bluuebunny/crossref_metadata_2025 huggingface dataset:
Time Course of Astrocyte Activation in Mouse Nucleus Tractus Solitarius During Exposure to Hypoxia
Authors: Howe Jacqueline Stenger , Moya Esteban , Howe Gabrielle , Mesarwi Omar , Powell Frank
Published: April 2020

Chronic sustained hypoxia (CH) occurs in populations living at high altitude and in patients with chronic pulmonary disease. Exposure to CH produces ventilatory acclimatization to hypoxia (VAH) and increases the hypoxic ventilatory response (HVR) by mechanisms that involve areas of the brainstem that control breathing. The nucleus tractus solitarius (NTS) is a sensory integrative center in the medulla receiving carotid body afferents and known to be crucial for VAH. Our previous results demonstrated that glia cells in the rat NTS contribute to VAH but these mechanisms have not been studied in mice exposed to CH beyond 24 hours. We hypothesized that CH produces an early transient activation of astrocytes and microglia in the mouse brainstem as observed in rats. To study the activation of glial cells with CH, we exposed mice to normobaric hypoxia (10% FiO2) for 0.5, 1 and 4 hours and 1 and 7 days. Mice were perfused with 4% paraformaldehyde and brainstem sections were obtained. We quantified astrocyte activation by measuring glial fibrillary acidic protein (GFAP) intensity with immunofluorescence, and microglial activation by measuring branch morphology using Iba‐1 marker. Exposure to CH significantly increased GFAP intensity after 30 minutes and 1day while microglia morphology did not change significantly. The results differ from those in rats (J. Neurophysiol.117: 1625–1635, 2017) and suggest that time‐dependent astrocyte activation in the NTS may contribute to VAH.

Support or Funding Information

Supported by NIH RO1 HL‐081823
EFFECTS OF MODERATE ETHANOL CONSUMPTION ON EXPRESSION OF GENES RELATED TO COLONIC CELL GROWTH IN YOUNG ADULT AND OLDER RATS
Authors: Wells Nicole , Quigley Jacqueline , Pascua Jeremy , Pinkowski Natalie , Almaiman Lama , Brasser Susan M. , Hong Mee Young
Published: April 2020

Our previous research showed that moderate ethanol consumption reduced colonic cell proliferation regardless of age. To examine the underlying mechanism, this study examined gene expression related to colonic cell growth. Twenty‐four nondeprived young adult (109 days old) Wistar rats and twenty‐four nondeprived middle‐aged (420 days old) Wistar rats were randomly assigned to an ethanol‐exposed or a non ethanol‐exposed (water control) group (n = 12 per group). The ethanol group was provided voluntary access to a 20% v/v ethanol solution on alternate days for 13 weeks (45 ethanol drinking sessions total), a paradigm which results in mean blood alcohol levels of ~30–50 mg/dl (0.03–0.05 mg%) in the outbred Wistar strain when measured 30–120 min into a standard drinking session. The control group was provided with standard drinking water during the same period. Gene expression of cyclin D1, cyclin dependent kinase 2 (CDK2), cyclin dependent kinase 4 (CDK4), p21, E‐cadherin and p53 were determined by real time polymerase chain reaction in colonic scraped mucosa. CDK2 was downregulated in ethanol‐fed rats compared to the control group (P = 0.017). Cyclin D1 trended toward lower expression (P = 0.095), and p21 trended toward higher expression (P = 0.095) in ethanol‐fed rats compared to controls, with no observed effect of age for either gene. Older rats showed significant decreases in CDK4 (P = 0.001), E cadherin (P = 0.013), and p53 (P = 0.005), with no observed effect of ethanol exposure. These results indicate that moderate ethanol consumption may improve expression of some genes related to important parameters of colonic cell growth and tumorigenesis.

Support or Funding Information

NIH AA023291
MICRORNA‐21 EXPRESSION IN DIAGNOSIS OF PANCREATIC ADENOCARCINOMA.
Authors: DE ASSIS CIRINO MUCIO LUIZ , FADEL PATRICIA CICCILIATO , NOVAIS PAULO CEZAR , DA CUNHA TIRAPELLI DANIELA PRETTI , KEMP RAFAEL , DOS SANTOS JOSE SEBASTIAO , PERIA FERNANDA MARIS
Published: April 2020

Introduction

The differential diagnosis between pancreatic adenocarcinoma (PA) and chronic pancreatitis is still a challenge and the use of biomarkers association can mitigate these difficulties.

Objectives

To evaluate the effect of combining miRNA‐21 and serum CA 19‐9 in the diagnosis of PA.

Patients and Methods

We studied 23 PA samples and 20 samples as controls: 13 from normal pancreatic tissue adjacent to the tumor (TPR) collected during pancreatic resections and 7 from normal pancreas (NP) from cadaveric donors or patients undergoing pancreatectomy for trauma. Serum CA 19‐9 was measured and miRNA‐21 expression were verified in PA, NP and TPR by quantitative real‐time polymerase chain reaction (PCR) amplification. The usefulness of miR‐21 in the diagnosis of PA, isolated or associated with CA 19‐9 were analyzed by constructing ROC curves.

Results

There was a significant difference in miR‐21 expression in the comparison between NP and PA groups (p <0.05). However, there was no significant difference in miRNA‐21 expression between NP and TPR groups. The combination of miRNA‐21 with CA 19‐9 was more effective in discriminating PA from NP (AUC‐ROC: = 0.94; sensitivity: 78%; specificity: 99%) when compared with isolated evaluations.

Conclusion

The expression of miRNA‐21 was effective in discriminating between PA and NP and its association with serum CA 19‐9 increased the sensitivity and accuracy in diagnosing PA in the sample studied.

Support or Funding Information

This study was financed in part by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior ‐ Brasil (CAPES) ‐ Finance Code 001.
Inflammation Biomarkers in Older Adult Rats Consuming Moderate Levels of Ethanol
Authors: Morales Lani , Quigley Jacqueline , Pascua Jeremy , Pinkowski Natalie , Almaiman Lama , Brasser Susan M. , Hong Mee Young
Published: April 2020

Previous epidemiological studies and experimental data from rodent models have reported a non‐linear relationship between consumption of alcohol and cardiovascular disease risk and colorectal health. These studies indicate that light‐to‐moderate alcohol consumption, in contrast to excessive alcohol use, may provide cardiovascular health benefits as well as a protective effects on risk for colorectal cancer. Although prior studies have examined the effects of moderate ethanol consumption primarily in young adult animals, it is unknown if the results can be extrapolated to older adults or if these effects may be more pronounced in aging animals due to declining indices of health.. This study examined the effects of moderate ethanol consumption on lipid profile and colonic gene expression in older rats. Twenty‐four non deprived middle aged (420 days old) Wistar rats (n=12/group) were allowed to voluntarily consume a 20% v/v ethanol solution on alternate days for 13 weeks or were given access to water as their sole source of fluid (non‐ethanol exposed control). Moderate ethanol consumption significantly increased HDL cholesterol levels (P = 0.016) and ApoA1 (P = 0.048) compared to control. There were no differences in serum triglyceride, total cholesterol, LDL cholesterol, oxidized LDL and glucose between the groups. Blood C‐reactive protein (CRP) was decreased significantly (P = 0.034) in ethanol‐exposed animals. Blood high‐mobility group protein 1 (HMGB1) showed a decreased trend in the ethanol group (P = 0.083). In colon, gene expression of aldehyde dehydrogenase (Aldh) displayed an increased trend in ethanol‐exposed rats (P = 0.073). Colonic superoxide dismutase (Sod) was upregulated with ethanol intervention (P = 0.050). Colonic gene expression of cyclooxygenase 2 (Cox‐2), RelA, b‐catenin, CD68, and Pparr were not different between the groups. These results suggest that moderate consumption of ethanol in older rats may potentially contribute to improved cardiovascular and colorectal cancer risk by improving blood cholesterol, decreasing blood markers of inflammation in and increasing antioxidant enzyme expression in colon.

Support or Funding Information

NIH AA023291
Effects of the Astrocyte Endozepine Octadecaneuropeptide (ODN) on Hypoglycemia Patterns of Ventromedial Hypothalamic Nucleus (VMN) Metabolic‐Sensory Neuron Transmitter Signaling and Energy Sensor Activation in Male and Female Rats
Authors: Mahmood ASM Hasan , Uddin Md. Main , Ali Md. Haider , Napit Prabhat Ranjan , Briski Karen P.
Published: April 2020

The astrocyte gliotransmitter octadecaneuropeptide (ODN) is a cleavage product of diazepam‐binding inhibitor (DBI) and an endogenous central benzodiazepine receptor ligand. ODN regulation of feeding responses to excess or diminished glucose availability in the brain infers ability to modulate neural metabolic‐sensory function [Lanfray et al., 2013]. The ventromedial hypothalamic nucleus (VMN), a principal component of the neural glucostatic network, integrates nutrient and other cues to shape glucose counter‐regulation. Our studies show that VMN gluco‐stimulatory nitric oxide (NO) and gluco‐inhibitory g‐aminobutyric acid (GABA) neurons express the ultra‐sensitive energy sensor 5′‐AMP‐activated protein kinase (AMPK). This research examined the premise that ODN exerts sex‐specific effects on hypoglycemic patterns of VMN nitrergic and/or GABAergic nerve cell sensor activation and neurotransmitter signaling. Testes‐intact male and ovariectomized, estradiol‐replaced female rats were pretreated by intracerebroventricular administration of the ODN octapeptide (OP) fragment or vehicle prior to insulin injection. VMN NO and GABA cells were identified by immunocytochemistry prior to combinatory laser‐catapult dissection/high‐sensitivity Western blotting. Results identified nitrergic cells exhibiting OP‐reversible effects of hypoglycemia on expression profiles of the NO marker protein neuronal nitric oxide synthase (nNOS), AMPK, and phospho (activated)‐AMPK in distinctive regions of the male and female VMN. In each sex, OP effects on GABA neuron AMPK protein content and activation were also observed at specific levels of the VMN. Outcomes provide novel evidence that ODN may critically impact VMN substrate fuel screening. Further studies are needed to characterize neuroanatomical connectivity of OP‐sensitive nitrergic and GABAergic neurons within central metabolic pathways, and to examine whether metabolic cues that regulate VMN astrocyte ODN release, astrocyte reactivity to ODN‐controlling signals, and metabolic‐sensory nerve cell receptivity to ODN are sex‐dimorphic.

Support or Funding Information

NIH DK 109382
Genome‐Wide Methylation in Patterns in Idiopathic Pulmonary Arterial Hypertension
Authors: Gunn Brady Luke , Memar Shayan , Markand Shanu , Abeyrathna Prasanna
Published: April 2020

Objective

Epigenomic regulation is important in the pathology of many diseases. Evidence suggests epigenetic modification of individual genes in idiopathic pulmonary arterial hypertension (IPAH), and understanding these epigenomic mechanisms involved in vascular remodeling in IPAH may be beneficial. The objective of the current study was to assess the genome‐wide methylation of IPAH lungs. We hypothesized that genome‐wide methylation would have an essential role in vascular remodeling in IPAH.

Methods

Lung tissues from 8 age‐matched and sex‐matched IPAH lungs and 8 healthy lungs were analyzed in the current study. Genome‐wide methylation of cytosine was assessed using whole genome bisulfite sequencing. Gene functional pathway analysis was performed on identified differentially methylated genes.

Results

Based on bioinformatics analysis of the methylation sequences, differentially methylated genes were identified in IPAH lungs. Further, there were distinct methylation patterns in the pathophysiology of the IPAH lungs.

Conclusion

Identification of epigenetically regulated pathways in IPAH may offer novel therapeutic opportunities for IPAH.

Support or Funding Information

ATSU Start Up Fund
Use of a spectrophotometric assay to evaluate amino acid activation and tRNA charging
Authors: Mobisa Japheth , Samuel Kyra , Okeke Idiuso , Castaneda Jacquelyn , Trinh Thanh , Enewali Chinazom , Bell Anthony Jerome
Published: April 2020

Conventional assays that investigate amino acid activation and tRNA‐charging rely on using radiolabeled amino acids (14C) and tRNAs (32P) to detect the presence of activated amino acid adenylate(s) and acylated tRNAs. There are also several assays that monitor activation and acylation products by using (32P) radiolabeled ATP and inorganic pyrophosphate. The radioisotope based assays offer unmatched sensitivity. However, the experiments require the use of hazardous reagents and often laborious protocols. More recently a colorimetric based assay has been developed to detect the level of inorganic free phosphate in aqueous solution. The assay is based on the complexation of malachite green molybdate and inorganic phosphate – the complex absorbs at 620 nm. The assay, referred to as the malachite green assay, has been modified to measure amino acid activation and tRNA charging. Our short‐term goal is to use the malachite green assay to measure the ability of aminoacyl‐tRNA syntheses (aaRSs) to activate and charge analog amino acids (AAs). Analog or “unnatural” AAs are non‐proteinogenic residues beyond the set of natural 20 L‐AAs. Bacteria and fungi use large multi‐subunit enzyme complexes, nonribosomal peptide synthetases (NRPs), to generate peptides composed of analog AAs. The resulting analog peptides typically possess enhanced bioactivity. Analog peptide bioactivity is generally linked with enhanced: i)membrane permeability, ii)protease stability and iii)chemical complexity. These advantages make analog peptide ideal for biological therapeutics. Our long‐term goal is to use the cell free expression system – the PURE system (
Protein synthesis
Using
Recombinant
Elements) to synthesize analog peptides. The resulting peptides will be directed against protein‐protein interactions known to exacerbate disease. Our initial studies focus on using the malachite green assay to screen analog amino acids for activation and tRNA charging using purified aaRS and tRNA (total tRNA and single isoacceptors). Analogs identified via screening will be assembled into a library that will be used to generate analog peptides.

Support or Funding Information

University of San Diego
Activation of Bradykinin‐Sensitive Pericardial Afferents Increases Splanchnic Nerve Activity
Authors: Martin Doug , Liu Xuefei , Zheng Hong
Published: April 2020

Sympathetic drive plays a key role in control of cardiovascular function. The heart performs an important sensory function via a variety of cardiac receptors that modulate sympathetic drive. Bradykinin‐sensitive sympathetic afferent nerves from the heart can trigger marked increases in efferent sympathetic nerve activity to some cardiovascular targets such as the kidney. We reported previously that pericardial injection of bradykinin increased venous tone and cardiac output. The splanchnic vascular compartment plays an important role in control of venous return and cardiac output. This work tested the hypothesis that activation of bradykinin‐sensitive pericardial afferents increases postganglionic splanchnic sympathetic nerve activity.

Sprague Dawley rats were anesthetized with a cocktail of alpha chloralose (80 mg/kg) and urethane (800 mg/kg). Catheters were placed in the femoral artery and vein to record arterial pressure (MAP) and heart rate (HR). A PE10 catheter was placed in the pericardial space for the injection of bradykinin (BK), an agent known to stimulate cardiac sympathetic afferents. The chest was closed and the rat allowed to breathe spontaneously. The splanchnic sympathetic nerve was isolated via a dorsal approach and placed on recording electrodes. After stabilization, the blood pressure, heart rate, and integrated splanchnic nerve activity (SPLNA) responses to pericardial injection of BK or vehicle were recorded. Responses were also recorded for intravenous injection of dimethylphenylpiperazinium (DMPP 150 ug/kg), a ganglionic stimulant, and a ganglion blocker (hexamethonium 20 mg/kg).

Pericardial injection of saline vehicle had little effect (MAP: 0.1±0.4 mm Hg; HR: 1.2±1.4 bpm; SPLNA 1±2%). In contrast, intravenous injection of a ganglionic stimulant, DMPP, increased MAP (23±1 mm Hg) and SPLNA (95±46%). Pericardial injection of BK (10 ug/kg) increased MAP (21±3 mm Hg), HR (28±7 bpm) and SPLNA (93±37%). Ganglionic blockade with hexamethonium reduced SPLNA (−75±6%) and abolished the responses to pericardial injection of BK (MAP: −1±3 mm Hg; HR: −2±1 bpm; SPLNA: 7±30 %).

We interpret these data to indicate that activation of bradykinin‐sensitive pericardial afferents increases postganglionic splanchnic sympathetic nerve activity. This finding is consistent with the view that activation of the cardiac sympathetic afferent reflex elicits a venoconstrictor response to increase venous return from the splanchnic vascular compartment.

Support or Funding Information

Supported by NIH R01 HL136741‐03 (DM), R01 DK114663‐1 (HZ), and the Basic Biomedical Sciences program of the Sanford School of Medicine, University of South Dakota.
Wheat Germ Acid Phosphatase Activity in High Percentages of Organic Solvents
Authors: Moorman Veronica R. , Kindl Evan D. , Summers Fiona N.
Published: April 2020

While enzymes canonically operate in aqueous environments, several have been shown to also function in non‐aqueous solvents and/or solvent mixes. Phosphatases, for example, have been previously shown to hydrolyze phosphates in mixtures containing modest amounts of certain organic solvents. Here, detecting the fluorescent dephosphorylation product naphthol AS‐MX with fluorescence spectroscopy (ex: 388 nm, em: 512 nm) has shown commercial wheat germ acid phosphatase to be active in high amounts (up to ~70% by volume) of 1,4‐dioxane, 1,2‐ dimethoxyethane, 2‐methoxyethanol, dimethyl sulfoxide, and acetonitrile when the aqueous component was comprised of Tris buffer (pH 7). These solvent mixtures were also physically characterized to better understand differences in both relative naphthol AS‐MX fluorescence and wheat germ acid phosphatase activity across the different solvent mixtures investigated. These results suggest that phosphatase activity can be detected with less water that previously published.

Support or Funding Information

Acknowledgment is made to the Donors of the American Chemical Society Petroleum Research Fund and to the National Science Foundation’s Research Experience for Undergraduates program for partial support of this research.
Relationship Between Team Dynamics and Performance in Solo‐Then‐Collaborative (Hybrid) High‐Stakes Testing in Pre‐Clerkship Medical Students
Authors: Yarbrough Tracy Lynnette , Ely Susan
Published: April 2020

Objectives

To characterize student perceptions of team dynamics and the relationship between team dynamics and performance within a novel hybrid exam platform.

Methods

Medical students enrolled in pre‐clerkship year 1 at CNUCOM during academic year 2017–2018 were recruited for participation in this study. Data was collected at two timepoints— at the mid‐course exam in the final course of year 1 and at the mid‐course exam in the initial course of year 2. All participants completed a 100‐item selected‐response exam individually and then as part of a pre‐formed student team of 8–9 members. Following the team exam, students completed a 15‐item survey reflecting on their team interactions. In addition, faculty proctors of the team exam during course 1 completed a 15‐item survey reflecting on their observations of team dynamics. Finally, student performance (both individual and team exam scores) were collected for all participants. Team dynamics scores were calculated based on responses to survey questions regarding team interactions during the exam as identified by students and proctors (for course 1). This human subjects protocol was reviewed and approved by the CNUCOM Institutional Review Board.

Results

95 and 94 students completed the first and second hybrid exam surveys, respectively. Average individual scores (+/− SD) on the first and second exams were 69 (+/11) and 80 (+/− 10), and average team scores on the first and second exams were 92 (+/−3) and 99 (+/−1). At the first exam, student‐derived team dynamics score was highly correlated with team exam score, while team member average individual exam score did not strongly correlate with team exam score. {It is noted that teams for the first exam were formed 8 months prior to the exam and had been used extensively in other team‐based learning sessions.} Conversely, teams utilized in the second exam had been formed less than 1 month prior to the exam and had been functioning as a team for a limited number of sessions prior to the exam—on the second exam, team dynamics score was not well‐correlated with team exam score, and instead team member average individual exam score correlated more strongly with team score. Proctor’s assessment of team dynamics correlated moderately well with students’ assessment of team dynamics in course 1 (r = 0.24).

Conclusions

Our data suggest that team dynamics play a role in team performance in hybrid testing utilized for high‐stakes exams among undergraduate medical students. This relationship may depend more on student comfort and familiarity within a team than on the individual knowledge and specific contributions of team members. More investigation on long‐term knowledge retention as related to dynamics within teams is desirable to further characterize outcomes utilizing this novel testing format.
The Effect of Long Distance Running on the Pattern of Production of Salivary Cortisol on Rest Days
Authors: Hill Gabrielle Marie , Baumbach Jordan , Barry Alla
Published: April 2020

INTRODUCTION

Cortisol is a glucocorticoid synthesized from cholesterol by the zona fasciculate of the adrenal cortex and is controlled by way of a negative feedback pathway within the hypothalamic‐pituitary‐adrenal (HPA) system. HPA axis receives strong circadian input and plays an essential role in the regulation of stress responses. Cortisol spikes in the morning upon waking up, and then decreases throughout the day, but exercise modifies this pattern of production. Physical exercise activates HPA axis to maintain proper blood glucose levels during these times of energy expenditure. According to previous studies endurance‐trained athletes possess a significantly higher level of circulating cortisol than power trained athletes immediately after exercise. Cortisol is known to have debilitating effects on factors related to bone mineral density (BMD). Consequently, the chronic increase in cortisol in long‐distance runners can lead to adverse effects such as decreased BMD and can contribute to stress fractures. The purpose of this study is to evaluate cortisol levels during days of rest to determine the possible change in the pattern of cortisol production stimulated by repetitive prolonged previous exposure to the physical activity rather than actual stimulus.

METHODS

Missouri Southern State University (MSSU) Institutional Review Board approval was gained before data collection. A participant group was gathered including 12 males from the men’s cross‐country team at MSSU in the fall of 2019. Participants completed a demographic questionnaire stating the number of hours per week actively participating in sport specified workouts, average hours of sleep per night, and a list of supplements being consumed. Participants gave saliva samples within pre‐determined time intervals before, during, and after their workouts as well as during the same times during days of rest. Samples were collected on two training days and one rest day a week on three separate weeks for a total of 27 samples per athlete. In addition to saliva samples, a stress assessment was filled out by every athlete each day of collection. All samples were placed in a freezer at 0 degrees Celsius until collections were completed and analyzed. Samples were evaluated via ELISA by a competitive immunoenzymatic colorimetric method for the quantitative determination of cortisol concentration. ELISA absorptions were analyzed with linear regression to calculate cortisol concentrations.

CONCLUSION

The result of this study will assist in a better understanding of the hypothalamic‐pituitary‐adrenal axis function. Furthermore, while allowing the endurance‐trained athlete to efficiently and properly recover, it will aid in reducing the risk of injury and overtraining syndrome.
Suppression of Organismal Death Pathway in the Heart Tissues of Diabetic Mice by NP‐ 6A4, A Peptide Agonist of the Angiotensin II Receptor AT2R
Authors: Pulakat Lakshmi , Belenchia Anthony , Mooney Brian , DeMarco Vincent G.
Published: April 2020

Background

Diabetes accelerates cardiac dysfunction and aging. Angiotensin II type 2 receptor AT2R is a member of the anti‐inflammatory arm of the renin‐angiotensin‐aldosterone (RAAS) system. NP‐6A4 is an AT2R peptide agonist with the FDA orphan drug designation for pediatric cardiomyopathy. To test the hypothesis that NP‐6A4‐AT2R signaling is cardioprotective in chronic diabetes, we investigated the changes in cardiac proteome of 63‐week old diabetic mice after a 55‐week treatment with NP‐6A4.

Methods and Results

All studies were conducted in accordance with the FASEB Statement of Principles for the use of Animals in Research and Education. Diabetes was induced in 8‐week old male c57Bl6 mice via intraperitoneal delivery of streptozotocin (STZ: 50mg/kg/day: 5 days; n=11). Fasting insulin levels were decreased in the STZ‐treated mice (~0.2±0.03 ng/mL versus ~0.94±0.08ng/mL in control mice; p<0.05), but fasting glucose was increased (313±29mg/dL p<0.05). STZ‐treated mice also showed a reduction in body weight by ~10–14% compared to control mice (p<0.05). NP‐6A4 treatment (5mg/kg/day; n=5) of STZ‐treated mice did not change these metabolic parameters. Whole proteome analysis of the cardiac tissues of these 63‐week old mice identified a total of 2760 protein groups in 15 samples with the following criteria: >=2 unique peptides per protein, peptide spectrum FDR <1%. For quantification, a threshold minimum of 4 spectral counts (average of each group of replicates) was used. This resulted in a quantified proteome of 1,569 proteins. NP‐6A4 treated mice hearts exhibited 109 significant differences (p<0.01) compared to vehicle treated diabetic mice hearts with 100 proteins showing increased and 9 proteins showing decreased abundance. Analysis of these data using Ingenuity Pathway Analysis (IPA) showed that 32 of deferentially expressed proteins in diabetic mouse heart induced a pathway designated ‘suppression of organismal death’ (Z score −4.706). Comparing NP‐6A4‐treated mice hearts to healthy mice hearts resulted in 311 significant differences (p<0.01), with 253 proteins increased and 58 proteins decreased. Moreover, 82 of these differentially expressed proteins in the NP‐6A4‐treated mice hearts, compared to healthy control mice hearts, also induced the pathway ‘suppression of organismal death’ (Z score‐3.415).

Conclusion

A 55‐week treatment with NP‐6A4 induced a cardioprotective pathway in diabetic mouse heart that has the potential to suppress organismal death when compared to age‐matched healthy mice or vehicle treated diabetic mice. This observation suggests that continuous treatment with NP‐6A4 in chronic diseases such as diabetes has the potential to confer cardiac protection and prevent organ failure.

Support or Funding Information

This work was supported by NIH NHLBI 1R01HL118376‐01 (LP), and NIH NHLBI 1R01HL138988‐01A1 (LP) and a small grant from Novopyxis Inc. (LP).
Vagal afferent nerve ghrelin signaling influences energy balance and episodic memory
Authors: Davis EA , Suarez AN , Liu CM , Cortella AM , de Lartigue G , Kanoski SE
Published: April 2020

Vagal afferent nerve (VAN) signaling sends sensory information from the gut to the brain and participates in the regulation of meal size and blood glucose control. Recent findings reveal that VAN signaling also plays a critical role in cognitive processes, including hippocampus(HPC)‐ dependent memory function. The nodose ganglia (VAN neurons) express receptors for various gut‐derived endocrine signals, however, the function of these receptors with regards to feeding behavior, glucose regulation, and HPC‐dependent memory is poorly understood. We hypothesized that these VAN‐mediated behavioral and physiological processes are influenced by ghrelin, a stomach‐derived orexigenic hormone, via communication to its receptor (growth hormone secretagogue receptor [GHSR]) expressed on gut‐innervating VAN terminals. To examine this hypothesis, adult male rats received bilateral nodose ganglion injections of an adeno‐associated virus (AAV) expressing short hairpin RNAs targeting GHSR (or a scrambled control AAV) for RNA interference‐mediated VAN GHSR knockdown. Meal‐pattern analyses revealed a significant increase in meal frequency in the VAN GHSR knockdown group compared with controls, coupled with a nonsignificant trend in reduced average meal size such that cumulative 24h food intake did not differ between groups. Results from an intraperitoneal glucose tolerance test revealed impaired glucose tolerance in VAN GHSR knockdown animals vs. controls. Despite no differences in cumulative 24h food intake, the VAN GHSR knockdown group also had significantly increased body weight gain compared with controls. Lastly, to examine the role of VAN GHSR signaling in HPC‐dependent memory function, the rats were tested in novel object in context task that measures contextual episodic memory. Results reveal that VAN GHSR knockdown impairs memory performance in this task, yet has no effect on measures of anxiety‐like behavior or levels of general activity. Collectively, these results reveal that VAN GHSR signaling is important for the control of both energy balance as well as HPCdependent memory function.
Unbiased topological parameters to characterize the signatures of GPCR conformational changes
Authors: Khleif Rafeed , Serrano Erik , Abrol Ravinder
Published: April 2020

G protein coupled receptors (GPCRs) are integral membrane proteins that allow a cell to convert extracellular stimuli like light, small molecules, peptides, and proteins into intracellular signals. These receptors are conformationally very malleable, which is necessary for their different functional states. Recent developments in the structural biology of membrane proteins and utilization of other biophysical techniques for GPCRs is generating rich evidence of the existence of multiple receptor conformations. Different receptor conformations are thought to be responsible for enabling distinct intracellular signaling outcomes. The structures are now being generated for the inactive, active, and intermediate states for many GPCRs, however, there is no unbiased way to characterize the functional states of GPCRs. We are developing general higher‐order GPCR topology parameters (TMHTOP2) that can find functional‐state specific structural signatures and can be applied to any alpha‐helical transmembrane protein. The newly developed TMHTOP2 topology parameters were able to identify unique conformational signatures of the pre‐active state of the adenosine A2A receptor compared to its inactive and active states, that can now be used to identify the pre‐active states of other receptors as well. This method can be used in identifying and classifying structures from experiments or simulations as active, inactive, and intermediates.
Day and Night Comparison to Maximize Learning in Sprague Dawley Rats (Rattus norvegicus)
Authors: Bagoyo Editha Angela , Drew Kelly , Jenkins Mackenzie
Published: April 2020

Domesticated mice and laboratory rats have been traditionally viewed as nocturnal creatures, the vast majority of their daily activity occurring during the “dark” phase of their circadian cycle. Behavioral studies of rats have been traditionally performed during their “light” period when rats should be sleeping. The purpose of this study is to examine whether learning using a spatial, novel object recognition paradigm is enhanced if rats are trained and tested during the dark phase of their cycle rather than the light phase. 10 Sprague Dawley rats were placed in an open field apparatus and observed for 6 minutes at a time during their “light” phase. Measurements were made of distance traveled within the open field, speed of travel, time spent within different areas of the field, time spent mobile and immobile, time spent on grooming behavior, and time spent “rearing” (standing upright). Learning was assessed by observing differences in time spent with a novel toy and a familiar toy. All tests were repeated 1 week later during the rats’ “dark” phase, and results for the two phases were compared using a t‐test. Results showed no evidence of learning in either the light or dark phases, indicated by an increased time spent interacting with the non‐novel object compared to the novel object. Locomotor activity, total time spent interacting with both objects, and rearing behavior were significantly increased during the dark phase, while total distance traveled was significantly lower, which is likely related to the increased rearing. Though these results do not indicate enhanced learning in the rats during the dark phase, the observed differences in activity and behaviors compared with the light phase warrant further study as they could have implications for other behavior experiments performed on rats during each phase.

Support or Funding Information

Research reported in this publication was supported by the NIH Common Fund, through the Office of Strategic Coordination, Office of the NIH Director with the linked awards: TL4GM118992, RL5GM118990, UL1GM118991. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. UAF is an affirmative action/equal employment opportunity employer and educational institution:
www.alaska.edu/nondiscrimination.
Adaptation of enhanced crosslinking and immunoprecipitation (eCLIP) for the high‐throughput, high‐resolution mapping of N6‐methyladenosine modifications
Authors: Rabano Ines , Shishkin Alexander A. , Shen Kylie , Foster Heather M. , Chu Peter , Van Nostrand Eric L. , Yeo Gene W.
Published: April 2020

N6‐methyladenosine (m6A) is the most prevalent epitranscriptomic RNA modification found in eukaryotes and has been shown to regulate many stages of RNA biology including splicing, secondary/tertiary structure, localization, stability, and translation. Adenosine modifications are actively added, recognized, and removed by a series of “writer”, “reader”, and “eraser” proteins, making m6A a dynamic and reversible system for regulating RNAs. Further, there are now clear links between m6A writer, reader, and eraser proteins as well as m6A state of specific mRNAs to cancer pathogenesis, indicating that both global and specific dysregulation of m6A can have targeted effects that lead to disease. Thus, there is a need for methods to robustly profile m6A‐modification states in both cell lines as well as tissues, and in normal as well as diseased or modified states. Advancements in high‐throughput sequencing have led to the development of methods to map m6A transcriptome‐wide, but there remains a need for improved methods that are scalable across conditions and treatments and with low abundance samples, such as blood or patient tissue. To address this need, we adapted enhanced crosslinking and immunoprecipitation (eCLIP) to create an optimized protocol for profiling m6A modification sites with 20–50‐fold less starting RNA material (m6A‐eCLIP). Using m6A‐eCLIP, we recover traditional m6A motif enrichment and positioning enrichment at stop codons, validating the accuracy of the approach. Further, the improved handling and efficiency enabled us to profile m6A status across cell types and tissues, showing that m6A‐eCLIP can be a robust method that is widely applicable for epitranscriptomic profiling across a range of conditions.
Oxygen‐Directed Laboratory Evolution and Conserved Mechanisms Underlying Hypoxia Tolerance
Authors: Zhou Dan , Iranmehr Arya , Zhao Helen , Stobdan Tsering , Bafna Vineet , Haddad Gabriel G.
Published: April 2020

Oxygen is essential for metazoan life on earth. In this work, we have taken advantage of Drosophila melanogaster as a genetic model system to dissect mechanisms of genomic adaptation to extreme oxygen conditions. We conducted a ~15‐year laboratory evolution experiment over >290 generations by chronically exposing multiple fly populations to normal, decreasing, or increasing O2 levels to determine the effect of selection pressure on their genomes, and to understand the mechanisms of adaptation to O2 imbalance. We have obtained Drosophila melanogaster populations and strains that can tolerate and perpetually survive environments containing extremely low (<4%) or high (>90%) level of O2, which are lethal for naïve controls. A time series of whole genome sequencing (WGS) analysis of multiple generations of the Low (L‐O2) and high (H‐O2) populations and normoxia control (N‐populations) demonstrated clear genomic changes along evolution between environments and generations. Furthermore, four genomic regions in the H‐O2 populations and five genomic regions in the L‐O2 populations were identified, which contain selection sweeps generated by selections with low or high oxygen condition. A total of 433 candidate genes and 215 candidate genes were identified in the selection sweeps in the L‐O2 populations and H‐O2 populations, respectively. This suggested a number of evolutionarily conserved mechanisms (e.g., the Notch signaling pathway) that may play a role in regulating adaptation to the extreme oxygen conditions. We believe that these mechanisms have a strong potential to be translated into humans and serve as novel targets for developing therapeutic strategies to prevent and treat hypoxia‐related diseases and oxidative stress‐induced injuries.
The Effects of Christianity on Whole Body Donation within the African American Community
Authors: Triska Jeffrey Scott , Barremkala Malli , Farr A. Celeste
Published: April 2020

INTRODUCTION

The goal of this project is to understand if there is a relationship between Christian perspectives relative to the practice of whole body donation. Previous studies have pointed to a racial disparity among the rates of whole body donation between Caucasians and African Americans (AA). As a majority of AAs identify as Christians, there may be a connection between the religious beliefs and perspectives of body donation among individuals of the AA community. According to the Pew Research Center, 87% of this racial group claims a religious affiliation; 53% said they attend church at least once per week.

METHODS

AA adults were recruited from two majority AA Christian churches in Southeastern Michigan. Focus groups were held with 5–7 participants each. Five focus groups were conducted with a total of 26 participants between the ages of 21–80 (avg 48, median 51). Prior to the focus groups, participants answered a short survey on demographics and social characteristics including questions about how large of a role religion plays in their lives and how often they attend church. The focus groups were conducted for 1.5‐hours in a conversational manner to elucidate the participants’ thoughts, feelings, and perspectives on whole‐body donation.

RESULTS

The majority of participants conflated the whole‐body and organ donation processes. They were generally unknowledgeable about whole‐body donation, but were supportive of the idea, stating that it was “beneficial for advancement and science knowledge,” and they “commend those who donate their body.” Several were knowledgeable about organ donation and said they were open to that option, but others expressed fears and concerns that healthcare professionals would allow people to die rather than provide life‐saving treatment if they knew they were a body donor. Christianity seemed to mostly be an encouraging factor for donating with participants stating “Scripture says we will get a new body, we are spiritual beings,” and “It’s [my body] on loan from him. It is being loaned to me.” Others seemed to be opposed to the process due to the necessity that the donor bodies be cremated after use. Most agreed that whole‐body donations are not religious convictions, but rather a matter of personal preference.

CONCLUSION

Overall, the largest barriers to body donation seemed to be lack of knowledge and trust of the medical establishment. Christianity seems to mostly be an encouraging factor for donation. There should be more educational campaigns, particularly in AA churches to spread awareness of the process, methods, and benefits of whole body donation and its difference from organ donation. There needs to be more research done on this topic and a comparison to a group of non‐religious AAs to see if these effects hold.
Reproducibility of Whole‐Body Cooling Response Using Laser Doppler Flowmetry
Authors: Schwartz Kelsey S. , Shirazi Behnia R. , Lang James A.
Published: April 2020

Laser Doppler Flowmetry (LDF) has been used as a non‐invasive tool to measure cutaneous microvascular function. While previous studies have assessed reproducibility with perturbations that increase skin blood flow, it is unclear as to the reproducibility of the whole body cooling response. We hypothesized that the reflex vasoconstriction response to whole‐body cooling would be reproducible. Eight healthy, young adults participated in two cooling bouts, each separated by 48 hours to one week. Participants donned a whole‐body suit and were cooled from a baseline skin temperature (Tsk) of 34°C to 30.5°C over a 30 min period. The final Tsk was maintained for an additional ten minutes. Mean Tsk was calculated as the unweighted average temperature of the calf, thigh, abdomen, upper chest, back, and upper arm. LDF flux was measured at the ventral forearm and cutaneous vascular conductance (CVC) was calculated at each 0.5°C reduction in skin temperature, CVC = flux/MAP and expressed as percent changes from baseline (%ΔCVC). The coefficient of variation (CV) for each 0.5°C reduction in skin temperature were determined (33.5°C = 44.3, 33.0°C = 27.9). The data indicate poor reproducibility at the onset of cooling. Reproducibility steadily improved with lower skin temperature ranging from moderate at 32.5°C to 30.5°C to good at the cooling plateau. The data suggest that skin blood flow measurements collected at colder skin temperatures are reproducible and may be used as a longitudinal indicator of microvasculature function.

Support or Funding Information

This study received funding from the Wilderness Medical Society Hackett‐Auerbach Grant, supported by the Academy of Wilderness Medicine(r).
Disrupting the protein signaling activity of MAPK pathway as target for metastatic colorectal cancer therapy
Authors: Nkembo Augustine T. , Lamango Nazarius S. , Jin Yonghoa , Tawfeeq Nada , Mensah-Mamfo Justin
Published: April 2020

Despite recent advances including the use colonoscopy for early detection of colorectal cancer (CRC), the 2019 incidence and death rates projection for the disease in the U S were still alarming, as they stood at 145,600 and 51,020, respectively. Patients with the metastatic colorectal cancer (mCRC) subtype remain without hope as they do not respond or quickly develop resistance to the current available therapeutics. Over 50% of patients with CRC harbor constitutively active K‐Ras and 10% with B‐Raf mutations. Men of African American descent are more likely to be diagnosed with mCRC and will equally die of it compare to other races. Also, the most aggressive and deadly subtype with constitutively active mutant B‐Raf is commonly diagnosed in women and patients of more advanced age. Therefore, investigating novel targeted therapy to inhibit and disrupt the activities of the driver mCRC remains a matter of urgency. Recent tests for biological activities of novel agents, polyisoprenylated cysteinyl amide inhibitor (PCAIs) designed and synthesized to target the hyperactivity of aberrant monomeric G‐Proteins such as Ras and Rho protein family show promising results for continued development as a drug molecule. We hypothesized that the PCAIs would target and suppress the hyperactivity of aberrant Mutant K‐Ras and/or B‐Raf function that drive the aggressive mCRC subtype. To test this hypothesis, we screened for the potency of PCAIs on inhibiting the cell viability of 2D and 3D cultures of different CRC cells including Caco‐2, HCT116 and HT‐ 29 cells. We also evaluated the mechanism by which they induce cancer cell death. Our results indicate that the PCAIs NSL‐BA‐040 and NSL‐BA‐055 inhibited 2D Caco‐2 cells cultured at very low concentrations of 3.8 and 1.4, respectively, and 2.2 and 1.5 μM, respectively for HCT116 cells. For the 3D cultures, NSL‐BA‐040 and NSL‐BA‐055 induced cell death of spheroids at EC50 of 20 and 5.5 μM, respectively. NSL‐BA‐055 induced cell death by apoptosis, blocks spheroid growth and shrinks their volumes. The effects of the PCAIs on HT‐29 2D and 3D cell cultures were even more dramatic. The PCAIs tested here demonstrate significant potency on CRC cells while sparing HEK293 cells. Since these PCAIs demonstrate a strong drug‐like property at this early stage of their development, they would be further tested, characterized and hopefully developed as drug for CRC therapy, targeting the drivers of the metastatic subtype.

Support or Funding Information

“Research reported in this publication was supported by the National Institute on Minority Health and Health Disparities of the National Institutes of Health under Award Number U54MD007582. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health”.
Resveratrol Prevents Hyperoxia‐Induced Lung Inflammation
Authors: Reçica Shkëlzen , Kryeziu Islam , Thaçi Qëndrim , Kurshumliu Fisnik , Mladenov Mitko , Hadzi-Petrushev Nikola , Sopi Ramadan B. , Basholli-Salihu Mimoza
Published: April 2020

Prolonged oxygen exposure of preterm infants results in the development of bronchopulmonary dysplasia (BPD), a chronic lung disease characterized with lung inflammation, remodeling and airway reactivity. Unfortunately, therapeutic options are limited. Resveratrol is known to have anti‐inflammatory and anti‐oxidant properties in different diseases, however it is not clear about its effects on BPD. In this study we investigated the effects of resveratrol in a rat pup model of BPD. We hypothesize that supplementation of rat pups with resveratrol prevents the lung inflammation induced by hyperoxia. At P5 rat pups were assigned either to room air (21% O2)‐ or hyperoxic (≥95% O2)‐groups and exposed for seven days. During this time, animals were supplemented daily with resveratrol (30 mg/kg of body weight; i.p.) or vehicle. At day P12 animals were euthanized and lungs were harvested, and processed for biochemical and immunohistochemistry studies. Pro‐inflammatory cytokines’ levels (IL‐1β and TNF‐α) were measured in whole lung homogenate using ELISA method. Immunostaining for T‐lymphocyte marker (CD3), B‐lymphocyte marker (CD20) and CD45 was performed on 5 μm lung sections. Levels of IL‐1β and TNF‐α in lung tissue of hyperoxic group of animals were increased significantly (p < 0.01) compared to room air group animals. Supplementation of hyperoxic animals prevented this increase of IL‐1β and TNF‐α, while did not change in room air animals. In hyperoxic animals there was an abundance of CD3, CD20 and CD45 stained cells in the lungs of rat pups exposed to hyperoxia compared to room air exposed pups. Supplementation of hyperoxic animals with resveratrol reduced the number of CD3, CD20 and CD45 stained cells. We conclude that resveratrol prevents lung inflammation induced by neonatal hyperoxia and we speculate that resveratrol might be an effective therapeutic approach to prevent the adverse effects of neonatal hyperoxia.

Support or Funding Information

Supported by MEST