bluuebunny/crossref_metadata_2025_split huggingface dataset:
Molecular Mechanisms of Hypoxia‐Induced Extracellular Vesicle Release
Authors: Mora Silvia , Muniz-Garcia Ana , Zorzano Antonio 
Published: April 2020

BackgroundExtracellular vesicles (EVs) are released by many cells and provide a mechanism for intercellular communication in autocrine and paracrine fashion. EVs of various sizes, namely microvesicles (ranging from 100–1000 nm) or exosomes (with sizes between 30–150 nm) are released by most eukaryotic cells. EVs are known to contain bioactive molecules including proteins lipids and nucleic acids including miRNAs and LNC RNAs that regulate gene expression and cell functions in recipient cells. For example, tumour released exosomes remodel the microenvironment allowing cancer cells to survive and thrive. Low oxygen (hypoxia) is known to increase the release of exosomes in cancer cells. Hypoxia leads to the activation of hypoxia inducible factor (HIF) transcription factors, considered master regulators of the hypoxic response. The objective of this study was to investigate whether hypoxia induced EV (exosome) release is dependent on hypoxia inducible factor (HIF) proteins.MethodsWe isolated exosomes and microvesicles by differential centrifugation of conditioned media of cells exposed to normoxia or hypoxia (1% Oxygen) for different lengths of time ranging from 1–24hrs. We determined, size distribution and concentration of vesicles by nanoparticle tracking analysis (NTA). Expression of HIF1 and HIF2 was monitored by western blot of cellular lysates. We expressed oxygen‐stable mutated HIF1 and HIF2 isoforms either alone or in combination and determined EV release by NTA. We used chemical inhibitors of dioxygenase (PHD) and von Hippel–Lindau (VHL) proteins, the two proteins involved in HIF degradation under oxygen conditions, to stabilize HIF expression in normoxic cells and analysed EV release and HIF expression. We also analysed EV release in a cell line lacking VHL and after a VHL knock‐in. Western blotting of cellular lysates was used to monitor HIFs, VHL, and VEGF proteins. NTA was used to determine EV concentration and size. ANOVA statistical analysis was performed to analyse the data.ResultsHypoxia increased the amount extracellular vesicles in the conditioned media of all cells tested. The release of small vesicles (exosomes) was most increased following the exposure to hypoxia. HIF protein expression increased steadily during the time course of hypoxia, reaching a plateau after 2hrs following exposure. Expression of mutated (oxygen stable) HIF1 and HIF2 proteins in normoxic cells, either alone or in combination, did not increase exosome release. Chemical inhibition of PHD or VHL did not affect EV release. Exosome release was not different in VHL KD or KI cells.Conclusionthese data suggest that hypoxia driven exosome release is HIF independent.Support or Funding InformationSponsored by Fundacion Ramon Areces
A Course‐based Undergraduate Research Experience (CURE) in Biochemistry II Lecture
Authors: Ragan Emily 
Published: April 2020

Course‐based Undergraduate Research Experiences (CUREs) aim to involve all undergraduate students in a course in an authentic research question. CUREs increase the number of students exposed to research and provide students an opportunity to apply concepts. While many CUREs have been used in laboratory classes, I developed and implemented a CURE on iron uptake in insects in a second semester biochemistry lecture course. Students used common database and bioinformatics tools to explore an assigned Drosophila melanogaster gene and its expression data and found related protein sequences in other insects. After making a multiple sequence alignment, students used the alignment and a related crystal structure to identify key amino acid residues and make predictions about protein structure and function. The project culminated in a formal memo to a research collaborator, who gave students feedback about how their findings fit in the larger project. In the second half of the semester students expanded on skills developed in the CURE to explore the sequence and structure of a protein of interest. From five semesters of iteration I share strategies for incorporating research and bioinformatics into a lecture classroom environment. Student feedback using a modified CURE assessment survey indicates recent versions of the project had student self‐reported scores on discovery and iteration comparable to laboratory‐based CURE projects.Support or Funding InformationNSF IOS‐1656407
Targeting Epithelial Expressed Sialylated Lewis glycans improves colonic mucosal wound healing and protects against colitis
Authors: Kelm Matthias , Quiros Miguel , Nusrat Asma , Cummings Richard D. , Parkos Charles A. , Brazil Jennifer C. 
Published: April 2020

Delayed healing of mucosal wounds is a hallmark of many pathological conditions including inflammatory bowel disease (IBD). Mucosal wound healing involves a complex interplay between epithelial cells and infiltrating immune cells that trigger cell signaling events and epithelial cell migration, proliferation and differentiation that are necessary for restoration of mucosal homeostasis. Specific alterations in surface glycosylation have been reported in intestinal epithelial cells following injury and we have shown that targeting the inflammation induced glycan sialyl Lewis A (sLea) on epithelial CD44v6 (with a specific mAb termed GM35) results in inhibition of neutrophil (PMN) trafficking in the inflamed intestine. Despite this observation, the role of glycans in intestinal wound healing has not been examined. Here we report that selective targeting of sialylated Lewis glycans on epithelial glycoprotein CD44v6 increases human intestinal epithelial cell proliferation, migration and wound healing. Furthermore, direct injection of GM35 into biopsy‐induced colonic wounds as well as systemic injection resulted in a significant increase in epithelial proliferation and mucosal wound closure in vivo. These glycan mediated increases in epithelial proliferation were found to be mediated through the activation of Src, Fak and Akt (kinases previously shown to signal downstream of CD44v6 engagement). Finally, systemic administration of GM35 to C57BL/6J mice followed by induction of colitis with dextran sodium sulfate resulted in reduced disease activity, as assessed clinically and histologically, when compared to mice treated with an isotype control mAb. Given our observations of robust upregulation of sialylated Lewis glycans during intestinal inflammation in mice and humans, these data suggest that targeting sLea on epithelial CD44v6 may represent a promising new target for improved recovery of epithelial barrier function and restitution of intestinal homeostasis following inflammation or injury.Support or Funding InformationThis work was funded by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation – 410855404), a senior Research Award from the Crohn’s and Colitis Foundation (CCF) and R01 awards from the NIH (DK072564 and DK079392).
Neurons in the rat superior colliculus translate visual threat into autonomic responses via a relay in the rostral ventromedial medulla
Authors: McMullan Simon , Lynch Erin , Redmond William John , Dempsey Bowen , Monteiro Eloise , Dampney Roger A.L. , Goodchild Ann K. 
Published: April 2020

The superior colliculus (SC) is a sensory integration hub in the dorsal brainstem where multimodal information is combined and, depending on the saliency of the competing sensory inputs, appropriate motor commands and supportive autonomic changes initiated. In rodents, the SC is indispensable for initiating behavioral responses to stereotypical visual stimuli that resemble approaching objects, such as looming (an expanding overhead black circle). Here we report that presentation of overhead looming or naturalistic stimuli drove acute surges in blood pressure in telemetered conscious rats, an effect that was replicated by optogenetic stimulation of the deep SC (dSC). dSC stimulation also evoked increases in respiratory rate and tail vasoconstriction in the absence of detectable anxiety‐like behaviors and continued to exert excitatory effects on heart rate, respiratory rate, and sympathetic nerve activity under urethane anesthesia. The objective of the current study was to identify the central pathways responsible for mediating these physiological effects.Anterograde labeling of dSC neurons revealed a previously uncharacterized axonal projections to brainstem cell groups associated with arousal and autonomic control, including the locus coeruleus, A5 group and, most extensively, neurons within a region that spanned the medullary gigantocellular and raphe cell groups, collectively called the rostral ventromedial medulla (RVMM). Optogenetic stimulation of dSC terminals within the RVMM recapitulated some of the sympathetic and respiratory effects evoked by dSC stimulation, and optogenetic dSC activation evoked powerful excitatory effects on extracellular recordings of putative RVMM sympathetic premotor neurons, suggesting that elements of the physiological response dSC stimulation are mediated by direct activation of medullary autonomic neurons.To investigate the contribution of environmental stimuli to the excitability of this pathway we conducted single‐unit recordings of SC neuronal responses to visual and acoustic stimuli using high‐density silicon probes. In addition to responding to stereotypical audio‐visual looming stimuli, we report the presence of SC neurons with higher‐order visual capabilities relating to object detection that differ by subregion and are several orders of magnitude more complex than previously recognized. These tuning properties were also found in subpopulations of opto‐tagged SC neurons that project to the RVMM. Our data suggest that the SC is not only capable of nuanced object recognition, but can translate naturalistic visual cues into fast‐acting autonomic changes via direct medullary projectionsSupport or Funding InformationResearch was supported by the NHMRC and Hillcrest Foundation
Evaluation of Mitochondrial Energy Metabolism and Translation in Clear Cell Renal Cell Carcinoma
Authors: Koc Fatih C. , Frear Benjamin C. , Koc Emine C. 
Published: April 2020

Clear cell renal cell carcinoma (ccRCC) is one of the most common renal cell carcinomas. The defining morphological hallmark of ccRCC is the accumulation of glycogen and lipid droplets in the cytoplasm of the cells due to the reprogramming of glucose and fatty acid metabolism and oxidative phosphorylation (OXPHOS). Energy metabolism by OXPHOS is supported by both nuclear and mitochondrial‐encoded genes. Alterations in the expression of mitochondrial genes involved in OXPHOS have been well documented in ccRCC.We proposed that the mitochondrial translation machinery that exists solely for the synthesis of 13 mitochondrially‐encoded subunits of OXPHOS complexes is also implicated in the progression of ccRCC.To test this hypothesis, immunoblotting analyses of 14 ccRCC biopsies and their matched normal tissues were performed to investigate the role of mitochondrial translation machinery in the remodeling of OXPHOS complexes. Along with changes in the expression of nuclear and mitochondrial encoded subunits of OXPHOS complexes, we discovered that the expression of several mitochondrial translation factors such as mtEF‐Tu (TUFM), mtIF2 (MTIF2), and mtEF‐G (GFM1) and ribosomal proteins were also significantly reduced in ccRCC biopsies. To identify the key factors involved in mitochondrial translation further, genomics and proteomics data mining analyses of publicly available ccRCC databases at the Cancer Genome Atlas (TCGA) and The Clinical Proteomic Tumor Analysis Consortium (CPTAC) were also performed.Based on the evidence provided in our studies, we propose that changes in the expression of mitochondrial translation components, specifically translation factors and mitochondrial ribosomal proteins, are part of the remodeling of mitochondrial energy metabolism and resistance to apoptosis in ccRCC.
Myristoylation of LMCD1 leads to its species‐specific derepression of E2F1 and NFATc1 in the modulation of CDC6 and IL‐33 expression during development of vascular lesions
Authors: Govatati Suresh , Pichavaram Prahalathan , Janjanam Jagadeesh , Guo Liang , Virmani Renu , Rao Gadiparthi N. 
Published: April 2020

Majority of translational research is focused on the use of rodent models to understand the pathophysiology of human disease. However, many experimental findings of rodent models fail to translate to human. In this regard, our previous studies show that LIM and cysteine‐rich domains 1 (LMCD1) plays a role in thrombin‐induced vascular smooth muscle cell (VSMC) proliferation and migration only in human but not rodents. Based on these findings, we asked the question whether LMCD1 exhibits any species‐specific role in the development of vascular lesions. A combination of genetic, molecular, cellular and disease models were used to test species‐specific role of LMCD1 in the pathogenesis of vascular lesions. Here, we report species‐specific regulation of LMCD1 expression in mediating VSMC proliferation and migration during vascular wall remodeling in human versus mice. Thrombin induced LMCD1 expression in human aortic smooth muscle cells (HASMCs) but not mouse aortic smooth muscle cells (MASMCs). We found association of ‘rapid promoter evolution’ in this expression divergence of LMCD1 in human versus mice. In exploring the mechanism, we found that Par1‐Gαq/11‐PLCβ3‐NFATc1 signaling axis mediates thrombin‐induced LMCD1 expression in HASMCs. Furthermore, although LMCD1 mediates thrombin‐induced proliferation and migration of both HASMCs and MASMCs via influencing CDC6 and IL‐33 expression, respectively, in humans it acts as an activator and in mice it acts as a repressor of their promoters. Interestingly, its repressor activity was revoked by its myristoylation in mouse. Besides, we found increased expression of LMCD1 in human stenotic arteries as compared to non‐stenotic arteries. On the other hand, LMCD1 expression was decreased in neointimal lesions of mouse injured arteries as compared to non‐injured arteries. Together, these observations reveal that LMCD1 acts as an activator and repressor of E2F1 and NFATc1 in human and mice, respectively, in the induction of CDC6 and IL‐33 expression during development of vascular lesions. Based on these findings, LMCD could be a potential target for drug development against restenosis and atherosclerosis in humans.Support or Funding InformationThis work was supported by a grant HL069908 from NIH to Dr. Gadiparthi N. Rao
Advanced volatilome analysis for assessing gut microbial metabolism
Authors: Zhu Jiangjiang (Chris) 
Published: April 2020

The olfactory sense of “smell” has long been used to detect disease. Hippocrates from ancient Greece characterized the pungent stench of melena as early as the Age of Pericles, and later, the traditional Chinese and Arab physicians documented distinct urinary scents in diseases such as diabetes. In 1971, Nobel laureate Linus Pauling quantified volatile organic compounds (VOCs) from breath and urine samples with modern analytical tools, and launched a new era of volatile and smell research. Mass spectrometry‐based techniques have greatly driven biomedical research in the past decade, and have generated excitement in biomarker discovery, disease diagnosis, and effective public health monitoring. A group of largely untapped metabolites, however, is the easily accessible biological VOCs that can be detected from the headspace of many biological specimens. While incremental progress has been made in this field, sensitivity, selectivity, and broad coverage of volatile metabolites are still suffering from underdeveloped measurement technologies. Fecal odor, driven in large part by the gut microbiota, is established to change in the presence of gastrointestinal disorders. Understanding these changes could help to diagnose various diseases. Shifts in microbiota composition, or overt gut dysbiosis, is associated with metabolic dysregulation (e.g., obesity, inflammatory bowel disease), although a causal role into impaired metabolism requires validation. Compared with clinical analysis of other body fluids and biological specimens, VOC analysis from the headspace of feces for gut dysbiosis detection has the potential to save analysis time (from hours to minutes) and significant reduce costs otherwise incurred from expensive blood chemistry or genomic approaches. However, VOC‐detection techniques are still far from reaching the maturity needed to widely support clinicians in making day‐to‐day patient care decisions. To address these critical needs, we developed and applied an innovative biological VOC detection technology (i.e. Secondary Electrospray Ionization‐High Resolution Mass Spectrometry; SESI‐HRMS) to sensitively and specifically detect metabolic VOCs of gut microbial metabolism during gut dysbiosis. Further, validation will be supported using a green tea polyphenols (GTP) intervention in an in vitro gut model during the process of rescuing the gut dysbiosis with the support of GTP. The results of the in vitro study were further compared with our controlled animal studies demonstrating the benefit of GTP intervention on gut dysbiosis that otherwise occurring in obesity.Support or Funding InformationThis work is supported by The Ohio State University James Comprehensive Cancer Center, The Food for Health Initiative, and the NIGMS grant R35GM133510.
Downregulation of KCNQ channels in the CeA contributes to elevated sympathetic outflow in hypertension
Authors: Sheng Zhao-Fu , Zhang Hua , Zheng Peiru , Pan Hui-Lin , Li De-Pei 
Published: April 2020

Elevation of sympathetic outflow is associated with many hypertensive conditions. The central nucleus of amygdala (CeA) is crucial in the regulation of emotion, pain, and cardiovascular functions. The M‐channel is a non‐inactivating voltage‐dependent K+ channel stabilizing the membrane potential and mainly consists of heterotetrameric Kv7.2 and Kv7.3 subunits. However, the role of M channels in the CeA in heightened sympathetic vasomotor tone in hypertension remain unknown. Microinjecting of the specific M‐channel blocker XE‐991 into the CeA significantly increased arterial blood pressure (ABP) and renal sympathetic nerve activity (RSNA) but did not alter heart rate (HR) in anesthetized Wistar Kyoto rats (WKY) rats. In spontaneously hypertensive rats (SHRs), microinjection of the same dose of XE‐991 into the CeA had no effect on ABP, RSNA, or HR in SHRs. On the other hand, microinjection of the M‐channel opener QO‐58 into the CeA decreased ABP and RSNA in WKY rats but failed to alter these cardiovascular variables in SHRs. The protein and mRNA levels of Kv7.2 and Kv7.3 in the CeA were significantly lower in SHRs than in WKY rats. Lowering ABP via celiac ganglionectomy in SHRs did not altered the expression levels of Kv7.2 and Kv7.3 in the CeA. In a brain slice preparation, the basal firing activity of labeled CeA‐RVLM projection neurons was significantly greater in SHRs than in WKY rats. Blocking M‐currents with XE‐991 increased both spontaneous and current injection‐evoked firing rate of CeA‐RVLM projection neurons in WKY rats but had no such effect in SHRs. Also, the M‐currents triggered by a membrane depolarization in CeA‐RVLM neurons were significantly smaller in SHRs than in WKY rats. These data suggest that M‐channel activity is impaired and leads to hyperactivity of CeA‐RVLM projection neurons, which contributes to elevated sympathetic vasomotor activity in hypertension.Support or Funding Information SupportThis study was supported by NIH grant HL139523 and HL142133.
Nicotinamide mononucleotide (NMN) supplementation promotes anti‐aging miRNA expression profile in the aorta of aged mice, predicting epigenetic rejuvenation and anti‐atherogenic effects.
Authors: Kiss Tamas , Giles Cory , Tarantini Stefano , Yabluchanskiy Andriy , Balasubramanian Priya , Gautam Tripti , Csipo Tamas , Farkas Eszter , Nyúl-Tóth Ádam , Lipecz Agnes , Zsabo Csaba , Wren Jonathan , Csiszar Anna , Ungvari Zoltan 
Published: April 2020

Understanding molecular mechanisms involved in vascular aging is essential to develop novel interventional strategies for treatment and prevention of age‐related vascular pathologies. Recent studies provide critical evidence that vascular aging is characterized by NAD+ depletion. Importantly, in aged mice, restoration of cellular NAD+ levels by treatment with the NAD+ booster nicotinamide mononucleotide (NMN) exerts significant vasoprotective effects, improving endothelium‐dependent vasodilation, attenuating oxidative stress, and rescuing age‐related changes in gene expression. Strong experimental evidence shows that dysregulation of microRNAs (miRNAs) has a role in vascular aging. The present study was designed to test the hypothesis that age‐related NAD+ depletion is causally linked to dysregulation of vascular miRNA expression. A corollary hypothesis is that functional vascular rejuvenation in NMN‐treated aged mice is also associated with restoration of a youthful vascular miRNA expression profile. To test these hypotheses, aged (24‐month‐old) mice were treated with NMN for 2 weeks and miRNA signatures in the aortas were compared to those in aortas obtained from untreated young and aged control mice. We found that protective effects of NMN treatment on vascular function are associated with anti‐aging changes in the miRNA expression profile in the aged mouse aorta. The predicted regulatory effects of NMN‐induced differentially expressed miRNAs in aged vessels include anti‐atherogenic effects and epigenetic rejuvenation. Future studies will uncover the mechanistic role of miRNA gene expression regulatory networks in the anti‐aging effects of NAD+ booster treatments and determine the links between miRNAs regulated by NMN and sirtuin activators and miRNAs known to act in the conserved pathways of aging and major aging‐related vascular diseases.Support or Funding InformationThis work was supported by grants the Oklahoma Center for the Advancement of Science and Technology, the Oklahoma Shared Clinical and Translational Resources (OSCTR) program funded by the National Institute of General Medical Sciences (GM104938), the NIA‐supported Geroscience Training Program in Oklahoma (T32AG052363), the Oklahoma Nathan Shock Center (P30AG050911), the Cellular and Molecular GeroScience CoBRE (1P20GM125528, sub#5337), and the NIGMS supported Center of Biomedical Research Excellence (CoBRE).
Dirty Mice Have Altered Influenza A Virus Clearance And Lesions Compared To SPF Mice: A Proposed Model For Study Of Vaccine Response In Humans
Authors: Meyerholz David K. , Fiege Jessica E. , Block Katharine E. , Pierson Mark , Pape Kathryn A. , Jenkins Marc K. , Hamilton Sarah E. , Masopust David , Langlois Ryan A. 
Published: April 2020

Influenza A virus (IAV) is a seasonal pathogen that causes annual morbidities and mortalities. Current vaccines often exhibit reduced efficacy to seasonal strains and offer limited protection to novel pandemics. Additionally, vaccines that are successful in mice often fail to translate to humans. This could be due to the nature of the mouse model housed in specific pathogen free (SPF) conditions. Humans are exposed to a variety of pathogens that SPF mice are protected from. Unlike SPF mice, the immune system of pet store mice more closely recapitulates the immune system of humans. When SPF mice are co‐housed with pet store mice harboring natural mouse pathogens, termed dirty mice, these mice obtain phenotypes observed in the human immune system. In order to determine if dirty mice are an improved mouse model for vaccine development/testing, we sought to study IAV disease in dirty mice. We infected control or dirty mice with IAV and assessed viral clearance and pathology. We observed reduced IAV clearance and enhanced airway pathology characterize by denuded and sloughed/necrotic airway epithelium in dirty mice. These data demonstrate the importance of studying IAV in the dirty mouse model to facilitate more effective vaccine development and testing to improve translation to humans.Support or Funding InformationNIH