bluuebunny/crossref_metadata_2025_split huggingface dataset:
Selection and Characterization of Novel of RNA Aptamer Recognizing Homocysteine which Ameliorates Oxidative Stress in Mouse Brain Endothelial (bEND3) Cells
Authors: Bala Jyoti , Behera Jyotirmaya , Kelly Kimberly , Tyagi Neetu 
Published: November 2023

Neurodegenerative diseases are characterized by a progressive deterioration of brain function, with a significant consequent decrease in the quality of patient's lives. Recently pathological mechanisms operating in cerebrovascular diseases mediated via homocysteine (Hcy) have gained increased attention. It has also been known that Hcy causes endothelial dysfunction by inducing oxidative stress in most cerebrovascular disorders. This dysfunction is highly correlated with reactive oxygen generation (ROS). Therefore, it is crucial to develop potential novel measures to slow down or prevent the onset of these diseases mediated by increased level of Hcy. Therefore, we proposed to select and characterize novel RNA aptamer recognizing Hcy to inhibit and modulates the Hcy activities involved in cerebrovascular disease. RNA Aptamers are specific bio‐molecules selected by the process called Systematic Evolution of Ligands by Exponential Enrichment (SELEX). Aptamer folds into 3‐dimensional shapes which are capable of binding non‐covalently with high affinity and specificity to their target molecule. We have selected and characterized Hcy RNA aptamers using SELEX technology. The structural characterization was done by bioinformatics tools such as Clustal W, Mfold, IFold and HADDOCK. Binding characterization were done by CD and RNA EMSA studies. The best Hcy RNA aptamers were further investigated for their biological and functional characterization. Our results demonstrate that the Hcy RNA aptamer undergoes a conformation change upon ligand binding. These aptamers exhibit high specificity to ligand over nonspecific competitor. In our studies, bEND3 cells were exposed to Hcy treatment in the presence or absence of Hcy RNA aptamers for 24 hrs. We also demonstrated that these aptamers are novel class of RNA aptamer which ameliorates the effects of ROS thereby reducing the oxidative stress in bEND cell lines. Apoptotic and autophagic cell death were measured with 3‐(4, 5‐Dimethylthiazol‐2‐yl)‐2, 5‐ Diphenyltetrazolium Bromide (MTT) and monodansylcadaverine (MDC) staining assay. ROS production was determined using fluorescent probes 2, 7‐dichlorofluorescein acetate (DCFH‐DA. Autophagy flux was assessed using a conventional GFP‐microtubule‐associated protein 1 light chain 3 (LC3) dot assay. Mitochondrial fusion and fission was evaluated using Mfn‐2 and Drp‐1 RT‐PCR. Treatment of bEnd3 with Hcy RNA aptamer attenuated the production of free radicals and protected the cells from oxidative damage. Altogether, our studies strongly suggest that Hcy‐RNA aptamer has beneficial effects on cerebrovascular diseases and could be developed as a potential therapeutic agent against hyperhomocysteinemia (HHcy).Support or Funding InformationFinancial support from National Institute of health grant AR‐067667 and HL‐107640 are greatly acknowledged.
Dynamic Bayesian Graphical Modeling to Predict Regulatory Networks in Hypertensive Rats
Authors: Dayton Alex , Ahn Kwang Woo , Liu Pengyuan , Laud Purushottam , Stingo Francesco , Vannucci Marina , Bukowy John D , Liang Mingyu , Cowley Allen W 
Published: November 2023

We have previously used Bayesian graphical methods to detect networks of interacting genes relevant to the development of salt sensitive hypertension. However, these previous approaches are “static” or time‐insensitive methods that cannot infer causality to gene interactions. Here, we present a dynamic Bayesian graphical modeling approach which is used to connect potential target genes to other genes and networks.Dynamic Bayesian graphical networking approaches require large amounts of time‐series data, in addition to being computationally complex. Data was generated by isolating medullary thick ascending limb (mTAL) cells from 3 rat strains: the Dahl Salt‐sensitive rat (SS), the Fawn Hooded Hypertensive rat (FHH) and the Spontaneously Hypertensive Rat (SHR). After isolation, the freshly isolated mTAL cells were exposed to one of two stimuli: H2O2 (500 nM) or TNF‐α (50 ng/mL). RNA was then collected at 30 minute intervals for 8 hours, resulting in a 16‐point time‐series for each strain and stimulus. Each time‐series was duplicated, and duplicates were pooled to reduce overall measurement noise. As there are 6 combinations of strains (3) and stimuli (2) this procedure resulted in 96 individual time‐points. RNA was then isolated and sequenced on an Illumina Hi‐Seq 2500.The Dynamic Bayesian graphical model attempts to find connections between genes by looking at the sequence of activation/inactivation of genes across the entire 8 hour time‐series. For example, in a time‐series generated by treatment of SS mTAL cells with 500 nM H2O2, we find that the gene Dusp5 undergoes a 4‐fold increase in RNA expression one hour before a similar 4‐fold increase in the gene Rasgrp3, a GWAS gene for hypertension that has no known function in the mTAL. The Bayesian graphical modeling algorithm looks at such changes across each time‐series for the different rat strains and stimuli, and detects the most probable connections between the analyzed genes.This analysis represents the first attempt to create a dynamic Bayesian graph in non‐cancer mammalian cells. The use of such a graphical modeling approach in mTAL, a cell type of particular relevance to the pathophysiology of hypertension, will give us insight into the regulation of critical genes in hypertension and allow us to better understand, and potentially treat, this important disease.Support or Funding InformationA. Dayton supported by NIH Fellowship: 1F30HL127979‐01A1.Work funded by NIH grant: 4P01HL116264.
Modeling of calcium‐induced swelling in spatially divergent populations of cardiac mitochondria
Authors: Chapa Xavier R , Makarov Vladimir , Javadov Sabzali 
Published: November 2023

In addition to ATP synthesis, mitochondria are involved in high cytosolic calcium (Ca2+) waves necessary for the regulation of the cardiac excitation‐contraction coupling, a focal point in the determination of cardio‐pathophysiology. Depending on mitochondrial location within subcellular compartments, mitochondrial functions expands from the buffering of cytosolic Ca2+ necessary for cellular signaling to the initiation of cell death via necrosis or apoptosis depending on ATP availability. The exposure of mitochondria to Ca2+ plays a dual role in determining metabolic activity; the sequestering of low Ca2+ concentration ([Ca2+]) accompanied by a minor matrix swelling enhances ATP production through the upregulation in activity of TCA cycle enzymes, ETC complexes and oxidative phosphorylation. However, excessive matrix swelling at high [Ca2+] can induce the opening of non‐selective permeability transition pores (PTP), sustained (irreversible) opening of which allow for the release Ca2+ to propagate from one mitochondria to the other leading to the wide spread of mitochondrial swelling, thus initiating cell death. Studies have suggested that different mitochondrial sub‐population differ in functional properties such as membrane potential, calcium retention capacity (CRC), and ROS production. In this study, we designed a biophysical model that can explain the kinetics of the transition from a reversible to an irreversible Ca2+‐induced swelling state in heterogenic mitochondrial population including subsarcolemmal (SSM) and intermyofibrillar mitochondria (IFM) of cardiac cells.Model developmentIn this project, a heterogenic population of mitochondria were isolated and purified via differential centrifugation. CRC was determined by the application of numerous Ca2+ pulses until Ca2+ release via PTP opening measured through spectrophotometry. Kinetics model was developed utilizing the 4th order Runge‐Kutta approximation method and fitted to experimental mitochondrial swelling data using the least square method. Reversible and irreversible mitochondrial swelling induced by low and high [Ca2+] in isolated cardiac mitochondria have been measured. Reversibility of mitochondrial swelling was determined in the presence of EGTA, a Ca2+‐chelating agent. Based on experimental data, the development of a kinetic model that simulates the swelling dynamics at low and high [Ca2+] in cardiac mitochondria has been created with a correlation coefficient of r >0.95.ConclusionOur model describes transition from reversible to irreversible swelling in different subcellular populations of mitochondria that is important for understanding the role of mitochondrial heterogeneity in PTP‐induced cell death.Support or Funding InformationSupported by NHLBI NIH (SC1HL118669)
Brain inflammation and oxidative stress in the developmental programming of metabolic syndrome in adult offspring to maternal nutrition insult
Authors: Chan Julie YH , Chao Yung‐Mei 
Published: November 2023

Early life exposure to adverse environments can lead to a variety of adult diseases by a process referred to as developmental origin of adult health and disease (DOHaD) or developmental programming. Maternal high fructose diet (HFD) promotes programmed hypertension in adult offspring, although the underlying mechanism is not fully understood. Brain inflammation and oxidative stress have been reported to play pivotal roles in neural mechanism of hypertension. We tested in this study the hypothesis that brain inflammation and oxidative stress contribute to the programmed hypertension in adult offspring to maternal HFD. In normotensive Sprague‐Dawley rats, maternal HFD during gestation and lactation led to insulin resistance, hypertriglyceride, increased sympathetic activity and hypertension in the 3‐month‐old young offspring. The increase in blood pressure was associated with the augmented neurogenic sympathetic vasomotor tone, increase in plasma norepinephrine level, production of proinflammatory cytokines and high tissue levels of reactive oxygen species (ROS) at the brain stem. Maternal melatonin treatment or minocycline treatment beginning at the age of two months significantly ameliorated neuroinflammation and oxidative stress in the brain stem; alongside the delay in the programming of hypertension in adult offspring. At the same time, intracisternal infusion with a superoxide scavenger, tempol, abrogated sympathetic overexcitation and prevented the development of programmed hypertension in the 3‐month‐old offspring to maternal HFD. Together, these results suggest that brain inflammation and stem oxidative stress may contribute to sympathoexcitation and the programmed hypertension in offspring to maternal HFD.Support or Funding InformationThis study was supported by grant (CMRPG8C0051‐053) from Kaohsiung Chang Gung Memorial Hospital.
Effects of Culture Time, Fetal Bovine Serum, Progesterone, Testosterone and Estradiol on Alkaline Phosphatase Activity, Extracellular Matrix Mineralization and Osteoblast Viability
Authors: Lemos José E.S. , Brancaglião Lucas F.C. , Bonfá Ana L.O. , Rocha Nícolas F. , Gonçalves Vítor M. , Montrezor Luís H. 
Published: November 2023

Understanding the interactions between ovarian and plasma changes induced by polycystic ovary syndrome (PCOS) and bone metabolism is essential for the comprehension of the pathophysiological mechanisms involving these organs. Ovarian steroids are involved in activities related to bone synthesis and reabsorption. Knowledge of the actions of the main ovarian steroids on the viability of osteoblasts in culture, which mimic the conditions of PCOS, is relevant to a better understanding of the functions of the cells in this endocrine disease that affects more than 10% of women of reproductive age. The goal of this study was to develop a cell culture model mimicking polycystic ovary syndrome that can be used for studies on culture conditions and the effects of steroids progesterone (P4), testosterone (T) and estradiol (E2) on cell viability, alkaline phosphatase activity and extracellular matrix mineralization of osteoblasts. We plated 5 × 103 cells in each well of 96‐well culture plates with 100 μL of modified Eagle's medium supplemented or not with 10% fetal bovine serum (FBS), penicillin (100 U/mL) and streptomycin (100 μg/mL). Cells were maintained at 37° C under a humid atmosphere of 5% CO2 and 95% air for 72 h. P4, T and E2 were used in different concentrations: control (without hormones), 10−8 M and 10−7 M mimicking the normal plasma concentrations in adult female rats and 10−6 M and 10−5 M, mimicking plasma concentrations in adult female rats with induced PCOS. To this end, each of the steroids were added to the wells of culture both separately and together (all in the same well). Our results show the cell viability of undifferentiated and differentiated osteoblasts in the osteogenic medium, the alkaline phosphatase activity and mineralization of the extracellular matrix in cells cultured for different periods of time and challenged with different concentrations of P4, T, E2, and the combination of all three added to the culture medium. Furthermore, FBS influences the culture conditions. From these results, we propose that the culture system described can be used to study the viability of osteoblasts, the activity of alkaline phosphatase and mineralization of the extracellular matrix in conditions similar to PCOS and, thus, it can be suggested that ovarian steroids, in isolation and in combination, modulate bone formation in vitro.Support or Funding InformationCNPq and FUNADESP
Effects of Perinatal Nicotine Exposure on Cholinergic Signaling in the Airway Epithelium
Authors: Borrero Nathan , Fregosi Ralph , Boitano Scott 
Published: November 2023

Up to 20% of pregnant women in the USA smoke or use nicotine delivery devices (e‐cigarettes, water pipes, nicotine patches or gum). While addictive, many believe nicotine to be otherwise safe, causing the use of nicotine delivery devices to double between 2008–2012. However, there is growing evidence that nicotine can alter development of the lungs and airways, as well as pulmonary immune function and brainstem neurons that control breathing and heart rate. In this study, we propose to test the hypothesis that in utero exposure to nicotine leads to alterations in cholinergic signaling in the airway that are evident at birth and persist into adulthood. Importantly, dysfunction of acetylcholine (ACh) signaling in the airway epithelium can result in compromised airway surface liquid and subsequent loss of innate immune function in the airways. This signaling is mediated primarily by muscarinic acetylcholine receptors (mAChRs) and nicotinic acetylcholine receptors (nAChRs), both of which may be affected by developmental nicotine exposure. We have used rat neonate tracheal mounts and primary cultured rodent airway epithelium in Ussing Chamber studies to evaluate airway epithelial cholinergic signaling in animals with and without developmental nicotine exposure. Initial results show that transtracheal resistance (TTR), transepithelial resistances (TER), and short circuit current (Isc) responses to apical application of ACh are not altered by nicotine exposure. However, airway epithelial signaling is dominated by mAChRs‐dependent signaling. When preparations are exposed to nicotine, unexposed rat trachea and primary mouse cultures demonstrate limited, but clear responses. These responses appear to be altered in animals exposed to nicotine in utero. Our data suggest that perinatal nicotine exposure adversely affects the development of cholinergic signaling in the airway epithelium.Support or Funding Information1R25ES025494NIEHS/SBRPP ES04940
Sex Differences and Role of Sex Hormones in the Expression of Inflammatory Mediators and Lung Function in Response to Ozone
Authors: Fuentes Nathalie , Cabello Noe , Nicoleau Marvin , Silveyra Patricia 
Published: November 2023

BackgroundBreathing ground‐level ozone, a reactive oxidant gas, results in a number of negative health effects that affect men and women disproportionally. Previous studies have shown that ozone induces a differential inflammatory response in males vs. females, but the mechanisms associated with these differences remain unclear. Here, we hypothesized that circulating sex hormone levels can modulate lung function and the inflammatory response to air pollutants such as ozone.MethodsTo test this, we performed gonadectomy and hormone replacement (estradiol, 2 weeks) in adult male and female mice. We exposed these mice to 1 ppm of ozone or filtered air (FA) for 3 hours, and we compared lung function 24 h after exposure with the FlexiVent system. We also measured the levels of lipocalin, albumin, and cell differential counts in bronchoalveolar lavage fluid (BALF); as well as the expression of the pro‐inflammatory cytokine interleukin‐6 (IL‐6) in lung tissue by Real‐Time PCR.ResultsOur preliminary results indicate that males showed higher lung compliance and elasticity than females, and treatment with estradiol for two weeks in gonadectomized mice increased lung expansion in both sexes. Furthermore, gonadectomy decreases IL‐6 expression in the female lung, but not in the male lung. Estradiol replacement in females restored IL‐6 levels, but decreased it in males. Exposure to ozone resulted in higher neutrophil counts in BALF, with females higher than males. Gonadectomy and estradiol treatment had opposite effects in BALF cell counts in males vs females.ConclusionsTogether, our preliminary results suggest that both lung function and the inflammatory response to ozone can be influenced by circulating sex hormone levels (estradiol). Future studies will investigate the specific role of other sex hormones (testosterone, progesterone) in these responses.Support or Funding InformationThe authors thank the Pennsylvania State University College of Medicine Genome Sciences Core Facility for Bioanalyzer analysis and real‐time PCR equipment. The authors also thank Todd Umstead and Susan Di Angelo for assistance with training and experiments, and Dr. Joanna Floros for the ozone equipment. NIH Grant K01HL133520.
TNFα Induces Endoplasmic Reticulum Stress in Human Airway Smooth Muscle Cells
Authors: Yap John Qian , Delmotte Philippe , Sieck Gary C 
Published: November 2023

Airway inflammation is a key aspect of diseases such as asthma. Several inflammatory cytokines (e.g., TNFα and IL‐13) enhance cytosolic Ca2+ concentration ([Ca2+]cyto) triggering airway smooth muscle (ASM) hyperreactivity (enhanced contractile state). Airway inflammation also induces ASM proliferation and remodeling (enhanced ASM synthetic state). In various diseases inflammation induces sarcoplasmic/endoplasmic reticulum (SR/ER) stress, which decreases SR/ER‐mitochondria interactions, with downstream effects on Ca2+ regulation. In vascular smooth muscle cells, SR/ER stress promotes smooth muscle contraction and proliferation by disrupting SR/ER‐mitochondrial coupling. We hypothesized that inflammation induces SR/ER stress in human ASM, thereby altering expression of mitochondrial fission/fusion proteins (dynamin‐related protein ‐ Drp1 and mitofusin ‐ Mfn2) with downstream impact on ASM Ca2+ regulation and contractility as well as proliferation.Human ASM cells were isolated from lung specimen incidental to patient surgery and exposed to TNFα or IL‐13 for 24h. Expression of SR/ER stress markers including PERK (protein kinase‐like endoplasmic reticulum kinase), Bip (Binding immunoglobulin protein also known as 78 kDa glucose‐regulated protein), PDI (protein disulfide isomerase), and mitochondrial fission/fusion proteins Drp1 and Mfn2 were evaluated by Western blot. The expression of the spliced isoform of the transcription factor XBP1 was investigated by quantitative real‐time PCR (RT‐PCR). For mitochondrial morphology (fission/fusion), ASM cells were loaded with Mitotracker Green and images analyzed using ImageJ and Matlab to quantify mitochondrial Form Factor and Aspect Ratio. The effect of inflammation induced SR/ER stress on human ASM proliferation was evaluated using CyQUANT assay.We found that after 24 h exposure to TNFα and IL‐13: 1) expression of SR/ER stress protein markers (PERK, BiP and XBP1) increased; 2) Drp1 expression increased and Mfn2 expression decreased; 3) mitochondrial fragmentation increased; 4) [Ca2+]cyt and [Ca2+]mito responses to ACh stimulation were uncoupled; 5) net [Ca2+]cyt increased; 6) contractility increased; and 7) ASM cell proliferation increased.Together these results suggest that inflammation induces SR/ER stress that leads to changes in Mfn2 and Drp1 expression, which alter mitochondrial dynamics and Ca2+ regulation resulting in ASM hyperreactivity and remodeling.Support or Funding InformationNIH grant HL126451 (GCS).
Reflex bronchoconstriction evoked by inhaled nicotine aerosol in guinea pigs: role of the nicotinic acetylcholine receptor
Authors: Lin Ruei‐Lung , Khosravi Mehdi , Xu Fadi , Lee Lu‐Yuan 
Published: November 2023

We have previously reported that stimulation of vagal bronchopulmonary C‐fibers by nicotine via its action on neuronal nicotinic acetylcholine receptor (NnAChR) is primarily responsible for the acute bronchoconstriction evoked by inhaled cigarette smoke. Recent studies have reported that nicotine at high concentrations can also activate the transient receptor potential ankyrin 1 receptor (TRPA1) expressed in these sensory nerves. This study was carried out to investigate the mechanisms involved in the bronchoconstrictive effect of inhaled nicotine, and the relative roles of NnAChR and TRPA1 in this effect. Guinea pigs were anesthetized and mechanically ventilated; nicotine aerosol (2% solution; 1 × VT) was diluted by air to 2 × VT and delivered directly into the lung via trachea tube. Our results showed: 1) Delivery of one breath of nicotine aerosol immediately triggered a pronounced airway constriction; the increase in airway resistance (Raw) reached a peak of 588 ± 205 % in 10–40 sec, which gradually returned to baseline after 1–5 min. The response was reproducible in the same animal tested 20 min later. 2) Challenge with aerosolized alkaline solution of the same pH as that in the nicotine solution (10.5) did not cause any change in Raw. 3) Pretreatment with atropine (0.1 mg/kg; i.v.) and mecamylamine (Mec; an antagonist of NnAChR) (2%; 4 breaths of aerosol) almost completely abolished the nicotine‐induced bronchoconstriction: ΔRaw was reduced by 83.1 ± 8.2 % and 92.5 ± 1.9 %, respectively. The Mec pretreatment did not block the bronchoconstriction and bradycardia generated by electrical stimulation of the distal end of one sectioned vagus nerve, indicating its minimal systemic effects. 4) Pretreatment with HC‐030031 (0.5 mg/kg; i.v.), a selective TRPA1 antagonist, abolished the bronchoconstriction induced by allyl isothiocyanate (1.7 mg/kg, i.v.), a TRPA1 agonist, but did not render any attenuation of the nicotine‐evoked bronchoconstriction. In conclusion, inhalation of a single puff of nicotine aerosol evoked acute bronchoconstriction mediated through the cholinergic reflex pathway, which was triggered by activation of NnAChR located in airway sensory nerves. In contrast, TRPA1 receptor did not play a significant role in this response.Support or Funding InformationSupported by NIH grants HL96914, HL107462 and UL1TR001998