bluuebunny/crossref_metadata_2025 huggingface dataset:
Ethanol Shifts Myoblast Bioenergetic Phenotype: Implications for Muscle Regeneration
Authors: Levitt Danielle E. , Simon Liz , Molina Patricia E.
Published: May 2022

At‐risk alcohol use is nearly twice as prevalent among people living with HIV (PLWH) as the general population. Alcohol and HIV can independently and synergistically increase risk for metabolic diseases, including myopathy. Decreased functional skeletal muscle (SKM) mass is associated with morbidity and all‐cause mortality. A major focus of our research is to investigate the independent and combined effects of alcohol and HIV on SKM regenerative capacity and its contribution to myopathy development. Previously, we showed that in vitro ethanol decreases myoblast extracellular acidification rate (ECAR), indicative of decreased glycolytic function, and that the decrease in ECAR was associated with decreased myoblast differentiation. The objective of this study was to determine effects of prior and recent alcohol exposure and simian immunodeficiency virus (SIV) on bioenergetic function in myoblasts from naïve and SIV‐infected rhesus macaques. Female rhesus macaques (6‐10 years of age) were administered chronic binge alcohol (CBA; peak blood alcohol concentration=50‐60 mM, 5 days per week) or isovolumetric water vehicle (VEH) beginning 3 months prior to infection with SIVmac251. Daily antiretroviral therapy (ART) was initiated 2.5 months after SIV infection, and macaques were humanely euthanized 9 months later. SKM samples were collected for myoblast isolation prior to CBA or VEH (naïve, N=6) and at study end point (VEH/SIV, N=6; CBA/SIV, N=6). Myoblasts from each group were incubated with ethanol (0 and 50 mM) for 3 days prior to Mito Stress Test, Glycolysis Stress Test, and ATP Rate Assay using an XFe96 Extracellular Flux Analyzer. There were no significant differences due to CBA or SIV on mitochondrial or glycolytic function for any measure. During the Mito Stress Test, ethanol exposure significantly (p<0.05) decreased ECAR at baseline and after the addition of oligomycin (ATP synthase inhibitor), indicative of decreased glycolytic function, without significant changes in mitochondrial function. Similarly, ethanol exposure decreased all parameters in the Glycolysis Stress Test (non‐glycolytic acidification, glycolysis, glycolytic reserve, and glycolytic capacity). An ATP rate assay confirmed that ethanol exposure significantly decreased glycolytic ATP production, contributing to a decrease in total ATP production. These results indicate that recent exposure to an intoxicating concentration of ethanol shifts myoblast bioenergetic phenotype, impairs glycolysis, and produces a bioenergetic deficit regardless of prior alcohol exposure or SIV/ART. The shift away from glycolysis occurs under basal and stressed conditions. We propose that altered glycolytic enzyme activity underlies the alcohol‐mediated decrease in glycolytic function, negatively impacting myoblast differentiation and SKM regenerative capacity.
FOXI1 Promotes Expression of V‐ATPase and GPR116 in M‐1 Cells
Authors: Kui Mackenzie , Pluznick Jennifer L. , Zaidman Nathan A.
Published: May 2022

G protein‐coupled receptors (GPCRs) are a diverse family of integral membrane proteins that have significant roles in numerous physiological systems. We previously reported that GPR116 (ADGRF5), an adhesion‐class GPCR, is a critical regulator of vacuolar‐type H+‐ATPase (V‐ATPase) surface expression in A‐type intercalated cells (AICs) in mouse kidney cortical collecting ducts. The V‐ATPase is a multi‐subunit proton pump that localizes to the plasma membrane in specialized acid‐secreting epithelial cells. FOXI1 is a transcription factor that regulates V‐ATPase expression in ICs and other mitochondria‐rich cells. Recently, FOXI1 was identified as a key regulator of CFTR‐rich pulmonary ionocytes which express both V‐ATPase and GPR116. Therefore, we hypothesized that FOXI1 is a transcription factor in AICs that upregulates V‐ATPase as well as GPR116. To test this hypothesis, we cloned FOXI1 from whole mouse kidney into a pME18s expression vector (pFOXI1). Transfection with pFOXI1 significantly increased GPR116 expression (qPCR) in M‐1 mouse cortical collecting duct cells (n=3, ΔCt ±SEM: untransfected = 13.8±0.6, pFoxi1 = 10.7±0.2, p<0.05), but not in HEK293T cells (n=3, not significant). RNA‐scope hybridization on murine kidney sections and pFOXI1‐transfected M‐1 cells revealed colocalization of FOXI1 and GPR116 mRNA in the same cell. Treating pFOXI1‐transfected M‐1 cells with a synthetic agonist peptide for GPR116 results in an increase of [Ca2+]iin a subset of cells (n=3, ΔF340/380 ±SEM: pFoxi1 = 0.296±0.017). Internal calcium mobilization is never seen with the agonist peptide in untransfected control cells, indicating that pFOXI1 is sufficient for the production of physiologically responsive GPR116 on the cell surface (n=3, ΔF340/380±SEM: untransfected = 0 ±0.0009). Moreover, only M‐1 cells transfected with pFOXI1 express GPR110 (ADGRF1), an adhesion‐GPCR with unknown function in the kidney, as well as three V‐ATPase subunits, ATP6V1B1, ATP6V1G3, ATP6V0D2, indicating a larger transcriptional program initiated by pFOXI1 (n=3, untransfected: Ct ~ 40 for all genes, pFoxi1 ΔCt±SEM: ADGRF1 = 13.2±0.21, ATP6V1B1 = 11.4±0.3, ATP6V1G3 = 10±0.1, ATP6V0D2 = 12.1±0.03). Additionally, these pFOXI1 cells also upregulate the expression of SLC26A4 (pendrin), a known marker of B‐type intercalated cells, suggesting that FOXI1‐driven regulation is upstream of AIC fate determination (n=3, ΔCt ± SEM: untransfected = 19.3±0.7, pFoxi1 = 16.1±0.2, p<0.01). These data reveal that FOXI1 upregulates both GPR116 and V‐ATPase in M‐1 cells, generating IC‐like cells. Finally, our study suggests that GPR116 may be a universal and genetically‐coded regulator of V‐ATPase surface expression in FOXI1‐positive cells.
Enalapril associated with combined training is more effective in reducing SBP and improve baroreflex sensitivity than drug therapies alone in hypertensive rats
Authors: de Araujo Amanda A. , Shecaira Tânia P. , Paixão Camila , Ferreira Maycon J. , da Silva Dias Danielle , Bernardes Nathália , Irigoyen Maria Claudia , De Angelis Kátia
Published: May 2022

High blood pressure is the leading risk factor to development of cardiovascular disease worldwide. Diuretic and angiotensin‐converting enzyme blockers treatment is widely used for its efficiency in reducing blood pressure (BP). Combined physical training (aerobic + resistance) is recommended for hypertensive patients; however, it is not yet clear whether the combination of antihypertensive treatment with this training modality results in additional benefits. Therefore, the aim of this study was to compare the effects of combined training with different drug treatments on systolic BP (SBP) and baroreflex sensitivity (BRS) of spontaneously hypertensive rats (SHR). Twenty‐eight males SHR rats were divided into four groups (n=7/group): HS (sedentary hydrochlorothiazide), HT (trained hydrochlorothiazide), ES (sedentary enalapril) and ET (trained enalapril). Drugs were administered in drinking water (enalapril 3 mg/kg/day and hydrochlorothiazide 30 mg/kg/day). All groups underwent exercise testing before and after 2 months. The trained groups performed combined training 3 times a week, being aerobic and resistance at same session, moderate intensity. The SBP was analyzed by caudal plethysmography before starting the drugs and 24h after the last training session. BRS was analyzed by response to phenylephrine and sodium nitroprusside. Both trained groups increased maximal aerobic exercise test (p<0.0001) and resistance exercise test (HT vs. HS, p=0.02; ET vs. ES, p<0.0001) versus the sedentary groups at the end of the protocol. There was no difference in the initial SBP between the groups (HS 176 ± 4.3, HT 175 ± 3, ES 173 ± 1.4 and ET 183 ± 3 mmHg). There were reductions in SBP in the HT (161 ± 1.3 mmHg), ES (159 ± 4.4 mmHg) and ET groups (147 ± 3.3 mmHg) at the end of the protocol. In addition, the trained group associated with enalapril treatment further reduced SBP when compared to the trained hydrochlorothiazide group (ET vs. HT, p = 0.02). Regarding BRS, there was no difference in tachycardic response between groups; however, ET group (‐2.8 ± 0.5 bpm/mmHg) showed increased bradycardic responses comparing with ES group (‐1.8 ± 0.1 bpm/mmHg). Therefore, the data support that the association of combined exercise training with enalapril treatment and is more effective in reducing SBP and improving BRS than the combination with hydrochlorothiazide treatment. It is noteworthy that the training proposed in this study was performed only three times a week, getting closer to clinical recommendations, and being enough for induce additional benefits to classical drug therapies in functional capacity and blood pressure control in an experimental model of hypertension.
Lowering of Sympathetic Burst Frequency in Post‐Pubertal Female but not Male Rats
Authors: Mirza Umme Aiman , Desai Shivansh A. , Mueller Patrick J.
Published: May 2022

INTRO

Cardiovascular disease (CVD) leads to an overall reduction in the quality of life and is the leading cause of mortality in the United States. The risk of CVD increases with age; however, women of reproductive age have a lower CVD prevalence. CVD is associated with elevations in sympathetic nerve activity, which has been analyzed in humans by quantifying sympathetic burst frequency. Whether sympathetic burst frequencies are different between pre‐ and post‐pubertal males and females is unknown.

PURPOSE

The purpose of this study was to analyze sympathetic burst frequencies for potential differences between male and female rats.

HYPOTHESIS

We hypothesized that resting sympathetic burst frequency would be lower in post‐pubertal females compared to age‐matched males. We also predicted lower burst frequency in post‐ versus pre‐pubescent females.

METHODS

We analyzed splanchnic nerve (SSNA) recordings from Inactin‐anesthetized male and female Sprague‐Dawley rats at 4‐weeks (pre‐pubertal) and 8‐ and 16‐weeks (post‐pubertal) of age. Sympathetic bursts were obtained by rectifying and integrating raw SSNA. Burst frequency was calculated using the Peak Parameters extension in LabChart software.

RESULTS

Burst frequency was higher in 4‐week (n=8) and 8‐week (n=9) old female rats compared to 16‐week old (n=6) female rats (5.6+1.2; 4.8+0.4 and 2.4+0.3 Hz, p<.001 and p=0.02, respectively). At 16 weeks of age, female rats also had a significantly lower burst frequency when compared to male rats (n=4) (2.4+0.3 vs, 4.8+0.4 Hz, respectively, p=0.01). Finally, there was no significant difference found in burst frequencies between male rats at any age (p>0.05 for all), suggesting sex‐dependent differences in the development of sympathoexcitation.

CONCLUSION

Preliminary results suggest that following the onset of puberty in female rats, sympathetic burst frequency decreases over time, changes not seen in age‐matched male rats. Given CVD is significantly lower in premenopausal women, lower sympathetic nerve activity as a result of puberty may provide a mechanistic basis by which decreased sympathetic burst frequency protects against CVD. Future experiments may be conducted to explore other burst characteristics (e.g. burst amplitude and width), and the role of specific female sex hormones, to further understand sex‐dependent development of CVD.
Diaphragm EMG activity during hyperbaric oxygen exposure in spinal injured rats
Authors: Bindi Victoria E. , Sunshine Michael D. , Benevides Ethan S. , Fuller David D.
Published: May 2022

Cervical spinal cord injury can cause significant respiratory dysfunction and in severe cases necessitates mechanical ventilation. Accumulating evidence indicates that hyperbaric oxygen (HBO) therapy, initiated shortly after spinal cord injury (SCI), can mitigate spinal cord pathology. Further, a recent study from our group demonstrated that HBO therapy can attenuate diaphragm atrophy after SCI. The primary purpose of the present investigation was to evaluate diaphragm electromyogram (EMG) activity during HBO therapy in rats with cervical SCI. HBO therapy has not been widely adopted after SCI, and if HBO is to be translated to human use, we reasoned that it is important to determine how breathing is impacted during HBO exposure. Adult female Sprague‐Dawley rats were implanted with chronic indwelling EMG electrodes in the mid‐costal diaphragm two weeks prior to SCI. Following hemicontusion injury of the C4 spinal cord, one experimental group was treated with HBO (n=3), and another group was left untreated (n=3). The HBO treatment consisted of a one hour exposure to 100% O2 at 3 atmospheres (ATA). In all experiments, diaphragm EMG activity was recorded in freely moving rats, without anesthesia. Recordings were made for 10 minutes prior to treatment, throughout the HBO exposure, and for 10 minutes after. Diaphragm EMG recordings in the normobaric normoxia control group conditions were completed for the same duration of time as the treated group. Daily HBO treatment and EMG recordings were done until 10‐days post‐SCI. Peak diaphragm EMG burst amplitude was quantified before SCI, over the 10 days of treatment, and at 1 and 2 weeks post‐treatment. Prior to SCI, body weight was similar between groups (control: 227±4 g; HBO: 228±3 g). All rats with tolerated HBO treatment with no evidence of adverse events or impact on weight gain over the 10 day period (10 day body weight: control: 198 ± 14 g; HBO: 197 ± 13 g). The acute HBO exposure caused an increase in the area under the curve of the rectified and integrated inspiratory burst amplitude of the diaphragm recording made ipsilateral to the lesion. This increase averaged 126 ± 18% of baseline across the 10 HBO treatments. The ipsilateral EMG burst amplitude also remained elevated at 10 min post‐HBO exposure (115 ± 32%) compared to pre HBO exposure. At the conclusion of the 10 day treatment paradigm, ipsilateral diaphragm EMG output during quiet breathing was elevated in HBO (156 ± 50% of post‐injury baseline) as compared to the control group (111 ± 6% of baseline). However the ipsilateral diaphragm EMG burst amplitude of the two groups were similar when evaluated 2 weeks after the daily HBO sessions concluded. This ongoing study indicates that acute exposure to HBO therapy is associated with an increase in diaphragm EMG activity, and 10 days of HBO therapy accelerates the recovery of diaphragm activation following cervical contusion injury.
Vascular anatomical variants found in the VH Dissector Pro software. Descriptive anatomical study
Authors: Bruna Alejandro , Orellana Mathias , Santana Andres , Valenzuela Juan Jose , Nova Pablo
Published: May 2022

Introduction

With advances in technology over the past decades, reductions in teaching time,

increasing class sizes, and increasing costs of cadaver‐based instruction, computer‐

based learning resources are used every increasingly in anatomy curricula to encourage

student learning, and its use has been found to increase anatomy teaching, enhance

independent learning, problem solving, and provide flexibility.

Methods

Using the VH Dissector Pro software version 6.0.16, a group of researchers through dissection tools reached the vascular planes of the cadaver in all body regions, in which the presence of some type of arterial vascular variant was searched.

Results

After having carried out the search in the VH Dissector Pro software version 6.0.16, it was found that in the upper limbs the deep brachial artery presented its origin at the level of the third portion of the right axillary artery and the left subscapular artery. An accessory renal artery was found bilaterally in the abdominal region. In the pelvic region, an origin was found bilaterally at the level of the posterior trunk of the internal iliac of the obturator artery. All these findings were found after analyzing all the body regions of the cadaver in the arterial vascular plane.

Conclusion

The knowledge of normal anatomy and anatomical variants is of great importance for anatomists who teach in different careers in the health area, as well as for clinical professionals who could find this type of variations both in diagnostic processes and surgical. By presenting this knowledge they will be able to transmit and differentiate normality versus the presence of anatomical variants that not only can be in cadavers, in imaging samples but could also be present in virtual anatomy. In addition, this study leaves the concern of analyzing other systems that could also present some other anatomical variant.
Sex Differences in Hydrogen Peroxide‐Induced K+ Channel Activation in Arterioles of Ischemic Myocardium after Exercise Training
Authors: Johnson Kalen , Bray Jeff F. , Heaps Cris F.
Published: May 2022

The objective of this study was to determine sex differences in potassium (K+) channel contribution to H2O2‐mediated dilation after exercise training in arterioles distal to chronic coronary artery occlusion. We tested the hypothesis that females would have enhanced H2O2‐mediated vasodilation compared to males through enhanced coupling of H2O2 with large‐conductance, calcium‐dependent K+ (BKCa; iberiotoxin 100 nM) channels and 4‐aminopyridine (4‐AP; 1 mM)‐sensitive voltage‐gated K+ (Kv). Adult Yucatan miniature swine were surgically instrumented with an Ameroid constrictor around the proximal left circumflex coronary artery, which gradually induced occlusion and created a collateral‐dependent vascular bed. Arterioles from the left anterior descending artery myocardial region served as nonoccluded control vessels. Pigs of each sex were separated into two groups: one that underwent a progressive exercise training program (treadmill run; 5 days/week for 14 weeks) and another that remained sedentary and was limited to normal pen activity. Collateral‐dependent arterioles from female, sedentary pigs were significantly less sensitive to H2O2‐induced dilation compared with nonoccluded arterioles (EC50: ‐4.55 ± 0.15 vs. ‐4.91 ± 0.15 log M, respectively; P<0.05), whereas exercise training reversed the impaired dilation so that collateral‐dependent and nonoccluded arterioles responded similarly to H2O2 (EC50: ‐4.86 ± 0.16 vs. ‐4.95 ± 0.18 log M, respectively). Conversely, male pigs demonstrated enhanced vasodilation to H2O2 in collateral‐dependent compared with nonoccluded arterioles (‐4.92 ± 0.16 vs. –4.35 ± 0.16 log M, respectively) of sedentary pigs, which persisted after exercise training (‐4.94 ± 0.11 vs. ‐4.50 ± 0.21 log M, respectively). In female sedentary pigs, H2O2‐mediated dilation in nonoccluded or collateral‐dependent arterioles was not altered by inhibitors of BKCa or Kv channels. In contrast, after exercise training, Kv channels contributed significantly to dilation in nonoccluded arterioles and both BKCa and Kv channels played a significant role in dilation of collateral‐dependent arterioles in female pigs. Blockade of BKCa or Kv channels in nonoccluded or collateral‐dependent arterioles from male pigs had no effect on H2O2‐mediated vasodilation, independent of exercise‐training. Taken together, our studies suggest that coronary arterioles in females compensate for impaired H2O2‐mediated vasodilation by modulating BKCa and 4‐AP‐sensitive Kv channels after exercise‐training. Males do not appear to utilize the particular pathway involving H2O2 action on BKCa or 4‐AP‐sensitive Kv channels.
Pulmonary endothelial cells from different vascular compartments exhibit unique recovery from acidification and Na+/H+ exchanger isoform expression
Authors: Sayner Sarah L. , Choi Chug‐Sik , Adams Dylan
Published: May 2022

Sodium‐hydrogen exchangers (NHEs) tightly regulate intracellular pH (pHi), proliferation, migration and cell volume. Heterogeneity exists between pulmonary endothelial cells derived from different vascular segments, yet the activity and isoform expression of NHEs between these vascular segments has not been fully examined.

Utilizing the ammonium‐prepulse and recovery from acidification technique in a buffer lacking bicarbonate, pulmonary microvascular and pulmonary artery endothelial cells exhibited a similar rate of recovery from the acid load regardless of the concentrations of the sodium transport inhibitor, amiloride; however, pulmonary artery endothelial cells required a higher dose of amiloride to inhibit sodium‐dependent acid recovery compared to pulmonary microvascular endothelial cells, suggesting a unique complement of NHEs between the different endothelial cell types. While NHE1 has been described in pulmonary endothelial cells, all NHE isoforms have not been accounted for. To address NHE expression in endothelial cells, qPCR was performed. Using a two‐gene normalization approach, Sdha and Ywhag were identified for qPCR normalization and analysis of NHE isoforms between pulmonary microvascular and pulmonary artery endothelial cells. NHE1 and NHE8 mRNA were equally expressed between the two cell types, but NHE5 expression was significantly higher in pulmonary microvascular and pulmonary artery endothelial cells.

Thus, pulmonary microvascular and pulmonary artery endothelial cells exhibit unique NHE isoform expression and have a unique response to acid load revealed through recovery from cellular acidification.
Anti‐S2 Protection in COVID‐19 Infection and SARS‐CoV‐2 Spike Vaccination
Authors: Canziani Gabriela A. , Tang Jackie , Nangarlia Aakansha , Zhang Shiyu , Fazloon‐Hassen Farah , Taludker Choya , Connors Jennifer , Cusimano Gina , Bernui Mariana , Bell Matt , Kutzler Michele , Haddad Elias , Cairns Charles , Chaiken Irwin
Published: May 2022

The overall goal of this project is to define the magnitude, quality, and duration of the primary immune response elicited against SARS‐CoV‐2 Spike by measuring domain‐specific antibody abundance and binding characteristics in plasmas after infection and vaccination. This investigation has enabled initiation of the screening of convalescent plasma polyclonal antibody (pAb) abundance and specificity through the IMPACC (Immunophenotyping Assessment in a COVID‐19 Cohort) at Drexel U College of Medicine (DUCOM) in collaboration with Tower Health Hospitals. We measured the active concentration of pAbs specific for RBD, S1 and S2 domains using SPR (surface plasmon resonance) molecular interaction analysis. By adopting a kinetic format, a complementary SPR analysis step was optimized to determine the binding rates and affinities of elicited antibodies targeting each domain of the Spike using the same plasma dilution aliquot. Most importantly, we found that the abundance of S2 reactive antibodies was comparable to that of anti‐S1 and RBD in convalescent plasmas. Plasmas obtained up to 6 months post‐vaccination are also becoming available through the TTC (Vaccination TetraCore cohort) assessment at DUCOM, and screening for these has demonstrated that anti‐S2 pAbs are also elicited, though intriguingly in lower abundance than after infection. To assess the importance anti‐S2 antibodies from convalescent plasmas, we purified anti‐S2 fractions by an SPR‐based microaffinity method and used the recovered antibodies in pseudovirus infection inhibition assays of ACE2 expressing cells to measure neutralization activity. Evidence for sustained generation of S2 antibodies up to 6 months post‐infection and occurrence of neutralizing anti‐S2 pAbs has begun to emerge with the possibility that antibodies targeting the S2 domain of the SARS‐CoV‐2 spike protein complex could provide pan‐coronavirus protection against COVID‐19, emerging variants, and other coronaviruses with conserved spike structures. Targeting the more conserved fusion machinery in the virus spike ultimately can lead to therapeutic antibodies or small molecule inhibitors effective on escape variants that occur mainly in S1 as well as other coronaviruses.
Spectral Changes in Skin Blood Flow Do Not Reflect Pressure Manipulations
Authors: Lima Natalia S. , Tzen Yi‐Ting , Clifford Philip S.
Published: May 2022

Skin blood flow is commonly assessed by laser Doppler flowmetry. It has been suggested that pathophysiological conditions can be assessed by analysis of frequency domains of the laser Doppler signals. The frequency range of 0.06‐0.15 Hz has been associated with myogenic control of skin blood flow. We tested the hypothesis that changes in blood pressure would be detectable by short‐time Fourier transform of the myogenic portion of the laser Doppler signal. Laser Doppler sensors were placed on the right ventral forearm of fourteen healthy young volunteers (8 males, 6 females, age 30±6 years, mean ± SD) and a photoplethysmography sensor on the middle finger of the right arm. Measurements of skin blood flow were made with the right arm supported at heart level. Intravascular pressure of the forearm was manipulated by positioning the arm ~50˚ above the heart level to decrease pressure and lowering the arm ~50˚ below the heart level to increase pressure. Short‐time Fourier analyses were computed over the range 0.06 to 0.15 Hz. There were no statistically significant differences (p = 0.526) in spectral densities for baseline (5±7 x 10‐4dB, median ± SD), arm above heart (7±54 x 10‐4dB), and arm below heart (7±14 x 10‐4dB). The results demonstrate that spectral density in the myogenic frequency range is unchanged by manipulation of intravascular pressure. We conclude that spectral analysis of laser Doppler signals in the myogenic range does not reflect changes in blood pressure. These findings suggest that myogenic changes in the skin vasculature cannot be inferred from spectral analysis of laser Doppler signals.
Role of ACAT1 in Atherosclerosis and Alzheimer's Disease
Authors: Link Tina , Reddy Megan , Agasthi Sarayu , Handa Samir , Manikkoth Shria , Ray Allison
Published: May 2022

The acyl‐coenzyme A: cholesterol acyltransferase (ACAT) intracellular membrane protein is located in the endoplasmic reticulum and is composed of 427 amino acids. ACAT is a part of the membrane‐bound O‐acyltransferase enzyme family (MBOAT). MBOATs are recognized by their most carried residues, histamine, and arginine, and are used in lipid synthesis, phospholipid remodeling, and peptide acylation. The enzyme itself is structured as a tetramer split into two homodimers. Nine transmembrane helices or domains (TMs) are on each monomer, the most notable being a cavity formed through TMs 4‐9 harboring histidine, an amino acid tied to catalysis and enzymatic activity.

The ACAT1 protein‐coupled with its ACAT2 counterpart catalyze the storage of cholesteryl esters. ACAT2 differs from ACAT1 in function and structure. ACAT2 contains 2 domains and is mainly expressed in intestinal cells and controls lipoprotein particle secretion. The enzyme transfers an acyl group to 3β‐hydroxyl of cholesterol, a cholesterol‐binding site, to form cytosolic lipid droplets that are used for reducing membrane fluidity of the cell. Lipo‐protein‐induced ACAT in microphages is key to the creation of atheroma foam cells, cells characterized by the aggregation of these fatty plaque deposits on the walls of arteries, thus appearing like foam. The aggregation of this plaque to eventually narrow and harden arteries to obstruct blood flow is atherosclerosis. ACAT1 also maintains homeostasis during periods of cholesterol overload in the ER. Other functions of the protein include aiding lipoprotein synthesis and dietary LDL absorption.

Recent studies have seen a pathogenic connection between cholesterol and Alzheimer’s disease or other neurodegenerative diseases. Cells contain a membrane that contains microdomains rich in cholesterol. The microdomains assist in the maintenance and growth of the cell and cell membrane; however, it is the disruption of these microdomains through individual disease‐causing toxins that cause them to inactivate ACAT1 and block cholesterol storage, disrupting brain activity.

ACAT is currently being studied for its targeted drug therapy countering atherosclerosis. Current studies have proved ACAT inhibition to be ineffective, but there still remains potential immunotherapies to neurodegenerative disorders through the ACAT enzyme. Further research remains to be done to analyze the link between neurodegenerative disorders and the ACAT1 protein.
A Mechanistic Role for RECK in the Regulation of Hepatocellular Inflammation
Authors: Dashek Ryan J. , Diaz Connor J. , Chandrasekar Bysani , Rector R Scott
Published: May 2022

RECK (Reversion Inducing Cysteine Rich Protein with Kazal Motifs), a membrane‐anchored glycoprotein, modifies the extracellular matrix involved in various cancers, including hepatocellular carcinoma (HCC). Its role in regulating inflammatory and fibrogenic processes has also been postulated. Both inflammation and fibrosis contribute to progression of nonalcoholic steatohepatitis (NASH) to HCC. Here, we tested the hypothesis that inducing or sustaining RECK expression will inhibit proinflammatory amphiregulin and epidermal growth factor receptor (EGFR) signaling, progression of NASH and the development of HCC using an in vitro cell culture model employing isolated adult mouse primary hepatocytes. Ectopic RECK overexpression (gain‐of‐function) was achieved by the adenoviral (Adv) transduction of murine RECK cDNA. Adv.GFP served as a control. RECK expression was silenced (loss‐of‐function) by transducing RECK‐specific siRNA using lipofectamine. Nonspecific siRNA served as a control. Hepatocytes were exposed to TNFα in the presence or absence of inhibitors against ADAM 10 (A Disintegrin and Metalloproteinase Domain‐Containing Protein 10) and/or ADAM17 – two well‐established pro‐inflammatory sheddases. Results show that RECK overexpression inhibited TNFα‐induced ADAM10/17 activity, and amphiregulin (an EGFR ligand) and EGFR expression. In the presence of ADAM10/17 inhibitors, no further reduction was observed in amphiregulin and EGFR activity, suggesting a maximal inhibitory effect of RECK on EGFR activity. In contrast, silencing RECK significantly increased amphiregulin secretion, and this effect was reversed by inhibition of ADAM10/17, indicating RECK signals via regulation of these sheddases. Thus, RECK not only regulates sheddase activity of ADAM10 and 17, but also downstream amphiregulin and EGFR signaling under pro‐inflammatory stimuli. Because increased and sustained EGFR activity contributes to progression of NASH to HCC in preclinical models, our results indicate that inducing RECK has the potential to inhibit hepatocellular inflammation in the setting of NASH and HCC.
Moderate Intensity Exercise‐Induced Shear Stress Upregulates eNOS mRNA Expression
Authors: Gurovich Alvaro N. , Cabral Shelsea , Rodriguez Carmen , Conde Daniel , Garcia Mario
Published: May 2022

Background

Physical activity has been shown to reduce the risk of cardiovascular disease. Exercise‐induced endothelial shear stress (ESS) produces a mechanical force on the surface of endothelial cells that triggers an intracellular signaling response regulating vascular function. SIRT‐1 and eNOS are mechanosensitive genes that have been shown to improve vascular function and their expression levels may be upregulated in response to higher ESS levels. Although it has been reported that higher physiological levels of ESS may improve vascular function, the role of pulsatile exercise induced ESS varying in exercise intensities has not been studied. Therefore, the purpose of this study was to determine the effects of varying pulsatile exercise‐induced ESS intensities on NOS3 and SIRT‐1 mRNA expression.

Methods

Commercially available human umbilical vein endothelial cells (HUVEC; Sigma‐Aldrich, St Louis, MO) were cultured until 90–100% confluence. Exercise‐induced ESS in vivo was obtained from our previous reports. Cultured HUVEC cells were exposed to pulsatile resting ESS (18 dynes/cm2) for 2 hours, followed by 1 hour at low‐intensity exercise‐induced ESS (35 dynes/cm2), moderate exercise‐induced ESS, or high‐intensity exercise‐induced ESS (75 dynes/cm2) on a closed circuit pump and channeled slide (Ibidi pump system (PumpControl Software 1.5.4) and slides, Ibidi Inc., Fitchburg, WI). Total RNA was extracted and reverse transcribed followed by measurement of SIRT‐1 and eNOS mRNA expression via qRT‐PCR. qRT‐PCRs were performed in duplicates and changes in gene expression were calculated using the ΔΔ‐CT method with GAPDH used as the normalizing control gene. Statistical analysis was performed using graphpad prism 8 software (Graphpad Software.). Significance was considered at p<0.05.

Results

There were no alterations in mRNA expression of SIRT‐1 in response to varying exercise intensities. Moderate intensity exercise‐induced ESS produced significantly higher eNOS mRNA expression compared to low and high intensity exercise‐induced ESS (p<0.05).

Conclusions

Our data shows that moderate intensity of exercise‐induced ESS may promote an anti‐atherogenic phenotype due to higher expression levels of eNOS. Although SIRT‐1 mRNA levels were not altered in response to varying exercise intensities, it is possible that SIRT‐1 is chronically activated and one bout of exercise will not alter its expression. Further research needs to be performed on the role of exercise and molecular pathways for endothelial health.
Male Mice Expressing Human APOE4 Genotype Exhibit Greater Baseline Respiratory Rate and Less Robust Hypoxic Ventilatory Response Than APOE3 Mice as Measured by Whole Body Plethysmography
Authors: Taylor Chase E. , Newton Jessica S. , Calulot Chris M. , Silverstein Aaron L. , Strattan Lydia E. , Johnson Lance A. , Alilain Warren J.
Published: May 2022

Breathing is vital for life and is both uniquely complex and vulnerable to disruption. Disorders of breathing such as those that occur in sleep apnea and even high cervical spinal cord injury (SCI) can impair quality of life and potentiate the ill effects of comorbidities like Alzheimer’s disease (AD). Indeed, studies have shown greater cognitive and cardiovascular impairments in subjects with sleep apnea and AD. Apolipoprotein E (ApoE) is a ubiquitous protein found throughout the body and CNS which aids in the regulation and transport of cholesterol and lipids. Moreover, the E4 allele confers a higher risk of early onset AD than either the E2 or E3 alleles. Previous research in our lab has shown the interaction between ApoE genotype and sex differentially affects breathing motor plasticity following SCI in mice. However, it is unknown how baseline respiratory pattern and capability during hypoxic challenge may differ between uninjured individuals expressing APOE4 and E3 alleles. Thus, with APOE genotype as our independent variable, we measured the respiratory parameters of male E3 and E4 targeted replacement mice using whole body plethysmography (WBP), hypothesizing that E3 animals would display a more adaptive, heightened response to hypoxic exposure than those expressing E4. Preliminary data comparing E3 animals (n=9) and E4 animals (n=8) demonstrated differences across several key measurements. At baseline, E4 animals were shown to breathe in an energy inefficient manner as exemplified high measures in tidal volume and frequency. During sustained hypoxia, frequency, tidal volume, and minute volume initially increased across all animals, but APOE3 animals mounted a much more robust and sustained response with APOE4 values falling well below baseline measures prior to termination of hypoxia. These preliminary results suggest a critical role of the APOE genotype in regulation of baseline breathing and in response to hypoxic conditions and that possessing the APOE4 allele may confer vulnerabilities in respiratory function and during environmental challenges leading to cognitive decline. Future directions include examining anatomically critical CNS respiratory regions and neural cell function.
Polycystin 1 ciliary localization is regulated by its aGPCR activity
Authors: Gresko Nikolay , Caplan Michael
Published: May 2022

Autosomal dominant polycystic kidney disease (ADPKD) develops due to loss of function mutations in the PKD1 and PKD2 genes, which encode PC1 and PC2, respectively. PC1 is a 460kD multi‐spanning membrane protein that undergoes multiple proteolytic cleavages, at least two of which release C‐terminal fragments. Polycystin 1 and Polycystin 2 both prominently localize to the primary cilium where they contribute to cellular mechano‐sensation and also form a cation‐permeable heterotetrameric channel that may contribute to Ca2+ flux into the cilia. Recent studies demonstrate that PC1 can function as an atypical adhesion GPCR that is activated by Wnt ligands. Wnt ligand binding leads to dissociation of the PC1 extracellular N terminal fragment and allows the PC1 receptor function to be activated by a tethered agonist peptide that resides at the N terminus of the PC1 transmembrane domains.

Here we report that Polycystin 1 cilia and cell surface localization is regulated by its activity status. We demonstrate that a constitutively active form of the Polycystin 1 protein is absent from the primary cilia whereas a constitutively inactive version of the PC1 protein that lacks its tethered agonist peptide resides in the primary cilia. Moreover, we show that interruption of the cellular receptor desensitization process by β‐arrestin inhibition permits the constitutively active PC1 construct to accumulate in the cilium. We also studied the surface localization of the full length PC1 and found that extended Wnt9b ligand treatment or mechanical stimulation both lead to reduced quantity of the full length PC1 at the cell surface and in the primary cilium, a process that can be disrupted by inhibition of β‐arrestin. In vivo, we find that reduction of the luminal flow in renal tubules by ureter obstruction leads to accumulation of the PC1 in the primary cilia of the tubule cells.

Taken together, our data suggest that PC1 surface trafficking is regulated by receptor‐activating stimuli including ligand binding and mechanical stress and, similar to the regulation of aGPCR receptors, is subject to an activity‐dependent receptor desensitization process. Our findings indicate that the aGPCR receptor‐like properties of Polycystin 1 offer new insights into this protein’s physiological function and the regulation of its trafficking. Understanding these processes may suggest targets for the development of new therapeutic interventions for ADPKD.
A Model of Potassium‐Assisted Olfactory Sensory Neuron Response to Odorant
Authors: Singletary Melissa , Murphy Karen E. , Pustovyy Oleg , Globa Ludmila , Vodyanoy Vitaly
Published: May 2022

Odorants interact with specialized G‐protein coupled receptors lining the cilia of the olfactory sensory neuron initiating an intracellular cascade of signal transduction events resulting in cAMP‐gated ion channel opening and generation of ion currents. These ion currents polarize the cilia membrane resulting in the voltage transition across the olfactory epithelium, which can be recorded as an electroolfactogram (EOG). The observed dose‐dependent change of the EOG amplitude after stimulation of olfactory epithelium with the mixture of odorant and electron donors and acceptors indicates a potential involvement of electrons in the initial events of signal transduction. Our model of signal transduction consists of a dimer of the G‐protein coupled olfactory receptors positioned vertically in the phospholipid bilayer between olfactory extracellular mucus layer and the intracellular cytoplasm, within the cilia of the olfactory sensory neuron. In this model, two identical receptors of the dimer form an odorant activated potassium ion channel. Prior to odorant binding at the receptor, the ion channel gate is closed. Each receptor has a single biding site for the odorant molecule on the extracellular end located within the mucus layer, and a binding site for the heterotrimeric G‐protein complex within the cytoplasm. The brief binding of odorant molecules to the mucosal part of the receptors causes the ion channel to open and pass intracellular potassium ions to the extracellular mucus layer. The potassium‐coupled electrons polarize the cytoplasmic portion of the receptors and facilitate activation of the bound G‐protein complex thereby influencing the subsequent signal cascade.
Acute Hypernatremia Increases Functional Connectivity of Sodium Sensing Regions in the Brain
Authors: Stock Joseph M. , Burciu Roxana G. , Chung Jae Woo , Romberger Nathan T. , McMillan Ronald K. , Patel Darshil , Salhab Ibrahim , Stocker Sean D. , Farquhar William B.
Published: May 2022

Rodent studies have identified specialized sodium chloride (NaCl)‐sensing neurons in the circumventricular organs (CVOs), which mediate NaCl‐induced changes in sympathetic nerve activity, arginine vasopressin (AVP), thirst, and blood pressure (BP). In humans, acute hypernatremia has been shown to increase sympathetic nerve activity, AVP, thirst and BP. However, few human studies have investigated the network of NaCl sensing regions of the brain using functional magnetic resonance imaging (fMRI).

Objective

To determine how the functional connectivity of sodium sensing regions of the brain change during an acute hypernatremic perturbation.

Methods

Resting‐state fMRI was performed on 9 healthy young adults (29±4 yrs, 5M) at baseline and during a 30 min 3% NaCl intravenous infusion at a rate of 0.15 ml/kg/min. Venous blood samples and perceived thirst (Likert scale, cm) were obtained before and immediately following the infusion. Serum electrolytes, plasma osmolality, hematocrit, and hemoglobin were assessed. Beat‐to‐beat blood pressure was measured continuously. Seed‐based voxel‐wise functional connectivity analysis was performed in AFNI using spherical seeds placed bilaterally in the subfornical organ (SFO), the organum vasculosum lamina terminalis (OVLT) as well as cortical sensory regions such as the insula. Functional connectivity was compared at baseline vs. early infusion (min 0‐15) and baseline vs. late infusion (min 16‐30) at p < 0.01, corrected for multiple comparisons with 3dClustSim.

Results

Thirst (∆1.9±2.0 cm), serum sodium (∆3.2±2.1 mmol/L), and plasma osmolality (∆6.9±4.7 mOsm/kg H2O) all increased from baseline to post‐infusion (p<0.05). Head motion was minimal and did not change throughout the stages of the infusion (mean motion = 0.11±0.10 mm, p>0.80). Hypertonic saline infusion increased local functional connectivity between the SFO and OVLT (Z score baseline=0.07±0.01, Z score late phase=0.17+0.01, p<0.01). A similar increase in functional connectivity was seen between the insula and somatosensory cortex.

Conclusion

Acute hypernatremia increases local functional connectivity within the CVOs and between cortical sensory regions of the brain.
Reduced expression of SOD2 in the Retrotrapezoid nucleus is associated with enhanced ventilatory chemoreception: implications for breathing disorders in heart failure
Authors: Díaz‐Jara Esteban , Toledo Camilo , Schwarz Karla G. , Pereyra Katherin , Diaz Hugo , Del Rio Rodrigo
Published: May 2022

Enhanced chemoreflex drive and irregular breathing patterns are both hallmarks in heart failure (HF). The Retrotrapezoid nucleus (RTN), a main area for central chemoreception, contribute to maintain both heightened chemoreflex drive and irregular breathing in experimental HF. Recently, we found that high levels of reactive oxygen species (ROS) within the RTN of HF rats were closely linked to the enhanced central chemoreflex and the generation of breathing disorders. Despite this evidence, the effects of ROS on RTN‐mediated chemoreception and the precise mechanism(s) associated with ROS generation in the RTN has not been previously studied. However, pro/anti‐oxidant enzymes gene screening in RTN from HF rats revealed reduce expression of super oxide dismutase 2 (SOD2). Therefore, in the present study we aimed to determine whether reductions in SOD2 expression in healthy subjects resembles i) high RTN ROS levels, ii) heightened chemoreflex drive, and iii) generation of breathings disorders observed in HF animals. Accordingly, breathing function and central chemoreflex drive were assessed in freely moving heterozygous SOD2+/‐ mice by unrestrained whole‐body plethysmography. ROS levels were determined in brainstem sections containing the RTN using dihydroethidium staining. SOD2 protein expression and tissue localization were determined by immunoblot and immunofluorescence, respectively. SOD2+/‐ mice displayed reduced SOD2 protein expression (65.6 ± 3.2 vs. 100.0 ± 5.8 %; SOD2+/‐ vs. WT, respectively) and high ROS levels at the RTN compared to WT littermates (2.50 ± 0.46 vs. 1.00 ± 0.05 a.u.; SOD2+/‐ vs. WT, respectively). In addition, SOD2+/‐mice displayed an exacerbated ventilatory response to hypercapnia compared to WT mice (3.5 ± 0.2 vs. 1.9 ± 0.2 ml/min/10g; SOD2+/‐ vs. WT, respectively). Importantly, the latter was closely linked to RTN ROS production since a positive correlation (p<0.05) between ROS levels and the hypercapnic ventilatory response was found only in SOD2+/‐animals. Furthermore, SOD2+/‐ mice showed overt signs of disordered breathing while resting at eupneic conditions compared to WT mice as evidenced by large increases in ventilatory instability and apneas/hypoapneas incidence (AHI: 7.7 ± 0.9 vs. 3.4 ± 0.6 events/hr; SOD2+/‐ vs. WT, respectively). Lastly, we found that SOD2 expression is preferentially confined to RTN neurons rather than to medullary astrocytes. Our results shows that reductions in the SOD2 gene expression in healthy rodents mimic the respiratory phenotype observed in experimental HF and suggests that reduced expression of SOD2 within the RTN of HF animals may account for the local increases in ROS production, the enhanced central chemoreflex drive and the generation/maintenance of irregular breathing patterns.