bluuebunny/crossref_metadata_2025 huggingface dataset:
Depletion of Cranial Suture Stem Cells by Pharmacological Exposures May Precipitate Premature Suture Fusion
Authors: Durham Emily , Howie R. Nicole , LaRue Amanda , Cray James
Published: June 2021

The CDC National Birth Defects Prevention Study has published data suggesting that “environmental” exposures including maternal thyroid diseases, use of selective serotonin reuptake inhibitors (SSRIs), and maternal nicotine use may increase the incidence and or severity of craniofacial anomalies including craniosynostosis. Craniosynostosis is defined as the premature fusion of the suture(s) of the skull occurring in 1:1800–2500 births. A proposed mechanism of craniosynostosis is the disruption of proliferation and differentiation of stem cells in the perisutural area. Here, we hypothesize that the aforementioned teratogenic exposures can cause a depletion of stem cells within the suture resulting in premature suture fusion. To determine if in utero pharmacological exposures deplete stem cells within the calvarial sutures, we exposed pregnant wild‐type mice to levothyroxine, citalopram (SSRI), and nicotine and investigated suture fusion and the presence of stem cell markers ex vivo. Additionally, we exposed primary calvarial suture derived cells to clinically relevant doses levothyroxine, citalopram, and nicotine in vitro and assessed the presence of stem markers via flow cytometry. Micro‐CT assessment of coronal and posterior interfrontal suture fusion revealed that in utero exposure to nicotine and citalopram increased the risk of premature posterior interfrontal suture fusion and exposure to citalopram resulted in an increased likelihood for coronal suture fusion at post‐natal day 15. Further, we confirmed a reduction in Gli1+ cells ex vivo in correlation with in utero teratogen exposure. Our in vitro analysis also indicates a depletion of stem cell populations with teratogen exposure via flow cytometry. Teratogenic exposures including maternal thyroid disorder, maternal use of SSRIs and maternal nicotine use may target calvarial stem cell populations for depletion precipitating an increased risk for craniosynostosis. Investigating the newly defined stem cell niche of the calvarial sutures and its relationship to suture maintenance will provide insight into future manipulation of these cells for therapeutic benefit.

Support or Funding Information

National Institute of Dental and Craniofacial Research [F31DE026684 to ELD; R03DE023350A and R03DE02619‐01A to JC; 5T32DE017551 to MUSC]

Cleft Palate Foundation Cleft/Craniofacial Anomalies Grant Award to JC

Plastic Surgery Education Foundation Pilot Award to JC

National Institute on Aging (NIA) 1P01AG036675 (Augusta University)

National Institutes of Health National Institute of General Medicine [P30GM103331]

South Carolina Clinical and Translational Research Institute (NIH/NCATS NIH/NCATS UL1TR000062)

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Critical role of claudin‐7 in maintaining intestinal crypt stem cell functions
Authors: Xing Tiaosi , Sabu Stephiya , Benderman Lesley Jasmine , Chen Yanghua
Published: June 2021

Intestinal epithelial integrity and self‐renewal are two main features that maintain intestinal homeostasis. Tight junctions control epithelial integrity while intestinal crypt stem cells fuel epithelial self‐renewal. Claudin‐7 is a tight junction membrane protein present along the entire intestinal epithelium. Using our global (gCldn7−/−) and inducible intestinal‐specific (cCldn7−/−) claudin‐7 knockout mouse models, we found that claudin‐7 deletion induces rapid crypt stem cell loss and increases cell proliferation. We hypothesized that claudin‐7 regulates intestinal epithelial self‐renewal. To elucidate the underlying mechanism, we employed ex vivo organoid culture using Cldn7+/+ and gCldn7−/− small intestines. We found that claudin‐7 deletion not only decreases the survival of organoids and crypt stem cells, but also induces the formation of spheroids without villi compared to normal organoids with villi derived from Cldn7+/+ small intestines. In addition, immunostaining of Cldn7+/+ and gCldn7−/− organoids demonstrated that claudin‐7 deletion increases the proliferative epithelial cells and decreases the number of enterocytes, Paneth cells, enteroendocrine cells, and tuft cells. Interestingly, the number of goblet cells was unaffected which comports with our in vivo findings. Western blotting analysis revealed that β‐catenin (a key molecule in the Wnt signaling pathway) is upregulated in claudin‐7‐deficient intestines. Furthermore, immunostaining revealed that β‐catenin localizes predominately to the epithelial cell cytoplasm. More importantly, we found that activation of the Wnt signaling pathway increases the size of the claudin‐7‐deficient spheroids compared to claudin‐7‐positive organoids. These findings suggest that claudin‐7 plays a novel, but as yet unidentified function in the regulation of epithelial cell renewal in intestines. Claudin‐7 is required for regeneration of intestinal epithelium, survival of crypt stem cells, and differentiation of intestinal epithelial cells. Claudin‐7 also maintains the balance of epithelial cell proliferation and suppresses Wnt signaling pathway during normal intestinal epithelial renewal.

Support or Funding Information

This study is supported by the National Institute of Health grant DK103166.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Endosomal Escape of ASOs Internalized by Stabilin Receptors Is Regulated by Rab5C and EEA1.
Authors: Miller Colton , Harris Edward N. , Seth Punit P.
Published: June 2021

One of the major drug classes that has been gaining traction in the pharmacological world has been the emergence of Antisense oligonucleotides (ASOs). Increased modifications of these drugs have led to major breakthroughs leading to a boost in this drug class being adapted to clinical trials. Second generation (Gen 2) Antisense oligonucleotides show increased nuclease stability and affinity for their RNA targets which has translated to improved potency and therapeutic index in the clinic. Gen 2 ASOs are typically modified using the phosphorothioate (PS) backbone modification which enhances ASO interactions with plasma, cell surface and intracellular proteins. This facilitates ASO distribution to peripheral tissues and also promotes cellular uptake after injection into animals. Recent work of ours has shown that a group of class H Stabilin receptors specifically internalize PS‐ASOs in the sinusoidal cells of the liver and the spleen. This work identifies a breakthrough candidate for the determination of a molecular pathway for these drugs to reach their desired target in the cell. Further exploration of the endocytotic uptake by these proteins has led to a rudimentary understanding of this pathway. By modulating expression of specific proteins involved in the trafficking and maturation of the endo‐lysosomal compartments, we show that the proteins Rab5C and EEA1 play a direct role in the early endosomal pathway. These proteins, along with Rab7A and LBPA (lyso‐lipid) in the late endosomal pathway, are important for trafficking of PS‐ASOs and facilitate their escape from endo‐lysosomal compartments after Stabilin‐mediated internalization. In conclusion, this research identifies key interacting partners involved in ASO‐containing endosomal maturation, as well as late endosomal partners involved in PS‐ASO endosomal release. This work also identifies the limiting proteins in the pathway for PS‐ASO translocation and escape from the endosome.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Use of gradient gel filtration as a refolding, buffer exchange and fine‐tuning step in denaturing protein purification
Authors: Tovar Carmel N. , Nguyen Samantha K. , Odunuga Odutayo O.
Published: June 2021

Purification of recombinant proteins in denaturing conditions has many practical advantages. Chemical denaturants such as urea and guanidine hydrochloride are used to solubilize recombinant proteins from inclusion bodies. Proteins purified in denaturing conditions generally contain fewer contaminants compared to their counterparts purified in native buffers; thus reducing the number of steps in the purification scheme. Chemically denatured proteins can be stored for long time in buffer containing the denaturant and then refolded as required. Refolding is commonly performed by equilibrium dialysis and sometimes on metal affinity columns by diluting out the denaturant and allowing the protein to fold back into its native state. The refolding steps often result in significant loss of protein through aggregation. Using a modified gel filtration technique, we successfully refolded a spectrum of proteins that are either highly aggregation‐prone or contain several disulfide bonds or both, back to their native states. Some of the proteins are well known to precipitate significantly during refolding. However, using this technique, precipitation was either minimal or non‐existent. Secondary and tertiary structural analysis using circular dichroism and fluorescence microscopy coupled with functional assays confirmed that these proteins were correctly refolded and biologically active. This strategy provides a robust approach to refolding denatured proteins that can also be used simultaneously as buffer exchange and additional purification steps.

Support or Funding Information

Welch Foundation Grant # AN‐0008

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
A Novel Monomodular and Multifunctional Processive β‐1,4‐Endoglucanase Has Been Identified and Characterized from Porcine Gut Microbiome
Authors: Wang Weijun , Archbold Tania , Lam Joseph S. , Kimber Matthew S. , Fan Ming Z.
Published: June 2021

Cellulose‐degrading enzymes play an important role in dietary fibre digestion of humans and animals, as well as in multiple industrial applications. Herein, a plasmid metagenomic expression library was constructed for mining cellulose‐degrading enzymes from the pig hindgut microbiota that was modulated by feeding a purified crystalline cellulose supplemented diet (Journal of Animal Science, 2012, 90 (Suppl. 4):400–402). One of the cellulase genes screened from this library was localized to a 2949‐base pair insert fragment p4818. Within the sequence preceding this enzyme‐coding region, ‐10 and ‐35 promoter elements were identified along with three transcription‐factor binding sites. The enzyme possesses a catalytic domain consistent with those of the glycoside hydrolase family 5 subfamily 2 (GH5_2), thus named as p4818Cel5_2A. The protein was overexpressed, purified, and characterized; and it displayed optimal enzymatic activity at pH 6.0 and 50 °C. The enzyme also exhibited resilience to trypsin digestion, where >70 % activity was retained after 6.0 h incubation with trypsin at 37 °C. p4818Cel5_2A exhibited a broad substrate profile with the highest specific activity towards β‐glucan, carboxymethyl cellulose (CMC), followed by hydroxyethyl‐cellulose and regenerated amorphous cellulose (RAC). The enzyme was also active on other substrates including crystalline cellulose substrates such as Avicel and Solkafloc, as well as the hemicelluloses of glucomannan and xyloglucan. Furthermore, based on viscosity and hydrolysis product analyses, the enzyme behaved like a typical endo‐β‐1,4‐glucanase, and displayed processivity towards RAC. In summary, p4818Cel5_2A exhibited novel enzyme properties for potential industrial applications.

Support or Funding Information

Related research activities have been supported by grants (to M.Z.F.) from the Ontario Ministry of Agriculture, Food and Rural Affairs (OMAFRA) New Directions & Alternative Renewable Fuels Research Program; and the University of Guelph Gryphon's LAAIR (Leading to Accelerated Adoption of Innovative Research) program.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
A molecular validation of serotonin signaling systems association with major depressive disorder (MDD) in South Indian population
Authors: Rashmi Subramanian , Sangilimuthu Alagaryadav , Srinivasan Dinesh Kumar , Balachandar Vellingiri
Published: June 2021

Major depressive disorder (MDD) is a multi factorial disease that affects morbidity, mortality and quality of life. It is chronic and recurrent phenotype of complex etiological relationship between genetic and environmental factors. The serotonergic system plays an important role in the modulation of brain function. Tryptophan hydroxylase (TPH) converts tryptophan in the brain to 5‐hydroxytryptophan (5‐HTP). TPH is the rate‐limiting enzyme in 5‐HT synthesis. Most tissues synthesize 5‐HT, a relatively unstable substance at low levels. Minor changes in TPH abundance or the activity can dramatically alter 5‐HT content and serotonergic function. With regard to MDD, there is a strong evidence for serotonergic abnormalities. The present study was performed to validate the association of single nucleotide polymorphisms (SNPs) in several functional variants of serotonergic genes [serotonin‐transporter‐linked polymorphic region (5HTTLPR), TPH1 and TPH2] with MDD in South Indian population. We have analyzed genotypic polymorphism in well characterized sample of MDD patients and controls with PCR‐RFLP methods. We identified a significant difference in the frequency of 5HTTLPR, TPH1 and TPH2 gene alleles between patients and controls. In addition, there was a significant difference in genotypic distribution of 5HTTLPR and TPH2 alleles between these two groups. The distribution of 5HTTLPR genotype frequencies SS, SL, LL and TPH2 GG, TT, GT and SS, GG allele showed strong association with MDD. To our knowledge, the present study reveals for the first time that polymorphism in 5HTTLPR and TPH2 genes may play a role in susceptibility to MDD in South Indian population.

Support or Funding Information

This work was supported by research grant from the Karpagam Research Foundation, Karpagam Faculty of Medical Sciences and Research, Coimbatore, India.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
“Regulator of G‐protein Signaling 10 (RGS10) inhibits NF‐κB signaling, COX‐2 and inflammatory cytokine production in ovarian cancer cells”
Authors: Almutairi Faris , Hooks Shelley B.
Published: June 2021

Epithelial Ovarian Cancer (EOC) is the fifth leading cause of cancer‐related deaths among females in the United States. NF‐κB signaling and production of inflammatory mediators have been strongly linked to ovarian cancer progression and chemoresistance. Regulator of G‐protein Signaling 10 (RGS10) regulates ovarian cancer survival and chemoresistance, and has been linked to NF‐κB signaling in immune cells. The goal of the current study was to determine the ability of RGS10 to inhibit NF‐κB signaling and subsequent inflammatory effects in ovarian cancer. RGS10 is a negative regulator of Gαi signaling and multiple Gi coupled GPCRs are implicated in ovarian cancer. Therefore, we predicted that RGS10 regulates NF‐κB signaling and inflammatory cytokines downstream of Gαi mediated‐pathway. We found that knock‐down of RGS10 enhanced NF‐κB phosphorylation and produced significantly higher levels of COX‐2 and TNF‐α mRNA compared to cells transfected with control siRNA in SKOV‐3 EOC cells. Surprisingly, we found that Pertussis toxin (ptx), which is a Gαi inhibitor, had no effects on upregulation of p‐NF‐κB, COX‐2 and TNF‐α expressions‐mediated by RGS10 knock‐down. These results provide insight in the mechanism that RGS10 utilizes to regulate ovarian cancer survival and chemoresistance.

Support or Funding Information

National Institutes of Health

Rivkin Center for Ovarian Cancer.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Characterizing The Effects of Ascorbic Acid and Hyperbaric Oxygen on Glioblastoma Cells
Authors: DeBlasi Janine , Poff Angela , Ward Nathan , Koutnik Andrew , Rogers Christopher , Diamond David , Moss Sara , D'Agostino Dominic
Published: June 2021

There is emerging evidence supporting the use of intravenous ascorbic acid (AA) as a pro‐oxidative, non‐toxic adjuvant therapy. Before AA is implicated in the clinic, however, more research needs to be conducted to better understand AA's mechanisms of action, and to determine what cancers would be most amenable to this adjunctive therapy. When administered intravenously, AA can achieve pharmacological, millimolar concentrations (~0.3–20 mM) and act as a pro‐oxidant in tumorous tissue, contrary to its antioxidant effects observed with oral administration, which produces micromolar, physiological concentrations (≤0.1 mM). Our study aims to evaluate the effects of pharmacological AA on glioblastoma cells (VM‐M3 and U87), and to see if another pro‐oxidative therapy, hyperbaric oxygen (HBOT), can sensitize cells to treatment. We hypothesize that 1‐hour treatment with AA will decrease VM‐M3 and U87 cell viability, and that co‐treatment with antioxidant N‐Acetylcysteine (NAC) will attenuate cell death. We also hypothesize that hydrogen peroxide mediates AA‐induced VM‐M3 and U87 cell death, and thus that exogenous catalase treatment will attenuate this effect. It was found that 1‐hour treatment with pharmacological AA decreased viability in both VM‐M3 and U87 cells, with VM‐M3 cells exhibiting greater sensitivity to treatment (assessed with crystal violet staining). HBOT did not enhance AA‐induced cytotoxicity in U87 cells; however, HBOT did sensitize VM‐M3 cells to AA treatment. Additionally, 1‐hour co‐treatment with AA and NAC, as well as AA and catalase, rescued cell death in U87 and VM‐M3 cells, suggesting that AA‐induced oxidative stress is mediated by hydrogen peroxide. This study also investigates the effects of AA and HBOT on HIF‐1 alpha, catalase, and transferrin receptor protein expression, yielding further insight into the pro‐oxidative role of AA in cancer.

Support or Funding Information

Work supported by: American Physiological Society Undergraduate Research Excellence Fellowship (APS UGREF); Foundation (501c3); Metabolic Therapy and Cancer Research Account (#250244); Scivation: Florida High Tech Corridor Funding (#MED052‐0061361).

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Citrullinated Histone 3 Contributes to Endothelial Barrier Dysfunction During Sepsis
Authors: Meegan Jamie , Yang Xiaoyuan (Sherry) , Jannaway Melanie , Chatterjee Victor , Yuan Sarah Y.
Published: June 2021

Circulating extracellular histones are known to contribute to tissue injury in systemic inflammatory responses to infection, trauma, and burns. The mechanisms of histone modifications and their effects on inflammation are currently being investigated. One such modification, namely citrullination of histone 3, is catalyzed by the peptidylarginine deiminase 4 (PAD4) enzyme and is thought to initiate the process of NETosis (the release of neutrophil extracellular traps) in response to infection. NETs are composed of a chromatin web with attached proteases and histones, including citrullinated histone 3 (citH3). This study aimed to identify the effects of citH3 on endothelial barrier dysfunction during inflammation. We identified increased citH3 in the tissues and plasma of mice subjected to cecal ligation and puncture, a clinically relevant animal model of polymicrobial sepsis. In vivo, stimulation of mesenteric microvessels with citH3 elicited plasma protein leakage into the surrounding tissue. In cultured endothelial cell monolayers, treatment with citH3 induced barrier dysfunction as indicated by reduced transendothelial electrical resistance. Endothelial monolayer dysfunction partially resulted from disrupted adherens junction proteins VE‐cadherin and β‐catenin, as well as disturbed actin cytoskeletal homeostasis, as demonstrated by immunofluorescence with confocal microscopy. Taken together, the data suggest citH3 induces endothelial barrier dysfunction, which can exacerbate the inflammatory response during sepsis.

Support or Funding Information

This work is supported by NIH R01s HL070752 and HL126646.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Improved methods for characterizing ion channel expression in smooth muscle, endothelial and adventitial cells from collecting lymphatic vessels
Authors: Gui Peichun , Zawieja Scott , Li Min , Castorena‐Gonzale Jorge , Davis Michael J.
Published: June 2021

Lymph is propelled centrally by collecting lymphatic vessels though active, twitch‐like contractions, which are initiated by a pacemaker that generates action potentials to drive calcium influx through voltage‐gated calcium channels in lymphatic smooth muscle cells (LMCs). Whether the pacemaker is intrinsic to LMCs or to another cell type in the vessel wall is not yet established. Both the frequency and amplitude of lymphatic contractions are regulated in turn by a number of additional factors, including the transmural pressure difference, wall shear stress, neurotransmitters, locally‐ and remotely‐generated vasoactive factors, and endothelial‐derived products. Difficulties in identifying the complex interactions of the various cell types within the vessel wall limit discernment of the mechanisms underlying lymphatic pacemaking and the factors that modulate it. Cells within the wall include LMCs, lymphatic endothelial cells (LECs), dendritic cells, macrophages, mast cells, fibroblasts, nerve endings and possibly glia. We developed methods for purifying specific cell populations and determining the expression and function of important ion channels with a focus on identifying the ion channels that control and modulate lymphatic function. Our strategy was to utilize fluorescent reporter mice (SMMHCGFP, Prox1GFP and PDGFRaGFP, respectively) to 1) identify the distribution of LMCs, LECs and a population of adventitial fibroblast‐like cells in mouse inguinal‐axillary lymphatic (IALs) vessels; 2) enzymatically dissociate IALs and utilize fluorescent activated cell sorting of GFP+ cells to obtain pure cell populations for transcriptional analysis of key ion channels; 3) confirm the presence and functionality of identified ion channels through whole cell patch clamp of GFP+ cells. LMCs expressed message for SMA, TMEM16A, SK3, BK and Cav1.2 but were negative for eNOS, Prox1 and VE‐caderin. LECs were positive for VE‐cadherin and eNOS but negative for SMA, SMMHC, PDGFRa, Cav1.2, TMEM16A and all SK‐IK‐BK channels. PDGFRa+ cells were positive for VE‐cadherin, SMA, SMMHC, TMEM16A, Cav1.2 and SK1, but negative for eNOS and all other SK‐IK‐BK channels. Single GFP+ cells used for patch clamping revealed spindle‐shaped LMCs expressing functional TMEM16A, Cav1.2, Cav3 and variable BK currents, LECs (also spindle‐shaped) that did not exhibit inward cation current or SK‐IK current, and rounded PDGFRa+ cells with SK currents. Our techniques improve on whole‐vessel and explant analyses, align with the results of recent functional studies, and show the feasibility of using these methods to more accurately determine the functional roles of the various cell populations in the lymphatic wall.

Support or Funding Information

NIH HL122578

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Postconditioning Effect of PDE5 inhibitor, Sildenafil in Normal and Diabetic Rabbits following Myocardial Ischemia/Reperfusion injury.
Authors: Ockaili Ramzi , Samidurai Arun , Filippone Scott M. , Cain Chad K. , Das Anindita , Kukreja Rakesh C.
Published: June 2021

Background

Several studies have demonstrated cardioprotective effects of the phosphodiesterase 5 inhibitor, sildenafil (SILD), in the setting of myocardial ischemia and reperfusion (MI/R) in mice and rabbits. However, it is unclear whether sildenafil exerts postconditioning effect in normal and diabetic rabbits.

Methods and Results

To induce diabetes, alloxan monohydrate (125 mg/kg, i.v.) was administered in New Zealand white male rabbits (n=10; ~3 kg, 4 months old). Blood glucose level increased to 342±21 mg/dL following 2 weeks of alloxan administration. Balloon occluders were implanted on the top of the coronary artery and 7 days later, conscious rabbits were subjected to 45‐min ischemia (I) and 3 days of reperfusion (R) by inflating/deflating the balloon. Saline (as control) or Sildenafil (0.71 or 1.4 mg/kg; i.v.) was infused for 60 min starting 5 min before reperfusion. Myocardial infarct size was determined by triphenyltetrazolium chloride staining and troponin I (cTnI) was measured using ELISA method in the plasma. The result showed that sildenafil (both concentrations) significantly reduced myocardial infarct size (Figure A) and peak plasma levels of cardiac troponin I. Similarly, sildenafil reduced infarct size in diabetic rabbits (DM) following MI/R (Figure C). There were no differences in hemodynamics during MI/R or myocardial area‐at‐risk (AAR) (Figure B and D).

Conclusion

Sildenafil exerts significant postconditioning effect in normal and diabetic rabbits of conscious I/R model that is potentially is free of confounding factors associated with open‐chest preparations.

Support or Funding Information

NIHRO1HL134366 (AD & RCK), RO1HL118808 (RCK), RO1HL133167 (RCK)

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Bilirubin, a novel endocrine hormone with fat burning properties
Authors: Hinds Terry D. , Gordon Darren M. , Combs Shauna D. , Stec David E.
Published: June 2021

Plasma bilirubin levels have been shown to be inversely related to obesity, diabetes, and cardiovascular events. Numerous clinical and population studies have demonstrated that increased serum bilirubin levels protect against metabolic diseases such as obesity and diabetes. Bilirubin is a potent antioxidant, and the beneficial actions of moderate increases in plasma bilirubin have been thought to be due to the antioxidant effects of the bile pigment. In the present study, we found that bilirubin has a new function as an endocrine hormone that can signal to the fat burning nuclear receptor, peroxisome proliferator‐activated receptor α (PPARα). The bilirubin‐PPARα mediated effects cause a significant decrease in lipid accumulation in adipose and liver of mice. Bilirubin is conjugated in the liver by UDP‐glucuronosyltransferase 1‐1 (UGT1A1) protein and then excreted into bile. Furthermore, we found that blockade of the UGT1A1 by either anti‐sense morpholinos or by genetic mutation caused significantly raised plasma unconjugated bilirubin levels which also heightened PPARα transcriptional activity in liver and adipose. Ultimately, increasing plasma unconjugated bilirubin decrease adiposity and lower blood glucose levels. These data indicate a new function for bilirubin as an endocrine hormone that can be regulated in the liver to control adiposity and insulin resistance in peripheral tissues. Targeting of UGT1A1 in the liver may offer future implications in the treatment of obesity and type II diabetes.

Support or Funding Information

This research was supported by the National Heart, Lung, and Blood Institute of the National Institutes of Health under Award Number K01HL125445 (T.D.H.) and PO1HL‐51971 (D.E.S) and National Institute of General Medical Sciences P20GM‐104357 (D.E.S.).

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Excessive βeta‐adrenergic receptor stimulation induces cardiomyocyte necroptosis via a RIP3‐dependent pathway
Authors: Wu Penglong , Wang Yibin , Wang Xuejun
Published: June 2021

Background/Hypothesis

Excessive β‐adrenergic receptor activation causes maladaptive cardiac remodeling in humans with myocardial infarction. Cardiomyocyte apoptosis and necrosis are both considered as the main modes of cell death in β‐adrenergic receptor agonist isoproterenol (ISO)‐induced myocardial injury but the mechanism underlying necrosis remains elusive. Necroptosis is a newly discovered form of regulated cell death that exhibits morphological characteristics of necrosis but is regulated by the receptor interacting protein kinase (RIP) 3‐dependant pathway. Necroptosis has been suggested to play a role in the pathogenesis of common cardiovascular diseases. During necroptosis, the formation of necroptosomes consisting of proteins including RIP3 and RIP1 is critical. More recently, Ca2+/calmodulin‐dependent kinase II (CaMKII), which can be phosphorylated and thereby activated by β‐adrenergic receptor activation, was also reported to be involved in cardiomyocyte necroptosis by directly binding to and being phosphorylated by RIP3. However, whether ISO‐induced cardiomyocyte death involves necroptosis requires further investigation. Hence, we sought to test the hypothesis that RIP3‐depedent cardiomyocyte necroptosis contributes to ISO‐induced cardiac injury.

Methods and Results

Young adult mixed‐sex wild type (RIP3+/+) or RIP3 global knockout (RIP3−/−) C57BL/6 mice were subject to two consecutive subcutaneous injections of ISO (85mg/kg body weight/injection, 24h interval) or vehicle, myocardial tissues were collected at 24h after the 2nd ISO injection. Evan's blue dye (EBD) was administered (100ug/g body weight, i.p.) at 18h before tissues collection for the EBD‐based detection of increased cardiomyocyte membrane permeability, a hallmark of necrosis. In RIP3+/+ mice, the ISO treatment significantly increased myocardial protein expression of RIP3 (both in the soluble and insoluble fractions), RIP1, RIP3 bund RIP1 (as detected by immunoprecipitation), and phosphorylated CaMKII and resulted in increased EDB uptake by a large number of cardiomyocytes (p<0.01). By contrast, the elevation of RIP1 and the number of EBD‐positive cardiomyocytes, but not phosphorylated CaMKII, were significantly less in the RIP3−/− mice subject to the same ISO treatment (p<0.01).

Conclusion

Induction of myocardial injury by excessive β‐adrenergic receptor activation involves cardiomyocyte necroptosis via a RIP3‐dependent but CaMKII‐independent pathway.

Support or Funding Information

This work is supported in part by grants from the NIH [HL072166, HL085629, and HL131667] and the National Natural Science Foundation of China [81570278]

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Ascorbate Rescues Peroxide Induced Insulin Resistance in Skeletal Muscle
Authors: Eccardt Amanda , Mattathil Lyn , Bell Thomas , Patel Rishi , Mannino Mark , Fisher Jonathan
Published: June 2021

Ascorbate plays a key antioxidant role in protection of cells from damage by reactive oxygen species, which have been implicated in causing metabolic dysfunction such as diabetes. Thus, we hypothesized that ascorbate could aid in rescue from insulin resistance brought on by exposure to reactive oxygen species. In addition, we hypothesized that the peroxidase mimetic Fe(III)tetrakis(4‐benzoic acid)porphyrin (FeTBAP) could act synergistically with ascorbate in defense against oxidative stress. In order to measure the protective effect of ascorbate, we pretreated C2C12 myotubes with ascorbate. We then exposed the myotubes to glucose oxidase (GO), an enzyme that utilizes glucose to produce hydrogen peroxide, thus mimicking a state of oxidative stress prior to exposure to insulin. Data were quantified via western blot analysis monitoring phosphorylated AKT and total AKT. In order to assay the ability of FeTBAP in conjunction with ascorbate to protect against insulin resistance, C2C12 myotubes and isolated soleus were first pretreated with ascorbate and FeTBAP and then exposed to glucose oxidase followed by insulin. We found that ascorbate rescues insulin resistance brought on by exposure to reactive oxygen species. In addition, preliminary data from C2C12 myotubes and soleus illustrated a trend in which FeTBAP in combination with ascorbate protected against insulin resistance. Taken together, these data demonstrate a functional role of ascorbate in which the antioxidant on its own and in conjunction with FeTBAP rescues cells from peroxide induced insulin resistance.

Support or Funding Information

This project was supported by United States Public Health Service award R15DK102122 from the National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK).

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Promoting ubiquitination‐mediated degradation of Orai1 protein by high glucose in glomerular mesangial cells
Authors: Chaudhari Sarika , Hui Jiang , Zou Shubiao , Huang Linjing , Ma Rong
Published: June 2021

Glomerular mesangial cells (MCs) are an important target of metabolic abnormalities in a diabetic environment. The Orai1‐mediated store‐operated calcium entry (SOCE) is associated with many physiological processes in a variety of cells, including MCs. However, whether SOCE in MCs is involved in diabetic kidney disease is not clear. High glucose (HG) is the principal cause of MC pathogenesis in diabetes and altered MC function by HG is central to the pathogenesis of progressive diabetic glomerulopathy. The present study was carried out to determine if HG treatment altered the protein content of Orai1 and the protein‐mediated SOCE in MCs. Western blot was conducted to estimate abundance of Orai1 protein and Fura‐2 fluorescence ratiometry was used to analyze SOCE. We found that treatment of rat MCs with HG (25 mM) for time periods ranging from 2 hours to 24 hours decreased the abundance of Orai1 protein. A significant decrease was observed at the time point of 8 hours, which sustained at least for additional 16 hours. Consistently, HG treatment for 8 hours significantly reduced SOCE. HG treatment for the same time periods did not alter the level of Orai1 transcript. In the presence of cycloheximide, a protein synthesis inhibitor, the HG effects on the level of Orai1 protein still existed, suggesting posttranslational mechanisms involved. Furthermore, both MG132 (the ubiquitin‐proteasome inhibitor) and NH4Cl (the lysosomal pathway inhibitor) significantly attenuated the HG‐induced reduction of Orai1 protein abundance. Moreover, HG treatment for 8 hours stimulated ubiquitination of Orai1 protein. We further found that HG treatment elevated the level of cellular hydrogen peroxide (H2O2) in a time‐dependent manner. Treatment of cells with PEG‐catalase significantly blunted the HG‐induced reduction of Orai1 protein abundance. In addition, H2O2 itself also significantly decreased the abundance of Orai1 protein and increased the level of ubiquitinated Orai1. Taking together, these results suggest that HG treatment for a short‐term decreased abundance of Orai1 protein in MCs by promoting its degradation through the ubiquitination‐proteosome and –lysosome mechanisms. This HG‐stimulated posttranslational regulation of Orai1 protein was mediated by H2O2.

Support or Funding Information

1) NIH/NIDDK: R56 DK108761; 2) NIH/NIDDK: 1RO1DK115424‐01; 3) AHA Southwest Affiliate: 16GRNT27780043; 4) The Harry S. Moss Heart Trust.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Splicing Variation of TTN Novex Isoforms Across Species and RBM20 Does Not Regulate Splicing of Novex Isoforms in Cardiac Muscle.
Authors: Chen Zhilong , Guo Wei
Published: June 2021

TTN is a major disease‐causing gene in cardiac muscle. TTN contains 363 exons in human encoding various sizes of TTN protein due to alternative splicing regulated mainly by RNA binding motif 20 (RBM20). Three isoforms of TTN protein are produced by alternative exon usages of exon 45 (Novex 1), exon 46 (Novex 2) and exon 48 (Novex 3). It is unknown how these exons are alternatively used in Novex isoforms across species and whether Novex isoforms are associated with heart disease. In this study, we compared alternative exon usage between species with the mVISTA online tool, and found that exon 45 is conserved across all species, but exons 46 and 48 only found in some species. Interestingly, we found a conserved region between exons 47 and 48 across species which has never been characterized before. RT‐PCR and DNA sequencing confirmed that this is a new exon named as 48′ in Novex 3. In addition, with primer pairs for Novex 1, we found two new splicing forms, which one has intron 44 retention and the other has intron 45 retention. With Novex 2 primer pairs, we found that Novex 2 is lack of expression in pig, mouse and rat. A new truncated isoform with intron 46 retention is mainly expressed in human and frog heart, and another new truncated isoform with exon 45 and 46 are co‐expressed predominantly in chicken and frog heart. Using RBM20 knockout heart, we revealed that RBM20 doesn't regulate splicing of Novex isoforms. Finally, we examined the expression levels of Novex isoforms in human heart with cardiomyopathies, and we didn't find any difference. These results suggest that exon usage of Novex isoforms varies across species which is not associated with cardiomyopathies. Hence, their functional role in the heart need be further studied in the future, which is beyond the scope of this study.

Support or Funding Information

This work was supported by the NIFA‐USDA 1009266, National Institutes of Health NIGMSP20GM103432, AHA BGIA and Faculty‐Grant‐in‐Aid from University of Wyoming.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Evaluating the importance of pseudoknot formation to HTLV‐1 pro‐pol ‐1 programmed ribosomal frameshift stimulation.
Authors: Cooper‐Sansone Andrew , Williams Marcus , Chadeayne Devon , Contreras Uriah , Dailey Leandrew , Joe Natalie , Mylroie Elena , Mouzakis Kathryn
Published: June 2021

The Human T‐cell Lymphotropic Virus Type 1 (HTLV‐1) encodes two ‐1 programmed ribosomal frameshift (PRF) sites, which allow for the translation of enzymes critical to viral replication. The frequency, or frameshift efficiency, of this event is also thought to be important in the viral life cycle. Each PRF site includes a heptanucleotide “slippery” sequence, a spacer, and a downstream structure. The downstream structure in the HTLV‐1 pro‐pol frameshift site is predicted to be a pseudoknot. The importance of this structure to the efficiency of frameshifting has not yet been established. To improve our understanding of the role this structure has in frameshift stimulation, we designed frameshift site variants that disrupt the pseudoknot structure. The frameshift efficiency for these sites was then measured using an in vitro dual‐luciferase assay. Preliminary data suggests that the removal or mutation of sequences important to pseudoknot formation significantly reduce the HTLV‐1 pro‐pol frameshift efficiency. These findings are significant because they suggest that the HTLV‐1 pro‐pol frameshift site pseudoknot structure plays a criticial role in frameshift stimulation.

Support or Funding Information

IH NIGMS SCORE SC2 Award (1SC2GM121197‐01), Research Corporation for Science Advancement Cottrell Scholar Award (23983), Fort Lewis College Undergraduate Scholarly and Creative Activities Award

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Mechanistic interrogation of the entry‐ and exit‐channel arms of eIF3
Authors: Aitken Colin Echeverría , Beznoskova Petra , Dong Jinsheng , Yourik Paul , Valasek Leos , Hinnebusch Alan G. , Lorsch Jon R.
Published: June 2021

Eukaryotic translation initiation factor 3 (eIF3) is a pivotal player in the recruitment of mRNA to the translation pre‐initiation complex (PIC). Biochemical and genetic studies have identified interactions of eIF3 with the PIC near the mRNA entry and exit channels of the ribosome; structural studies have revealed that eIF3 binds the solvent face of the small (40S) ribosomal subunit, projecting arms into both mRNA channels and ultimately encircling the PIC. We recently probed the effects of a library of S. cerevisiae eIF3 functional variants using an in‐vitro reconstituted system. These experiments demonstrated that mutations throughout eIF3 disrupt its interaction with the PIC and diminish its ability to accelerate recruitment of a native yeast mRNA. Using a series of model mRNAs to control the amount of nucleotide sequence in either the mRNA entry or exit channel, we further revealed a critical role for eIF3 in stabilizing mRNA at the exit channel. Together, these experiments are consistent with the entry‐ and exit‐channel arms of eIF3 playing distinct roles during PIC formation, mRNA recruitment, and scanning. We have also investigated the collaboration of eIF3 with Rps3/uS3 in stabilizing mRNA within the mRNA entry channel. We found that Rps3/uS3 residues previously implicated in the helicase activity of the elongating ribosome stabilize mRNA binding within ribosomal entry channel and enhance the effects of eIF3 on mRNA binding in both channels. More recently, we have observed that eIF3i and eIF3g contribute to the PIC‐dependent enhancement of eIF4A ATPase activity, which recent work suggests may function to remodel the mRNA entry channel. Our current work is focused on dissecting the interactions between eIF3, the PIC, and other initiation factors in both the mRNA entry and exit channels, as well as on the inter‐subunit face of the PIC, using both in‐vitro and next‐generation sequencing approaches.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Transcriptional and Post‐Transcriptional Regulation of mtlA in Vibrio cholerae
Authors: Wang Jessica , Zhang Mark
Published: June 2021

Vibrio cholerae, the bacterium that causes cholera, performs a complex regulation of genes encoding carbon‐source transporters in order to survive in both aquatic reservoirs and the human small intestine. One such transporter is MtlA, which operates within the phosphotransferase system (PTS), a highly‐conserved system for carbohydrate uptake across bacteria. MtlA concomitantly phosphorylates and transports mannitol, the six‐carbon sugar alcohol produced by algae, into the cell. In addition to mtlA, the mtl locus contains mtlS and mtlR, which encode two major regulators of MtlA. mtlS encodes a 120‐nucleotide small regulatory RNA (sRNA) that posttranscriptionally regulates MtlA synthesis by binding to the mtlA mRNA 5′ untranslated region. Our objective is to identify regulators of mtlS expression. We have constructed a V. cholerae strain harboring a chromosomal mtlS‐lacZ transcriptional fusion. Using a V. cholerae genomic library, we identified putative regulators of mtlS expression. The second regulator of mtlA, encoded by mtlR, showed repressive effects in Escherichia coli, but had not been well characterized in V. cholerae. We studied how MtlR regulates MtlA activity in V. cholerae grown in a variety of carbon sources. Biofilm assays demonstrated that mtlR overexpression results in a greater than 50% decrease in biofilm formation (p<0.001 by unpaired two‐tailed t‐test), an activity known to be positively affected by MtlA. qRT‐PCR showed that a ΔmtlR mutant has higher mtlA RNA levels than a wild‐type control when grown in glucose‐supplemented minimal medium (p<0.0001 by unpaired two‐tailed t‐test), and even higher mtlA RNA levels when grown in maltose‐ and mannose‐ supplemented minimal medium (p<0.01 by unpaired two‐tailed t‐test). Western blotting analysis reflected these increases; MtlA protein levels were more elevated in ΔmtlR compared to wild‐type when grown in glucose, maltose, and mannose. The ΔmtlR mutant was partially complemented, as demonstrated by qRT‐PCR (p<0.01 by unpaired two‐tailed ttest) and western blotting analysis. This shows that the observed increases in MtlA were a direct effect of the gene product of mtlR. These results contribute to a model in which MtlR acts as a transcriptional repressor of mtlA in non‐mannitol conditions, working synergistically with MtlS to regulate mtlA.

Support or Funding Information

NIH R15 AI090606

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Determination of the PIP2 binding sites on ENaC
Authors: Archer Crystal R. , Chen Yinghua , Stockand Alexis M. , Stockand James D.
Published: June 2021

Hypertension is a major contributor to cardiovascular disease. Much of the details about the molecular mechanisms that regulate blood pressure, though, are obscure. Activity of the Epithelial Na+ Channel (ENaC) is rate limiting for Na+ reabsorption in the distal nephron. Consequently, ENaC activity fine‐tunes plasma volume and blood pressure. ENaC activity is facilitated by plasma membrane‐bound phosphatidylinositol 4,5‐bisphosphate (PIP2). Phospholipase C (PLC)‐mediated depletion of PIP2 reduces ENaC activity. Specific PIP2 binding sites within ENaC, however, remain as yet undefined. We hypothesize that ENaC has multiple PIP2 binding sites within its beta and gamma subunits. In this study, we investigate the direct biochemical binding of PIP2 to specific sites within ENaC subunits by biochemical and electrophysiology methods. We designed synthetic peptides that correspond with regions in the N‐termini of ENaC subunits that are enriched in basic residues. Binding affinities of these channel peptides are tested with fluorescent labeled PIP2 analogs using biophysical methods like microscale thermophoresis. To verify these findings, pulldown of transfected CHO cell lysates using ENaC antibodies followed by dot blot analysis is used to compare the binding of PIP2 to wild type ENaC, with the binding of ENaC containing mutations in the proposed binding sites. To simultaneously analyze the functional importance of these proposed PIP2 sites, we also compare macroscopic currents of wild type and mutant ENaC in responses to activation of PLC. We expect these results to improve our understanding of the regulation of ENaC by PIP2 as we aim to define the specific binding sites and the binding affinity of PIP2 for ENaC subunits.

Support or Funding Information

NIH/T32 HL007446

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.