bluuebunny/crossref_metadata_2025 huggingface dataset:
Role of S‐nitrosoglutathione reductase (GSNOR) Dysregulation and Nitrosative Stress in Heart Failure With Preserved Ejection Fraction
Authors: LaPenna Kyle B. , Li Zhen , Sharp Thomas E. , Mose Karl H. , Doiron Jake E. , Xia Huijing , Goodchild Traci T. , Lefer David J.
Published: May 2022

Background

Heart failure with preserved ejection fraction (HFpEF) is the most predominant form of heart failure in the United States. This multi‐system pathology results in a 5‐year mortality rate for which there are very few treatment options. The obese ZSF1 rat model has previously been shown to recapitulate much of the human phenotype including obesity, metabolic syndrome, diabetes, and hypertension. In this study, we investigated the complex and largely unknown role of abnormal nitric oxide signaling and nitrosative stress in the pathobiology of HFpEF.

Materials and Methods

Male ZSF1 rats and normotensive controls of Wistar Kyoto (WKY) background strain (n=7 each group) were studied at 10, 14 and 18 weeks of age to monitor disease progression and alterations in nitric oxide signaling. Cardiac protein nitrosylation (RxNO) was measured by chemiluminescence. Analysis of circulating plasma nitrite bioavailability was performed using high performance liquid chromatography. GSNOR, one of the enzymes that degrade nitrosothiols, mRNA expression was measured and plotted as relative fold difference to WKY expression in this model of cardiometabolic HFpEF.

Results

ZSF1 rats exhibited significantly increased cardiac protein nitrosylation as the HFpEF phenotype progressed from 10 to 18 weeks of age as compared to the WKY rat group. GSNOR mRNA expression was significantly decreased in the ZSF1 rat relative to the WKY at all time points. During the later 18‐week timepont, cardiac GSNOR activity was found to be decreased compared to that of the WKY. Circulating plasma nitrite, a measure of eNOS function, was decreased in the ZSF1 rat at 14 and 18 weeks compared to the WKY at subsequent aging timepoints. These data correlate to an increasing left ventricular end diastolic pressure (LVEDP) and decreased vascular relaxation as the ZSF1 animals age and develop more severe HFpEF disease pathology.

Conclusion

Our data suggests that nitric oxide signaling is dysregulated in the setting of HFpEF resulting in a significant increase in cardiac protein nitrosylation coupled with a reduction in circulating nitrite and NO signaling in the vasculature. Excessive cardiac nitrosative stress resulting in pathological myocardial signaling has been previously implicated as a driver of HFpEF. Reduced vascular nitrite bioavailability resulting in attenuated physiological NO signaling could account for the profound vascular dysfunction observed in HFpEF patients. Future studies are aimed at the further elucidation of the role of various NO storage pools in HFpEF.
The New “Normal”: Online Tools for In‐Person Learning in the Molecular Life Sciences
Authors: Vega Quinn , Huff Mary , Booth Rachell
Published: May 2022

In the spring of 2020, the world experienced one of the worst pandemics in a century, forcing educators to almost immediately shift from on campus to online instruction. Given that most universities were largely built for teaching in person, the transition to distance learning required a rapid expansion of internet infrastructure and the mid‐semester implementation of online instructional tools. After the initial shock, confusion, and adjustment experienced in the spring of 2020, the continuation of the pandemic led many institutions to maintain a predominantly online or hybrid instruction model for the 2021 academic year. Although not as rushed as the previous semester, many faculty members were still trying to navigate the online landscape and adjust their curriculum to meet course objectives as defined during pre‐pandemic times. With universities inviting students back into the classroom and departments shifting more activities back to campus in the current academic year, we wanted to see what, if any, processes or activities implemented because of the pandemic would be retained or expanded post‐pandemic, specifically looking from the perspective of biochemists, molecular biologists, and science administrators. This feedback included information about alterations to in‐person instruction, communication with students outside of the classroom or laboratory, student assessment, additional educational opportunities that were not easily accessible previously, and enhanced research collaborations. Our conclusion is that, while few faculty members were interested in maintaining a primarily online format in future semesters, some distance learning tools were transferable to in‐person instruction and, in fact, enhanced the in‐person experience.
Compromised endothelial progenitor cell exosomal communication with endothelial cells in hypertension ischemia conditions
Authors: Wang Jinju , Chen Shuzhen , Polaki Venkata , Sawant Harshal , Bihl Ji
Published: May 2022

Background

We have previously demonstrated that EPC‐exosomes (EPC‐EXs) can protect endothelial cells (ECs) against hypoxia injury in vitro. What's more, we found that extracellular vesicles released from starved EPCs have opposite functions to those released in inflammation conditions, suggesting that EPC‐EXs‐mediated communication is functional and modulable by factors that affect the status of their parent cells. Given clinical studies showing the function of EPCs is declined in patients with hypertension, we speculate the EPC‐EX mediated communication with ECs is impaired in hypertension‐ischemia conditions.

Methods

EPC‐EXs were prepared from the bone marrow EPCs of wild type (WT) and hypertensive renin transgene (R+) mice and were denoted as WT‐EPC‐EXs and R‐EPC‐EXs, respectively. To mimic hypertension‐ischemia injury, ECs were treated with angiotensin II (10‐6 M) plus hypoxia (1% O2 for 6 hrs) and reoxygenation (21% O2 for 24 hrs). To determine the function of EPC‐EXs, ECs were co‐cultured with EXs for 24hrs. EX uptake efficiency, cellular viability and function were assessed.

Results

1) The incorporation efficiency of EPC‐EXs from R+ mice by ECs were decreased. 2) Angiotensin II plus hypoxia reoxygenation‐injured ECs displayed decreased cell viability, increased cell apoptosis and compromised angiogenic ability. 3) R‐EPC‐EXs displayed the impaired capability of rescuing ECs and improving their angiogenic ability on ECs as compared to that of WT‐EPC‐EXs did.

Conclusion

Our data have suggested that EPC‐EXs mediated communication of EPC/EC is compromised in R+ mice hypertension‐ischemia conditions. These findings suggest that impairment of EPC exosomal communication might contribute to the exaggerated cerebral ischemia injury in hypertension‐associated ischemic stroke.
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.
Complex Cognitive Functions and Neurovascular Coupling are Protected by Carbon Dioxide during Hypoxia
Authors: Bloomfield Peter M. , Green Hayden , Fisher James P. , Gant Nicholas
Published: May 2022

Reductions in environmental oxygen availability can impair the performance of cognitively demanding tasks. The compensatory increase in ventilation during hypoxia can have a counterproductive effect on the brain, as reduced arterial carbon dioxide (CO2) leads to cerebral vasoconstriction and a consequent decline in cerebral oxygen delivery. Providing supplemental CO2 exerts a protective effect on basic cognitive functions; however, the effects on complex real‐world tasks with a greater cognitive demand have yet to be examined. We tested the hypotheses that complex task performance and neurovascular coupling (NVC) would be impaired by poikilocapnic hypoxia, but comparatively protected during isocapnic and hypercapnic hypoxia. Accordingly, twenty‐one participants completed a 10‐minute simulated driving task whilst breathing room air (normoxia) or hypoxic air (PETO2 = 45 mmHg) under poikilocapnic, isocapnic, and hypercapnic conditions (PETCO2 = not manipulated, clamped at baseline, and clamped at baseline +10 mmHg, respectively). Simulated driving performance, assessed using a fixed‐base motor vehicle simulator, provided a complex cognitive task. Cortical oxygenation in the frontal cortex was measured using functional near‐infrared spectroscopy (fNIRS). The number of road speed limit exceedances was greater during the poikilocapnic than normoxic, hypercapnic, and isocapnic conditions (mean exceedances = 8, 4, 5, and 7, respectively, all p ≤ 0.05). Mean vehicle speed was greater in the poikilocapnic than normoxic and hypercapnic conditions (mean difference = 0.35 km.h‐1 and 0.67 km.h‐1, respectively). Compared to normoxia, prefrontal oxygenation decreased in all hypoxic conditions. During simulated driving, total haemoglobin in the right frontal cortex decreased to a greater extent in poikilocapnic hypoxia compared to normoxic, isocapnic, and hypercapnic conditions (mean decline = 390 %, 280 %, and 200 %, respectively), suggesting that cerebral blood volume was attenuated to a greater degree in the poikilocapnic condition (i.e., NVC blunted). Collectively, these findings demonstrate that controlling CO2 availability during severe hypoxia exerts a protective effect on complex cognitive task performance and NVC.
A Novel Technological Breakthrough in Effective Body Recomposition and Weight Management: A Therapeutic Strategy
Authors: Bagchi Debasis , Downs Bernard W. , Bagchi Manashi , Kushner Steve W. , Morrison Bruce S.
Published: May 2022

Obesity and overweight are now defined as an epidemic. An array of evidence substantiates that existing weight loss strategies and programs have not worked. Fat is the lightest of macromolecules, the highest energy reserve of the body, and the last reservoir of survival insurance to be expended. Conventional weight loss concepts and therapies, for the most part, are based on two primary factors; people eat too much and don’t exercise enough. However, with the increase in gym memberships there has also been a significant increase in the purchase of more wholesome foods. The trajectory of obesity therefore should be decreasing; but it’s not. Furthermore, even the culturally ingrained term “weight loss” itself establishes “heaviness” as the primary criteria for evaluating body composition and size, i.e., scale weight, an inaccurate and erroneous measuring criterion owing to additional variables. An additional 10 factors that contribute to a reduction in metabolic rate and an increase in fat storage have been identified. Individually or in combination, these factors are almost totally ignored or omitted from considerations regarding the etiology of obesity. We developed TrimRox, a stimulant‐ and sugar‐free Prodosomed® TRCAP21 nutraceutical technology, utilizing six standardized extracts comprising of D‐ribose nicotinamide complex, Gynostemma pentaphyllum leaf extract, Myrciaria Dubia whole‐berry extract, Ilex guayusa Loes. (Guayusa) leaf extract, Purple Tea Camellia sinensis extract (PurpleForce™), and alpha Glyceryl phosphoryl choline that effectively addresses all the contributing factors. A 21‐day concept validation physician‐supervised pilot study on the TRCAP21 was conducted in 9 subjects to assess changes in various body parts, including chest, upper arms, waist/belly, hips, and thighs, as well as body weight. The results demonstrated that all subjects experienced a significant reduction in size of one or more of these body parts and significant reduction in body weight. A randomized double‐blind placebo‐controlled study is in progress.
Effect of loop variants on enzymatic activity in acyl‐protein thioesterases
Authors: Harris William J. , Johnson R. J.
Published: May 2022

Acyl‐protein thioesterases (APT) depalmitoylate, or remove fatty acid modifications, from proteins attached to membranes. In humans, APT1 and APT2 are known to control the palmitoylation of Ras, a signaling oncogene whose mutations are linked to cancer. My goal was to analyze the relative importance of specific loop residues hypothesized to regulate the membrane binding and catalytic activity of APT1. To accomplish this goal, wild‐type and loop variants of APT1 were expressed in Escherichia coli and purified to homogeneity. Purified APT proteins were then assayed for changes in thermal stability to confirm that mutations did not significantly shift the folding of the APT. Changes in enzyme function were measured using fluorescent kinetic measurements on substrates with various alkyl chain lengths. From each kinetic assay, a Michaelis‐Menten plot was created and the catalytic constant (kcat) and catalytic efficiency (kcat/KM) were recorded. Multiple APT loop variants have been successfully engineered and purified. Enzyme activity results indicate that substrates with smaller methyl chains are hydrolyzed more quickly by wild‐type APTs while APT1 variant W71A showed hinderance of enzymatic function in these substrates. In the future, we plan to analyze a wider library of APT1 and APT2 variants for shifts in biological activity of APT upon loop substitution.
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.

References

Holmström, 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.
Strategies for Identifying Important Residues in the tRNA Modification Protein Trm732
Authors: Fraley Morgan , Mullins Alexandra , Funk Holly , Guy Michael
Published: May 2022

Post‐transcriptional tRNA modifications are required for efficient protein translation. Proteins involved in forming tRNA modifications are being studied to understand their role and function. In yeast, the Trm7 methyltransferase forms a complex with Trm732 to modify tRNA at position 32. In humans, lack of the Trm7 homolog, FTSJ1, has been linked to intellectual disabilities. The human Trm732 homolog, THADA, is associated with type 2 diabetes, PCOS, and cancers. Little is known about the function of Trm732, although we have shown that one conserved motif in Trm732 is important for tRNA modification. We are identifying other residues important for function using two strategies. First, we compare Trm732 proteins of different organisms using protein alignments to determine conserved regions to mutate for testing. Using this strategy, AIN801 was determined to be an important region for the function of Trm732 and we are continuing to look at other regions that may be of importance. Second, we are expressing randomly mutated Trm732 variants in a sick strain that lacks Trm732. Lack of rescue of strain indicates that the mutation present is harmful to protein function. DNA from these colonies is extracted and sequenced to determine the mutation that causes loss of Trm732 function. Understanding the functions of Trm7 and Trm732 in yeast could aid in our understanding of their human homologs, potentially providing insight into the causes of diseases.
Normal and Abnormal Development of Left Vertebral Artery and its Implications in Cervical Region Surgery
Authors: Hilt Elizabeth , Patil Pallavi , Schreiber Olivia , Shahzad Ahmed , Gill Tanner , Vasan Nagaswami
Published: May 2022

Anomalies occur in branches arising from the aortic arch (AA) in approximately 3‐5% of cadaveric cases (Lazaridis et al. 2018; Natsis et al. 2021), and these atypical branches remain a point of discussion in cervical neck surgery. Here, we describe a 73‐year‐old Caucasian female who died of renal failure following a complicated urinary tract infection whose left vertebral artery (VA) originated from the AA between the left common carotid and subclavian arteries. The preforaminal part of the left VA was 3.5 mm in diameter and entered the C5 transverse foramen while the right VA arising from the right subclavian artery was 4.2 mm in diameter and entered the C5 transverse foramen normally. Embryologically, the VAs are formed during weeks 4 through 8 by development of longitudinal anastomoses linking the cervical intersegmental arteries (ISA). The ISA regresses except the seventh, which becomes the proximal subclavian artery, the point of origin of the adult VA. Persons with left VAs that arise from the AA may be asymptomatic; however, secondary dilatation of the right VA may be implicated in the development of cerebrovascular disorders and atherosclerotic changes due to increased blood flow. Additionally, the recognition of the anomalous origin and anatomical positioning of a left VA is important when considering an anterior approach for cervical spine surgery (Tardieu et al. 2017) and other head‐neck procedures when soft structures such as arteries, veins and muscles are retracted to reach the cervical spine.
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

Introduction

Obesity 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.

Methods

We 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.

Results

Chemogenetic 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.

Conclusion

Leptin 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.
Effect of rate of intravenous cocaine infusion on psychomotor sensitization and striatal AMPAR expression in female rats
Authors: Maitland Alexander D. , Ferrario Carrie R.
Published: May 2022

The speed at which addictive drugs reach the brain influences the magnitude of their effects and increases abuse liability. For example, in rats, fast rates of i.v. cocaine infusion (5 s) induce greater cocaine intake and self‐administration, and enhance psychomotor sensitization compared to slower infusion rates (90 s). Psychomotor sensitization is a behavioral manifestation of drug‐induced neural plasticity thought to underlie addiction. However, most rate of infusion (ROI) studies included only males and were limited to acute experimenter‐administered infusions. Furthermore, increases in cell‐surface expression of AMPA‐type glutamate receptors within the nucleus accumbens (NAc) are associated with psychomotor sensitization and enhanced cocaine‐seeking, but potential effects of ROI on NAc glutamate plasticity had not been examined. Therefore here, we examined psychomotor sensitization and glutamatergic plasticity in female Long‐Evans rats (N=22) given either fast (5 s, n=9) or slow (90 s, n=9) cocaine infusions (50 μl, 2.0 mg/kg i.v.) or saline infusions (5 s, n=2; 90 s, n=2). Rats were given one infusion per session, three sessions per week, for 8 total infusions. To examine effects on AMPAR expression, tissue from the NAc and dorsolateral striatal (DLS) will be collected after an additional 21 days of withdrawal. Briefly, tissue will be prepared using established BS3 crosslinking approaches, and surface expression of AMPAR subunits GluA1 and GluA2 will be examined. Psychomotor sensitization is typically quantified by measuring locomotion using automated beam break measures or by hand scoring behavior. However, beam break measures are inaccurate in small enclosures, and hand scoring is time‐consuming. Here, we developed a novel approach to measuring locomotion using DeepLabCut, a markerless pose estimation method. In pilot studies, tracking locomotion using this method provided reliable measures of locomotion that were comparable to hand‐scored videos. This approach also provided more detailed analysis of the entire time course (rather than using time sampling), and provided additional quantitative measures including velocity. In pilot studies, females in the fast infusion group showed locomotor sensitization, whereas rats in the slow infusion group did not. To our knowledge, this is the first demonstration that repeated slow infusions of cocaine do not produce sensitization, and suggests that although total drug exposure is identical, the speed of drug delivery strongly influences cocaine‐induced plasticity in females. Ongoing studies are being conducted to examine effects on glutamate plasticity. We predict that enhancements in AMPAR surface expression may occur selectively in the 5 s group, based on previous sensitization studies in males, but that this effect may be more pronounced in DLS than NAc. Together these data will reveal how varying pharmacokinetics influence cocaine‐induced neurobehavioral plasticity in females.
Development of a Novel ELISA for Sensitive Quantitation of Glycogen and Polyglucosan Bodies
Authors: Cantrell Alex R. , Manauis Elena , Webb Maddison , Rondon Alberto , Bruntz Ronald C. , Sun Ramon C. , Vander Kooi Craig W. , Gentry Matthew S.
Published: May 2022

Many glycogen storage diseases, e.g., Lafora disease and Pompe disease, are characterized by aberrant glycogen‐like aggregates known as polyglucosan bodies (PGBs). Despite the clinical relevance of PGB accumulation, there are limited options to rapidly assess PGB levels in tissues and biofluids. Additionally, there are limitations to current pre‐clinical methods that rely on enzymatic degradation assays and mass spectrometry in quantitating PGBs. We recently developed novel sandwich ELISAs for the quantitation of both glycogen and PGBs. By utilizing the specificities of glycogen and PGB antibodies, we can specifically capture glycogen or PGBs and detect them utilizing a unique carbohydrate binding module (CBM). We establish the specificity of the assays and the sensitivity of the sandwich ELISAs compared to other techniques used for quantitation. We also demonstrate the applicability of the ELISA using multiple mouse models of Lafora disease and Pompe disease to quantify PGB loads in the brain. Moving forward, these ELISAs could be used in both research and clinical settings to accurately determine PGB levels.
Mass Spectrometry Imaging Reveals Distinct Differences in Glycogen Accumulation in Lung Tumors from Appalachian Patients
Authors: Buoncristiani Michael , Conroy Lindsey R. , Clarke Harrison , Young Lyndsay E. , Sun Ramon C.
Published: May 2022

Lung cancer is the leading cause of cancer related death in the United States, with patients from rural Appalachia having drastically higher incidence and mortality rates, driven by largely unknown mechanisms. Identifying molecular features that contribute to this health disparity is a critical step in lung cancer research that could lead to predictive biomarkers and personalized therapy for the Appalachia population. Recently, aberrant glycogen accumulation in lung tumors has been reported to promote lung cancer progression. However, the full clinical implications of dysregulated glycogen metabolism and its role in lung cancer health disparities has yet to be explored. Matrix‐assisted laser desorption/ionization‐mass spectrometry imaging (MALDI‐MSI) is a new and innovative technique that combines mass spectrometry with high resolution imaging, enabling the detection of hundreds of analytes with spatial distribution. We recently developed an advanced method to analyze glycogen content in formalin‐fixed paraffin‐embedded tissues using MALDI‐MSI. With this technique, we analyzed glycogen chain length and glycogen phosphate content from tissue microarrays containing over 100 lung cancer patient samples banked at the University of Kentucky. Our patient cohort included adenocarcinoma and squamous cell carcinoma patients from Appalachian and non‐Appalachian counties, with diverse cytopathological features. We found significant glycogen accumulation and higher glycogen phosphate content in lung tumor tissue compared to match normal lung across all patients. Further, we found glycogen accumulation is markedly high in adenocarcinoma patients compared to other lung cancer subtypes. Interestingly, Appalachian adenocarcinoma patients exhibit increased glycogen accumulation and higher glycogen phosphate content than patients from non‐Appalachian counties. These data suggest differential glycogen accumulation is a unique feature of lung tumors from Appalachian adenocarcinoma patients. Our study highlights the potential clinical applications of MALDI‐MSI for digital pathology, biomarker applications, and reveals molecular features that contribute to the health disparity in Appalachian lung cancer patients that warrant further molecular analysis.
H3K9me3 binding analysis of the isolated PHD and TTD domains of UHRF2
Authors: Miller Isaak , Khuansanguan Panida , Ginnard Shane , Winkler Alyssa , Heyl Deborah , Trievel Ray
Published: May 2022

UHRF1 and UHRF2 are multi‐domain epigenetic proteins similar in amino acid sequence and structure. These proteins play an important role in regulating gene expression via binding histones and methylated DNA. Both proteins contain TTD and PHD domains, which bind histone H3 methylated at lysine 9 (H3K9me3). While the isolated TTD and PHD domains of UHRF1 are well characterized, the same isolated domains of UHRF2 have yet to be analyzed quantitatively. We used fluorescence polarization to quantify the binding affinities of the isolated TTD and PHD to fluorescent H3K9me3 tail peptides. We discovered that the TTD domain is the main contributor to histone binding in UHRF2. In contrast, the PHD domain is the main contributor to binding in UHRF1. Thus, we show that UHRF1 and UHRF2 have distinct mechanisms of histone binding.
Crosstalk in the Central Autonomic Neurocircuitry Underlying Cardiovascular and Metabolic Control
Authors: Williams Paul A. , Rahmouni Kamal
Published: May 2022

Obesity associated cardiovascular risks such as hypertension are serious medical conditions that increase mortality. The brain autonomic networks play an important role in the regulation of cardiovascular function and metabolism. Therefore, obesity may represent a cardiovascular‐metabolic coupling and possible crosstalk in the central autonomic networks. We and others have previously shown the existence of shared nuclei in the autonomic networks innervating cardiovascular organs such as the kidney and metabolic organs such as the liver and brown adipose tissue (BAT). However, whether there are shared neurons within these nuclei associated with these three organs is unknown. To address this, two groups of mice received injections of pseudorabies virus (PRV) expressing a green fluorescent protein (GFP) into both kidneys and PRV expressing a red fluorescent protein (RFP) in either the interscapular BAT (iBAT) or the left lobe of the liver. The animals were sacrificed 5‐7 days post‐injection and perfused. The brains were extracted, sectioned at 50 µm thickness, stained, and imaged with confocal microscopy. Sections were matched to the mouse atlas (Franklin & Paxinos) and neurons co‐expressing GFP and RFP throughout the brain were identified. We found several nuclei in which neurons were co‐labeled with GFP (kidney) and RFP (BAT or liver). Interestingly, co‐expression does not appear limited to nuclei of specific regions in the brain. Instead, co‐expression was observed in many nuclei from cortical and subcortical regions to hypothalamic areas, midbrain, and brainstem nuclei. Indeed, co‐expressing neurons were observed in areas such as the cortical regions (motor cortex and amygdala), hypothalamic nuclei (paraventricular nucleus (PVN), lateral hypothalamus (LH), and dorsomedial hypothalamus (DMH)), midbrain regions such as the periaqueductal gray, and brainstem nuclei such as the locus coeruleus (LC). In general, there appeared to be two scenarios for shared nuclei: 1) in regions such as LH, nucleus of the solitary tract, and dorsal motor nucleus of vagus there was very little co‐expression, and 2) in regions such as PVN, DMH, LC, and motor cortex there was moderate to high levels of co‐expression. This is the first study to provide anatomical evidence for crosstalk between the autonomic networks regulating cardiovascular and metabolic functions. Moreover, our data demonstrate that crosstalk between the autonomic networks controlling cardiovascular and metabolic functions is distributed throughout the central nervous system.
HUNK Regulation of Interleukin 4 in Triple Negative Breast Cancer
Authors: Ramos Solis Nicole , Yeh Elizabeth S.
Published: May 2022

Triple‐negative breast cancer (TNBC) is a type of breast cancer that does not express hormone receptors (estrogen receptor or progesterone receptor) or human epidermal growth factor receptor 2. Therefore, contrary to other types of breast cancer, TNBC is not sensitive to endocrine therapy or HER2 targeted inhibitors. Some new FDA approve as treatments for TNBC include immunotherapy targets such as immune checkpoint inhibitor (ICI). Unfortunately, inadequate anti‐tumor T‐cell effector function and high abundances of tumor‐associated macrophages (TAMs) has limited the efficacy of ICI therapy. TAMs constituted one of the most abundant immune cell population in mammary tumors. Within the tumor TAMS can be polarized to classicallyactivatedM1‐like and alternativelyactiveM2‐likephenotypes.M2‐likemacrophagescontribute to cancer progression by inducing tumor angiogenic responses, promoting tumor growth and metastasis. In cancer the polarization of macrophages toward a M2 phenotype is directed by cancer‐cell derived factors such as pleiotropic cytokines like interleukin‐4 (IL‐4). New knowledge suggests that IL‐4 expression in cancer cells is regulated by the signal transducer and activator of transcription 3 (STAT3) transcription factor. However, it is still undescribed what signaling pathways are responsible for mediating IL‐4 in breast cancer cells. Intriguingly, we observed that Hormonally Up‐Regulated Neu‐Associated Kinase (HUNK), is responsible for IL‐4 production in the 4T1 mammary tumor cell line, which is considered a metastatic TNBC model. We hypothesis that HUNK regulation of IL‐4 drives TAM’s polarization in triple negative breast cancer cells. We engineered4T1 cells expressing HUNK and HUNK knockdown by shRNA. Our current data shown that4T1 cells expressing HUNK, have elevated levels of STAT3 phosphorylation, IL‐4 production and secretion compared to 4T1 cells where HUNK has been downregulated by shRNA. Furthermore, the loss of IL‐4 secretion in 4T1 HUNK knockdown cells corresponds to a reduced ability of conditioned medium from HUNK knockdown cells to induce alternative activation of macrophages. We also observed that HUNK has a significant effect on M2‐likeTAMspresence in the tumor microenvironment, where tumors derived from HUNK knockdown 4T1 cells have reduced TAMs compared to control tumors. Therefore, our results proposed the identification of a HUNK signaling pathway that is responsible for pro‐metastatic TAM function in TNBC. Our study will evaluate HUNK as a therapeutic target for TNBC metastasis by modulating the TAM population within the tumor microenvironment.
Medullary overexpression of sphingosine‐1‐phosphate receptor 1 transgene attenuates sodium retention and hypertension in deoxycorticosterone acetate (DOCA) salt‐treated mice
Authors: Hu Gaizun , Xie Dengpiao , Chen Chaoling , Ritter Joseph K. , Wang Weili , Li Pin‐lan , Li Ningjun
Published: May 2022

Our previous studies have showed that sphingosine‐1‐phosphate (S1P) agonist stimulates urinary sodium excretion and the S1P type 1 receptor (S1PR1) knockout in the collecting duct led to increased sodium retention and enhanced salt‐sensitive hypertension under the treatment of deoxycorticosterone (DOCA) and high salt. We hypothesized that the increase in medullary S1PR1 would attenuate sodium retention by promoting sodium excretion in such model. Male C57BL/6J mice around 25g bodyweight received intramedullary transfection of S1PR1 plasmids to achieve renal medullary S1PR1 overexpression (OE). The mice were then implanted with the silastic DOCA sheet subcuntaneously and 1% NaCl drinking water to induce salt‐sensitive hypertension (SS‐HTN). Thereafter, the mice underwent either acute or chronic assessments on blood pressure and urinary sodium excretion. Under DOCA‐salt challenge, mice with S1PR1‐OE showed increased urine volume (UV) and urinary sodium excretion (UNAV) in responses to the acute sodium loading (IV of normal saline at 5% body weight in 30 min); the UV and UNAV were 12.51±2.31 vs. 28.50±4.23mL/min/gKW and 0.93±0.27 vs. 4.72±1.12mmole/min/gKW (p<0.05) in control and S1PR1‐OE mice, respectively. The mice with S1PR1‐OE also showed enhanced pressure natriuretic responses (UV, 18.73±7.57 vs. 40.61±6.43mL/min/gKW, p<0.05; UNAV, 3.58±1.77 vs. 9.52±1.38nmole/min/gKW, p<0.05). In the long‐term DOCA‐salt treatment, S1PR1‐OE mice showed less daily sodium balance with the greater sodium excretion (2.72±0.56 vs. 5.60±0.72mmole/day, p<0.05) and ameliorated sodium retention (3.05±0.39 vs. 1.65±0.39mmole/72hr, p<0.05), which was accompanied with the attenuation of SS‐HTN (SBP 134.2±6.79 vs. 109.8±3.54mmHg, p<0.05). The level of alpha subunit of epithelial sodium channel (αENaC) was not changed. However, the beta subunit (βENaC) was significantly decreased (55%, p<0.01). Although ENaCγ was significantly increased (350%) in the S1PR1‐OE group, immunostaining showed that the DOCA‐stimulated translocation of γENaC onto the apical membrane was inhibited. These results suggested that the overexpression of S1PR1 in the renal medulla attenuates sodium retention by the inhibition of ENaC function in the DOCA‐induced SS‐HTN.
Zebrafish as a Model for Evaluating Coilin‐Mediated microRNA Biogenesis
Authors: McLaurin Douglas , Davis Madelyn , Lett Katheryn , Hebert Michael
Published: May 2022

Cajal bodies (CBs) are nuclear bodies that are hosts for the modification and assembly of ribonucleoproteins (RNPs). The nuclear protein coilin is known as the CB marker protein as it is the scaffold for CB assembly and vital for structural integrity. However, there is still not much understood about the function of coilin. Recent studies by our lab implicate coilin in the biogenesis of microRNAs (miRNAs). Currently, there are sparse options in animal models for studying coilin and CB function that account for limitations such as off‐target effects of coilin depletion or incomplete protein knockdown. Due to this, we sought to develop novel coilin mutations in zebrafish to advance functional studies of this protein and CBs. In considering how we would approach this, we opted for the innovative CRISPR/Cas9 technology as an optimal strategy and hypothesized that the implementation of this genome editing technology would allow us to develop multiple lines of coilin‐modified zebrafish for studies investigating coilin’s role in miRNA biogenesis. We selected a suitable Protospacer Adjacent Motif (PAM) sequence and designed guide RNAs for our mutations of interest in silico, which we then generated in vitro, and injected into fertilized zebrafish embryos at the one‐cell stage. An initial screening for insertions or deletions (indels) took place at 72 hours post fertilization to determine mutation frequency and once again when the zebrafish reached adulthood. Through our efforts, we have managed to obtain zebrafish that are heterozygous for coilin‐null mutations and are currently in the midst of screening for additional lines of coilin mutations which could serve to characterize the functional domains of coilin in vivo. Utilizing CRISPR/Cas9 technology, we have generated the first coilin‐null mutation in a zebrafish line allowing our lab to expand our investigations into the role of coilin and CBs in the regulatory network of the nucleus.
Acute Effect of Intermittent Hypoxia on Peripheral Vascular Function in Young Healthy Adults
Authors: Stray‐Gundersen Sten , Wojan Frank , Massoudian Sahar D. , Tanaka Hirofumi , Lalande Sophie
Published: May 2022

Continuous exposure to hypoxia elicits a chemoreceptor‐mediated increase in sympathetic nerve activity and a simultaneous peripheral vasodilation due to an increase in nitric oxide production. The aim of the present study was to determine whether short, repeated bouts of hypoxia induce brachial artery vasodilation and acutely improve endothelial function. Ten young healthy adults (4 women, age: 24±3 years, height: 176±9 cm, body weight: 80±13 kg) visited the laboratory on two separate occasions. Endothelium‐dependent vasodilation was assessed by brachial artery flow‐mediated dilation using a semiautomated diagnostic ultrasound system before and after exposure to either intermittent hypoxia (IH) or intermittent normoxia (IN). Intermittent hypoxia consisted of eight 4‐min hypoxic cycles at a targeted arterial oxygen saturation of 80% interspersed with breathing room air to resaturation, and intermittent normoxia consisted of eight 4‐min normoxic cycles separated by one minute of breathing room air. Air was made hypoxic by titrating nitrogen into a breathing circuit. Brachial artery diameter was assessed via ultrasound throughout the protocols. Hemodynamics, arterial oxygen saturation, and pulmonary gas exchange were also continuously assessed during the protocols. By design, intermittent hypoxia resulted in an arterial oxygen saturation of 81±2%, corresponding to oxygen levels of 11.9±2.2%. Exposure to intermittent hypoxia, but not intermittent normoxia, elicited a brachial artery vasodilation (IH: 0.38±0.06 to 0.40±0.06 vs. IN: 0.38±0.06 to 0.38±0.06 cm, p<0.01). However, neither intermittent hypoxia nor intermittent normoxia acutely affected brachial artery flow‐mediated dilation (IH: 5.8±1.9 to 5.3±1.6% vs. IN: 6.1±2.4 to 6.5±1.7%, p=0.96). Intermittent hypoxia increased cardiac output (5.2±0.9 to 5.9±1.4 L·min‐1, p=0.03) mainly due to an increase in heart rate (57±9 to 68±11 bpm, p<0.01). In conclusion, exposure to intermittent hypoxia triggered a brachial artery vasodilation but did not affect endothelium‐dependent vasodilation in young healthy adults. Multiple sessions of intermittent hypoxia may be necessary to improve nitric oxide synthesis and endothelial function.