bluuebunny/crossref_metadata_2025_split huggingface dataset:
Intron 5 of RUNX1 Gene Harbor a Putative Promoter Region.
Authors: Gutierrez Soraya , Hinojosa Marcela , Schnake Nicolas , Martinez Milka 
Published: November 2023

The t(8:21) is one of the most frequent chromosomal translocation found in leukemia. To date all the break points mapped for this translocation, are located in intron 5 of RUNX1 gene. Interestingly, this intron exhibit structural chromatin elements like Topoisomerase II and DNase I hypersensitive sites, which have been wildly associated to presence of transcriptional cis regulatory elements. Therefore, we hypothesize that regulatory elements are harbored in intron5 of the RUNX1 gene. In order to test this hypothesis, we performed bioinformatics analysis of ChIPseq data available from the ENCODE database to identify regions of intron 5 enriched in epigenetics marks characteristic of regulatory elements. Our results shown that in hematopoietic cells there is one region highly enrich in K4H3me, K4H3me3 and K27H3ac that colocalizes with DNase I hypersensitive sites. These marks have been associated with promoters modules. Indeed, when we cloned this region in the pGL3 basic reporter vector, we found that it activates expression of the reporter gene luciferase in an orientation dependent manner. Using ETS databases we have assemble a putative messenger for this promoter. Moreover, by RT‐PCR we have shown that this RNA is expressed both in Jurkat and HL60 cells. Taken together our results demonstrate the presence of a putative promoter region located in intron 5 of RUNX1 gene that is active in hematopoietic cells.Support or Funding InformationFONDECYT 1130697
Mechanisms underlying hPMSC‐based protection on human brain endothelial cells
Authors: Barzegar Mansoureh , Minagar Alireza , Wang Yuping , Yun Jungmi W , Boyer Christen , Couraud Pierre‐Olivier , Alexander J.Steven 
Published: November 2023

Stroke remains among the leading causes of neurologically‐based morbidity and long‐term disability and results from thromboembolism (85% of strokes) or hemorrhage (15%). Several events lead to neuronal death in the stroke‐affected brain. Initially, production of local pro‐inflammatory mediators following brain ischemia/reperfusion promote stroke injury by decreasing blood‐brain barrier and by enhancing leukocyte binding and infiltration.Stem cell‐based therapies are a revolutionary treatment for several neurodegenerative diseases including stroke acting through neuronal cell trans‐differentiation but also provide acute protection properties by releasing substances which suppress inflammation, induce angiogenesis, and regulate vascular reactivity.We hypothesized that human placental‐derived mesenchymal stem cells (hPMSCs), a type of extraembryonic stem cell, might enhance acute protection against injury by releasing mediators which suppress the immune cell infiltration and maintain blood brain barrier.To investigate this, we examined the extent to which hPMSC conditioned media (CM) could mobilize junctional barrier‐creating proteins in human brain endothelial cells (hCMEC‐D3). We performed FITC‐Dextran permeability assays as well as junctional protein western blotting to evaluate the expression of junctional protein following treatment with hPMSC‐CM. We have also assessed the influence of hPMSC‐CM on contractility of human brain vascular smooth muscle cell (hBVSMC) using collagen gel contraction assay. Finally, we have used Matrigel Basement Membrane Matrix to follow up the formation of tubes from hCMEC‐D3 following treatment hPMSC‐CM. We found that passage four hPMSC‐CM improved the barrier function of hCMEC‐D3 and increased the expression of ZO‐1 and a‐Claudin‐1 proteins involved in tight junction. We also observed less contraction of hBVSMC treated with hPMSC‐CM. Moreover, hPMSC‐CM could accelerate the tube formation of hCMEC‐D3.We conclude that PMSC release stable factors which beneficially and acutely influence barrier structure and integrity. Therefore, PMSC may affect both acute as well as chronic phases of recovery in stroke.
Exploiting Delta Cell Paracrine Interactions to Curb Glucagon Hypersecretion in Diabetes
Authors: Noguchi Glyn M , Donaldson Cynthia J , Mawla Alex M , Saghatelian Alan , Huising Mark O 
Published: November 2023

Diabetes is first and foremost a disease of insulin impairment, but is often accompanied by aberrant glucagon secretion. Uncontrolled alpha cell activity leads to further complications such as diabetic hyperglycemia, and we currently lack good options to manage this aspect of the disease. Within healthy pancreatic islets, delta cells exert an inhibitory influence over the alpha and beta cells via somatostatin; thus, they are uniquely positioned as the principal source of local inhibitory control preventing hormone signaling from going awry. Recent studies from our lab and others have identified local signals (such as Urocortin3 from the beta cells) and external factors (such as ghrelin from the GI tract) that act to directly stimulate somatostatin secretion. These observations cast delta cells as central hubs that are essential to maintain homeostatic control of insulin and glucagon. The objective of this study is to test if the negative feedback from pancreatic delta cells can be targeted to restore control over excess glucagon release in diabetes.MethodsWe have combined transcriptomics, chemicogenetics, and functional imaging of alpha, beta and delta cells in intact islets to help showcase the role of the delta cell within the islet.ResultsOur results indicate that Urocortin3 transiently silences alpha cell activity by activating delta cell‐dependent negative feedback.ConclusionBy targeting the delta cells, we can take advantage of the local feedback mechanism and restore inhibition of alpha cells. Our findings suggest that the pancreatic delta cell is a viable target to normalize the excess glucagon secretion that is partially responsible for hyperglycemia in diabetes.Support or Funding InformationGlyn Noguchi is supported by a NIGMS‐funded Pharmacology Training Program (T32GM099608). The Huising Lab is funded by awards from the Juvenile Diabetes Research Foundation (JDRF) and a Hartwell Individual Biomedical Research Award.
The Effect of the Endocrine Disrupting Chemical Bisphenol A on Pro‐opiomelanocortin, Circadian Rhythm and Neuroinflammatory Marker Gene Expression in Hypothalamic Cell Models
Authors: Salehi Ashkan , Belsham Denise D. 
Published: November 2023

Obesity is a global health crisis that has doubled in prevalence since 1980 and is associated with co‐morbidities, such as type 2 diabetes mellitus and circadian rhythm disruption. Therefore, understanding the cellular mechanisms responsible for obesity has relevance for improving human health. A number of endocrine‐disrupting chemicals, such as bisphenol A (BPA), an industrial chemical present in 95% of human urine samples, have been linked to obesity. However, the effect of BPA on the pro‐opiomelanocortin (POMC) neurons of the hypothalamus has yet to be explored. POMC neurons produce a precursor polypeptide that is cleaved into the anorexigenic peptide MSH, known to inhibit feeding behaviour. Altering POMC expression could therefore disrupt energy homeostasis in the hypothalamus, ultimately leading to weight gain. We hypothesized that BPA induces a neuroinflammatory response to alter the gene expression of POMC, thereby impairing feeding regulation. We also hypothesized that BPA may alter the expression of circadian clock genes. This study utilized two characterized hypothalamic neuronal models, the adult‐derived mHypoA‐POMC/GFP‐2 and embryonic mHypoE‐43/5 cell‐lines. Both cell‐lines were chosen for their expression of inflammatory markers, circadian genes and POMC transcripts. We examined the effect of 100 M BPA on the transcription of POMC, pro‐inflammatory genes IL6, NFκB, IκB, and the endoplasmic reticulum (ER) stress marker CHOP, over a 24 h timecourse by real‐time quantitative PCR (RT‐qPCR). Analysis of circadian clock genes Bmal1, Per2 and Rev‐Erb was also undertaken. In the mHypoA‐POMC/GFP‐2 cell‐line, BPA significantly upregulated POMC, IL6, IκB and CHOP mRNA levels. Furthermore, BPA significantly altered the mRNA levels of Bmal1 and Per2 at specific time points, indicating a disruption of the circadian clock. Similar to the adult‐derived POMC line, BPA significantly altered the mRNA levels of POMC, IL6, IκB, NFκB and CHOP, Bmal1, Rev‐Erb and Per2 in the mHypoE‐43/5 cell‐line. Given the altered expression of feeding‐related peptides, BPA could have an inflammation‐mediated mechanism of action that is being explored. Additionally, the disruption of circadian genes by BPA could further alter cellular function necessary for proper energy homeostasis. In ongoing studies, we are employing anti‐inflammatory compounds to determine whether induction of inflammation causes POMC and circadian gene expression disruption. In conclusion, our study is the first to explore the direct effects of BPA on POMC neuronal cell models, demonstrating altered expression of genes associated with obesity. We therefore have the necessary tools to understand this process at a more mechanistic level.Support or Funding InformationThis research was supported by grants from the CIHR.
The Role of Androgens in the Exercise Induced Decrease in Protein Breakdown
Authors: Rossetti Michael L , Gordon Bradley S 
Published: November 2023

Androgens play an important role in the regulation of muscle protein balance. The decrease in androgen production and/or bioavailability observed in various atrophic conditions is associated with increased rates of muscle protein breakdown in the fasted metabolic state. While the increase in muscle protein breakdown is accompanied by a decrease in oxidative capacity, the contribution of muscle oxidative capacity to muscle protein breakdown in the absence of androgens is unclear. To shed light on this, male mice that were 10 weeks of age were subjected to a sham or castration surgery. Ten days post‐surgery, mice from both groups were further randomized to remain sedentary or perform aerobic treadmill exercise (6 days a week for 8 weeks) to increase oxidative capacity. At the conclusion of the 8‐week training, mice underwent a run to fatigue to assess training effectiveness. Mice were fasted overnight prior to sacrifice to induce a fasted metabolic state. All mice subjected to the training ran significantly longer compared to the sedentary groups with no effect of castration observed. Plantaris muscle mass was significantly higher in sham exercised mice compared to the castration exercised group. While exercise training increased expression of the mitochondrial protein, cytochrome c oxidase subunit IV (COX IV), in both sham and castration conditions, this measure was significantly lower in castrated mice relative to sham values. Interestingly, exercise training decreased the LC3 II/I ratio, a marker of autophagy activation, only in sham mice which could not be explained by corresponding changes in putative autophagy regulatory factors. However, a strong inverse relationship between COX IV and the LC3 II/I ratio was observed, suggesting that muscle oxidative capacity contributed to the change in autophagy activation. In all, these data support a role of muscle oxidative capacity in the androgen‐mediated regulation of autophagy activation during the fasted metabolic state.Support or Funding InformationUCF Office of Research and Commercialization Grant to Bradley Gordon
The Influence of CYP1A2 Genotype on Caffeine Consumption Habits and Athletic Performance Enhancement in Collegiate Distance Runners
Authors: Ye Jiayu Jennifer , Nagy Erzsebet Emily , Millard Julie T. 
Published: November 2023

The liver enzyme CYP1A2, which is responsible for metabolizing caffeine, is a member of the cytochrome P450 family. CYP1A2 activity is highly variable across individuals, depending on factors such as gender, personal habits (e.g., smoking status and diet), and genotype. A single nucleotide polymorphism (A/C) in intron 1 of the CYP1A2 gene leads to differences in gene inducibility, with AA individuals reportedly having higher activity than C‐allele carriers. Several reports have suggested that genotype can modulate the physiological effects of caffeine. For example, “fast metabolizers” with the AA genotype benefit more from caffeine supplementation before cycling time trials than C‐allele carriers. We investigated whether CYP1A2 genotype influences self‐reported caffeine consumption and performance effects in collegiate distance runners. DNA samples and questionnaires were collected from 65 subjects. PRC‐RFLP was performed to determine genotype. Preliminary statistical analysis based on the questionnaires indicates that there was no significant difference in self‐reported daily caffeine consumption and degree of performance enhancement between the AA homozygotes and C‐allele carriers, suggesting that environmental factors may modulate gene effects of CYP1A2.Support or Funding InformationResearch reported in this publication was supported by an Institutional Development Award (IDeA) from the National Institute of General Medical Sciences of the National Institutes of Health under grant number P20GM0103423.
Evidence for Direct Interactions between RNA Polymerase and Ribosomes.
Authors: Blaha Gregor M , Fan Haitian , Conn Adam B , Diggs Stephen , Hahm Joseph , Williams Preston B , Wang Yinsheng 
Published: November 2023

Transcription and translation are coupled in prokaryotes. Coupling originates from close proximity of the RNA polymerase and the ribosome due to their concurrent binding to the same nascent RNA. A similar proximity between RNA polymerase and the ribosome occurs during ribosome biogenesis; when ribosome assembly ensues while the rRNA is being transcribed. Here we report the existence of direct physical interactions between RNA polymerase core enzyme and vacant ribosome in the absence of mediating factors. The interactions appear to be of ionic nature and persist in the presence of an excess of BSA, RNA, or phosphate. The binding of ribosomes to RNA polymerases saturates at a one to one stoichiometry, while that of RNA polymerase to ribosomes saturates at a two to one. The dissociation constant of these complexes is within the physiological concentration range of free RNA polymerase and ribosomes. When bound to ribosomes, the RNA polymerase adopts a conformation similar to that under high salt which appears to be distinct from that of the transcription elongation complex and holoenzyme. Thermus thermophilus RNA polymerase and ribosomes can form complexes with each other as well as mixed‐species complexes with E coli RNA polymerase and ribosomes. Our results suggest that the interface between RNA polymerase and ribosome is conserved and coordinates the interactions between RNA polymerase and ribosome during transcription‐translation coupling and/or during ribosome biogenesis.Support or Funding InformationUCR collaborative seed grant
Ammonium Conductance in a Mouse Thick Ascending Limb Cell Line
Authors: Lee Soojung , Park Jonathan , Choi Inyeong 
Published: November 2023

NH4+ is a key buffer component that regulates blood pH. The kidneys excrete NH4+ to urine as they produce HCO3−, and the mechanism by which NH4+ excretion results in net acid excretion involves a series of sophisticated NH4+ transport processes in different parts of the nephron. One of the nephron segments that play key roles in NH4+ excretion is the thick ascending limb (TAL). In the luminal membrane of the TAL tubules, the Na/K/2Cl cotransporter is the major fraction of the active NH4+ flux. Nonetheless, in vitro studies reveal that K/NH4 exchange and NH4+ conductance, whose molecular identities are presently unknown, can contribute to the TAL NH4+ transport by 35–50%. The two pathways exhibit biophysical and pharmacological characteristics that distinguish them from other NH4+‐transporting proteins. Despite such physiological and functional significance, our understanding of these pathways is limited. Especially, the electrophysiological properties of the NH4+ conductance have been unknown.In this study, we examined the NH4+conductance in the TAL cell line ST‐1 to determine its basic electrophysiological properties such as the amounts of NH4+current produced in cells, current‐voltage (I–V) relationship, and reversal potential of the current. Whole cell patch clamp was performed to measure the current evoked by NH4Cl in the presence of BaCl2, tetraethylammonium and BAPTA. Application of 20 mM NH4Cl induced an inward current with mean amplitude of −272 ± 79 pA (n = 9). In I–V relationships, NH4Cl application caused the I–V curve to shift down in an inward direction. The difference in curves before and after NH4Cl application, which corresponds to the NH4Cl‐mediated currents at different voltages, was progressively larger at more negative potentials. The reversal potential was +15 mV, more positive than a calculated equilibrium potential for Cl−, thus indicating that the current is not due to Cl−but to NH +. We then expressed ST‐1 total proteins in Xenopus oocytes and performed two‐electrode voltage clamp. Application of NH4Cl in the presence of BaCl2 caused the I–V curve to be steeper in an inward direction. Interestingly, removing Na+ from the bath solution caused the NH4+ conductance to disappear. Na+removal additionally led to a partial reduction of endogenous oocyte conductance, which was abolished by bumetanide. At pH 6.4, the NH4+conductance was still produced while endogenous oocyte conductance was not detected. In conclusion, we, for the first time, report the electrophysiological properties of the NH4+conductance in the ST‐1 cells. This conductance is Na+‐dependent, different from the previously reported Cl−‐dependent NH4+conductance in isolated TAL tubules.Support or Funding InformationThis work was supported by the Emory physiology department bridge fund to I.C.
Experimental oxidative stress‐induced death of erythrocytes
Authors: Senchenkova Elena Y. , Skvertchinskaya Elisaveta , Dobrylko Irina , Sudnitsyna Julia , Gambaryan Stephan , Mindukshev Igor , Gavins Felicity 
Published: November 2023

BackgroundExcessive erythrocyte (red blood cells, RBCs) hemolysis is observed in different pathological states including sepsis, sickle cell disease and diabetes. Prior to senescence RBCs undergoing membrane injury due to reactive oxygen species (ROS) possess a damaged membrane coupled with impaired function and increased hemolysis. Oxidative stress plays a major role in alteration of membrane integrity and fragility of RBCs causing a functional propensity of erythrocytes to flow into microcirculatory vascular beds where erythocytes can adhere to endothelial monolayers and hemolyse. The mechanisms of oxidative stress‐induced death of RBCs relate to Ca2+‐influx and caspase activation coupled with an increase in apoptotic markers, such as cell shrinkage, membrane blebbing, leading to phosphatidylserine (PS) exposure and hemoglobin oxidation.ObjectiveTo understand the mechanisms by which ROS promotes dysfunction of RBC membrane integrity and RBC cell death.MethodsFlow cytometry, confocal microscopy, spectroscopic and lazer partical analysis techniques were appled to assess osmotic fragility of RBCs, cell morphology, apoptotic markers (esterase activity, Band 3 clasterization, caspase 3 activation, PS externalization), and hemoglobin oxidation state (methemoglobin (MetHb) and hemichrom (FeIII)).Results
Ca2+‐influx (A23187) is associated with decreased cell volumes, PS externalization and increased cell rigidity. Influx of Ca2+ resulted in no changes in either hemoglobin oxidation state (oxyHb~95%), band 3 clustering or caspase 3 activation.
Tert butyl hydroperoxide (tBHP) administration caused a dose‐dependent alteration of membrane integrity and fragility of RBCs. High dose of tBHP (>3 mM) resulted in a robust decrease in cell volume without cell membrane transformations. tBHP administration at 0.5–2mM mediated an alteration of membrane deformatility of RBCs, along with an initial increase in cell volume, hemolobin oxidation with increased MetHb and FeIII formation, caspase 3 activation, band 3 clustering, PS externalization, and formation of microparticles. These membrane alterations are Ca‐independent processes.
NO (S‐nitrosocysteine, SNC) causes increased MetHb formation associated with no changes in membrane structures.
ConclusionOur results illustrate that oxidative stress induces RBCs death associated with an increase in apoptotic markers (caspase 3 activation, PS exposure, formation of microparticles) which is, Ca‐independent and results in FeIII binding to band 3, band 3 clustering, erythrocyte membrane vesiculation, and oxidized haemoglobin containing microparticles.
Role of the Free Fatty Acid 4 Receptor in Endothelial Cell Responses to Oleic Acid
Authors: Rogers Kelsey , Davis Danielle , Kurjiaka David 
Published: November 2023

The impact of fatty acids on the vasculature is a complicated story: some appear to have pro‐inflammatory effects (trans and saturated) whereas others have anti‐inflammatory effects (cis and omega 3). The discovery of a G protein coupled free fatty acid receptor (FFAR) has provided a potential explanation for these disparate vascular responses to fatty acids of different structures. In particular, the FFAR‐4 is activated by fatty acids that are expected to be anti‐inflammatory. Based upon the size and structure of oleic acid (18 carbon, cis fatty acid), we expect the anti‐inflammatory effects of oleic acid on the vasculature involve activation of the FFAR‐4. We tested the hypothesis that oleic acid binds to the FFAR‐4 receptor to reduce the inflammatory response in vascular endothelial cells. Western blot confirmed the presence of the FFAR‐4 in bEnd.3 cells (cultured mouse endothelial cells: ATCC). After treating bEnd.3 cells for 12 hrs with 30 μM oleic acid, the expression of connexin 43 (a marker of endothelial cell damage/inflammation) was reduced (n=5). As the concentration of oleic acid was increased from 0.3 to 300 μM, connexin 43 expression decreased in a dose dependent manner (n=4). When treated with an agonist for the FFAR‐4 (5 μM TUG‐891: Tocris), bEnd.3 cell expression of connexin 43 was reduced (n=3). Thus, preliminary data are consistent with the FFAR‐4 mediating the anti‐inflammatory effects of oleic acid on endothelial cells.Support or Funding InformationThis work was funded by Grand Valley State University (Presidential Research Grant: D Davis and Student Summer Scholars: K Rogers and D Kurjiaka).