bluuebunny/crossref_metadata_2025_split huggingface dataset:
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 InformationR01 from the NHLBIThis 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 InformationThis 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 ResultsThe 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.ConclusionThe 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 InformationAlbion College Foundation for Undergraduate Research, Scholarly, and Creative Activity, and the Albion College Biology DepartmentThis 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 InformationFAPESPThis 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 InformationThis 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 InformationSupported by P01 DK 41315This 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 InformationFunded by the Ministry of Business, Innovation and Employment, New ZealandThis 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 InformationThis work was supported by NIH R01 1R01DK097361 to Julio AyalaThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.