bluuebunny/crossref_metadata_2025 huggingface dataset:
Blood Pressure Lowering Effect of Electro‐Acupuncture in Middle‐aged and Elderly Patients with Mild to Moderate Hypertension: Does Inflammation Play a Role
Authors: Xie Lifang , Fu Liangwu , Tjen‐A‐Looi Stephanie , Nguyen Lan Nguyen , Nadeswaran Bavani , Malik Shaista
Published: May 2022

Our previous findings have shown that Electroacupuncture (EA) at P5‐6 and ST36‐37 for 8 weeks reduces Blood Pressure (BP) in patients with mild to moderate hypertension (HTN) through sympathetic inhibition. Responsiveness to EA (defined as ≥‐6mmHg in peak or average systolic BP) was 80% in patients younger than 50 years, 71% in males older that 50 years, and 44% in females older than 50 years. It is well known that inflammation is increased in aging and may play a critical role in age related conditions like HTN. In this study, we formulated a targeted treatment strategy reducing inflammation and sympathetic activity named immuno‐sympathoinhibitory EA treatment that includes acupoints targeting both immune and sympathetic (P5‐6, ST36‐37, and SP6‐7) systems to address the low‐grade chronic inflammation in older hypertensive patients. High sensitivity C‐reactive protein (hs‐CRP), a marker of systemic chronic low‐grade inflammation was assessed. We hypothesized that immuno‐sympathoinhibition by EA treatment decreases BP and hs‐CRP in middle‐aged and elderly patients with HTN. Participants were not on antihypertensive medications and randomly allocated to either sympathoinhibitory (n=12) or immuno‐sympathoinhibitory (n=9) EA treatment. After completion of the 8‐week course of treatment, the BP lowering responsiveness to the immuno‐sympathoinhibitory EA was 88% (P=0.016) and to the sympathoinhibitory EA was 58% (P=0.042). The hs‐CRP likely could be reduced by immuno‐sympathoinhibitory EA treatment (decreased by 6.7 %, pre‐EA vs post‐EA P=0.08, n=5) while hs‐CRP did not decrease in patients treated with sympathoinhibitory EA (increased by 0.9 %, pre‐EA vs post‐EA P=0.42, n=15). Therefore, combining EA targeted at the immune and sympathetic systems may be important in lowering BP and chronic low‐grade inflammation and improving responsiveness to EA treatment in middle‐aged and elderly hypertensive patients.
A Mouse Model Tracking Cardiac Exosomes Spatially and Temporally
Authors: Chang Jiang , Luo Weijia , Dai Yuan , Chen Zhishi
Published: May 2022

Exosomes are 30‐150 nm secreted extracellular vesicles containing DNA, RNA, and proteins with lipid bilayer membranes. They exist in most organs and body fluids, including blood, tears, urine, amniotic fluid, breast milk, etc. The profiles of DNA, RNA, and proteins contained in exosomes are distinct from the exosome‐releasing cells. Recent studies revealed that different pathological conditions or stimuli could alter exosome sorting preferences, which resulted in the changes of phenotypes or functions in exosome recipient cells or organs. They are emerging as a new form of messengers facilitating cross‐talk between cells, tissues, and organs. Therefore, a critical demand arises for developing a sensitive and non‐invasive tracking system for endogenous exosomes. Tracking the biodistribution of endogenous exosomes and kinetics of endogenous exosome release and uptake should significantly impact basic and translational studies of exosome‐mediated pathways.

Based on the exosome membrane structure, two major strategies have been applied for exosome labeling: by a lipophilic reagent‐conjugated reporter that incorporates into the exosome membrane and by exosome surface marker fusion reporter proteins. However, several challenges to monitoring endogenous exosomes currently remain due to the limitation of these exosome‐labeling techniques.

In this study, we chose Nano‐luciferase (NanoLuc) as a reporter. NanoLuc is more stable and smaller in molecular size (19 kD) and generates a 150‐fold stronger signal compared to traditional Firefly and Renilla luciferases. The half‐life of the NanoLuc luminescence signal is more than 2 hours, the longest amongst all known luciferases. Its ultra‐stable and highly sensitive signal makes it an ideal reporter for endogenous exosome labeling to achieve a safe, non‐invasive, and quantifiable exosome tracking objective.

We exploited these NanoLuc properties by fusing it to the exosome surface marker CD63 for exosome labeling and generated a proof‐of‐concept mouse model to enable tracking of endogenous exosomes. To achieve spatial control of exosome labeling, CD63NanoLuc reporter expression was controlled by the cardiomyocyte‐specific αMHC promoter in the animal heart. Endogenous exosomes released from the transgenic mouse cardiomyocytes were labeled and tracked. Moreover, the loxP‐STOP‐loxP CD63NanoLuc cassette was introduced to achieve inducible expression of the CD63NanoLuc reporter in vivo upon tamoxifen treatment, allowing temporal control of exosome labeling and tracking and making such mice more versatile to satisfy diversified research goals. The specific labeling and tissue distribution of endogenous exosomes released from cardiomyocytes were demonstrated by luciferase assay and non‐invasive bioluminescent live imaging. Finally, when bred with gene‐specific floxed mice, this model can be used to study gene‐specific exosome functions. This endogenous exosome tracking mouse provides a valuable tool for a range of research applications.
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

Background

The 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.

Methods

CRISPR‐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.

Results

Sex (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).

Conclusion

Preliminary 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.
Protective Effects of Pegylated Choline Acetyltransferase in a Murine Model of DSS Colitis
Authors: Tsaava Téa , Tynan Aisling , Hepler Tyler D. , Gabalski Arielle H. , Li Jian Hua , Hide Daniel M. , Chang Eric H. , Tracey Kevin J. , Chavan Sangeeta S.
Published: May 2022

Inflammatory bowel disease (IBD), including Crohn’s disease and ulcerative colitis, affects up to 3 million people in the United States alone, significantly worsens a patient’s quality of life, and is associated with a high financial burden. Current treatments for IBD are mainly biologicals that have significant side effects. No efficient pharmacological treatments are currently available for IBD. Previous studies established that activation of the vagus nerve‐based inflammatory reflex inhibits cytokine production and regulates immune responses through cholinergic signaling. Acetylcholine released by a T cell subset expressing choline acetyltransferase (ChAT), the enzyme that catalyzes the biosynthesis of acetylcholine, inhibits cytokine production via signaling through α7 nicotinic acetylcholine receptors (α7nAChR). However, the possibility of controlling inflammation by systemic administration of ChAT is unexplored. Here we examined the efficacy of ChAT in improving disease activity in a preclinical model of colitis. To prolong the half‐life of circulating ChAT, the protein was modified by covalently attaching repeating units of polyethylene glycol (PEG), resulting in enzymatically active PEG‐ChAT. We utilized a well‐established model of colitis ‐ dextran sulfate sodium induced colitis. Wild type C57/black 6 mice were exposed to 4% DSS (M.W 36 000‐50 000) in drinking water for 7 days prior to administration of PEG‐ChAT. Starting on day 8, animals received PEG‐ChAT or vehicle intraperitoneally twice per day for 7 days. PEG‐ ChAT treated mice showed a significantly lower disease activity index as compared with vehicle treated controls with the disease (d11 p=0.006 ChAT mice: 0.17DAI±0.05, n=9 versus vehicle mice: 0.66DAI±0.13, n=10). The decreased severity of colitis in PEG‐ChAT‐treated mice as compared with vehicle‐treated controls is further demonstrated by a significant improvement in body weight gain (d14 p=0.01 ChAT mice: ‐5.1%±1.1, n=10 versus vehicle mice: ‐15.5%±2.9, n=10) and colon length (p=0.006 ChAT mice: 7.15cm±0.2, n=10 versus vehicle mice: 6.2cm±0.2, n=10). In conclusion, PEG‐ChAT reduces disease activity in a preclinical model of colitis. Together these studies provide new insights into the role of PEG‐ChAT as an experimental anti‐inflammatory therapeutic modality for the treatment of IBD.
The Effects of Dexamethasone Treatment and Dietary Fat Composition on Liver Lipid Metabolism in Mice Consuming High Fat Diets
Authors: Powell Zereque , Brown Katie , Son Wangkuk , Lin Jason , Puppa Melissa
Published: May 2022

Omega‐3 fatty acids are essential fats that the body cannot produce on its own and play a very integral role in the body in regard to inflammation and hormone production. Glucocorticoids are hormones that are also effective in inflammation reduction and suppressing the immune system; however, often have many detrimental side effects such as adipose accumulation, muscle atrophy and metabolic dysfunction. The purpose of this research experiment was to examine the effects dexamethasone treatment in combination with diets high in either omega‐3 fatty acids or omega‐6 fatty acids on markers of lipid metabolism in the liver. For a span of 4 weeks, two groups of mice were placed on a diet consisting of 45% fat containing either high omega‐3 or omega‐6 fatty acids. On the fifth week mice were randomized to two groups per diet and received either PBS placebo or Dexamethasone (Dex) injections daily. We hypothesized that Dex treatment would induce liver lipid accumulation and an omega‐3 rich diet would prevent this. At the completion of the 5 weeks, tissue was harvested and liver triglycerides were measured. There was a main effect of omega‐3 diet to have lower liver triglyceride levels (p=0.01). Fasn, Crat, Hsl, Cd36, and Fabp mRNA levels were measured in liver tissue. The resulted show that CratmRNA (p=.0153) and HslmRNA (p=.0417) was increased with omega‐3 rich diet, but there was no effect of Dex treatment. The Cd36 (p=.0495) and Fabp(p=.0229) mRNA increased in omega‐6 rich diets treated with Dex and omega‐3 high fat diet attenuated this increase. It can be concluded that one week of Dexamethasone treatment altered liver lipid signaling primarily by increasing lipid import and transport regulators, and omega‐3 high fat diet was able to prevent this increase. Additionally, omega‐3 high fat diet increase markers of lipid metabolism that were unaltered with one week of dex. These data suggest that omega‐3 fatty acids may help prevent the detrimental side effects of glucocorticoid treatment.
Single‐Molecule Analysis of in vivo DNA Replication Origin Licensing Stoichiometry
Authors: Rhind Nick , Chung Johnson , Friedman Larry , Gelles Jeff
Published: May 2022

The organization of DNA replication location and timing is regulated by the establishment of replication origins. Origins are established in G1 by the loading of a pair of MCMs, the catalytic core of the replicative helicase, as an inactive double‐hexamer complex. Activation of MCM during S phase is the rate‐limiting step for replication initiation, and therefore regulates where and when replication occurs. We have previously shown that origins at which multiple MCM complexes are loaded have a higher probability of initiation and therefore replicate earlier, on average. Bulk biochemical experiments suggest the ARS1, a early‐firing budding yeast origin, loads, on average, about three MCM complexes in G1, whereas ARS316, a late‐firing origin, loads less than one. However, these bulk measurements have two major drawbacks. First, the required normalization controls make our absolute stoichiometry estimates uncertain. Second, they only reveal the average numbers of MCM's loaded; they provide no insight into the distribution of MCM stoichiometry, information which could elucidate how MCM loading stoichiometry is controlled.
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.

Methods

C57BL/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.

Results

Nuclear 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.

Conclusion

These 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.
Identification and Characterization of Small Regulatory RNA in Streptococcus
Authors: Hoxha Klea , Oakes William C. , Ward Nicole M. , Busby L. B. , Pérez‐Alvarado Gabriela C. , Lee Brian M.
Published: May 2022

The human gut is a rich habitat for a diverse population of bacteria. These bacteria play a vital role in a multitude of functions by interacting with each‐other and the human host. They can play roles in immune responses, metabolism, and even neurological activities. Some inhabitants of the gut biome include the lactic acid bacteria: Streptococcus thermophilus, Lactobacillus acidophilus, and Lactobacillus bulgaricus. These bacteria are essential members of the flora and are non‐pathogenic, however, some of their relatives like Streptococcus pyogenes cause severe diseases like toxic shock syndrome and flesh‐eating disease. The metabolic functions of these bacteria are mediated by small regulatory RNAs (sRNAs), which are noncoding RNA sequences that fold into complex stem‐loop structures. The goal of this project was to identify and characterize the small regulatory RNAs that mediate bacterial metabolism and host interactions in non‐pathogenic, lactic acid bacteria. A small regulatory RNA that is conserved among different streptococcal species was identified. In S. pyogenes, this conserved sRNA, named MarS, is associated with virulence, however, the function of its ortholog found in non‐pathogenic bacteria, AsdS sRNA, has not been well‐characterized. Computational methods were used to elucidate the function of this sRNA as well as predict its 2D and 3D structures. The gene containing the target sRNA was isolated and RNA constructs were designed to characterize regions that are part of it. Future work will be focused on structure determination and characterizing interactions to target sRNA through biophysical and biochemical methods.
Different human placental epigenetics changes in pregnancies affected with preeclampsia and intrauterine growth restriction
Authors: Norton Carter , Holmstrom Joshua , Reynolds Paul , Jenkins Tim , Arroyo Juan
Published: May 2022

The placenta facilitates nutrient and gas exchange between the mother and the fetus. Intrauterine growth restriction (IUGR) and preeclampsia (PE) are placental pathologies known to cause pre‐term delivery and diverse disorders in neonates. Placental adversities during complicated pregnancy may cause epigenetic changes that can altered fetal development. DNA methylation is a heritable epigenetic process that can regulate important genetic mechanisms and processes critical for optimal placental and fetal development. Our objective was to determine differential placental DNA methylation from normal, PE and IUGR human patients. Placental DNA was extracted from PE, IUGR, and control patients. Extracted DNA was then bisulfite converted for DNA methylation analysis. Bisulfite‐converted DNA was hybridized to Illumina’s EPIC 850K methylation array for analysis. Methylation data was SWAN normalized and significant regions were identified using USEQ’s “methylation array scanner” and “enriched region maker” applications (with a threshold of significance of an adjusted p‐value < 0.001). UCSC’s Genome browser was used to identify potentially impacted gene promoters associated with differentially methylated regions. Additionally, Stanford’s GREAT analysis tool was used to perform a GO term analysis to find biological processes and cellular components associated with differentially methylated regions. We observed nine significantly hypomethylated regions, six for PE (including the HIST1H4L and NAPRT‐1) and three for IUGR (including CRABP1). Two regions significantly hypomethylated for both PE and IGUR gene promoters. We conclude that although we identified a unique methylation profile for both PE and IUGR, the overlap that was seen with some methylation alterations between the two pathologies could explain some of the clinical similarities observed during these obstetrics complications. Our results may provide some insights into placenta cell regulatory response to developmental syndromes. Information in these cellular components and biological pathways could be useful in determining cellular level responses to complicated diseases associated with PE and IUGR.
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.

Methods

Triglycerides, 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.

Results

Efferocytosis 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.

Conclusion

These results suggest that a loss of GCLM and GSH during efferocytosis impairs adequate efferocytosis.
A phosphorylation network controls the stability of alpha‐arrestins Aly1/Art6 and Aly2/Art3
Authors: Jordahl Eric M. , Bowman Ray W. , Davis Sydnie , Ozbaki‐Yagan Nejla , Chiang Annette , Hedayati Stefanie , Barsouk Hannah , O'Donnell Allyson F.
Published: May 2022

Selective protein trafficking controls the repertoire of membrane proteins at the cell surface. This contingent of surface proteins regulates nutrient/metabolite balance and response to extracellular cues. Environmental changes trigger cellular signaling that drives transitions in the plasma membrane proteome. These alterations are achieved in no small part through regulation of the α‐arrestins, an emergent and powerful class of protein trafficking adaptors. The α‐arrestins are largely cytosolic proteins that are transiently recruited to membranes via binding to membrane protein motifs. α‐Arrestins bring with them a ubiquitin ligase, which stimulates ubiquitination and subsequent endocytosis of membrane proteins. Phosphorylation of α‐arrestins is key to regulating their trafficking function; dephosphorylated α‐arrestins are generally ‘active’ endocytic adaptors. While some kinases and phosphatases for α‐arrestins are known, the degree of α‐arrestins phosphorylation is staggering, with >40 phosphorylation sites identified for a single α‐arrestin, and suggests we still have much to learn about α‐arrestins regulation. We sought to determine what kinases and phosphates regulate paralogous α‐arrestins Aly1 and Aly2 using a genetic screen in yeast. Using an array of all known non‐essential kinase and phosphatase deletions, we determined which of these influenced α‐arrestin‐mediated resistance to rapamycin, an inhibitor of the TORC1 nutrient‐sensing kinase. We identified a large cohort of kinases and phosphates that influenced Aly‐dependent phenotypes, causing electrophoretic mobility changes and, in many cases, diminishing the abundance of these α‐arrestins. We focused our studies on the Sit4 protein phosphatase, a key regulator downstream of TORC1 signaling able to influence other α‐arrestins, identified in our screen. Strikingly, Aly1 and Aly2 were hyperphosphorylated and destabilized in the absence of Sit4, suggesting that excessive phosphorylation may promote degradation of these α‐arrestins. For Aly2, but not Aly1, degradation in the sit4∆ cells could be reversed by loss of the vacuolar protease Pep4, indicating that vacuolar degradation predominates for Aly2 under these conditions. Loss of the Npr1 kinase in sit4∆ cells restored Aly protein abundances and reversed the hyperphosphorylation, demonstrating that this kinase is responsible for the excess Aly phosphorylation in sit4∆ cells. This is a remarkable finding as typically Npr1 is considered inactive in sit4∆, however, we suggest that Npr1 is selectively active in the absence of Sit4, able to modify some substrates but not others. We define new features of TORC1 signaling in regulating α‐arrestin phosphorylation and stability.
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.
Elucidating the Anticancer Mechanism of Arachidin‐1 in Triple‐Negative Breast Cancer Cells
Authors: Weaver Alexx P. , Mohammadhosseinpour Sepideh , Medina‐ Bolivar Fabricio
Published: May 2022

Triple‐negative breast cancer (TNBC) is one of the deadliest forms of breast cancer that affects women worldwide. TNBC is unresponsive to standard hormonal cancer treatments, leading to high mortality rates. Treatment resistance and low survival rates necessitate investigating alternative treatment options for this type of cancer. Plant natural products, such as the prenylated stilbenoid arachidin‐1 from peanut, have shown cytotoxicity to certain cancer cells. To this end, the goal of this study was to examine the apoptotic effects of arachidin‐1 in TNBC cell lines MDA‐MB‐231 and MDA‐MB‐436. To produce arachidin‐1, peanut hairy root cultures were co‐treated with elicitors, and arachidin‐1 was purified from extracts of the culture medium by semi‐preparative high performance liquid chromatography. The apoptotic effects of arachidin‐1 were studied via western blotting by checking the protein levels of PARP, caspase‐8, caspase‐9, and survivin. Vinculin and GAPDH were used as protein loading controls. Results showed that as arachidin‐1 concentration increased, the amount of cleaved PARP also increased. Similarly, the levels of cleaved caspase‐9 increased as concentrations of arachidin‐1 increased. Whereas the level of survivin, an apoptosis inhibitor protein, decreased at increasing concentrations of arachidin‐1. The increased expression of cleaved PARP, cleaved caspase‐9, and reduced surviving expression in TNBC cells treated with arachidin‐1 represents the activation of intrinsic apoptosis in the cells. This accentuates the importance of expanding our understanding of the connection between stilbenoids and triple‐negative breast cancer cells. In future research, we will study the effect of arachidin‐1 on signaling pathways in TNBC cell lines.
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

Purpose

Angiogenesis, 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.

Methods

Using 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.

Results

Our 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.

Conclusions

We conclude that IL‐33/ST2L signaling plays a significant role in angiogenesis and neovascularization by regulating endothelial permeability and BBB integrity.
Effects of Senescent Human Endothelial Cells on Neuronal Viability and Neurite Outgrowth Are Not IL‐8‐ or Serpin E1‐Dependent
Authors: Grodzki Ana C. , Lin Yun , Knowlton Anne , Lein Pamela
Published: May 2022

It is estimated that more than 50% of adults over 80 will develop dementia, including Alzheimer’s disease (AD), and the primary risk factor for dementia is aging. Aging provides the substrate for disease through increased inflammation and cellular dysfunction, which in turn sets the stage for neurodegenerative disease. In a previous study we validated replicative senescence, a model of cell senescence that permits the study of cells as they progress to senescence, in human coronary artery endothelial cells (HCAEC). Here, we tested the hypothesis that senescent endothelial cells, which accumulate with aging and release senescence‐associated secretory phenotype (SASP) and extracellular vesicles, create a pro‐inflammatory environment that adversely affects the viability and morphology of neurons, which are endpoints implicated in AD. We categorized the phenotypes of HCAEC by passage number and identified them as EP‐ early passage (non‐senescent), ES‐ early senescence and LS‐ late senescence. HCAEC at these varying stages of senescence were co‐cultured with LUHMES cells, a human neuronal cell line. Co‐culturing HCAEC LS with LUHMES significantly decreased LUHMES viability, while co‐culturing HCAEC ES with LUHMES decreased the number of neurites extended by LUHMES in 24h and decreased the length of LUHMES’ neurites in 48h, when compared to LUHMES grown in the absence of HCAEC for the same period of time. HCAEC EP or LS co‐cultured with LUHMES did not affect the number or length of the LUHMES’ neurites, when compared to LUHMES grown in the absence of HCAEC. Using a cytokine array containing probes for 105 analytes, we detected 17 inflammatory mediators released by the co‐cultures of HCAEC (EP, ES and LS) with LUHMES. Of these 17 mediators, 14 were also found at supernatants of HCAEC (EP, ES and LS) grown in the absence of LUHMES, and none of the analytes were detected in supernatants from LUHMES grown in the absence of HCAEC. Statistical analysis of the intensity of the positive reaction for inflammatory mediators in the array was run for all the 17 positive analytes, and only IL‐8 and Serpin E1 release into the supernatants of the co‐cultures were significantly increased in co‐cultures of LUHMES and senescent HCAEC (ES or LS), when compared to the levels of the same mediators released by co‐cultures of LUHMES and EP HCAEC. However, exposing LUHMES to IL‐8 and Serpin E1, in the absence of HCAEC, did not affect LUHMES viability, the number or length of LUHMES’ neurites or LUHMES’ NF‐kB activation. The results from the LUHMES and HCAEC co‐cultures study supports a role for aging endothelial cells in the toxicity and morphological remodeling of the surrounding neurons, which may contribute to the development and progression of neurodegenerative diseases. However, our data exclude IL‐8 and Serpin E1 as direct independent mediators of senescent HCAEC on neuronal cytotoxicity and neurite remodeling.
The Fragile X Syndrome‐Related Protein, FMRP, Modulates Innate Immune Gene Expression in Macrophages
Authors: Vail Krystal J. , Patrick Kristin L. , Watson Robert , Fischer Tracy
Published: May 2022

Fragile X mental retardation protein (FMRP) is an RNA‐binding protein with a wide range of functions, including mRNA translation, mRNA transport, and mitochondrial organization. Loss of FMRP due to silencing of the FRM1 gene is associated with intellectual disability and has been implicated in viral pathogenesis and cancer risk. However, the contribution of FMRP to innate immunity in response to bacterial infection remains understudied. Given the function of FMRP in post‐transcriptional regulation, we hypothesized that loss of FMRP would impair repression of inflammatory cytokines in response to infection with the bacterial pathogen Listeria monocytogenes. We used RNA‐seq as an unbiased approach to identify global gene expression changes in FMRP‐deficient murine macrophages, both at rest and during infection with Listeria monocytogenes. We identified 22 differentially expressed genes that were upregulated in resting FMRP KO macrophages, including aconitate decarboxylase 1 (ACOD1, IRG1), erythroid differentiation regulator 1 (Erdr1), and Cxcl10, as well as 6 downregulated genes, including microRNA 6236 and mitochondrially encoded ATP synthase 8 (MT‐ATP8). Unbiased canonical pathway analysis of the upregulated genes in resting FMRP KO macrophages revealed significant enrichment of genes related to phagosome maturation and immunity. Within the downregulated genes, unbiased canonical pathway analysis revealed pathways related to EIF2 signaling and BEX2 Signaling. Analysis of L. monocytogenes infected FMRP KO macrophages revealed 31 upregulated genes, including the E3 ubiquitin ligase gene midline 1 (Mid1, Trim18) and the complement genes C1QA and C1QB, as well as 8 downregulated genes including plasminogen activator urokinase (PLAU). Canonical pathway analysis of the downregulated genes included DNA‐damage signaling and role of pattern recognition receptors in recognition of bacteria and viruses. Surprisingly, when comparing differentially enriched genes in infected macrophages to uninfected macrophages, a greater number of innate immune transcripts were upregulated in resting macrophages lacking FMRP. Together, these data suggest that FMRP may also have a role in innate immunity.
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.