bluuebunny/crossref_metadata_2025 huggingface dataset:
Nonselective Cation Channels Maintain Alveolar Fluid Clearance After Exposure to Toxins from Respiratory Pathogens
Authors: Galarza Paez Lilian D. , Cespedes Victoria E. , Moseley Auriel , Yue Qiang , Lucas Rudolf , Eaton Douglas
Published: May 2022

Normal gas exchange in the alveoli is facilitated by a thin fluid layer. This layer is carefully regulated by two ion channels: highly selective channels (HSC or ENaC) and nonselective cation channels (NSC). Previous research found that both channels contribute about equally to alveolar fluid clearance (AFC). Since the molecular composition of NSC is a recent discovery, not much is known about NSC’s response to various respiratory pathogens. NSC are composed of an alpha‐ENaC subunit and at least one acid‐sensing ion channel 1a (ASIC1a) subunit. Our study aims are to understand how NSC contribute to alveolar epithelial permeability and how the lungs respond to toxins from respiratory pathogens. We hypothesize that AFC will be lower in the absence of ASIC1a (ASIC1 KO) when exposed to toxins from respiratory pathogens: Lipopolysaccharide (LPS) from E. coli and Pneumolysin (PLY) from P. pneumoniae. Additionally, we hypothesize that ASIC1 KO animals will have increased baseline alveolar epithelium permeability. All animal experiments were approved by Emory’s IACUC.

We introduced pathogen toxins via intratracheal instillation to ASIC1 KO (KO) and wildtype (WT) mouse models (both on a C57Bl6 background). After a four‐hour incubation period, we euthanized the mice by isoflurane overdose. We determined AFC by instilling their lungs with 0.7ml of a 2.0% Evans Blue (EB) or Fluorescein isothiocyanate–dextran (FITC) solution via the trachea. We took samples at t=0 and t=30 minutes after instillation. We determined the EB/ FITC concentration of both samples using spectrophotometry and calculated AFC from the change in dye concentration.

Our preliminary findings suggest that LPS and PLY inhibit all AFC associated with HSC leaving only AFC associated with NSC. The AFC of LPS exposed KO animals is low compared to AFC for LPS exposed WT animals (6.04 ± 3.02, n= 15 vs 15.8 ± 5.61, n= 11, p< 0.001). Similarly, AFC of PLY exposed KO mice is lower than AFC for PLY exposed WT mice (2.78 ± 0.96, n= 10 vs 12.4 ± 4.36, n= 4, p< 0.001). In the toxin exposed KO groups, neither HSC nor NSC are fully functioning or contributing to AFC. We are still collecting data on the permeability of the alveolar epithelium.

There is evidence to suggest that the regulation of HSC and NSC are drastically different. Given the critical nature of the alveoli fluid layer, it would be logical that NSC channels serve as a compensatory mechanism when HSC are inhibited by pathogens. These findings can help guide treatment for alveolar flooding caused by toxins from different respiratory pathogens.
Unraveling the labyrinth of our balance system: Visualizing bony otolith organ structure through novel 3D modeling
Authors: Smith Christopher M. , Curthoys Ian , Mukherjee Payal , Wong Christopher , Laitman Jeffrey T.
Published: May 2022

The organs of balance, the semicircular canals (SCCs) and otolith organs (utricle and saccule) of the inner ear labyrinth, are essential to basic human movement and function. While the SCCs (both external and internal structure) are well visualized and understood, the seemingly vague bony structure of the utricle and saccule remains largely unexplored. Consequently, relationships between this bony structure and the internal otolith organs themselves remain unknown. As a result, a comprehensive understanding of the morphology of our balance organs and relationships to function has eluded us.

As a first step to understand the relationships of the utricle and saccule (that is the membranous otolith organs responsible for detecting linear motion) to their external bony coverings, we have developed a morphometrically‐based modelling system to visualize bony otolith organ structure. The indeterminate shape of this capsule has precluded a standard metric approach to capture such relationships. Our method combines osmium tetroxide contrast enhanced micro‐computed tomography (μCT) imaging of human temporal bones (n=13), digital visualizations, and landmark‐based 3D shape analyses of bony and membranous structure. This was done in order to: 1) test the hypothesis that the bony form surrounding the otolith organs covaries with internal membranous anatomy (including the utricle and saccule); and 2) pinpoint specific arrangements of landmarks on the bone that best reflect the internal structure of the utricle and saccule. This approach allows us to extract the bony otolith organ structure from an otherwise nebulous bony form.

Tests of integration on 3D shape data between bony and membranous tissue demonstrate a significant association between the hard and soft tissues of the otolith organs (p<0.05). Relative orientation of the otolith organs also significantly correlates with surrounding bony structure (p<0.005). These data support our initial hypothesis and inform the creation of our 3D visualization of human otolith organ structure, establishing the configuration of the otolith organs to bony structure of the labyrinth. Furthermore, this model enhances our understanding of spatial relationships between the otolith organs and cranial structure and provides the foundation to assess the evolution of a prime sensory system essential for providing a gravitational frame of reference for all vertebrate life.
A Western Diet Influences Ventilation in a Sex‐Specific Manner and Prevents Expression of Respiratory Neuroplasticity in Adult Rats
Authors: Barok Rebecca , Dougherty Brendan
Published: May 2022

Estrogen impacts neuroplasticity in multiple areas of the central nervous system including within respiratory neural networks. Previous research from our group established that estrogen is necessary to permit expression of phrenic long‐term facilitation (pLTF), a form of respiratory neuroplasticity induced by acute, intermittent hypoxia (AIH). Many factors influence circulating estrogen levels such as age, gender, and diet. Specifically, the Western Diet is associated with increased levels of circulating estrogen in post‐menopausal women. Common to cultures of the Western Hemisphere, the Western Diet is characterized by high intakes of red and processed meats, sweets, fried foods, and refined grains with the addition of a high intakes of sugar, salt, omega 6 fatty acids, and a reduction of omega 3 fatty acids. Since the Western Diet is associated with high estrogen levels, and estrogen appears to play a critical role in expression of respiratory neuroplasticity, we assessed whether rats fed a Western Diet would have altered respiratory function and expression of AIH‐induced pLTF. Experimental rats of both sexes were fed a Western Diet in pellet form for 12 weeks and compared to rats consuming standard pellet chow. Weekly whole body plethysmography was used to measure ventilation and changes in ventilatory responses over time. At the conclusion of 12 weeks, AIH‐induced phrenic LTF was measured in anesthetized, vagotomized, and mechanically ventilated rats. Surprisingly, neither sex exhibited a change in body weight in response to the 12‐wk Western Diet, nor was baseline ventilation different between experimental groups within the same sex. However, with hypoxic challenge, male rats on the Western Diet showed lower breathing frequency and higher tidal volumes, while females on the Western Diet exhibited the opposite effects. Also, AIH‐induced pLTF was abolished in male and female Western Diet rats despite an increase in circulating estrogen levels in Western Diet Rats as measured by ELISA assay. These preliminary data suggest that the Western Diet may induce progressive changes in ventilation and loss of respiratory neuroplasticity independent of increased circulating estrogen levels.
Evaluation of a Cardiovascular Therapeutics Case‐Based Learning Activity
Authors: Guillory Ashley N. , Marciano Veronica
Published: May 2022

Physician Assistant students at the University of Texas Medical Branch often struggle with learning how patient characteristics affect cardiovascular therapeutic treatment choice. We implemented a case‐based learning (CBL) activity in the cardiovascular therapeutics module to address this problem.

The pre‐work for the CBL was the module lectures. Students completed an individual pre‐activity quiz then worked in small groups to discuss six cardiovascular cases. Each case included questions related to choosing the most appropriate treatments based on patient characteristics such as age or comorbidities, as well as the pharmacological characteristics of the drugs. Groups recorded their answers in a worksheet that was submitted for grading. The CBL concluded with an individual post‐activity quiz. Optional pre‐ and post‐session surveys evaluated their level of confidence in their knowledge as well as their satisfaction with the CBL.

Prior to the CBL, 74% of survey respondents indicated they lacked confidence in their knowledge of cardiovascular therapeutics. Following the activity, only 26% of respondents indicated they still lacked confidence in their knowledge of these topics. Students enjoyed working and discussing in their groups, having realistic complex patient cases, figuring out how patient characteristics alter medication choice, and thought the cases helped guide their studying for the subsequent exam. Ideas for improvement of the CBL included utilizing smaller learning groups, reducing the number of cases, adding additional time to the activity, and adding a large group discussion to review the case answers.

Overall, the response to the CBL was largely positive and the feedback will be useful for improving the session when it is next deployed. This type of activity can be used to reinforce material for other pharmacology topics students find difficult.
Spatial transcriptomics reveals differences among genetic models of disruption in the Wnt‐beta‐catenin signaling in hepatocytes
Authors: Monga Satdarshan P. , Cao Catherine , Hu Shikai , Singh Sucha , Poddar Minakshi , McGaughey Jackson
Published: May 2022

Wnt‐beta‐catenin signaling plays key roles in liver biology. One such role is in regulating the process of metabolic zonation within a liver lobule where Wnt‐beta‐catenin axis controls the expression of many genes expressed in zone‐3 hepatocytes. The cell‐molecule circuitry of the Wnt‐beta‐catenin signaling in this process has been identified and consists of basal expression of Wnt2 and Wnt9b in endothelial cells lining the central vein, which then are secreted by the Wntless (Wls) protein to act in a paracrine manner on Frizzled‐LRP5‐6 co‐receptors on hepatocytes in zone‐3 to activate beta‐catenin. This axis has been revealed through the generation of key genetic knockout (KO) mice including endothelial cell specific Wnt2‐Wnt9b double KO (EC‐Wnt2/9b‐KO), endothelial cell specific Wls KO (prevents secretion of all Wnts from endothelial cells lining central vein) (EC‐Wls‐KO), hepatocyte‐specific‐LRP5‐6 DKO (HC‐LRP5‐6‐DKO) and hepatocyte‐specific‐beta‐catenin KO (HC‐beta‐Cat‐KO). In all 4 genetic mouse models, we have identified notable decrease to complete loss of many zone‐3 genes. However, it remains unclear if all the 4 KO mice are exactly identical in the loss of all well recognized zone‐3 hepatocyte specific targets or are there specific differences since disruption of the pathway occurs at different levels. Likewise, since zonal expression of genes is thought to be dynamic, it is of high interest to determine if loss of zone‐3 target gene expression perturbs the expression of zone‐1 and zone‐2 genes and if that occurs uniformly and similarly in all 4 genetic KO models. Using single cell spatial transcriptomics with Molecular CartographyTM, we are querying the expression of one hundred zonated genes across the hepatic lobule involved in various functions. The current study will allow us to for the first time identify any differences in target genes of Wnt‐beta‐catenin, Wnt‐independent‐beta‐catenin‐dependent, and Wnt‐dependent‐beta‐catenin‐independent target genes. At the same time, we will be able to address if loss of zone‐3 genes in each of the 4 KO models also indirectly perturbs the expression of genes normally expressed in zone‐1 and zone‐2 of the liver. Such novel information will be critical to both liver biologists and pathologists who study alterations in metabolic zonation in many liver pathologies including fatty liver disease and cholestatic liver injury.
Human Cdc14 Phosphatase Stimulation by an Intramolecular Pseudosubstrate Motif
Authors: Waddey Benjamin , Milholland Kedric , DeMarco Andrew , Hall Mark
Published: May 2022

The Cdc14 protein phosphatase family regulates diverse cellular processes, including mitotic exit, cytokinesis, and DNA repair. Cdc14 is a member of the dual‐specificity subfamily of protein tyrosine phosphatases (PTPs), but has evolved to selectively recognize specific phosphoserine sites deposited by cyclin‐dependent kinases. In humans, it has been linked to hearing loss and male infertility conditions. In several fungal species, Cdc14 has been linked to pathogenesis. The Hall lab identified a novel pseudosubstrate motif in the disordered region of all fungal Cdc14 orthologs. Studies with S. cerevisiae Cdc14 revealed this motif interacts with the Cdc14 active site to stimulate the rate‐limiting catalytic step and is functionally important for fungal cell wall integrity. The motif may allow cells to dynamically control Cdc14 activity. Metazoan Cdc14 orthologs do not share this sequence motif. However, we demonstrate here that the human Cdc14A (hCdc14A) C‐terminal region also contains a similar catalytic enhancer. AlphaFold structural predictions suggest that a conserved alpha helix in the otherwise disordered C‐terminus makes extensive contacts with the active site region. Using site‐directed mutagenesis, recombinant protein expression and purification, and steady‐state kinetic assays, we show that the hCdc14A pseudosubstrate motif accelerates phosphoenzyme hydrolysis by nearly two orders of magnitude, similar to the fungal motif. Using a mass spectrometry‐based kinetics assay, we also demonstrate that hCdc14A substrate specificity is nearly identical to fungal Cdc14s, and that the pseudosubstrate motif in hCdc14A does not influence specificity or substrate binding. Current work is 1) testing the hypothesis that the hCdc14A pseudosubstrate motif also binds directly to the active site to accelerate the rate‐limiting catalytic step and 2) identifying the specific amino acids required for active site binding. Future work will focus on characterizing the biological significance of this motif and testing its conservation in human Cdc14B. Our results suggest that fungi and metazoa independently evolved pseudosubstrate motifs to similarly regulate Cdc14 catalytic activity. The direct contribution of sequences outside the core phosphatase domain to catalysis appears to be unique among PTP enzymes.
Hepatocyte β‐catenin is regulated by endothelial cell‐derived Wnt2 and Wnt9b to govern liver metabolic zonation and regeneration
Authors: Hu Shikai , Poddar Minakshi , Singh Sucha , Bell Aaron , Monga Satdarshan
Published: May 2022

Background

Wnt/β‐catenin signaling pathway regulates hepatic metabolic zonation (MZ) and liver regeneration (LR) after surgical or toxicant injury. Central vein endothelial cells (CVECs) are the main source of Wnts regulating MZ, while liver sinusoidal ECs (LSECs) are the major source of Wnts regulating LR after 70% partial hepatectomy (PH). Of the 19 Wnt genes, Wnt2 and Wnt9b were reported to be expressed in CVECs and the same 2 Wnts were also upregulated in LSECs isolated from regenerating livers at 12h post PH. However, conclusive evidence of the specific roles of Wnt2 and Wnt9b in regulating MZ and LR, is lacking.

Methods

Here, we report generation of Wnt2‐floxed mice and generation of Wnt2‐Wnt9b‐double‐floxed mice, which were then bred to lymphatic vessel endothelial hyaluronan receptor 1 (Lyve1)‐Cre mice to delete Wnt2, Wnt9b or both from both CVECs and LSECs referred to as EC knockouts (KO). We characterize EC‐Wnt2‐KO, EC‐Wnt9b‐KO, and EC‐Wnt2‐9b‐double KO (DKO), and assess for baseline phenotype, MZ, and LR after PH.

Results

Using reporter mice, we first reaffirmed that Lyve1‐Cre induces recombination of floxed alleles in both CVECs and LSECs. Liver weight to body weight ratio (LW/BW) was lower in EC‐Wnt2‐9b‐DKO mice. Subsets of these mice showed an abnormal increase in one liver lobe size and mass at the expense of other lobes, along with mild periportal fibrosis. Male but not female EC‐Wnt2‐KO and EC‐Wnt9b‐KO mice had partial loss of pericentral expression of glutamine synthetase (GS), cytochrome P450 2e1 (Cyp2e1), and cytochrome P450 1a2 (Cyp1a2). Both male and female EC‐Wnt2‐9b‐DKOs lacked GS, Cyp2e1 and Cyp1a2, phenocopying EC‐Wntless‐KO mice in which all Wnts secretion from ECs were inhibited. After PH, EC‐Wnt2‐KO and EC‐Wnt9b‐KO mice showed decreased expression of Cyclin D1, Ki67 and incorporation of BrdU at 40h, which was even lower in EC‐Wnt2‐9b‐DKOs. The subset of EC‐Wnt2‐9b‐DKO mice with abnormal lobar morphology developed multiple necrotic foci infiltrated with immune cells, had a detrimental delay of LR and exhibited increased mortality after PH.

Conclusion

Wnt2 and Wnt9b from CVECs play additive roles in controlling β‐catenin pericentral target gene expression. Additionally, Wnt2 and Wnt9b from LSECs play additive roles in regulating β‐catenin activation during LR after PH. Further studies are needed to address how and why ECs are the chief source of these two Wnts to regulate MZ and LR.
The Influence of Sleep Duration on Cerebrovascular Reactivity
Authors: DeConne Theodore M. , Sanjana Faria , Martens Christopher R.
Published: May 2022

The impaired ability of the cerebrovasculature to dilate in response to a vasoactive stimulus (i.e., cerebrovascular reactivity, CVR) is associated with an increased risk of cognitive impairment. The association between sleep duration and dementia risk is “U‐shaped”, where adults who achieve seven hours of sleep have the lowest risk of cognitive impairment, compared to adults who achieve greater than or less than seven hours of sleep. In otherwise healthy adults, poor sleep quality and duration impairs peripheral vascular function, but its effect on CVR in generally healthy adults has remained largely been unexplored. The purpose of this study was to assess the relation between subjective sleep quality and CVR in healthy adults. Because the relation between sleep duration and cognitive impairment is an inverted U‐shape, we hypothesized that CVR would be lower in adults who slept longer than or shorter than the recommended 7‐8 hours of sleep. Data from thirty‐five individuals across the adult lifespan (21 males / 14 females, age: 44 ± 17 years; BMI: 26.8 ± 4.8 kg/m2; blood pressure, BP: 114 ± 12 / 71 ± 10 mmHg; heart rate, HR: 62 ± 8 bpm) were included in this analysis. CVR was assessed by measuring the change in middle cerebral artery blood flow velocity (MCAv) at baseline and following a 3‐minute hypercapnic stimulus (+9 mmHg increase in the end‐tidal partial pressure of CO2) using transcranial Doppler ultrasound. MCAv was normalized to beat‐to‐beat BP (MCACVC), and CVR was calculated as the percentage change in MCACVC and normalized to the absolute change in end‐tidal CO2.Sleep duration was negatively predictive of lower CVR (R2=0.17, β1± SE = ‐0.45 ± 0.18 %/mmHg/hour, P=0.014). A correlation matrix was performed to determine if any variables other than sleep duration (e.g., age, sex, etc) might also impact CVR. Age (P=0.53), BMI (P=0.93), systolic BP (P=0.22), diastolic BP (P=0.84), HR (P=0.071), and mean arterial pressure (P=0.57) were not associated with CVR. Only sex was significantly associated with CVR (R= ‐0.36, P=0.036), where men had augmented CVR compared to women (2.20 ± 0.73 %/mmHg vs. 1.59 ± 0.93 %/mmHg, P=0.036). Importantly, sex (β1± SE= ‐0.18 ± 0.27 %/mmHg, P=0.51) did not influence the relation between CVR (β1± SE= ‐0.41 ± 0.16 %/mmHg/hour, P=0.013) and sleep duration (model adjusted for sex: R2= 0.17, P=0.042). In conclusion, longer sleep duration was independently associated with lower CVR in healthy adults. Future studies should include more individuals with shorter sleep duration to test the possibility of an inverted U‐shaped association with CVR.
Regulation of BK channel expression by Paraoxonase 3
Authors: Shi Shujie , Montalbetti Nicolas , Kleyman Thomas
Published: May 2022

Paraoxonase 3 (PON3) is a molecular chaperone that is primarily expressed in the aldosterone‐sensitive distal nephron (ASDN) where the fine tuning of Na+ and K+ homeostasis occurs. The flow‐induced K+ secretion in the distal nephron is mediated by large conductance Ca2+‐activated K+ (BK) channels in intercalated cells (ICs). As PON3 co‐localizes with BK in the ICs and Pon3 KO mice have altered blood [K+] at baseline, we hypothesize that PON3 has a role in regulating BK expression and functional activity.

To determine whether the functional expression of BK channels is regulated by PON3, the pore‐forming α subunits of mouse BK (αBK) was expressed in HEK293 cells with or without mouse PON3. We performed co‐immunoprecipitation (co‐IP) to examine whether PON3 interacts with αBK. We also examined the effects of PON3 on αBK expression and BK channel activity using biochemical and electrophysiological approaches.

When expressed in HEK293 cells, PON3 was pulled down by the αBK‐IP while αBK was detected in the PON3‐pulldown, only in cells expressing both proteins. Our data suggest that there are direct or indirect interactions between PON3 and αBK, the pore‐forming subunit of BK channels. We performed whole cell patch clamp to measure BK channel activity in HEK293 cells. we found that IBTX‐sensitive whole cell K+ currents were reduced approximately 50% by PON3. Consistently, both whole cell and surface abundance of αBK was significantly lower in cells co‐expressing PON3. Together, our data suggest that PON3 negatively regulates the expression and activity of BK channels.

In conclusion, we identified the BK channel as a novel target of the chaperone PON3. Future studies will determine mechanisms by which PON3 regulates BK channel functional expression as well as physiological consequences of this regulatory interaction in mouse kidney.
Interactions of the intrinsically disordered selenoprotein S with a small GTPase regulator
Authors: Gonzalez‐Arias Fabio A. , Rozovsky Sharon
Published: May 2022

Selenoproteins are a family of enzymes that employ the rare amino acid selenocysteine to catalyze chemical reactions. Among them, selenoprotein S stands out because the selenocysteine is positioned in an intrinsically disordered segment. The physiological function of this enzyme is unknown, although it was shown to take part in ER homeostasis by mediating protein degradation and may also have a role in vesicle trafficking, lipid metabolism, and management of oxidative stress. To elucidate its function in vesicle trafficking, we demonstrate that selenoprotein S binds the nucleotide exchange regulator of small GTPases, SmgGDS, a regulator of the Ras and Rho family members. Curiously, both selenoprotein S and SmgGDS are hijacked by non‐structural proteins of SARS‐CoV‐2, along with other proteins involved in maintaining ER homeostasis and regulation of the secretory pathway. To investigate this link, we have characterized the interactions between selenoprotein S and SmgGDS. We show that the interaction requires the hydrophobic segment of selenoprotein S, previously thought to be transmembrane. We describe biochemical assays to examine the putative role of selenoprotein S in modulating SmgGDS function and whether it accelerates the rate release of GTPases from SmgGDS.
Home‐Based Leg Heat Therapy Does Not Improve Vascular Function in Patients with Symptomatic Peripheral Artery Disease
Authors: Monroe Jacob C. , Parker Jason G. , Perkins Susan M. , Han Yan , Joon Pae Byung , Klein Janet , Motaganahalli Raghu L. , Roseguini Bruno T.
Published: May 2022

INTRODUCTION

Patients with peripheral artery disease (PAD) suffer from severe exercise intolerance and accelerated rates of functional decline, resulting, in part, from significant endothelial cell dysfunction and the consequent impairment in leg blood flow. Unfortunately, few non‐invasive therapeutic interventions have been shown to ameliorate the microvascular dysfunction in patients with PAD. Repeated exposure to heat therapy (HT) improves endothelial function in sedentary young individuals, but the effects of this therapy on vascular function in patients with PAD remain poorly defined. The objective of the present study was to test the hypothesis that 8 weeks of home‐based leg HT would improve leg vascular function in patients with symptomatic PAD.

METHODS

Thirty‐four patients with symptomatic PAD (66 ± 8 years, ankle‐brachial index: 0.68 ± 0.12) were randomized into one of two groups: home‐based leg HT (HT, n=18) or a control treatment (CON, n=16). Patients in both groups were provided with identical water‐circulating trousers and a portable heating pump and were asked to apply the therapy daily (7 days/week, 90 min per session) for 8 consecutive weeks. The pump issued to participants circulated water through the trousers at 43ºC in the HT group and 33ºC in the control group. Laser‐Doppler flowmetry was used to measure red blood cell flux, an index of skin blood flow, during local skin heating to 39°C for 40 min, followed by 43ºC for 20 min. Cutaneous vascular conductance (CVC) was calculated as red blood cell flux divided by mean arterial pressure and expressed as a percentage of maximal vasodilation (%CVCmax). Calf skeletal muscle perfusion was assessed using arterial spin labeling magnetic resonance imaging (MRI) following circulatory occlusion of the thigh for 5 min.

RESULTS

Five participants were either withdrawn prior to study completion or failed to complete the 8‐week follow‐up visit. The remaining 29 participants were included in the final analysis (CON: n=14, HT: n=15). Two participants from CON and 4 from HT were unable to undergo the MRI scans. At baseline, local skin heating to 39ºC increased %CVCmax to 55.8 ± 12.9% in CON and 52.5 ± 18.7% in the HT group. The change from baseline to 8 weeks in %CVCmax was similar between CON and HT (CON: ‐2.6 ± 13.9%, HT: ‐0.7 ± 16.3%, p=0.74). Peak calf blood flow during reactive hyperemia was comparable between groups at baseline (CON: 48.0 ± 24.6 ml/min/100g, HT: 43.8 ± 20.4 ml/min/100g, p=0.76). The change from baseline to 8 weeks in peak calf perfusion was also similar between CON and HT (CON: ‐4.8±21.9 ml/min/100g, HT: ‐2.8±11.0 ml/min/100g, p=0.84).

CONCLUSION

Eight weeks of home‐based leg HT does not improve leg vascular function in patients with symptomatic PAD.
Identification of Novel Cas Proteins in Metagenomic Datasets of Microbiomes in Puerto Rican Ecosystems
Authors: Jimenez‐Pagan Gabriel , Detres‐Martinez Diego , Rios‐Velazquez Carlos
Published: May 2022

CRISPR‐Cas9 technology has become one of the most groundbreaking discoveries of the 21st century. CRISPR, which stands for Clustered Regularly Interspaced Short Palindromic Repeats, along with CRISPR‐associated (Cas) proteins, function as an adaptive immune system for many prokaryotic organisms. As a gene editing tool, CRISPR‐Cas9 allows for sequence‐specific targeting, which scientists have exploited in favor of biomedicine, as a way to alter and remove genes and genomic regions altogether in almost all eukaryotic cells. In spite of its great implications in biomedical research, and as a means of studying gene function, CRISPR‐Cas9 has important limitations. Perhaps, most notably, Cas9 interacts with a specific DNA recognition site, known as the protospacer adjacent motif (PAM). In the absence of the correct PAM sequence, Cas9 will be unable to bind DNA and, hence, it will be unable to perform its endonuclease function. While the PAM sequences for the commonly used S. pyogenes Cas9 (SpCas9) is commonly found throughout the human genome, they are not always efficiently positioned to target a particular gene. Our current understanding of Cas9 orthologs is deficient, and the characterization of novel PAM sequences will continue to improve the gene‐editing efficiency of CRISPR technology as we know it. Moreover, the isolation of novel Cas9 and other CRISPR‐associated proteins has and will continue to increase the number of CRISPR applications. Throughout this project, we aim to use functional metagenomics as a vehicle for bioprospecting novel CRISPR‐Cas systems within different environmental samples of Puerto Rico. We analyzed two metagenomic datasets from the microbial and functional diversity of distinct water systems: the Guajataca water reservoir, and the Clara Cave and Empalme Sinkhole waters. Sequencing data was analyzed regarding CRISPR‐Cas analogy through CRISPR databases via CRISPRCasFinder and NCBI‐BLAST. Multiple sequence alignments were conducted to describe conserved regions in Cas genes using the ConSurf server. Several CRISPR‐Cas‐like regions were identified within the metagenomic datasets. Candidate prospects will be analyzed concerning Cas protein identity and function with in vivo and in vitro approaches. Throughout this approach, we expect to unravel previously unexplored CRISPR‐Cas systems, as well as characterizing and describing Cas protein orthologs within these environments respecting PAM sequence specificity and other functional and structural characteristics.
Physical and Mechanistic Characterization of Tardigrade Cryptobiotic States in Response to Environmental Stressors
Authors: Joseph Kara M. , O'Dell Hayden M. , Clark Trace A. , Crislip Jessica R. , Smythers Amanda L. , Hicks Leslie M. , Kolling Derrick
Published: May 2022

Tardigrades, known more commonly as ‘water bears,’ are semi‐microscopic invertebrates that possess a remarkable ability to withstand extreme stress. This stress tolerance is inferred via cryptobiosis, a collection of quiescent states enabling long‐term survival to extreme stressors through temporary metabolic suspension and upregulation of protective biomolecules. While cryptobiotic abilities span taxa across all kingdoms, the tardigrades ability to survive desiccation, freezing, oxygen starvation, fluctuating osmotic pressure, and ionizing radiation (using anhydrobiosis, cryobiosis, anoxybiosis, osmobiosis, and irradiation‐induced dormancy, respectively) is matched by none.

Tardigrade cryptobioses, particularly those that form tuns, seem to occur via two distinct but connected processes, one of which facilitates and/or initiates muscular contraction and one of which expels internal water stores. However, the mechanisms through which these cryptobioses are initiated, maintained, and regulated are still unknown. Additionally, while research has shown distinctions between cryobiosis and anhydrobiosis, it is unclear to what extent tun‐forming cryptobioses (i.e. anhydrobiosis and osmobiosis) are distinctly regulated. Furthermore, while anhydrobiosis and cryobiosis have been well characterized, other forms of cryptobiosis have not been empirically explored.

Our work has focused on charactering the physiological and metabolic distinctions between cryptobioses in Hypsibius exemplaris,a model tardigrade species. We have mapped all known cryptobiotic states through the implementation of confocal microscopy and biochemical characterization. Together we have shown structural and metabolic distinctions between tun‐forming and non‐tun cryptobiotic states. These quantitative measurements provide insight into mechanisms corresponding to protective physiological changes and represent the first known empirical analysis of the five cryptobiotic states.
Sex‐Selective Effects of Transient ACE Inhibition on Angiotensin II‐Stimulated Fibrogenic Responses
Authors: Floyd Dana B. , Garvin Alexandra M. , Nunez Wendy S. , Carroll Chad C. , Hale Taben M.
Published: May 2022

Hypertensive heart disease is characterized by cardiac fibrosis in the left ventricle (LV). Angiotensin II (Ang II) promotes cardiac fibrosis through direct actions on cardiac fibroblasts. Inhibition of endogenous Ang II production with angiotensin converting enzyme (ACE) inhibitors has been shown to reduce interstitial collagen deposition in the LV. We have previously shown that transient treatment of male spontaneously hypertensive rats (SHRs) with an ACE inhibitor suppresses the fibrogenic capacity of cardiac fibroblasts and this persists even after stopping treatment. In this study, the goal was to investigate the impact of transient ACE inhibition on subsequent Ang II‐stimulated fibrogenic responses in the LV of male and female SHR. Male and female SHR (11‐week‐old) were treated with an ACE inhibitor (enalapril, 30mg/kg/day) or vehicle for 2 weeks followed by a 2‐week washout period. At the end of the washout, rats were given Ang II (400ng/kg/min, s.c.) or vehicle (saline) for 2 weeks (n=8‐11 per group per sex). Collagen I, III, and IV gene expression was assessed via RT‐qPCR, total collagen was quantified using a hydroxyproline assay, and immunoblotting for lysyl oxidase (LOX) and periostin (Postn) was performed. In males, the Ang II‐stimulated increase in collagen gene expression (i.e. Col1a1, Col3a1, and Col4a1) was significantly attenuated in SHR previously treated with enalapril. In female rats, Ang II significantly increased the expression of Col1a1 and Col3a1 similarly, regardless of prior enalapril treatment. We found a positive correlation between the degree of increase in Col1a1 and Col3a1 as well as with Col1a1 and Col4a1 in male controls with Ang II stimulation. However, in Ang II‐stimulated male rats transiently treated with enalapril, there was a negative correlation between Col1a1 and Col3a1 and no correlation between changes in Col1a1 and Col4a1. In female rats, there were significant positive correlations in all cases regardless of prior transient ACE inhibition. Total collagen content in both male and female rats was significantly increased by Ang II in control animals, but not in those previously treated with enalapril. Additionally, in both male and female rats stimulated with Ang II, cross‐linking proteins Postn and LOX showed lower expression in SHR that were transiently treated with enalapril. These data reveal that even after stopping treatment, ACE inhibition produces persistent changes in the myocardium that render it resistant to the effects of Ang II. The effects seen in females suggests a possible dysregulation between collagen production and extracellular matrix cross‐linking events following transient ACE inhibition. Future studies will elucidate the mechanisms underlying the sex‐specific response to ACE inhibition and the long‐term protective effect in male LV, with a goal of identifying novel therapeutic targets.
Naringenin antihypertensive power is mediated via abrogation of mineralocorticoid receptor (MCR)/ angiotensin converting enzyme (ACE)/ kidney injury molecule (Kim‐1) signaling pathway
Authors: Oyagbemi Ademola A. , Omobowale Temidayo O. , Adejumobi Olumuyiwa A. , Owolabi Abiodun M. , Ogunpolu Blessing S. , Falayi Olufunke O. , Hassan Fasilat O. , Ogunmiluyi Iyanuoluwa O. , Ola‐Davies Olufunke E. , Saba Adebowale B. , Adedapo Adeolu A. , Oguntibeju Oluwafemi O. , Yakubu Momoh A.
Published: May 2022

Hypertension is a condition with chronic elevation of blood pressure and a common preventable risk factor for cardiovascular disease with attendant global morbidity and mortality. The present study investigated the novel antihypertensive action of Naringenin on NƱ‐Nitro‐L‐arginine Methyl Ester (L‐NAME)‐induced hypertension together with possible molecular mechanism of action. Rats were divided into four groups. Rats in Group A were normotensive. The hypertensive group (Group B) received 40 mg/kg) of L‐NAME alone while Groups C and D were concurrently administered Naringenin (50 mg/kg) or Lisinopril (10 mg/Kg) together with L‐NAME orally for 3 weeks. Blood pressure parameters, markers of oxidative stress and renal damage were measured. The immunohistochemistry of kidney injury molecule 1, mineralocorticoid receptor and angiotensin converting enzyme were also determined. Results indicated significant increase in oxidative stress markers and decrease in serum nitric oxide bioavailability in hypertensive rats. Furthermore, there were significant increases in serum myeloperoxidase activity, urinary creatinine, albumin, and blood urea nitrogen levels in hypertensive rats in comparison to hypertensive rats treated with Naringenin. Immunohistochemistry revealed higher expression of kidney injury molecule 1, mineralocorticoid receptor and angiotensin converting enzyme in hypertensive rats. However, co‐treatment with Naringenin mitigated renal oxidative stress, normalized blood pressure and lowered the expression of kidney injury molecule 1, mineralocorticoid receptor, and angiotensin converting enzyme. Combining all, Naringenin offered a novel antihypertensive action through the down regulation of kidney injury molecule 1, deactivation of mineralocorticoid receptor and inhibition of angiotensin converting enzyme.
Beta‐agonist Profiling Reveals Novel Biased Signaling Phenotypes for the Beta 2‐adrenergic Receptor with Implications in the Treatment of Asthma
Authors: De Pascali Francesco , Ippolito Michael , Wolfe Emily , Komolov Konstantine E. , Hopfinger Nathan , Lemenze Douglas , Armen Roger S. , An Steven S. , Scott Charles P. , Benovic Jeffrey L.
Published: May 2022

Asthma is characterized by chronic airway inflammation and airway smooth muscle (ASM) cell contraction. Current treatments for asthma include beta‐agonists which activate the beta 2‐adrenergic receptor, a G protein‐coupled receptor (GPCR) that promotes Gs‐dependent production of cAMP leading to ASM relaxation. Unfortunately, beta‐agonists can also promote severe side effects including an increased risk of having a severe asthmatic attack that can result in death. Such side effects have been correlated with the action of beta‐arrestins, scaffolding proteins that mediate GPCR desensitization and internalization as well as G protein independent signaling. In this scenario, biased ligands that promote selective receptor interaction with Gs should be beneficial in promoting airway relaxation while attenuating beta‐arrestin‐induced side effects. To test this, we have used high‐throughput screening to identify Gs‐biased beta‐agonists. The initial lead candidates were further analyzed for their ability to modulate receptor induced Gs and Gi interaction, cAMP production, and beta‐arrestin interaction using bioluminescence resonance energy transfer (BRET) analysis. We identified three compounds with Gs‐biased properties which showed minimal beta‐arrestin recruitment. These compounds were also able to decrease beta‐arrestin mediated outputs including receptor internalization and ERK activation. These compounds also showed reduced GRK‐mediated phosphorylation of the receptor as well as decreased agonist‐promoted desensitization in a physiological model of asthma (human ASM cells). In conclusion, these Gs‐biased beta‐agonists may pave the way for the development of more effective drugs for the treatment of asthma and may help to clarify the structural determinants of receptor mediated biased signaling.
Characterization of An Inducible p65 Knockout Mouse Model for Studying Glioblastoma
Authors: Trang Amy , Alptekin Ahmet , Rajpurohit Surendra , Arbab Ali , Bradford Jennifer
Published: May 2022

Glioblastoma (GBM) is a malignant brain cancer that results in patient death within two years following diagnosis. The GBM tumor microenvironment has an important impact on the formation, progression, and drug resistance of this lethal disease. The GBM tumor microenvironment is composed of a variety of cell types that can support tumor growth like microglia. Microglia are immune cells that can switch from a classical, tumoricidal M1 phenotype to an alternative, tumor promoting M2 phenotype which encourages tumor growth. However, there are limited studies about the specific effect(s) that microglia have on GBM thus requiring further research into how these cells affect GBM tumors.

Given the importance of the tumor microenvironment to GBM tumor progression, targeting key signaling pathways in tumor‐associated cells that are involved in sustaining GBM tumors could be an effective way to reduce tumor progression. One signaling pathway involved in causing different cancers is the nuclear factor‐kappa B (NF‐κB) pathway, which has been implicated in M1 to M2 phenotype polarization. Therefore, this study focuses on characterization of the p65fl/fl/CX3CR1creER/+ mouse model, which is an inducible p65 knockout mouse model for studying how inhibition of canonical NF‐κB signaling in microglia affects GBM tumors. After tamoxifen is administered to the mouse, p65, a transcription factor of the canonical NF‐κB pathway, should be deleted in microglia due to the C‐X3‐C chemokine receptor 1 (CX3CR1) promoter. Tamoxifen binds to the estrogen receptor on the cre recombinase (creER) so that the cre recombinase can recombine the floxed p65 (p65fl/fl) gene in microglia. Characterization of this particular mouse model is necessary to determine if the floxed p65 gene is effectively deleted in microglia from these mice. Liquid chromatography‐mass spectrometry analysis has shown that the orally administered tamoxifen was present and quantifiable in the brains of tamoxifen‐treated mice to induce recombination. Based on data from PCR and western blots, tamoxifen given to the p65 knockout mice induced partial deletion of the floxed p65 gene in isolated microglia compared to p65 knockout mice given the vehicle control. Preliminary flow cytometry data suggested that p65 deletion in microglia occurred in p65fl/fl/CX3CR1creER/+ mice that received GBM implantations and tamoxifen treatment compared to a vehicle control group. Overall, the data from this study suggested that the floxed p65 gene is partially deleted in microglia of the tamoxifen‐treated p65 knockout mice compared to the vehicle control mice.
Left Ventricular Systolic‐Diastolic Coupling: Insight from Magnetic Resonance Dobutamine Stress
Authors: Oneglia Andrew P. , Jaffery Manall , Zaha Vlad G. , Nelson Michael D.
Published: May 2022

Left ventricular systolic and diastolic function are intrinsically linked, such that diastolic relaxation is influenced by the preceding systolic contraction – similar to how a springs recoil to its original shape is influenced by the degree of compression. Several clinical investigations have proposed quantifying the ratio between diastolic and systolic function to assess the coupling efficiency between the two, however, these cross‐sectional comparisons may be influenced by additional confounding factors, such as differences in tissue characteristics. Therefore, to extend our understanding, and directly examine the relationship between left ventricular systolic and diastolic coupling, we performed cardiac magnetic resonance imaging in six healthy volunteers (4F/2M, 22 ± 3 years, 24.4 ± 3.0 kg/m2) at rest and during low‐dose intravenous dobutamine infusion (2 µg/kg/min) to increase cardiac inotropy without affecting other, potentially confounding, regulators of diastolic function. Systolic and diastolic function was measured using gold‐standard tissue tagging, from three evenly spaced short axis images spanning the left ventricle from base to apex. As expected, dobutamine infusion increased peak circumferential strain (‐19.8 ± 1.6 % vs ‐21.3 ± 2.0 %, p = 0.002), systolic circumferential strain rate (CSRs: ‐96 ± 2 %/s vs ‐117 ± 7 %/s, p = 0.001), peak twist (8.2 ± 0.9 ° vs 10.7 ± 1.1 °, p = 0.002) and peak rate of systolic twist (TR: 40 ± 7 °/s vs 58 ± 8 °/s, p = 0.004). Independent of any changes in heart rate (59 ± 9 bpm vs 58 ± 9 bpm, p = 0.303) or end‐diastolic volume (171 ± 26 mL vs 170 ± 24 mL, p = 0.882), early diastolic circumferential strain rate (CSRd: 141 ± 15 %/s vs 163 ± 9 %/s, p = 0.019) and peak untwisting rate (UTR: ‐67 ± 24 °/s vs ‐88 ± 16 °/s, p = 0.018) subsequently increased. As a result, the ratio between diastolic and systolic measurements remained unchanged (CSRd/CSRs: 1.47 ± 0.14 vs 1.40 ± 0.15, p = 0.266; UTR/TR: 1.69 ± 0.54 vs 1.57 ± 0.37, p = 0.190). Taken together, these data support a growing body of evidence linking left ventricular systolic function with subsequent early diastolic relaxation and suggest that future investigations examining diastolic function should consider the influence of preceding systolic contractions.
The Training‐Induced Increase in Critical Power Is Strongly Related to Changes in Vascular Function in Young Adults
Authors: Gifford Jayson R. , Wallace Taysom , Marchant Erik , Kaluhiokalani Jamie , Hyldahl Robert , Hancock Chad
Published: May 2022

Homeostatic or steady‐state conditions are necessary to sustain exercise. When steady state cannot be achieved, muscle fatigue quickly grows, and task failure promptly occurs. Critical Power (PCRIT) is the highest power output at which steady‐state conditions can be achieved during a given exercise and is strongly related to aerobic capacity. Endurance training increases PCRIT, but the extent to which changes in vascular function, exercise blood flow and mitochondrial function are related to training‐induced changes in PCRIT is unknown.

Purpose

Determine the relationship between training‐induced changes in blood flow, mitochondrial function and PCRIT.

Methods

20 untrained adults participated in either 6 weeks of high‐intensity knee extension exercise (3x per week, n=10) or 6 weeks of sham therapy (3x per week of sham heat therapy, n=10). PCRIT and work‐prime (W′) were determined during single‐leg knee extension (KE) before and after training. Maximum exercise blood flow (Doppler Ultrasound during maximum KE), resistance artery function (passive‐leg‐movement (PLM) technique) and maximum mitochondrial respiration (i.e., State 3 respiration of permeabilized muscle fibers biopsied from the vastus lateralis) were also assessed before and after training.

Results

PCRIT increased by 28% in the exercise group (Figure A, P=0.01), but did not change in the sham group (Figure A). W′ did not significantly change with the intervention (P=0.23). Maximum exercise blood flow (P=0.03), peak flow during PLM (P=0.02), and maximum coupled mitochondrial respiration (i.e., State 3 Respiration, P=0.03) all tended to increase in the exercise group, but not the sham group (P=0.82, 0.18, 0.76, respectively). As illustrated in Figures B, C and D, Pearson correlation indicated that the change in PCRIT was related to the changes in maximum exercise blood flow (R2=0.29, P=0.01), PLM Peak Flow (R2=0.54, P<0.01), and State 3 respiration (R2=0.29, P=0.03).

Conclusions

While changes maximum exercise blood flow and maximum coupled mitochondrial respiration were related to training‐induced changes in PCRIT, the change in resistance artery function best predicted the training‐induced increase in PCRIT. While not tested in this study, resistance artery function may potentially relate to PCRIT by improving the precision of oxygen delivery and reducing oxygen deficit at the start of exercise. Interventions seeking to improve PCRIT and exercise tolerance, should consider focusing on strategies to enhance resistance artery function in the exercising muscles.