bluuebunny/crossref_metadata_2025 huggingface dataset:
Synchronous biventricular pressure‐volume loops in rodents using a closed‐chest approach
Authors: West Christopher R. , Ahmadian Mehdi , Wainman Liisa
Published: May 2022

Introduction

The assessment of left‐ and right‐sided cardiac function via closed‐chest pressure‐volume loops is important to understanding and diagnosing a wide range of cardiovascular pathologies. In the vast majority of pre‐clinical studies, either left‐ or right‐sided heart function is assessed in isolation, or, more recently, by sequentially right‐ and left‐sided catheterization1. Synchronous biventricular catheterization would offer a significant advancement for pre‐clinical research. To date, closed‐chest synchronous biventricular pressure‐volume loops have only been obtained in a few studies, limited to porcine models2. Here, we describe initial results from what we believe to be the first synchronous biventricular pressure‐volume loops obtained in the rodent using a closed‐chest approach.

Aim

To obtain biventricular pressure‐volume loops in the anesthetized rat using a closed‐chest approach

Methods

A total of 8 Wistar rats were anesthetized with urethane (1.5mg/kg), tracheotomised, ventilated, and subsequently had a solid state catheter placed in the femoral artery to measure blood pressure, and admittance pressure‐volume catheters placed in the right‐ and left‐ventricle to obtain synchronous biventricular pressure‐volume loops. Admittance probes were set to different frequencies to avoid cross‐talk. All catheters were left to stabilize for a minimum of 10 min after which we extracted and averaged 1 minute of basal pressure‐volume indices.

Results

Reliable ventricular pressure waveforms were obtained from both the left (i.e., left‐ventricular end‐systolic pressure = 106±12mmHg) and right (i.e., right‐ventricular end systolic pressure = 28±8mmHg) side in all eight animals, and we were able to obtain standard left‐sided pressure‐volume loops from all eight animals (i.e., stroke work = 22±5mmHg/ml). We were only able to obtain reliable right‐sided pressure‐volume loops in two of eight animals, due to poor volume signals in the remaining 6 animals. In the two animals we were able to obtain synchronous pressure‐volume loops there was clear evidence of ventricular dependence across the respiratory cycle (Fig.1).

Conclusions

Obtaining synchronous biventricular pressure‐volume loops using a closed‐chest approach in rats is possible but technically challenging. Further work is needed to better obtain right‐sided ventricular volumes.

References

Potus F. et al., J. Vis. Exp (160), e61088, doi:10.3791/61088 (2020).

Lyhne MD. et al., J. Vis. Exp.(171), e62661, doi:10.3791/62661 (2021).
Dietary Zinc Deficiency Increases Bisphenol A Toxicity in Diabetic Rat: Studies on the Testicular and Epididymal Pathophysiology
Authors: Sahu Chittaranjan , Jena Gopabandhu
Published: May 2022

Bisphenol A (BPA), an endocrine disrupter extensively used in plastics products, that causes male reproductive dysfunction in humans and experimental systems. Type 1 diabetes (T1D) associates hyperglycaemia dysregulate the spermatogenesis and antioxidant status in the testes, which negatively impact on male fertility in adults. Dietary zinc (Zn) deficiency is a global health concern, because majority of the individuals are suffering from certain range of Zn‐deficiency. Zn plays an important role in the regulation and maintenance of testicular physiology and the overall reproductive health. The present study was aimed to investigate the influence of Zn‐deficiency on BPA induced germinal cell and male gonadal toxicity in diabetic rat. Sprague Dawley rats (4 weeks old) were randomly divided into eight different groups; like control (standard pellet diet and aquapure drinking water), BPA (10 mg/kg/day), Zn‐deficient diet (ZDD; Zn‐deficient diet), diabetic (DIA), BPA+ZDD, BPA+DIA, ZDD+DIA and BPA+ZDD+DIA for four weeks. Diabetes was induced by injecting single dose of streptozotocin (55 mg/kg) freshly prepared in cold citrate buffer at pH 4.5. The evaluation of various end‐points such as assessment of body and organ weights, biochemical estimations, sperm parameters. testes and epididymis histopathology, quantification of testicular DNA damage and sperm apoptosis were conducted. Testicular 8‐OHdG immunoexpression as well as catalase and OCT4 expressions were carried out by western blot analysis. The results showed that dietary Zn‐deficient condition significantly increased the BPA toxicity by decreasing body and organ weight, serum albumin and increasing glucose, SGPT, SGOT and BUN significantly in diabetic condition. Further, significant decrease in sperm count, sperm motility and increase in sperm deformities were observed. The Zn‐deficiency significantly increased the BPA induced testicular and epididymal tissue injury, elevated testicular 8‐OHdG immunoexpression and sperm apoptosis in diabetic rat. Moreover, BPA exposure significantly decreased the testicular catalase and OCT4 expressions in dietary Zn‐deficient diabetic rat. The present results concluded that dietary Zn‐deficiency exacerbated the BPA induced gonadal toxicity by hypogonadism, increased sperm abnormalities, testicular and epididymal cellular damage, testicular DNA damages, sperm apoptosis as well as decreased the testicular catalase and OCT4 expressions in diabetic rat.
Alanine Modulates Lactyl‐CoA Abundance in HepG2 Cells
Authors: Singh Pankaj K. , Bostwick Anna , Pepper Hannah , Snyder Nathaniel W.
Published: May 2022

The acyl‐coenzyme A (CoA) thioester lactyl‐CoA has been described as an acyl‐donor for histone post‐translational lactylation. Understanding the biology and functional significance of lactylation is hindered by a lack of knowledge of contexts that modulate lactyl‐CoA abundance and direct evidence of potential substrates that generate lactyl‐CoA. Using liquid chromatography‐ high resolution mass spectrometry and isotope tracing, we found that alanine provides carbons for the acyl‐group of lactyl‐CoA. Alanine supplementation increased lactyl‐CoA in a dose responsive manner. However, to our surprise, this effect was dependent on glucose availability as treatment with exogenous alanine decreased cellular lactyl‐CoA abundance after glucose withdrawal. This alanine induced decrease was rescued by cyclosporine, an inhibitor of alanine to pyruvate conversion by alanine transaminase, but not by an inhibitor of mitochondrial pyruvate import. Together, these findings provide a rationale to examine the function of lactyl‐CoA in settings where alanine, lactate, and pyruvate metabolism converge such as gluconeogenesis.
Coffee intake mitigated high fat diet‐induced whitening of brown adipose tissue in obese mice
Authors: Daleprane Julio B. , Cadete Bruna , Moura‐Nunes Nathália
Published: May 2022

The aim of this study was to evaluate the preventive and therapeutic effect of coffee consumption on metabolic and molecular aspects of adipose tissue (AT) remodeling in a high‐fat diet‐induced obesity mice. Our hypothesis is that coffee consumption mitigates brown adipose tissue (BAT) whitening and prevent obesity related comorbidities. 50 male mice of 90 days of the C57BL6 lineage divided into 3 groups: Control (C); High‐fat (HF) and prevention 1% w/w coffee (HF‐CP). After 10 weeks, the HF group gave rise to the treatment 1% w/w coffee group (HF‐CT), totaling 4 different groups for another 4 weeks, totaling 14 weeks of experiment. The HF‐CP group has lower body mass than the high fat (HF) group (‐8%, p < 0.05), better AT distribution, with smaller adipocytes and smaller mean sectional area (p<0.05). Both groups that received coffee associated with a high‐fat diet (HF‐CP and HF‐CT) improved glucose metabolism when compared to the HF group, the glucose tolerance test in the HF‐CP group was similar to the C. Coffee consumption prevented hyperinsulinemia and hyperleptinemia in the HF‐CP group, this group did not develop insulin resistance and in the group that consumed coffee after induction of obesity (HF‐CT) the hyperinsulinemia was attenuated. The plasma concentration of IL‐6 was reduced (p<0.05) in HF‐CP and HF‐CT when compared to the HF group. The immunofluorescence expression of UCP‐1was more intense in BAT of HF‐CP compared to others groups. Thermogenesis and mitochondrial biogenesis in BAT were higher with increased gene expression of UCP‐1, PPARγ, PRDM16, PCG1α, Rβ3A, OPA‐1in HF‐CP. Coffee has better effects when consumed preventively with better AT remodeling, with maintenance of the brown phenotype, thermogenic activity in BAT. Furthermore, coffee consumption was able to attenuate body mass gain, improve glucose and leptin homeostasis. When consumed after obesity induction, as a treatment, it was not as effective as prevention, however, there was also better BAT remodeling with attenuation of lightening, greater expression of thermogenic genes and reversal of hyperinsulinemia.
The role of Kisspeptin, KISS1 and KISS1R genes in primary female infertility: a case‐control study of a Nigerian population
Authors: Okafor Izuchukwu A. , Saanu Oluseun O. , Olayemi Oladapo , Omigbodun Akinyinka
Published: May 2022

Introduction

There is growing evidence that genetic changes are present in as many as 10% of infertile females. The Kisspeptin system plays a critical regulatory role in female reproductive functions. However, the extent of its role is not yet evident in primary infertility.

Objective

This study investigated the relative gene expression levels of Kisspeptin, KISS1, and KISS1R in primary infertile and fertile Nigerian females.

Materials and Methods

This is a case‐control study of 50 fertile and 54 primary infertile Nigerian females (20 to 40 years) attending the gynecology and family planning clinics of University College Hospital, Nigeria, and met the study’s inclusion criteria between August and November 2020. Basic clinical and sociodemographic information was collected from each participant using a clinical proforma. Five ml of blood samples were also collected by venepuncture from both the cases and control subjects for Kisspeptin, KISS1, and KISS1R genes analysis, using ELISA and RT‐qPCR techniques.

Results

BMI of cases was significantly different from the study controls, though both are still in the overweight category (27.98±0.87 vs. 28.18±0.59; p=0.004). Menarcheal age was not significantly different (p=0.573) but shows a significant strong association with ovarian factor infertility (p=0.008, χ2=13.839, φc= 0.458) and tubal factor infertility (p=0.025, χ2=11.111; φc=0.527). There is no change in the relative expression of Kisspeptin, Kiss1, and Kiss1R genes (P>0.05). However, Kisspeptin levels in primary infertile women showed a weak negative correlation (r = ‐.305; p=0.039) and a good predictive model for Kiss1 gene expression (p=0.001; r=0.458).

Conclusion

Females with primary infertility may present with overweight in no significantly different way than fertile females. Changes in the Kisspeptin levels and relative expressions of Kiss1 and Kiss1R may not be useful in diagnosing primary infertility. Menarcheal age should be investigated as an additional diagnostic indicator for tubal and ovarian factor infertility for primary infertile women.

Significance

Changes in the Kisspeptin system alone may not explain the primary female infertility due to the complexity of the disease. A look into the role of the Kisspeptin system in the specific causative factors of primary female infertility could be more revealing.
Sex Differences in ROS Production and SOD Activity Following Induced Inflammation
Authors: Mascone Sara E. , Kim Katherine I. , Evans William S. , Prior Steven J. , Ranadive Sushant M.
Published: May 2022

Vascular dysfunction is closely related to and caused by oxidative stress and chronic inflammation. Oxidative stress may be augmented by inflammation due to overproduction of reactive oxygen species (ROS) and concomitant impairments in ROS clearance. Premenopausal women experience a lower incidence of vascular dysfunction compared with age‐matched men. This sex difference is, in part, attributed to differences in estrogen and androgen concentrations and actions on endothelial cells. However, limited knowledge exists regarding sex differences in ROS production and ROS clearance in endothelial cells following induced inflammation. Because male human umbilical vein endothelial cells (HUVECs) are exposed to higher androgen concentrations in umbilical cord blood than female HUVECs, we sought to evaluate sex differences in ROS production and superoxide dismutase (SOD) activity (a major contributor to ROS clearance) in male and female HUVECs. We hypothesized that female HUVECs would exhibit lower ROS production and higher SOD activity than male HUVECs. Inflammation was induced in HUVEC cell lines (n=4/group) using tumor necrosis factor‐alpha (TNF‐α, 50ng/ml). Cell lysate samples from time‐matched control and TNF‐α treatments were collected at 4 (4H) and 24 hours (24H) post‐stimulus. Fluorescence detection was used to quantify ROS production and viable cells alive at 4H and 24H post‐stimulus in cells stained for ROS (CellROX) and living cells (Hoechst). Data are presented as a ratio of ROS to live cells (CellROX/Hoechst ratio). Cell lysate samples were subsequently assayed for SOD activity and normalized to protein concentration. TNF‐α treatment significantly increased ROS production in all HUVECs at 24H compared with 4H (CellROX/Hoechst ratio‐ 4H TNF‐α: 1.37 ± 0.21, 24H TNF‐α: 1.44 ± 0.20, p=0.0015). Notably, female HUVECs exhibited significantly lower ROS production than male HUVECs in time‐matched Control samples and following TNF‐α‐ induced inflammation (CellROX/Hoechst ratio‐ Female HUVECs: 4H TNF‐α: 1.20 ± 0.17, 24H TNF‐α: 1.33 ± 0.14, Male HUVECs: 4H TNF‐α: 1.32 ± 0.14, 24H TNF‐α: 1.56 ± 0.20; p=0.0040). Further, female HUVECs exhibited significant increases in SOD activity with increased exposure time to TNF‐α‐ induced inflammation while male HUVECs did not (female HUVECs: 4H TNF‐α: 2.93 ± 1.10, 24H TNF‐α: 4.82 ± 1.60, p=0.0098; male HUVECs: 4H TNF‐α: 3.30 ± 1.87, 24H TNF‐α: 3.72 ± 1.06 U/mg; p<0.05). Compared with female HUVECs, male HUVECs exhibit a pro‐inflammatory state following induced inflammation, potentially due to differences in exposure to androgen concentrations in fetal umbilical cord blood.
Improving Emotional Well‐Being and Cardio‐Metabolic Health with Continuous Glucose Monitoring
Authors: Walson Francis , Thomas Natalya , Poff Angela M. , Alnajar Reeman , Johnson Ayesha , Ari Csilla , Visovski Constance , Matthew Kelly , Hull Allison , D'Agostino Dominic P.
Published: May 2022

Continuous glucose monitoring (CGM) is extensively used in diabetic populations but understudied in non‐diabetic patients. Glycemic variability is a risk factor for cardiovascular disease, and emerging evidence suggests close monitoring of glycemic control may have a positive impact on chronic disease. CGM increases awareness of glycemic dysregulation and empowers patients to create sustainable changes in eating behaviors. The purpose of this study (ClinicalTrials.gov Identifier: NCT04920058) was to determine if access to continuous blood glucose data using a CGM‐Levels software system improves cardiometabolic health, sleep quality, and emotional well‐being in nondiabetic patients undergoing a comprehensive wellness program.

In this 12‐week randomized clinical trial, patients enrolled in a low carbohydrate‐based wellness program were randomly assigned to monitor glucose using a CGM with Levels software (CGM, N=14) or classic glucometer (Ctrl, N=11). Weight, BMI, waist circumference, liver fat (via abdominal ultrasound), and a cardiometabolic blood panel were measured pre‐and post‐program. Ketones were tested weekly, and glucose tested daily using a glucometer in both groups (CGM and Ctrl). Psychological measures of emotional well‐being, depression, anxiety and sleep quality were performed with a subjective well‐being survey, Patient Health Questionnaire‐9 (PHQ9), generalized anxiety disorder‐7 (GAD‐7) and Pittsburgh Sleep Quality Index (PSQI), respectively, pre‐program, week 6, and post‐program.

Differences were observed between the groups for liver fat which decreased over time in the CGM but not Ctrl group (p<0.05). Other cardio‐metabolic biomarkers improved in both groups over the course of the study, including weight loss, BMI, waist circumference, triglycerides, VLDL, and fasting insulin, but no differences between groups were observed. Total cholesterol, Non‐HDL, LDL, and HDL ratio increased in both groups over time, but did not differ between groups. Assessments of emotional well‐being, depression, anxiety, and sleep quality also improved in both groups over time, again with no intergroup differences.

In a non‐diabetic population, a 12‐week comprehensive wellness program inclusive of glycemic monitoring using CGM was effective at improving liver fat while several biomarkers of cardio‐metabolic health, sleep quality, and emotional well‐being improved regardless of the type of glycemic monitor used. CGM was endorsed by patients to be more comfortable and easier to use than finger stick glucometers, and its use did not impair trial results compared to Ctrl. Thus, CGM with Levels software appears to be a viable and effective method for monitoring glycemic variability in a non‐diabetic population seeking to improve cardio‐metabolic health.
Aging results in endothelial cell telomere uncapping that induces senescence and physiological dysfunction
Authors: Bloom Samuel I. , Tucker Jordan R. , Liu Yu , Abdeahad Hossein , Machin Daniel R. , Thomas Tyler G. , Bramwell R Colton , Islam Md Torikul , Lesniewski Lisa A. , Donato Anthony J.
Published: May 2022

Advancing age leads to an accumulation of senescent endothelial cells (ECs) within arteries. Senescent cells have undergone permanent cell cycle arrest, are pro‐oxidative and pro‐inflammatory, and therefore represent a likely cause of age‐related EC dysfunction. Yet, the molecular mechanisms and physiological consequences of EC senescence remain incompletely understood. Telomeres are repeat DNA sequences that cap chromosomes. Telomeres shorten with each cell division and are highly susceptible to oxidative damage. When telomeres become critically short or damaged, they become uncapped, which activates the DNA damage response and leads to cellular senescence. Here, we tested the hypothesis that aging results in EC telomere uncapping that induces senescence, leading to physiological hallmarks of aging. To assess whether aging results in EC telomere uncapping, we compared ECs from young (~3 mo) and old (~27 mo) mice. Aging resulted in ~4‐fold greater EC telomere uncapping (p<0.001, Figure 1A). To determine if EC telomere uncapping induces senescence, we deleted the telomere capping protein, TRF2, in ECs of young (~3.5mo) mice (TRF2‐ecKO). Compared to wildtype (WT) littermate controls, TRF2‐ecKO mice had a ~78% reduction in TRF2 gene expression (p<0.0001). TRF2 deletion reduced EC division by ~47% (p<0.0001, Figure 1B), indicative of senescence. To examine the physiological consequences of EC senescence, we examined hallmarks of vascular aging including perfused microvascular density and endothelium‐dependent dilation (EDD). TRF2‐ecKO mice had an ~18% reduction in perfused mesenteric microvessels between 5‐25 µM (p<0.001, Figure 1C). Likewise, TRF2‐ecKO displayed ~26% reduction in mesenteric artery EDD compared to WT mice (p<0.05, Figure 1D). Furthermore, TRF2‐ecKO arterial EDD was ameliorated by the superoxide scavenger, TEMPOL (p>0.05, Figure 1D). Endothelium‐independent dilation to the exogenous nitric oxide donor sodium nitroprusside was not different between WT and TRF2‑ecKO mice (p>0.05). These data suggest EC telomere uncapping leads to senescence that reduces perfused microvascular density, and to elevated oxidative stress that suppresses EDD, similar to advanced age. To assess metabolic and muscle function, we performed an intraperitoneal glucose tolerance test (GTT, 2g/kg body mass) and a forelimb grip strength test. TRF2‐ecKO mice had ~21% greater area under the curve during GTT compared to WT mice (p<0.05, Figure 2A) as well as a ~14% reduction in grip strength (p<0.05, Figure2B). Taken together, these data provide evidence that aging results in EC telomere uncapping that induces senescence and age‐related physiological dysfunction.
Yap and Taz promote osteogenesis in neural crest cells by preventing chondrogenesis
Authors: Zhao Xiaolei , Tang Li , Nguyen Bao H. , Le Tram P. , Chen Wen , Zheng Mingjie , Yamaguchi Hiroyuki , Martinez‐Traverso Idaliz M. , Wang Jianxin , Li Min , Martin James F. , Lee Brendan H. , Komatsu Yoshihiro , Wang Jun
Published: May 2022

The neural crest (NC) is a unique migratory stem cell population only existed during vertebrate embryogenesis. It has remarkable multipotency to give rise to various cell types including osteoblasts and chondrocytes, which contribute to majority of the cranial skeleton development. Defects in NC development led to many congenital diseases, including cleft palate and craniofacial abnormities. Recently, the Hippo‐Yap pathway, a conserved fundamental pathway has been indicated to play a key role in NCCs. Yet, the roles of Yap and Taz, function as Hippo signaling effectors, in NCC cells are still poorly understood.

To understand the roles of Yap and Taz, we performed both in vitro, ex vivo and in vivo studies using O9‐1 NC cells and Yap/Taz conditionally knockout (CKO) mice driven by Wnt1creSOR. We found that Yap/Taz are required for the osteogenic versus chondrogenic cell fate decision of NC cells. Yap/Taz prevent O9‐1 NC cells from differentiating into chondrocytes to favor osteogenesis, and Yap/Taz deficiency in NC cells resulted in chondrogenesis instead of osteogenesis. Notably, in vivo, we observed NC‐derived cranial bone defects accompanied by ectopic cartilage formation. From RNA‐seq and CUT/RUN datasets we found that Yap/Taz directly regulate key genes that govern osteogenesis (Dlx5, Runx2,…) and chondrogenesis (Sox9/5/6,…). Interestingly, many of those genes are known Wnt target genes. Moreover, our data from ATAC‐seq, PLA and Co‐IP demonstrated that Yap/Taz directly interact with β‐catenin to coordinately regulate related genes expression in NCCs to orchestrate the osteogenic or chondrogenic cell fate, such as the key chondrogenesis gene Sox9.

In summary, our data indicated that Yap/Taz play pivotal roles in determining osteogenesis or chondrogenesis of NC, partly through interactions with the Wnt‐β‐catenin pathway, which reveal potential novel diagnostic and therapeutic targets for the NC‐derived diseases.
Vascular and Skeletal Muscle Remodeling: Beyond Physical Inactivity
Authors: Pedrinolla Anna , Cavedon Valentina , Milanese Chiara , Barbi Chiara , La Ginestra Fabio , Giuriato Gaia , Martignon Camilla , Venturelli Massimo
Published: May 2022

Vascular remodeling refers to the changes in the structure and function of vessels. This adaptability of the arteries is essential to successfully handle vascular distress and arterial disease. Both, skeletal muscle mass and the level of physical activity are independent mechanisms that plays a role in this physiological phenomenon. However, the direct impact of the skeletal muscle mass remodeling on the structure and function of the vessels is not clear.

PURPOSE

To determine the impact of skeletal muscle mass remodeling on lower limbs arterial diameter, blood flow and arterial stiffness.

METHODS

Nine young (33±13 yrs) physical active individuals with a single‐leg amputation (transtibial amputation) were recruited for the study. Vascular size (femoral artery diameter), hemodynamics (femoral artery blood flow) and arterial stiffness (carotid‐femoral pulse wave velocity) were measure with an ultrasound doppler device at the level of the common femoral artery in both amputee (AL) and whole limbs (WL). Skeletal muscle mass of both limbs was measured with a DEXA system. Both AL and WL were equally utilized by the individuals for ambulation and recreational sports.

RESULTS

The skeletal muscle mass of the AL was significantly reduced in comparison to the WL (3.2±1.2 Kg Vs 9.4±2.1 Kg; P=0.0011). Also, the diameter of the femoral artery was significantly reduced in the AL 0.5±0.1 cm in comparison to the WL 0.9±0.2 (p=0.0012). However, femoral artery blood flow normalized for the muscle mass (mean difference 49.07 ml۰min‐1۰kg‐1; p = 0.11) and carotid‐femoral Pulse Wave Velocity (mean difference 0.91 cm۰s‐1; p =0.21) were similar between the two legs. Interestingly, linear regression analysis shows a significant correlation between skeletal muscle mass and femoral artery diameter (p=0.0031).

CONCLUSION

The results of this study revealed that the massive skeletal muscle reduction caused by a transtibial amputation, but independent form the level of physical activity, is coupled by a dramatic arterial diameter decrease. Interestingly, local circulation and arterial stiffness seems not impacted by these structural changes.
Hypoxia Regulates Endothelin‐1 Production and Microglial Cell Activation
Authors: Bailey Blake E. , Ojeda Carlos T. , De Dios Luz , Vazquez Ricardo , Inostroza‐Nieves Yaritza
Published: May 2022

Oxidative stress and inflammatory responses play critical roles in hypoxic‐ischemic brain injury.

Microglial cells are rapidly activated in response to injury and stressful stimuli, including hypoxia.

Endothelin‐1 (ET‐1) is a potent vasoconstrictor that has been associated with cerebrovascular diseases. Hypoxia stimulates endothelial ET‐1 production. However, the role of ET‐1 in microglia under hypoxia is not clear. The aim of this project was to characterize the effect of hypoxia in a human microglial cell line, HMC3. We induced hypoxia using a chamber (1% O2, 5% CO2, and 92 % N2) at 37 °C for 4h. MUSE Oxidative Stress Assay was performed to measure reactive oxygen species (ROS) formation, ELISA to determine TNF, IL‐6, and ET‐1 levels, qPCR to measure gene expression of ET‐1, and immunofluorescence staining to visualize and compare the presence of ET‐1 in hypoxic and control

group cells. It was observed that, when compared to normoxic HMC3 cells, hypoxic HMC3 exposure significantly increased the ROS by a factor of 2.5 (p<0.001, n=3), the pro‐inflammatory cytokine TNF‐α increased 3.8 times (p<0.01, n=4), and IL‐6 increased by a factor of 1.6 (p<0.01, n=4). In addition, hypoxia stimulates ET‐1 gene expression 5.0‐fold (p<0.001, n=4) and increased protein production 1.3 times (p<0.01, n=4). Consequently, treatment with ET‐1 increased the amount of ROS, TNF‐α, and IL‐6 in HMC3 cells by a factor of 1.4 (p<0.05, n=4), 1.6 (p<0.001, n=4), and 1.9 (p<0.05, n=4), respectively. All these events were blocked by ET‐1 receptor A (ETRA) antagonist, BQ123. Our results suggest that hypoxic conditions create a cycle of microglial cell activation leading to increased ROS and ET‐1 production that further stimulate microglial cells. Thus, we posit that the ET‐1 receptor blockade represents a promising therapeutic approach to regulate microglial cell responses in hypoxic‐ischemic brain injury.
OGT/CDK5/ACSS2 Axis Regulates Breast Cancer Brain Metastasis Growth
Authors: Ciraku Lorela , Esquea Emily , Jackson Joshua , Reginato Mauricio
Published: May 2022

Onset of brain metastases (BMs) in breast cancer patients is considered an end‐stage event, with no effective drug treatment and a median survival after diagnosis measured in months. Thus, there is an urgent need to develop novel treatment strategies. Metastatic breast cancer cells colonizing the brain encounter adverse ‘nutritional environment’, as the levels of key metabolic fuels such as glucose are much lower, in part because they are avidly consumed by neurons. To overcome this challenge, brain‐metastatic cancer cells rely on acetate as a fuel source and convert it into acetyl‐CoA via upregulation of acetyl‐CoA synthetase 2 (ACSS2) enzyme. We have previously shown that brain tumors increase the nutrient sensing post‐translational modification O‐GlcNAcylation and O‐GlcNAc transferase (OGT) levels to regulate acetate metabolism into acetyl‐CoA via increased phosphorylation of ACSS2 on Ser‐267 in a cyclin dependent kinase 5 (CDK5)‐dependent manner, increasing brain tumor growth. Here, we show that human breast cancer cells selected to metastasize to the brain contain increased levels of O‐GlcNAc, OGT and ACSS2‐Ser267 phosphorylation compared to parental cells and show that human breast cancer brain metastatic patient samples contain elevated ACSS2‐Ser‐267 levels. Moreover, overexpression of OGT or ACSS2‐S267D phospho‐mimetic mutant confer a growth advantage on brain metastatic breast cancer cells in an in vivo intracranial xenograft model, may be due to an increase in lipogenic proteins. Additionally, we show that pharmacologically targeting CDK5 and ACSS2 with small molecule inhibitors reduces tumor growth in a novel orthotopic ex vivo brain slice model. These results suggest a crucial role for OGT/CDK5/ACSS2 signaling in transducing nutritional state to regulate acetate metabolism in breast cancer BM cells and identify CDK5 and ACSS2 as novel therapeutic targets for treatment of breast cancer brain metastasis.
Suitability of Thermal Indices in Describing Heat Stress Compensability
Authors: Vecellio Daniel J. , Wolf S. T. , Cottle Rachel M. , Kenney W. L.
Published: May 2022

Multiple thermal indices have been created to categorize the impact of the ambient environment on human physiology and health. Many of these indices promulgate thresholds that are used to delineate between safe and unsafe thermal environments at rest and during physical activity (exercise, sport, industry, etc.). While some of these thresholds were empirically derived, others were established based on models or theoretical construct with no objective physiological measurements or rationale. In a world where heat is already the leading weather‐related cause of death and climate change will exacerbate the effects of extreme heat in the future, it is important to determine if commonly used indices and associated thresholds are able to accurately describe ambient conditions which are thermally safe or unsafe. One physiologically‐ and biophysically‐determined definition of safe environments is based on the upper limit of thermal balance (PSU H.E.A.T. Project). In the present study, young, healthy adults were exposed to a progressive heat stress protocol in an environmental chamber in both hot‐dry and warm‐humid conditions to determine critical environmental limits of dry‐bulb temperature and vapor pressure which could then be compared to other widely used critical heat stress index values. That is, upper environmental limits for human heat balance were compared to common heat stress indices, including wet‐bulb temperature (Twb), Heat Index (HI), Wet Bulb Globe Temperature (WBGT), and Universal Thermal Comfort Index (UTCI). Under activities of daily living, HI and WBGT thresholds very nearly match up with heat stress compensability curves based on critical heat balance limits while large variation was found in with UTCI. Interestingly, the theorized Twbthreshold of 35°C, used increasingly by mainstream media sources in describing extreme heat, was significantly higher than the critical limit for heat stress compensability. This work highlights the need for continued empirical work in determining and validating existing critical heat stress index thresholds as well as the need for better communication to the general public of what each heat stress index means for their environmental health and safety in a warming world.
Serum Estradiol Level Correlates With Molecules Regulating Phrenic Motoneuron Plasticity
Authors: Lurk Carter , Nair Jay , Marciante Alex B. , Mitchell Gordon S.
Published: May 2022

Acute Intermittent Hypoxia (AIH) elicits phrenic long‐term facilitation (pLTF), an important model of respiratory motor plasticity. Although moderate AIH‐induced pLTF exhibits profound age‐dependent sexual dimorphism, little is known concerning the age or sex‐dependent changes in molecules regulating the Q or S pathways to phrenic motor facilitation. AIH elicits pLTF via distinct signaling pathways in phrenic motor neurons initiated by 5HT2A/2B(Q pathway) or 5HT7 & Adenosine 2A receptors (S pathway); these pathways interact via powerful crosstalk inhibition. Moderate AIH (mAIH)‐induced pLTF declines ~50% from 3 to 12 months of age in male rats (Zabka et al., 2001). Gonadectomy abolishes pLTF in young adult males, and it is restored by supplemental testosterone; pLTF is restored due to testosterone conversion into estrogen in the CNS (Zabka et al., 2006). Thus, CNS estrogen must impact keys molecules regulating the Q and/or S pathways to phrenic motor facilitation. Here, we tested the hypothesis that serum estradiol is correlated with keys molecules known to regulate the Q and S pathways in rats. We assessed the mRNA of molecules known to regulate pLTF in ventral cervical spinal homogenates containing the phrenic motor nucleus from young (3 month) and middle‐aged (12 month) male and female Sprague‐Dawley rats; mRNA levels were correlated with serum estradiol levels assessed via ELISA. Spinal cord tissues were preserved in “RNA Later” solution <10 min from harvest, and stored at ‐80°C until mRNA extraction. RT PCR was used to quantify mRNA levels for: serotonin 2A (Htr2a) & 2B (Htr2b) and adenosine 2A receptors (Adora2a); exchange protein activated by cAMP (EPAC); PKA catalytic subunit (Prkaa1); PKA regulatory subunit (Prkar1a); fractalkine (CX3CL1); phosphodiesterase type 4 (pde4b); NOX genes (gp91 and p47); and PKCδ. We found that serum estradiol was positively correlated with mRNA expression of EPAC (r2=0.28, p=0.002), pde4b mRNA (r2=0.15, p=0.029) and adora2a (r2=0.15, p=0.030), but not the other molecules studied. We conclude that serum estradiol may shift the Q‐S pathway balance, although we have not yet demonstrated a causal relationship. An important limitation of this study is that serum estradiol concentration may differ from that in the ventral cervical spinal cord. In future studies, it is important to test the significance in these neurochemical shifts in mechanisms giving rise to the age‐dependent sexual dimorphism in AIH‐induced pLTF.
Elevated Urinary Arginine Vasopressin Concentrations during Preeclamptic Pregnancies do not Persist Postpartum
Authors: Santillan Mark K. , Funk Meghan L. , Brandt Debra , Nuckols Virgina , Stroud Amy , Pierce Gary , Santillan Donna A.
Published: May 2022

Background

Preeclampsia (PreE) is a common hypertensive disorder during the second and third trimesters of pregnancy. It is estimated to affect 5‐7% of all U.S. pregnancies; about 400,000 pregnancies per year. PreE leads to increased risks of fetal abnormalities, pregnancy complications, chronic hypertension, and cardiovascular disease. Arginine vasopressin (AVP) plays an important role in controlling osmotic balance, regulating blood pressure, maintaining sodium homeostasis, and functioning of the kidney. AVP is secreted by the hypothalamus as a proprotein with copeptin. Because copeptin and vasopressin are secreted in a 1:1 molar ratio, copeptin measurements can be used a surrogate measure of vasopressin. Our lab has previously demonstrated that vasopressin, as measured by copeptin, is elevated in plasma and urine throughout preeclamptic pregnancies; even before PreE can be clinically diagnosed. However, it is unknown whether urine vasopressin is elevated chronically postpartum. Our objective was to determine whether there is a difference in copeptin concentration between women with and without a history of preeclampsia at 1‐4 years postpartum. We hypothesized that AVP concentrations of subjects who had received a PreE diagnosis would continue to be elevated postpartum.

Methods

Inclusion criteria for this study (IRB# 201808705) included being 1‐4 years postpartum, not currently pregnant, non‐prisoner, and able to provide informed consent. Women without (N=26; Age 34 +/‐ 3.4 yrs, BMI 25.99 +/‐ 6.34 kg/m2) and with a history (N=25; age 33 +/‐ 4.5 yrs, BMI 30.32 +/‐ 9.12 kg/m2) of preeclampsia in their most recent pregnancy were enrolled. Women fasted, did not smoke, and did not have caffeine for 4 hours prior to their study visit, nor did they have alcohol or exercise for the prior 24 hours. Urine samples were assayed for copeptin concentrations via an automated immunoassay (Brahms KRYPTOR), creatinine concentrations via a QuantiChrom assay, total protein concentrations via a bicinchoninic acid (BCA) protein assay, osmolality by freezing point depression, and specific gravity using a hydrometer.

Results

There were no differences between the cohorts in race or ethnicity. There was no statistically significant difference in urinary copeptin, as a marker of AVP, in the postpartum period between women with vs. without a history of preeclampsia (4.5 vs. 3.2 mg/dL, P=0.4). No significant differences in copeptin were detected in women without a current hypertension diagnosis (5.49 mg/dL, N=43) and those being treated for hypertension (4.79 mg/dL, N=7, P=0.35).

Conclusions

This study suggests that copeptin, a surrogate measure for AVP, is not chronically elevated in urine after a preeclamptic pregnancy.
Comparison of Lectins as Staining Biomarkers for GNE Myopathy Gene Therapy
Authors: Crowe Kelly E.
Published: May 2022

GNE myopathy (GNEM) is an autosomal recessive disease in which mutations in the GNEgene lead to skeletal muscle weakness and progressive wasting. GNE encodes an essential enzyme of the sialic acid (SA) biosynthetic pathway; thus, decreased SA levels in muscle is a hallmark of GNEM. While several therapeutic approaches to increase SA levels in GNEM patients are under development, these efforts have been hindered by the lack of robust biomarkers to assess SA incorporation into affected muscle tissue at the cellular and molecular level. We sought to address this unmet need by comparing the capacity of glycan‐binding proteins known as lectins to effectively monitor SA levels via fluorescent staining. Here, we compare the efficacy of a panel of SA‐detecting lectins by staining samples from multiple models of GNEM. These models include skeletal muscle of the GNED207VTgGne‐/‐ mouse model of GNEM and cell‐based in vitro models, such as those in which SA is removed by enzymatic activity or its production is precluded by GNE mutation. Together, these results indicate the relative ability of each lectin to detect SA alterations in models of GNEM, revealing specific lectins that can act as predictive biomarkers for this disease that may facilitate the development of novel therapeutic approaches.
PDZ‐Binding Kinase Drives Pulmonary Artery Smooth Muscle Proliferation and Vascular Remodeling in Pulmonary Arterial Hypertension
Authors: Bordan Zsuzsanna , Batori Robert , Chen Feng , Li Xueyi , Meadows Mary‐Louise , Haigh Stephen , Sellers Hunter , Su Yunchao , Barman Scott , Fulton David
Published: May 2022

Pulmonary Arterial Hypertension (PAH) is a progressive disease that is caused by high blood pressure in the lungs that originates from increased resistance of small pulmonary arteries (PA). A defining characteristic of PAH is vascular smooth muscle cell (VSMC) proliferation and remodeling of PA, that eventually results in right ventricular failure and death. The hyperproliferative nature of vascular cells in PAH shares many characteristics with cancer cells such as sustained proliferative signaling, changes in hypoxia signaling and cellular metabolism. Using microarray analysis, we discovered a novel gene, PDZ‐Binding Kinase (PBK) that is upregulated in hypertensive PA. PBK is a serine/threonine kinase that has important roles in mitosis and cell proliferation. Also, elevated expression of PBK is associated with numerous aggressive cancers suggesting that it may be an important contributor to PAH. Our hypothesis is that PBK has a functional role in the pathogenesis of PAH but the therapeutic utility of targeting PBK in PAH and the mechanisms by which PBK promotes pulmonary vascular remodeling have not been studied. To investigate the role of PBK in PAH, we performed immunostaining, and found that PBK was highly upregulated in pulmonary arterial smooth muscle in the lungs of two experimental PAH rat models (MCT; Su/H), in the Su/H mouse model, as well as in human PAs and lungs with PAH. Furthermore, human pulmonary arterial smooth cells (HPASMCs) overexpressing PBK showed increased proliferation, while silencing PBK or in vitro pharmacological inhibition significantly reduced HPASMC proliferation. To determine whether PBK plays an important role in PA remodeling and PAH in vivo, we next utilized PBK inhibitor (OTS‐514) to treat MCT‐induced PAH rats in a reversal treatment method and Su/H Rats and Mouse in a preventative treatment. In all of the PAH animal models, right ventricle (RV) thickness, velocity time integral (VTI, index of PA stiffness), right ventricular systolic pressure (RVSP), and the Fulton Index were significantly improved in the presence of the PBK inhibitor. To complement our pharmacological studies with a genetic approach, we have generated PBK KO rats to investigate whether genetic ablation of PBK protects against the development of PAH. Taken together, these data support the hypothesis, that PBK drives pulmonary artery smooth muscle proliferation leading to abnormal vascular remodeling and the development of PAH. Our findings advance the utility of novel therapeutic approaches targeting PBK to improve the morbidity and mortality associated with PAH.
Gas Phase Diffusion Does Not Limit Lung Volatile Anesthetic Uptake Rate
Authors: Peyton Philip J.
Published: May 2022

Background

Inefficiency of lung gas exchange during general anesthesia is reflected in alveolar (end-tidal) to arterial (end-tidal–arterial) partial pressure gradients for inhaled gases, resulting in an increase in alveolar deadspace. Ventilation–perfusion mismatch is the main contributor to this, but it is unclear what contribution arises from diffusion limitation in the gas phase down the respiratory tree (longitudinal stratification) or at the alveolar–capillary barrier, especially for gases of high molecular weight such as volatile anesthetics.

Methods

The contribution of longitudinal stratification was examined by comparison of end-tidal–arterial partial pressure gradients for two inhaled gases with similar blood solubility but different molecular weights: desflurane and nitrous oxide, administered together at 2 to 3% and 10 to 15% inspired concentration (FiG), respectively, in 17 anesthetized ventilated patients undergoing cardiac surgery before cardiopulmonary bypass. Simultaneous measurements were done of tidal gas concentrations, of arterial and mixed venous blood partial pressures by headspace equilibration, and of gas uptake rate calculated using the direct Fick method using thermodilution cardiac output measurement. Adjustment for differences between the two gases in FiG and in lung uptake rate (VG) was made on mass balance principles. A 20% larger end-tidal–arterial partial pressure gradient relative to inspired concentration (PetG – PaG)/FiG for desflurane than for N2O was hypothesized as physiologically significant.

Results

Mean (SD) measured (PetG – PaG)/FiG for desflurane was significantly smaller than that for N2O (0.86 [0.37] vs. 1.65 [0.58] mmHg; P < 0.0001), as was alveolar deadspace for desflurane. After adjustment for the different VG of the two gases, the adjusted (PetG – PaG)/FiG for desflurane remained less than the 20% threshold above that for N2O (1.62 [0.61] vs. 1.98 [0.69] mmHg; P = 0.028).

Conclusions

No evidence was found in measured end-tidal to arterial partial pressure gradients and alveolar deadspace to support a clinically significant additional diffusion limitation to lung uptake of desflurane relative to nitrous oxide.

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Advances in Conceptual and Methodological Issues in Symptom Cluster Research
Authors: Harris Carolyn S. , Dodd Marylin , Kober Kord M. , Dhruva Anand A. , Hammer Marilyn J. , Conley Yvette P. , Miaskowski Christine A.
Published: May 2022

Two conceptual approaches are used to evaluate symptom clusters: “clustering” symptoms (ie, variable-centered analytic approach) and “clustering” patients (ie, person-centered analytic approach). However, these methods are not used consistently and conceptual clarity is needed. Given the emergence of novel methods to evaluate symptom clusters, a review of the conceptual basis for older and newer analytic methods is warranted. Therefore, this article will review the conceptual basis for symptom cluster research; compare and contrast the conceptual basis for using variable-centered versus patient-centered analytic approaches in symptom cluster research; review their strengths and weaknesses; and compare their applications in symptom cluster research.
Treatment outcomes of hips in patients with epiphyseal dysplasia
Authors: Lindgren Amelia M. , Bomar James D. , Upasani Vidyadhar V. , Wenger Dennis R.
Published: May 2022

Hip containment surgeries in multiple epiphyseal and spondyloepiphyseal dysplasia (MED/SED) patients aim to improve the mechanical environment of the hip joint. The purpose of this study was to determine if surgical intervention to improve femoral head coverage improved radiographic and clinical outcomes. A retrospective study identified patients with MED/SED seen in clinic between May 2000 and September 2017, with a minimum of 2-year follow-up. Patient charts/radiographs were reviewed for radiographic hip measurements, pain, and gait. Sixty-nine hips in 35 patients were identified. Forty-four hips were treated nonoperatively and 25 were treated surgically. The mean age at diagnosis was 6.2 years. The mean follow-up was 7.7 years for the surgical group and 7.1 years for the nonsurgical group. The mean postoperative follow-up was 5.4 years. Acetabular index decreased from initial to final visit by 9.0° in the surgical group and 1.6° in the nonsurgical group. Tonnis angle decreased by 13.5° in the surgical group and 1.5° in the nonsurgical group. Center edge angle increased by 19.0° in the surgical group and 7.1° in the nonsurgical group. Hips in the surgical group were 6.1 times more likely to experience an improvement in pain compared with hips in the nonsurgical group. Gait at the final follow-up was similar among the two groups. In this study cohort, containment surgery provided increased femoral head coverage; however, there was equal femoral head deformation despite intervention. Hips treated surgically were more likely to experience an improvement in pain; however, gait alterations did not improve.