bluuebunny/crossref_metadata_2025 huggingface dataset:
Cerebrovascular Reactivity in Young Adults with Major Depressive Disorder
Authors: Richey Rauchelle E. , Akins John D. , Campbell Jeremiah C. , Saunders Erika F.H. , Brothers R. Matthew , Greaney Jody L.
Published: April 2020

Cerebral vasodilatory responsiveness is blunted in older adults (~70 yrs) with depressive disorders and thought to contribute to the link between depressive symptomology and increased risk for neurocognitive (e.g., dementia) and cerebral vascular (e.g., stroke) diseases. In young adults with major depressive disorder (MDD), peripheral vascular endothelial dysfunction is evident; however, to date, limited investigations have examined cerebral vasodilatory function in young otherwise healthy adults with MDD. We tested the hypothesis that cerebral vasodilatory responsiveness to a hypercapnic stimulus would be blunted in otherwise healthy young adults with MDD compared to healthy non‐depressed adults (HA). Four HA (22±2yrs) and 6 adults with MDD (23±2yrs; n=2 tested during a major depressive episode) participated. Beat‐to‐beat mean arterial pressure (MAP; finger photoplethysmography), middle cerebral artery blood velocity (MCAv; transcranial Doppler ultrasound), internal carotid artery (ICA) diameter and blood flow (Doppler ultrasound), and end‐tidal carbon dioxide concentration (PETCO2; capnograph) were continuously measured during baseline (i.e., normocapnia) and rebreathing‐induced hypercapnia. Cerebral vascular conductance index (CVCi=MCAv•MAP−1) and ICA blood flow (Vmean•π(d•20−1)2•60) and conductance (CVC=ICA blood flow•MAP−1) were calculated at baseline and at the highest common magnitude of hypercapnia achieved by all subjects during rebreathing (ΔPETCO2 = 9 Torr). At baseline, there were no differences in any outcome variable between groups (all p>0.05). During hypercapnia, there were no group differences in the increase in MAP (Δ3±3 HA vs. Δ3±4 mmHg MDD; p=0.98). The increase in MCAv was blunted in adults with MDD (Δ33±4 HA vs. Δ23±7 cm•s−1 MDD; p=0.03). Both the absolute (Δ0.30±0.02 HA vs. Δ0.20±0.07 cm•s−1•mmHg−1 MDD; p=0.01) and relative (Δ42±7 HA vs. Δ26±9 %baseline MDD; p=0.01) increase in CVCi during hypercapnia were reduced in adults with MDD. There were no group differences in ICA diameter (Δ−0.04±0.22 HA vs. Δ0.05±0.17 mm MDD; p=0.51) or blood flow responses (Δ143±20 HA vs. Δ127±95 mL•min−1 MDD; p=0.49). Although the dilatory response in the ICA was ~50% less in adults with MDD, the increase in CVC was not significantly different between groups (Δ51±26 HA vs. Δ27±22 %baseline MDD; p=0.17). In adults with MDD, there was no relation between habitual physical activity and cerebral vasodilatory responsiveness (R2=0.09; p=0.57). These preliminary data suggest that cerebral vasodilatory reactivity to a hypercapnic stimulus may be blunted in young otherwise healthy adults with MDD, providing further support for the potential mechanistic link between cerebrovascular dysfunction and depression in increased neurocognitive and cerebral vascular disease risk.

Support or Funding Information

HL133414 (JLG)
Exercise Improved Cognitive Impairment in Aging Mice via Skeletal Irisin
Authors: Behera Jyotirmaya , Ison Jessica , Tyagi Neetu
Published: April 2020

The progressive decline of neurological learning and memory is rising as the lifespan increases in humans. The mechanistic basis of aging‐associated memory impairment is not still clearly understood. It is also currently unknown whether irisin is secreted by bone tissue after exercise or whether it regulates brain memory function via distant paracrine signaling in vivo. Here we show that age‐related suppression of irisin expression is linked with skeletal bone loss and memory disturbances in the aging brain. However, daily treadmill exercise (Ex) was found to restore the bone and memory loss in the aging mouse. To test the hypothesis, we employed young and aged male/female C57BJ/L6 wild‐type (WT), which were grouped as follows: Young (Yg, 3month);(ii) Yg+Ex(iii) Aged(Ag.18–22 month old); (iv) Ag+Ex. The selected mice were exercised for this 12‐week duration on a treadmill, with controlled speeds of 7 meters/min for the first week, 9 meters/min for the second week and 11 meters/min the following weeks for a total of 330 meters every day. The results show that treadmill exercise‐induced high levels of irisin mRNA transcript as well as protein expression in murine femoral bone tissues. This resulted in increased trabecular and cortical bone formation in aging mice (AG+EX) in vivo. On the other hand, AG+EX mice displayed an increased plasma and hippocampal level of bone‐derived Irisin (FNDC5). Interestingly, AG+EX mice showed higher behavioral proficiency and more robust long‐term and short‐term memory compared to AG mice. The mechanistic study shows that AG+EX mice display an increased irsin dependent upregulation of neuronal derived neurotropic factor (NDNF) in mouse hippocampus probably via epigenetic inhibition of H3K27me3 methylation at the promoter. Hippocampal overexpression of irisin by Lentiviral FNDC5/irisin administration rescues age‐memory impairment, whereas blockade of brain irisin attenuates the neuroprotective actions of physical exercise on memory in AG mice. Taken together, our findings suggest that physical exercise increases irisin production in bone and that an increase in paracrine irisin signaling in the brain enhances memory function in aging mice.

Support or Funding Information

Acknowledgment

Financial support from National Institute of Health grant AR‐067667‐NT is greatly acknowledged.
PMSF, SFN, and Metformin Reduce Alpha‐synuclein Aggregation in a Yeast Model of Parkinson’s Disease
Authors: Kozub Noah Joseph , Van Brysgel Taylor , Yerxa Christopher Anthony , Moss Stephen , Haak Victoria Melina , Austriaco Nicanor
Published: April 2020

Parkinson’s Disease (PD) is the second most common neurodegenerative disease in humans. PD is marked by Lewy body formation in the brain, which disturbs the dopamine transfer system across neurons. Previous studies have shown that the protein, α‐synuclein, is a major contributor in the formation of Lewy bodies. In this study, we modeled α‐synuclein aggregation in the budding yeast, Saccharomyces cerevisiae. Using this model, we treated cells, in separate trials, with phenylmethylsulfonyl fluoride (PMSF), sulforaphane (SFN), metformin, and methylene blue (MB). Our data suggests that a 4mM concentration of PMSF, 200μg/ml of SFN, and 50mM of metformin significantly reduce protein aggregation. Different concentrations of MB had no effect on α‐synuclein aggregation.

Support or Funding Information

Our laboratory is supported by NIH Grant NIGMS R15 GM110578 awarded to N. Austriaco and P20GM103430 awarded to the RI ‐ INBRE Program for SURF training
Simple and Rapid Slide‐based Mass Spectrometry Workflows Applied to N‐glycan Analysis of Cells, Biofluids and Antibody Arrays
Authors: Drake Richard R. , Black Alyson P. , Blaschke Calvin , Mehta Anand S. , Angel Peggi M.
Published: April 2020

Alterations in glycosylation have long been associated with the development and progression of many types of chronic and acute diseases. Despite major advances in technology for analysis of the glycome, there remain barriers to both non‐glycomic and glycomic researchers for implementing large scale glycan analysis workflows applied to biofluids and cells. Most current liquid chromatography and mass spectrometry workflows require extensive sample purification and processing prior to analysis, expensive reagents and significant technical expertise. Our group has recently adapted tissue‐based mass spectrometry imaging methods to develop streamlined and simplified approaches to efficiently profile N‐glycan constituents in biofluids and cultured cells. A recombinant PNGaseF Prime enzyme is sprayed on to glass slides containing the biological sample of interest, followed by digestion, coating with chemical matrix and analysis of released N‐glycans by matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry (MALDI‐TOF MS). This is accomplished with a solvent sprayer, and the reactions are essentially a solid‐phase format with minimal solvent or sample diffusion. One application example is an antibody slide array approach to directly profile N‐glycans of captured serum glycoproteins like immunoglobulin G. This method requires only a few microliters of sample, rapid processing workflows that require no purification or sugar modifications prior to analysis, and the data readout can be configured as a bar‐code format. Multiplexed slides arrays with seven serum glycoprotein‐targeted antibodies are currently in evaluation, and there is no limit to how many antibodies could be included. Another application is for the N‐glycan analysis of cultures cells on glass slides. Our current method using 8‐well chamber slides is rapid and can be done within 4 hours of completing cell culture, requires as few as 3000 cells per replicate and can include stable isotopic labeling of N‐glycans. The method is amenable to any experimental application that uses cultured cells, can be easily inserted into conventional cell function studies, and depending on the cell type, can generate single cell glycomic information. Both the antibody and cell culture approaches are being adapted to the analysis of immune cell isolates, as well as direct profiling of biofluids like serum and urine. The long‐term goals of these approaches are to provide validated workflows such that any research or core laboratory with a MALDI mass spectrometer will be able to perform routine N‐glycan analysis on the most common types of samples.

Support or Funding Information

U01CA242096 NIH Common Fund in Glycoscience
Saccharomyces cerevisiae Cell Wall Isolates are Potential Inhibitors of Bacterial Growth
Authors: Gudell Evan Victor Judd , Callahan Kevin
Published: April 2020

Saccharomyces cerevisiae cell wall components are thought to bind and help pass pathogenic bacteria, such as E. coli, through the intestines
1. The binding between bacteria and S. cerevisiae is thought to occur between type‐1 mannose specific fimbriae, a common feature of pathogenic bacteria, and the mannoproteins that comprise 30–50% of the cell wall of S. cerevisiae by weight
2,
3,
4. With the fimbriae bound to mannoproteins, bacteria are unable to adhere to the walls of the intestine and thus, no colonization occurs. This interaction provides a potential antibiotic free method to prevent E. coli infection. The goals of this project are two‐fold. First, to develop a cell wall harvesting procedure and characterize the cell wall fractions. Second, to evaluate the binding interaction of S. cerevisiae with E. coli and its effect on E. coli proliferation. A cell wall isolation procedure has been optimized using temperature induced autolysis of a cell culture at 50 °C and pH 5.0 for 24 hours followed by purification washes with salt solutions. Cell wall fractions have been characterized using the Bradford assay, Coomassie stained SDS gels, and Western blot analysis employing the lectin Concanavalin A antibody to detect mannose. The cell wall fractions have been found to contain high levels of mannoproteins. An in‐vitro binding assay, adapted from Ganner et al. 2013, has been employed to evaluate the interaction between the cell wall fractions and E. coli. Cell wall coated microplate wells display a faster progression to log phase than controls due to the cell wall‐fimbriae binding interaction increasing retention of initial colony forming units. In‐vivo, a stronger binding interaction would inhibit bacterial infection by disrupting initial attachment of E. coli to the intestinal lining. Work is currently being performed to better understand factors influencing the binding mechanism as previous studies have indicated an unclear connection between total mannoprotein content and binding capacity.
2

Support or Funding Information

SJFC Internal funds
Apoptosis in Neuronal Populations of C. Elegans through Exposure to Caffeine
Authors: Leeman Jacob T. , Fong Ethan , Madejski Sophia , Iyer Ananya , De Luke
Published: April 2020

Caffeine is the most widely used psychoactive drug in the world and forms a mild dependency in individuals who overuse the drug for an extended period. Other stimulant drugs, including methamphetamines, have been shown to induce apoptosis in the brain’s reward pathways, suggesting that stimulants may lead to apoptosis to lasting neuronal damage. Caffeine has been shown to induce apoptosis in gastric cancer cells in vitro as well as neonatal rat brain and cortical cell cultures. In C. elegans, the homolog of the p‐53 pathway is the CEP‐1 pathway. Caffeine induces apoptosis in human osteoblasts via the p53, Bax, and caspase 3 pathways. However, other studies suggest that caffeine produces inverse effects; caffeine exposure has been reported to increase longevity of Caenorhabditis elegans through activation of the IGF‐1 signaling pathway. Our current data clarifies the relationship between caffeine exposure and CEP‐1 via TG‐12, a C. elegans strain with CEP‐1:: GFP fusion gene.

Here we discuss the results of caffeine exposure on TG‐12 worms. Early data indicates that exposure to high levels of caffeine leads to apoptosis in the nerve ring region of the worm. Here we attempt to identify the affected neuronal populations. We plan to further investigate the relationship between CEP‐1 and caffeine exposure in a dose‐dependent fashion via qRT‐PCR.

Support or Funding Information

This work was supported and funded by the Nueva School. The Shen Lab at Stanford University provided lab equipment and OH11061 worms. Worms and technical support were provided by Caenorhabditis Genetics Center (CGC) ‐ College of Biological Sciences
Oral Contraceptives Do Not Alter Sympathetic Neural Control of Blood Pressure in Young, Healthy Women
Authors: Pletsch Michaela C. , Limberg Jacqueline K. , Cupeiro Rocío , van Helmond Noud , Harvey Ronée E. , Joyner Michael J. , Curry Timothy B. , Baker Sarah E.
Published: April 2020

Background

Sympathetic nerve activity and its effect on blood pressure are altered by female sex hormones; however, this effect differs between exogenous and endogenous hormones. With oral contraceptive (OC) use, cross‐sectional data has demonstrated higher blood pressure (BP) with no corresponding change in muscle sympathetic nerve activity (MSNA). This results in a higher BP/MSNA ratio with OC use although contributing mechanisms are unclear. The higher BP/MSNA ratio in women on OC suggests OC use may limit the ability of sympathetic baroreflex (sBRS) to suppress MSNA. Alternatively, a “burst” of MSNA could result in a greater change in BP (augmented neurovascular transduction).

Objective

The goal of this study was to determine if OCs alter blood pressure control through autonomic mechanisms in young women.

Hypotheses

We hypothesized (1) sympathetic baroreflex sensitivity would be decreased and (2) neurovascular transduction would be augmented in women on OCs compared to naturally cycling women.

Methods

We conducted a retrospective analysis of data from healthy young women taking OCs (n=30, 25±4yrs) and with natural menstrual cycles (n=23, 27±4yrs). Women were studied during the placebo phase of OC use or early follicular phase of the menstrual cycle. Heart rate (HR, 3‐lead ECG), BP (arterial line or photoplethysmography for both groups), and MSNA (peroneal nerve microneurography) were measured over 5 min of quiet rest and data were analyzed for measures of sBRS and neurovascular transduction (Ensemble, Elucimed Inc., Dunedin, New Zealand).

Results

Groups did not differ in mean BP (Control: 94±9, OC:92±8mmHg, p=0.60), or HR (Control: 61±8, OC: 62±9 bpm, p=0.57), but women on OCs had lower MSNA than controls (Control: 16±6, OC: 12±5 bursts/min, p=0.006). There were no significant differences in TPR (Control: 0.022±0.005, OC: 0.020±0.005 mmHg min/L, p=0.12), however there was a trend toward increased cardiac output in women on OCs (Control: 4.45±0.86, OC: 4.94±1.02 L/min, p=.068). There were no significant group differences in sBRS (Control: −4.3±2.1, OC: −4.0±2.6 bursts ·100 heartbeats−1 ·mmHg−1, p=0.79), or neurovascular transduction (Control: 0.093±0.042, OC: 0.091±0.058 ΔmmHg/% burst area, p=0.87). Furthermore, there were no significant correlations between BP and BRS, MSNA, or transduction in either group.

Conclusions

Our data strengthen previous findings which have shown women taking OCs have higher BP for a given MSNA. The analysis we performed build on these results and demonstrate that the increase in BP for a given MSNA in women taking OCs is not due to attenuated sBRS or augmented neurovascular transduction. This work has advanced our understanding of the alteration of blood pressure with OC use in young women.

Support or Funding Information

NIH HL083947
‘SPARC:The Structure of Vagal Nociceptive Nerve Fibers in the Mouse Esophagus’
Authors: Patil Mayur J. , Harsanyiova Jana , Kim Seol-Hee , Taylor-Clark Thomas E. , Kollarik Marian
Published: April 2020

The vagus nerves provide afferent nerve fibers with nociceptive sensory properties to the esophagus. However, it is unknown which esophageal layers are innervated by these vagal nociceptive fibers. We hypothesized that the vagal nociceptive TRPV1‐positive afferent fibers innervate esophageal mucosa. We evaluated our hypothesis by using genetic neuronal tracing in several mutant mice, including two new strains we generated. The wholemount preparations of separated esophageal mucosa and muscle were immunostained for appropriate fluorescent proteins and imaged by spinning‐disk confocal microscopy. In the TRPV1‐Flp/RC: FLTG mice(N=8) in which TRPV1‐expressing cells express tdTomato the TRPV1+ fibers with varicose appearance densely innervated both the mucosa and the muscle. In the mucosa the TRPV1+ fibers are located just beneath the epithelium, run in parallel registers of two or more fibers, have craniocaudal orientation and branch repeatedly. In the muscle the TRPV1+ fibers form a dense network in the myenteric plexus innervating virtually all myenteric ganglia forming intraganglionic varicose endings (IGVEs). Many TRPV1+ fibers connecting IGVEs run in parallel to the fibers of outer and inner striated muscle. In the TRPV1‐Flp/Tac1‐Cre/RC:FLTG mice(N=4) in which GFP is selectively expressed in cells positive for both TRPV1 and Tac1 (the marker of esophageal nociceptors derived from neural crest) the esophageal innervation by TRPV1+/Tac1+ fibers mimicked the dense innervation by TRPV1+ fibers. In contrast, in the TRPV1‐Flp/P2X2‐Cre/RC:FLTG mice(N=4) in which GFP is selectively expressed in cells positive for both TRPV1 and P2X2 (the marker of esophageal nociceptors derived from placodes) the TRPV1+/P2X2+ fibers were almost absent in the esophagus. Consistent with this finding, in the P2X2‐Cre mice unilaterally injected with AAV9‐FLEX‐EGFP virus into vagal ganglia(N=2) in which GFP is selectively expressed in vagal P2X2‐positive fibers, the nodose TRPV1‐negative tension mechanosensor nerve terminals (intragangionic laminar endings) were visualized in the esophageal muscle. Finally, in the TRPV1‐Cre mice unilaterally injected with AAV9‐FLEX‐EGFP virus into vagal ganglia(N=3), in which only the vagal TRPV1‐positive fibers express GFP, the vagal TRPV1+ fibers had the same pattern as TRPV1+/Tac1+ fibers, but with lower density and patchy distribution. Our data indicate that vagal nociceptive TRPV1+ fibers innervate both the esophageal mucosa and myenteric ganglia and originate from neural crest‐derived jugular portion of the vagal jugular‐nodose ganglia complex.

Support or Funding Information

NIH SPARC
The Acetylcholinesterase Inhibitor Galantamine Ameliorates Oxidative Stress in Subjects with the Metabolic Syndrome
Authors: Consolim-Colombo Fernanda M. , Sangaleti Carine T. , Katayama Keyla Y. , De Angelis Kátia Y. , de Moraes Tércio L. , Araújo Amanda A. , Lopes Heno F. , Camacho Cleber Y. , Bortolotto Luiz M. , Michelini Lisete M. , Irigoyen Maria M. , Barnaby Douglas P. , Tracey Kevin J. , Pavlov Valentin A.
Published: April 2020

The metabolic syndrome (MetS) is an obesity‐driven disorder with pandemic proportions and limited treatment options. Oxidative stress, low‐grade inflammation and altered autonomic regulation, are important components of MetS pathophysiology. We recently reported that galantamine, an acetylcholinesterase inhibitor and an FDA‐approved drug (for Alzheimer’s disease) alleviates the inflammatory state in MetS subjects. Here we examined the effects of galantamine on oxidative stress in parallel with inflammatory and cardio‐metabolic parameters in subjects with MetS. The effects of galantamine treatment, 8 mg daily for 4 weeks, followed by 16 mg daily for 8 weeks or placebo were studied in randomly assigned subjects with MetS (n=22 per group) of both genders. Oxidative stress, including superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase activities, lipid and protein peroxidation, and nitrite levels were analyzed before and at the end of the treatment. Plasma cytokine and adipokine levels, insulin resistance (HOMA‐IR) and other relevant cardio‐metabolic indices were also determined. Autonomic regulation was also examined by heart rate variability (HRV) before treatment, and at every 4 weeks of treatment. Galantamine treatment significantly increased antioxidant enzyme activities, including SOD (+1.65 USOD/mg protein, P=0.004) and CAT (+0.93 nmol/mg, P=0.011), decreased lipid peroxidation (thiobarbituric acid reactive substances, −5.45 pmol/mg, P=0.053) and systemic nitrite levels (−0.05 nit/mg protein, P=0.038) compared with placebo. In addition, galantamine significantly alleviated the inflammatory state and insulin resistance, and decreased the low frequency/high frequency ratio of HRV, following 8 and 12 weeks of drug treatment. In conclusion, a low‐dose galantamine treatment alleviates oxidative stress, alongside beneficial anti‐inflammatory, and metabolic effects, and modulates autonomic regulation in subjects with MetS. These findings are of considerable interest for further studies with galantamine to ameliorate MetS pathophysiology.
Transcriptome analysis of Rrp6p’s role in intron retention during the stationary phase of Saccharomyces cerevisiae
Authors: Villalta Oscar , Callahan Kevin , Myres Jacquelyn , Ashton John
Published: April 2020

Introns are non‐coding RNAs that are readily degraded into single nucleotides within the nucleus where they can be recycled in another transcription cycle. Unspliced introns form 3‐D structures at their 5′ end, never separate from their splicing factors and are stable during and after pre‐mRNA modifications
(4). Recently it was observed that retention of introns in Saccharomyces cerevisiae increases when the cells are in a nutrient deficient environment, specifically stationary phase
(3,
4,
5). Transcriptomic and genetic analyses showed that intron retention promotes resistance to starvation by downregulating ribosomal protein genes downstream of nutrient‐sensing TORC1 and PKA pathways
(3). Yeast cells that cannot synthesize introns or have a low concentration of them fail to survive in stationary phase, suggesting that introns are necessary for the survival of the organism during starvation. Rrp6p is a 3′→5′ RNA exonuclease that associates with the nuclear exosome
(1). This complex participates in 3′ end formation and degradation of a variety of non‐coding RNAs and unstable transcripts
(1,
2). We hypothesized that yeast cells lacking Rrp6p will retain more introns than a wild type strain, thus transcriptome analysis of this strain during starvation conditions will lead to a better understanding of intron retention. We monitored the growth of WT and rrp6‐Δ strains during prolonged stationary phase and the subsequent lag, log phases following placement into fresh media. This analysis identified the time frame at which each strain exited stationary phase and entered log phase. We collected RNA from each strain during stationary phase and early log phase and performed transcriptome analysis. Data analysis was performed using the scripting program, R, to identify introns whose expression significantly changes. Introns identified from this analysis will be confirmed using quantitative PCR.

Support or Funding Information

SJFC internal funds
Evaluation of Pulse Arrival Time Model to Estimate Systolic Blood Pressure during Exercise
Authors: Hedge Eric T. , Amelard Robert , Hughson Richard L.
Published: April 2020

Pulse arrival time (PAT) is a method used to estimate systolic blood pressure (SBP), as the amount of time it takes for a pulse to travel from the heart to a peripheral location is inversely related to blood pressure. However, the validity and accuracy of this method’s blood pressure estimation has been questioned. Therefore, the purpose of this study was to evaluate the accuracy of a PAT model’s estimation of SBP over a range of exercise intensities. Six participants (5 men, 1 woman; age: 26 ± 4 yrs) completed three cycling exercise tests (25 Watt/min ramp incremental test, and moderate and heavy pseudorandom binary sequence exercise) during three separate laboratory visits. PAT was calculated as the time difference between the R‐wave of an electrocardiogram and the pulse arrival at the forehead measured with a pulse oximeter. SBP was estimated using the following equation: SBPPAT = 64.578/(PAT) – 43.957, and was compared to calibrated brachial blood pressure measured at the finger (FBP) by photoplethysmography during the exercise trials. Participant blood pressure responses were pooled, and Bland‐Altman analysis was conducted to evaluate the accuracy of estimated SBP for each exercise test. PAT model estimation of SBP had the smallest bias during heavy (−2.8 mmHg), then moderate (8.1 mmHg), and the largest bias during ramp incremental exercise (−11 mmHg), with their limits of agreement being between −56 and 50 mmHg, −29 and 45 mmHg, and −74 and 53 mmHg, respectively. Linear regression of the ramp incremental SBP response revealed that SBP estimated from the PAT does not increase by the same magnitude as the pressure measured at the finger (SBPPAT = 0.295 · SBPFBP + 106.236, r2 = 0.350, p < 0.05). Overall, these findings support that SBP estimated by PAT is linearly related to brachial SBP measured at the finger; however, this PAT model is a poor estimator of absolute SBP during exercise ranging from moderate to maximal intensities.

Support or Funding Information

Supported by NSERC.
Mammalian Target of Rapamycin Mediates Expression and Activity of ADAM 17 in Diabetic Kidney Disease
Authors: Abraham Sachin , Ford Bridget Marie
Published: April 2020

Diabetic kidney disease is a serious complication faced by type 1 and type 2 diabetic patients alike. Albuminuria and extracellular matrix accumulation are prominent features of the disease and this accumulation of extracellular matrix is a contributing factor to renal fibrosis and decline in renal function. The mechanisms involved in the pathogenesis of diabetic kidney disease have not been completely identified.

Previous data have illustrated a role for the matrix metalloprotease A Disintegrin And Metalloprotease 17 (ADAM17), known to cleave growth factors and cytokines, in renal cell injury in diabetes. The goal of this study was to identify upstream regulators of ADAM17 in the cascade of events contributing to extracellular matrix accumulation in diabetic nephropathy. Using the mTOR complex 1 inhibitor rapamycin, it was determined that increased ADAM17 enzymatic activity and ADAM17 protein expression is dependent on mTORC1 in streptozotocin‐induced type 1 diabetic rats. Inhibition of mTORC1 with rapamycin abrogated the increase in collagen IV α 2 protein expression observed in diabetic rat cortex. Additionally, this study is the first to provide evidence that mTOR complex 1 activates ADAM17 contributing to extracellular matrix accumulation in diabetic nephropathy.

Support or Funding Information

This project was funded by the University of the Incarnate Word, Office of Research and Graduate Studies
Mice Expressing Fluorescent Protease‐Activated Receptor‐2 as a Novel Tool to Visualize Intracellular Receptor Trafficking
Authors: Teng Shavonne , Latorre Rocco , Jensen Dane , Schmidt Brian L. , Bunnett Nigel
Published: April 2020

Protease‐activated receptor‐2 (PAR2) is a G‐protein coupled receptor (GPCR) that plays a critical role in mediating the proinflammatory and pronociceptive actions of serine and cysteine proteases in many tissues. In common with a growing number of GPCRs, PAR2 can signal from endosomes as well as the plasma membrane, and sustained endosomal signaling underlies disease‐relevant processes. Our knowledge of the role of PAR2 in health and disease states is hampered by an inadequate understanding of its cellular and subcellular localization. Since antibodies to GPCRs often lack sensitivity and specificity, and are unsuitable for studying receptor trafficking in living cells, we used a knockin approach to generate mice expressing PAR2 fused to monomeric ultrastable GFP (muGFP). To investigate whether mouse PAR2 fused at the C‐terminus to muGFP was functional, we compared agonist‐evoked signaling and trafficking of PAR2‐muGFP and PAR2 with a C‐terminal HA11 epitope expressed in KNRK cells. Trypsin and the PAR2 selective peptide agonist 2‐Furoyl‐LIGRLO‐NH2 similarly stimulated calcium signals and PAR2 endocytosis. Thus, muGFP does not disrupt PAR2 signaling and trafficking. We then generated knockin mice expressing mouse PAR2 fused at the C‐terminus to muGFP. RT‐PCR revealed similar levels of expression of PAR2 in PAR2‐muGFP and wild‐type mice. Western blotting of colonic extracts using a GFP antibody revealed expression of PAR2‐muGFP at the protein level. We localized PAR2‐muGFP by immunofluorescence and confocal microscopy using the GFP antibody. PAR2‐muGFP was highly expressed in epithelial cells in the digestive tract and skin, and was detected at lower levels in the enteric nervous system and pancreatic islets. In the unstimulated state, PAR2‐muGFP was principally localized to the plasma membrane of colonocytes. Exposure to trypsin or 2‐Furoyl‐LIGRLO‐NH2 induced redistribution of PAR2‐muGFP from the plasma membrane to endosomes. PAR2‐muGFP mice provide a valuable tool to study the localization, regulation, and trafficking of PAR2 in intact animals in healthy and diseased conditions.

Support or Funding Information

NIH
Synthesis and Testing of The Antibiotic Properties of Schiff Base Ligands and Their Cobalt Complexes
Authors: Bryant Timothy C. , Deifel Nicholas P.
Published: April 2020

The ability to design and synthesize new antibiotic compounds has become necessary in recent years due to the emergence of antibiotic resistant bacteria from such species as E. coli, K. pneumoniae, and S. aureus, among others. Schiff base ligands and their metal complexes, namely those complexes formed with cobalt (II), have displayed promising potential as a means to synthesize new compounds for antibiotic use in a straightforward and efficient manner. Specifically, cobalt complexes of tetradentate Schiff base ligands have been shown to be effective against both Gram‐positive and Gram‐negative bacterial strains (Shaheen et al., 2019); however, there is as of yet limited information as to how a variety of specific bacterial strains will be affected by such compounds. This project is centered on the synthesis of Schiff base ligands from O‐phenylenediamine and various salicylaldehyde precursors in order to form tetradentate complexes and to test their antibiotic properties through the Kirby‐Bauer disc diffusion test. The proper synthesis of the Schiff base ligands is currently be confirmed by NMR, IR, and MS analysis. Following this confirmation, we will add cobalt acetate to facilitate metal complex formation and confirm expected complexing by Microwave Plasma‐Atomic Emission Spectrometry (MP‐AES), cyclic voltammetry, and Electron Spin Resonance (ESR) analyses. Overall, we expect that this project will shed new insights on the specific antibacterial range for these cobalt Schiff base complexes as well as the efficacy of these complexes as effective antibiotics for general medical use.
The Terrestrial Migration of Vertebrates Drove the Evolution of ENaC Activation by Cleavage α
Authors: Kashlan Ossama B , Wang Xue-ping , Balchak Deidra M , Gentilcore Clayton , Clark Nathan L
Published: April 2020

The epithelial Na+ channel (ENaC) is critical to regulating extracellular fluids through its activity in several epithelia, including the aldosterone‐sensitive distal nephron and colon, and the airway and alveoli. Human ENaC subunits are subject to activating proteolysis. The ENaC α and γ subunits may each be cleaved twice at polybasic tracts in their respective extracellular GRIP domains. This liberates imbedded inhibitory tracts, which activate the channel. Activation through either subunit requires double cleavage. To examine the evolution of ENaC cleavage sites, we generated a phylogenetic tree of ENaC and ENaC‐related subunits to identify key branch points in the evolution of ENaC subunits. The four ENaC subunits arose from three gene duplication events. First, a single gene diverged to an α/δ precursor and a β/γ precursor. Second, the β/γ precursor diverged to β and γ subunits. These two events occurred before the emergence of jawless fishes, as lampreys have α, β, and γ subunits. After the emergence of jawed fishes, α and δ diverged from the α/δ precursor; the lobe‐finned coelacanth is earliest rooted species to have all four subunits. We observed that the activating cleavage sites in the α and γ subunits appeared around the transition to terrestrial life. Among the fishes, only the Australian lungfish γ subunit had two cleavage sites, and all tetrapod α and γ subunits had two apparent cleavage sites. We ran nested likelihood models in BayesTraits and found a significant relationship between terrestrial status and having two cleavage sites in the α and γ subunits. Both sites showed a statistically significant coevolutionary pattern with the terrestrial state (proximal site, p = 0.004; distal site, p = 0.006). The sites also showed a coevolutionary pattern with each other (p = 0.0003). In contrast, the C‐terminal PY motif, which is important for aldosterone regulation through Sgk1/Nedd4‐2, did not show a dependence on terrestrial status (p = 0.58). We also performed ancestral reconstructions to determine whether these protein sequences arose through divergent or convergent evolution. Competing evolutionary models, where common ancestors were constrained to states reflecting each model, were compared using marginal likelihoods from Markov chain Monte Carlo simulations of each model. Our analysis strongly supports a convergent evolutionary model for the cleavage sites, and a divergent evolutionary model for the PY motifs. Our data suggest that aldosterone signaling adopted both long existing and newly evolved mechanisms to regulate ENaC function.
Valproic Acid Alters Activities of Key Enzymes of Glucose Oxidative Metabolism, Citric Acid Cycle and Neurotransmitter Synthesis in Embryonic Rat Brain
Authors: Triyasakorn Korawin , Lai James C.K. , Awale Prabha
Published: April 2020

Although studies have investigated the role of gamma‐aminobutyric acid (GABA)ergic signaling in rodent neural development and behaviors relevant to autism, the energetics that underlie neural developmental changes are poorly understood. Here, we employed a valproic acid (VPA) rat model of autism to investigate the effects of VPA treatment on key enzymes of glucose oxidative metabolism and citric acid cycle (namely lactate dehydrogenase (LDH), malate dehydrogenase (MDH) and glutamate dehydrogenase (GDH)) in rat brain. Pregnant Wistar rats were administered 600 mg/kg of VPA by intraperitoneal injection on day 12.5 of gestation and embryonic (E) day 15 and E17 rat brains were assayed for the activities of the enzymes. Both E15 and E17 male and female rat models of VPA‐induced autism showed significantly changed whole brain activities of the enzymes studied compared to the corresponding values in control rats. The dysregulated GDH activity could alter the excitatory‐inhibitory balance in the brain of embryonic rats during development. Thus, our findings may have pathophysiological/pathogenic implications in autism and may ultimately lead to the discovery of new therapeutic target(s) for autism.

Support or Funding Information

Seed Grant Idaho State University College of Pharmacy
Respiratory Dysfunction in a Mouse Model of Spinocerebellar Ataxia 7
Authors: Fusco Anna F. , Pucci Logan , McCall Angela L. , Dhindsa Justin , Kahn Amanda , Switonski Pawel , Strickland Laura , Spada Albert La , ElMallah Mai K.
Published: April 2020

Spinocerebellar ataxia type 7 (SCA‐7) is a neurodegenerative polyglutamine disease within the family of spinocerebellar ataxias. Classically SCA7 is a disease of ataxia and vision loss. However, patients with SCA also have difficulty breathing, swallowing, coughing and maintaining a stable open airway which often result in fatal respiratory failure. While respiratory measures have been reported in other SCA mouse models, ventilation in a SCA‐7 mouse has not yet been studied. Using the SCA‐7 mouse model (Atxn7266Q/+) we sought to characterize the respiratory pathology of SCA‐7.

Whole body plethysmography (WBP) was used to assess respiratory dysfunction in the Atxn7266Q/+ mouse both at baseline breathing (FiO2: 0.21; nitrogen balance) and during a hypercapnic and hypoxic challenge (Fi:CO2: 0.07, FiO2: 0.10; nitrogen balance). Neurobehavioral testing and strength was assessed using the inverted screen test and the grip strength test. Post mortem histological analysis was performed on the phrenic and hypoglossal motor neurons and nerves.

At 4 weeks of age, the SCA‐7 mice are indistinguishable from their unaffected littermates. However, by 8 weeks of age, SCA‐7 mice perform poorly on both neurobehavioral and strength tests as compared to their WT littermates. 50% of the SCA‐7 mice died by 9 weeks, while littermate controls thrived. SCA‐7 mice have elevated respiratory rate at baseline, as well as reduced tidal volume, minute ventilation, peak inspiratory flow, peak expiratory flow, and inspiratory time during the respiratory challenge. These results indicate that the SCA‐7 mice have respiratory dysfunction. To understand the respiratory deficits, ongoing studies are histologically analyzing the diaphragm neuromuscular junction, phrenic and hypoglossal nerves, as well as phrenic and hypoglossal motor nuclei. Preliminary nerve analysis of the hypoglossal and phrenic nerves reveals significant axonal pathology. In conclusion, WBP analysis and preliminary histological nerve data reveals respiratory dysfunction in the Atxn7266Q/+ mouse model.

Support or Funding Information

Funding: R01 HD099486‐01; 1R21NS098131‐01
Interrogating the Activity of Environmental Contaminants Against the Druggable GPCR‐ome
Authors: Wilkinson Joshua , Lehmler Hans-Joachim , Roman David
Published: April 2020

Exposure to environmental contaminants can result in a myriad of conditions including developmental disorders, pulmonary conditions, and neurological pathologies. Literature precedent and new, emerging data, demonstrates specific GPCRs can be activated by some classes of environmental contaminants, including PCBs, pesticides, chloropropanols, and others. When interrogating GPCR’s with small‐molecule based screens, the traditional paradigm is to test hundreds of thousands of compounds against a single target. In recent literature, an open‐source method “PRESTO‐TANGO” (Parallel Receptor‐ome Expression and Screening via Transcriptional Output – TANGO) has been described as a feasible approach to simultaneously screen the entire druggable human GPCR‐ome against a smaller collection of compounds via a G protein‐independent β‐arrestin recruitment assay. In this work, we have adopted a similar approach to the PRESTO‐TANGO methodology, enabling us to screen activity of a number of environmental contaminates against the known druggable GPCR‐ome of 314 GPCRs. From our initial screen, we identified several potential ligand‐receptor interactions. Secondary dose response experiments uncovered what we believe to be a novel ligand‐receptor relationship between PCBs and the Sphingosine‐1‐phoshpate receptor (S1PR) family. Experiments to validate the relationship between the PCBs and S1PR family, as well as investigate the relevant signaling cascades impacted are currently underway. Ultimately, this work utilizes a modified form of the PRESTO‐TANGO assay as a novel screening platform to elucidate bioactivity of environmental contaminants.

Support or Funding Information

This work was supported by the University of Iowa Environmental Health Science Research Center (NIEHS P30 ES05605 D.L.R)
Inflammation and the Cardiovascular Responses to Exercise in Patients with Peripheral Artery Disease
Authors: Craig Jesse C. , Hart Corey R. , Layec Gwenael C. , Kwon Oh Sung C. , Richardson Russell S. , Trinity Joel D.
Published: April 2020

Background

Patients with peripheral artery disease (PAD) demonstrate an exaggerated exercise pressor response (EPR). Heightened inflammation critically contributes to this exaggerated EPR in animal models of PAD, however, this relationship remains largely unexplored in humans. Therefore, we tested the hypotheses that the blood pressure response to exercise would be exaggerated in patients with PAD compared to controls; and that markers of inflammation would be positively related to this response.

Methods

Eight patients with PAD (ABI: 0.67±0.11) and eight healthy controls (HC; ABI: 1.16±0.07) matched for age, sex, physical activity, and physical characteristics completed plantar flexion exercise at both absolute (2.7 W) and relative (40% max; PAD: 2.1±0.2 vs HC: 4.3±0.4 W, p<0.001) intensities. Popliteal artery blood flow (Flow) was measured via Doppler ultrasound and systemic mean arterial pressure (MAP) via Finapres. Resting systemic and intramuscular markers of inflammation and oxidative stress were quantified from plasma and muscle biopsies.

Results

The exercise‐induced ΔMAP was greater for PAD at both the absolute (PAD: 20±3 vs HC: 9±2 mmHg, p<0.01) and relative (PAD: 23±5 vs HC: 11±2 mmHg, p=0.02) exercise intensities. The ΔFlow was lower for PAD compared to HC during both absolute (PAD: 251±53 vs HC: 545±66 ml/min, p<0.01) and relative intensities (PAD: 242±61 vs HC: 595±89 ml/min, p<0.01). Inflammatory markers including plasma tumor necrosis factor alpha (TNFα: ↑134%), interleukin‐6 (IL6: ↑92%), and intramuscular IL6 (↑77%) were elevated in PAD compared to HC (p<0.05). Additionally, markers of oxidative stress including intramuscular protein carbonyls (PC: ↑57%) and 4‐hydroxynonenal (HNE: ↑80%) were elevated in PAD (p<0.05). TNFα, plasma IL6, and PC were positively correlated with ΔMAP (r: 0.57–0.82, all p<0.05) while plasma and intramuscular IL6, PC, and HNE were negatively correlated with ΔFlow (r: 0.64–0.78, all p<0.05).

Conclusions

The present findings indicate an association between markers of inflammation with the cardiovascular responses to exercise in humans with PAD. Specifically, heightened inflammation and oxidative stress appear to contribute to the exaggeration the exercise pressor reflex and attenuated blood flow response during lower limb exercise in PAD. Therapeutically, targeting systemic and local inflammation may be a viable option to improve the blood pressure and blood flow responses to exercise in patients with PAD.
Brominated Flame Retardants alter Cytokine Production in Mast Cells
Authors: King Ethan , Mahoney Jonathan M. , Krajewski Dylan , Mathias Clinton , Bose Diptiman D.
Published: April 2020

Polybrominated diphenyl ethers (PBDEs), extensively used as flame‐retardants have been classified as persistent environmental pollutants. PBDEs have also been shown to cause neurotoxicity, immunotoxicity and endocrine disruption; however, the exact mechanisms are largely unknown. In this study, we propose to investigate PBDE‐induced immunotoxicity in mast cells. We examined the effects of two of the most commonly occurring brominated diphenyl ether (BDE) congeners, BDE‐47 and −99, and a metabolite of BDE‐47, 6‐OH‐BDE‐47 on MC/9 mast cells. MC/9 cells treated with BDE‐47, −99 and 6‐OH‐BDE‐47 (0.25, 0.5, 5, 20 and 40μM, 24h) were stimulated with IgE/antigen (1μg/ml), and levels of IL‐6 and TNF‐α were measured by ELISA. Our results indicate that PBDEs inhibit the IgE‐mediated cytokine release in MC/9 cells in a concentration‐dependent manner. Interestingly, exposure to BDE‐47 and 6‐OH‐BDE47 decreased IL‐6 release in a concentration‐dependent manner; however, BDE‐99 decreased IL‐6 release only at concentrations above 20 μM. These results demonstrate that tetra‐brominated BDE‐47 and its 6‐OH metabolite can suppress cytokine release with increasing concentrations, while penta‐brominated BDE‐99 congener inhibits IL‐6 release only at higher concentrations. Mast cell degranulation measured by β‐hexoaminidase also showed that BDE congeners decreased degranulation in a concentration‐dependent manner in IgE stimulated mast cells. In order to test effects on cell viability, we exposed the MC/9 cells to increasing concentrations of the BDE congeners and measured cytotoxicity at 24 and 48h by MTS assay. We found that tetra‐brominated BDE‐47 exhibited more potent cytotoxicity (EC50 μM =42.38 at 24h and 32.33 at 48h) in comparison to the penta‐brominated BDE‐99 (EC50 μM =48.41 and 35.04 at 48h) in the MC/9 cells. However, the 6‐OH metabolite of BDE‐47 was more cytotoxic in comparison to its parent compound BDE‐47 as well as penta‐brominated BDE‐99 (EC50 μM =25.82 at 24h and 20.16 at 48h). We are further characterizing development of apoptosis in the MC9 cells in the presence of BDE congeners. Our results suggest that PBDE congeners can modulate cytokine release in mast cells and the extent of inhibition is dependent on the bromination of PBDEs. These results address the need to better understand the molecular and cellular mechanisms by which PBDE exposure can mediate immunotoxicity.
HIV‐1 Matrix Protein Interactions with Cellular tRNAs
Authors: Ejimogu Jason , Parker Connor , Bauerle Mahlet , Palm Cheyenne , Brown Janae Baptiste , Summers Michael
Published: April 2020

The human immunodeficiency virus (HIV) is a retrovirus that infects roughly 40 million people globally that attacks CD4+ T‐cells, thus impairing the immune system. Highly active antiretroviral treatment against the virus is available; however, the viral genome frequently mutates, which gives rise to evasion of current therapies and highlights the need for new drugs. Better understanding molecular mechanisms of viral replication may lend to this effort. One process necessary for HIV replication is membrane trafficking of the Gag polyprotein (Gag) to the plasma membrane via Gag’s N‐terminal matrix domain (MA). MA mediates membrane targeting by means of electrostatic interaction and anchoring to the plasma membrane using a covalently linked myristoyl moiety. Recent virology studies demonstrated that MA interacts with cellular tRNAs, including tRNALys3 and tRNAGlyGCC, before binding to the plasma membrane, but the role of tRNA on assembly is poorly understood. The long‐term goal of this work is to study the MA:tRNA complex and elucidate its 3D structure as characterization of this interaction may shed light on the function of tRNA during membrane targeting. To achieve this goal, conditions to stabilize MA and the tRNA independently and in complex must be established. Nuclear magnetic resonance (NMR) and gel electrophoresis were employed to identify conditions that stabilized the tRNA and MA independently. Electrophoretic mobility shift assays (EMSAs) were carried out to probe MA:tRNA interactions under these conditions and showed complex formation upon addition of a stoichiometric ratio of MA to tRNA. This work lays a foundation to study thermodynamics of the MA‐tRNA interaction and determine the three‐dimensional structure of the complex.

Support or Funding Information

This research was sponsored by NIH/NIGMS MARC U*STAR T34 HHS 00026 National Research Service Award to UMBC, HHMI, and NIH grants R01GM042561 and 8R01AI50498.