bluuebunny/crossref_metadata_2025 huggingface dataset:
Blocking the entry of HIV into host cells through co‐receptor inhibition
Authors: Ralli Garyfallia , Pedzisa Lee
Published: May 2022

The goal of our project is to synthesize a dual CXCR4/CCR5 co‐receptor inhibitor to block the entry of HIV into host cells.

In the early phase of the HIV‐1 replication cycle, HIV‐1 binds to host cells through the CD4 protein present on the host cell surface. To infect the cell, HIV‐1 requires further interactions that promote fusion of the viral and cellular membranes. This can occur through binding to the chemokine co‐receptors such as CXCR4 and CCR5. We are choosing dual inhibition since under selective pressure of a CCR5 antagonist, CXCR4‐using strains have been shown to predominate. Through dual inhibition, we want to account for a possible resistance development that could occur when inhibiting only one of the two co‐receptors.

Using a computational screen, a compound predicted to bind to both CCR5 and CXCR4 was identified (Hit 1). We established a reaction scheme to synthesize an analog of this compound (target compound 4) through solid phase peptide synthesis (Scheme 1). We completed the synthesis of compound 4 and we confirmed the addition of the first two amino acids (Fmoc‐phenylalanine and Fmoc‐proline) through LC‐MS and HPLC.

We are currently expecting LC‐MS and HPLC results for the rest of the synthesis steps. Before the time of presentation, we plan to use our established methodology to synthesize multiple analogs through varying amino acids and evaluate them for biological activity. The goal of synthesizing analogs is to optimize CXCR4/CCR5 dual inhibition.
Expression and Purification of Corticosterone Binding Globulin in E. colifor Quantification
Authors: Ridley Lauryn A. , Malisch Jessica L. , Odunuga Odutayo O. , Mertz Pamela S.
Published: May 2022

Corticosterone (CORT) is analogous to the mammalian stress hormone cortisol and is involved in the acute stress response in birds. Birds can be used as a model of the stress response due to the evolutionarily conserved activation of the hypothalamic‐pituitary‐adrenal (HPA) axis and sympathetic adrenomedullary (SA) system across vertebrate species. Corticosterone binding globulin (CBG) is a protein that binds and carries approximately 80% of corticosterone in the plasma in both avian and mammalian species. There are limitations in the most common technique for quantification of CBG due to the use of radioactive binding assay. The ultimate goal of this project is to develop a robust and safe method to quantify CBG levels. This abstract reports the first part of that goal – the expression and purification of variously‐tagged zebra finch CBG from E. coli. CBG was expressed either as histidine‐tagged or in fusion with glutathione‐S‐transferase (GST) or maltose‐binding protein (MBP). Purified CBG was confirmed and assessed by SDS‐PAGE and western blot using antiserum to zebra finch CBG. Following purification, circular dichroism will be used to analyze the secondary structure of the recombinant CBG protein. The project aims to develop an ELISA‐based method to measure the amount of CBG in samples.
Brain dysmorphologies associated with Alzheimer disease in Down syndrome
Authors: Martínez Abadías Neus , Pardina Helena , Echeverry Luis M. , Carmona‐Iragui María , Sevillano Xavier , Heuzé Yann , Fortea Juan
Published: May 2022

Down syndrome (DS) is a genetic disorder occurring in 1 out of every 700 to 1,000 live births caused by trisomy of chromosome 21. The overexpression of the Amyloid Precursor Protein (APP) gene located on this chromosome leads to an early onset and rapid accumulation of ß‐amyloid (Aß) in the brain that invariably results in Alzheimer Disease (AD) in people with DS. Therefore, AD, the most common form of dementia, is currently one of the most relevant medical issues in people with DS. In this study, we assessed brain morphological patterns to quantify dysmorphologies associated with AD in Down syndrome.

Brain shape and size analyses were performed using Geometric Morphometrics in a sample of 243 adults, including 153 euploid healthy controls (EU) and 90 individuals with DS at different stages of dementia: asymptomatic DS with no signs of AD‐related cognitive impairment (N=58), individuals with DS and prodromal AD (N=21) and individuals diagnosed with AD dementia (N=11). A set of 38 3D brain landmarks representing the overall shape of the brain and internal brain structures (ventricles, corpus callosum, cerebellum and pons) were located on magnetic resonance images of the head.

Results showed a significant separation between EU and DS populations based on both global and local brain shape and size analyses. Individuals with DS displayed a smaller cerebellum, a protruding posterior midbody in the corpus callosum, a frontally projected brain and expanded ventricles. Significant brain shape differences were detected in individuals diagnosed with AD, which mainly affected the shape of enlarged brain ventricles. Remarkably, individuals at advanced stages of the disease displayed more severe dysmorphologies as compared to those individuals at prodromal stages and individuals with no signs of dementia, confirming a phenotypic continuum associated with the severity of dementia that highlights the impact of this neurodegenerative disorder in brain morphology.
Prefrontal‐medullary circuit activation attenuates stress reactivity
Authors: Pace Sebastian A. , Lukinic Ema , Wallace Tyler , Schaeuble Derek , Moore Jacob , Myers Brent
Published: May 2022

Organismal survival and adaptation to stress rely on brainstem catecholaminergic neurons. In particular, catecholaminergic neurons in the rostral ventrolateral medulla (RVLM) drive sympathetic activity and enable physiological adaptations, including vasoconstriction, corticosterone release, and glycemic mobilization. However, it is unclear how brain regions involved in the cognitive appraisal of stress regulate the activity of RVLM neurons. Our previous studies found that the rodent infralimbic prefrontal cortex (IL) integrates behavioral and physiological responses to stress. Thus, a potential IL‐to‐RVLM connection would represent a crucial link between stress appraisal and sympathetic reactivity. In the current study, we investigated a direct IL‐to‐RVLM circuit by targeting a genetically‐encoded anterograde tracer under the control of the CaMKIIα promoter to the IL of adult male and female rats. Analysis revealed that IL terminals apposed RVLM neurons expressing the catecholamine‐synthesizing enzyme dopamine beta hydroxylase. Further, IL input to catecholamine cells was widespread throughout the rostral to caudal extent of the VLM in both male and female rodents. Quantification of innervation density revealed that males had a larger proportion of VLM catecholamine neurons receiving IL inputs relative to female rats. Additionally, IL appositions were identified on GABAergic and glycinergic neurons in both sexes. Accordingly, we hypothesized that IL projections may activate local RVLM inhibitory cells to limit sympathetic output. To test this hypothesis, we injected a viral vector coding for channelrhodopsin‐2 (ChR2) in the IL of males and females. Next, a fiber optic cannula was implanted dorsal to the RVLM to evoke IL synaptic glutamate release. Animals then received photostimulation during restraint stress with blood sampled to determine stress reactivity. Compared to controls, male rats expressing ChR2 on IL terminals had suppressed glycemic stress responses (p < 0.05). In contrast, stimulation of the IL‐RVLM circuit in females did not affect glucose mobilization (p < 0.05). However, ChR2 decreased corticosterone responses to stress relative to control rats in both sexes (males, p < 0.01; females, p < 0.05). Thus, both male and female rats have a direct circuit from the IL portion of the mPFC to catecholamine‐synthesizing cells of the RVLM that limits glucocorticoid stress responses, likely through the activation of local inhibitory neurons. However, the density of this circuit is greater in males, potentially accounting for reduced glycemic responses. Ultimately, excitatory/inhibitory balance at IL synapses in the RVLM may be critical for the health consequences of stress.
Biological Consequences of HIV‐1 Interactions with Bacteria
Authors: Heindel Daniel W. , Chan Kun‐Wei , Petrova Mariya I. , Kong Xiang‐Peng , Lebeer Sarah , Bensing Barbara A. , Mahal Lara K. , Hioe Catarina E.
Published: May 2022

Microbiota dysbiosis due to bacterial vaginosis and other venereal diseases has been associated with an increased risk of HIV‐1 transmission and acquisition. The prevalent idea is that microbial perturbations compromise the mucosal integrity and create inflammatory conditions in the mucosa which results in the recruitment and stimulation of immune cells that harbor or are targeted by HIV‐1. However, it is also possible that HIV‐1 virions bind bacteria or bacterial products in a manner that impacts virus infectivity and transmissibility. This study aims to examine direct interactions between HIV‐1 and bacteria and to better understand the biological consequences of such interactions. Specifically, we focus on HIV‐1 interactions via glycan‐binding proteins, or lectins, expressed on bacterial surfaces. We investigated multiple lectins expressed by bacteria: FimH from uropathogenic Escherichia coli, MsL from vaginal and oral non‐pathogenic Lactobacillus plantarum, and three Siglec‐like adhesins (SLBR‐B, SLBR‐H, and SLBR‐N) from oral Streptococcus gordonii. FimH and MsL bind high mannose N‐glycans while the SLBRs bind different α‐2,3 sialylated O‐glycan structures. N‐ and O‐glycans have been identified on the surface of HIV‐1 on Env or host‐derived glycoproteins. Both FimH and MsL had no impact on HIV‐1 infectivity, while SLBR‐N increased HIV‐1 infection by up to 3‐fold. The observed effects of SLBR‐N were recapitulated by plant lectins specific for similar glycan epitopes, further validating these findings. We hypothesize that this enhancement effect could be due to accumulation of viral particles and/or bridging effects between the cell surface and HIV‐1 virions. Studies are in progress to decipher the molecular mechanisms for this enhancement effect. This data will help us better understand the importance of direct interactions between viral particles and bacteria in HIV‐1 acquisition.
Glucose consumption of vascular cell types in culture; toward optimization of experimental conditions
Authors: Torimoto Keiichi , Okuno Keisuke , Cicalese Stephanie , Eguchi Kunie , Elliott Katherine , Kawai Tatsuo , Eguchi Satoru
Published: May 2022

Any experimental outcomes are potentially influenced by extracellular glucose availability and its cellular metabolism. However, there is a lack of attention paid to the supply and utilization of glucose in many cultured experiments. Surveillance of vascular‐related journals for the past 5 years demonstrated that less than 20% of published articles declared the medium glucose concentration. The present studies were designed to seek ideal medium glucose concentration(s) in various cell types with particular attention paid to changes in glucose consumption. By using distinct glucose concentrations in Dulbecco's Modified Eagle's medium (DMEM), we identified ideal glucose media for stimulation experiments. We have compared glucose consumption in three vascular cell types, endothelial cells (EC), vascular smooth muscle cells (VSMC) and adventitial fibroblasts (AF) with or without angiotensin II stimulation. In all cell types after a 48‐hour incubation in relatively low glucose media (1 g/L), medium glucose concentration was reduced to almost 0 (EC 0.01±0.01 g/L, VSMC 0.13±0.05 g/L, AF 0.02±0.01 g/L). Whereas medium glucose concentration remained significantly higher (EC 2.77±0.05 g/L, VSMC 3.87±0.05 g/L, AF 3.32±0.01 g/L) when cells were incubated for 48 hours in high glucose (4.5 g/L) media. In middle glucose (2.75 g/L) media, medium glucose concentration remained in physiological ranges (EC 0.62±0.18 g/L, VSMC 1.98±0.07 g/L, AF 1.17±0.17 g/L). AngII treatment enhanced glucose consumption in AF and VSMC but not in EC. Enhanced extracellular acidification rate by AngⅡ was observed in AF as well as VSMC. In AF, AngII induction of target proteins at 48 hours varied depending on the glucose concentration used. In low glucose media induction of Grp78 or hexokinase II was highest, whereas induction of vascular cell adhesion molecule‐1 was lowest. However, no glucose preference was seen in AngII‐induced c‐Fos induction at 1 hour in AF. Utilization of specific inhibitors further suggest essential roles of AT1 receptor and glycolysis in AngII‐induced fibroblast activation. Overall, the present study demonstrates a high risk of hypo‐ or hyperglycemic conditions when standard low or high glucose media is used with vascular cells. Moreover, these conditions may significantly alter experimental outcomes. Medium glucose concentration should be monitored during any culture experiments and utilization of middle glucose media is recommended for all vascular cell types.
Effect of Mediators in the Plasma of E‐Cigarette Users on Endothelial and Epithelial Cell Metabolism
Authors: Chitteti Ramamurthy , Hertz Juan P. Z. , Masso‐Silva Jorge A. , Shin John , Alexander Laura E. C. , Patel Hemal H. , Roth David M.
Published: May 2022

Background

Tobacco smoking is a major risk factor for cardiovascular and lung disease. Inhalation of aerosols formed by electronic nicotine delivery systems (ENDS) such as E‐cigarettes (E‐cigs) may expose the user to harms beyond those of nicotine alone. Data on the cardiovascular risks of E‐cigs and vaping devices remain inconclusive. However, exposure to E‐cigs induces the release of multiple substances, including exosomes and metabolites, into the bloodstream. There is limited information on the role of these exosomes and metabolites on cellular homeostasis. Mitochondria are highly sensitive to cigarette smoke, however E‐cig aerosol induced changes in mitochondrial function are not well studied. We investigated the effects of E‐cig plasma mediators on metabolism and function in human vascular endothelial (EA.hy 926) and lung epithelial cells (A549).

Hypothesis

Plasma from E‐cig users will induce alterations in the metabolism and mitochondrial function of endothelial and lung epithelial cells.

Methods

A longitudinal cohort study was conducted on subjects who exclusively used E‐cigarettes (n=21) and 10 non‐smoking, non‐vaping subjects. We included active E‐cig use without known lung disease and with normal lung function, between the ages of 18‐30 years. E‐cig use was confirmed with in‐person interviews and plasma cotinine levels. Active E‐cig use was defined as use of >0.5‐1 mL e‐liquid/day or 3.5‐7 mL/week for >6 months. Plasma was isolated and stored at ‐70°C until exosomes were profiled. The effects of E‐cig plasma (1%, 1h conditioning) mediators were assessed in vitro on endothelial cells (8x104/well) and epithelial cells (5x104/well) using the Seahorse XF Cell Mito Stress Test.

Results

Results were standardized for cell number and FCCP concentrations for both EA.hy 926 and A549 cells. Maximal mitochondrial respiration and spare capacity were decreased in human vascular endothelial cells and epithelial cells treated with E‐cig plasma relative to plasma from non‐smoker/non‐vapers. Basal respiration and ATP‐linked respiration was not different after treatment with plasma from E‐cig users versus non‐smoker/non‐vapers in either cell type.

Conclusions

To our knowledge, our results are the first to suggest that factors in the plasma of E‐cig users lead to reduction in maximal metabolic rates of human endothelial and lung cells in vitro. These results have important implications regarding the effects of E‐cig use on cellular metabolism and mitochondrial function and eventual public health recommendations. We are currently investigating the underlying mechanisms of action of E‐cig plasma components on mitochondrial biology.
Triple isozyme LDH Inhibition in breast cancer cells does not alter energy production but negatively regulates cell proliferation independent of the absence of lactic acid
Authors: Mazzio Elizabeth , Mack Nzinga , Badisa Ramesh , Larson Brad , Barnes Andrew , Soliman Karam F. A.
Published: May 2022

Several aggressive human malignant tumors are characterized by an intense glycolytic rate, over‐expression of lactic acid dehydrogenase A (LDHA), and subsequent lactate accumulation, all of which contribute toward an acidic peri‐cellular immunosuppressive tumor microenvironment (TME). While recent focus has been directed at how to inhibit LDHA, it is now becoming clear that the multiple isozymes of LDH must simultaneously be inhibited to suppress lactic acid and halt glycolysis. In this study, we explore the biochemical and genomic consequences of the administration of triple LDH isozyme inhibitor (A, B &C) (GNE‐140) in MDA‐MB‐231 triple‐negative breast cancer cells (TNBC) cells. The findings confirm that GNE‐140 does, in fact, fully block the production of lactic acid, which also results in a block of glucose utilization and severe impedance of the glycolytic pathway. Without a fully functional glycolytic pathway, breast cancer cells continue to thrive, sustain viability, produce ample energy, and maintain mitochondrial potential (ΔΨM). The only observable negative consequence of GNE‐140 in this study was the attenuation of cell division, evident in both 2D and 3D cultures and occurring in fully viable cells. Of important note, the cytostatic effects were not reversible by the addition of exogenous lactic acid. The effects of GNE‐140 on the whole transcriptome were mild (12 up‐regulated differential expressed genes (DEGs) / 77 down‐regulated DEGs) out of the 48,226 evaluated, and downregulated DEGS collectively centered around a loss of genes related to mitosis, cell cycle, G0/G1; G1/S transition, and DNA replication. These data were observed by digital fluorescence cytometry and flow cytometry, both corroborating a G0/G1 phase blockage. In conclusion, the findings in this work suggest that there is an unknown element linking LDH enzyme activity to the cell cycle and that this factor is completely independent of lactic acid. The data also establish that complete inhibition of LDH in cancer cells does not affect cell viability or energy production.
Cardamonin Antioxidant Effects Through the Modulation of Proteins and Genes Expressions in LPS‐Activated BV‐2 Microglial Cells
Authors: Barber Kimberly , Mendonca Patricia , Soliman Karam F.
Published: May 2022

Neurodegeneration is characterized by slow and progressive loss of neuronal cells and synapses in different brain regions. These pathologic effects are the major feature of Alzheimer's disease (AD), leading to a decline in learning and memory function. The main hallmarks of AD are the aggregation and deposition of amyloid‐β peptides on the extracellular surface of neuronal cells. Hyperphosphorylation of tau protein and the formation of neurofibrillary tangles is another feature of AD. Neuroinflammation and oxidative stress follow the accumulation of amyloid‐β and neurofibrillary tangles. Continuous activation of microglia cells, known as the resident, innate immune cells in the brain, participates in producing numerous neurotoxic factors, including nitric oxide and reactive oxygen species (ROS), which induce neurodegeneration. Numerous natural compounds, such as cardamonin, a bioactive compound found in many plants, have been reported to exert multiple therapeutic properties, including anti‐cancer, anti‐inflammatory, antioxidant, antiviral antibiotic, antifungal, and antiallergic activities. This study examined the effects of cardamonin on LPS‐activated BV‐2 microglial cells. Results of this study showed that cardamonin concentrations of 6.25 to 50 µM reduced over 95% the release of NO in LPS‐activated BV‐2 cells, compared to the cells treated with LPS only (P<0.001). Moreover, cardamonin significantly decreased the cellular production of SOD 3‐fold (P < 0.05) and increased the levels of expression of CAT 2.5‐fold (P < 0.05) and glutathione 2‐fold (p < 0.05) in the LPS‐activated BV‐2 cells. In RT‐PCR assays, cardamonin increased the mRNA expression of CAT and decreased the levels of NOS2, the enzyme responsible for the production of NO and associated with inflammatory processes. Furthermore, cardamonin downregulated the mRNA expression of CCL5/RANTES (5‐fold), which promotes the recruitment and activation of inflammatory cells. Also, cardamonin downregulated SLC38A1 (3‐fold), whose expression is upregulated during oxidative stress, and TXNIP (2‐fold) that regulates thioredoxin, an important redox protein that controls levels of ROS in cells. The data show cardamonin efficacy in modulating proteins and genes involved in inflammation and the control of elevated intracellular levels of ROS. In conclusion, these findings suggest that cardamonin may have potential in the therapy of microglia‐derived neurodegeneration, helping to prevent or slow the progression of the disease in the CNS.
Thermoregulatory dysfunction in post‐weaning congenic corpulent rats
Authors: Tulp Orien L. , Outtrim Deborah , Einstein George P. , Awan Aftab R.
Published: May 2022

To assess the capacity for cold induced thermoregulation in congenic corpulent rats, groups of normally fed post‐weaning lean and obese LA/Ntul//‐cp rats 6‐9 weeks of age were subjected to a 24°C vs. 4°C environment for < 14 hours. Measures of resting VO2 (RMR) and the thermoregulatory responses to cold exposure, the thermic response to noradrenaline, thyroidal parameters, and measures of core and peripheral body temperatures following cold exposure were obtained. Body weights of obese were greater than in lean rats (p=<0.01), and 14 hours of 4°C exposure demonstrated phenotype effects on both core and rectal temperatures (lean > obese, rectal p=0.0012, core p= 0.0038). The RMR at thermal neutrality and the thermic response to 45 minutes of acute 4°C cold exposure was greater in lean than obese phenotype at all time points measured. The thermic responses to 100 vs 200 µg NE s.c. were greater in lean than in obese rats. Cold exposure induced a significant increase in circulating T3 and the T3:T4 ratios but T4 concentrations were unchanged by cold exposure challenge. Phenotype effects on outer ring T4‐5’ deiodinase activity/mg protein were present in Kidney and Liver (Lean > Obese) while Interscapular brown adipose tissue (IBAT) demonstrated both phenotype and cold induced effects (Obese > Lean). In contrast, T4‐5’deiodinase activity in gastrocnemius muscle tissue was similar in both phenotypes and lacked cold induced effects (p= n.s.). The results of this study indicate that an impaired capacity for thermoregulation occurs early in the lifespan of the corpulent rat, despite a robust increase in total IBAT 5’deiodinase activity and in circulating T3 concentrations following cold exposure in both lean and obese phenotypes. While the physiologic basis for the elevated T3 concentrations following cold exposure remains unclear and are likely secondary to the combined effects of optimizing peripheral T4 to T3 conversion rates in both phenotypes, and possible changes in kinetics of T3 receptor binding and clearance in IBAT and other tissues and which may contribute to a greater caloric efficiency and propensity for weight gain in the obese phenotype.
“Effects of Metformin on Attenuating Renal Dysfunction Through the Modulation of AMPK/PPARα Dependent Pathways in Obese Rats”
Authors: Lungkaphin Anusorn , Thongnak Laongdao , Promsan Sasivimon , Phengpol Nichakorn , Sutthasupha Prempree
Published: May 2022

Insulin signaling and lipid metabolism are disrupted in a condition of long‐term high‐fat diet (HF) consumption inducing insulin resistance, dyslipidemia and subsequent renal dysfunction as a consequence of the inactivation of AMP‐activated protein kinase (AMPK) and peroxisome proliferator‐activated receptor‐α (PPARα) pathway. We investigated the effects of metformin on renoprotective effect through the modulation of AMPK/PPARα dependent pathway in HF induced insulin resistant rats. Male Wistar rats were fed with HF for 16 weeks. After insulin resistance had been confirmed, metformin (30 mg/kg) or gemfibrozil (50 mg/kg) was given orally for 8 weeks. Insulin resistance, dyslipidemia, lipid accumulation and kidney injury were demonstrated in HF rats. Impairment of lipid oxidation, energy metabolism and renal organic anion transporter 3 (Oat3) expression and function were also exhibited in HF rats. Metformin could stimulate AMPK/PPARα pathway to regulate lipid metabolism. Moreover, renal inflammation markers, NF‐κB, COX2, iNOS expression, and renal fibrosis induced by HF were reduced in metformin treatment with the higher efficacy than gemfibrozil. Interestingly, renal Oat3 function and expression as well as kidney injury were improved in metformin and gemfibrozil treatment groups. Renal CD36 or SGLT2 expression were not different after treatment with metformin or gemfibrozil. In conclusion, metformin and gemfibrozil could restore the impairment of AMPK/PPARα signaling induced by HF to improve metabolic disturbance. Metformin demonstrated a greater efficacy than gemfibrozil in attenuating renal inflammation and fibrosis. The protective effects of metformin on renal injury induced by HF were involved in AMPK/PPARα dependent pathway.
Does Parkinson's Disease Affect Peripheral Circulation and Vascular Integrity?
Authors: Martignon Camilla , Pedrinolla Anna , Laginestra Fabio Giuseppe , Barbi Chiara , Giuriato Gaia , Schena Federico , Venturelli Massimo
Published: May 2022

Previous studies demonstrated that aging, neurodegeneration, and the level of physical activity are associated with vascular alterations. However, in Parkinson’s disease (PD) only cerebral vascular function has been investigated; instead, the contribution of PD on systemic vascular function and skeletal muscle circulation remains a matter of debate. In this study, the hyperemic response during the single passive leg movement test (sPLM) was examined at the level of the common femoral artery with an ultrasound Doppler system to assess systemic vascular function in 10 subjects with PD (PDG), compared with 10 aged‐sex and physically active matched healthy elderly (EHG), and 10 physically active young healthy individuals (YHG). Interestingly, femoral blood flow at rest was similar in PDG (64±15 mL min‐1 L‐1), EHG (44±8 mL min‐1 L‐1) and YHG (58±11 mL min‐1 L‐1). Our working hypothesis was that PDG would present an altered blood flow to the skeletal muscle, a reduced vascular function due to the pathology compared with the other groups. However, the sPLM‐hyperemic response appeared markedly lower in PDG and EHG compared to YHG (8.3±0.1 vs 9.8±0.8 vs 17±3 mL min‐1 L‐1; p < 0.05) but the difference between PDG and EHG was negligible (p > 0.05). The results of our study indicate that peripheral circulation and vascular function are not reduced in physically active patients with PD, suggesting that these vascular changes could resemble the physiological adjustments of aging, without any impact from the disease.
A Retrospective Medical History Based on Cadaveric Dissection
Authors: Macek Sarah , Sitto Hannah M.
Published: May 2022

The purpose of this case study was to form a well‐researched evaluation of the potential procedures and disease states of a donor from Western Michigan University during a cadaveric dissection program at Central Michigan University College of Medicine. The donor was an 83‐year‐old male with a primary cause of death of acute respiratory failure secondary to chronic obstructive pulmonary disease (COPD). Over 6 weeks, using a systematic dissection approach, we identified various surgical remnants and abnormalities within our donor’s body. Based on the surgical interventions and evidence of pathology, we formulated a retrospective diagnosis of the donor. We hypothesized the following procedures and disease states were present: bilateral percutaneous femoral‐popliteal bypass with a reverse saphenous vein anastomosis of the left leg, left carotid endarterectomy, and femorofemoral bypass with an aortobifemoral bypass graft, all of which were thought to be secondary to vascular disease, tobacco use, diabetes and hypertension, with the additional presence of knee arthroplasty and autosomal dominant polycystic kidney disease. At the end of the program, we compared our predictions to the donor's medical history, provided to us by Western Michigan University. Our methods included researching the surgical procedures as we discovered them and using these procedures to predict underlying disease states of our donor. Utilizing resources from PubMed and our program directors, we were able to narrow down the bilateral percutaneous femoral‐popliteal bypass, left carotid endarterectomy, and femorofemoral bypass with an aortobifemoral bypass graft. These vascular surgical interventions led us to believe our donor experienced symptoms of claudication due to underlying vascular disease including peripheral artery disease (PAD), aortoiliac occlusive disease (AIOD), and carotid artery disease (CAD). Common risk factors and causes for vascular disease include male sex, smoking, diabetes mellitus and hypertension; these are risk factors present in our donor that we later confirmed. Additionally, due to the age of our donor and the carotid endarterectomy, we predicted our donor likely experienced a cerebrovascular accident (CVA) during his lifetime. Our results, based on the provided medical history, revealed that we correctly predicted our donor's underlying vascular disease, our donor's risk factors of smoking, diabetes and hypertension, and our donor's surgical interventions including bilateral percutaneous femoral‐popliteal bypass, femorofemoral bypass with an aortobifemoral bypass graft, knee arthroplasty and left carotid endarterectomy. Additional procedures and disease states that we did not explicitly predict include spinal stenosis surgery, bilateral cataract extraction, coronary stent placement, deformity of the proximal femoral shaft, and myocardial infarction.
TMPRSS11a as a new target in aging
Authors: Fernandez Christian , Morales Diego , Caceres Monica
Published: May 2022

Aging is a natural process, that is associated with changes in dynamic biological, physiological, environmental, psychological, behavioral, and social processes. In fact, advancing age is the major risk factor for several chronic diseases in humans. During our study of impaired wound healing in aged models, we analyzed skin lysates of young and old mice by LC‐MS/MS to identify age‐related changes in the proteome. Within the proteins whose levels vary during aging we identified TMPRSS11a, a protein that is overexpressed in aged skin. Transmembrane protease serine 11a (TMPRSS11a, gene ECRG1) is an enzyme, which play a role in cellular senescence and may play a role in SARS‐CoV2 infection. This protein has been described at the enzymatic level, but its three‐dimensional structure and interactome, also its physiological role in the cell, are still unknown. This protease features three clearly defined domains: Peptidase S1, SEA and cytoplasmic domain. Interestingly, within the structure we have detected two sequences of interest, such as the RGD motif, necessary to interact with integrins and an ITIM sequence involved in immunological activation. Through computational analysis we found that the Peptidase domain contains the hydrophobic pocket that inside it presents the classic catalytic triad (H230, D275 and S371) and next to the catalytic serine there is a RGD motif that meets the characteristics of the canonical motif. Interestingly, using molecular dynamics, docking and proximity ligation assay, we found that the RGD motif is functional and allows direct interaction with integrin β1 and this RGD motif of TMPRSS11a binds directly to the site adjacent to MIDAS in the integrin β1. Finally, we evaluated cellular spreading of HEK293 cells, seeded over a fibronectin matrix, and cell migration through trans well migration assays. And in this way, TMPRSS11a may affect processes such as migration or cell adhesion in an RGD‐dependent manner. The expression of this protease could affect processes of vital importance such as wound healing in old animal models. Considering our results, the study of this protein can be used as new therapeutic target of aging.
Cardiopulmonary protection against nicotine‐induced pulmonary hypertension and right ventricular remodeling in mice is not mediated by ovarian hormones
Authors: Fried Nicholas D. , Whitehead Anna K. , Garcia‐Sanchez Idanis Z. , Lazartigues Eric , Yue Xinping , Gardner Jason D.
Published: May 2022

Introduction

Electronic cigarette use has increased globally prompting a joint committee of international cardiovascular research organizations to call for improved understanding of the health effects of nicotine. Our group has previously demonstrated that chronic, inhaled nicotine induces pulmonary hypertension (PH) and right ventricular (RV) remodeling in male but not female mice, suggesting sex differences in nicotine‐related pathology. Clinically, biological females develop PH more often, but have less severe disease than biological males, likely due to the cardiopulmonary protective effects of estrogen. Nicotine is also metabolized more rapidly in biological females due to sex hormone‐mediated differences in cytochrome P450 activity. These findings led us to hypothesize that female mice are protected against nicotine‐induced PH by a sex hormone‐dependent mechanism. Here, we assessed the role of ovarian hormones in a murine model of nicotine‐induced PH.

Methods

Female C57BL/6J mice were ovariectomized (OVX) one week prior to nicotine exposure. Intact and OVX mice were exposed to nicotine vapor or room air for 12 hours per day for 8 weeks. Nicotine exposure was measured using serum cotinine, which was comparable to human electronic cigarette users, and was not significantly different between intact (421 ± 101 ng/mL) and OVX (314 ± 61 ng/mL) mice. Cardiac structure and function were assessed using echocardiography and RV catheterization.

Results

In contrast to previous findings in male mice, inhaled nicotine does not cause PH (measured by RV systolic pressure, RVSP) in intact female mice (24.0 ± 0.5 mmHg, n=7 in air vs. 22.7 ± 0.4 mmHg, n=9 in nicotine). RVSP was also unaffected by OVX (24.4 ± 0.9 mmHg, n=9 in air vs. 23.6 ± 0.6 mmHg, n=10 in nicotine). RV free wall thickness during diastole (RV FWT;d) was not significantly different between air‐exposed intact mice (0.328 ± 0.023 mm, n=8), nicotine‐exposed intact mice (0.292 ± 0.015 mm, n=10), air‐exposed OVX mice (0.315 ± 0.021 mm, n=8), and nicotine‐exposed OVX mice (0.293 ± 0.013 mm, n=10). RV internal diameter during diastole (RVID;d) was, likewise, not significantly different between air‐exposed intact mice (1.30 ± 0.03 mm, n=8), nicotine‐exposed intact mice (1.34 ± 0.04 mm, n=10), air‐exposed OVX mice (1.23 ± 0.05 mm, n=8), and nicotine‐exposed OVX mice (1.24 ± 0.05 mm, n=10).

Conclusion

In this study, we found no differences in serum cotinine, RVSP, RV FWT;d, or RVID;d between intact and OVX females, leading us to conclude that ovarian hormones do not mediate cardiopulmonary protection against nicotine‐induced PH. Due to profound sex differences in clinical PH pathogenesis and nicotine metabolism, further studies (including exploration of androgen‐driven RV remodeling) are necessary to illuminate mechanisms underlying protection from nicotine‐induced pathology in female mice.
Cellular and Molecular Mechanisms Regulating the Normal Physiological Function of the Epithelial Sodium Channel
Authors: Abd El‐Aziz Tarek M. , Soares Antonio , Mironova Elena , Stockand James
Published: May 2022

High blood pressure is a significant risk factor for cardiovascular disease. Many details about the molecular mechanisms that regulate blood pressure are unknown. The Epithelial Na+ Channel (ENaC) is the final arbiter of Na+ excretion in the kidneys (see attached Figure). Appropriate renal sodium excretion is a key factor in the regulation of blood pressure. Abnormalities in ENaC function have been directly linked to several human diseases of blood pressure. The kinase, casein kinase II (CKII), phosphorylates ENaC but the physiological importance of this is obscure. The CKII phosphorylation site within ENaC resides within a canonical “anchor” ankyrin binding motif. Phosphorylation of Nav and KCNQ channels by CKII acts as a molecular “switch” favoring the binding of ankyrin‐3 (Ank‐3). The binding of Ank‐3 facilitates the proper membrane localization of these channels increasing their activity. Here, we tested the premise that phosphorylation of ENaC by CKII within “anchor” motifs is necessary and sufficient for Ank‐3 binding to the channel, which is required for normal channel locale and function, and the proper regulation of renal Na+ excretion and blood pressure (see attached Figure). We tested this hypothesis using endpoint measurements including analyses of ENaC activity and trafficking to the plasma membrane, and quantification of CKII‐dependent ENaC‐Ank3 binding. This was addressed by combining total internal reflection fluorescence microscopy with fluorescence recovery after photo bleaching (TIRF/FRAP) to follow movement of ENaC toward the plasma membrane in living cells and electrophysiology of ENaC activity in split‐open collecting ducts from principal cell‐specific CKII and Ank3 knockout mice. We also used whole animal physiological studies of sodium excretion and blood pressure in knockout mice to understand the physiological consequences of CKII and Ank3 regulation of ENaC. In addition, scanning mutagenesis was used to identify key residues in the overlapping CKII and Ank3 sites in ENaC. Furthermore, we tested whether CKII influences Ank‐3 binding to β‐ENaC subunit using fluorescence resonance energy transfer (FRET). Findings showed that disrupting CKII signaling and abrogation of the CKII phosphorylation and Ank3 binding site within ENaC decreases channel trafficking toward the plasma membrane and activity, promotes improper sodium excretion, and decreases blood pressure in KO mice. These results showed how and when CKII phosphorylation of ENaC functions as a “switch” to favor Ank‐3 binding to increase channel activity to include having a detailed understanding of the mechanisms mediating this regulation, and a rich appreciation of the physiological consequences of such regulation.
Effect of Mild Acidosis on CA2+ Signaling in Rat Carotid Body Chemoreceptor Cells: Role of CAV, ASIC, and Task
Authors: Kim Donghee , Hogan James O. , White Carl
Published: May 2022

Carotid body (CB) glomus cells serve as peripheral chemoreceptors that detect changes in arterial O2 pressure (pO2) and pH, and adjust the level of ventilation to help maintain a normal level of blood pO2 and pH. Severe levels of hypoxia and acidosis are well known to cause sustained rise in cytosolic Ca2+ concentration ([Ca2+]c) in CB glomus cells. We showed recently that isolated gloms cells exhibit spontaneous [Ca2+]c oscillations and that mild levels of hypoxia increase the frequency and amplitude of Ca2+oscillations to augment the secretory response. To understand how mild levels of acidosis regulate Ca2+signaling in glomus cells, we studied the effects of small changes in extracellular pH (pHo) on the kinetics of Ca2+oscillations and the roles of Cav, ASIC and TASK. A decrease in pHo from 7.4 to 7.3 (mild acidosis) and 7.2 (moderate acidosis) produced significant increases in the frequency and amplitude of Ca2+ oscillations without affecting the resting basal [Ca2+]c. These effects on Ca2+ oscillations were not blocked by 0.1 mM amiloride (an ASIC inhibitor) that blocked Na‐H exchange in glomus cells. Mild acidosis was associated with a small but significant inhibition of TASK current and cell depolarization. The increase in Ca2+oscillations produced by mild acidosis was strongly inhibited by nifedipine (1 µM; L‐type Cav inhibitor), as well as by TTA‐P2 (20 µM; T‐type Cav inhibitor). Mild acidosis (0.1 pH unit decrease) and mild hypoxia (~5% O2) produced similar levels of change in the kinetics of Ca2+oscillations and produced an additive effect on Ca2+oscillations. Our study shows that mild to moderate levels of acidosis inhibit TASK to increase Ca2+ influx via both L‐ and T‐type Cav, resulting in increased frequency and amplitude of Ca2+ oscillations. Our study also indicate that ASICs are not involved in the stimulatory effects of mild acidosis on Ca2+oscillations.
NOR‐1 Knockdown Reduces Mitochondrial Function in C2C12 Myotubes
Authors: Paez Hector G. , Pitzer Christopher R. , Ferrandi Peter J. , Mohamed Junaith S. , Alway Stephen E.
Published: May 2022

Introduction

Skeletal muscle is critical for whole‐body metabolic health and a prime determinant of quality of life. Mitochondrial health is compromised by several pathological states that alter muscle metabolism. Exercise is among the most effective means of preserving muscle mitochondrial health, yet rigorous exercise is often impossible in patients suffering from muscle‐related pathologies. Thus, the investigation of exercise responsive molecular targets is a requisite to identify therapeutics that improve muscle metabolic health. Neuron‐derived orphan receptor‐1 (NOR‐1) is highly responsive to exercise and is reduced in preclinical models of obesity and diabetes. The objective of the current study was to investigate the impact of NOR‐1 loss on C2C12 metabolic signaling. We hypothesized that knockdown (KD) of NOR‐1 in myotubes would perturb mitochondrial‐associated gene expression.

Methods

C2C12 myotubes were transfected with control or NOR‐1 targeting siRNA to knock down NOR‐1. Protein and RNA were isolated from cells for immunoblotting and qPCR analysis, respectively. Cells were also stained with JC‐1 to evaluate alterations in mitochondrial membrane potential (MMP). NAD+ and NADH+ content was also measured in myotubes. Gene expression was normalized to ß‐actin mRNA whereas protein levels were normalized to total protein. Data were analyzed by an unpaired T‐Test with significance set at p < 0.05 and presented as means ± SEM.

Results

NOR‐1 KD cells had lower gene expression of PGC‐1α, TFAM, and MFN2 and several proteins involved in energy production were altered (ND2, CYCS, CPT‐1ß, FABP4). Myosin protein content and gene expression of MYHI, MYHIIA, and MYHIIX were also reduced. Protein abundance of mitochondrial electron transport system complexes were reduced and both BAX and DRP1 abundance were enhanced after NOR‐1 KD, which was coincident with a diminished NADH content, and a shift towards the green monomeric spectra after JC‐1 staining, indicating a reduced mitochondrial membrane potential. Surprisingly, NOR‐1 KD also reduced mTORC1 signaling, evidenced by reduced P70S6K and S6K phosphorylation.

Conclusions

These data suggest loss of NOR‐1 reduces oxidative gene expression, induces mitochondrial depolarization, and reduces NADH content, implicating NOR‐1 as an important regulator of energy homeostasis and muscle metabolic signaling. Investigations into the molecular hierarchy between NOR‐1 and PGC‐1α will delineate between primary and secondary effects of NOR‐1 deficiency.
Factors Contributing to Cognitive Enhancement (CEM) and Psychostimulant Medication (PSM) Use Among First Year Medical Students
Authors: Pavlick Kayla V. , Schaefer Audra
Published: May 2022

Medical students perceive elevated levels of stress within their pre‐clerkship years in part due to select curricular factors such as grading scale or method of content delivery (e.g. traditional or integrated). Medical students cope with these stressors in a variety of ways. Alcohol and nicotine use are among the top coping methods. There are many changes being made to the medical curriculum to alleviate this perception of stress and increase effectiveness of the curriculum. An additional manifestation of perceived stress is the exhibition of non‐medical prescription drug use. Non‐medical prescription drug use (NMPDU) of cognitive enhancement (CEM) and psychostimulant medications (PSM) (i.e. Adderall) is associated with elevated perceived levels of stress within both the undergraduate college and practicing physician populations. The prevalence of non‐medical prescription drug use of CEM/PSMs among medical students is scarcely documented, while the perception of stress is heavily documented. This pilot study sought to address this gap in the literature by describing the prevalence of NMPDU of CEM/PSMs among first year medical students at an American allopathic institution in addition to perceived stress levels and the potential factors contributing to it. Ten students (less than 10% response rate) completed a questionnaire regarding CEM/PSM use, alcohol and nicotine use, sleep and study habits, and perceived stress. Student responses indicated presence of CEM/PSM use, with four of those students reporting non‐prescription use. “School” was the top reason for taking CEM/PSMs and the number one contributor to perceived stress. Additionally, half of the respondents reported an increase in use within their first year of medical school. The results, while limited, confirm presence of NMPDU of CEM/PSMs within this population, with potential correlations with levels of perceived academic stress. This work provides initial insights from which future work can build to analyze the relationship between NMPDU and curricular factors which may shed light on additional benefits to curricular redesign or evidence to necessitate proactive measures to prevent the phenomenon of NMPDU within the medical student population.
Pharmacological characterization of kratom alkaloids at opiate receptors: binding affinities, in vitro and in vivo functional assessments
Authors: Hemby Scott E. , McIntosh Scot , Graf Tyler N. , Rangel‐Grimaldo Manuel , Flores‐Bocanegra Laura , Coover Robert , Oberlies Nicholas
Published: May 2022

Kratom (Mitragyna speciosa) is consumed by 10‐15 million individuals in the US for its mood‐elevating effects, as an alternative to traditional opiates for pain relief and to attenuate opiate withdrawal symptoms. Complex natural products, such as kratom, contain a variety of chemicals whose combined effects can only be understood by the meticulous identification and characterization of the individual chemicals and their effects in preclinical model systems. Twenty‐five indole and oxindole kratom alkaloids were evaluated for mu (MOR), delta (DOR) and kappa (KOR) opiate receptors binding affinities, opiate receptor mediated G‐protein activation, inhibition of forskolin stimulated cAMP accumulation and b‐arrestin2 recruitment. Several indole and oxindole alkaloids exhibited partial agonism at the MOR (7‐hydroxymitragynine, corynoxine A > epiallo‐isopaynantheine, isospeciofoline, speciociliatine, corynoxine B > mitragynine, 3‐epicorynoxine B > paynantheine and speciogynine) – all of which also exhibited strong G protein bias. Mitraciliatine and isopaynantheine were weak MOR antagonists. Speciophylline exhibited positive allosteric modulatory activity at the MOR as evidenced by increased affinity of DAMGO binding and potentiation of [met]‐enkephalin‐induced inhibition of forskolin stimulated cAMP activity in the absence of intrinsic activity. Corynoxine A induced displayed similar affinity at the mu opiate receptor as 7 hydroxymitragynine but was less potent in the nociceptive hot plate assay and the mechanical allodynia von Frey test. Corynoxine did not significantly alter respiration or heart rate or induce a conditioned place preference. Results demonstrate the pharmacological complexity of kratom alkaloids and have important implications for determining the combined effects of less abundant indole and oxindole alkaloids with regard to the therapeutic as well as potential harmful effects of kratom.