bluuebunny/crossref_metadata_2025 huggingface dataset:
Molecular Mechanisms of Hypoxia‐Induced Extracellular Vesicle Release
Authors: Mora Silvia , Muniz-Garcia Ana , Zorzano Antonio
Published: April 2020

Background

Extracellular 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.

Methods

We 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.

Results

Hypoxia 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.

Conclusion

these data suggest that hypoxia driven exosome release is HIF independent.

Support or Funding Information

Sponsored 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 Information

NSF IOS‐1656407
Exploring the Biosynthesis of O‐acetylated Sialic Acids using CASD1‐null Mice
Authors: Diaz Sandra L. , Ji Yang , Srivastava Saurabh , Mahajan Vinay S. , Pillai Shiv , Varki Nissi , Varki Ajit
Published: April 2020

Sialic acids (Sias) are a family of nine‐carbon backbone monosaccharides naturally occurring at the outer ends of vertebrate cell surface glycoconjugates. O‐acetyl esters at C4, C7, C8 or C9 of Sias are very common modification of Sias, and can play key roles in a number of biological processes. Cell‐, tissue‐ and individual‐specific variations in Sia O‐acetylation have been reported. Several studies also showed that different cell types incorporated 9(7)‐O‐acetylated Sias differentially into different classes of glycoconjugates. Sialate:O‐acetyltransferase activities were also studied in various systems. But the genetic basis and the regulation mechanisms involved remain largely unknown until the discovery of CASD1 as the first candidate gene responsible for sialate:O‐acetylation in eukaryotic systems, and in vitro studies confirmed the enzymatic activity on CMP‐Neu5Ac. Systemic CASD1 knockout (KO) mice, (Casd1tm1b/tm1b ) showed a marked reduction of 9‐O‐acetylation on blood cells. These mice now provide a tool for us to explore the role of CASD1 in sialate:O‐acetylation of Sias in other tissues, and existence of potential O‐acetyltransferase other than CASD1. A combination of different analytical approaches were used to detect residual O‐acetylation in various tissues and cell types of the CASD1‐deficient mice. 9‐O‐acetyl‐Sia distribution was analyzed by histochemistry staining of wildtype (WT) and CASD1 KO mouse tissues with the recombinant 9‐O‐acetyl‐Sia recognizing probe originated from porcine torovirus (PToV) and its non‐binding mutant (PToV‐mut). Significant loss of PToV staining was seen in multiple tissues from CASD1 KO mice, compared to those from WT mice. This is further confirmed by western blot analysis on tissue samples from the WT and CASD1 homozygous KO mouse probing with PToV. O‐acetyl‐Sia levels in various tissue homogenates from CASD1 KO mice in comparison to WT mice was analyzed by mild acid or sialidase release, 1,2‐diamino‐4,5‐methylenedioxybenzene‐2HCl (DMB) derivatization and HPLC and compared to CASD1 heterozygous mice. Base treatment was also applied to Sias released with acetic acid or sialidase to provide a more accurate quantification of O‐acetyl‐Sias. Marked reduction of 9‐O‐acetyl‐Sias was observed in homozygous CASD1 KO tissue (particularly colon, brain and testis and blood) with a small amount of residual 9‐O‐acetyl‐Sias still found in certain tissue like adrenals and bladders. This work suggested that CASD1 plays a major role in the biosynthesis of 9‐O‐acetyl Sias in different mouse tissue‐ and cell‐types. Whether CASD1 participates in the 4‐O‐acetylation of sialic acids and whether different O‐acetyltransferases work on glycolipid and glycoprotein‐bound sialic acids needs further study. These mice also provide a model system to study the in vivo functions of sialic acid 9(7)‐O‐acetylation.
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 Information

This 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 projections

Support or Funding Information

Research 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.
Hyperoxia results in HMGB1 production and subsequent inflammatory signaling in human pulmonary microvascular endothelial cells
Authors: Jin Yi , Pool Caitlyn , Liu Yusen , Nelin Leif Daniel
Published: April 2020

We have previously shown that neonatal mice exposed to 85% oxygen have substantial elevations in bronchoalveolar lavage levels of HMGB1 protein and that inhibiting TLR4 signaling using TAK‐242, which prevents TLR4 interaction with adaptor proteins like HMGB1, attenuated hyperoxia‐induced lung injury. To begin to understand the molecular and cellular mechanisms underlying these in vivo findings we studied HMGB1 mediated signaling in cultured cells. We hypothesized that hyperoxia would induce HMGB1 production in cells key to lung injury and that HMGB1 would interact with TLR4 to cause pro‐inflammatory cytokine production. We exposed human pulmonary microvascular endothelial cells (hPMVEC), A549 cells (a human lung epithelial cell line), and RAW264.7 cells (a murine macrophage‐like cell line) to hyperoxia, and examined the effect on HMGB1 levels. We found that exposure to 85% oxygen for 48 hours resulted in 16‐fold, 2‐fold, and 14‐fold increases in HMGB1 protein levels in these cells, respectively, relative to the same cells in normoxia. When HMGB1 (100 ng/ml) was added to the media of hPMVEC, there was significant (p<0.005) induction of IL‐8 and MCP‐1 protein levels. Furthermore, HMBG1 added to the media of hPMVEC resulted in significant induction of cleaved caspase‐1 and IL‐1β protein levels compared to vehicle treated hPMVEC. The addition of HMGB1 to the media of hPMVEC also resulted in a significant time‐dependent induction of pNFκB, pIKKα/β, and pIκBα. Hyperoxia exposed hPMVEC had a 3‐fold greater expression of phosphorylated p38 then did normoxic cells and this pp38 induction was significantly attenuated by TAK‐242. In hPMVEC hyperoxia resulted in 2.5‐fold greater IL‐8 protein levels than in normoxia exposed cells (p<0.05) and this effect was completely blocked by TAK‐242. In hPMVEC exposure to hyperoxia resulted in 8‐fold greater levels of cleaved caspase‐1 than in normoxia exposed cells (p<0.05), and in 4‐fold greater levels of cleaved IL‐1β than in normoxia exposed cells (p<0.005). In conclusion, our results suggest that hyperoxic exposure results in HMGB1 mediated TLR4 activation, leading to caspase‐1 activation and induction of pro‐inflammatory cytokines including cleaved IL‐1β. These results suggest that targeting HMGB1‐TLR4 interactions may confer protection from hyperoxia‐induced injury.
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 Information

This 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 Information

This 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 Support

This 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 Information

This 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 Information

NIH
Effects of Temperature and Nutrition on Growth Efficiency of the South American Locust (Schistocerca cancellata) During the Sixth Instar
Authors: Elliott Deanna B. , Youngblood Jacob P. , Angilletta Michael J. , Cease Arianne J.
Published: April 2020

Throughout history, locust swarms have caused ecological and economic destruction in agricultural communities. Today, there are 20 species responsible for plagues on every continent excluding North America and Antarctica. In recent years, the South American species Schistocerca cancellata has wrought such devastation that Argentina and Bolivia were forced to declare states of emergency. Temperature and nutritional content of crops are known to be important variables in where a swarm will occur, but how these phenomena interact has not been studied. This experiment was intended to determine which temperature and dietary composition cause the highest growth rate in S. cancellata, based on the assumption that swarms may very well occur in places where the locusts are most likely to maximize their fitness. To this end, 180 locusts were separated into treatments composed of all combinations of three temperatures and three diets. Each treatment lasted the length of each animal’s 6th instar. Temperatures were held at 25°C, 35°C, and 42.5°C, and diets with protein‐to‐carbohydrate ratios of 0P:42C, 14P:28C, and 35P:7C were fed. Once completed, waste was collected and weighed, and then compared to the mass of diet consumed in order to determine how much mass was retained. Additionally, the caloric content of the diets and waste was measured to calculate energy efficiency between treatments. The hottest treatments tended to result in the highest, fastest growth rate, while the cold treatments grew less and took significantly longer to reach adulthood. The diet highest in protein showed the most weight gain, while the on with no protein had very few survivors. The results of this study may help to predict where and when locust swarms will occur, based on weather and crop content in South America, and allow for precautions, such as chemical control measures, to take place in the probable affected areas.

Support or Funding Information

NSF LEAP Scholars Program
Sex Differences in Offensive and Defensive Investment in Crayfish Claws
Authors: Vargas Cindy , Graham Zackary A. , Palaoro Alexandre , Angilletta Michael
Published: April 2020

Many animals possess weapons that have evolved to inflict damage to opponents during territorial encounters. Although the offensive capabilities of these weapons are important determinants of contest success, the defensive capabilities of the weapons are also important. However, current theory only accounts for investment into offensive performance, and not defensive endurance. To help better understand these dynamics, we investigated claw strength, cuticle thickness, and injury rates in male and female virile crayfish (Faxonius virilis). To measure the offensive capacity of a claw, we collected the max pinch of 3–5 pinches of each claw using a force transducer. To measure the defensive capacity of the claw, we dissected and measured the cuticle thickness of the carapace and seven sections of the claw: merus, dorsal manus, ventral manus, dorsal dactyl, ventral dactyl, dorsal pollex, and the ventral pollex. Injury rate was determined by visual inspection of the claw. Both male and female virile crayfish engage in aggression over access to both food and shelter. Thus, we expected each sex to invest similarly into the offensive and defensive abilities of their weapons. Interestingly, we found that despite males having proportionally larger claws, the average strength for a given size of the claw was similar between the sexes. Furthermore, we did not detect any sex differences in defensive ability as measured by cuticle thickness and injury rates. Overall, we demonstrated that both male and female virile crayfish invest similarly into the offensive and defensive ability of their claws.

Support or Funding Information

National Science Foundation (NSF) LEAP Scholars program
The Pattern of SOX11 During Chicken Limb Development Suggests a Role in Myogenesis
Authors: Ly Van Dai N. , Damoah Ruth-Love , Pira Charmaine U. , Cooper Allen M. , Oberg Kerby C.
Published: April 2020

SOX11 has been implicated in several aspects of development including epithelial to mesenchymal interactions, neuronal migration, skeletogenesis, joint maintenance, and hedgehog signaling. As a first step in evaluating its role in limb development, we determined the expression pattern for SOX11 between Hamilton‐Hamburger (HH) stages 19–35 chicken embryos. Initally (HH19), SOX11 expression is detected in the the emerging limb bud, with strong ventral expression centrally. At HH22, dorsal anterior and posterior proximal expression is evident. As the limb develops (HH24), SOX11 expression shifts distally and wanes proximally. At HH25, expression surrounds condensing cartilage, and by HH29, SOX11 expression surrounds and accentuates the elbow and distal phalanges. By HH35, SOX11 has clearly shifted to selected muscles and tendons but is poorly expressed in joint spaces. This is the first report to implicate SOX11 in myogenesis. These findings suggest that SOX11 may play multiple roles during skeletogenesis, including the regulation of selected muscle tracts. Further work is needed to determine whether there is a common role for SOX11 during these various components of musculoskeletal development or whether SOX11 adapts to multiple pleiotropic roles that occur during limb development.
Estradiol’s Permissive Role in Social Stress Susceptibility in Female Rats
Authors: Pope Brittany , Mott Sarah E. , Fadel James R. , Wilson Marlene A. , Wood Susan K.
Published: April 2020

Social stress is a common risk factor for both depression and cardiovascular disease (CVD), and individuals suffering from depression are at increased risk of CVD‐related mortality. Additionally, women are more likely than men to suffer from depression and anxiety along with comorbid CVD, however this increased risk is confined to the range between the beginning of puberty and end of menopause. Therefore, while specific systems regulating heightened susceptibility in females are unclear, there is evidence to suggest it may be regulated in part by ovarian hormones. Using a social stress model in female rats whereby a female is forced to witness a social defeat encounter between two males (15 mins, 5 days), we have previously shown that intact females are highly susceptible to this form of stress as evidenced by anxiety‐and depressive‐like behaviors, brain cytokines, and diminished vagal tone. Importantly, ovariectomized (OVX) females are resistant. Here we conducted two separate sets of studies to determine the role that 17‐β estradiol (17‐βE, 10μg/rat, sc) plays in susceptibility to social stress in females. In study #1, following ovariectomy and surgical implantation of cardiovascular transmitters (HD‐S11, DSI), rats were treated with either 17‐βE or vehicle and subjected to control or witness stress during cardiovascular recording. In study #2 all females were implanted with an i.v. catheter and either left intact (sham, and treated with vehicle) or OVX (and treated with vehicle or 17‐βE), followed one hour later by witness stress or control handling. Blood samples were collected (0, 15 and 60 mins) and behavior was video recorded. OVX females exhibited a blunted tachycardic response to stress that was reversed by 17‐βE administration. Moreover, epinephrine release in response to witness stress was highly reactive in intact females above that of OVX females, despite similar corticosterone levels. Anxiety‐like responses were also shown to be regulated by 17‐βE; witness stress‐evoked burying that emerged in intact females was blunted by OVX and reinstated by 17‐βE. Taken together these studies identify that proestrous levels of estradiol are critical for acute behavioral and cardiovascular responses to social stress in females. Future studies are aimed at determining the neural systems that are regulated by estradiol and precipitate stress susceptibility.

Support or Funding Information

MH113892‐01A1, NARSAD Young Investigator Award 26809
Chronic sodium bicarbonate may promote metabolic dysregulation in a rodent model of CKD
Authors: Mannon Elinor , O'Connor Paul
Published: April 2020

Chronic kidney disease (CKD) has no cure, and few treatment options targeted at slowing the decline in glomerular filtration rate (GFR). Several small clinical trials have indicated that oral sodium bicarbonate (NaHCO3) targets and slows the decline in GFR in CKD. The reported beneficial effects of NaHCO3 in this setting have led to its now common use clinically. It’s known however, that chronic alkali ingestion can promote potassium (K+) wasting, and that K+ depletion has been linked with metabolic dysregulation. Further, CKD patients who are supplemented chronically with oral NaHCO3 display significantly decreased plasma K+ levels relative to control treated counterparts, although these decreases in plasma K+ remain within normal range. No study has investigated the metabolic impacts of chronic NaHCO3 treatment and K+ wasting in CKD. To this end, we tested the hypothesis that in the setting of high tubular flow, chronic NaHCO3 ingestion would promote K+ wasting and metabolic dysfunction. In order to test our hypothesis, 9 male, 8 wk old Sprague Dawley (SD) rats underwent a 2/3 nephrectomy (Nx) and telemetry implantation. The 2/3 Nx is a model of reduced renal mass that has increased single nephron GFR in the absence of metabolic acidosis, similar to most human patients with CKD. Following 4 weeks of recovery, animals were given either 0.1M NaHCO3 (Bicarb, n=3), 0.1M NaCl (Vehicle, n=3) or 0.2M NH4Cl‐NaHCO3 (AC/B, n=3) in the drinking water for 12 weeks. Following 9 weeks of drinking water treatment, we observed a significantly higher hemoglobin A1c in our bicarb treated group compared to vehicle and AC/B treated groups (Bicarb 4.6 ± 0.1 vs Veh 4.2 ± 0.3 vs AC/B 4.2 ± 0.2; One‐way ANOVA, p=0.05). Following another 3 weeks of drinking water treatment, we performed blood gas measurements to assess plasma K+. In contrast to our hypothesis we observed the highest levels of plasma K+ in our bicarb treated animals compared to our vehicle or AC/B groups (Bicarb 5.18 ± 0.28 vs Veh 4.48 ± 0.66 vs AC/B 4.5 ± 0.21; One‐way ANOVA, p=0.16). Our data show that oral NaHCO3 could worsen blood glucose control in the setting of high tubular flow. Additionally, plasma K+ may not be a reliable measure for assessment of K+ status in CKD patients supplemented with NaHCO3. As the majority of K+ is stored intracellularly, further studies characterizing the effect of chronic alkali ingestion on total body K+ are warranted to assess the impact of NaHCO3 on K+ depletion in this patient population.

Support or Funding Information

NIH PO1H134604 and DK099548
ER‐associated Regulation of Astrocyte Mitochondrial Function during METH and HIV‐1 Comorbidity
Authors: Proulx Jessica , Borgmann Kathleen
Published: April 2020

Astrocytes are key regulators of central nervous system (CNS) health and neuronal function. However, astrocyte mitochondrial dysfunction, such as induced by METH and HIV‐1, threatens the ability of astrocytes to provide the essential metabolic and antioxidant support to neurons. Thus, being able to determine regulatory pathways that can be targeted to prevent aberrant mitochondria activity in astrocytes will be imperative for ensuring neuronal fitness/survival against CNS pathologies. The current investigation examined the endoplasmic reticulum (ER)‐mitochondrial interface in HIV‐1+ primary human astrocytes with and without METH exposure to characterize changes in mitochondrial bioenergetics, ER stress, Ca2+ signaling, and the regulation of mitochondria associated membranes (MAMs). Then, we explored the role of ER‐associated mechanisms in regulating astrocyte mitochondrial function. The effects of METH treatment were explored in both acute and chronic paradigms while responses to HIV‐1 were determined by transfecting astrocytes with a plasmid expressing whole HIV‐1 genome. Mitochondrial bioenergetics were then assessed using Seahorse Assay. Changes in the expression of unfolded protein response (UPR) and MAM mediators were determined using RT‐PCR and protein expression assays. Finally, pharmacological inhibition of Ca2+ signaling and UPR pathways were used to delineate the regulatory mechanisms mediating the changes in mitochondria bioenergetics. These findings illustrate the importance of ER‐mitochondria communication in regulating astrocyte mitochondrial function during METH/HIV‐1 pathogenesis.

Support or Funding Information

This project was supported by: R01 DA039789 & T32 AG020494‐16 A1 Neurobiology of Aging Fellowship
Aminoglycoside conformation and ring substituents dictate evasion from bacterial resistance mechanisms
Authors: Dey Debayan , Hoffer Eric D. , Francis Dillion , Dunham Christine M. , Conn Graeme L.
Published: April 2020

Ribosomal RNA (rRNA) methylation by aminoglycoside‐resistance methyltransferases is presumed to reduce antibiotic activity by sterically occluding part of the drug binding site in helix 44 of 16S rRNA. However, for 4,6‐disubstituted deoxystreptamine (4,6‐DOS) aminoglycosides, the m1A1408 aminoglycoside resistance modification results in a wide range of bacterial resistance phenotypes with structurally similar drugs, from complete resistance to highly susceptible. Thus, for currently undefined reasons, some 4,6‐DOS aminoglycosides appear to be able to “evade” the effects of rRNA methylation at A1408. In contrast for the m7G1405 aminoglycoside resistance modification,
all 4,6‐DOS aminoglycosides are rendered inactive. Why these same drugs are unable to evade this methylation is also currently not mechanistically understood. We will describe the development of a framework for understanding methylated rRNA‐antibiotic interactions through the evaluation of energetics of drug‐methylated rRNA complexes for a collection of 4,6‐DOS aminoglycosides. With computational calculations using Monte Carlo simulations and free energy maps of aminoglycosides, we probed the flexibility around each ring’s dihedral angles (ϕ/ψ) in rRNA bound poses and the contributions of Ring I substituents to drug binding. These analyses revealed three key molecular features that may contribute to evasion of m1A1408 modification: 1) The polarity of atoms of the Ring I 6’ substituent; 2) the number of Ring I substituents; and 3) the presence of a L‐hydroxyaminobuteroyl amide (HABA) group on the 2‐DOS core ring. These studies lay a foundation for understanding aminoglycoside‐methylated rRNA interactions at the molecular level and could, for example, directly facilitate design of new aminoglycoside derivatives that retain activity against pathogens harboring aminoglycoside‐resistance rRNA methyltransferases.

Support or Funding Information

This work was supported by National Institutes of Health grants R01‐AI088025 (to GLC) and Cystic Fibrosis Foundation postdoctoral fellowship DEY18F0 (to DD).
Mutations In The Furin Cleavage Site To Improve Cytotoxicity In Anti‐Mesothelin Recombinant Immunotoxins Based On Pseudomonas Exotoxin A
Authors: Butters Keeley , Baker Jillian , Reifer Danielle , Weldon John
Published: April 2020

A recombinant immunotoxin (RIT) is a fusion protein composed of an antibody and a toxin. The antibody binds to receptors on target cells and promotes internalization. Once internalized, the toxin kills the cell. RITs based on Pseudomonas exotoxin A (PE) have been created to target and kill cancer cells. In order to do this, internalized PE must reach the cytosol through a retrograde trafficking route, during which it encounters and is cleaved by the protease furin. Furin has been established as an integral part of PE‐based RIT intoxication pathways. Mutations in the PE furin cleavage site have been shown to influence cleavage efficiency and RIT cytotoxicity. In order to explore the role of furin in RIT intoxication further, we have constructed anti‐mesothelin/PE‐based RITs with mutations on either side of the furin cleavage site. Based on previous results, we initially hypothesized that mutations N‐terminal to the cleavage site would increase both cleavage efficiency and cytotoxicity in these RITs, while mutations C‐terminal to the cleavage site would increase cleavage efficiency without significant improvements to cytotoxicity. We expressed, purified, and tested the cytotoxicity of these constructs in tissue culture and found that mutations N‐terminal to the cleavage site decreased cytotoxicity relative to the wild‐type RIT, while mutations C‐terminal to the cleavage site had minimal effect. We next plan to explore cleavage efficiency of the mutant RITs.

Support or Funding Information

Towson University Department of Biological Sciences