammatory cross‐talk along the gut‐liver axis and identify intestinal bile acid signaling as a potential therapeutic target for the treatment of PSC‐IBD.Support or Funding InformationUniversity of Colorado GI and Liver Innate Immune Program (GALIIP)
Comparisons between Animal Models and Intestinal Crypt Cultures when Investigating the Impact of Fatty Acids on Gene Expression in Intestinal Epithelial Cells
Authors: Yang Xiaojing , Mennillo Elvira , Tukey Robert , Chen Shujuan 
Published: April 2020

Developmental delay of UDP‐glucuronosyltransferase 1A1 (UGT1A1) expression is central toward the development of neonatal hyperbilirubinemia, observed clinically as increased total serum bilirubin (TSB) in newborns. UGT1A1 is the sole enzyme responsible for the glucuronidation and detoxification of bilirubin. We have developed a murine model that resembles neonatal hyperbilirubinemia. In humanized UGT1 (hUGT1) mice, the expression of intestinal but not hepatic UGT1A1 is inversely associated with TSB levels. In addition, the expression of intestinal UGT1A1 is closely associated with proliferation and maturation of intestinal epithelial cells (IECs). It is known that fatty acids (FAs), the major component of breast milk, plays an important role in intestinal development. We have treated hUGT1 neonatal mice with oleic acid (OA, 16:1), a monounsaturated fatty acid. Following OA exposure, TSB levels in hUGT1 neonatal mice were reduced significantly, and RT‐qPCR analysis demonstrated that intestinal human UGT1A1 was induced, along with increased expression of Cyp4a10, a target gene of peroxisome proliferator‐activated receptor alpha (PPARα). In addition, IEC maturation markers including sucrase‐isomaltase (Sis) and alkaline phosphatase 3 (Akp3) are also induced. To examine the mechanism of OA induction, intestinal crypt cells were isolated from hUGT1 mice. The organoids were exposed to different fatty acids, including OA, palmitoleic acid (PA), arachidonic acid (AA), linoleic acid (LA), and docosahexaenoic acid (DHA). Gene expression parameters were monitored 24 hours after exposure. We found that OA‐ and PA‐treated organoids have similar survival rates in comparison to controls, but at similar concentrations of LA, DHA or AA cell death and decreased organoid viability was observed. At lower FA concentrations, RT‐qPCR analysis demonstrated that the UGT1A1 gene was not induced in any of the samples, including the IEC maturation markers Sis and Akp3. However, increased expression of the Cyp4a10 gene resulted in DHA, AA, and LA‐treated organoids. By comparing the in vivo and in vitro experiments, it suggests that unlike Cyp4a10, the regulation of UGT1A1 by fatty acids is associated with the development of the intestinal tract. Under current culture conditions, the application of intestinal organoid cultures in the study of developmental‐associated events is limited.Support or Funding InformationSupported in part by NIH grants GM126074 and ES010337
ROLE OF GABAA AND NMDA RECEPTORS OF RAPHE IN THE CONTROL OF TERMOEFFECTORS OF PRECOCIOUS BIRDS
Authors: Silva Caroline Cristina , Gargaglioni Luciane Helena , Bícego Kenia Cardoso 
Published: April 2020

It is demonstrated in mammals, at least in rats, that caudal brain regions, such as raphe nuclei, are involved in the modulation of thermoeffectors for heat loss and heat production, such as peripheral vasodilation/vasoconstriction, nonshivering and shivering thermogenesis. If the same scenario occurs in the other group of endothermic vertebrates, i.e., birds, it is still completely unknown. The aim of the present study was to verify the effect of GABAA and GLU inhibition in the Raphe on body temperature (Tb), oxygen consumption and ventilation of precocious birds under neutral (31°C) and cold (25°C), using 7‐day‐old chicks as animal models. For this, the Tb, oxygen consumption (index of thermogenesis) and ventilation were measured in chicks implanted with a mini temperature sensor in the coelomatic cavity, before and each 20 min up to 2hs after intra‐raphe microinjections of the GABAA receptor antagonist, bicuculline (0.05 mM and 0.5 mM), and the NMDA receptor antagonist, AP5 (0.5mM and 5mM), or vehicle. Protocols were approved by the local Animal Care Committee (CEUA of FCAV/Unesp, n° 013907/17). No treatment or condition affected ventilation. Bicuculline (0.5 mM) at 31°C significantly decreased (P <0.05) Tb (vehicle: 41.2±0.1°C; Bicuculline 0.05mM: 40.9±0.1°C; Bicuculline 0.5mM: 40.6±0.2°C) and oxygen consumption (13.9±0.7% decrease) of the animals when compared to the vehicle group. Bicuculline (0.05 mM) at 25°C significantly reduced (P< 0.05) Tb (vehicle: 41.4±0.1°C; Bicuculline 0.05mM: 40.6±0.1°C) and oxygen consumption (12.6±1.4% of decrease) of the animals when compared to the vehicle group. AP6 (5 mM) at 31°C significantly decreased (P< 0.05) Tb (vehicle: 41.3±0.1°C; AP5 0.5mM: 40.9±0.1°C; AP5 5mM: 40.3±0.2°C) and oxygen consumption (14.7±0.6% decrease) of the animals when compared to the vehicle group. AP5 (0.5 mM) at 25°C significantly reduced (P <0.05) Tb (vehicle: 41.2±0.2°C; AP5 0.5mM: 40.7±0.1°C) and oxygen consumption (17.4±3.1% of decrease) of the animals when compared to the vehicle group. Our preliminary results suggest that GABAA and NMDA receptors inhibition in Raphe neurons show an inhibitory effect on Tb of chicks affecting thermogenesis under neutral (31°C) and cold (25°C) conditions. Other thermoeffectors possibly involved in this response is currently under investigation.Support or Funding InformationFAPESP (2017/17278‐2)
Triclosan (TCS) Exposure in Neonatal Mice Leads to ER Stress, Inflammation and Lipid Accumulation in Liver
Authors: Weber Andre Alberto , Mennillo Elvira , Ding Jeffrey , Jain Mohit , Chen Shujuan , Tukey Robert 
Published: April 2020

Triclosan (TCS) is an antimicrobial agent used in multiple personal care products and is represented as an emerging environmental contaminant worldwide. Recent findings have demonstrated that TCS can accumulate in human breast milk. Neonatal humanized UGT1 (hUGT1) mice, which exhibit severe levels of total serum bilirubin (TSB), were breastfed from females fed with a diet containing 120 ppm TCS. Newborn mice exhibited levels of TCS in their serum that were comparable to those quantitated in human blood samples. After 14 days, TSB levels decreased dramatically in TCS exposed neonates and UGT1A1 was significantly induced (13‐fold) in liver. The nuclear receptors, CAR, PXR and PPARα, when activated can induce UGT1A1 expression, were upregulated in TCS breastfed mice. The environmental sensor NRF2 is also increased in TCS breastfed mice along with target genes, Ho‐1 and Nqo‐1. At day 21, TCS neonates showed an increase in endoplasmic reticulum (ER) stress markers such as p‐eIF2α and ATF4 when compared to control mice. Caspase‐1 and p20 are also increased, together with a host of cytokines indicating pyroptosis in liver. TCS neonates at 21 days also present a reduction in fatty acid oxidation genes (Pparα, Cyp4a10, Cyp4a14, Cpt1a) and an increase in fatty acid synthase genes (Fasn and Srebp1‐c). At this age TCS mice presented accumulation of lipids in liver with an increase in triglyceride contents. These results suggest that activation of nuclear receptors induce hepatic UGT1A1 following TCS exposure by lactation while eliciting early signs of oxidative stress that leads to ER stress and inflammation that may be linked to fatty liver.Support or Funding InformationSupported by National Institute of Health Grants (ES010337 and GM126074) and seed grant from the UCSD BreastMilk MoMI CORE.