bluuebunny/crossref_metadata_2025 huggingface dataset:
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 Information

NIH‐HL148844 and RED account funds
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 Information

NSF 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 Information

NIEHS Grant # P30 ES027792‐02
Use of a short, in‐class, open‐ended free response activity to assess student understanding of the cell membrane in an undergraduate physiology course
Authors: Walton Kristen L.W.
Published: April 2020

With an ever‐increasing body of information about human and animal physiology, it can be difficult for an instructor to decide what topics to focus on and what students exiting a single, stand‐alone physiology course should have learned. One valuable tool to assist in making these decisions is the set of 15 core concepts in physiology published by Michael and McFarland (Adv Physiol Educ 2011). Students in the upper division, stand‐alone physiology course at Missouri Western State University are required to have completed a freshman‐level introductory cell biology course and a sophomore‐level genetics course, but no other prerequisites are required. The prerequisite courses include discussion of the eukaryotic cell membrane and basic properties and functions of the membrane. However, many students remember the basic information about membrane structure but have difficulty relating that structure to membrane function and related topics in our physiology course, including vesicular transport, primary and secondary active transport, osmosis, and current flow across the membrane. To gain a better understanding of what students recall about the cell membrane before beginning in‐class discussion about membrane structure and function, students were given an open‐ended prompt, “What do you know about the animal cell membrane? Include structural characteristics and functions.” The students were allowed to work in small groups or alone if they preferred, and could generate their response as drawings, written statements, or both. They were given 10 minutes to construct a response. These student responses were analyzed using the conceptual framework for the “cell membrane” core concept recently published by Michael and Modell (Adv Physiol Educ 2019). 100% (19 out of 19) submissions included a drawing or description of the cell membrane as a phospholipid bilayer, and 84% of submissions included integral and peripheral membrane proteins. However, other components of the “cell membrane” conceptual framework were included much less frequently or not at all. While 58% of the responses listed diffusion or osmosis, only 32% listed active transport, and less than 10% mentioned vesicular transport, cell‐cell or cell‐matrix junctions, maintenance of solute concentration inside versus outside the cell, or cell‐to‐cell communication. No responses explicitly included simple diffusion of lipid‐soluble molecules, membrane receptors, ion channels, or physical separation/compartmentalization of the cell. Several responses had errors, such as incorrect labeled of the fatty acid tails of phospholipids as hydrophilic. This quick activity provides valuable information about what students recall from prior coursework and what concepts need to be revisited before moving into more detailed discussion of cell membrane function.
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 & Objective

Primary 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 & Results

Mice 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.

Conclusions

These 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 Information

University 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 Information

Supported 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 Information

FAPESP (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 Information

Supported by National Institute of Health Grants (ES010337 and GM126074) and seed grant from the UCSD BreastMilk MoMI CORE.
NLRP3 Inflammasome Products IL‐1beta and IL‐18 Have A Direct Effect on Cardiomyocytes
Authors: Enni Jacqueline Bella Anderson , Ninh Van , Miyamoto Shigeki , Brown Joan Heller
Published: April 2020

Objective

We previously demonstrated a significant role for NLRP3 inflammasome activation in cardiomyocytes in cardiac inflammation, remodeling and contractile dysfunction in response to stressors such as Angiotensin II infusion and pressure overload. There is limited data regarding responses of cardiomyocytes to interleukin‐1 beta (IL‐1beta) and interleukin 18 (IL‐18), which are generated as a result of NLRP3 activation. The goal of this study was to determine the effects of IL‐1beta and IL‐18 on cardiomyocytes.

Methods

Isolated Neonatal Rat Ventricular Myocytes (NRVMs) were treated for various times with a range of doses of recombinant IL‐1beta or IL‐18. Western Blotting was used to measure the activation state of protein kinases and downstream signaling pathways. Quantitative Polymerase Chain Reaction (q‐PCR) was used to assess the expression of pro‐inflammatory cytokines and chemokines in cardiomyocytes.

Results

Western blotting showed a significant increase in phosphorylation of multiple protein kinases and molecules that regulate apoptosis and other inflammatory functions after IL‐1beta and IL‐18 treatment, with maximal responses seen at 1ng/ml IL‐1beta and 10ng/ml IL‐18. Phosphorylated mitogen‐activated protein kinases (MAPKs) including JNK and P38 as well as p‐STAT3 were increased several fold at 10 and 30 mins of treatment with either cytokine. q‐PCR analysis of NRVM mRNA revealed increases in gene expression of multiple pro‐inflammatory cytokines and chemokines in cells treated with either IL‐1beta and IL‐18. CXCL‐10, TGF‐beta, IL‐18, and NLRP3 were all upregulated by both IL‐1beta or IL‐18. Interestingly, upregulation of CCL2, IL‐6, CXCL1, and TNF‐alpha resulted from treatment with IL‐1beta but not with IL‐18. We are currently analyzing the effects of IL‐1beta and IL‐18 on cell viability to further determine the direct effect of these products of the NLRP3 inflammasome in regulating cardiomyocyte apoptosis basally or in response to stressors.

Conclusions

We have previously shown that the cardiomyocyte is a source of proinflammatory cytokines and chemokines, including IL‐1beta and IL‐18. Our results suggest the possibility that IL‐1beta and IL‐18 activate a pro‐inflammatory positive feedback loop in cardiomyocytes which enhances and sustains cardiac inflammation, contributing to cardiac remodeling under stress conditions.
R‐(‐)‐2,5‐dimethoxy‐4‐iodoamphetamine (DOI)‐induced Vocalizations and Observable Behavior in Nonhuman Primates
Authors: Wakeford Alison G.P. , Kohut Stephen J.
Published: April 2020

Many psychiatric disorders include elements of dysfunctional social behavior. Understanding the pharmacological underpinnings of social behavior in a nonhuman primate species that demonstrates a repertoire of complex behavior (e.g., aggression or affiliative behavior) may provide insights into novel medication approaches aimed at restoring social deficits. Specifically, squirrel monkeys (Samiri boliviensis) emit a rich array of vocalizations that provide information on socio‐emotional and behavioral states. These vocalizations often co‐occur with readily observable behaviors that are easily quantifiable, permitting analyses of various aspects of social behavior. Previous studies have indicated that affiliative, or prosocial, vocalizations (pulsed calls and chucks) and behavior (huddling) may be modulated by serotonin 2A (5HT2A) receptors. To begin to understand how 5HT2A receptor activation influences social behavior, we examined the effects of R‐(‐)‐2,5‐dimethoxy‐4‐iodoamphetamine (DOI), a potent and moderately selective 5HT2A agonist, on affiliative vocalizations and observable behavior in nonhuman primates. Pair‐housed squirrel monkeys (2 males, 2 females) were transferred to an observation chamber outside of the colony and vocalizations and huddling were recorded during test sessions. Saline or DOI (0.01 or 0.06 mg/kg, IM) was administered prior to 30 min sessions. Results show that vocalizations, in particular pulsed calls, were increased following administration of 0.01 mg/kg DOI (438 ± 28) relative to saline (190.5 ± 21.5); a higher dose of 0.06 mg/kg DOI elicited a number of vocalizations that were not different from saline (201.5 ± 88.5). Frequency of huddling was unaffected by saline or DOI administration, however, increases in general activity were observed following DOI administration at both doses in comparison to saline. A preliminary examination of potential sex differences suggests that there were no differences between males and females in huddling or total number of vocalizations, though males produced more alarm calls at the highest dose of DOI tested. These data suggest that 5HT2A receptor activation is sufficient for increasing affiliative vocalizations, but may not change other indices of prosocial behavior, such as huddling. Future studies will expand the list of behaviors captured to include positional analyses of each subject, as well as evaluating the contribution of other 5HT receptors to specific dimensions of prosocial behavior.

Support or Funding Information

Research support provided by R01DA048150 (SJK).
High‐yield production of hematopoietic growth factors by tobacco BY‐2 cells for ex vivo production of red blood cells
Authors: Xu Jianfeng , Wang Xiaoting , Wright Tristen
Published: April 2020

Ex vivo generation of clinically available red blood cells (RBCs) from hematopoietic stem cells (HSCs) represents a promising approach for overcoming the limitations associated with the use of donor’s blood, such as shortage of blood and risk of transfusion‐transmitted infections. Both expansion and differentiation of stem cells are highly reliant on growth and differentiation factors, with the activity and purity of these molecules being critical to successful research and clinical applications. The overarching goal of this project is to develop a novel plant cell‐based bioproduction platform for high‐quality (i.e. animal‐free and endotoxin‐free) erythropoietic growth factors (eGFs) that facilitate mass production of RBCs from HSCs at significantly reduced cost. In addressing the low productivity bottleneck of plant cell culture technology, this proposal aims to leverage a unique, plant‐specific, O‐glycosylation process called “hydroxyproline (Hyp)‐O‐glycosylation” for de novo design and engineering of
Hyp‐O‐
glycosylated
peptides (HypGPs”) that presumably function as a molecular carrier in boosting secretion of conjoined eGFs from cultured plant cells. This will dramatically increase the secreted yields of plant cell‐derived eGFs and simplify the product purification process. Three key eGFs essential for HSCs expansion and differentiation: erythropoietin (EPO), stem cell factor (SCF) and interleukin 3 (IL‐3), are expressed in tobacco BY‐2 cells as fusion with a strategically designed HypGP tag. The secreted protein yields, molecular structure, and Hyp‐O‐glycosylation of the HypGP‐tagged eGFs are characterized. The bioactivities of individual HypGP‐tagged eGF in stimulating proliferation and differentiation of relevant hematopoietic cell lines, includingTF1 erythroleukemic cell and umbilical cord blood‐derived CD34+ cell, are evaluated to establish a suitable design of HypGP tag (size and orientation) for each eGF. A scalable cell culture system fortified sorely with plant cell‐produced eGFs is developed for production of RBCs from CD34+ HSC cells. This project provides a proof‐of‐concept for ex vivo manufacturing of stem cell‐derived RBCs with plant cell‐produced eGFs.

Support or Funding Information

National Science Foundation Directorate for Engineering [1605564]; National Institutes of Health‐Arkansas INBRE [P20GM103429]
Kisspeptin Amplifies GLP‐1 Potentiation of Insulin Secretion by a cAMP Independent Pathway
Authors: Lynch Ronald M. , Aragaki Kai A. , Harnois Emily , Vagner Josef , Weber Craig
Published: April 2020

Glucagon Like Peptide‐1 (GLP‐1) and Kisspeptin (KP) receptors are co‐expressed on pancreatic b‐cells, and activation of both receptors potentiates glucose stimulated insulin secretion (GSIS). Unlike GLP‐1, KP has a low affinity for receptor activation (EC50 GLP‐1 ~20 nM vs KP >1 uM). The effects of GLP‐1 and a 10 amino acid analog of KP (KP10) on insulin secretion and cell signaling were studied in the INS 832/3 cell line. By itself KP10 has no effect on insulin secretion in the absence of glucose, but doubled GSIS (GSIS = 37.7 +/− 6.7; +KP10 = 69.2 +/− 1.9 ng/ml/80min) with an EC50 of ~5–10 mM. At saturating doses, KP10 (100 mm) and GLP‐1 (100 nM) had similar effects on GSIS, and in combination had an additive effect (GLP‐1 = 49.0 +/− 2.0, GLP‐1 + KP10 = 63.5 +/− 5.6 ng/ml/80min) consistent with activation of independent downstream signaling pathways. Neither GLP‐1 or KP‐10 altered intracellular Ca2+ levels at basal or activating glucose concentrations. GLP‐1 increased cAMP production (165.0 +/− 17.4 % over control), whereas KP‐10 was without effect. Conversely, pretreatment with KP‐10 enhanced glucose stimulated oxygen consumption and mitochondrial NADH fluorescence, although both responses were inconsistent. Therefore, the effects of KP on b‐cell metabolic activity needs to be investigated in more detail.

We also developed a bivalent ligand composed of GLP‐17‐36 and KP10 binding domains, linked with a 3‐mer of proline/glycine repeats, to evaluate if linking KP10 to a high affinity ligand could promote KP10 activity at low concentrations by presumably increasing its local membrane concentration after GLP‐1 binding. The dimer also did not activate secretion in the absence of glucose, but doubled GSIS much like the individual monomers with an EC50 ~200nM; that is, an additive response was not obvious. Currently, we are determining the ability of GLP‐1/KP10 to initiate downstream signaling from both ligands to resolve the question: is KP active in the GLP‐1/KP at concentrations where monomeric KP10 is not.

Support or Funding Information

Funded by the American Diabetes Association and Juvenile Diabetes Research Foundation
Endothelial sodium channel (EnNaC) activation contributes to mineralocorticoid receptormediated increases in coronary artery and cardiac fibrosis/stiffness leading to diastolic dysfunction in obesity
Authors: Aroor Annayya Rao , DeMarco Vincent , Bender Shawn , Jia Guanghong , Habibi Javad , Yang Yan , Manrique-Acevedo Camila , Lastra Guido , Whaley-Connell Adam , Jaisser Frederic , Sowers James , Hill Michael
Published: April 2020

Background

While obesity, induced by an industrialized or Western diet (WD), has been shown to impact structural and functional properties of both coronary arteries and myocardium, the exact mechanisms by which these interact to cause diastolic dysfunction (DD) are uncertain. We hypothesized that the mineralocorticoid receptor (MR) activation in coronary vascular endothelial cells (ECs) impairs endothelial function via EC Na+ channel, EnNaC, activation. Further, we posited that such coronary dysfunction initiates increased myocyte stiffness and fibrosis ultimately leading to DD. The objective was to demonstrate that WD‐induced DD is dependent on EnNaC activation.

Methods

To cause MR activation, aldosterone was administered to female or male mice (C57BL6) by osmotic minipump for 3 wks. Further, female wild‐type and EC MR deficient mice (2–3 mths of age) or low aldosterone implanted as subcutaneous pellet were fed a WD, high in saturated fat and fructose (16 wks). In an additional experiment, female wild‐type and EnNaC α subunit deficient mice (3–4 mths of age) were fed the WD (12–16 wks). Vascular function was assessed in isolated coronary arteries by pressure myography, cardiac and perivascular fibrosis was evaluated by picosirius red (PSR) staining and diastolic function was assessed by echocardiography. Using in vitro cellular approaches, intrinsic cardiomyocyte stiffness was measured by atomic force microscopy (AFM) and EnNaC activity (Na+ current) determined by whole cell patch clamp.

Results

Aldosterone infusion caused impaired endothelium‐dependent relaxation and coronary vascular fibrosis, which were prevented by the MR antagonist, spironolactone (1 mg/kg/day). WD feeding was associated with DD, but not systolic dysfunction, that was prevented by either spironolactone or EC‐specific MR deletion. Furthermore, DD was accompanied by increased vascular EnNaC currents and increased stiffness of both ECs and cardiomyocytes, which were attenuated by EC MR deletion. Finally, WD‐induced increases in activation of coronary EnNaC, EC stiffness and cardiomyocyte stiffness, together with DD, were suppressed in EnNaC α deficient mice.

Conclusions

Collectively, these results demonstrate that MR activation (aldosterone infusion and diet‐induced) elicits coronary vascular dysfunction associated with tissue and cellular cardiac changes and DD. Further, activation of EnNaC, a known target of MR signaling, promotes coronary EC‐dependent cardiomyocyte stiffness, cardiac fibrosis and impairs DD. Therapeutic targeting to prevent EnNaC activation may be a viable approach for the management of obesity‐associated diastolic dysfunction.
Uncovering Sex‐specific Roles of Farnesoid X Receptor in Heme Metabolism and Liver Proliferation
Authors: Dean Angela Elizabeth , Anakk Sayeepriyadarshini
Published: April 2020

Nuclear receptor, Farnesoid X Receptor (FXR), regulates bile acid homeostasis and has been shown to promote liver regenerative response. Serendipitously, we uncovered a distinct role for FXR in mediating liver injury response in female compared to male mice challenged with 3,5‐diethoxycarboncyl‐1,4‐dihydrocollidine (DDC, a heme biosynthesis disruptor). As expected, the DDC diet led to liver injury as measured by elevated serum ALT and AST levels in both control and FxrKO mice. Furthermore, accumulation of bile plugs and ductular reaction was visible in the WT mice, whereas both of these histological hallmarks were dramatically reduced in FxrKO mice. Despite similar histology, the proliferative response in the hepatocytes were different between both the sexes of FxrKO animals. Loss of Fxr resulted in increased hepatocyte size and low‐grade Ki‐67 positive hepatocyte nuclei even under basal conditions. But upon DDC challenge, FxrKO female livers displayed a dramatic induction of Ki‐67 positive staining even though no bile duct injury was visible. This finding correlated well with increased expression of cyclins. On the contrary, Ki‐67 staining was comparable between male FxrKO livers fed either chow or DDC diet, which matches with lower injury. We are actively investigating the crosstalk of estrogen signaling with FXR in controlling liver proliferation. At the molecular level, genes regulating the cell cycle (Ccnd1, Ccna, Ccnb1) were induced upon injury in the male livers in an Fxr‐dependent manner, whereas this response was not observed in female livers. Expression of Cyp2b10 and Cyp3a11, which are known detoxification genes, were induced with DDC treatment. To test if the absence of bile plugs in FxrKO mice is secondary to reduced heme synthesis in these livers, we examined the heme biosynthetic pathway. We mined previously published data and found strong Fxr occupancy and binding to 6 out of the 8 genes involved in the heme synthesis pathway including Alas1, which is the rate‐limiting step in heme biosynthesis. In summary, FXR may regulate heme metabolism and modulate hepatocyte proliferation in a sex‐specific manner.

Support or Funding Information

Research Scholar Grant TBE 132315
A single residue exerts two contrasting effects on autoinhibition of HCN channels
Authors: Page Dana A. , Li Jessica , Jung Matthew , Ela-Alim Allaa , Young Edgar C.
Published: April 2020

Hyperpolarization‐activated cation (HCN) channels regulate electrical activity in the brain and heart in a cAMP‐dependent manner. The voltage‐gating of these channels is mediated by a transmembrane (TM) region but is additionally regulated by direct binding of cAMP to a cyclic nucleotide‐binding (CNB) fold in the cytoplasmic C‐terminal region. In the unliganded form, the CNB fold mediates an autoinhibitory mechanism that kinetically and thermodynamically stabilizes the closed state, and cAMP binding relieves this inhibition. This autoinhibition is an important regulatory mechanism, relief of which provides a molecular mechanism for how cAMP potentiation occurs. The magnitude of this autoinhibition can be calculated by comparing thermodynamic or kinetic parameters of a channel to the parameters from a channel that is autoinhibition‐free due to truncation of the CNB fold. Recently, I have found that the magnitude of autoinhibition is regulated by interdomain interactions between the C‐terminal region and TM region structures. Specifically, the magnitude of both kinetic and thermodynamic autoinhibition was augmented by replacing the TM region of the HCN2 channel with the TM region of HCN4. I hypothesized that interdomain interactions between the HCN4 TM region and charged residues in the C‐linker, which connects the CNB fold to the TM region, can modify the magnitude of autoinhibition. I used site directed mutagenesis to identify that the E457 residue formed an electrostatic interaction with the HCN4 TM region to stabilize the open state. This residue also has an important role in both the kinetic and thermodynamic components of the autoinhibition mechanism. A charge reversal mutation (E457R) limited the thermodynamic effect of autoinhibition while augmenting the kinetic effect of autoinhibition. Overall, this work suggests that the molecular mechanisms of autoinhibition must include participation of TM region structures and provides functional evidence for interdomain interactions that facilitate HCN channel regulation by CNB fold‐mediated mechanisms.

Support or Funding Information

NSERC, Simon Fraser University
Sensory Receptors are Found in a Non‐sensory Organ: The Bladder
Authors: Smith Tilmira , Berkowitz Dan , Matoso Andres , Pluznick Jennifer
Published: April 2020

Recent research has found that “sensory” receptors (including olfactory receptors (Olfr), taste receptors (Tas), and opsins (Opn)) play functional roles throughout the body. However, the expression of these receptors has not yet been carefully examined in the bladder. In this study, we utilized customized Taqman low density gene array cards to perform quantitative PCR and identify novel sensory receptors in mouse bladder tissue. We extracted RNA from murine bladder (2 male, 2 female), and then screened for the presence of sensory receptors using custom Taqman arrays which included probesets for 43 ORs, 35 bitter TRs, 3 TRs for umami/sweet, 5 opsins, and 7 G‐proteins and/or accessory proteins associated with sensory signaling pathways, as well as a Gapdh control. Using these arrays, we found the expression of 30 sensory transcripts in all male and female bladder samples, with 10 receptors being relatively well‐expressed (Ct values<~30). These moderately expressed receptors included 2 opsins (Opn1sw, Opn3), 3 ORs (Olfr558, Olfr99, Olfr78), and 5 TRs (Tas1r3,Tas2r143, Tas2r135, Tas2r108, Tas2r126. We did not note any significant sex differences, and the top hit (Opn1sw) was the same for all four arrays. We have localized these top receptors within the bladder using two different methods: quantitative PCR on microdissected bladder tissue, and RNAscope fluorescent imaging. Although some receptors have higher relative expression in either urothelium or muscle, all of the receptors are expressed in both of these cell types. Notably, two of the ORs found in the bladder (Olfr78, Olfr558) are known to respond to bacterial metabolites, which may be relevant in the setting of cystitis/urinary tract infections (UTIs). We then screened by qRT‐PCR for a subset of five of these receptors as well as two control receptors (not detected in bladder in the initial screen) in various tissues: brain, colon, eye, heart, kidney, skeletal muscle, and tongue. We found that all five of the receptors of interest were expressed in at least one tissue in addition to bladder, with varying patterns of expression for each. Further investigation could help us better understand why these receptors are expressed in these tissues. Moreover, determining the role of these novel sensory receptors in the bladder has the potential to enhance our understanding of bladder function, and may lead to novel treatments for conditions such as cystitis/UTIs.
Calcium‐Sensing Receptor (CaSR) Modulates Hyperoxia‐Induced Airway Hyperreactivity In a Neonatal Mouse Model of Bronchopulmonary Dysplasia
Authors: Sopi Ramadan B. , Mayer Catherine A. , Martin Richard J. , Pabelick Christina B. , Prakash Y. S. , MacFarlane Peter M.
Published: April 2020

Supplemental O2 is a primary mode of respiratory therapy for preterm infants. Numerous studies, however, have shown that supplemental O2 may contribute to the pathophysiology of airway hyperreactivity (AHR) associated with wheezing disorders such as asthma later in childhood. The calcium‐sensitive receptor (CaSR) is a potent modulator of smooth muscle contractility and may be an underlying feature of childhood asthma. In the present study, we investigated whether CaSR is involved in neonatal hyperoxia induced AHR in a mouse model of BPD. We hypothesize that inhibition (CaSR antagonism), downregulation of expression (siRNA), or deletion (CaSR KO mice) of CaSR will attenuate the AHR induced by hyperoxia in neonatal mice. Newborn C57BL/6 wild‐type (background) or CaSR−/− (knockout) mice were randomized on the second day of life and assigned to room air (21% O2) or hyperoxic (40% O2) groups and exposed for seven days, then recovered in room air for two additional weeks. At day P21, AHR was assessed in vitro using precision‐cut living lung slice preparations in response to increasing doses of methacholine (MCh, 0.25–8 μM). Wild‐type lung slices were pre‐incubated for 48 h in a CaSR siRNA sequence (40 nM) or scrambled RNA as a negative control. In another set of experiments, slices were pre‐incubated in a CaSR antagonist – NPS 2143 (10 nM) for 1 h. Airways were imaged and airway reactivity was expressed as percent change from baseline airway lumen area (± SEM). Neonatal hyperoxia significantly increased airway contractile responses to MCh in wild‐type mice with a mean maximal effect (Emax) of 68.4±8.7% decrease in airway lumen area compared to room air controls (47.3±5.8%, p<0.001), indicating AHR. Pre‐incubation (48hrs) with siRNA targeting CaSR or pharmacologic inhibition (NPS 2143, 1hr incubation) of CaSR reversed the hyperoxia‐induced AHR and the contractile responses were normalized to control level, with hyperoxic groups Emax of 44.0±5.2% and 41.3±3.1%, respectively. AHR was not observed in CaSR knockout mice suggesting resistance to hyperoxia exposure. These data suggest that CaSR plays an important role in the long‐term effects of neonatal hyperoxia on AHR. We speculate that CaSR may be a novel therapeutic target to reverse airway hyperreactivity in former preterm infants who had received prior supplemental O2 therapy.

Support or Funding Information

Funding: NIH: HL R01 138402 and NIH R01 HL 056470
Exploring the Autoinhibitory Domain (AID) of the Electrogenic Na/HCO3 Transporter NBCe1‐B, and Binding Motifs for the Cytosolic Protein IRBIT
Authors: Lee Seong-Ki , Boron Walter F.
Published: April 2020

The electrogenic Na/HCO3 cotransporter NBCe1 is expressed widely throughout the body, and plays crucial roles in trans‐epithelial HCO3− transport (e.g., renal proximal tubule, PT) and regulation of intracellular pH (e.g., cells of central nervous system, CNS). The five known NBCe1 variants are: NBCe1‐A variant, mainly in PT; B, ubiquitous; C, CNS; and D and E, murine reproductive organs. For each variant, the extreme NH2‐terminus (Nt) markedly affects transport activity. For the A and D variants, the initial 41 amino acids (aa) are unique and, at least in A, confer high transport activity. For the B, C, and E variants, the initial 85 aa are likewise unique and, at least in B and C, confer low transport activity. Among these variants, at least B interacts with IRBIT [inositol trisphosphate (IP3)‐receptor binding protein released with IP3], which binds to the unique Nt and markedly enhances transport activity. In a previous study, we examined NBCe1‐B (e1B) residues 28–62, inclusive, and found that the cationic cluster (residues 40–48, inclusive) is an essential element of the AID that confers low activity to e1B. The purpose of the present study is to: (a) determine whether other portions of the unique Nt of e1B (i.e., residues 2–27 and 63–85, inclusive) contribute to the AID, (b) better characterize the cationic cluster, and (c) refine the structural requirements of the IRBIT‐e1B interaction under physiological conditions. Our approach is to co‐express in Xenopus oocytes one of several e1B constructs ± super‐IRBIT (sIRBIT, which lacks the PP1 binding site), followed by two‐electrode voltage‐clamping, extracellular surface biotinylation, and NeutrAvidin pull‐down of plasma‐membrane proteins. As assayed by western blot, we find that sIRBIT co‐purifies with biotinylated e1B protein, but only for e1B constructs with an intact AID (i.e., low‐activity constructs that sIRBIT strongly stimulates). For example, sIRBIT does not copurify with the Δ940‐48 e1B construct, which lacks the cationic cluster (i.e., AID disrupted). We find that residues 63–85, inclusive, are unnecessary for both the AID and for sIRBIT binding. Currently, we are using alanine scanning to explore residues 2–27, inclusive. When we replace the normal cation cluster—mostly made of arginine (R) residues—with increasing numbers (N) of lysine residues (KN) or histidine residues (HN), we observe that (a) the KN and HN constructs also produce autoinhibition, (b) IRBIT relieves the autoinhibition, and (c) sIRBIT co‐purifies with the KN and HN constructs, and (d) the amount of co‐purified sIRBIT gradually increases with increasing N for both KN and HN constructs, as is the case with RN constructs. Thus, the cationic cluster—whether made up of RN, KN, or HN—is essential both for autoinhibition and sIRBIT binding.

Support or Funding Information

The Office of Naval Research, N00014‐15‐1‐2060,N00014‐16‐1‐2535