bluuebunny/crossref_metadata_2025 huggingface dataset:
Pannexin channels regulate cell‐cell calcium mobilization and motility during epithelial wound repair
Authors: Gonzales Selina Grace , Lee Yoonjoo , Trinkaus‐Randall Vickery
Published: June 2021

The cornea is a transparent avascular tissue that transmits light to the retina and covers the lens and iris. According to the World Health Organization, blindness due to injury and disease in the cornea is the fourth leading cause of preventable blindness. Previous research demonstrates that epithelial cells release nucleotides that activate purinergic receptors P2X7 and P2Y2, which induces cell‐cell calcium mobilization. This calcium mobilization is reported to potentially play a role in collective cell migration and wound healing. To better understand this specific mobilization, we studied pannexin, a channel forming glycoprotein. ATP is able to move through the Pannexin channel and binds with P2X7. We want to investigate the mechanism of the calcium signal propagation and whether pannexins are involved. We hypothesize that pannexin channels mediate cell‐cell calcium mobilization and regulates actin activity in response to injury or agonist stimulation. Experiments were performed using 10Panx, an inhibitory peptide for pannexin channels, and scramble Panx, a control peptide. Human Corneal Limbal Epithelial (HCLE) cells were cultured to confluence on glass bottom dishes. Twenty‐four hours prior to experimentation, growth supplements were removed. Cells were preincubated with either 100uM 10Panx or 100uM scramble Panx to inhibit pannexin channels. In order to visualize the change in calcium, cells were pre‐loaded with 5 μM Fluo‐3AM, a fluorescent dye, at a final concentration of 1% (v/v) DMSO and 0.02% (w/v) pluronic acid for 20 minutes at 37°C and 5% CO2. Images were collected every 3 seconds on a Zeiss Axiovert LSM 880 confocal microscope for a period of 45 minutes after either agonist stimulation or injury. The agonist used for stimulation was BzATP, a synthetic nucleotide that selectively activates P2X7 and UTP, an agonist for P2Y2. Baseline and post‐wound frames were captured. Analysis was performed using FIJI/ImageJ and MATLAB programs. We found that 10PanX correlated with dampened calcium mobilization after injury. In uninjured cultures the decrease was only detected in response to BzATP and not to UTP. Actin activity was also inhibited by 10PanX after injury. MATLAB analysis using heatmap indicates that 10PanX reduces correlation of cells during calcium mobilization (Scr BzATP mean: 0.030 10PanXBzATP mean:0.079). Our data indicates that P2X7‐mediated Pannexin channels play a role in the injury response. This provides greater insight as to the level of involvement of pannexin in calcium signal propagation in response to injury in HCLE cells.

Support or Funding Information

NIH 5R25HL118693‐05, NIH RO1 EY06000, NIH R21 EY024392, The Mass Lions Foundation New England Corneal Transplant Fund

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Dialysis of an Unknown Cytosolic Cofactor Results in Partial, Delayed Activation of TRPA1
Authors: Parks Thomas
Published: June 2021

In many organ systems, a subset of sensory nerve fibers termed nociceptors are stimulated by noxious stimuli and evoke defensive sensations, behaviors and reflexes. Transient Receptor Potential Ankyrin 1 (TRPA1) is a non‐selective cation channel that is expressed in sensory neurons and evokes action potentials in nociceptive fibers when activated. Agonists of TRPA1 are electrophilic compounds that covalently modify reactive cysteine residues on TRPA1 subunits, which result in activation. Electrophiles can modify cysteines through reversible or irreversible reactions based on the structure of the compound. Whole‐cell patch clamp and live‐cell Ca2+ imaging have been used to characterize human TRPA1 (hTRPA1) channel activity (expressed in HEK293 cells) in the presence of electrophiles. Ca2+ imaging experiments show that irreversible electrophiles like N‐ethylmaleimide (NEM) rapidly increase Ca2+ influx, which suggests TRPA1 activation. However, whole‐cell patch clamp studies show a delayed, partial activation of TRPA1 with NEM treatment, but display rapid activation by reversible electrophiles. Furthermore, inside‐out single channel patch clamp experiments exhibit a reduction in open probability with NEM treatment that is comparable to the whole‐cell patch clamp experiments. To determine the mechanism of TRPA1 activation, the cause for the disparity of TRPA1 activation between whole‐cell patch clamp and Ca2+ imaging assays must be identified. We hypothesized that dialysis of cellular components in whole‐cell patch clamp that promote rapid, robust TRPA1 activation with NEM. To test our hypothesis, I performed whole‐cell patch clamp and gramicidin‐perforated patch clamp, a non‐invasive patch clamp method, to observe and compare the rate‐of‐change of NEM‐induced TRPA1 responses. Both methods contain 5mM Na‐polyphosphates in the pipette solution to prevent rundown of the TRPA1 channel. Studies using gramicidin‐perforated patch clamp exhibit a significant increase in the rate‐of‐change of NEM‐induced TRPA1 responses compared to activation in whole‐cell experiments. We also hypothesize that glutathione is the unidentified cofactor that is dialyzed out of the cell in whole‐cell experiments. Future whole‐cell experiments with glutathione (5mM) in the pipette solution will be performed to determine if rapid TRPA1 activation can be rescued. Identification of the unknown cytosolic cofactor will promote a complete understanding of the electrophilic cysteine modification events that underlie TRPA1 activation.

Support or Funding Information

R01 from the NHLBI

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
MicroRNA‐944 inhibits migration and invasion in melanoma cells by targeting Notch2
Authors: Kim Sang Hoon
Published: June 2021

Melanoma is a malignant tumor of melanocytes. The incidence of melanoma is increasing in worldwide. miR‐944 is a primates‐specific microRNA, which plays a role as putative tumor suppressor in cancer cells. However, the function of miR‐944 in melanoma is not reported yet. In this study, we observed that the migration and invasion in melanoma cells transfected with miR‐944 was decreased compared to that in control cells. The expression of cellular invasion‐related genes was decreased in miR‐944 transfected cells. By using bioinformatics program, Notch2 was selected as a target of miR‐944. The overexpression of miR‐944 inhibited the expression of Notch2 in melanoma cells. By reporter assay, we found miR‐944 binding to the 3′UTR of Notch2. Knockdown of Notch2 in melanoma cells also suppressed cellular migration and decreased the expression of cellular invasion‐related genes. Taken together, our results show that miR‐944 suppressed migration of melanoma cells by targeting Notch2. These finding may have important implications concerning the molecular mechanisms of invasion and migration in melanoma.

Support or Funding Information

This research was supported by a grant from Basic Science Research Program through the National Research Foundation of Korea (NRF) (No. 2017R1A2B4009615).

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Binding Potential between miR‐126 and Klotho to Inhibit Inflammation in U937 Monocytes
Authors: Budd Tristan , Rabquer Bradley James
Published: June 2021

Rheumatoid Arthritis (RA) is a chronic, autoimmune disease that features inflammation of synovial tissue in the joints. Monocytes play a key role in the pathophysiology of inflammation and have been found to be activated in patients with RA. Micro RNA (miRNA) can bind to messenger RNA (mRNA) and prevent protein translation. Previous work in our laboratory has revealed specific miRNA molecules, including miR‐126, that are overexpressed in monocytes isolated from RA patients. miR‐126 is a miRNA molecule that can be regulated to impact signaling pathways involved with RA. The KLotho gene has previously been shown to play a role in inflammatory monocytes. We hypothesized that miR‐126 binds to the 3′ UTR of KLotho mRNA, thus inhibiting the translation of inflammatory proteins.

Methods and Results

The database on microrna.org lists potential gene targets for miRNA molecules, predicted by the program miRanda. The binding scores between the target mRNA and miRNA are powered by mirSVR. Using microrna.org, the KLotho gene was selected as a potential target gene to be bound by miR‐126 based on the two mirSVR scores of −0.0602 and −1.1269 within KLotho's 3′UTR. U937 monocytes were grown, monocyte mRNA was isolated and was used to make cDNA. Forward and reverse primers were designed for the 3′UTR of KLotho mRNA. These primers were used in the qPCR reactions along with the U937 cDNA in order to amplify the 3′UTR of the KLotho mRNA insert. A double digest was performed on both the KLotho insert and a pmirGLO dual luciferase vector using Sac1 and Sal1. The cut 3′UTR of KLotho mRNA was cloned into the cut pmirGLO vector with ligation and transformation with competent E.Coli cells. A 1% agarose gel electrophoresis was performed to analyze the presence of KLotho insert in the pmirGLO vector. The gel analyzed the qPCR products, amplifying the transformation product using sequencing primers. A band on this gel around 750 base pairs confirms the presence of the KLotho insert in the pmirGLO vector. Sequencing of the transformation product is used to further confirm successful cloning of the KLotho insert into the pmirGLO vector. After transfection of the cloned insert in vector, the presence of the insert KLotho gene in the pmirGLO vector can be confirmed by performing a Dual‐Glo□ Luciferase Assay.

Conclusion

The KLotho gene is expressed in U937 monocytes. To analyze the 3′ UTR KLotho mRNA cloning into the pmirGLO vector, a 1% agarose gel was performed, and the presence of KLotho mRNA was confirmed in the pmirGLO dual luciferase vector.

Support or Funding Information

Albion College Foundation for Undergraduate Research, Scholarly, and Creative Activity, and the Albion College Biology Department

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Superoxide‐Dependent Redox Signaling in the Supraoptic Nucleus Is Associated with the Neuroendocrine Response to Water and Electrolyte Imbalance
Authors: de Lima Juliana Bezerra Medeiros , de Morais Sharon Del Bem Velloso , Schlichte Sarah L. , Debarba Lucas Kniess , Coletti Ricardo , Veanholi Vechiato Fernanda M. , Antunes‐Rodrigues Jose , Zucker Irving H. , Zimmerman Matthew C.
Published: June 2021

In models of altered osmolality and water balance, including two‐day salt loading and water deprivation, previous studies have suggested that intracellular redox signaling in the supraoptic nucleus (SON) contributes to the neuroendocrine response. However, the precise redox‐dependent signaling pathways and reactive oxygen species (ROS) involved in this response remain unclear. Herein, we tested the hypothesis that water deprivation increases specific ROS, particularly superoxide, and stimulates the redox‐sensitive transcription factor, nuclear factor erythroid 2‐related factor 2 (Nrf2) in the SON and that these responses are dependent on the duration of water restriction. To test this hypothesis, we used rodent models deprived of water for 24 and 48 hours (24h WD and 48h WD, respectively). We observed an increase in Nrf2 mRNA in the SON of rodents subjected to 24h WD (Control: 0.84 ± 0.08 vs 24h WD: 1.16 ± 0.09 arbitrary units) suggesting the activation of redox signaling during dehydration. In addition, at 48h WD, levels of hemeoxygenase‐1 (HO‐1), a Nrf2 target gene, were elevated in the SON (Control: 0.98 ± 0.1 vs 48h WD: 1.46 ± 0.1 arbitrary units). HO‐1 activity was also increased at both time points of water restriction (F2,34= 7.718, p < 0.05). To determine if levels of specific ROS are altered during water deprivation, we used the electron paramagnetic resonance (EPR) spectroscopy. After 24h or 48h of water deprivation in C57BL/6 mice, fresh non‐fixed brains were collected and the SON was harvested via micropunch. Superoxide levels were measured in the SON micropunches by EPR. We also measured plasma osmolality and volume. We observed a dehydration period‐dependent increase in superoxide levels (reported at EPR spectra amplitude in arbitrary units) in the SON (Control: 7150 ± 3856; 24h WD: 14240 ± 9931; 48h WD: 15795 ± 7400 arbitrary units/mg of tissue) along with plasma osmolality (Control: 317 ± 2.0; 24h WD: 326.8 ± 1.3; 48h WD: 330.7 ± 1.3 mOsm/kg H2O); protein (Control: 3.56 ± 0.16; 24h WD: 4.36 ± 0.16; 48h WD: 3.94 ± 0.14 g/dL plasma); and hematocrit (Control: 35.8 ± 5.1; 24h WD: 42.4 ± 0.9; 48h WD: 45.7 ± 0.9 %). Together, these data suggest that the neuroendocrine system is associated with superoxide‐dependent redox signaling and activation of Nrf2 in the SON to cope with dehydration.

Support or Funding Information

FAPESP

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
The Effects of 4‐weeks of Prolonged Nighttime Fasting on Biomarkers of Health and Longevity in Healthy Males
Authors: Dirks‐Naylor Amie J. , Wilson Jennifer A. , Bush Mark , Waghel Rashi , Dresser Johanna , Gandhi Nidhi , Hanseler Gregory
Published: June 2021

Caloric restriction and intermittent fasting have shown to confer a plethora of health benefits. However, traditional regimens of caloric restriction (restricting caloric intake by 30–40% every day) and intermittent fasting (fasting every other day), have proven difficult to adhere. Prolonged nighttime fasting is a type of intermittent fasting which may be an easier regimen to maintain, but has been scarcely studied. Thus, the aim of this study was to determine the effects of this regimen on biomarkers of health and longevity. Eleven healthy men ranging in age from 21–39 years fasted for 16‐hrs (~6pm–10am) each night for 4‐weeks. Subjects were instructed to eat their normal diet outside of the suggested fasting hours. Pre‐ and post‐ fasting blood samples were collected. 24‐hr food and drink intake was self‐recorded periodically before and during the fasting period using the Automated Self‐Administered 24‐hour Recall (ASA24) website. Subjects also completed a survey pertaining to compliance and feeding behavior in response to the fasting protocol. Prolonged‐nighttime fasting decreased systolic blood pressure (SBP), but did not have a statistically significant effect on other biomarkers: diastolic BP, body mass index, resting heart rate, or fasting plasma levels of glucose, lipids, beta hydroxybutyrate, fructosamine, C‐reactive protein, insulin, insulin‐like growth factor (IGF), fibroblast GF21, growth differentiation factor 11, myostatin, adiponectin, or sirtuin 3. Average daily caloric intake did not differ before and during the fasting protocol. When surveyed, 63.6% of subjects felt it was very easy or easy to adhere to the fasting protocol, while 9.1% felt it was difficult or very difficult. While the majority of subjects believed that they decreased their food intake during the fasting protocol, their cravings for unhealthy food increased. In conclusion, the prolonged nighttime fasting regimen did not show an effect on most biomarkers of health and longevity. This may have been due to the small number of subjects who were also mostly young and healthy, short study duration, changes to unhealthy food choices, and/or lack of compliance.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Luteolin Relaxes Cholecystokinin‐Induced Tension in Guinea PIg Gallbladder Strips
Authors: Kline Loren
Published: June 2021

Luteolin, a flavone, is found in celery, broccoli, green pepper, parsley, carrots, and olive oil. Luteolin was shown to relax rat aortic rings by stimulating the release of nitric oxide (NO) and rat thoracic aortic strips by blocking intracellular Ca2+ release and activating K+ channels. The purpose of this study was to determine if luteolin had an effect on gallbladder motility. An in vitro technique was used to determine which system(s) mediated the relaxation. Both paired t‐tests and analysis of variance were used for statistical analysis; differences between mean values of p<0.05 were considered significant. Luteolin relaxed cholecystokinin octapeptide (CCK)‐induced tension in male guinea pig gallbladder strips in a concentration‐dependent manner. Adding luteolin prior to either CCK or KCl produced a significant decrease in the amount of tension (0.99±0.07 g vs. 0.82+0.09 g CCK [p<0.02]; 1.1±0.09 g vs. 0.97±0.08 g KCl [p<0.001]. The protein kinase A blocker, PKA inhibitor 14–22 amide myristolated, significantly (p<0.01) reduced the amount of luteolin‐induced relaxation (53.9±3.9% vs. 46.5±4.3%). The blocker of intracellular Ca2+ release, 2‐APB, significantly (p<0.001) decreased the amount of luteolin‐induced relaxation (55.9±5.3% vs. 28.9±4.6%). Neither tetraethylammonium (TEA), a non‐selective K+ channel blocker; NO synthase blocker, L‐NAME; KT5823, blocker of protein kinase G; bisindolymaleimide IV and chelerythrine Cl− were used together and are blockers of PKC; fulvestrant, an estrogen receptor blocker; nor genistein, a protein tyrosine kinase blocker had a significant effect on the luteolininduced relaxation. The luteolin‐induced relaxation of CCK‐ or KCl‐induced tension was mediated by blocking extracellular Ca2+ entry which affected downstream events such as intracellular Ca2+ release, and the activation of protein kinase A. Th.

Support or Funding Information

This research did not receive any specific grant from funding agencies in the public, commercial, or not‐for‐profit sectors.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
The effect of mitochondrial inhibitors on Ca2+ signalling and pacemaking conductances in interstitial cells of Cajal in the mouse small intestine
Authors: Drumm Bernard T. , Sung Tae Sik , Baker Salah A. , Koh Sang Don , Sanders Kenton M.
Published: June 2021

Myenteric interstitial cells of Cajal (ICC‐MY) in the mouse small intestine are pacemaker cells that generate and propagate electrical slow waves. Conduction of slow waves to smooth muscle cells activates voltage‐gated Ca2+ channels and coordinates rhythmic contractions of intestinal muscles. Slow waves are activated by intracellular Ca2+ release events firing asynchronously from multiple release sites within ICC‐MY to activate the Cl− channel Ano1, the resulting depolarization of which forms slow waves. 1 Propagation of slow waves through the ICC‐MY network depends upon activation of a voltage dependent, T‐type Ca2+ conductance (Cav3.2). Previous studies have suggested that mitochondria play an important role in the Ca2+ signaling responsible for slow waves, and slow waves in mouse small intestine, guinea‐pig stomach and canine colon were blocked by pharmacological agents that inhibit the electron transport chain, reduce mitochondrial transmembrane potential and prevent mitochondrial Ca2+ uptake (FCCP, CCCP and Antimycin). 2 We used in‐situ Ca2+ imaging from the small intestines of mice expressing a genetically encoded Ca2+ indicator (GCaMP3) exclusively in ICC (GCaMP3 activated using Cre‐LoxP approach, driven off the Kit promoter) to visualize Ca2+ transients in ICC‐MY and used spatio‐temporal particle‐based analysis techniques to quantify the effects of mitochondrial inhibitors on Ca2+ transients in ICC‐MY. The protonophores FCCP (1 μM, n=6) and CCCP (1 μM, n=6), the ETC inhibitor, Antimycin (10 μM, n=5), and the mitochondrial Na+/Ca2+ exchanger inhibitor CGP‐37157 (30 μM, n=6) either abolished or drastically reduced Ca2+ transients in ICC‐MY. This effect was not due to ATP depletion, since oligomycin (1 μM, n=6) had no effect on Ca2+ transients. We also used the patch‐clamp technique to measure Ano1 and T‐type Ca2+ (Cav3.2) currents in HEK 293 cells to examine the effects of these drugs on key membrane conductances that are essential for slow wave propagation in ICC‐MY. Antimycin (1–10 μM, n=5) blocked Ano1 and reduced Cav3.2 currents (n=4). CCCP (1–3 μM, n=5) had no effect on Ano1 but significantly reduced Cav3.2 currents (n=5). Both Ano1 and Cav3.2 currents were inhibited by CGP‐37157 (10–30 μM, n=5). Thus, the inhibitory effects of mitochondrial drugs on ICC‐MY Ca2+ signalling and slow wave generation can be explained by direct antagonism of the conductances that generate and propagate slow waves and their effects are not necessarily due to mitochondrial effects. Our findings suggest that ICC pacemaker models, which emphasize a role for mitochondrial Ca2+ handling based on similar pharmacological evidence, should be interpreted with caution.

Support or Funding Information

Supported by P01 DK 41315

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Exploring the link between Irritable Bowel Syndrome and the microbiome
Authors: Young Wayne , Gearry Richard , Cotter Paul D. , Fraser Karl , McNabb Warren C. , Roy Nicole C.
Published: June 2021

Irritable Bowel Syndrome (IBS) is a functional gastrointestinal (GI) disorder featuring chronic or recurrent abdominal discomfort, usually with changes in GI habit. However, the mechanisms responsible for IBS are poorly understood. Although alterations in the GI microbiome has been implicated in IBS, there is a lack of consensus on what the exact role of the microbiome is, and how it changes, in IBS. To gain a better understanding of the link between the microbiome and IBS, we undertook shotgun metagenomic sequencing of faecal samples from a case‐control study. The overall aim of the study was to identify microbial and metabolomic factors that provide mechanistic insights into functional GI disorders and increase the predictability of phenotypes for use in nutrition intervention studies. Faecal samples from 112 individuals with functional GI symptoms (cases) or those that were asymptomatic (controls) were shotgun sequenced using the Illumina NextSeq platform. Taxonomic classifications were determined using Metaxa2 and the SILVA 128 database. Gene functions were assigned with the MG‐RAST pipeline. Bacterial genera that discriminated case‐controls from IBS groups included Faecalibacterium, Blautia, Roseburia, Bilophila, and Streptococcus, which were relatively more abundant in certain IBS subtypes. For example, Faecalibacterium was relatively more abundant in individuals presenting with constipation associated IBS (IBS‐C; P<0.001), while Blautia was more prominent in those with the diarrhea form of IBS (IBS‐D; P=0.015). Gene functions that best separated groups included those related to carbohydrate metabolism (higher in IBS compared to case‐controls), protein metabolism, and virulence factors. Our results suggest that carbohydrate fermentation by the microbiome may be an important factor in IBS. This work shows that efforts to understand the role of the microbiome in IBS need to include consideration of microbial function rather than just microbial composition.

Support or Funding Information

Funded by the Ministry of Business, Innovation and Employment, New Zealand

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Central Fructose Impairs Exendin 4‐Mediated Anorexia in the Ventromedial Hypothalamus in Male C57Bl/6 Mice
Authors: Burmeister Melissa , Ayala Julio Enrique
Published: June 2021

Glucagon‐like peptide‐1 (Glp1) is a gut hormone that stimulates insulin secretion but also promotes satiety via actions on the central nervous system. As such, Glp1 receptor (Glp1r) agonists are currently being developed as therapeutics for body weight control. We have previously shown that pharmacological activation of the Glp1r in the ventromedial hypothalamus (VMH) by the Glp1r agonist Exendin‐4 (Ex4) suppresses food intake via stimulation of intracellular glucose metabolism and inhibition of the cellular nutrient sensor AMP‐activated protein kinase (AMPK). This raises the possibility that interference with these nutrient‐sensing mechanisms may attenuate the anorectic effect of Glp1r agonists in the brain. The dietary monosaccharide fructose is an AMPK activator, and intracerebroventricular (ICV) fructose attenuates the anorectic effect of ICV Ex4. The relevance of this finding is underscored by evidence supporting an association between excessive dietary fructose consumption (e.g., high fructose corn syrup, sucrose) and the obesity epidemic. The aim of these studies was to assess whether fructose also impairs Ex4‐mediated anorexia in the VMH. Male C57Bl/6 mice were maintained on a chow diet and were instrumented with bilateral cannulae targeting the VMH for delivery of Ex4 following (1) VMH‐targeted or (2) orally‐gavaged pre‐treatment with fructose, glucose or vehicle. In additional studies, animals were challenged with a high fructose (vs. carbohydrate control) diet for 15wks prior to treatment with Ex4 in the VMH. 18‐h food intake, energy expenditure (EE) and locomotor activity were assessed using a Comprehensive Lab Animal Monitoring System. VMH‐targeted fructose significantly attenuated the food intake‐suppressive effect of VMH Ex4 but had no effect on EE. VMH‐targeted glucose, however, did not potentiate the anorectic effect of Ex4. Orally‐dosed fructose did not impair the anorectic effect of Ex4 in the VMH, nor did dietary fructose. Similarly, orally‐dosed glucose did not potentiate the anorectic effect of Ex4 in the VMH. In conclusion, although fructose impairs the ability of Ex4 to reduce food intake when targeted to the VMH, it is not an inhibitor of VMH Glp1r action on food intake when it is administered along an ingested route (i.e., either orally or dietary). However, these observations validate the role of AMPK as a mediator of the anorectic effect of Ex4 in the VMH. Further elucidation of the interactions between fructose and Glp1r‐mediated signaling in the brain could be beneficial in developing more effective anti‐obesity therapies.

Support or Funding Information

This work was supported by NIH R01 1R01DK097361 to Julio Ayala

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
The effect of zinc deficiency and diet restriction on gene expression of hypoxia‐inducible factors in rat's kidney
Authors: Konomi Aki , Yokoi Katsuhiko
Published: June 2021

We reported that plasma erythropoietin (EPO) concentration, kidney EPO concentration and kidney EPO mRNA level were changed by moderate zinc deficiency or diet restriction. It is not established how moderate zinc deficiency or diet restriction induces these alterations. We investigated whether moderate zinc deficiency or diet restriction affects renal hypoxia‐inducible factor genes expression in rats.

Thirty 3‐week‐old male Sprague‐Dawley rats were assigned into 3 dietary groups (n=10 each) and fed on dietary regimens for 4 weeks: control group (AIN‐93G; Control), moderate zinc‐deficient group (4.5 mg Zn/kg) and pair‐fed group (AIN‐93G; diet restriction). Kidney was separated into cortex and medulla for RNA isolation. Quantitative real time‐PCR using TaqMan proves was performed for mRNA measurement of hypoxia‐inducible factor (Hif) genes (Hif‐1a, Hif‐1an and Hif‐3a). Statistical analysis was performed by ANOVA and PLSD with separate variance approach (when normality was satisfied) or the Kruskal‐Wallis and Conover‐Iman tests (when normality was not satisfied). P value less than 0.05 was considered significant. Kidney medulla mRNA expressions per organ weight of Hif‐1a and Hif‐1an were significantly decreased by zinc deficiency or diet restriction compared with Control. Kidney medulla Hif‐3a and cortex Hif‐1a, Hif‐1an and Hif‐3a mRNA expressions per organ weight were not significantly different among all groups.

These results suggest that kidney hypoxia‐inducible factor gene expressions are decreased by zinc deficiency and diet restriction.

Support or Funding Information

This work was supported by KAKENHI grant‐in‐aid for Young Scientists (B) (23700928) and (15K16216).

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Artificial sweeteners and dietary disaccharides promote the release of glucagon‐like peptide‐1 in GLUTag cells, an in vitro model of the enteroendocrine cell
Authors: Mehat Kirnjot , Corpe Christopher
Published: June 2021

Due to the rise in diabetes rates across the world, the enteroendocrine cells (EEC) of the small intestine have gathered much interest in recent years because of their roles in secreting the incretin hormones, glucagon‐like peptide 1 (GLP‐1) and glucose‐dependent insulinotropic peptide (GIP) in response to ingested carbohydrates. Both GLP‐1 and GIP promote insulin synthesis and secretion and therefore play an important role in controlling blood glucose levels. While glucose induced incretin secretion by EEC cells has been well established, the response of EEC cells to other dietary carbohydrates, such as the dissacharides, and non‐nutritive sweeteners, such as artificial sweeteners (AS), is less well understood. Thus, to investigate the effects of the AS, aspartame, acesfulfame‐K and Canderel® (1.4% aspartame;0.95% acesulfame‐k) and the disaccharides, maltose, sucrose, trehalose and lactose on GLP‐1 secretion, we used the mouse EEC line, GLUTag, a well established model of the L cell, which is predominantly found in the distal regions of the small intestine. GLUTag cells were incubated with the AS (at 0, 0.25, 0.4 and 5mM), and the disaccharides and glucose (at 0, 0.25, 0.5, 2.5, 5, 12.5, 25 and 50mM) for 2 hours at 37° C and the supernatant collected. GLP‐1 was measured from the supernatant using a GLP‐1 (Active) ELISA assay (EMD Millipore®, UK). Our results showed that at dietary relevant doses (0.25–5mM) the individual AS, aspartame and acesulfame‐k, and the AS mixture, Canderel® significantly induced GLP‐1 secretion in GLUTag cells when compared to baseline (except for 0.25mM acesulfame‐k) and values were similar to glucose‐induced GLP‐1 secretion. Cells incubated in a combination of Canderel® and glucose (0.4mM and 5mM) showed the highest secretion of GLP‐1; indicating a potential synergistic effect of AS and other nutrients in EEC function. Of the disaccharides tested, maltose showed a significant dose‐responsive increase in GLP‐1 secretion from 0–5mM with values nearly similar to glucose induced secretion over the same concentration range. Gene expression studies by RT‐PCR showed the sweet taste receptors (T1R3) and maltase‐glucoamylase (MGAM) gene to be expressed in the cell line; suggesting the potential mechanism behind AS and maltose sensing in EEC. These in vitro results indicate a possible role for AS and the disaccharide maltose in promoting incretin hormone secretion and having an impact on glycaemia.

Support or Funding Information

Self‐funded project

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Kvβ subunit interacts with NEDD4 leading to decreased mouse skeletal muscle size.
Authors: Tipparaju Srinivas , Tur Jared , Brotto Marco , Chapalamadagu Kalyan
Published: June 2021

Background

The voltage‐gated potassium channel subunits 1–3 play significant physiological roles in excitable and non‐excitable tissues. However, the precise nature of its expression and functional identify in skeletal muscle remains unknown.

Hypothesis

Therefore we hypothesized that Kvβ subunits play a significant physiological role in the skeletal muscle.

Methods and Results

In the present study we identified that the voltage gated potassium channel subunit Kvβ interacts with NEDD4 in skeletal muscle. Previous research indicated a possible interaction with Kvβ and NEDD4 through pathway analysis. Utilizing immunoprecipitate pulldown technique, wild type skeletal muscle homogenates were incubated with Kvβ2 coated magnetic beads and run on SDS‐PAGE and immunoblotted with NEDD4 antibody demonstrating a visible band. Using a genetic deletion of kcnab we identified that the size of the major hind limb muscle is significantly decreased in Kvβ KO mice compared with wild type mice. Therefore we explored the underlying causes for this phenotype since Kvβ is ubiquitously expressed in skeletal muscle. Based on our investigation Kvβ plays a major role in muscle differentiation and overall size, however the proliferation aspect in skeletal muscle at least in the in vitro level remains unaffected. We utilized targeted siRNA knockdown approached to confirm these findings. In addition, tissue analysis reveals that the MHCI fiber type is significantly increased in Kvβ KO group compared with wild type.

Conclusion

Overall, this is the first study demonstrating that Kvβ2 plays a significant role in determining the size of the skeletal muscle and phenotype.

Support or Funding Information

This Research work was funded by National Institutes of Health (NIH) Grant R01‐HL‐102171 (S. M. Tipparaju).

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Protein Synthesis throughout the Progression of Cancer Cachexia in Tumor‐Bearing Mice
Authors: Brown Jacob Levi , Lee David , Rosa‐Caldwell Megan , Perry Richard , Haynie Wesley , Benson Conner , Washington Tyrone , Wiggs Michael , Greene Nicholas
Published: June 2021

Cancer cachexia occurs in approximately 80 percent of cancer patients and is a key contributor to cancer‐related death. The mechanisms controlling development of tumor‐induced muscle wasting are not fully elucidated. Specifically, the progression of cancer cachexia is underexplored. We have previously reported that muscle wasting occurs 4 wks following tumor implantation in these mice.

Purpose

To examine the rate of muscle protein synthesis throughout the progression of cancer cachexia in tumor‐bearing mice.

Methods

Lewis Lung Carcinoma (LLC) was injected into the hind‐flank of C57BL6/J mice at 8 wks age with tumor allowed to develop for 1, 2, 3, or 4 wks and compared to PBS injected control. PBS control animals were age matched to the 4 wk tumor bearing group. Cumulative fractional protein synthetic rates (FSR) were determined using deuterium oxide (2H20). Briefly, a (20 μL/g body weight) bolus priming dose of deuterium oxide was injected intraperitoneally approximately 24 hours before tissue collection. Drinking water was thereafter supplemented with 4% deuterium oxide in order to maintain the plasma pool of deuterium oxide. Fractional synthetic rate (FSR) of mixed muscle fraction (soluble, mitochondria, sarcolemmal, and contractile) and myofibrillar (contractile) rich fraction from gastrocnemius muscle were measured us a GC/MS. Data were analyzed by one‐way ANOVA. When significant F‐ratios were found, a SNK post hoc was used to compare differences between experimental groups. Significance was established at p<0.05.

Results

Plasma enrichment of 2H20 for all groups was 2.91 ± 0.073% and no significant differences existed in means between groups. Mixed muscle FSR remained consistent in 1 wk and 2 wk animals and was ~40% lower 4 wks following tumor implantation (p<0.05) when compared to PBS control. The myofibrillar rich fraction of FSR followed a similar pattern, but changes did not reach statistical significance between groups.

Conclusions

These data indicate muscle protein synthesis decreases concomitantly with muscle loss and may be a major contributor of cancer cachexia in tumor‐bearing mice. More research needs to be conducted to analyze the mechanism for the loss of protein synthesis in cachectic mice.

Support or Funding Information

This study was supported by The Arkansas Bioscience Institute and National Institutes of Health R15AR069913.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Participation of Toll‐like Receptor (TLR) 9 in Obesity‐Induced Benign Prostatic Hyperplasia (BPH) in Mice: Implication of Periprostatic Fat
Authors: Calmasini Fabiano Beraldi , McCarthy Cameron G. , Wenceslau Camilla F. , Priviero Fernanda BM. , Antunes Edson , Webb RC
Published: June 2021

Introduction

Clinical and experimental studies indicate a strong correlation between obesity and benign prostatic hyperplasia (BPH)[1]. Recently, prostatic inflammation and innate immune system have been shown to play a pivotal role in BPH pathogenesis/progression [2]. In the present study we explored the presence and participation of TLR9 signaling pathway on obesity‐induced BPH in mice.

Methods

Using prostate from C57BL6/JUnib mice, we performed immunofluorescence analysis for TLR9 localization. We also evaluated the effects of the in vitro TLR9 activation by ODN 2395 on functional (concentration‐response curves) and molecular (MYD88, TRAF6 and COX2 ‐ TLR9 pathway protein expression) protocols. Besides, male C57BL6/JUnib (Wild type) and TLR9 mutant mice were fed for 12 weeks with either a standard chow or high‐fat diet (HFD) to induce obesity. Fasting glucose levels and body, epididymal and periprostatic fat weight were evaluated. Concentration‐response curves to phenylephrine (PE; α1‐adrenoceptor agonist) and potassium chloride (KCl; depolarizing agent), as well as neurogenic contractions produced by electrical‐field stimulation (EFS; 1–32 Hz, 50 V, 10 s) were also obtained.

Results

The immunofluorescence assay revealed the presence of TLR9 in epithelial layer of the mouse prostate. The in vitro TLR9 activation with ODN 2395 (TLR9 agonist; 5 μM) led to a significant decrease (P<0.05) in prostate smooth muscle (PSM) contractions to PE and EFS along with increases in MYD88, TRAF6 and COX2 protein expression in prostate from C57BL6/JUnib mice. After 12 weeks of HFD, the TLR9 mutant obese mice exhibited the same body weight compared with WT obese group, however, fasting glucose (FG) and epididymal fat (EF) were reduced on TLR9 mutant obese mice. Despite improvements in FG and EF, TLR9 mutant obese group showed increases in prostate weight and PSM reactivity to PE, KCl and EFS compared with WT and TLR9 mutant lean group. Interestingly, the periprostatic fat (PF) showed augmented (P<0.001) in TLR9 mutant lean group compared with WT lean group. Under obesity conditions, an increase in PF was seen in WT obese group, which was further increased in TLR9 mutant obese group.

Conclusion

Despite amelioration in fasting glucose and reduction in epididymal fat, TLR9 mutant obese mice still exhibit BPH, which can be, at least in part, due to augmented periprostatic fat.

Support or Funding Information

Financial support: FAPESP (2016/20592‐8); NIH (NHLBI 001961)

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
STAT1 (Signal Transducer and Activator of Transcription 1) Deficiency in T Cells Upregulates MHC Class II and Co‐stimulation Receptors, Suggesting that STAT1 deficiency in T Cells May Increase the Plasticity and Convert in to Atypical Antigen Presenting Cells
Authors: Yang William Y. , Shen Haitao , Wu Na , Nanayakkara Gayani K. , Fu Hangfei , Drummer Charles , Shao Ying , Wang LuQiao , Yang Qian , Xu Keman , Hu Wenhui , Choi Eric T. , Wang Hong , Yang Xiaofeng
Published: June 2021

It is well known that antigen presenting cells (APC) express co‐stimulation receptors (CSR) and co‐inhibition receptors (CIR) that play important roles in modulating T cell activation status. The CSR and CIR can affect priming, T‐helper (Th) cell differentiation, effector and memory function of T‐cells. However, the issue of whether the types and number of APCs vary across different tissues is not known. Therefore, we hypothesize that the types and number of APCs vary across different tissues, which create different stimulatory/suppressive environments depending on the tissues and determine the four functional aspects of T cells mentioned above. Therefore, to validate this hypothesis, we conducted an extensive data mining study. In our analysis, we included 14 CSRs and 14 CIRs. Our study revealed following interesting findings. 1) The CSRs and CIRs are differentially expressed in human tissues. Amongst all the co‐stimulation receptors included in our analysis, CSRs such as CD40, CD48, HVEM and B7‐H2 are highly expressed in many types of human tissues. SEMA4A is the only CIR that is highly expressed in human tissues. Moreover, lymph node, placenta, thymus and trachea had the highest co‐stimulation and co‐inhibition potential. 2) Two CSRs including CD40 and CD70 can affect all the four functional aspects of T‐cells. CSRs such as CD48, CD58, TL1A, OX40L, LGHT, B7‐H2 and one co‐inhibition receptor (CIR) Galectin 9 regulates only three functions of T‐cells. 3) Co‐signaling potential varies between tissues, therefore T‐cell functions may undergo differential regulation. 4) Human tumors have increased co‐stimulation potential than correlated healthy human tissues. This may predict the improved prognosis of patients with tumor. 5) Inflammasome components and tissue hyper‐/hypo‐methylation status can significantly regulate the expression of co‐stimulation receptors in the tissues. 6) STAT1 deficiency in T cells upregulates MHC class II and co‐stimulation Receptor. This suggest that STAT1 deficiency in T cells may increase the plasticity and promote the conversion of T‐cells into APCs. Based on our findings, we conclude that tissue environment can regulate the expression of co‐stimulation receptors, and thereby significantly affect the functions of T‐cells; and key transcription factors such as STAT1 not only plays important roles in regulating cytokine pathways and T cell function, but also determine T cell identity and roles of atypical T cells in immune responses.

Support or Funding Information

RO1 ‐ HL132399‐01A1

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Redesigning a Physiology Course Using Core Concepts and Active Learning Methods
Authors: Jensen Murray , Malmquist Sarah , Hull Kerry
Published: June 2021

The findings of discipline based education research support the transition away from lecture, towards active learning, and away from details, towards core concepts. In response, a junior level physiology course at the U of Minnesota has been transformed in terms of where it is taught, how it is taught, and what is taught.

In 2013 the course was moved from a large auditorium to an active learning classroom. This physical change, however, was not sufficient to induce transformation. Lecture remained the primary mode of instruction, reflecting the instructors' unfamiliarity with teaching methods required to facilitate active learning as well as the lack of active learning curriculum materials for physiology.

In August 2017, the course was assigned to two professors who were familiar with Process Oriented Guided Inquiry Learning (POGIL), a well‐established teaching and learning method that utilizes guided inquiry and small cooperative groups. The instructors developed relevant curriculum materials; some were modified forms of existing POGIL curriculum, but most were new. The course now features regular group activities, with minimal class time devoted to traditional lecture.

The instructors modified the course content as well as the classroom practices, bypassing the traditional body systems approach in favor of the Core Concepts of Physiology developed by Michael et al. They used core concepts such as homeostasis, flux, and communication to introduce topics such as blood pressure regulation, blood flow dynamics, neuronal physiology, and endocrine regulation. While students took the Homeostasis Concept Inventory on a pre‐ and post‐ basis, the detected learning gains were quite modest because of their strong scores in the pretest.

In anonymous open‐ended surveys, students voiced concern about the emphasis on group work over traditional lecture and the lack of reliable study materials. Several expressed their preference for PowerPoint slides over information derived via group activities. Students also remarked that the summative assessments (primarily multiple choice exams) were not reflective of the course practices and content, since they seemed to address information that was not covered in class.

While the instructors in the course will continue to use the modified format, they note the importance of better alignment between the core concepts‐based curricular materials and the assessments. The lack of alignment noted by the students largely reflects the difficulty of constructing conceptually‐based assessments. Thus, question databases testing core concepts as well as validated concept inventories are clearly needed to facilitate increased incorporation of core concepts into physiology courses.

Support or Funding Information

None

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Student Perspectives on Active Learning Activities in a Human Anatomy and Physiology Course
Authors: DiFrancesca Heidi M.
Published: June 2021

An inverted (flipped) teaching model is a blended‐learning pedagogical approach that focuses on learner‐centered instruction. Active learning strategies that facilitate collaboration‐based discussion and small group work that ask students to engage in higher order thinking are paramount to the learner‐centric classes. Evidence that supports the effectiveness of a learner‐centered approach on student learning is well established, and a growing body of research has shown this approach to have a positive impact on student engagement, motivation, and retention. Thus, learner‐centered instruction using a blended‐learning model has gradually become a fundamental part of higher education. Yet, only recently has this approach become a more widely used pedagogy strategy in pre‐health professions and STEM classrooms. Few studies have reported on how students feel about the use of active learning activities as it relates to their learning. Therefore, the purpose of this study was to gather student perspectives on active learning techniques used in an inverted classroom. Undergraduate students (n=88) enrolled in an undergraduate Human Anatomy and Physiology II course taught by one instructor during three separate semesters were asked to evaluate their perception on how various activities impacted their understanding of concepts and helped them to learn. Analysis of the reflection data revealed that 73.86% (n=65) felt strongly that the activities enhanced their learning, while 13.64% (n=12) and 15.9% (n=14) felt that the activities either enhanced their learning slightly or not at all. The results indicate that significantly more students had a positive perspective on how collaboration‐based discussion and small group activities improved their learning (p< 0.000344). Thus, these results support the use of active learning strategies as a way to help increase student learning.

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