bluuebunny/crossref_metadata_2025 huggingface dataset:
Interactions between β3‐adrenergic receptors and bFGF‐induced effects in rat aortic smooth muscle cells
Authors: Chang Yingzi , Dagat Lei Alena , Yusuf Aisha , Zahriya Yusuf , Staputyte Kotryna , Worley Emma , Holt Alex , Canuteson Natalie , Messieha Vereena , Halila Kasey
Published: May 2022

Background

Basic fibroblast growth factor (bFGF)‐mediated vascular smooth muscle cell (VSMC) proliferation and migration play an important role in vascular injury‐induced neointima formation and subsequent vascular restenosis, a major event that hinders the long‐term success of angioplasty. The function of β3‐adrenergic receptors (β3‐ARs) in vascular injury‐induced neointima formation has not yet been defined.

Objectives

Our current study explored the possible role of β3‐ARs in vascular injury‐induced neointima formation by testing its effects on bFGF‐induced VSMC migration and proliferation.

Methods

β3‐AR expression in rat carotid arteries was examined at 14 days following a balloon catheter‐induced injury. The effects of β3‐AR activation on bFGF‐induced rat aortic smooth muscle cell proliferation, migration, and signaling transduction (including extracellular‐signal‐regulated kinase/ mitogen activated protein kinase, ERK/MAPK and Protein kinase B, AKT) were tested.

Results

We found that vascular injury induced upregulation of β3‐ARs in neointima. Pretreatment of VSMCs with a selective β3‐AR agonist, CL316,243 significantly potentiated bFGF‐induced cell migration and proliferation, and ERK and AKT phosphorylation. Our results also revealed that suppressing phosphorylation of ERK and AKT blocked bFGF‐induced cell migration and that inhibiting AKT phosphorylation reduced bFGF‐mediated cell proliferation.

Conclusion

Our results suggest that activation of β3‐ARs enhances bFGF‐mediated effects on VSMCs by interacting with bFGF‐mediated ERK and AKT phosphorylation and β3‐ARs may play a role in vascular injury‐induced neointima formation.
A Qualitative Study on the Benefits of Tumescence in Cadaveric Dissection with Faculty & First‐year Medical Students at SHSU‐COM
Authors: Hines Jonathon H. , Reynolds Amberly , Loomis Mario
Published: May 2022

Hands‐on cadaver dissection is considered essential to the optimal education of future physicians, and the quality of students’ dissections has been directly related to their performance on national board exams. Some centers, however, have reduced or even eliminated cadaver dissections due to time constraints and safety concerns. To address such concerns while optimizing the benefits of hands‐on learning, we introduced tumescence into our first‐year anatomy lab. This involved the manual infusion of cadaver wetting solution into subcutaneous and interstitial tissue planes using a syringe and a blunt‐tipped canula. This technique was utilized by our students and faculty during the Clinical Anatomy course. A qualitative survey of students and faculty regarding the benefit, if any, of the tumescence was subsequently carried out and a thematic analysis of the responses was performed. The most frequent theme which surfaced in the survey was an increased ease and efficiency of dissection with the use of tumescence. In addition, the themes of separation, spaces, and planes, were common, reflecting in the respondents, an increased appreciation of tissue planes and relationships. The survey lent support to the conclusion that cadaver tumescence can improve the quality and speed of dissections safely, while increasing the appreciation of natural tissue planes.
Run for Your Life: A Meta‐Analysis of the Effects of Exercise Intensity on Cardiovascular Outcomes in a Rat Model of Hypertension
Authors: Meredith Kayla M. , Luckey Stephen W.
Published: May 2022

Objective

Hypertension increases the risk of pulmonary embolism, stroke, heart attack, and death. To prevent these events in hypertensive patients, regular exercise has been recommended as a nonpharmacological intervention for decades, but it is still not well understood whether cardiovascular benefits vary with exercise intensity. In this meta‐analysis, we explored the effect of treadmill running intensity on systolic blood pressure (SBP), mean arterial pressure (MAP), and resting heart rate (RHR) in a rat model of hypertension, known as spontaneously hypertensive rats (SHR).

Methods

A SciFinder Scholar database search returned 817 comparative studies, of which 72 (1505 rats) were included for quantitative analysis. Studies were characterized as low, moderate, or high intensity based on treadmill speed setting or, where available, %VO2max. Aggregate means and standard errors for SBP, MAP, and RHR were extracted and subjected to one‐way ANOVA with post‐hoc Tukey’s test using GraphPad Prism 9.

Results

When compared to sedentary hypertensive controls, both low and moderate‐intensity treadmill exercise yielded significant reductions in SBP, MAP, and RHR, but high‐intensity exercise did not. Although the greatest improvement in SBP, MAP, and RHR resulted from low‐intensity treadmill exercise, no statistically significant difference was found between training intensity levels for these cardiovascular outcomes.

Conclusions

Low and moderate‐intensity, steady‐state treadmill sessions over several weeks reduced blood pressure and resting heart rate in hypertensive subjects. The failure of high‐intensity exercise to improve cardiovascular outcomes may suggest a role played by oxidative stress and merits further investigation. Overall, these preliminary results suggest that even low‐intensity exercise may serve as a beneficial tool that positively impacts cardiovascular outcomes in chronic hypertension.
Phosphoproteomic profiling of skeletal muscle twitch torque potentiation in ovarian hormone deficient female mice
Authors: Peyton Mina , Yang Tzu Yi , Higgins LeeAnn , Parker Laurie , Lowe Dawn
Published: May 2022

Skeletal muscle makes up ~40% of total body mass in a healthy adult. Muscle strength begins to decline with age. In females, preclinical and clinical studies have shown that reduction in estrogen reduces muscle force (i.e., strength). Previously, we showed the skeletal muscle phosphoproteome in a basal, non‐contracting state was remodeled in estrogen‐deficient females. Therefore, we questioned how estrogen deficiency would impact the skeletal muscle phosphoproteome after force generation. We performed two label‐free quantification phosphoproteomic analyses of the tibialis anterior muscle in ovariectomized (Ovx/Sham) and ovarian senescent (Old/Young) female mice after in vivo post‐tetanic potentiation protocol. We identified 25 and 60 differentially regulated phosphoproteins (p‐value<0.05 and ≥1.4‐fold change) in Ovx/Sham and Old/Young mice, respectively. Comparative analysis using Ingenuity Pathway Analysis’s activation Z‐score found similar patterns of predicted inhibition and activation of canonical pathways, such as inhibition of calcium signaling and activation of 14‐3‐3‐mediated signaling in both datasets. Likewise, parallel patterns in functional analysis were found relating to muscle contraction, fibrogenesis, etc. Overall, our findings suggest that the similarities identified in both datasets could elucidate the molecular characteristics of muscle proteins that might contribute to decrements in muscle function observed in Ovx and Old females due to the loss of estrogen.
Pyrazole Reduces the Effects of Oxidative Stress in Cardiomyocytes Induced by Rotenone and Doxorubicin
Authors: Shum Alika , Ara Hosne , Bess Ezra , Colvin Chase , Sharma Sudha , Murru Siva , Panchatcharam Manikandan , Miriyala Sumitra
Published: May 2022

Background

In the United States, cardiovascular disease accounts for approximately 1 in every 4 deaths. One factor that has been identified as a source for the development of cardiovascular disease is oxidative stress in the mitochondria of cardiomyocytes. Oxidative stress is a condition where the number of reactive oxygen species (ROS) is greater than the number of antioxidants. The consequences of oxidative stress in cardiomyocytes include tissue damage through cell death and the formation of 4‐hydroxynenal (4‐HNE) aggresomes through lipid peroxidation. These known effects have led to this study in which we determined the antioxidant function of a synthetic drug called pyrazole. We specifically tested the antioxidant property of two pyrazoles known as S9 and S10 in the HL‐1 cardiomyocyte cell line.

Methods

Oxidative stress was induced by using a compound known as rotenone and a chemotherapy agent known as doxorubicin (DOX). Both have been found to cause cardiotoxicity partly due to their ability to induce oxidative stress within cardiomyocytes. MitoSOX®and high pressure liquid chromatography (HPLC) were used to measure mitochondrial superoxide production. Immunocytochemistry (ICC) was used to measure the formation of 4‐HNE aggresomes. Western blot analysis was used to measure the expression of the antioxidant enzyme manganese‐dependent superoxide dismutase (SOD2). Seahorse XF24 analyzer was used to measure oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) to determine mitochondrial function.

Results

DOX and rotenone induced mitochondrial superoxide production was found to be reduced in cells treated with S9 or S10. From ICC, S9 and S10 treatments showed reduced formation of 4‐HNE aggreosomes compared to the level of 4‐HNE aggresome formation following treatment with DOX or rotenone alone. Western blot analysis results confirmed that both S9 and S10 were capable of restoring expression of SOD2 which was reduced by rotenone or DOX in HL‐1 cells. Seahorse analysis showed that cells treated with either DOX or rotenone and either S9 or S10 resulted in increased OCR, ATP turnover, reserve capacity, and glycolysis & glycolytic reserve capacity compared to the cells treated with rotenone or DOX alone.

Conclusion

The results from our study indicate that pyrazoles S9 and S10 have the ability to reduce the oxidative stress in cardiomyocytes and therefore have the potential to serve as therapeutics for cardiovascular disease.
Chronic Binge Alcohol Differentially Affects Extracellular Matrix Phenotype in Omental and Subcutaneous Adipose Tissue of SIV‐infected Macaques
Authors: Poret Jonquil M. , Simon Liz , Molina Patricia
Published: May 2022

The incidence of at‐risk alcohol use among people living with HIV (PLWH) is two times greater than in the general population. Though antiretroviral therapy (ART) has significantly reduced HIV‐associated mortality, increased survival of PLWH on ART is associated with increased incidence of metabolic comorbidities. Adipose tissue maladaptation is central to metabolic alterations in PLWH and at‐risk alcohol use. This involves adipokine disruption, adipocyte dysfunction, inflammation, and dysregulation of the extracellular milieu. Using a relevant preclinical model of HIV‐infection, the objective of our study was to investigate the effects of chronic binge alcohol (CBA) administration on omental adipose tissue (OmAT) and subcutaneous adipose tissue (SAT) extracellular matrix (ECM) phenotype. Rhesus macaques were administered CBA or isovolumetric water (VEH) through an intragastric catheter for 30 min, 5 days a week, with blood alcohol concentrations reaching 50‐60 mM. Three months after initiation of CBA or VEH administration, macaques were inoculated with SIVMac251, and 2.5 months later animals were initiated on ART. CBA or VEH administration was continued throughout the duration of the study (14.5 mo). Animals were euthanized at 11.5 mo of SIV infection, and adipose (OmAT and SAT) samples excised. Collagen content, adipocyte diameter and adipocyte numbers were measured by Picrosirius Red Staining (PSR). A custom PCR Array was used to assess differential expression of 41 ECM‐related genes. CBA administration significantly increased collagen expression and decreased adipocyte diameter in the OmAT without altering adipocyte numbers. There was a strong negative correlation of OmAT adipocyte diameter and collagen expression. CBA administration did not result in significant differences in collagen expression, adipocyte diameter or number in SAT. CBA administration significantly upregulated expression of transforming growth factor beta‐1 (TGFB1), platelet‐derived growth factor receptor alpha (PDGFRA) and secreted protein acidic and rich in cysteine (SPARC), mediators of a pro‐fibrotic milieu in OmAT. In contrast, CBA did not significantly alter expression of ECM‐related genes in the SAT. These data suggest that the maladaptive adipose changes associated with CBA in SIV infection are depot specific. We speculate that changes in the ECM contribute to altered gene expression and impaired adipocyte metabolic capacity.
Intestinal Epithelial PTPN2 Restricts Adherent‐Invasive E. coli Colonization and Promotes Anti‐Microbial Peptide Responses in Mice
Authors: Chatterjee Pritha , Spalinger Marianne , Shawki Ali , Santos Alina , Gries Casey , Sayoc Anica , Canale Vinicus , Crawford Meli'sa , Lei Hillmin , Borneman James , McCole Declan
Published: May 2022

Background

In addition to a physical barrier, the intestinal epithelium generates a chemical barrier to the luminal microbiota by secreting various antimicrobial factors. Alterations in the gut microbiota and expansion of pathobionts such as adherent‐invasive Escherichia coli (AIEC) are associated with inflammatory bowel disease (IBD) pathogenesis. Our previous work showed, mice deficient for the expression of the IBD risk gene, Ptpn2, exhibit pronounced expansion of a novel murine adherent‐invasive Escherichia coli (mAIEC) strain. This study aimed to investigate how PTPN2 expression in intestinal epithelial cells restricts mAIEC colonization in vivo.

Methods

We generated tamoxifen‐inducible, intestinal epithelial cell‐specific knockout mice (Ptpn2∆IEC). Ptpn2∆IEC and control littermates (Ptpn2fl/fl) were infected with either PBS, non‐invasive E. coli K12, or fluorescent‐tagged mAIEC (mAIECred)for 4 consecutive days. After euthanasia, bacterial colonies were enumerated in mouse tissues. mRNA and protein expression were assayed in intestinal epithelial cells (IECs) by PCR and Western blot.

Results

Ptpn2∆IEC mice exhibited intestinal region‐specific higher mAIECred ‐ but not K12 ‐ bacterial load in distal colon tissue compared to Ptpn2fl/fl mice (P=0.038; n=9‐12). To identify if the higher susceptibility to mAIECred infection was associated with altered host defenses, we measured levels of anti‐microbial peptide (AMPs) in these mice. Ileal RNA expression of the alpha‐defensin AMPs, Defa5 and Defa6, were significantly lower (P=0.008, 0.0182 respectively; n=3‐5) in Ptpn2∆IEC vs. Ptpn2fl/fl mice, after mAIECred infection but not K12 infection. Further, expression of the protease matrilysin‐7 (MMP7) ‐ which is responsible for the proteolytic cleavage of alpha‐defensins – was significantly decreased in ileum and distal colon of Ptpn2∆IEC mice post mAIECred infection compared to Ptpn2fl/fllittermates (P=0.042, 0.007; n=4‐6). REG3γ, an AMP that acts largely against gram‐positive bacteria, was unchanged in all conditions. However, ileal IEC lysozyme protein expression was lower in PBS and K12, but not mAIECred, infected Ptpn2∆IECmice (P= 0.067, 0.0165 respectively; n=8) compared to control Ptpn2fl/fl mice.

Conclusion

Intestinal epithelial PTPN2 plays a major role in regulating intestinal bacterial defenses and gut regional variations in pathobiont colonization.
Oxygen Consumption Rates Between Skeletal Muscle Cells Derived From Young and Old Human Donors Elucidate Mitochondrial Dysfunction
Authors: Park Lindsay (. Y. , Arevalo Jose A. , Brooks George A.
Published: May 2022

Mitochondrial oxidative phosphorylation plays a significant role in cellular functions such as nutrient metabolism, ATP synthesis, and respiratory capacity. Mitochondria’s branching network, the reticulum, is active through the fusion (connection) and fission (fragmentation) dynamics. As humans age, however, there is a loss of mitochondrial fusion which increases fragmentation and this results in the loss of mitochondrial function. Therefore, the purpose of this study is to investigate mitochondrial dysfunction in human skeletal muscle derived cells (SkM). The discrepancies of the oxygen consumption rate (OCR) of the mitochondria between the young versus old human cells may reflect the driving factor behind aging. In order to determine mitochondrial dysfunction, OCR was measured between a primary human SkM from an 18 year‐old‐male (18M) and 66 year old male (66M) were purchased from Cook MyoSite Inc. (Pittsburgh, PA). OCR was measured using the Cell Mito Stress Test by SeaHorse Analytics XFp Analyzer (Agilent Technologies; Santa Clara, CA). Data generated by Seahorse Report Generator (Mean±SEM). Basal OCR and Maximal OCR were higher in 18M compared to 66M (Basal: 28.51 ± 1.61 and 20.43 ± 2.18; Maximal: 54.98 ± 6.74 and 28.68 ± 3.91 pmol/min). In addition, the results reflected that ATP production as well as Spare Respiratory Capacity (SRC) were higher in 18M compared to 66M (ATP Production: 23.88 ± 1.37 and 16.84 ± 2.04; SRC: 26.47 ± 5.13 and 8.25 ± 1.73 pmol/min). Basal OCR, Maximal OCR, ATP Production, and SRC of the 18M cells were higher than those of the 66M cells, revealing a greater mitochondrial function in the primary skeletal muscle‐derived cells derived from the young compared to the old. This could be accounted for by the fact that the young cells have a higher stress adaptability compared to the old cells. The discrepancies in the OCR between the young versus the old human cells, therefore, augment our understanding of how mitochondrial dysfunction may serve as a driving force behind aging in humans. Combining data from additional samples to be obtained, this study will elucidate the various mechanisms that propel age‐related mitochondrial dysfunction and provide crucial information to prevent skeletal muscle pathologies. Therefore, this project will unveil the effects of aging on skeletal muscle mitochondrial dysfunction and mitigate age‐related muscle pathologies.
Mitochondrial Transplantation as a Novel Therapeutic for Retinal Metabolic Dysfunction‐Driven Disease
Authors: Butcher Erik R. , Shu Daisy Y. , Frank Scott , Saint‐Geniez Magali
Published: May 2022

Metabolic dysfunction and mitochondria defect are implicated in several age‐associated diseases including age‐related macular degeneration (AMD), a leading cause of blindness in the elderly. In AMD, mitochondrial oxidative stress in the retinal pigment epithelium (RPE) drives disease progression and growth of atrophic lesions. Mitochondria release and uptake has been recently identified as a novel mechanism of intercellular communication but its implication in ocular diseases such as AMD has never been investigated. Here, we examined the role of mitochondrial transfer as a new mechanism of metabolic crosstalk between RPE cells. Diseased mitochondria were purified from RPE cells treated with the AMD‐associated cytokine TNFα (10 ng/mL), administered to host RPE cells and the effects of MitoTNFA compared to MitoCtrl, isolated from control RPE, or exposure to exogenous TNFα. We showed that treatment of healthy RPE with MitoTNFA, and not MitoCtrl, triggers mitochondrial network fragmentation and transcriptional upregulation of inflammatory factors RelB, IL6, IL8, and repression of PGC1α, mirroring the effect of direct TNFα treatment. ELISA assay confirmed that the effects observed were not caused by presence of soluble TNFα within the mitochondrial fraction. Metabolic profiling using the Seahorse XFe96 bioanalyzer further validated the ability of MitoTNFA to phenocopy TNFα‐induced metabolic reprograming in RPE. Finally, we demonstrated that transfer of MitoCtrl improved the bioenergetic functions of both healthy and diseased RPE with significant increases in basal, maximal, and spare respiratory functions. Our results showed that mitochondria transfer recapitulates the context‐specific phenotype from donor to host cells in RPE. This new paradigm in RPE biology may not only explain the centrifugal expansion of RPE lesions in AMD but also presents a promising therapeutic avenue for mitochondria‐driven disorders such as AMD.
Detection of cofactor conformer exchange in the active sites of methyltransferases with vibrational spectroscopy
Authors: Bradley Brianna , Hamilton Grace , Douglas Teri , Pillai Anjali , Fick Robert , Hargrove Drew , Le Brendan , Unjia Maitri , Trievel Raymond , Stelling Allison
Published: May 2022

SAM (S‐adenosyl‐L‐Methionine) is the universal methyl donor used by cells, and is also the second most used cofactor after ATP. Methyltransferases catalyze the methylation of numerous biomolecules including DNA, protein, and RNA; and have a broad array of functions ranging from cell signaling to gene transcription. Abnormal levels of methyltransferases have been linked to malignant cellular states, cardiovascular diseases, and neurological diseases. Therefore, methyltransferases are an attractive drug target. Several inhibitors of histone PMTs (protein methyltransferases) have been used in the clinic, signifying PMTs as a target class. The SAM binding pocket in protein methyltransferases has been utilized by in drug design strategies. Yet, there is not high specificity when it comes to these drugs, making the drugs imperfect. Recent NMR work reveals that unbound SAM has high populations of different conformations in dynamic equilibrium, which means there may be multiple conformations when SAM is bound to methyltransferases. Having multiple conformations of SAM may allow for specific drugs to be designed to fit the unique pockets, allowing for higher drug specificity. Here we use vibrational spectroscopy, which is sensitive to molecular conformation and immune to timescale, to determine if these conformational dynamics persist when SAM is bound to PMTs. Molecular dynamic studies and small molecules studies support the hypothesis that various SAM conformations exist both when free and when bound to the proteins.
Evaluating Anticancer Activity of Isomeric Analogues of the 3,4‐Diarylfuranone, PY‐407‐C
Authors: McNulty Matthew , Mowery Patricia
Published: May 2022

Microtubules are filamentous proteins that comprise the cytoskeleton of eukaryotic cells and play an essential role in cell structure, mitosis, migration, and signaling. The function of these proteins is dependent on their ability to exhibit dynamic instability, or the ability to spontaneously depolymerize and polymerize. Microtubule targeting agents are promising anti‐cancer therapeutics, that inhibit the dynamic instability of microtubules by destabilizing or stabilizing the proteins. Our lab previously showed that the 3,4‐diarylfuranone, PY‐407‐C, exhibits nanomolar toxicity on U937 cells. The National Cancer Institute has confirmed this activity on several other cell lines (NCI‐60 assay), and algorithms suggested that it binds tubulin, which we confirmed. The goal of this project is to assess the potency of isomeric analogues of PY‐407‐C on U937 cells using a cytotoxicity assay. Results suggest that particular substitution patterns lead to low micromolar and nanomolar effectiveness. The results of this next generation of compounds will provide a better understanding of how to maximize potency and guide the synthesis of future analogues.
Monoamine Receptor and Transporter Interaction Profiles of 4‐Alkyl‐Substituted 2,5‐Dimethoxyamphetamines
Authors: Luethi Dino , Rudin Deborah , Hoener Marius C. , Liechti Matthias E.
Published: May 2022

Background

Various ring‐substituted phenethylamines and amphetamines are used recreationally due to their mind‐altering effects. Furthermore, such psychedelic substances have recently gained increased interest as prospective therapeutics. Here, we studied the pharmacological properties of 4‐alkylated 2,5‐dimethoxyamphetamines in vitro. Specifically, we assessed the effect of the 4‐alkyl chain length on interactions with monoaminergic targets. We aimed to test the hypothesis that the derivatives with the highest clinical potency display the most potent serotonin (5‐HT) type 2A receptor interactions, which is the key driver of psychedelic effects.

Methods

We determined binding affinities of 2,5‐dimethoxyamphetamine (2,5‐DMA), 4‐methyl‐2,5‐dimethoxyamphetamine (DOM), 4‐ethyl‐2,5‐dimethoxyamphetamine (DOET), 4‐butyl‐2,5‐dimethoxyamphetamine (DOBU), and 4‐amyl‐2,5‐dimethoxyamphetamine (DOAM) at serotonergic, adrenergic, and dopaminergic receptors and transporters, as well as at the trace amine‐associated receptor 1 (TAAR1). For this, we assessed radioligand displacement using cell membrane preparations expressing the respective targets. Furthermore, we determined the functional activity of the derivatives at the 5‐HT2A and 5‐HT2B receptors by measuring calcium mobilization and at the TAAR1 by measuring cAMP accumulation. In addition, we assessed whether any of the derivatives inhibit transporter‐mediated uptake of radiolabeled monoamines.

Results

Unlike 2,5‐DMA, its 4‐alkylated derivatives displayed potent (i.e., nanomolar) affinity at 5‐HT2A and 5‐HT2C receptors. Increasing length of the 4‐alkyl chain increased the selectivity for 5‐HT2A vs. 5‐HT1A and 5‐HT2C receptors. Whereas DOM and DOET activated the 5‐HT2B receptor as partial agonists in the nanomolar range (EC50: 68–128 nM, Emax: 73–85%), DOBU and DOAM did not activate the receptor at investigated concentrations (EC50 > 10,000 nM). In contrast to the potent interactions with 5‐HT2 receptors, the 4‐alkyl‐substituted 2,5‐dimethoxyamphetamines did not or only weakly (i.e., in the micromolar range) interact with other monoaminergic targets.

Conclusion

Among the tested substances, user reports suggest DOM and DOET to be the only derivatives with a distinct psychedelic effect profile. Our data therefore indicate that 5‐HT2A and 5‐HT2C receptor affinity alone is not a sufficient predictor for the clinical and psychedelic potency of 4‐substituted 2,5‐dimethoxyamphetamines. To assess the clinical potential of the tested substances, future studies should focus in more detail on pharmacokinetics and qualitative differences in 5‐HT2 receptor activation.
Prodromal Physiologic Changes During Copper Sulfate‐Induced Nausea and Vomiting: Potential Use of Intestinal EMG Recordings as a Marker for Nausea
Authors: Murphey Charles P. , Shulgach Jonathan A. , Amin Pooja R. , Douglas Nerone K. , Bielanin John P. , Horn Charles C. , Yates Bill J.
Published: May 2022

Although emetic responses are present during many disease states, there is no consensus regarding the physiologic changes that signal the onset of nausea. In this study, 10 adult felines (6 males and 4 females) were instrumented to chronically record, during the awake state, the electrocardiogram, diaphragm and abdominal muscle electromyographic (EMG) activity, and EMG activity from the stomach and duodenum before and after the gavage or intragastric infusion of saline or copper sulfate (CuSO4, doses ranging from 83 to 322 mg). Intragastric CuSO4 is a prototypical emetic stimulus that triggers emesis primarily by activating a gastrointestinal vagus nerve‐to‐brain pathway. CuSO4 infusion elicited a significant increase in heart rate, decrease in respiratory rate, and a disruption in gastric (baseline~6 CPM, cycles‐per‐minute) and intestinal (baseline~20 CPM) EMG activity several minutes prior to vomiting (see Fig. 1). The change in EMG activity was most consistent in the intestine. Administration of saline did not induce these physiologic changes. Increasing the dose of CuSO4 did not alter the physiologic changes induced by the treatment. In some animals, a second bout of vomiting occurred following the first episode, when prodromal changes of the intestinal EMG were less pronounced. It is postulated that the intestinal EMG activity was related to a retrograde movement of chyme from the intestine to the stomach. These findings suggest that monitoring of intestinal EMG activity may be the best indicator of the onset of nausea following treatments and in disease conditions associated with emesis.
Detection of prohibited growth factors (GFs) in doping control: serum concentration ranges in a reference population of athletes
Authors: Botre Francesco , de la Torre Xavier , Donati Francesco
Published: May 2022

Growth factors (GFs) are a wide class of polypeptides that can regulate cellular proliferation, migration and differentiation. Due to their ability to extensively impact on tissue regeneration, the use of GFs in sports is currently prohibited in and out of competition and included in the 2022 List of Prohibited Substances of the World Anti‐Doping Agency (WADA). At present, methods of detection of prohibited GFs for doping control purposes have been developed exclusively for recombinant Growth Hormone (recGH) and Insulin‐like growth factor1 (IGF‐1).

Since GFs are endogenously produced substances, their abuse can be detected by fixing “reporting threshold” to differentiate their normal, physiological levels and the exogenous administration. Such a strategy imposes the determination of “normality ranges” for the athletic population.

We considered the principal banned GFs, that are fibroblast growth factor (FGF), platelet‐derived growth factor (PDGF), vascular endothelial growth factor (VEGF), and hepatocyte growth factor (HGF), measuring their levels – including those of their major isoforms – in 92 serum samples, collected from elite athletes in the framework of doping control tests. Preliminarily, we confirmed all the above considered growth factors are actually measurable in serum, defining also their respective limits of detection. Furthermore, we were able to identify differences in concentration levels possibly related to gender and age. Finally, we established normal serum concentration ranges, that can be used as reference normal population values necessary to fix threshold values able to discriminate the physiological levels and the recourse of doping practices.
LC/MS coupled to PODIUM establishes the amino acid metabolomes of Sorghum bicolor
Authors: Tuinstra Caden R. , Luo Zhiwei , Simpson Jeffrey , Dilkes Brian , Chapple Clint
Published: May 2022

Untargeted metabolomics is a well‐known and reliable method to globally profile small molecules. A number of analytical tools can be used to accomplish this goal, but liquid chromatography mass spectrometry (LC/MS) is most often used due to its precision and sensitivity. Although the use of LC/MS results in large amounts of data, much of it is difficult and time consuming to analyze. Most importantly, LC/MS cannot conclusively identify small molecules; additional analytical tools are needed to address these limitations. Effort has been put into streamlining the analysis of data from LC/MS in untargeted metabolomics with recently created analytical pipelines like XCMS, CAMERA, and PODIUM. The advent of these technologies makes it possible to efficiently analyze large amounts of data and conclusively identify small molecules, thus expanding our knowledge of metabolites and their role in various biochemical pathways. In this research, we attempted to use PODIUM to analyze the data from an untargeted metabolomics experiment in order to identify metabolites derived from amino acids. The aim of the experiment had two parts: (1) to feed Sorghum bicolor seedlings with isotopically labeled amino acids and analyze them using PODIUM and (2) to tentatively identify small molecules derived from the amino acids. To do this, plants were fed individually with one of 15 amino acids, which were expected to be direct precursors for natural products. These amino acids were labeled with Carbon‐13 (13C) so that labeled products could be distinguished and traced back to their precursor amino acid. Metabolites were quantified by identifying isotope peak pairs using LC/MS and the PODIUM analytical pipeline. Carbon‐12 (12C) amino acids were also fed as controls. Overall, this research will utilize PODIUM to identify metabolites and will determine the relationship between metabolites and the amino acids responsible for their synthesis.
Structure‐based Computational Modeling of Germline PTEN Mutations in Cancer and Autism Risk: Implications for Therapeutic Targeting
Authors: Smith Iris N. , Eng Charis
Published: May 2022

The Phosphatase and TENsin homolog deleted on chromosome ten (PTEN) is a dual‐specificity phosphatase encoded by a tumor suppressor gene frequently somatically mutated in human cancer. Individuals with germline PTEN mutations are diagnosed with PTEN hamartoma tumor syndrome (PHTS). Clinically, PHTS has a variable phenotype spectrum, including distinct subsets of patients with autism spectrum disorder (ASD) and/or cancer. It remains unclear why mutations in one gene can lead to seemingly disparate phenotypes. Our pilot studies identified structural stability and functional differences in germline PTEN variants that contribute to cancer or to autismsuggesting distinct conformational disease states and a potential basis for allosteric communication via the inter‐domain region of PTEN. Thus, we hypothesized that key inter‐domain sites will delineate novel structurally correlated areas for potential drug targeting. Moreover, PTEN is inactivated by phosphorylation of C‐tail cluster sites (Ser/Thr residues 380‐385) which is known to play a critical role in the function and stability of PTEN. However, very little is known about the mechanism underlying such inactivation. We therefore sought to further understand whether C‐tail phosphorylation sites represent a possible phosphodegron motif that influences distinct conformational ensembles and inter‐domain interface specific to PTEN‐ASD and PTEN‐cancer phenotypes. By integrating in silico modeling, molecular simulations, and allosteric communication analyses we reveal the role of C‐tail phosphorylation on conformational dynamics and allosteric regulation of PTEN‐ASD and PTEN‐cancer phenotypes that exhibited the most exaggerated disease‐specific effects in our pilot studies. We show that C‐tail phosphorylation in PTEN‐ASD induces fluctuations in degradation‐sensitive sites (K13 and K289) that render a closed conformation. Furthermore, allosteric analysis reveals inter‐domain effector residues that strengthen the structural communication pathway. In contrast, a higher intrinsic flexibility and less stable conformational state emerges in PTEN‐cancer. Importantly, this reveals a less dense inter‐domain structural communication pathway that lacks effector degradation‐sensitive residues. Our integrative approach offers new insight into structurally correlated areas for potential drug targeting and will serve as the basis for designing novel therapeutics to attenuate PHTS‐associated cancer and ASD phenotypes.
Characterization and Chemical Optimization of the D2 Dopamine Receptor‐Selective Antagonist, ML321, Identifies Lead Compounds for the Clinical Treatment of Neuropsychiatric Disorders
Authors: Nilson Ashley N. , Dulcey Garcia Andres , Free R. B. , Boldizsar Noelia , Pearlstein Hannah , Rocereta Julia A. , Rodriguiz Ramona M. , Lane J. R. , Lee Kuo H. , Shi Lei , Wetsel William C. , Marugan Juan J. , Sibley David R.
Published: May 2022

Schizophrenia is a devastating illness characterized by both positive (hallucinations, delusions) and negative (flat affect, decreased motivation) symptoms coupled with cognitive impairment. Current antipsychotic medications are effective in treating the positive symptoms through antagonism of the D2 dopamine receptor (D2R). However, antipsychotic treatment is also hindered by side‐effects due to off‐target activities at other GPCRs and unfavorable binding kinetics at the D2R. We have identified and characterized a novel D2R antagonist with high selectivity against other GPCRs – ML321. In functional profiling screens of up to 168 different GPCRs, ML321 showed little activity beyond potent inhibition of the D2R, and to a lesser extent the D3R, demonstrating exceptional GPCR selectivity. Schild‐type functional assays revealed that ML321 acts as a competitive antagonist of the D2R while kinetic studies showed that ML321 exhibits slow‐on and fast‐off receptor binding rates; properties that are believed to limit extrapyramidal side‐effects that are commonly observed with antipsychotics. In fact, using doses that were maximally effective in antipsychotic‐predictive behavioral assays in rodents, ML321 promoted little to no catalepsy, suggesting that ML321 may produce less extrapyramidal side‐effects in patients. Importantly, no other D2R antagonist exhibits this pharmacological and behavioral profile supporting its development into an advanced drug lead. While a promising therapeutic, ML321 has a short metabolic half‐life, impeding its clinical development. A metabolite study revealed that the primary site of metabolism involves oxidation of the alkyl‐thiophene portion of ML321. To create more metabolically stable derivatives, we iteratively designed and synthesized over 100 analogs with modifications focused on the alkyl‐thiophene moiety. These analogs were pharmacologically characterized for both D2R binding affinity and function, and were also tested for metabolic stability, permeability, and solubility. These efforts have led to the optimization of ML321 into a collection of lead candidates that show similar pharmacological characteristics of ML321, but with marked increases in metabolic stability and ADME properties. Molecular docking and mutagenesis studies have led to a better understanding of how ML321 binds to the D2R, which will further assist in analog design and development. Together, these findings have advanced our understanding of ML321 structure‐activity relationships, particularly around the alkyl‐thiophene moiety, and have identified lead candidates for in vivo pharmacokinetic studies, thus representing substantial progress in the development of a new antipsychotic treatment.
Plate‐Based Respirometry to Assess Zebrafish Embryo Bioenergetics in situ in Changing Environmental Conditions
Authors: Rollwitz Erik , Jastroch Martin
Published: May 2022

All species require bioenergetic adaptation to maintain life functions, especially in changing environmental conditions. Global warming and deteriorating water quality may pose threats to aquatic species by interfering with bioenergetic mechanisms. Mitochondrial oxidative metabolism is a key component for bioenergetic responses as it provides the major proportion of cellular energy in the form of adenosine triphosphate (ATP). In our studies, we aim to establish the assessment of zebrafish mitochondrial bioenergetics and investigate the mechanisms, adaptation, and plasticity to temperature, pollutants, and the combination of both to gain insights into the upcoming threats for their energy homeostasis and survival. Using plate‐based respirometry of the Seahorse XF96 analyzer, we measured zebrafish (Danio rerio) embryo oxygen consumption rates in situ. To understand how temperature alters oxidative phosphorylation, we exposed 48 hours post‐fertilization zebrafish embryos to five different water temperatures before measuring oxygen consumption at various assay temperatures. The oxygen consumption rates were challenged using respiratory chain inhibitors and chemical uncouplers to reveal bioenergetic mechanisms and adjustments of mitochondrial efficiency. We show that at 18°C assay temperature, basal oxygen consumption of the embryo is low due to decreased ATP‐linked respiration and not limited by maximum substrate oxidation. Basal oxygen consumption rates increase with assay temperature and remain stable up to 37°C, before rapidly deteriorating at 41°C and above. The pre‐exposure of embryos to 37°C, however, expands the thermal window to higher limits, indicated by stable basal oxygen consumption rates at 41°C. Proton leak respiration, which wastes nutrient energy in parallel to ATP production, increases at assay temperatures above 28°C, thereby reducing the efficiency of ATP synthesis. This is seen as a decrease in coupling efficiency, which is defined as the fraction of oxygen consumption that drives ATP synthesis. Collectively, temperature affects the performance of zebrafish embryos by reducing cellular ATP turnover at lower temperatures and by increasing proton leak at higher temperatures, resulting in an optimum energy conversion temperature that can be shifted by thermal adaptation. In current experiments, we measure how pollutants impact zebrafish bioenergetics and its relation to thermal adaptation.
Effect of Self‐Reported Anxiety Levels on Blood Pressure Responses to Slow Breathing
Authors: Howse Fiona M. , Chen Celine , Jette Evan D. , Coovadia Yasmine , Usselman Charlotte W.
Published: May 2022

Anxiety, an emotion involved in the development and presentation of anxiety disorders, is linked to elevated blood pressure (BP) which may contribute to the increased cardiovascular disease risk observed in individuals diagnosed with anxiety disorders. Slow breathing (SB), defined as a respiration rate below 10bpm, has been implemented successfully to reduce BP in several clinical populations. However, to the best of our knowledge, the efficacy of SB has yet to be investigated in individuals with anxiety. Therefore, the purpose of this study was to compare BP responses to an acute bout of SB between individuals with moderate to severe anxiety and individuals with no or mild levels of anxiety. We hypothesized that individuals with moderate/severe anxiety levels would exhibit greater SB‐induced decreases in BP compared to individuals with no/mild anxiety levels. We assessed 14 young (18‐25 years old) lean (body mass index < 24.9kg/m2) non‐smoking individuals with moderate/severe anxiety levels (n=7), as well as age‐ and body mass index‐matched individuals with no/mild anxiety levels during 15‐mins of rest and during 15‐mins of device‐guided SB (RESPeRATE). Anxiety levels were assessed using the Hospital Anxiety and Depression Scale, in which participants with a score of at least 8 were categorized as having a moderate/severe anxiety score (ANX) and individuals with scores less than 8 were classified as having no/mild anxiety levels (CTRL). Respiratory patterns were monitored via respitrace strain gauge. Beat‐by‐beat BP was measured using finger photoplethysmography calibrated to values obtained from manual sphygmomanometry. Respiratory and systolic BP (SBP) data were extracted averaged across 5‐mins of steady state rest and during the final 5‐mins of SB; differences between ANX and CTRL were evaluated using unpaired t‐tests. Despite similar resting SBP between ANX and CTRL (111.28 ± 11.40 vs 109.39 ± 7.05mmHg; p=0.75), the SBP response to SB differed between ANX and CTRL (‐2.18 ± 4.84 vs 3.57 ± 4.47mmHg; p=0.04). In fact, ANX experienced a clinically meaningful SB‐induced reduction in BP (defined as a change greater than or equal to 2 mmHg) that was absent in CTRL. Interestingly, resting respiration rates were higher in ANX than CTRL (13.33 ± 4.07 vs 7.74 ± 2.18bpm; p<0.01), and reductions in respiration rate during SB were greater in ANX than CTRL (‐9.27 ± 3.74 vs ‐2.66 ± 2.19bpm; p<0.01). Taken together, in a young and relatively healthy cohort of individuals with a range of self‐reported anxiety levels, it appears that SB is effective in lowering SBP in individuals with moderate or high levels of anxiety but has no effect in individuals reporting no or low levels of anxiety. Thus, SB may be an effective intervention to reduce BP in individuals with high anxiety levels, which may have long‐term implications in the treatment of elevated BP and reduction of cardiovascular disease risk in individuals diagnosed with anxiety disorders.
Increased Nuclear Localization of Engineered Hsp104 Variants Mitigates aS, FUS, and TDP‐43 Toxicity in Yeast
Authors: Mass Benjamin , Shorter James , Lin JiaBei
Published: May 2022

The cytoplasmic mislocalization and aggregation of aberrantly folded proteins is a hallmark of human neurodegenerative diseases, including Parkinson’s disease (PD) and frontotemporal dementia‐amyotrophic lateral sclerosis (FTD‐ALS). Endogenous human disaggregase machineries are insufficient to combat pathological protein aggregates. However, the yeast protein Hsp104 has shown enhanced activity in antagonizing oligomers and aggregates of the PD‐associated protein alpha‐Synuclein (aS) and the FTD‐ALS‐associated proteins Fused‐In‐Sarcoma (FUS) and TAR DNA binding protein 43 (TDP‐43). Hsp104 is a hexameric AAA+ ATPase and protein disaggregase which, in collaboration with the Hsp70/Hsp40 chaperone system, can extract peptides from higher‐order protein structures and mediate their proper refolding and cellular localization. Strategic missense mutations potentiate the ability of Hsp104 to mitigate aS, FUS, and TDP‐43 toxicity in yeast. However, on their own, some of the potentiated Hsp104 variants demonstrate off‐target toxicity which impairs yeast growth. Here, yeast survival assays show that increasing the nuclear localization of these already potentiated Hsp104 mutants reduces their off‐target toxicity and improves yeast growth. Additionally, fluorescence microscopy indicates that the resolution of cytoplasmic foci and the proper relocalization of disease‐associated proteins often corresponds to increased yeast survival. Together, these data suggest that increasing the nuclear localization of already potentiated Hsp104 mutants further enhances their rescue activity, in part through the increased resolution of cytoplasmic aS, FUS, and TDP‐43 foci. Ultimately, the delivery of these potent nuclear Hsp104 mutants to first animal models and then humans could represent a novel therapy for a variety of neurodegenerative diseases.
High‐intensity Interval Training Combined With Blood‐flow Restriction Predominantly Alters Anaerobic Capacity in Endurance‐trained Athletes
Authors: Billaut François , Bourgeois Hubert , Paradis‐Deschênes Pénélope
Published: May 2022

Blood‐flow restriction (BFR) training has gained popularity amongst athletes and sport practitioners to enhance training adaptations and performance. However, BFR has typically been associated with low‐intensity exercise, and there is still limited evidence of its impact when combined with high‐intensity training. This study investigated the impact of high‐intensity interval training (HIIT) with concurrent BFR on anaerobic and aerobic physical capacities and key physiological responses. In a pre‐post, parallel‐groups design, fifteen endurance‐trained males (VO2max 65.0±4.8 mL/min/kg) included three sessions of HIIT per week (sets of 15 s ON/15 s OFF at 100% maximal aerobic power) into their usual training for three weeks either with (BFR group, n=9) or without restriction (CTL group, n=6). In BFR, cuffs were worn proximal on both quadriceps and inflated progressively from 50 to 70% of arterial occlusion pressure across training weeks. Before and after training, athletes completed a maximal incremental step cycling test, a Wingate anaerobic test (WAnT) and a 5‐km cycling time trial. Blood samples were drawn during the time trials. Maximal aerobic power increased in BFR (364.8±60.7 vs 383.4±61.4 watts, p=0.003, Cohen’s effect size ES 0.27) but not in CTL (381.2±62.1 vs 385.5±64.4 watts, p=0.45, ES 0.05). Concomitantly, mean power output achieved during the WAnt also increased with BFR (23.8±4.0 vs 24.6±4.1 kJ, p=0.08, ES 0.28) but not in CTL (23.4±2.5 vs 23.4±2.4 kJ, p=0.95, ES 0.01). However, there was no change between groups in VO2max (BFR: 0.86% vs CTL: 1.77%, ES ‐0.15) and in both the mean power output (BFR: 1.81% vs CTL: 6.51%, ES ‐0.21) and completion time (BFR: ‐0.69% vs CTL: ‐2.55%, ES ‐0.21) of the 5‐km time trial. During the time trial, BFR also induced greater changes in pH (BFR: ‐0.05 vs CTL: ‐0.03 units, ES ‐0.43) and base excess (BFR: ‐1.55 vs CTL: ‐0.70 units, ES ‐0.37), and lowered the potassium ion concentration (BFR: ‐0.55 vs CTL: 0.44 mmol/L, ES ‐0.63). There was no change between groups in lactate production from pre‐ to post‐training. These findings suggest that short‐term HIIT combined with BFR improved anaerobic capacity in endurance‐trained athletes without meaningful effect on endurance exercise performance.