bluuebunny/crossref_metadata_2025 huggingface dataset:
Excessive Thiol Nitrosylation Predisposes to Western Diet‐Induced Redox Imbalance and Erectile Dysfunction
Authors: La Favor Justin , Soetkamp Daniel , Venkatraman Vidya , Bivalacqua Trinity , Van Eyk Jennifer , Burnett Arthur
Published: November 2023

Objectives

Binding of nitric oxide (NO) to a cysteine thiol is termed S‐nitrosylation, which is emerging as an important means of NO‐mediated signal transduction independent of stimulation of soluble guanylate cyclase. However, the impact of S‐nitrosylation on erectile function remains unclear. S‐nitrosylation is a redox sensitive post‐translational modification, which is thought to be promoted by reactive oxygen species (ROS). Protein S‐nitrosylation levels are regulated in part by S‐nitrosoglutatione reductase (GSNOR), which acts to de‐nitrosylate protein thiols. The objectives of this study were to determine the role of GSNOR on maintenance of redox balance and erectile function in response to a Western diet (WD) high in fat and sugar.

Materials and Methods

Young male wild type (WT; n = 110) and GSNOR−/− (n = 108) mice were fed either a control diet or a WD ad libitum for 3, 6, 9, or 12 weeks. Following the dietary intervention, erectile function was assessed by measuring intracavernosal pressure (ICP) and mean arterial pressure (MAP) during cavernous nerve stimulation. In separate mice, in vivo ROS production was measured in the penis utilizing a novel microdialysis approach. Microdialysis probes were inserted into the penis of anesthetized mice and perfused with saline containing 100 uM Amplex Ultrared, 1 U/ml horseradish peroxidase, and 10 U/ml superoxide dismutase. ROS convert the reagents to a fluorescent byproduct resorufin, which was measured in the outflowing dialysate. Penile tissue was harvested from mice and processed for enrichment of nitrosylated thiols. Nitrosylated thiols were labeled with iodoTMT, and nitrosylated proteins were detected by mass spectrometry.

Results

Neither ROS generation (WT: 0.66 ± 0.09 vs. GSNOR−/− 0.96 ± 0.12 μM H2O2; p = 0.41) nor erectile function (WT:8.44 ± 1.4 vs. GSNOR−/− 9.6 ± 1.6 ICP area/MAP; p = 0.97) were different between genotypes fed the control diet. However, ROS were elevated following just 3 weeks of WD consumption in GSNOR−/− mice (1.44 ± 0.19; p = 0.01), whereas ROS increased following 6 weeks of WD in WT mice (1.29 ± 0.19; p = 0.02). Subsequent to increases in ROS, ED was evident after 6 weeks of WD in GSNOR−/− mice (5.0 ± 1.1, p = 0.04) and after 12 weeks of WD in WT mice (3.1 ± 0.6, p = 0.02). 249 unique peptides corresponding to 136 different proteins with nitrosylated thiol groups were identified by mass spectrometry.

Conclusions

Mice lacking GSNOR demonstrated an impaired ability to maintain redox balance in the face of the pro‐oxidant stimulus of the WD, prompting accelerated development of erectile dysfunction. These data implicate a protective role for GSNOR in the penis and suggest that excessive S‐nitrosylation may negatively regulate erectile function.

Support or Funding Information

Supported by a mini grant from the National Kidney Foundation of Maryland and National Institutes of Health grant 1F32DK100082.
Endothelial Marker Expressing Stromal Cells Play a Critical Role in Recovery Following Acute Kidney Injury
Authors: Maringer Katie , Sims‐Lucas Sunder
Published: November 2023

Background

Acute Kidney Injury (AKI) is characterized by an abrupt decrease in renal function that can lead to renal failure, thus contributing to high percentages of morbidity and mortality. The kidney contains a complex and high degree of vascularization making it susceptible to ischemic injury, particularly the peritubular capillaries. We have identified a subset of endothelial marker expressing stromal (EMES) cells that contribute to the peritubular capillary endothelium. Subsequently, we hypothesize that EMES cells are important contributors to kidney recovery after AKI.

Methods

We confirmed the importance of EMES following ischemia reperfusion injury (IRI) via lineage tracing, using a TdTomato reporter (permanently labeling all EMES cells) and interrogated the percentage of EMES cells that were present in the IRI and contralateral control kidneys. Furthermore, we generated mice with a conditional deletion of Flk1 (Vegfr2, essential for vascular development) in the Foxd1cre positive renal stroma (Flk1ST−/−), and evaluated the tissue after both blood flow dependent AKI, utilizing ischemia reperfusion injury (IRI), and blood flow independent or nephrotoxic AKI utilizing the chemotherapy agent cisplatin and focused on the recovery phase (7, or 28 days) post injury.

Results

We determined in control mice following IRI that developmental stromal genes were re‐expressed suggesting that the stroma may be undergoing de‐differentiation and subsequently driving EMES proliferation. Furthermore, using lineage tracing we found that EMES preferentially increase during the repair phase (7 days) of IRI. To evaluate the role of EMES cells following AKI, we used Flk1ST−/− animals subjected to IRI, and found that mutants had less perfusion and an increase in hypoxia and increased expression of hypoxia inducible factor 1a (HIF1a) at 7 days. The hypoxic environment caused exasperated kidney damage in the mutant animals. Furthermore, when animals were allowed to recover for 28 days, there was significant fibrosis, and damage in the injured kidney compared to the contralateral control. When these animals were subjected to cisplatin injections we found that while perfusion of the kidneys appeared unaffected, HIF1a was similarly increased and the stromal genes were re‐expressed.

Conclusions

Taken together we determined that for repair after AKI the renal stroma requires de‐differentiation and re‐expression of developmental markers prior to proliferation and re‐differentiation. This cellular reprogramming event also requires EMES cell proliferation, which play an important role in mediating vascular perfusion, reestablishing normal oxygen concentrations and regulating HIF signaling to modulate repair and recovery.

Support or Funding Information

F32 DK111165‐01

K01 DK096996
Urine electrolyte excretion in a hypertensive population of East Africans
Authors: Dobrovolskaite Aiste , Rice Nancy A.
Published: November 2023

According to the World Health Organization (WHO), chronic non‐communicable diseases (NCDs) cause 38 million deaths annually of which 75 % occur in low and middle income countries. Hypertension is a primary risk factor for cardiovascular disease (CVD), a leading chronic NCD. Kenya, a country in Sub‐Saharan Africa, has a high prevalence of hypertension of which only ~ 3 % is pharmaceutically controlled. Previous work from our lab identified a population of Kenyans with a high prevalence of hypertension that does not statistically correlate with any known risk factor for the disease such as hyperlipidemia or obesity. Therefore, we hypothesized that consumption of a high Na+ and low K+ diet may be involved in the etiology of hypertension in this community. To test this hypothesis, systolic and diastolic blood pressure levels and spot urine samples from 135 people were collected in the morning and evening, and subsequently analyzed for Na+, K+ and Cl− excretion using the Smartlyte Electrolyte Analyzer. Overall mean urine electrolyte excretion values for Na+, K+ and Cl− were 80.2 ± 42.0 mmol/L, 32.0 ± 21.1 mmol/L, and 87.7 ± 42.1 mmol/L respectively. These values fall well within the suggested levels for Na+ (40–220 mmol/L) and K+ (25–125 mmol/L), but under normal excretion levels for Cl− (110–250 mmol/L). Females exhibited higher overall electrolyte excretion when compared to males, with significantly higher excretion levels of K+ (p<0.05). Additionally, there is a significant difference between normotensive and stage I hypertensive individuals in both Na+ (57.9 mmol/L vs. 88.9 mmol/L; p<0.05) and Cl− excretion (65.5 mmol/L vs. 96.7 mmol/L; p<0.05).

Support or Funding Information

This work was funded by a WKU Graduate Studies research award to A. D. and a WKU RCAP award to N.A.R.
Vasoactive effects of P2X7 receptors in the aorta of control and hypertensive mice
Authors: Howarth Amelia Rose , Menzies R.I. , Conway B.R. , Bailey M.A.
Published: November 2023

P2X7 receptors (P2X7R) are activated by extracellular ATP and have well defined pro‐inflammatory roles in immune cells. The receptor is also expressed in the vascular endothelium and we recently reported that systemic infusion of a P2X7R antagonist reduced blood pressure and increased renal perfusion in hypertensive rats (Menzies et al, Kidney International, 88, p1079, 2015). In the present study we performed a comprehensive immunlocalisation study of P2X7R expression in the mouse renal vasculature and examined the effect of P2X7R activation on the contractility of isolated aorta.

Kidneys from male C57BL6 mice were fixed and P2X7R immunofluorescence performed using a rabbit anti‐P2X7 primary antibody (Alomone, 1:500). Immunofluorescence staining was also performed using antibodies against CD31 (endothelium marker); αSMA (vascular smooth muscle marker) and PDGFRβ (pericyte marker). This approach localised P2X7 to the endothelium of mouse renal arteries and glomerular capillaries, but not to the vascular smooth muscle. P2X7 was also seen to co‐localise with PDGFRβ at focal points in the renal medulla.

To examine the functional role of vascular P2XR7 expression, we performed wire myography on thoracic aorta dissected from male C57BL6 mice (n=5). The P2X7R agonist, BzATP (50μM) induced a modest vasoconstriction, amounting to 20±5.2% of that induced by depolarisation with KCL. Preincubation with the P2X7R antagonist A438079 (10μM), blocked ~70% of the BZATP‐induced constriction.

In a separate group of mice, we induced hypertension (Hypertensive mice: 136.5±3.2mmHg Control mice: 113.3±1.9mmHg) through a 4 week administration of ANGII (100ng/kg/min) and deoxycorticosterone (50mg), combined with high salt feeding. This regimen induced perivascular fibrosis but no albuminuria. In this setting, BZATP also induced a constriction, similar to that observed in non‐treated mice. However, pre‐incubation with the P2X7R antagonist amplified the constrictive response to BZATP.

Our data confirm endothelial expression of P2X7R innormotensive mice and suggest that activation of the receptor exerts a modest vasoconstriction. In hypertensive animals, our data suggest that P2X7R exerts a vasodilatory tone.

Support or Funding Information

This work has been generously funded by the British Heart Foundation
EHD4 Regulates Renal Urea Handling In Mice
Authors: Rahman Shamma S , Storck Matthew , Band Hamid , Boesen Erika
Published: November 2023

Eps15 homology domain‐containing protein 4 (EHD4) is an important regulator of endocytic recycling and is abundantly expressed within the inner medullary collecting duct (IMCD). We have previously found that global EHD4 knockout (EHD4‐KO) mice have a higher urine flow and lower urine osmolality as compared to wild‐type (WT) C57Bl/6 mice, but no significant difference in urinary sodium excretion. Urea transport is important in generating the medullary interstitial osmotic gradient that is required for concentrating urine. Baseline urinary urea excretion in EHD4‐KO on normal rodent chow (19% protein) was significantly higher than that in the WT mice, despite similar abundance of the key urea transporter UT‐A1 in the inner medulla by Western Blot. These data may suggest that EHD4 plays a role in regulating the subcellular distribution or activity of UT‐A1 or other urea transporters in the kidney, rendering renal urea handling dysfunctional in EHD4‐KO mice. The Berliner hypothesis predicts low‐protein diet‐induced restoration of urine‐concentrating ability in the absence of urea transporters in IMCDs. Accordingly, we hypothesized that due to impaired urea transport, the polyuric phenotype observed in the EHD4‐KO mice would be ameliorated by low protein intake and exacerbated high protein intake. To test this hypothesis, we provided female EHD4‐KO (n = 4) and WT littermate (n = 3) mice with either 6% (low), 20% (normal), and 40% (high) protein diet for 7 seven days. On day 8, mice were placed in metabolic cages with the corresponding food and water for 48 hours to acclimate followed by a 24 hours urine collection period. Urine flow remained higher in EHD4‐KO mice than WT mice on the normal protein diet (1.99 vs 1.05 ml/day in EHD4‐KO vs WT), which further increased on the high protein diet (3.67 vs 1.50 ml/day in EHD4‐KO vs WT) but was similar to WT mice on the low protein diet (1.34 vs 1.13 ml/day in EHD4‐KO vs WT) (Pgenotype=0.06, Pdiet<0.01, Pinteraction<0.01). These results indicate that renal urea handling is regulated by EHD4, possibly via a mechanism that involves the trafficking of the UT‐A1 to the apical plasma membrane of the IMCDs rather than regulating UT‐A1 abundance. Future studies will be designed to explore the mechanism of how EHD4 regulates urea transporter activity or localization.

Support or Funding Information

•American Heart Association Pre‐doctoral Fellowship (SR) (15PRE25580003)

•Nebraska Center for Cellular Signaling Pilot Project Grant (EIB and HB)
Racial Differences in Serum Cystatin‐C Following Antenatal Corticosteroid Exposure
Authors: Zerihun Lillian M. , Nixon Patricia A. , Washburn Lisa K. , Gwathmey TanYa M.
Published: November 2023

Antenatal corticosteroid (ANCS) administration to mothers at risk of pre‐term delivery has been linked to both transient and long‐term renal and cardiovascular damage in the exposed offspring. Previously, we reported that exposure to ANCS was associated with elevated levels of renal angiotensin II, which may contribute to renal inflammation and fibrosis over time. Cystatin‐C, a protease inhibitor excreted by glomerular filtration, is used as a biomarker for early‐stage renal damage, as its concentration is inversely correlated with glomerular filtration rate and it is thought to be less influenced by gender, age, and muscle mass than other biomarkers. The purpose of this study was to compare serum levels of Cystatin‐C in a cohort of young adults (~ 19 years of age) born prematurely (<37 weeks gestation) with very low birth weight (< 1500 g), who were exposed or not to ANCS. Potential race and gender‐specific effects were also examined. ANCS‐exposed Caucasians showed a trend for higher Cystatin‐C levels [1.00 ±0.05 mg/L; N=52] compared with unexposed Caucasians [0.89 ± 0.05 mg/L; N=34; P=0.091], while no differences were observed between ANCS‐exposed and unexposed African Americans. Importantly, ANCS‐exposed Caucasian males had significantly elevated serum Cystatin‐C [1.04 ± 0.06 mg/L; N=26] compared with ANCS‐exposed African American males [0.80 ± 0.05 mg/L; N=15; P=0.003]. Similarly, a trend was observed for higher serum Cystatin‐C in ANCS‐exposed Caucasian females [1.00 ± 0.08 mg/L; N=26] compared with ANCS‐exposed African American females [0.80 ± 0.05 mg/L; N=10; P=0.055]. However, no significant difference was observed between Caucasians and African Americans for unexposed males or females. These data suggest exposure to ANCS may have a greater impact towards increasing risk for early renal injury in Caucasian individuals than in African Americans at this age. These observations align with recent reports in the literature revealing racial differences in Cystatin‐C levels, and remain consistent with our previous findings of race and gender‐specific programming effects of ANCS on health outcomes in persons born with very low birth weight. These findings provide an important perspective for the alleviation of renal health disparities following ANCS exposure.

Support or Funding Information

AHA 12CRP9390000 and HD047584
Chloroquine Is a Potent Pulmonary Vasodilator that Attenuates Hypoxia‐Induced Pulmonary Hypertension
Authors: Wu Kang
Published: November 2023

Sustained pulmonary vasoconstriction and excessive pulmonary vascular wall thickening are two major causes for elevated pulmonary vascular resistance and pulmonary arterial pressure in patients with pulmonary arterial hypertension. Chloroquine, as an anti‐malaria agent, is found to have inhibitory effects on autophagy and a relaxant effect on airway smooth muscle cells. In this study, we observed that chloroquine (100–200 μM) significantly attenuates agonist‐ and high K+‐induced contraction in isolated rat pulmonary artery (PA). Pretreatment with L‐NAME or indomethacin and functional removal of endothelium failed to inhibit chloroquine‐induced relaxation in isolated PA rings pre‐constricted with phenylephrine or 40 mM K+. In pulmonary arterial smooth muscle cells (PASMCs), extracellular application of chloroquine significantly attenuated store‐operated Ca2+ entry induced by passive depletion of intracellularly stored Ca2+ in the sarcoplasmic reticulum using cyclopiazonic acid and partially inhibited ATP‐induced Ca2+ entry. Furthermore, chloroquine was shown to effectively attenuate Ca2+ entry via voltage‐dependent Ca2+ channels (VDCC). In vivo experiments showed that 50mg/kg chloroquine treatment (ip injections) significantly attenuated the development of pulmonary hypertension in rats exposed to chronic hypoxia (10% O2 for 3 weeks). These data indicate that chloroquine is a potent vasodilator of PA and a potential blocker of VDCC, store‐operated Ca2+ channels and receptor‐operated Ca2+ channels in PASMCs. The therapeutic potential of chloroquine on pulmonary hypertension is likely due to the combination of its vasodilative, anti‐proliferative and anti‐autophagic effects.

Support or Funding Information

This work was supported in part by the grants from the National Natural Science Foundation of China (81630004, 81470246, 81220108001, 81520108001), Guangzhou Department of Education Yangcheng Scholarship (12A001S), and Guangdong Province Universities, Colleges Pearl River Scholar Funded Scheme of China and China Scholarship Council (201608440324), as well as from the National Heart, Lung and Blood Institute of the National Institutes of Health (HL115014, HL066012).
P2X3 Receptor Antagonism Reduces the Occurrence of Apnoeas in Newborn Rats
Authors: Katayama Pedro Lourenço , Charles Ian , Pijacka Wioletta , Abdala Ana Paula , Salgado Helio Cesar , Ford Anthony P. , Paton Julian F. R.
Published: November 2023

Apnoeas of prematurity (AoP) are a common problem in neonates. The mechanisms underlying this condition are not understood. We hypothesised hyperreflexia of afferent systems regulating breathing as a potential cause. Since P2X3 receptors are present on afferents controlling respiration and can mediated hyperreflexia (e.g. carotid body; Pijacka et al. 2016. Nat Med, 22, 1151–1159), we tested whether their antagonism would reduce AoP in new born rats. Rats were subjected to whole‐body plethysmography to record respiratory frequency (fR) and tidal volume (VT); minute ventilation (VE) was calculated (fR × VT). The frequency of apnoeas, which included both AoP and post‐sigh apnoeas, was counted over 10 min epochs at 5 and 15 min following administration of either a non‐brain penetrant P2X3 antagonist ‐ AF‐454 (15 mg/kg s.c.) or vehicle in separate rats. In 7 rats, basal apnoea frequency was 6.42 ± 1.32 per 10 min; AF‐454 reduced significantly the occurrence of these apnoeas at both 5 (2.42 ± 0.61; P>0.01) and 15 min post‐drug injection (3.4 ± 0.67; P>0.05). There was neither a change in fR, VT or MV after AF‐454 nor an effect from injection of vehicle (n=4). These data suggest that P2X3 receptor antagonism is a novel approach to reduce the occurrence of apnoeas in new born rats. We propose P2X3 receptor mediated afferent hyperreflexia as a mechanism triggering breathing dysfunction at birth.

Support or Funding Information

British Heart Foundation, FAPESP and Afferent Pharmaceuticals
Behaviorally Inhibited Individuals Exhibit Greater Minute Ventilation and Breathing Frequency Than Non‐Inhibited Individuals When Breathing 4.5% CO2
Authors: Patschorke Emma , Evenhouse Annika , Grove Amanda , Kathryn McAuliffe , Santos Nick , Masini Marissa , Harris Morgan , Robinson Kyle , Williams Jordyn , Ferdman Leonard , Buchholz Kirstyn , Nicosia Tim , Miller Justin R , Miller Daniel P , Martino Paul F , Servatius Richard J
Published: November 2023

Behavioral inhibition (BI) is a temperament in which an individual tends to avoid unfamiliar situations or environments. Individuals with high BI are more vulnerable to developing an anxiety disorder, such as Post‐Traumatic Stress Disorder (PTSD), when placed under stressful conditions. Behaviorally inhibited individuals learn faster and are slower to stop the response to a stimulus (Sheynin, 2005; Servatius, 2008). Our lab over the past several years has been investigating the relationship between a respiratory stress response to 4.5% CO2 and BI. BI is characterized by avoidance behavior, which is an active response aimed to reduce fear of a certain stimulus. In this study, a computer game was used as an aversive stimulus to detect BI. 4.5% CO2 was applied as a mild respiratory stimulant to detect any differences in those with BI compared to those without. One indication of PTSD is avoidance behaviors. 4.5% CO2 decreases the pH of the blood, hence causing an increase in frequency of breathing (fbr), tidal volume (VT), and inspired minute ventilation (VI). We hypothesize that individuals with high BI will exhibit greater increases in all parameters of breathing compared to individuals with low BI. The 4.5% CO2 was obtained by mixing room air with pure CO2 in a Tissot Spirometer and was validated using an Oxygraph O2 and CO2 analyzer. The mixed gas was then delivered from a 100 liter gas bag attached to a breathing tube attached to a Hans Rudolph two‐way nonrebreathing valve. The subjects placed the two‐way valve attached to a mouthpiece into their mouths and then had a nose clip placed over their noses to created a tight seal between the breathing gas bags and the participants. BI and non‐BI individuals were separated based on Adult Measure of Behavioral Inhibition (AMBI) and Retrospective Measure of Behavioral Inhibition (RMBI). Heart rate and respiratory data were collected using a BioPac computer data collection system while breathing room air and 4.5% CO2. In BI individuals, we observed a 35.9% (+/−33.3) increase in minute ventilation and a 32.4% (+/−32.1) increase in breathing frequency (n=4). In non‐BI individuals, we observed a −6.4% (+/−18.3) decrease in minute ventilation and a −15.1% (+/−2.9) decrease in breathing frequency (n=2). We observed no difference in tidal volume or heart rate for either group. In conclusion, individuals who scored high on the BI test have a greater response to a respiratory stimuli. Our future studies will include cortisol measurements by analyzing saliva samples taken before and after the test.

Support or Funding Information

Carthage College Research and Development Grants
Expiratory Muscle Activation via High Frequency Spinal Cord Stimulation
Authors: Kowalski Krzysztof E , Romaniuk J Richard , Brose Steven W , Richmond Mary Ann , Kowalski Tomasz , DiMarco Anthony F
Published: November 2023

Background

Lack of an effective cough may result in the frequent aspiration of airway secretions and foreign material and the development of recurrent respiratory tract infections. In persons with spinal cord injury, lower thoracic low frequency spinal cord stimulation (conventional LF‐SCS, 50Hz, 15mA) results in large positive airway pressure generation (P), and is a useful method to restore an effective cough mechanism. Unfortunately, activation of the expiratory muscles via LF‐SCS requires high stimulus amplitudes, which may also cause unwanted side effects including stimulation of sensory fibers and therefore cannot be applied in patients with intact sensation.

Objective

To evaluate the effectiveness a novel method of expiratory muscle activation, which involves the application of high frequency spinal cord stimulation with low stimulus amplitudes (HF‐SCS; 500Hz, 1mA).

Methods

Studies were performed on 13 anesthetized dogs. Multi contact stimulating electrodes were positioned over the dorsal surface of the lower thoracic and upper lumbar spinal cord. In one group of animals the effects of HF‐SCS and LF‐SCS on P were evaluated. SCS was applied at functional residual capacity (FRC) and also over a wide range of lung volumes. Given our previous success with LF‐SCS, these values were used as our gold standard to which all comparisons were made. In a second group of animals, the mechanism of expiratory muscle activation during HF‐SCS was evaluated. P and EMG recordings from the external oblique (EO) before and after spinal cord section at the T12 level were evaluated. Internal intercostal muscle (II) activation at the level of the stimulating electrode was also assessed via II EMG recordings, before and after T9 root section.

Results

For both HF‐SCS and LF‐SCS, the optimum site for pressure generation was in the region of T9 spinal level (62±5 and 66±8cmH2O). P declined progressively at more rostral and caudal levels. In response to increasing lung volume, P increased in linear fashion during both HF‐SCS and LF‐SCS over the entire vital capacity range. For both HF‐SCS and LF‐SCS, bilateral dorsal column section significantly reduced EO EMG activity recorded below the level of spinal section and also resulted in a substantial fall in P (from 56±6 to 28±1cmH2O, p<0.05). Subsequent lateral funiculi section completely abolished those activities and resulted in further reductions in P. However, onset time of II EMG activity during HF‐SCS was significantly longer than obtained during direct T9 motor root activation (p<0.05).

Conclusion

These results suggest that HF‐SCS produces a comparable level of expiratory muscle activation to that achieved with conventional stimulus parameters, but with much lower stimulus amplitudes and is more dependent upon synaptic pathways rather than direct motor root activation.

Support or Funding Information

Support: VA RR&D A1488‐R and NIH‐NINDS R01NS064157
Improved Respiratory Function Following Spinal Cord Injury in Male and Female Rats
Authors: Williams Kathleen , Khanbhai Nilofar , Zimmer M. Beth , Westerkamp Christopher
Published: November 2023

Dysfunctions below the site of spinal cord injuries (SCI) include loss of respiratory muscle function. Respiratory dysfunction is the primary cause of death of individuals with SCI. Approximately 17,000 people in the United States suffer from SCI each year, with males comprising 80% of this population. The intercostal muscles and the diaphragm are important for the inspiratory phase of respiration. Hemisection of the spinal cord second cervical vertebra causes paralysis of the ipsilateral hemidiaphragm and decreased EMG activity of the ipsilateral intercostal muscles. Past studies have reported complete or partial EMG activity is regained faster in the ipsilateral intercostal muscles of young male rats than old female rats after SCI. This study used male and female rats of the same age to determine if the regained EMG activity is sex specific. We hypothesized that there would be more EMG activity in the intercostal muscles of male rats of the same age compared to female rats three weeks post‐hemisection. Male and female Long‐Evans rats had bilateral phrenic and intercostal EMG activity recorded three weeks post‐C2 hemisection. The amount of regained ipsilateral intercostal muscle EMG activity was compared between male and female rats and samples collected confirmed complete hemisection.
Ventilatory Chemoreflexes and HMOX2 Variation in Individuals of Himalayan and Han Chinese Ancestry Residing at Sea Level
Authors: Heinrich Erica C , Djokic Matea , Powell Frank L , Simonson Tatum S
Published: November 2023

Humans have lived on the Tibetan Plateau (>4,000 m) for thousands of years and exhibit distinct patterns of adaptation to high altitude. Individuals with Tibetan ancestry living at high altitude for their entire life have a greater hypoxic ventilatory response (HVR) than Han Chinese and Andeans resident at comparable altitudes. Tibetan highlanders also exhibit lower average hemoglobin concentration, a trait associated with SNP variants in the heme oxygenase 2 gene (HMOX2). The genomic region containing HMOX2 displays a strong signal of selection in several independent studies in Tibetans and HMOX2 has been linked to hypoxic chemosensitivity via carbon monoxide‐sensitive hydrogen sulfide signaling in the carotid body. The goals of our study were (1) to determine if HVR remains elevated in ancestral Tibetans residing at sea level and (2) to determine if putatively adaptive HMOX2 variants are associated with HVR in populations at sea level. We measured isocapnic HVR, hypercapnic ventilatory response (HCVR), and hematocrits in individuals of Himalayan ancestry (HA, Tibetan or Nepalese, female = 4; male = 3) or Han Chinese (HC, male = 7; female = 17) ancestry residing at sea level. We found no difference in the HVR across HA and HC men or women at sea level (men: HA=0.17±0.25, HC: 0.14±0.13 L min−1 %SaO2−1, NS; women: HA: 0.13±0.08, HC: 0.27±0.22, NS). However, HA women show decreased HCVR (HA: 0.14±0.14, HC: 0.59±0.28 L min−1 torr−1, p<0.01) and HA men show increased resting ventilation (HA: 10.85±1.86, HC: 7.58±1.66 L min−1, p<0.02) compared to HC women and men, respectively. Both HA men and women exhibit a higher resting oxygen saturation compared to HC (HA: 99.02±1.02, HC: 97.93±1.07%, p<0.02). Similar to previous observations, hematocrits tend to be lower on average in both HA men (HA: 43.1 ± 1.99; HC: 45.6 ± 2.34, p<0.14) and women (HA: 37.4±3.9; HC: 40.2±2.4, p<0.07). Our data show that the HVR in sea level residents of HA and HC ancestry are not different, suggesting that increases in the HVR with ventilatory acclimatization or decreases hypoxic desensitization (i.e. “blunting”) differs between these populations. We are completing SNP genotyping of HMOX2 in these populations based on Tibetan‐specific adaptive signals to determine if the HVR is associated with those adaptive variants.

Support or Funding Information

This work was supported by an NIH Ruth L. Kirschstein National Research Service Award (NRSA).
Gender Differences on Heart Rate Variability of Subjects Exposed to Regular Resistance Training Routine
Authors: Ramalho Ellen Sonnaly Villar , Souza‐Junior Estevam Luiz , Magnani Marciane , Braga Valdir A
Published: November 2023

Heart rate variability (HRV) using frequency‐domain method is a tool for evaluating autonomic balance. In this cross‐sectional study, we investigated the low frequency (LF), high frequency (HF) and the LF/HF ratio components of the spectral analysis in men (n= 22) and women (n = 27) exposed to resistance training using the nerve express system analyzed by Student's t test. The study was approved by Ethics Committee of the Center for Health Sciences of the Federal University of Paraiba and was in accordance with the Helsinki Declaration of 1975, as revised in 2008. Our hypothesis was that there is a gender difference on HRV when subjects were exposed to resistance training. In men, the LF component was increased during supine (1171 ± 192 vs. 545 ± 74 ms2, P = 0.0067) and upright position after the orthostatic test (1824 ± 260 vs. 738 ± 133 v, P = 0.0011). In addition, LF/HF ratio, an index for sympathovagal balance, was increased in male subjects compared to females. These data suggest a possible gender difference on HRV of individuals exposed to resistance training.

Support or Funding Information

Supported by Capes and CNPq
Acute Spinal Cord Injury Alters the Acquisition Phase of Learning in Long Evans Rats
Authors: Klein Evan , Cole Andrew , Tran Cindy , Workman Madeline , Zimmer M. Beth
Published: November 2023

It is well known that spinal cord injury (SCI) affects regions of the body below the site of injury by damaging sensory and motor neural pathways. A few studies have also shown that SCI affects regions above the site of injury and affects higher cognitive processes such as learning and memory similar to traumatic brain injuries (concussions for example). Previous studies in our lab have shown that the acquisition phase of learning was altered two weeks after injury in young male rats. In addition, several studies have shown possible sex and/or age differences in neural responses after SCI. The current study was designed to determine if there are any differences between males and females on the effect of SCI on learning and memory. Young male and female Long Evans rats were anesthetized with ketamine and xylazine, and a C2 hemisection (=4), or a sham surgery (n=4) was performed. Male and female rats were given two weeks to recover, before using the Morris Water maze to examine learning and memory performance. Rats completed four trials a day for 5 days followed by a probe trial. This study will provide data to help our understanding of the impacts of SCI on higher brain function.

Support or Funding Information

Ferris Faculty Foundation, Ferris Faculty Research Grant, Ferris Dean's Grant (awarded to EK, AC, CT, MW).
Cerebral Edema and Blood Brain Barrier Dysfunction in the Dahl S Rat Model of Superimposed Preeclampsia
Authors: Maeda Kenji John , Warrington Junie P , Duncan Jeremy , Granger Joey P , Garrett Michael R , Ryan Michael J , Sasser Jennifer M
Published: November 2023

Preeclampsia, characterized by hypertension and renal dysfunction in pregnancy, is associated with placental ischemia, which has been linked to increased blood‐brain barrier permeability and cerebral edema. We recently demonstrated that pregnant Dahl salt sensitive rats (Dahl S) closely mimic the maternal syndrome of preeclampsia. In this study, we tested the hypothesis that pregnant Dahl S rats develop cerebral vascular dysfunction associated with encephalopathies commonly observed in preeclamptic women. To test this, pregnant Dahl S and Sprague Dawley (SD, healthy pregnancy control) rats were studied during late pregnancy (gestational day 20, n=4–6). Mean arterial pressure (MAP) was significantly greater on day 20 of pregnancy in the Dahl S rat compared to SD (153±11 vs. 107±3 mmHg, p<0.05), and there was no drop in MAP during pregnancy in the Dahl S compared to virgin littermates (141±12, p=0.48). To assess blood‐brain barrier function, Evan's Blue extravasation into the brain was measured. Evan's Blue concentration was increased in both posterior (0.04±0.012 vs. 0.018±0.001 ng/g tissue/plasma concentration, p=0.07) and anterior (0.021±0.001 vs. 0.034±0.006, p=0.06) brain in pregnant Dahl S rats compared to virgin littermates, respectively. No difference was observed in either brain region in SD rats. In a separate group of Dahl S pregnant and virgin rats (n=5), brains were harvested and weighed on day 20, heated at 50°C for 72 hours, and weighed again to determine the dry:wet weight ratio as a measure of brain water content (edema). Greater water content was found in the posterior brain of the pregnant Dahl S compared to virgin littermates (78.0±0.1 vs. 77.4±0.2, p<0.05) with no significant difference in the anterior brain (79.2±0.2 vs. 78.8±0.3, p=0.33). These results suggest that the Dahl S rat model of superimposed preeclampsia exhibits blood brain barrier dysfunction and cerebral edema. Therefore, pregnant Dahl S rats may be a useful model to investigate mechanisms leading to cerebral vascular abnormalities that occur during preeclampsia.

Support or Funding Information

National Institutes of Health, American Society of Nephrology, UMMCIRSP
Nuclear transport of fungal transcription factors mediated by Importin‐α and specificities of this interaction
Authors: Bernardes Natália Elisa , Litvac Daniel , Bertolini Maria Célia , Panté Nelly , de Matos Fontes Marcos Roberto
Published: November 2023

Importin‐α (Impα) is a protein that mediates nuclear import of macromolecules via recognition of nuclear localization sequences in the macromolecule that will be transported. Some proteins and transcription factors related with nitrogen and glycogen metabolism in fungus possess putative NLSs, suggesting that they can be transported into the nucleus via the classical nuclear import pathway, which is mediated by Impα and Importin‐β. This work presents biophysical and functional experiments that confirms the interaction between Impα from Neurospora crassa (NcImpα) and Mus musculus (MmImpα) complexed with potential NLSs of transcription factors NIT2; NCU1629 and PACC. Calorimetric analyses showed a high affinity of the NIT2_NLS, PACC_NLS and 1629_NLS peptides at the binding sites of NcImpα and a lower affinity with MmImpα. NcImpα/NIT2_NLS and MmImpα/NIT2_NLS complexes were cocrystallized and X‐ray diffraction data were collected with resolution of 2.5 and 1.5 Å, respectively. The final NcImpα/NIT2_NLS structure allowed unambiguously to observe that the NIT2_NLS peptide binds at major and minor binding sites of NcImpα. However, the final MmImpα/NIT2_NLS structure, showed the binding of the peptide just at the minor binding site. Nuclear import assays were performed with the potential NLS peptides conjugated with bovine serum albumin. Dextran was used to confirm the permeabilization of the cytoplasmatic membrane of cells by digitonin, without damaging the nuclear envelope. The nuclear import assays with NIT2‐NLS‐BSA‐Cy3 and 1629‐NLS‐BSA‐Cy3 indicated that a significant amount of these complexes were imported into the nucleus in digitonin‐permeabilized cells, incubated with cytosol from rabbit and an energy regenerating system. However the nuclear import was more efficient when a mammalian peptide was used, as confirmed by the quantification performed with Image J. The experiments confirmed that the peptides are functional NLSs, with high affinity to Impα and evidenced specificities of the interaction between NLS peptides with Impα from different organisms.

Support or Funding Information

FAPESP ‐ São Paulo State Foundation
The Folding Mechanism of an Artificial Knotted Protein Characterized by Optical Tweezers.
Authors: Bustamante Andrés , Rivera Maira , Molina José , Baez Mauricio
Published: November 2023

Protein folding occurs because establishment of weak interactions observed in the native state (native contacts) predominate over the formation of transient interactions that could promote alternative conformations (non‐native contacts). However, knotted proteins defy this view because their polypeptide chains would require to form non‐native contacts during its folding. To determine the role of non‐native contacts in such topologies, we explored the folding mechanism of an artificial knotted protein (2ouf‐knot). In this case, optimization of non‐native contacts should play a minor role during its folding. The folding of 2ouf‐knot was characterized maintaining the knotted topology in the unfolded state by pulling their C and N extremes by using optical tweezers. The molecular stretching of 2ouf‐knot obtained at constant velocity of pulling showed two transitions of unfolding occurring at 9 ± 1 and 14 ± 1 pN, followed by two transitions of refolding at 8 ± 1 and 12 ± 1 pN. This indicates the presence of an intermediate in both directions of the folding reaction of 2ouf‐knot. In terms of the molecular extension, the contour length (Lc) associated with the first transition at low force account for the 30% of the expected extension for the fully unfolded polypeptide. Notably, the intermediate of 2ouf‐knot was also apparent under equilibrium conditions. At constant force, 2ouf‐knot oscillates between the intermediate and the unfolded state extended by 5 and 16 nm with respect to the native state respectively. We observe that there was no full unfolding of the protein without visiting the intermediate state of 5 nm. Therefore, this specie is an obligate step (on‐pathway intermediate) for the complete unfolding of the protein. Nevertheless, we also observe that approximately 20% of the times the fully unfolded protein jump until reach a smaller intermediate of 4 nm with very short residence time. This short‐live off‐pathway intermediates backtrack to the fully unfolded state which refolds to the on‐pathway intermediate of 5 nm. These results contrast with the absence of intermediates (on and off‐pathway) in the case of a natural knotted protein (MJ0366 of Methanocaldococcus jannaschii) characterized under the same experimental setup. We suggest that the intermediates observed in the case of artificial knots arise by lack of specific non‐native contacts that could help to accommodate the knot during unfolding/refolding process. These specific interactions otherwise will help to smooth the energetic landscape of natural knotted proteins.

Support or Funding Information

Fondecyt 1151274, CONICYT fellowship No 21150966. CONICYT fellowship No 21130254.
Implications of Neuroglobin Structure and Function in Human Brain Degenerative Disorders
Authors: Demski Joshua Lee , Beekman Ryley , Ellcey Kaylee , Guenzel Brandi , Hollander Joshua , Jodarski Alyssa , Johnson Mckayla , Moore Dillon
Published: November 2023

Neuroglobin is a respiratory protein with three possible ligands; oxygen, carbon dioxide, and a distal histidine of the protein itself. The unique structure of this protein allows for greater oxygen carrying potential in hypoxic or ischemic conditions. The Laconia High School MSOE Center for BioMolecular Modeling MAPS Team used 3‐D modeling and printing technology to examine structure‐function relationships of human neuroglobin. Internal cysteines at CD7 and D5 are close enough together to form a disulfide bond which has been shown to greatly increase oxygen affinity. Neuroglobin also has a hexacoordinated iron in its reduced state. The E7 His breaks its bond with iron when oxidized and allows for the transport of oxygen. The high expression levels of this globin point to many functions including a role in apoptosis, detoxification, signaling, and oxygen transport. Among this proteins many functions is a role in slowing neurodegenerative disorders by delaying apoptosis which when fully understood may provide treatment opportunities for neurodegenerative disorders such as Alzheimer's disease. While hundreds of studies on neuroglobin have been conducted its role in the human body is still poorly understood. Further research needs to be conducted to fully understand this highly conserved and doubtless crucial member of the globin family.
Initial Crystallography of a Phage DNA Kinase
Authors: Gnann Andrew , Weigele Peter , Dowling Daniel
Published: November 2023

Enzymes capable of selectively modifying DNA comprise an attractive field of research. Modified nucleotides are involved in many biological phenomena ranging from differential gene expression to packaging of nucleic acids in viral capsids. Interesting examples of DNA modification have been found in bacteriophages of gram‐negative bacteria, which use hypermodification of DNA bases to prevent recognition of viral DNA by host restriction endonucleases during infection. Hypermodification of 5‐hydroxymethyl‐2′‐deoxyuridine, a noncanonical DNA base incorporated in some phage genomes in place of thymine, begins with phosphorylation of the C5 hydroxymethyl group. Structural information about the kinase responsible for this phosphorylation reaction will shed light on a largely unstudied class of DNA kinases that can modify duplex DNA (dsDNA) at the major groove. These enzymes may prove useful as molecular biological tools targeting DNA modification states in the DNA polymer. To acquire high‐resolution structural information, we are employing protein crystallography. Here we report biochemical characterization and crystallization of the kinase involved in this bacteriophage DNA hypermodification.
Characterization of a putative monooxygenase involved in climate regulation from Hyphomicrobium sulfonivorans
Authors: Gordon Jackson M. , Culpepper Matthew B. , Culpepper Megen A.
Published: November 2023

Dimethylsulfide (DMS) is a volatile organic sulfur compound that readily aerosolizes in the atmosphere. Aerosolized DMS products decrease solar radiation by increasing cloud albedo, resulting in overall climate cooling. DMS is degraded via bacterial metabolic pathways, including the enzyme DMS monooxygenase. DMS monooxygenase purified from Hyphomicrobium sulfonivorans has been identified as a two‐component enzyme in which a DmoA monooxygenase subunit couples with an NADH‐dependent FMNH2 oxidoreductase (DmoB) to catalyze DMS degradation. The putative DmoA monooxygenase subunit has been expressed recombinantly in E. coli, and purified to >99 % after gel filtration chromatography. A method has been developed to detect and quantitate enzymatic conversion of DMS to its product methanethiol by headspace gas chromatography coupled to a flame ionization detector. Ethanethiol is used as an internal standard. Peak resolution of the three analytes; DMS, ethanethiol, and methanethiol has been determined. Activity assays of the DmoA monooxygenase subunit are underway. In addition, the purified DmoA monooxygenase subunit has been crystallized by vapor diffusion under several sparse‐matrix screening conditions. Preliminary diffraction data supports that the crystals contain protein. Crystal optimization is underway to improve the resolution toward unraveling the three‐dimensional structure of the DmoA monooxygenase subunit.

Support or Funding Information

This research was funded by the Appalachian State University College of Arts and Sciences and Office of Student Research.
Diversity and evolutionary analysis of iron‐containing (Type‐III) alcohol dehydrogenases in the three domains of life
Authors: Julián‐Sánchez Adriana , Gaona‐López Carlos , Riveros‐Rosas Héctor
Published: November 2023

Alcohol dehydrogenase (ADH) activity is widely distributed in the three domains of life. Currently, there are three non‐homologous NAD(P)+‐dependent ADH families reported: Type I ADH comprises Zn‐dependent ADHs; type II ADH comprises short‐chain ADHs described first in Drosophila; and, type III ADH comprises iron‐containing ADHs (FeADHs). These three families arose independently throughout evolution and possess different structures and mechanisms of reaction. While types I and II ADHs have been extensively studied, type III (iron‐activated) ADHs have been scarcely analyzed. Therefore in this work, a global phylogenetic analysis of iron‐activated ADHs was performed to get insights into the evolution of this protein family.

Results showed that FeADHs are distributed in twenty two protein subfamilies, eight of them possessing protein sequences distributed in the three domains of life. Protein sequences from bacteria are present in all FeADH subfamilies, but protein sequences from archaea and eukarya are present in fourteen and thirteen subfamilies, respectively. Interestingly, none of these protein subfamilies possess protein sequences found simultaneously in the three main eukaryotic kingdoms (i.e., animals, plants and fungi).

Animal FeADHs are found in just one protein subfamily, the hydroxyacid‐oxoacid transhydrogenase (HOT) subfamily, which includes protein sequences widely distributed in fungi, but not in plants), and in several taxa from lower eukaryotes, bacteria and archaea. Fungi FeADHs are found mainly in two subfamilies: HOT and maleylacetate reductase (MAR), but some can be found also in other three different protein subfamilies. Plant FeADHs are found only in chlorophyta but not in higher plants, and are distributed in three different protein subfamilies.

In conclusion, FeADH is an ancient protein family that probably was present in the last common ancestor, and comprises a diversity of subfamilies that shares a common 3D scaffold that resulted with a patchy distribution in eukaryotes.

Support or Funding Information

Supported by DGAPA‐UNAM grant IN225016.