bluuebunny/crossref_metadata_2025 huggingface dataset:
Resistance Breathing and Sympathetic Nerve Activity During Simulated Hemorrhage in Humans
Authors: Davis Kenneth A. , Sprick Justin , Kay Victoria , Rickards Caroline A.
Published: May 2022

Background

Resistance breathing amplifies the respiratory pump during inspiration, so may be an effective intervention for treatment of hemorrhagic injuries. In animal studies of actual hemorrhage, and human studies of simulated hemorrhage, resistance breathing protects arterial pressure, and improves tolerance to this stress. Anecdotally, resistance breathing also increases the coupling between sympathetic nerve activity and arterial pressure. The impact of resistance breathing on overall sympathetic nerve activity, however, has not been examined. We tested the hypothesis that resistance breathing increases sympathetic nerve activity during simulated hemorrhage in healthy humans.

Methods

Lower body negative pressure (LBNP) was used to simulate hemorrhage in five human subjects (3M, 2F; 29.2 ± 6.8 y). Two experiments were conducted (randomized order): 1) a control condition in which LBNP was applied at 3 mmHg/min until the onset of presyncope, and 2) a resistance breathing condition in which the same LBNP protocol was used, but subjects breathed through a resistance device (set at ‐7 cm.H2O) during the final stages of the protocol. Arterial pressure and muscle sympathetic nerve activity (MSNA) of the radial nerve were monitored continuously. Bursts frequency (bursts/min) and burst incidence (burst/ 100 heart beats) were used to quantify MSNA. Coupling between diastolic arterial pressure (DAP) and MSNA was assessed by transfer function analysis coherence within the low frequency range (0.04‐0.15 Hz). Two‐way repeated measures ANOVAs were conducted for assessment of responses in the control and resistance breathing conditions, between baseline and at matched time points late in the LBNP protocol.

Results

While LBNP induced increases in both MSNA burst frequency (P=0.003) and burst incidence (P=0.06), there was no effect of resistance breathing on MSNA for either index during LBNP (control, 57.9 ± 25.9 bursts/min vs. resistance breathing, 50.6 ± 21.7 bursts/min, P=0.99; control, 55.6 ± 25.6 b/100 heart beats vs. resistance breathing, 42.3 ± 18.3 b/100 heart beats, P=0.42). Additionally, there was no effect of resistance breathing on DAP (control, 73.2 ± 9.9 mmHg vs. resistance breathing, 72.8 ± 4.4 mmHg; P=0.99), or coherence between MSNA and DAP during LBNP (control, 0.53 ± 0.21 vs. resistance breathing, 0.69 ± 0.17; P=0.46).

Conclusion

Contrary to our hypothesis, resistance breathing had no effect on sympathetic nerve activity during LBNP. A limitation of this study is the low sample size (N=5), and high variability of MSNA. Future investigations with a larger sample size are needed to determine if respiratory dynamics can influence the coupling between MSNA and arterial pressure.
Ciprofloxacin Permanently Accelerates Gastrointestinal Transit in a Rodent Model of Fluoroquinolones Associated Disability
Authors: Kerler Jacqueline , Getaneh Bitseat , Bove Cecilia
Published: May 2022

Fluoroquinolones (FQs) are a class of antibiotics intended to be prescribed for life‐threatening infections. However, they are often administered for less severe infections, which they are not meant to treat. In addition, studies suggest that FQs can lead to severe long‐lasting complications described as Fluoroquinolone Associated Disability (FQAD). Some of these permanent issues affect the central nervous system, cardiac, and musculoskeletal systems among others. Gastrointestinal issues have also been considered a possible side effect due to how FQs may negatively modulate the vagus nerve, which plays a major role in digestion and regulation of satiety. Indeed, the inhibitory neurotransmitter GABA regulates the motor portion of the vagus nerve, and FQs act as a selective inhibitor of GABAA receptors. This blockade of GABAA receptors is believed to contribute to the GI issues that seem to arise after taking FQs.

To measure possible changes in digestion, Sprague Dawley rats were treated daily for 14 consecutive days with either 0.3ml of 0.9% saline (CTL n=6) or 20mg/kg of ciprofloxacin (CPX) via oral gavage. On experiment day (day 14 or 28; n=6 for both), rats were administered phenol red dye, sacrificed, and the stomach, proximal, middle, and distal small intestine were harvested to measure dye recovery via spectrophotometry as a measure of gastrointestinal transit. Results showed that both male and female rats in the 28 day group had significantly less dye recovered from their stomach compared to the 14 day and CTL group, with female rats being disproportionately affected compared to males (P<0.05). This data provides strong evidence that FQs lead to permanent acceleration in digestive patterns. Further experiments will analyze how the phenotype of enteric motor neurons, as well as the phenotype of neurons within the Dorsal Motor Nucleus of the vagus in the medulla oblongata, is affected by this drug. This will contribute to determining the mechanism underlying the observed digestion change in rats treated with ciprofloxacin.
Ampakine‐Hypoxia Induced Phrenic Motor Facilitation is Not Prevented by Intrathecal Methysergide
Authors: Thakre Prajwal P. , Sunshine Michael D. , Fuller David D.
Published: May 2022

Hypoxia‐induced sustained increases in phrenic nerve burst amplitude lasting beyond the hypoxic stimulus are generally accepted to require repeated bouts of hypoxia (i.e., acute intermittent hypoxia, AIH). However, our laboratory recently reported that pretreatment with a drug (ampakine) which acts as a positive allosteric modulator of AMPA receptors lowered the number of hypoxic exposures required to trigger sustained phrenic motor facilitation. Our working hypothesis for the current study was that the combination of ampakine and hypoxia activates the same serotonin (5‐HT) dependent cellular mechanisms that are well established for AIH‐induced phrenic long‐term facilitation following repeated bouts of moderate hypoxia. Phrenic nerve output was recorded from vagotomized, mechanically ventilated, adult male Sprague‐Dawley rats under urethane‐anesthesia. A broad‐spectrum 5‐HT receptor antagonist (methysergide, 20mM, dissolved in DMSO) was delivered intrathecally over the C4 spinal dorsal surface. At least 15 min of stable phrenic burst amplitude was recorded post methysergide delivery. After a stable baseline (BL) was established, intravenous (i.v.) ampakine (CX717, 15mg/kg) was followed by a single episode of hypoxia (arterial partial pressure of O2 = 42±9mmHg). Phrenic nerve bursting was recorded for 60 min after hypoxia. Initial results indicate that intrathecal methysergide does not prevent the ampakine‐hypoxia induced phrenic motor facilitation as evidenced by a significant increase in burst amplitude at 60 min (89±54% BL, n=7). To verify the efficacy of intrathecal drug delivery method, additional experiments were conducted. Specifically, we determined if intrathecal methysergide prevented the phrenic long‐term facilitation induced by AIH, as has been shown by several prior investigations. These experiments used the same preparation, and AIH consisted of three‐5 min episodes of hypoxia with 5‐min intervals of baseline O2conditions. Intrathecal methysergide blocked pLTF (60 min post‐AIH, phrenic burst amplitude = ‐2±31% BL, n=3), but intrathecal delivery of the DMSO vehicle did not (60 min: 94±28% BL, n=3). Collectively, these results do not support our hypothesis that phrenic motor facilitation induced by the pairing of ampakine with a single bout of hypoxia is dependent on serotonin receptor activation.
Solid stress impairs lymphocyte infiltration into lymph node metastases
Authors: Jones Dennis , Wang Zixiong , Chen Ivy , Zhang Sue , Banerji Rohin , Lei Pin‐Ji , Zhou Hengbo , Xiao Victoria , Kwong Cecilia , Van Wijnbergen Jan W. , Pereira Ethel , Vakoc Benjamin , Huang Peigen , Nia Hadi , Padera Timothy
Published: May 2022

Metastasis remains the principal cause of cancer mortality. Lymph node metastases are common and indicate poor overall prognosis, likely due to both the metastatic potential of primary tumor cells and the contribution of lymph node metastases to distant metastases. Although antitumor T cells are generated in lymph nodes, metastatic cancer cells are able to grow in this seemingly hostile microenvironment. We hypothesized that lymph node metastases actively suppress anti‐tumor responses in lymph nodes. The overall goal of our study is to understand how cancer cells avoid immune detection in lymph nodes. We obtained patient samples, established a novel ER+ mammary carcinoma (MCa) cell line and employed 4T1 triple negative breast cancer cells to investigate the growth of spontaneous lymphatic metastases. In addition, we developed a lymph node compression apparatus to mimic the compressive forces, known as solid stress, generated by cancer cells in lymph nodes. We used immunofluorescence microscopy, intravital imaging, flow cytometry, and quantitative PCR to characterize the effect of cancer growth on adaptive immune responses in tumor‐involved lymph nodes. Using patient tissue from multiple cancers and preclinical models of spontaneous lymph node metastases, we show that cancer cells disrupt lymphocyte trafficking to lymph nodes, resulting in exclusion of lymphocytes from metastatic lesions. The presence of solid stress caused by lesion growth is associated with a reduction of functional blood vessels and lymphocyte exclusion in the nodes. Relieving solid stress in the mice increased the presence of lymphocytes in lymph node lesions. These findings reveal a novel mechanism for the lack of immune response against lymph node metastases and may inform strategies for immunotherapy.
Effects of passive heat exposure on myocardial blood flow in healthy young and older adults
Authors: Barry Hadiatou , Chaseling Georgia K. , D’Oliviera‐Sousa Caroline , Harel François , Pelletier‐Galarneau Matthieu , Gagnon Daniel
Published: May 2022

INTRODUCTION

Extreme heat is associated with a greater risk of cardiovascular mortality in older adults (≥60 years). However, the pathophysiology that links extreme heat and cardiovascular mortality remains unknown. One possibility is that myocardial oxygen requirements during heat exposure cannot be met by sufficient increased in myocardial blood flow (MBF) thereby predisposing to ischemia. However, the extent to which MBF increases during heat exposure, and whether age alters this increase, is unknown. The objective of this study was to test the hypothesis that passive heat exposure increases MBF and that this increase is attenuated in healthy older adults.

METHODS

MBF was measured during passive heat exposure (water‐perfused suit) in 15 young (27 years, range: 24‐30) and 14 older (68 years, range: 64‐72) healthy adults with 82‐Rubidium positron emission tomography. Four MBF measurements were performed: prior to heat exposure (baseline) and at an increase in rectal temperature of 0.5°C, 1.0°C, and 1.5°C. Heart rate (ECG) and blood pressure (automated auscultation) were also measured to calculate rate pressure product (RPP; heart rate x systolic blood pressure). MBF reserve (MFR) was calculated by dividing the MBF at 1.5°C by baseline MBF.

RESULTS

Baseline MBF was 0.76 ± 0.19 mL/min/g in young adults and 0.88 ± 0.21 mL/min/g in older adults (p=0.14). MBF increased similarly in both groups during heat exposure (interaction, p=0.31). Nonetheless, older adults displayed a lower MFR at an increase in rectal temperature of 1.5°C (young: 2.3 ± 0.7 vs. older: 1.8 ± 0.4, p=0.03). The lower MFR in older adults was paralleled by an attenuated increase in RPP during passive heating (at + 1.5°C, young: 6.8 ± 1.7vs. older: 3.6 ± 2.2 bpm·mmHg·103, p<0.01), due to a blunted increase in heart rate (young: 50 ± 12vs. older: 34 ± 14 bpm, p<0.01). When the change in MBF was plotted against the change in RPP, the slope of the regression lines did not differ between young (0.11 [95% CI; 0.07, 0.15]) and older (0.08 [95% CI; 0.04, 0.11] mL/g/min/ bpm·mmHg·103,p=0.24) adults.

CONCLUSION

These results show that passive heat exposure roughly doubles MBF in healthy young and older adults. Furthermore, the results demonstrate that the increase in MBF during passive heat exposure is not altered by age and is proportional to the increase in RPP.
Determining the Optimal Soft Tissue Preservation Techniques for Surgical Skills Training
Authors: Le Elaine , Wang Austine , Rockarts Jasmine , Palombella Andrew , Wainman Bruce , de Sa Darren , Nguyen Laura , Downer Naomi
Published: May 2022

Introduction

Competency Based Medical Education, overseen and implemented by the Royal College of Physicians and Surgeons of Canada, requires realistic simulation of specific tasks called Entrustable Professional Activities (EPAs). Mastery of these tasks is required to proceed in the surgical specialty. Due to the growing need for high fidelity training in postgraduate surgical education, there has been a shift from the use of hard‐fixed to soft‐fixed material for improved realism. Currently, there exists no standardized way of assessing the suitability of various fixation techniques for different EPAs.

Objective

The current study seeks to establish the most appropriate tissue fixation method for each EPA. Additionally, we aim to develop a standardized matrix to rate the effectiveness of soft‐fixation methods for the purposes of surgical training based on a number of factors such as the EPA, storage, longevity, biohazardous risk, cost, biomechanical properties, and realism.

Hypothesis

We hypothesize that different soft‐fixation methods possess varying suitabilities for each EPA, which could be deduced through the use of a testing matrix.

Methods

EPAs will be performed on cadaveric tissues embalmed with various soft‐fixation solutions. In order to establish the solutions that will be used, initial tests will be conducted on porcine material. Each pork hock (the joint between the tibia/fibula and the metatarsals) will be embalmed with one of 8 soft‐fixation solutions. Surgical residents will perform an EPA on each embalmed tissue and record their observations. The residents will respond to statements about each of the embalming solutions using a 5‐point Likert scale. The results from this testing will be used to select the embalming solutions to be used when the study is conducted on human donors.

Results

Testing of porcine material to determine the most useful fixation technique is expected to be completed in February of 2022. Testing on cadaveric tissue is expected to be completed in July of 2022.

Conclusion

Gaining an understanding of the suitability of various soft‐fixation methods allows for high fidelity surgical skills training and the maximization of the use of each donor.
SUMO Regulates Histone mRNA Processing and Polyadenylation
Authors: He Shuying , Skinner Marnie , Matunis Michael J.
Published: May 2022

Replication‐dependent histone genes encode the only non‐polyadenylated mRNAs in mammals. Histone pre‐mRNAs, which do not contain introns, therefore require only one processing step that involves 3’ end recognition and cleavage. Both histone gene expression and pre‐mRNA processing take place in specialized nuclear organelles known as histone locus bodies. Recent studies have revealed that polyadenylation of histone mRNAs does occur, however, under specific physiological conditions. These include during normal terminal cellular differentiation, tumorigenesis, and in disease patients with Aicardi–Goutières syndrome. Despite these links between polyadenylation of histone mRNAs, human physiology, and disease, the triggers, the mechanisms, and the consequences of histone mRNA polyadenylation remain poorly defined. In recent studies of small ubiquitin‐related modifier (SUMO) knockout cell lines, we have obtained evidence that SUMO1 and SUMO2 play roles in regulating histone pre‐mRNA 3’ end processing and polyadenylation. This evidence includes alterations in histone locus body size and structure in SUMO1 and SUMO2 knockout cells, as well as mislocalization of key pre‐mRNA processing factors required for normal processing. Collectively, our findings provide new insights into control of histone pre‐mRNA processing that involves posttranslational protein modifications by SUMO1 and SUMO2.
A Protein Tyrosine Phosphatase 4A3 (PTP4A3) inhibitor prevents and repairs pulmonary endothelial barrier dysfunction and ameliorates acute lung injury provoked by the SARS‐CoV‐2 Spike protein subunit 1 in k18‐hACE2 transgenic mice
Authors: Colunga Biancatelli Ruben Manuel Luciano , Solopov Pavel , Dimitropoulou Christiana , Lazo John , Sharlow Elizabeth , Catravas John
Published: May 2022

PTP4A3 regulates various inflammatory and cytoskeletal signaling pathways (e.g., STAT3, RhoA, ERK, AKT) that are involved in endothelial barrier dysfunction and acute lung injury (ALI). We have investigated the actions of JMS‐053, a small PTP4A3 inhibitor, in both in vitro and in vivo models of SARS‐CoV‐2 Spike protein subunit 1 (S1SP)‐induced endothelial inflammation and acute lung injury. S1SP (10nM) caused a time‐dependent endothelial barrier dysfunction in human lung microvascular endothelial cell monolayers that was ameliorated by JMS‐053 (12.5µM) administered either as pre‐treatment (4 hours prior to S1SP) or post‐treatment (5 hours after S1SP). In K18‐hACE2 transgenic mice, which express the human ACE2 receptor for SARS‐CoV‐2 S1SP, we instilled S1SP intratracheally, post‐treated with JMS‐053 (10mg/kg i.p. at 1, 24 and 48 hours after S1SP) or vehicle, and investigated molecular, functional and histological outcomes 72 hours later. JMS‐053 mice displayed lower alveolar cellularity, proteinosis and “cytokine storm” (i.e., IL‐6, IL‐1β, IL‐17), and lower levels of phosphorylated (activated) STAT3 and NF‐kB, compared to mice instilled with S1SP and treated with vehicle. Moreover, JMS‐053 greatly reduced quantifiable histological evidence of injury, compared to vehicle‐treated animals. This data suggests that inhibition of PTP4A3 could represent a critical target for novel therapeutic approaches towards endothelial dysfunction, inflammation and acute lung injury.
Estradiol Augments Sensory Nerve‐Mediated Vasodilation in Young Women
Authors: Schwab Allyson I. , Del Vecchio Angelica R. , Welti Laura M. , Haigh Katherine , Feinberg Ronald F. , McGinty Shane J. , Wong Brett J. , Wenner Megan M.
Published: May 2022

Local heating of the skin elicits biphasic vasodilation: a sensory nerve‐mediated initial peak (axon reflex) and an endothelial‐dependent plateau. We have previously shown that sex hormones, particularly estradiol (E2), improves cutaneous endothelial‐dependent vasodilation, but less is known about the effects of E2 on cutaneous sensory nerve‐mediated vasodilation.

PURPOSE

We tested the hypothesis that E2 enhances sensory‐nerve mediated vasodilation in response to local heating of the skin in young women.

METHODS

We retrospectively analyzed data in 15 healthy young women (age 24 ± 4 yr; MAP 84 ± 6 mmHg; BMI 24 ± 3 kg/m2). Endogenous sex hormone production was suppressed with daily administration of a gonadotropin‐releasing hormone antagonist (GnRHant; Ganirelix) for 10 days. E2 (0.1 mg/day, Vivelle‐Dot) was added back via transdermal patch on days 4–10. Cutaneous vasodilatory responses to local heating were measured on days 4 and 10 using laser Doppler flowmetry during microdialysis perfusions of lactated Ringer’s (control), and in a subset of women (n=6), L‐NAME (non‐selective nitric oxide synthase inhibition). Cutaneous vascular conductance (CVC) was calculated during the initial peak of the axon reflex during local heating (42°C) and normalized to maximal vasodilation achieved by perfusion of sodium nitroprusside (28mM) and heating to 43°C.

RESULTS

Sensory nerve‐mediated vasodilation increased during E2 administration compared to GnRHant (E2: 71 ± 15, GnRHant: 57 ± 18 %CVCmax; p=0.046). Blockade of nitric oxide with L‐NAME did not blunt the axon reflex during GnRHant (control: 57 ± 18 vs. L‐NAME: 50 ± 17 %CVCmax; p=0.401) or E2 administration (control: 71 ± 15 vs. L‐NAME: 52 ± 19 %CVCmax; p=0.152).

CONCLUSION

These preliminary findings suggest that E2 enhances sensory nerve‐mediated vasodilation in young women, and that this may occur through mechanisms independent of nitric oxide. Future mechanistic studies isolating the nitric oxide component and other pathways could help elucidate the role of E2 in sensory nerve‐mediated vasodilation.
Serum from rats subjected to traumatic brain injury and hemorrhagic shock induces profound endothelial barrier dysfunction
Authors: Ke Yunbo , Proctor Julie , Zhang Chenou , Medina Juliana , Miller Catriona , Junhyun Kim , Grisom Thomas , Birukova Anna , Fiskum Gary , Birukov Konstantin
Published: May 2022

Introduction

Traumatic brain injury (TBI) has been associated with development of indirect acute respiratory distress syndrome (ARDS). However, the causative relationship between TBI and lung injury remains unclear. To explore potential mechanism linking TBI with development of ARDS, we characterized effects of serum factors released during TBI and hemorrhagic shock (HS) in a rat model on the pulmonary endothelial cell (EC) barrier dysfunction, a key feature of ARDS.

Methods

The adult male rat polytrauma model consisted of controlled cortical impact (CCI)–induced TBI followed by 30 minutes of hemorrhage shock (HS) induced by blood withdrawal. The HS phase was followed by a 1‐hour “prehospital” Hextend fluid resuscitation phase and then a 1‐hour “hospital phase” when shed blood was reinfused. 48 hours after the TBI, blood was drawn from right ventricle and serum samples were obtained. Serum was collected from the TBI or sham rats, and its effects on barrier properties of pulmonary endothelial cell culture were assessed by measurements of transendothelial electrical resistance (TER) using the electrical cell impedance sensor and visualization of fluorescent tracer penetration through EC monolayer using XPerT assay.

Results

We found pronounced barrier‐disruptive effects of serum samples from animals exposed to controlled cortical impact (CCI) and HS, but not from sham operated rats. Rapid and sustained decrease in EC monolayer transendothelial resistance was observed within minutes after serum addition and remained below basal TER levels after incubation with CCI‐HS sera. Thrombin has been implicated in the early‐phase barrier disruptive activity of CCI‐HS serum, as both thrombin inhibitor and thrombin receptor antagonist attenuated the acute phase of serum‐induced TER decline, but did not affect TER decline at later time points. These findings suggest that thrombin elevation may play a role in early‐phase barrier disruptive activity of CCI‐HS serum. However, both the early and late phases of CCI‐HS‐induced EC permeability were inhibited by heparin. The late phase barrier disruptive effect of CCI‐HS was also prevented by serum preincubation with heparin‐sepharose. Pulmonary EC treated with serum from CCI‐HS rats for 3 hours have demonstrated significant decline in expression of EC junctional protein, VE‐Cadherin, and disassembly of peripheral EC adherens junction complexes monitored by immunostaining with VE‐cadherin antibody.

Conclusion

These results suggest that exposure to CCI‐HS leads to production of humoral factors causing early and late‐phase barrier disruptive effects in lung vascular endothelium. While thrombin‐PAR1 signaling has been identified as mechanism of acute EC permeability increase by CCI‐HS serum, the factor(s) defining long‐term EC barrier disruption in CCI‐HS model remain to be determined.
Proton‐Sensing Receptor GPR65 Dynamically Traffics To and Signals From Multiple Cellular Compartments
Authors: Morales Rodriguez Loyda M. , Puthenveedu Manojkumar
Published: May 2022

Proton‐sensing G protein‐coupled receptors (GPCRs) allow cells to sense and respond to changes in physiological pH. Over the last 20 years, proton‐sensing membrane proteins have been implicated in a variety of biological functions and disease states, including inflammation, and stimulation and inhibition of oncogenic signaling pathways. Proton‐sensing GPCRs are unique in that, upon internalization and trafficking to acidic intracellular compartments, they are exposed to environments that are more likely to activate the receptor. However, little is known about whether and how proton‐sensing receptors signal from intracellular compartments. Characterizing proton‐sensing receptor trafficking and signaling is critical to understand how these receptors integrate responses from multiple locations in the cell, and how these responses change during the progression of diseases associated with acidic microenvironments. Here we use proton‐sensing receptor GPR65 as a prototype to study how receptor location influences proton‐sensing receptor signaling. We use live imaging of GPR65‐expressing cells to show that GPR65 internalizes from the plasma membrane at both neutral and acidic pH. Further, GPR65 localizes to early and late endosomes irrespective of extracellular pH. However, GPR65 increases second messenger cAMP by coupling to Gs proteins upon exposure to acidic extracellular pH. To determine whether GPR65 at internal compartments contributes to cAMP production, we pre‐treated GPR65‐expressing cells with Dyngo4a, an endocytosis inhibitor. GPR65 internalization was required for a full cAMP response after acidic extracellular pH. Together, our findings show that GPR65 dynamically traffics and signals from multiple cellular compartments irrespective of extracellular pH. Importantly, our results suggest that, unlike for most known GPCRs, activation of GPR65 is uncoupled from receptor trafficking.
Calcium‐Dependent Dual Oxidase 2 is a Novel Source of Reactive Oxygen Species Implicated in Glomerular Mesangial Cell Fibrotic Response to Angiotensin II and Glucose
Authors: Hecker Jacqueline , Ford Bridget M.
Published: May 2022

The vasoactive peptide angiotensin II (Ang II) and hyperglycemia contribute to the initiation and the progression of glomerular fibrosis via activation of glomerular mesangial cells (MCs) and subsequent extracellular matrix expansion. We have previously shown that oxidative stress is critical for MC fibrotic response to Ang II or high concentrations of glucose (HG). Here, we demonstrate that Dual oxidase 2 (Duox2), a member of the Nox/Duox family of NADPH oxidases, is present in MCs and that its protein expression, along with its associated maturation factor, DuoxA2, are upregulated by Ang II and HG.

Small interfering RNA (siRNA)‐mediated downregulation of Duox2 significantly reduces Ang II‐induced increase in reactive oxygen (ROS) generation and prevents the stimulatory effect of Ang II on MC fibrotic injury (as assessed by measuring a‐smooth muscle actin and fibronectin expression).

To demonstrate that Duox2 activation is Ca2+‐dependent, we show that the extracellular Ca2+ chelator BAPTA prevented Ang II‐induced ROS generation and the stimulation of MC fibrotic injury by Ang II. Moreover, treatment of MCs with ionomycin resulted in increased ROS production and enhanced MC fibrotic injury. These effects were abrogated by siRNA targeting Duox2. Fura‐2 fluorescence was utilized as well to show calcium mobilization in response to Ang II and this effect was abrogated with siDuox2 transfection. These data indicate that Ang II‐stimulated Duox2 activation and ROS generation subsequently lead to MC fibrotic injury.

In summary, we have identified a novel role for Duox2 and its cognate maturation factor, DuoxA2, as a major source of ROS in response to Ang II and HG and established the significance of Duox2 in Ang II‐mediated MC activation and fibrotic injury. Therapeutic targeting of this pathway may prevent or reverse pathophysiologic manifestations of renal fibrotic diseases.
Role of Neurotensin Receptor‐1 Expression in Dopamine Neurons for Feeding, Locomotor and Anxiety Behaviors
Authors: Arriaga Sydney K. , Ramirez‐Virella Jariel , Leinninger Gina
Published: May 2022

The dopamine (DA) system is essential for motivated feeding and locomotion, but disruption of this system is implicated in the development of obesity and low body weight, namely anorexia nervosa. All DA neurons transiently express the neurotensin receptor‐1 (NtsR1) during development, suggesting that this receptor may contribute to the establishment and/or function of the DA system. Thus, we hypothesized that lacking NtsR1 expression in DA neurons impairs the function of the DA system and DA‐dependent feeding, locomotor activity, and anxiety behaviors. To test this, we crossed DATCre/+ and NtsR1flox/floxmice to generate mice with intact NtsR1 (Controls ‐ DAT+/+; NtsR1flox/flox) or mice in which NtsR1 was selectively deleted from DA neurons (DATCre/+; NtsR1flox/floxmice). We then assessed mice of each genotype and sex for feeding and physical activity, DA‐dependent behaviors and body weight. Feeding and body weight were similar in Control mice and those lacking NtsR1 from DA neurons. However, mice lacking NtsR1 in DA neurons exhibited more locomotor activity than Control mice. This altered locomotor activity was not due to altered stress or anxiety like behaviors, nor differences in dopamine‐ dependent behaviors, as these were similar between Control and NtsR1‐deficient mice. Thus, in contrast to our hypothesis, developmental deletion of NtsR1 deletion from dopamine neurons does not disrupt regulation of body weight or behavior, and is likely not a pathogenetic contributor to disordered body weight.
Ex vivo and In vivo Assays for Drug Discovery and Detection of Photoreceptor Survival Factors
Authors: Bernardo‐Colon Alexandra , Rebustini Ivan T. , Becerra S. Patricia
Published: May 2022

Retinitis pigmentosa (RP) is one of the most common inherited diseases of the retina (retinopathies) that is estimated to affect 1 in 4,000 people in the United States and worldwide. This collection of genetic diseases leads to progressive photoreceptor cell degeneration and ultimately vision loss, with more than 60 genes implicated in molecular pathways towards photoreceptor cell death. Currently, there are no treatments available and, due to its vast diverse genetic background, a putative gene‐based therapy is challenging. Consequently, mutation‐independent strategies to protect photoreceptors against continued damage and progressive vision loss are appealing approaches. One of them is the use of survival, neurotrophic and neuroprotective factors that target common signaling pathways of cell death in the different types of RP. The aim of this study is to optimize methods to evaluate the efficacy of compounds to protect photoreceptor cells against death.

We used PSVue® to optimize the cell death read‐out in cultured retinal explants and in retinas of a mouse model for a human inherited autosomal recessive RP, the rd10 mouse. Retinal degeneration in the rd10 mice is caused by a spontaneous mutation in the gene for the β‐subunit of phosphodiesterase. PSVue® is a robust probe for detecting phosphatidyl serine on the surface of cells undergoing death. We used retinal tissue explanted from wild type mouse eyes, and cultured it ex vivo. Induction of photoreceptor cell death was achieved with Zaprinast, an inhibitor of phosphodiesterase, to mimic the rd10 photoreceptor cell death. Low probe concentrations and short incubation times resulted in a reproducible detection of photoreceptor death in the retina by confocal super resolution microscopy. The PSVue® detection method yielded lower fluorescence background than the TUNEL detection method. Pigment epithelium‐derived factor (PEDF), a photoreceptor survival protein, was used as control to prevent photoreceptor cell death induced by Zaprinast. PEDF supplementation prior to Zaprinast treatment decreased cell death detection, as expected. We used the mouse model rd10 to evaluate photoreceptor death in vivo. PSVue® eye‐drops were administered to the left eye of rd10 mice of P17‐P25 postnatal ages, and fluorescence was detected by fundoscopy using MICRON III a day later. HBBS eye‐drops were administered to the right eye as a control reference. The fluorescence of the probe detected in the back of mouse eyes increased between ages P17 and P23 relative to that of HBBS‐treated eyes. The results revealed a peak of photoreceptor cell degeneration that occurred at P25 days of age in living rd10 mouse models.

We have optimized methods to determine photoreceptor cell death in the ex vivo and in vivo experimental systems described here using a fluorescent probe. The methodologies are simple, fast, and complementary, and together they will prove to be suitable for screening factors and drugs against RP.
Cardiac Adjustments During a Hot and Humid Heatwave Simulation in Older Adults
Authors: Foster Josh , Belval Luke N. , Watso Joseph C. , Cimino Frank A. , Orth Bonnie D. , Sarma Satyam , MacNamara James P. , Crandall Craig G.
Published: May 2022

Heatwaves cause disproportionately high morbidity and mortality rates in those aged ≥ 65 years. While the underlying pathologies are typically of cardiovascular origin, the cardiac specific responses to real‐world heatwave conditions are not well‐defined.

Purpose

We tested the hypothesis that three hours exposure to a hot and humid heat wave would increase left ventricular systolic contractility (s’) and early propagation velocity of mitral inflow (Vp) and decrease isovolumetric relaxation time (IVRT).

Methods

Eight healthy older adults (5 female/3 male; 70 ± 4 years; 26 ± 3 kg·m‐2) were exposed to 41°C, 40% humidity (replicating the 1995 Chicago heat wave) for three‐hours, interspersed with seven 5‐minute work bouts at three METS to replicate daily living activities. Core temperature (telemetric pill or rectal thermistor), skin temperature, blood pressure and heart rate were monitored continuously during heating. Water was provided throughout the protocol to minimize dehydration. Echocardiograms were taken at baseline (in a thermoneutral environment) and at three hours into heat stress to assess cardiac function. Pre to post heatwave differences in all variables were compared using paired samples t‐tests.

Results

Core temperature increased (36.7 ± 0.3 to 37.7 ± 0.5°C, p < 0.001), skin temperature increased (34.0 ± 1.1 to 36.2 ± 0.8°C, p = 0.002), heart rate increased (59 ± 9 to 78 ± 18 b·min‐1,p = 0.012), and mean arterial pressure decreased (90 ± 14 to 78 ± 12 mmHg, p= 0.034) from baseline to 3‐hours heat exposure. Left ventricular contractility (s’) increased (8.2 ± 1.0 to 10.4 ± 1.4 cm/s, p = 0.002), while measures of diastolic function were unchanged during heating (p > 0.14 for Vp, IVRT, E, A, E/A, e’, E/e’).

Conclusions

Our preliminary findings suggest that humid heatwave exposure increases left ventricular contractility without impacting diastolic function in healthy older adults.
Colistin Action Against Planktonic Pseudomonas aeruginosa
Authors: Paul Amisha
Published: May 2022

Pseudomonas aeruginosa is an opportunistic human pathogen that causes nosocomial, acute, and chronic infections in patients with conditions such as cystic fibrosis, severe burns, and cancer. P. aeruginosa is associated with increased patient morbidity, mortality, and healthcare costs and is known for its persistence of infections. P. aeruginosa is resistant to many antibiotics; however, most strains are susceptible to colistin. The World Health Organization has identified antimicrobial resistance as a major threat and Antimicrobial Stewardship Programs (ASP) have been initiated. ASP goals are to improve patient outcomes, optimize patient safety, reduce antimicrobial resistance, and control healthcare costs. Recommendations to reduce antimicrobial resistance include optimizing the use of antimicrobials and preventing the transmission of drug‐resistant organisms. Using antimicrobic agents wisely leads to optimization of their use. For these reasons, we sought to adopt a model system for testing minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) levels for standardized strains of P. aeruginosa (ATCC strains 10145, 27853, 1744, CRM‐9027, 9721) using standardized methods. This study sought to compare results of standardized analysis that used Mueller‐Hinton Broth (MHB), the recommended and standardized growth medium, or tryptic soy broth (TSB), the growth medium used in many labs with published MIC and MBC reports. Given the ASP recommendations of optimizing use of antimicrobial agents, it is important to know the impact of variations in and deviations from standardized MIC and MBC methodologies in reporting of research results relevant to antimicrobic agents. This comparison study tested the assumption that MIC and MBC results are the same regardless of whether MHB or TSB is used as the growth medium. Preliminary results suggest that MIC and MBC values obtained by using TSB or MHB may display some degree of variability.
Systems‐level Multi‐organ Modeling of Transcriptomic Data to Reveal Sex‐specific Shifts in Regulatory Control During the Development of Hypertension
Authors: Achanta Sirisha , Hornung Eden , Moss Alison , Schwaber James , Vadigepalli Rajanikanth
Published: May 2022

Hypertension is a multifactorial disease involving the dysregulation of several organs. The organ‐organ interactions and time course dynamics are yet to be well delineated. We analyzed pathway‐scale gene expression across five organs during the development of hypertension in female Spontaneously Hypertensive rats (SHR), at five time points from early onset (8,10,12 weeks of age) to established hypertension (16 and 24 weeks), and age‐matched normotensive Wistar Kyoto (WKY) rats. We studied the interactions within and across organs by integrating the gene expression analysis with a Hartley Modulating Function based system identification approach to reverse engineer data‐driven dynamic network models of multi‐organ gene regulatory influences. Our results indicate a dynamic dysregulation of gene expression spanning multiple organs over time in female SHR. Adrenal gland showed the most robust gene expression changes across multiple pathways, including the renin angiotensin system and catecholaminergic processes. These changes in the adrenal gland preceded the differential regulation of inflammation‐relevant processes in the kidney. The inferred multi‐organ network model suggests a diminished influence of the differential gene regulation in the central nervous system on the gene expression changes in spleen and lung. This was paralleled by an increased influence of adrenal gene regulation over the gene expression changes in multiple other organs. We compared the results to the available male SHR multi‐organ gene expression analysis and modeling. We found that the gene dysregulation patterns, temporal sequencing and putative organ‐organ influences are distinct to female SHR, highlighting the sex‐specific multi‐organ regulatory processes driving hypertension.
Identification of microRNA Targets in colorectal cancer through Data Sequencing
Authors: Rubio‐Guevara Susana , Olascuaga‐Castillo Karyn , Cáceres‐Andonaire Elena , Altamirano‐Sarmiento Dan , Caballero‐Aquiño Olga , Mantilla‐Rodríguez Elena , Hilario‐Vargas Julio , Berezovski Maxim , Morgado‐Díaz José Andrés
Published: May 2022

Colorectal cancer (CRC) is the third leading cause of cancer‐related mortality globally. MicroRNAs (miRNAs, miRs), a class of small non‐coding RNA molecules, have demonstrated important roles in carcinogenesis and its progression through the regulation of the epithelialmesenchymal transition (EMT), oncogenic signaling pathways, and metastasis. Despite the increase in miRNA studies and extensive analyzes of their expression, the role and function of many individual miRNAs in CRC remains poorly understood. The aim of this study was to identify miRNA targets in CRC through data sequencing, which could be a solid prognostic prediction tool and help clinical strategy.

Methods

CRC gene expression data sets were collected from the public database, The Cancer Genome Atlas (TCGA). In addition, web‐based tools were used to explore TCGA data, specifically the one developed by the Broad Institute TCGA GDAC Firehose, which provides data sets, algorithms, and analysis results standardized for TCGA. This pipeline has the following steps: The CLR (Context Likelihood of Relatedness) approach is applied to infer putative miRNA regulatory connections: gene; miRNA filtering: gene pairs based on Pearson's correlation (<= ‐0.3); and miRNA filtering: gene pairs based on predicted interactions in three sequence prediction databases (Miranda, Pictar, Targetscan). The CLR algorithm was applied on 617 miRs and 18012 mRNAs across 220 samples. After 2 filtering steps, the number of 9 miR:genes pairs were detected.

Results

The initial search with the term “Colorectal adenocarcinoma”, “COADREAD” yielded 631 cases. After the analysis of these samples, data on the significance miR:gene pairs were obtained and Table 1 shows the results of miR:gene pairs with corr < ‐0.30 and predicted interactions in three sequence prediction databases. About the miRNA connections, Table 2 shows all miRNA hubs with their associated genes in the putative direct target network. Finally, about gene connections, Table 3 shows all gene hubs with their associated miRNAs in the putative direct target network.

Conclusion

The use of miRNAs as biomarkers for CRC could provide a new and less invasive technique to detect CRC and help determine prognosis. These miRNAs and their targets require further evaluation for a better understanding of their associations, ultimately, with the potential to develop new therapeutic targets. Therefore, it is proposed to develop a screening panel that should consist of multiple miRNAs that would provide a more accurate and efficient screening tool for CRC.
Proximal Tubule‐Specific Transgenic Overexpression of Peroxisome Proliferator‐Activated Receptor Alpha Prevents the Development of Hypertension in Dahl Salt‐Sensitive Rats
Authors: Yunker Lauren , Zemaj Jeylan , Geurts Aron , Kriegel Alison
Published: May 2022

The Dahl salt‐sensitive rat (Dahl SS) is a well investigated model of hypertension that mimics human salt sensitive hypertension. These rats develop a significant increase in blood pressure and renal disease when challenged with a high salt diet compared to salt‐resistant strains. Prolonged high salt feeding results in extensive renal pathology, including glomerular and proximal tubular pathology, tubular casting, and interstitial fibrosis. It has been shown that clofibrate, a lipid lowering peroxisome proliferator‐activated receptor alpha (PPARα) agonist, can prevent the hypertensive phenotype in Dahl SS rats. PPARα is a ligand activated transcription factor that regulates β‐oxidation of fatty acids (FAO). The proximal tubule is a very metabolically active tubular segment that relies primarily on FAO to meet its high energy requirements. Reduced FAO can lead to proximal tubular damage and renal dysfunction. To investigate the impact of PPARα‐mediated FAO during a high‐salt challenge, we developed a novel transgenic Dahl SS strain with a proximal tubule‐specific promoter from the sodium/glucose cotransporter 2 gene (Sglt2). This resulted in the overexpression of Ppara only in the proximal tubule (SSTgPTPpara). At 9 weeks of age, male wild‐type rats and heterozygous transgenic SSTgPTPpara rats were placed on a 4.0% NaCl, high salt diet (n=6/group). Following a 14‐day high salt challenge, rats were anesthetized for carotid blood pressure recording. Proximal tubule‐specific transgenic overexpression of PPARα in heterozygous SSTgPTPpararats had lower systolic blood pressure when compared to wild‐type littermates (161.2.6 ± 12.0 mmHg vs.122.8 ± 4.2 mmHg; mean ± SEM; p <0.05, t‐test), as well as mean arterial pressure and diastolic blood pressure. Immunohistochemical analysis confirmed an increase in PPARA protein within proximal tubules of the renal cortex, relative to other nephron segments. In conclusion, proximal tubule specific‐transgenic overexpression of PPARα reduced salt‐sensitive hypertension, suggesting that proximal tubules may be one of the primary sites of anti‐hypertensive actions of clofibrate in Dahl SS rats. This also indicates that the SSTgPTPpara rat model could be useful for studying the contribution of dysregulated proximal tubule metabolism to salt‐sensitive hypertension and renal pathology in the Dahl SS rat.
Screening of Non‐Purine Ligands Binding to Adenosine Receptors A1, A2A, A2B, and A3
Authors: Fiala Mary M. , Pattabiraman Mahesh , Hamilton Luke , Chandra Surabhi
Published: May 2022

Adenosine receptors (ARs) are G‐protein coupled receptors (GPCRs) which have been shown to have therapeutic potential. ARs are made up of four subtypes, A1, A2A, A2B, and A3. A3AR ligands have previously been indicated to lower inflammation, prevent or treat cancer, and produce antihyperalgesic effects in many preclinical pain models, specifically for neuropathy. We have previously published in animal models that ferulic acid dimer, a non‐purine compound with the 3‐methoxy phenyl moiety, has non‐opioid antinociceptive properties through primarily binding to A3ARs. Current literature suggests that the antinociceptive properties of a natural compound, incarvillateine, which contains the 3‐methoxy phenyl moiety is mediated through ARs, however there is a lack of sufficient evidence of in vitro binding of such compounds at ARs. We thus hypothesized that compounds with the 3‐methoxy phenyl moiety show binding to ARs, with an affinity in the submicromolar range. For this study, we performed fluorescent competitive binding assays using cells transfected with ARs: Chinese Hamster Ovary (CHO) cells for A1 or A3 receptors and Human Embryonic Kidney (HEK) cells for A2A or A2B receptors. The compounds tested included trans cinnamic acid analogs and curcumin analogs. The compounds were synthesized using cavitand‐mediated photoisomerization and cavitand‐mediated photodimerization. Our fluorescent binding protocol employed the separation method, in which the unbound ligand was removed after equilibrium was reached, via PBS wash. The binding assays performed suggested that 3‐methoxy cinnamic acid dimer was the most effective in binding to A3ARs, while the monomer did not bind. There was very little to no binding observed at the other receptors. Other analogs of trans cinnamic acid and curcumin did not show binding at any of the ARs so far. In conclusion, 3‐methoxy cinnamic acid dimer binds to G‐inhibitory coupled adenosine receptor, A3. Future studies will investigate structure‐activity‐relationship of 3‐methoxy phenyl dimers to demonstrate why dimerization leads to superior binding compared to similar monomeric compounds.