bluuebunny/crossref_metadata_2025_split huggingface dataset:
Exposure to Repeated Cold Stress Influences Sympathetic and Cardiovascular Responses to Muscle Stretch in Decerebrated Rats
Authors: Hori Amane , Nasu Teruaki , Saito Ryuji , Katanosaka Kimiaki , Mizumura Kazue , Mizuno Masaki , Hotta Norio 
Published: May 2022

Chronic stress is known to cause adverse physical and mental effects such as pain, chronic fatigue, and depression, and it is strongly related to many diseases and syndromes (e.g., fibromyalgia, chronic fatigue syndrome, and post‐traumatic stress disorder). Evidence suggests that exercise and physical activity in leisure time are effective in improving stress‐induced symptoms. Repeated cold stress (RCS), in which an animal is repeatedly exposed to alternating room and low temperatures, is one of the chronic stress models that induces chronic pain and depression. Mechanical hyperalgesia through thin muscle afferent fibers has also been reported to occur from RCS exposure. Since the muscle afferents have a dual modulatory function in nociception and cardiovascular reflex, we hypothesized that RCS augments the mechanical component of the exercise pressor reflex (the skeletal muscle mechanoreflex), i.e., skeletal muscle afferents‐mediated increases sympathetic nerve activity (SNA) and arterial blood pressure (AP).[Purpose]The purpose of this study was to clarify the impacts of RCS on sympathetic and cardiovascular responses to stimulation of the skeletal muscle mechanoreflex in decerebrated rats.[Methods]Male Sprague‐Dawley rats (body weight: 410 ± 15 g, age: 12 weeks) were exposed to RCS using a homemade automated RCS device. The rats were alternately moved to room temperature (22°C) and cold temperature (4°C) compartments at 30‐min intervals for 5 days. To assess the skeletal muscle mechanoreflex function, we measured mean AP (MAP), heart rate (HR), and renal SNA (RSNA) responses to 30‐s static passive stretching of the hindlimb muscles by in vivo recording from unanesthetized decerebrated rats. Stretching was performed by tracing the maximum tension curve obtained from 30‐s electrical stimulation. Changes (Δ) from baseline to peak values of the measured parameters between control and RCS rats were compared.[Results]Peak tension during the Achilles tendon stretch from baseline was not significantly different between groups (control: Δ929 ± 63 g [n = 6] vs RCS: Δ765 ± 66 g [n = 4], p = 0.17). Importantly, the peak RSNA response in RCS rats (Δ236.3% and Δ242.7%, n = 2) tended to be greater than that in control rats (Δ37.1% and Δ23.9%, n = 2). Moreover, RCS had a significantly (p < 0.05) greater HR response (RCS: Δ3.7 ± 1.0 bpm [n = 4] vs control: Δ0.6 ± 0.5 bpm [n = 6]), and MAP response tended (P = 0.07) to be higher in the RCS group (Δ23 ± 6 mmHg, n = 4) than in the control group (Δ8 ± 4 mmHg, n = 6).[Conclusions]Our preliminary data demonstrate that RCS augments the skeletal muscle mechanoreflex. These results suggest that chronic stress can potentially cause exaggerated cardiovascular responses during physical activity.
Metal Chelators Ethylenediaminetetraacetic acid (EDTA) and N,N,N′,N′‐tetrakis(2‐pyridinylmethyl)‐1,2‐ethanediamine (TPEN) Interfere with Carbonic Anhydrase (CA) Activity in Wilbur‐Anderson Assay (WAa)
Authors: Snitsarev Vladislav , Lucas Madeline , Petroff Elena , Vega Quinn C. 
Published: May 2022

In our effort to set up a laboratory for undergraduate students with the tentative title ”Studying enzymatic activity of Carbonic Anhydrase”, we successfully determined carbonic anhydrase activity and demonstrated its inhibition by Acetazolamide, a potent CA inhibitor, using the Wilbur‐Anderson assay (WAa) using 6 experimental set‐ups with a class of 24 students.WAa hinges upon determining the time T (in seconds) required for 4 mL of saturated with carbon dioxide (CO2) water to lower pH from 8.3 to 6.3 of the mixture when added to 6 mL of 0.02M Tris buffer at 0°C without (T0) and with enzyme (TE). Activity (A) is defined as A = 2*(T0/TE‐1) assay units (AU). In n = 16 blank trials, T0 = 75.0±10.7s (Mean±Standard deviation), and with 0.001mg of CA in n = 12 trials, TE = 50.2±8.1s, A = 0.99±0.66 AU (p < 0.001). When for a test we used EDTA, a carboxylic high‐affinity metal ion chelator, to inactivate this Zn2+‐containing enzyme, we unexpectedly encountered that in the presence of the sodium salt of EDTA (Na2EDTA): 1) T was significantly slowed in a concentration‐dependent manner: for 5µM, 50µM, and 500µM, T was 84.3±7.9s (n = 10, p < 0.05), 108.5±.6s (n = 7, p < 0.001), and 130.8±22.7s (n = 10, p < 0.001), respectively; and 2) unexpectedly, in the context of the previous finding, Na2EDTA significantly enhanced CA activity (TE = 38.5±4.0s, A = 1.84 AU, n = 10, p < 0.001) in the presence of 5µM Na2EDTA that was abrogated by 10µM Acetazolamide, a potent CA inhibitor (T = 138.3±10.4s, n = 7, p < 0.001). TPEN, a pyridinic non‐carboxylic metal ion chelator that is more potent in metal ion chelation that EDTA, significantly slowed T0 (99.5±15.9s, n = 4) similar to Na2EDTA, and significantly enhance CA activity (TE = 41.1±3.8, n = 4, p < 0.05) with equal potency.This indicates that principally chemically different metal ion chelators EDTA and TPEN interfere with the determination of CA activity in the context of WAa. Studies to explain these phenomena are underway with the hypotheses that the contaminating metal ions that catalyze CO2 hydration/dehydration without CA are responsible for the observed phenomena.
Skeletal Muscle Deoxygenation Amplitude, Rather Than Splenic Emptying Contributes to Higher V̇O2max. During Supine Cycling in Healthy Active Men
Authors: Zubac Damir , Obad Ante , Šupe‐Domić Daniela , Bošnjak Ana , Zec Mirela , Ivančev Vladimir , Valić Zoran 
Published: May 2022

The aim of the present study was to investigate whether greater splenic emptying or higher skeletal muscle deoxygenation amplitude (determined by near‐infrared spectroscopy, NIRS) contributes to higher V̇O2 max. during supine cycling. We hypothesized that the reduction in splenic volume, and subsequent manipulations of O2 delivery by blood ejected from the spleen, would not contribute to higher V̇O2max. during supine cycling, because this transient increase in circulating red blood cells (RBC’s) would be insufficient to increase the O2 storage capacity in the blood. Rather, O2 uptake during supine cycling in healthy subjects is expected to be limited, at least in part, by skeletal muscle bioenergetics. Here, fourteen healthy young individuals completed all study procedures. On three separate days they underwent a medical examination, supine V̇O2 max. test, and three step‐transitions from 20 W to a moderate‐intensity power output equivalent to V̇O2 uptake at 90% gas exchange threshold. During these step‐transitions, pulmonary V̇O2, NIRS of the vastus lateralis (VL), and cardiovascular responses (via Finapres) were measured continuously. In parallel, minute‐by‐minute ultrasound measurements of the spleen were performed. Blood samples were taken before and immediately after the step‐transition cycling. Mean V̇O2 max. was 46.5±6.5 mL·kg·min‐1. In response to supine step‐transition cycling, spleen volume was significantly reduced (by ~38%, p=.001). Both hematocrit and hemoglobin concentration increased by ~4%, after exercise cessation. There was no correlation between: i)spleen volume at rest, ii) changes in spleen volume (%), iii) hematocrit at rest and V̇O2 max. However, a linear, near‐significant correlation was observed between VL deoxygenation amplitude and V̇O2 max. Apparently, individuals with a higher V̇O2 max. are capable of greater O2 extraction from the superficial knee extensors, whereas in parallel, greater splenic emptying does not correlate with higher V̇O2 max, regardless of the subsequent release of erythrocytes. Therefore, it is reasonable to suggest that maximal O2 utilization during supine cycling in healthy young men is limited, in part, by skeletal muscle bioenergetics.ReferencesHolmström, PK, Karlsson Ö, Lindblom H, McGawley K, & Schagatay, EK. (2021). Enhanced splenic volume and contraction in elite endurance athletes. J Appl Physiol, 131(2), 474‐486, DOI: 10.1152/japplphysiol.01066.2020;Zubac, D, Obad, A, Bosnjak, A, Zec, M, Ivancev, V, Valic, Z. (2021). Spleen emptying does not correlate with faster oxygen kinetics during a step‐transition supine cycling. Appl Physiol Nutr Metab. Accepted for publication in Jun 24. doi: 10.1139/apnm‐2021‐0294.2021.
Activation of Leptin‐Receptor‐Expressing Neurons in the Dorsomedial Hypothalamus Increases Hypercapcnic Ventilatory Response and Relieves Obstructive Sleep Apnea in Obese Mice
Authors: Amorim Mateus R. , Wang Xin , Pho Huy , Bevans‐Fonti Shannon , Anokye‐Danso Frederick , Escobar Joan , Dergacheva Olga , Branco Luiz G. S. , Mendelowitz David , Polotsky Vsevolod Y. 
Published: May 2022

IntroductionObesity leads to obstructive sleep apnea (OSA), characterized by loss of upper airway muscle tone during sleep leading to carbon dioxide retention and intermittent hypoxemia. OSA leads to high neurocognitive, cardiovascular, and oncologic morbidity and mortality. There is no effective pharmacotherapy for OSA. We have previously shown that an adipose‐produce hormone leptin acts in the brain to abolish hypoventilation and upper airway obstruction during sleep, and, therefore may be a promising therapeutic in obese patients with OSA. Our previous data also suggest that leptin stimulates breathing acting on the leptin receptor (LEPRb) positive neurons in the dorsomedial hypothalamus (DMH), but not in the medulla. Herein, we tested the hyphotesis that leptin stimulates control of breathing and improves upper airway patency during sleep acting on LEPRb positive DMH neurons and their projections to serotonergic neurons in raphe. Aim:In this study, we examined the contribution of LEPRb positive neurons in the DMH to control of breathing and upper airway function during sleep in diet‐induced obese mice.MethodsWe expressed designer receptors exclusively activated by designer drugs (DREADD) selectively in the LEPRb positive neurons of the DMH of Leprb‐Cre‐GFP mice with diet‐induced obesity (DIO) and examined the effect of DREADD ligand, J60, on breathing during sleep.ResultsChemogenetic activation of LEPRb positive DMH neurons increased inspiratory flow, respiratory rate, and minute ventilation during REM sleep, without significant changes in sleep architecture. The effect was particularly pronounced during inspiratory flow limitation, which suggest that upper airway obstruction during REM sleep was relieved. Activation of LEPRb positive DMH neurons with a DREADD ligand, J60, increased the hypercapnic ventilatory response and this response was blunted by a serotonin receptor antagonist, methysergide. In vitroexperiments revealed that DMH LEPRb neurons project to the serotonergic neurons in the dorsal raphe and that optogenetic stimulation of DMH LEPRb neurons evoked excitatory post‐synaptic currents in ChR2 expressing neurons.ConclusionLeptin up‐regulates the hypercapnic ventilatory response and upper airway patency during sleep acting on LEPRb neurons in the DMH. Respiratory effects of leptin are likely mediated via projections of these neurons to the dorsal raphe.
Stabilization of Meta‐I Rhodopsin Conformation by a Nanobody
Authors: Salom David , Wu Arum , Sander Christopher L. , Pardon Els , Steyaert Jan , Kiser Philip D. , Palczewski Krzysztof 
Published: May 2022

Binding of an extracellular signaling molecule to a G protein‐coupled receptor (GPCR) results in G protein activation, which in turn triggers the production of second messengers. Although many advances have been achieved in understanding GPCR activation, the early steps of GPCR of this process are still poorly understood. In the case of rhodopsin, the paradigmatic member of class A GPCRs, the endogenous ligand is retinal which is covalently bound to K296 through a Schiff base. The activation of rhodopsin is triggered by the absorption of a photon and the isomerization of 11‐cis‐retinal to all‐trans‐retinal. When rhodopsin in ground state (λmax= 500 nm) is photoactivated, it progresses through short‐lived intermediates leading to the Meta‐I state (λmax= ~478 nm), which then establishes an equilibrium with Meta‐II (λmax= 380 nm), the G‐protein binding state.We have developed a llama antibody (nanobody Nb2) that binds to bovine rhodopsin and stabilizes its early activation intermediate Meta‐I. Here we present the crystal structures of Nb2 in complex with ground‐state bovine rhodopsin, as well as Nb2 in complex with apo‐rhodopsin (opsin). The binding site of Nb2 includes rhodopsin’s N‐terminus and extracellular loops ECL2 and ECL3. Whereas rhodopsin’s structure in the ground‐state rhodopsin/Nb2 complex is virtually identical to that of rhodopsin alone, Nb2 binding induces a dramatic structural change in opsin, trapping it in a conformation similar to that of ground‐state rhodopsin. In a similar way, when Nb2 binds in solution to photoactivated, deprotonated rhodopsin (Meta‐II) it shifts the equilibrium towards a protonated Schiff base state corresponding to the Meta‐I conformation. Finally, co‐expression of Nb2 and P23H mutant rhodopsin in HEK293 cell lines is able to rescue the phenotype of misfolding and degradation in vitro, suggesting a therapeutic potential of this nanobody.In summary, we have developed a nanobody that traps photoactivated rhodopsin in the elusive Meta‐I state; in addition, Nb2 stabilizes a misfolding‐prone mutant of rhodopsin. Nb2 might also be a useful tool to facilitate the crystallization of wild type and mutants of rhodopsin in different conformational states bound to different ligands. Current efforts include solving the crystal structure of the Meta‐I rhodopsin/nanobody complex.
Galectin‐3 Regulates Oxidant Stress in Obesity
Authors: Padgett Caleb A. , Speese Andrew C. , Rosewater Cody L. , Corley Zachary L. , Mintz James D. , Fulton David J. , Stepp David W. 
Published: May 2022

ObjectiveInvestigate the mechanism by which overexpression of Galectin‐3 (GAL3) in the endothelium of obese mice contributes to excess reactive oxygen species (ROS) production and subsequent vascular dysfunction.Methodsdb/db mice, a well‐characterized model of human obesity, were crossed with mice lacking GAL3, a receptor for advanced glycation end products, to generate lean and obese mice with and without GAL3 deletion. Metabolic and cardiovascular endpoints were assessed by mRNA and protein expression in freshly isolated microvascular endothelial cells, microvascular reactivity assessed by pressure myography, and ROS production assessed by dihydroethidium (DHE) staining. Other previously characterized db/db colonies were utilized to further elucidate the mechanistic contributions of GAL3, including mice harboring deletions of NADPH Oxidase I (NOX1), Myostatin (MSTN), and Protein Tyrosine Phosphatase 1B (PTP1B).ResultsDeletion of GAL3 in obese mice had no observable effect on anatomical, metabolic, or plasma phenotype, except for normalizing levels of plasma ROS as evidenced by decrease in thiobarbituric acid reactive substances (TBARS). Obese mice exhibited profound endothelial dysfunction, which was rescued by deletion of GAL3. Our data demonstrate that NOX1, the predominant source of endothelial superoxide production, is significantly downregulated by GAL3 deletion, thereby restoring endothelial function and ameliorating endothelial ROS production in obesity. Furthermore, we demonstrate that GAL3‐mediated NOX1 overexpression is amenable to improvements in metabolic status, such as lowering blood glucose with metformin, improving glucose handling by augmenting muscle mass through deletion of MSTN, or improving insulin signaling through deletion of PTP1B.ConclusionTaken together, these data demonstrate that the overproduction of superoxide by endothelial NOX1 is regulated by the metabolic sensor GAL3 in obesity, leading to endothelial dysfunction and subsequent cardiovascular disease. This pathway presents an attractive target for therapeutic intervention to break the link between aberrant metabolism in obesity and its corresponding vascular pathologies.
Depolarized Mitochondrial Membrane Potential and Protection with Duroquinone in Isolated Perfused Lungs from Rats Exposed to Hyperoxia
Authors: Audi Said , Ganesh Swetha , Taheri Pardis , Zhang Xiao , Dash Ranjan , Clough Anne , Jacobs Elizabeth 
Published: May 2022

ObjectiveDissipation of mitochondrial membrane potential (Δψm) is a hallmark of mitochondrial dysfunction, which plays a key in the pathogenesis of Acute Respiratory Distress Syndrome (ARDS). Our objective was to use a previously developed experimental‐computational approach to estimate tissue Δψm in isolated perfused lungs of rats exposed to hyperoxia as a rat model of human ARDS, and to evaluate the ability of the ubiquinone analog duroquinone (DQ) to reverse any hyperoxia‐induced depolarization of lung Δψm.MethodsRats were exposed to hyperoxia (>95% O2) or normoxia (room air) for 48 hrs, after which lungs were excised and connected to a ventilation‐perfusion system. The experimental protocol consisted of measuring the concentration of the fluorescent dye rhodamine 6G (R6G) during three single‐pass phases: loading, washing, and uncoupling, in which the lungs were perfused with and without R6G, and with the mitochondrial uncoupler FCCP, respectively. For normoxic lungs, the protocol was repeated with 1) rotenone (mitochondrial complex I inhibitor), 2) rotenone and either DQ or its vehicle (DMSO), and 3) rotenone, glutathione (GSH), and either DQ or DMSO added to the perfusate. Hyperoxic lungs were studied with and without DQ and GSH added to the perfusate. A physiologically‐based pharmacokinetic (PBPK) computational model was used to estimate lung Δψm from R6G data.ResultsFigure 1 shows that for hyperoxic lungs, R6G venous effluent concentrations tended to be higher during the loading phase, but significantly lower during the uncoupling phase compared to normoxic lungs, consistent with less uptake of R6G by hyperoxic lungs due to partial depolarization of Δψm. Figure 1 also shows that adding DQ to perfusate during the three phases almost completely reversed differences in R6G concentrations between normoxic and hyperoxic lungs. The PBPK computational model analysis of the R6G data show that exposure to hyperoxia depolarized lung tissue Δψm from ‐140 ± 9 (SE, n = 5) mV to ‐107 ± 9 (n = 5) mV, and complex I inhibition depolarized lung Δψm from ‐140 ± 9 (n = 5) to ‐57 ± 2 (n = 4) mV. Computational model analysis results also demonstrate the efficacy of DQ to fully reverse both rotenone‐induced and hyperoxia‐induced lung Δψm depolarization, consistent with the ability of the lungs to reduce DQ to its hydroquinone form (DQH2), which in turn bypasses mitochondrial complexes I and II to reduce mitochondrial complex III and restores Δψm.ConclusionsThis study demonstrates hyperoxia‐induced Δψm depolarization in isolated perfused lungs, and the utility of this approach for assessing the impact of potential therapies such as exogenous quinones that target mitochondria in isolated perfused lungs.
Mice Exposed to Chronic low‐dose Ethanol Exhibit Reduced Sensitivity to Changes in Magnitude of Reward
Authors: Curran‐Alfaro Christina M. , Bryant Kathleen G. , Barker Jacqueline M. 
Published: May 2022

While approximately 2 billion individuals consume alcohol worldwide, the majority of these individuals to not exhibit drinking patterns consistent with alcohol use disorders (AUDs). Efforts have been made in investigating and characterizing the adverse effects on learning, memory and reward seeking with individuals afflicted with AUDs in a pursuit of therapeutic advancements. However, sub‐diagnostic alcohol effects have been understudied, despite the large number of low‐dose alcohol drinkers and the potential for low dose alcohol to impact both brain and behavior.Data from our lab has shown that low‐dose alcohol exposure enhances reward motivation in male, but not female, mice, but the mechanism of this is effect is unclear. Thus, we investigated the effects of low‐dose alcohol exposure on sensitivity to change in reward magnitude. A group of adult male C57BL6J mice were trained to self‐administer a 20 ul sucrose reward and received either a low‐dose ethanol (0.5g/kg) or saline injection 1 hour after training. To assess motivation, we used a progressive ratio (PR) task in which the effort required to earn the sucrose reward was increased linearly. Mice received multiple counterbalanced PR sessions in which the progressive schedule remained the same, but the reward magnitude was changed from 20ul (baseline) to 35ul, 25ul, 15ul or 5ul in counterbalanced sessions.Consistent with sensitivity to changes in reward value, saline control mice reduced or increased responding when reward volume was reduced or increased, respectively. Similar to saline controls, chronic low‐dose ethanol exposed mice increased their responding when reward volumes were increased compared to the 20ul baseline. However, ethanol exposed mice did not reduce responding to small reductions in reward magnitude. This observation in low‐dose ethanol mice suggests either a failure to detect reductions in reward magnitude or a dissociation between reduced reward magnitude and behavioral response. This lack of strategizing behavior to maintain effort‐to‐reward balance by ethanol exposed mice may imply that chronic low‐dose ethanol exposed mice may perceive greater reward value than controls even at reduced volume and implicates even low‐dose ethanol as a contributor to aberrant reward processing.
Examining the role of Drp1 in age‐related microvascular dysfunction
Authors: Gutierrez Huerta Cristhian A. , Hader Shelby N. , Beare Jason E. , Tracy Evan , Astbury Katherine , Jacobs Elizabeth R. , LeBlanc Amanda J. , Gutterman David D. , Beyer Andreas M. 
Published: May 2022

IntroductionAdvanced age is a well‐documented risk factor for coronary microvascular dysfunction. Recent evidence has underscored that advanced age is associated with a shift toward greater mitochondrial fission rather than mitochondrial fusion. Therefore, understanding key regulators of the balance of mitochondrial fission/fusion may help to identify the contribution of mitochondrial dynamics on microvascular endothelial function. DRP1 is a pro‐fission factor that has been linked to several disease manifestations including advanced age and cardiovascular disease (CVD).MethodsCoronary arteries were obtained from young control rats (YC, 15‐20 weeks) and old control rats (OC, 95‐100 weeks) and expression of DRP1 was assessed by immunofluorescence (IF). A novel rat model crossing flox‐stop DRP1‐GFP rats and VECAD‐Cre expressing rats was generated to assess the vascular and cardiac effects of endothelial‐specific DRP1 overexpression. Endothelial vascular function was determined by endothelial‐dependent flow‐mediated dilation while cardiac function was determined by echocardiography at 20 weeks old and compared to wildtype control group.ResultsData from IF comparing coronary arteries from YC and OC show that OC had higher DRP1 expression compared to YC (1.42 to 1.00, p=0.01). Max dilation measurements, taken at 20‐weeks of age, showed that DRP1‐overexpressing rats was decreased when compared to wildtype (WT) control rats (31.4 to 60.1, p=0.009). Echocardiography revealed that DRP1‐overexpressing rats showed a trend towards a decrease in coronary flow reserve and increased HR in response to a dobutamine stressor when compared to WT. Ejection fraction between the two groups remained largely preserved.ConclusionOur data demonstrate that there may be a critical role for DRP1 within in vitro vasodilation that mimics a CVD pro‐fission phenotype. Future studies will be performed to further evaluate the role of DRP1 in response to other stressors well‐established in age‐dependent CVD such as hypertension.