bluuebunny/crossref_metadata_2025_split huggingface dataset:
Phenotypic Characterization of Macrophage and Monocyte Populations in the NLRP3 KO DAHL/SS‐Nlrp3em2Mcwi Rat Model
Authors: De Jeronimo Diaz Cesar , Teng Jiajing , Flynn Ryan A. , Lin Matthew , Yuan Junyao , Rogers Connie J. , Korzick Donna H. 
Published: May 2022

BackgroundThe NLR family pyrin domain containing 3 (NLRP3) inflammasome is a multimeric protein complex that is activated in response to pathogen and damage associated molecular patterns (PAMPs & DAMPs) to initiate inflammatory responses through IL‐1ß, IL‐18, and pyroptosis. Pharmacological inhibition of NLRP3 attenuates inflammatory responses after myocardial infarction and hypertension in rodents. However, the effect of NLRP3 gene loss on basal macrophage and monocyte populations in rats is unknown. We hypothesized that NLRP3 gene loss would not alter macrophage and monocyte phenotype under basal conditions.MethodsCRISPR‐Cas9 was used to induce a 14 base pair deletion in exon 1 of the NLRP3 gene in the DAHL/SSJrHsdMcwi strain to produce NLRP3 KO DAHL/SS‐Nlrp3em2Mcwi rats. NLRP3 KO male (n=11), NLRP3 KO female (n=10), wild type (WT) male (n=5), and WT female (n=10) were studied at 4‐5 mo. Teklad 7034 low salt (0.12% NaCl) diet was provided ad libitum. Proinflammatory M1 (CD11b+CD68+) and anti‐inflammatory M2 (CD11b+CD206+) macrophages (Mɸs) were quantified in CD45+ cells from left ventricle (LV), peripheral blood monocytes (PBMCs), bone marrow (BMDMs), and splenocytes using density gradient centrifugation and flow cytometry.ResultsSex (p<0.01) and genotype (p<0.01) main effects were observed in several morphological characteristics. NLRP3 KO and male rats exhibited significantly increased rat, kidney and LV weights, and LV/tibial length vs WT and female, respectively. Males also displayed significantly increased spleen length and visceral adipose weight vs females (p<0.01). In LV, the percent of CD11b+CD68+ cells was 28% greater in NLRP3 KO vs WT (n=1‐2/group). In PBMCs, the percent of CD11b+CD206+ cells was 13% greater in WT female vs males and NLRP3 KO (n=2‐4/group), while NLRP3 KO females exhibited ~20% less CD11b+CD68+ in splenocytes (n=1‐4/group). In BMDMs, ~60% were positive for CD11b+CD68+, while ~2% were CD11b+CD206+ across all groups (n=1‐2/group).ConclusionPreliminary data suggest that NLRP3 gene loss on a Dahl salt sensitive background is associated with increased proinflammatory M1 Mɸs in LV concomitant with increased LV mass, independent of sex. We hypothesize that NLRP3 may possess an inflammasome independent role in regulating resident LV macrophages under basal conditions.
Alcohol intoxication stimulates antioxidant gene expression in skeletal muscle in a time dependent manner
Authors: Tice Abigail L. , Laudato Joseph A. , Gordon Bradley S. , Steiner Jennifer L. 
Published: May 2022

Alcohol is a known myotoxin that disrupts skeletal muscle function and health. Mitochondria play an important role in maintaining proper skeletal muscle function as they contribute to the production of ATP. Mitochondrial oxidative metabolism as well as xenobiotics like alcohol, lead to the production of reactive oxygen species (ROS) which can cause cellular damage if the antioxidant response is exceeded. Chronic alcohol ingestion has been linked to enhanced oxidative stress in the muscle and liver, while the temporal progression of antioxidant gene programs in the skeletal muscle following acute alcohol intoxication remains unknown. Therefore, the goal of this work was to examine the effects of an acute alcohol binge on skeletal muscle oxidative stress and antioxidant pathways.MethodsC57BL/6Hsd female mice (15wks old) received an IP injection of either saline (CON; n=39) or ethanol (EtOH; n=39). Gastrocnemius muscles were collected from baseline (untreated; n=3), CON (n=3), and EtOH (n=3) mice every 4‐hours for 48‐hours to determine the time course of changes following intoxication. RNA was isolated, cDNA synthesized, and RT‐PCR was performed. Protein expression was measured via Western blotting.ResultsNuclear factor erythroid 2‐related factor 2 (Nrf2) is a transcription factor central to the control of cellular antioxidant responses along with its inhibitor, Kelch like ECH associated protein 1 (Keap1). Alcohol increased the mRNA expression of Nrf2 as well as Keap1 starting 12 hours after intoxication. Downstream target genes of this antioxidant pathway were also increased after acute alcohol starting at 12‐16 hours post intoxication including Cytochrome P450 oxidoreductase (Por),Heme oxygenase 1 (Hmox1),Peroxiredoxin 6 (Prdx6),Glutamate‐cysteine ligase catalytic subunit (Glcl),Glutamate‐cysteine ligase modifier subunit (Gclm),and Glutathione‐disulfide reductase (Gsr). EtOH also enhanced Superoxide dismutase 1 (Sod1) mRNA expression from 24‐48hr following intoxication and NADPH Oxidase 4 (Nox4) expression 24hrs after the binge. Meanwhile, mRNA expression of Sod2and Nox2 was suppressed over the 48hr period following alcohol intoxication.ConclusionThese results suggest that binge alcohol stimulates ROS production and thereby antioxidant systems, to assist in the breakdown of ROS in the skeletal muscle. Future work is needed to determine the contribution of a transient increase in these pathways to longer term skeletal muscle health, especially in the context of repeat binges, a common drinking behavior.
Altered T Regulatory Cell Phenotype due to Chronic Hypoxia Exposure Contributes to the Pathogenesis of Pulmonary Hypertension
Authors: Lantz Benjamin J. , Jones David , Oyebamiji Olufunmilola , Guo Yan , Gonzalez Bosc Laura 
Published: May 2022

Hypoxic pulmonary hypertension (PH) can be caused by chronic obstructive lung diseases, acute lung injury, sleep apnea, and high altitude. Hallmarks for PH include recruitment of immune cells to the perivascular region of the lungs, thickening of the pulmonary arterial walls, elevated pulmonary vascular resistance and pressure, and right ventricular remodeling, ultimately causing right heart failure and death.We previously reported that chronic hypoxia (CH) leads to an upregulation of pro‐inflammatory Th17 cells but does not alter T regulatory (Treg) cell numbers within the lung. This change in Th17‐Treg ratio can upset the pro‐inflammatory/anti‐inflammatory balance required to maintain homeostasis leading to disease pathogenesis. We also demonstrated that these Th17 cells react to the cryptic self‐antigen collagen V (col V), which is upregulated following CH. Nasally instilling mice with col V attenuated CH‐induced PH, presumably by inducing mucosal tolerance to col V and restoring Treg‐Th17 balance. Tregs can lose their suppressive phenotype and transition into Th17 cells (deemed exTreg Th17 cells) in certain autoimmune conditions. However, it is unknown whether CH triggers this mechanism. We hypothesized that CH disrupts the Th17‐Treg cell balance due to the pathogenic conversion of Tregs into Th17 cells, contributing to PH development. Using a mouse model (Foxp3tm9(EGFP/cre/ERT2)Ayr/J x Ai14‐tdTomato) that allows the study of lineage stability and genetic mapping of Tregs, together with single‐cell RNA sequencing, we discovered that CH resulted in a gene expression profile consistent with exTregs or Tregs with less suppressive activity, such as downregulation of Il2ra and upregulation of Tnfrsf4 within CD4 memory T cell cluster (adjusted p‐value 0.03). The single‐cell sequencing data were processed with the conventional pipeline. Seurat (v4.0.5), R (v4.1.0), single R (v1.6.1) were used for downstream analysis and cell type prediction against the mouse Immunologic Genome Project. Differential gene expression was determined using the R package DESEq2, and significant genes were defined by adjusted p‐value ≤ 0.05. Our future studies include using multicolor flow cytometry to characterize these changes further and determine if and how CH decreases Treg suppressive activity, contributing to the phenotypic switch to Th17 cells. In conclusion, our data suggest that CH alters Treg cell phenotype, which aids in the pathogenesis of PH.
The Impact of Gclm on Efferocytosis and Atherosclerotic Progression
Authors: Delgadillo Luisa F. , Mackay Ryan P. , Rashdan Nabil A. , Yurdagul Arif , Pattillo Christopher B. 
Published: May 2022

Atherosclerosis is the leading cause of coronary artery disease (CAD) and stroke which are the primary contributors to cardiovascular disease related deaths in the world. This is an inflammatory disease characterized by the accumulation of lipid and fibrous elements in large arteries resulting in the development of a plaque. Progression of the disease leads to the accumulation of apoptotic cells and formation of a necrotic core making the plaque unstable and prone to rupture. Rupture can lead to artery occlusion resulting in stroke or myocardial infarction (MI). One of the main contributors to a large necrotic core is macrophages that are unable to clear the accumulated apoptotic cells and as a result undergo secondary necrosis. This clearance process, referred to as efferocytosis, is impaired in advanced atherosclerotic lesions. It has been shown that the process that leads to a thin fibrous cap (characteristic of an unstable plaque) and a large necrotic core are accelerated by a decrease in glutathione (GSH) availability due to a deletion on the modifier subunit of Glutamate Cysteine Ligase (GCLM). The objective of this study is to identify the role and mechanism of GSH during the formation of the necrotic core in atherosclerosis. We hypothesize that availability of GSH is important in macrophages for adequate efferocytosis of apoptotic cells during atherosclerosis progression.MethodsTriglycerides, total cholesterol, and HDL levels were measured on PCSK9 virus driven atherosclerosis in wild type and heterozygous GCLM mice. Sections of the aortic root from these mice were stained with Movat Pentachrome to measure plaque area and percent necrosis. An efferocytic index was measured using PHK26 labelled apoptotic cells (AC). AC were added to cultured bone marrow derived macrophages (BMDM) allowing for their uptake. Percent positive macrophages containing a fluorescently labelled AC were quantified. BMDM from WT, Het, and KO GCLM mice were collected for total protein, surface receptor expression, and mRNA analysis.ResultsEfferocytosis was affected in KO mice as compared to WT and Het BMDM. RNA analysis has showed a decrease in efferocytosis receptors such as MerTK, confirmed by cell surface expression.ConclusionThese results suggest that a loss of GCLM and GSH during efferocytosis impairs adequate efferocytosis.
Neuroanatomical Remodeling of Colonic Interstitial Cells of Cajal after Spinal Cord Injury
Authors: Werner Claire M. , Willing Lisa B. , Holmes Gregory M. 
Published: May 2022

After spinal cord injury (SCI), individuals often experience gastrointestinal comorbidities including neurogenic bowel, a condition where colonic dysmotility causes chronic constipation, difficulty with evacuation and overflow incontinence. Located intrinsic to the gastrointestinal (GI) tract, the anatomy of the enteric nervous system (ENS) is not directly damaged at the time of the initial spinal trauma, thereby serving as a potential pharmacologic target for neurogenic bowel. Enteric neuromuscular control of colonic slow‐wave propagation is a balance of excitatory and inhibitory inputs to Interstitial Cells of Cajal (ICC), pacemaker cells that are interposed between enteric neurons and smooth muscle cells. Reduction of colonic ICCs accompany colonic dysmotility in other disease states and our lab has previously shown a reduction in cholinergic and nitrergic enteric neurons following experimental SCI. Of the ICC subtypes, those of the myenteric plexus are strongly associated with driving colonic motility. We hypothesized that after SCI, the distal colon myenteric plexus ICCs will present with decreased cell bodies and projection length in both sexes, contributing to neurogenic bowel.This study aims to expand current understanding of neurogenic bowel neuroanatomy in acute 3‐day and chronic 3‐week SCI rats. Experimentally, male and female rats receive a surgical control or severe T3 contusion SCI (n = 19 M, 22 F). Three days or three weeks post‐operatively, the distal colon smooth muscle tissue is harvested and dissected for immunohistochemical (IHC) and electrophysiological analysis. C‐Kit antibody is then used to fluorescently label the ICCs, upon which we quantified myenteric plexus cell bodies and projection length. Unexpectedly, the myenteric plexus presented with a significant increase in cell count (p = <0.01) and projection length (p = <0.01) following SCI compared to control animals in both sexes. Additionally, a significant interaction effect was observed between animal time point and surgical group for cell count (p = <0.01) and a significant interaction between sex and surgical group for projection length (p = <0.03). The current data illustrates sex‐related differences in remodeling of the colonic neuromuscular interface after SCI, reinforcing the importance of sex‐specific treatment for neurogenic bowel to improve clinical outcomes. The enduring plasticity of the ICC in our chronic animal model demonstrates the importance of continual medication adjustments among the SCI population. Myenteric ICC network upregulation may reflect a compensatory response to diminished cholinergic enteric neuronal input, therefore our future studies will investigate cholinergic receptor neurophysiology after SCI.
Skull‐Brain Biomechanics of Headbutting. An XROMM Pilot Study on Goats
Authors: Ackermans Nicole , Williams Susan H. 
Published: May 2022

In humans, the coup‐contrecoup injury occurs opposite the site of impact when the brain meets the skull, causing intracerebral hemorrhage. It makes up a significant portion of traumatic brain injuries (TBI) and can often lead to neurodegeneration. Bighorn sheep and related bovids are known for their impressive headbutting rituals performed during the rut, the general belief being that these animals come away unharmed from these encounters. However, a recent immunohistochemical study revealed TBI in bighorn sheep and muskoxen in relation to headbutting. With a large gyrencephalic brain, repetitive headbutting, and a pathology pattern resembling early chronic traumatic encephalopathy in humans, bovids present themselves as a significant model for TBI. It is, however, uncertain whether bovids sustain coup‐contrecoup injury due to the oblong shape of their braincase and tight fit to the brain. We aimed to investigate whether cranial and neuroanatomical aspects of the bovid head anatomy contribute to TBI reduction, using goats as a model to measure brain movement independent of the skull upon impact.Four cadaveric goat heads were implanted with radiodense tantalum beads in the skull and brain. A weight‐drop system was used to create an impact within the field of view of a biplanar fluoroscopy system. The heads were CT scanned and the recording was reconstructed as an animated 3D model using x‐ray reconstruction of moving morphology (XROMM). The brain markers were tracked relative to each other to account for soft tissue deformation and marker migration and relative to the skull markers to account for brain movement. We were able to visualize how the brain moves in relation to the skull for the first time in bovids, providing a better understanding of skull‐brain biomechanics.
IL‐33/ST2L Signaling Regulate Blood‐Retinal Barrier Integrity via Affecting Cadherin‐Catenin Adheren Junction Complex
Authors: Sharma Deepti , Bisen Shivantika , Kaur Geetika , Singh Nikhlesh K. 
Published: May 2022

PurposeAngiogenesis, neovascularization and vascular remodeling are highly dynamic processes, where endothelial cell‐cell adhesion within the vessel wall controls a range of physiological processes, such as growth, integrity and barrier function. The cadherin–catenin adhesion complex is a key contributor to blood‐retinal barrier (BRB) integrity and dynamic cell movements. However, the pre‐eminent role of cadherins and their associated catenins in adheren junction structure and function is not fully understood. Also, the role of IL33/ST2L signaling in regulating BRB integrity is not known.MethodsUsing a murine model of oxygen‐induced retinopathy (OIR) and human retinal microvascular endothelial cells (HRMVECs), we are trying to understand the significance of IL‐33/ST2L‐signaling on endothelial barrier disruption, leading to abnormal angiogenesis and enhanced vascular permeability.ResultsOur in vivo genetic deletion studies revealed that deletion of IL‐33 resulted in reduced vascular tufts and vascular leakage. Therefore, in order to understand the molecular mechanism by which IL‐33 regulates the BRB integrity, we performed some in vitro studies using HRMVECs. The electric cell‐substrate impedance sensing (ECIS) analysis showed that IL‐33 (20 ng/ml) induce endothelial barrier disruption in HRMVECs. Likewise, FITC‐dextran permeability assay also showed that IL‐33 stimulate the endothelial dysfunction in HRMVECs. As we know, tight junctions (TJs) and adheren junctions (AJs) form the adhesion structure and maintain the BRB integrity, therefore we look for the junctional proteins involved in the IL‐33/ST2L mediated endothelial dysfunction. Furthermore, we found that IL‐33 mediates the phosphorylation of VE‐cadherin and a‐catenin in HRMVECs. Furthermore, mass‐spectroscopy (MS) analysis revealed the phosphorylation of specific Ser439 residue of a‐catenin upon IL‐33 inducement in HRMVECs. Consequently, mutation of a‐catenin at Ser439 residue restores the BRB integrity disrupted by IL‐33. In addition, we have observed that IL‐33 induce the generation of reactive oxygen species (ROS), thereby destabilize adheren junctions in HRMVECs.ConclusionsWe conclude that IL‐33/ST2L signaling plays a significant role in angiogenesis and neovascularization by regulating endothelial permeability and BBB integrity.
Interface investigation the protein interaction interface between malate dehydrogenase and citrate synthase
Authors: O’Murphy Fiona , Vigilante Danielle , Heras Olivia , Tarbox Harrison , Huynh Chris , Dowling Brenden , Faustino Kalii , Desalegne Sara , Gaylor Stephen , Tanaka Carina , Botros Natalie , Hixon Samuel , Kueng Lukas , Randolph Mitchell , Auth Marin , Bose Emma , Calzada Oscar , Enenwali Chinazom , Ghumman Parvinder , Infante Charlotte , Little Shelby , Maher Kendyl , Shi Jessica , Visinaiz Victoria , Williams Becca , Woodward Jon , Bell Ellis , Provost Joseph 
Published: May 2022

Enzymes complexes (metabolons) support the metabolic reactions through substrate channeling. Several enzymes in the Krebs cycle are found in protein‐protein metabolons including mitochondrial malate dehydrogenase (MDH2) and citric synthase (CS). While the weak transitory interaction between these enzymes has been demonstrated, the key residues of human MDH2 involved in the interface with CS has not been identified. The purpose of this study is to probe possible residues of MDH responsible for binding to CS. Because cytosolic MDH (MDH1) poorly binds to CS, we initially identified unique sequences that were involved in or adjacent to reported or predicted binding sites of MDH2 and CS. Four regions were selected and residues corresponding to the primary sequence of cytosolic MDH1 were substituted in place of mitochondrial MDH2. Computational models of all the monomeric constructs were made using phyre2 homology modeling, subsequent refinement of the monomers using Galaxy Refine and construction of the dimer forms using the appropriate template in PyMol. Dimers were then refined using Galaxy Refine Complex to give replicate structures of the final models. Potential metabolon formation was explored using HawkDock with the dimer versions of MDH and Citrate Synthase either with or without restraints imposed by published metabolon models.Wild‐type human genes of MDH1, MDH2, and CS were codon optimized for bacterial expression and cloned into the C‐terminus of each gene in a pET28a expression vector. Each MDH1‐MDH2 substitution (DS1‐DS4) were constructed by Gibson cloning. Resulting structure and functional impacts were determined by melting points and kinetic parameters (specific activity, Km, Vmax and Kcat) in purified proteins. Interactions between wild‐type MDH1/2 and CS were compared to MDH2 DS mutants. To qualitatively demonstrate interactions, pull‐down assays were performed in the absence and presence of crowding agents, glycerol, PEG or Ficoll 70. Finally wild‐type MDH 1 and 2 vs MDH2 mutants were subjected to competitive pull‐down assays to identify regions responsible for isozyme specific interactions. This work will show four of the potential interacting regions between MDH and CS and lead to a better understanding of dynamics of this metabolic pair.
Oxygen Dose: A new perspective
Authors: MacLaughlin Kent , Lamers Jacob J. , Braun Rudolf , Eldridge Marlowe W. 
Published: May 2022

IntroductionThis study followed up on our findings, published in 2019, of stem progenitor cell mobilization following intermittent low level hyperoxia (42%) in an animal model. This most recent study in human participants used a never before tested small dose of intermittent hyperbaric air that has been used for many years as a placebo control in hyperbaric oxygen (HBOT) research. Because of the reports of a placebo response using this small dose in HBOT research performed by the Department of Defense and conflicting reports disputing the placebo response, coupled with the lack of evidence in the literature that this dose had ever been tested, we questioned if this a placebo or a dose response.In this research human participants breathed room air in a portable hyperbaric chamber (mountain sickness chamber) (Vitaeris 320 Oxyhealth LLC) at 1.3 atmospheres absolute (ATA), daily for 10 days. This hyperbaric hyperoxia level is similar to 28% oxygen concentration in normobaria.We hypothesized that the low dose of intermittent hyperoxia would mobilize stem cells in humans similar to our animal study.MethodsEight adult subjects were exposed to 1.3 atmospheres absolute (ATA) in a mountain sickness bag daily for 60‐minutes, 10‐times (M‐F) over a 12‐day period. Venous blood draws were taken at four time points. (1) Immediately preceding the first treatment (control), (2) Immediately following the first treatment, (3) Prior to the 10th treatment, and (4) 72‐hours after final treatment. We analyzed blood samples using flow cytometry for changes in the expression of surface markers CD45, CD34 and CD133 and performed a statistical analysis using a Friedman test followed by a paired Wilcoxon signed‐rank test.ResultsWe found a nearly 2 fold increase in the expression of CD45dimCD34+CD133‐prior to the 10th treatment (9.8% to 18.9% = 194% increase) and a 3 fold increase 72 hours after the 10th treatment (9.8% to 29.6% = 303% increase). We also found CD45dimCD34‐133+ decreased by 85% between the control time point and prior to the 10th treatment (15% to 2.3% = 85% decrease) and further decreased to a decrease of 84%, 72 hours after the final treatment (15% to 2.0% = 87% decrease).ConclusionsWe conclude that breathing room air at 1.3 ATA daily for 60 minutes over a 2 week period has significant effects on the mobilization of stem progenitor cells and therefore is physiologically active. These findings suggest that 1.3 ATA hyperbaric air breathed daily is a dose treatment and not a placebo nor a sham. These data suggest a re‐evaluation of its use as a placebo control in scientific research is warranted.