bluuebunny/crossref_metadata_2025_split huggingface dataset:
Insights into the Role of Occluding Septate Junctions in the Maintenance of Salt and Water Balance in Mosquito (Aedes aegypti) Larvae
Authors: Jonusaite Sima , Kelly Scott Phillip , Donini Andrew 
Published: November 2023

Septate junctions (SJs) are specialized occluding junctions that function as paracellular diffusion barriers in the epithelia of invertebrates. SJs are composed of transmembrane and cytosolic proteins which are reported to be essential for epithelium integrity. This is of particular importance to aquatic animals because their habitat is often compositionally different from their internal body fluids. Furthermore, aquatic habitats are subjected to alterations in their solute composition by anthropogenic and climatic factors. In this regard, the contribution of SJ proteins to the regulation of paracellular solute movement across epithelia during and/or following changes in an aquatic invertebrate's environmental conditions is not known. It can however be hypothesized that epithelial SJ protein abundance is altered in response to environmental changes, reflecting their involvement in the regulated movement of solutes across the paracellular pathway. To begin testing this hypothesis, a role for SJ proteins in the maintenance of salt and water balance in larval mosquito Aedes aegypti was considered. These animals reside in freshwater (FW), a hypoosmotic environment, but are capable of tolerating saline environments that reflect isosmotic conditions. Genes encoding A. aegypti SJ proteins Megatrachea, Sinuous, Kune‐kune, Coracle, Scribble, Discs large, Neurexin IV, Lethal giant larva, Gliotactin, Mesh and Snakeskin were identified and their expression was examined in midgut, Malpighian tubules, hindgut and anal papillae, which are tissues that participate in osmoregulation. SJ proteins exhibited tissue specific differences in transcript abundance and immunolocalized to SJ domains between epithelial cells and in the apical membrane domain of the syncytial anal papillae epithelium. Upon rearing larvae in brackish water (30% seawater) tissue‐specific alterations in SJ transcript and protein abundance were observed. Furthermore, in the midgut, salinity‐induced differences in SJ protein abundance occurred in conjunction with differences in the paracellular permeability of this tissue. Taken together, data support the hypothesis that SJ protein abundance is altered in response to environmental change and that SJ proteins play an important tissue‐specific role in the maintenance of salt and water balance in the aquatic larvae of A. aegypti. Since A. aegypti is a vector of re‐emerging viral diseases, Dengue and Chikungunya, and has been shown to successfully breed in both salinated and freshwater in regions where these diseases are active, a complete understanding of the role of SJ proteins in larval mosquito salt and water balance is important.Support or Funding InformationNatural Sciences and Engineering Research Council of Canada Discovery Grants to S.P.K and A.D., an Ontario Graduate Scholarship to S.J
Urinary Excretion of Kidney Aquaporins as Possible Biomarker of Diabetic Nephropathy
Authors: Procino Giuseppe , Rossi Luigi , Nicoletti Maria Celeste , Carmosino Monica , Di Franco Antonella , Indrio Francesca , Lella Rosa , Laviola Luigi , Gesualdo Loreto , Svelto Maria 
Published: November 2023

BackgroundDiabetic nephropathy (DN) is is classified into four hierarchical glomerular lesions on the basis of histological analysis of kidney biopsies. This is clearly a highly invasive diagnostic approach numerous studies have been conducted to identify a non‐invasive biomarker of the disease, but none of these is considered to be sufficiently specific and sensitive Experimental evidences showed that the water channel aquaporins (AQP), expressed at the plasma membrane of epithelial cells lining in the kidney tubule, are often dysregulated during DN. Interestingly, apical membrane protein are excreted in the urinary space as nano‐scale exosome vesicles, to an extent proportional to their amount expressed at the plasma membrane, thus allowing their quantitation in urine samples.MethodsIn this work we compared excretion of AQP5 and AQP2 (uAQP5 and uAQP2) in the urine of 35 diabetic patients: 12 with histological diagnosis of DN, 12 with with normal renal function and normoalbuminuric (DM) and 11 with non‐diabetic nephropathy (NDN). Both proteins were quantified by ELISA method on urine samples and the results were validated by Western blotting analysis on the exosome fraction isolated by ultracentrifugation.ResultsBothELISA and Western blotting analysis independently showed that uAQP5 was three‐ to fourfold higher in DN patients (7458 ± 2576, p <0.05) compared to the other two groups (D 847,2 ± 70,51; NDN1882 ± 215). uAQP5 was not statistically different between D and NDN patients. Strikingly, uAQP5 dramatically increased in parallel with the clinical severity of DN. The same analysis showed comparable results for uAQP2.ConclusionsOur data showed, for the first time, that uAQP5 and uAQP2 dramatically and specifically increase in patients with DN and positively correlate with the histological class of DN. Taken together these data suggest a possible role of AQP5 and AQP2 as novel non‐invasive biomarkers to help diagnosing DN and classifying its histological stage.Support or Funding InformationNon U.S.
Acute Pancreatitis Involves Alterations in the Transport of Amino Acids Necessary for Glutathione Synthesis
Authors: Kuster Evelyne , Graf Rolf , Verrey François , Camargo Simone Mafalda 
Published: November 2023

Acute pancreatitis (AP) is a serious inflammatory disease of the exocrine pancreas. During its pathogenesis, reactive oxygen species (ROS) have deleterious effects and they can be neutralised by glutathione (GSH), an intracellular antioxidant. GSH is synthesised from glutamate, glycine and cysteine but amino acid transport into acinar cells remains poorly understood. It is the aim of this work to characterise the transport of glycine, glutamate and cysteine (as well as their precursors glutamine and cystine) for the production of GSH during AP in isolated pancreatic acinar cells. For this purpose, AP was induced in C57BL6/J mice using supraphysiological doses of cerulein (12 times hourly 50μg/kg intraperitoneally) and acinar cells were isolated 12, 24, 36 and 72 hours after the first injection.Glutamate, glycine and cysteine concentrations are rapidly depleted in acinar cells during AP. Concurrently, gene expression levels of enzymes necessary for GSH synthesis are increased. Levels of free GSH (oxidised and reduced) are initially diminished but they are increased during the following regeneration phase. Gene expression levels of the acinar glutamine transporters (LAT1 (slc7a5), LAT2 (slc7a8), SNAT2 (slc38a2), SNAT3 (slc38a3) and SNAT5 (slc38a5) as well as the glycine transporter GlyT1 (slc6a9) are impaired during AP, while the cystine/glutamate exchanger xCT (slc7a11) is remarkably increased during the early phase. Immunofluorescence analysis revealed low xCT expression on pancreatic acinar cells during the healthy state, whereas the protein level increases significantly during AP, as was also shown via Western blotting. Uptake experiments using radiolabelled cystine confirmed an enhanced capacity of isolated acinar cells to import cystine during AP, presumably via the up‐regulated xCT protein levels.Our data suggests that acinar cells adapt amino acid transport and enzymes necessary for GSH synthesis to match the cells’ needs in the oxidative stress situation occurring during AP. Specifically, xCT expression is up‐regulated in acinar cells to enhance cystine import, the rate‐limiting factor of GSH synthesis.
Smoking‐induced urothelial cell accumulation of platelet‐activating factor: Implications for interstitial cystitis/bladder pain syndrome
Authors: McHowat Jane , Marentette John , Isbell T. Scott , Campian E. Cristian 
Published: November 2023

Interstitial cystitis/bladder pain syndrome (IC/BPS) may encompass many diseases with causes such as stress, irritants, previous bladder infections, a dysregulated immune response, inappropriate diet or cigarette smoking. We have determined previously that cigarette smoke extract (CSE) results in accumulation of platelet‐activating factor (PAF) via activation of calcium‐independent phospholipase A2 and inhibition of PAF acetylhydrolase (PAF‐AH). We hypothesized that cigarette smoking may predispose smokers to IC/PBS via PAF accumulation in the bladder wall. Incubation of human urothelial cells (HUC) with cigarette smoke extract (CSE) resulted in a significant inhibition of PAF‐AH activity and PAF accumulation. Inhibition of HUC with CSE resulted in a decrease in electrical resistance and rate of wound healing. Histological examination of bladders removed from mice exposed to cigarette smoke showed urothelial cell thinning and sloughing. Immunohistological staining indicated an increase in PAF accumulation in the bladder wall of mice exposed to cigarette smoke when compared to room air. PAF content in the urine obtained from IC/BPS patients was greater than that observed in controls (PAF:creatinine ratio 13.5 ± 1.5 compared with 5.4 ± 0.6, n=3, p<0.05). PAF content was greater in IC/BPS patients who were current smokers (PAF: creatinine ratio 34.3 ± 10.4, n=3) than in IC/BPS patients who did not smoke. We conclude that cigarette smoking is associated with PAF accumulation in the bladder wall that could contribute to impairment of urothelial function in IC/BPS patients.Support or Funding InformationSaint Louis University President's Research Fund
Mutation in the PPARγ Ligand Binding Domain Impairs the Anti‐inflammatory Action of PPARγ
Authors: Mukohda Masashi , Ketsawatsomkron Pimonrat , Stump Madeliene , Liu Xuebo , Guo Deng F. , Rahmouni Kamal , Quelle Frederick W. , Sigmund Curt D. 
Published: November 2023

Peroxisome proliferator‐activated receptor gamma (PPARγ) is a ligand‐activated transcription factor with anti‐inflammatory properties. One of the anti‐inflammatory mechanisms is antagonism of nuclear factor kappa B (NFκB) activity. Synthetic activators of PPARγ are known to inhibit the formation of atherosclerotic lesions in animal models. In contrast, transgenic mice expressing a dominant negative (DN) mutant of PPARγ specifically in vascular smooth muscle cells (SMC) exhibited exacerbated atherosclerosis with increased expression of NFκB target genes in aortic tissue. Based on this, we hypothesized that the exaggerated atherosclerosis in these mice is due to increased NFκB‐mediated inflammation. We tested the role of SMC PPARγ in controlling inflammatory gene expression. Mesenteric SMCs were cultured from transgenic mice designed to conditionally express wild‐type (WT) or DN PPARγ (P467L) in response to Cre‐recombinase (delivered by adenovirus). PPARγ expression remained silent in control SMC infected with AdGFP but was induced in SMC infected with AdCRE. The PPARγ target gene aP2 was induced in WT‐Cre SMC but not in P467L‐Cre SMC. TNF‐α, IL‐1β or LPS all induced NFκB target gene (VCAM1, MCP‐1, iNOS and MMP9) expression in WT SMC infected with AdGFP (WT‐GFP). Expression of these genes was reduced in WT SMC infected with AdCRE (WT‐Cre). For example, TNF‐α induced MMP9 expression by 6.0±0.3‐fold in WT‐GFP vs 0.7±1.2‐fold in WT‐Cre (p<0.01). In contrast, the ability of TNF‐α to induce NFκB target gene expression was increased in P467L cells infected with AdCRE (P467L‐Cre) compared to those infected with AdGFP (P467L‐GFP, MMP9: 6.3±0.2 vs 3.2±0.5, p<0.01). Similarly, TNF‐α or LPS‐induced NFκB activity assessed by an adenovirus‐NFκB‐Luciferase reporter was blunted in WT‐Cre SMC but increased in P467L‐Cre SMC. Although expression of the NFκB subunit, p65, was not altered in any of these cells, nuclear export of p65 was accelerated in WT‐Cre SMC, but prevented in P467L‐Cre SMC. WT PPARγ co‐precipitated with p65, but the interaction between p65 and P467L PPARγ was severely impaired in HEK293 cells. Finally, to examine mechanisms in vivo, we crossbred transgenic mice expressing WT PPARγ specifically in SMC (S‐WT) with mice expressing luciferase under control of a NFκB‐responsive promoter. TNF‐α (500 ng/ml, 24 hr)‐induced NFκB activity was decreased in aorta and carotid artery from S‐WT mice compared to control mice (aorta: 4.7±1.1 vs 6.7±0.7, p<0.05, carotid artery: 4.0±0.6 vs 8.7±1.2, p<0.01). We conclude that PPARγ in SMC exerts anti‐inflammatory effects by inhibition of NFκB activity through a mechanism promoting nuclear export of p65. This mechanism is abolished by P467L mutation in PPARγ.Support or Funding InformationThis work was supported by National Institutes of Health Grants HL048058, HL062984 and HL08307 (to Curt D. Sigmund).
Toll‐like Receptor 4 Mutation Moderates Hyperhomocysteinemia‐ Induced Hypertension
Authors: Familtseva Anastasia , Chaturvedi Pankaj , Kalani Anuradha , Metreveli Naira , Kunkel George H , Tyagi Suresh C 
Published: November 2023

Hyperhomocysteinemia (HHcy) has been observed to promote hypertension, but the mechanisms are unclear. Toll‐like receptor 4 (TLR‐4) is a cellular membrane protein that is expressed on immune cells and non‐immune cells (endothelial cells, vascular smooth muscle cells). TLR‐4 activation has been known to promote innate immune system response with inflammatory cytokine up‐regulation. TLR‐4‐ driven inflammatory cascade has been shown to be involved in pathogenesis of hypertension and atherosclerosis. In this study, we hypothesize that HHcy‐ induced TLR‐4 activation promotes inflammatory cytokine up‐regulation (IL‐1β, TNF‐α) and initiation of mitochondrial‐ dependent apoptosis leading to cell death and chronic vascular inflammation that predispose to hypertension. To test this hypothesis, we used 12 week old C57BL/6J mice (WT); Cystathionine‐β‐synthase deficient mice (CBS+/−) with genetic HHcy; C3H/HeJ (C3H) mice with TLR‐4 mutation and CBS+/−/C3H mice. Blood pressure measurement, western blotting (Caspase‐9, Caspase‐3, HSP60, TLR‐4), q‐PCR (Bax, IL‐1β, TNF‐α, NF‐kB), immunohistochemistry (cleaved Caspase‐3) and TUNEL assay were used. Blood pressure was up‐regulated in CBS+/− mice (systolic: 156.54 ± 10.2 mmHg; mean: 132.4 ± 11.16 mmHg) compared to WT mice (systolic: 121.8 ± 3.6 mmHg; mean: 97.2 ± 6.5 mmHg). Interestingly, blood pressure values were significantly decreased in CBS+/−/C3H mice (systolic: 130.13 ± 9.3 mmHg; mean: 105.8 ± 12.6 mmHg) compared to CBS+/− mice. q‐PCR showed significant up‐regulation of Bax, IL‐1β, TNF‐α mRNA expression in the mesenteric artery of CBS+/− mice compared to control and C3H mice. In addition, Bax, IL‐1β, TNF‐α mRNA expression was decreased in the mesenteric artery of CBS+/−/C3H mice compared to CBS+/− mice. Western blotting validated increase of Caspase‐9 and Caspase‐3 protein expression in the mesenteric artery of CBS+/−mice compared to WT and C3H mice. Immunohistochemistry confirmed cleaved Caspase‐3 up‐regulation in the mesenteric artery of CBS+/− mice compared to control and C3H mice. TUNEL assay showed DNA fragmentation in the mesenteric artery of CBS+/− mice compared to control and C3H mice. In addition, active Caspase‐3 and DNA fragmentation were significantly decreased in the mesenteric artery of CBS+/−/C3H mice compared to CBS+/− mice. In conclusion, our data suggested that HHcy‐induced TLR‐4 activation promotes inflammatory cytokine (IL‐1β, TNF‐α) and Bax up‐regulation followed by mitochondrial apoptosis leading to cell loss and chronic vascular inflammation that contributes to hypertension. Therefore, TLR‐4 mutation abates vascular inflammation and mitochondrial‐dependent apoptosis that moderate hypertension.Support or Funding InformationThis work was supported by NIH grants: HL‐74185
Domain of hCLCA1 that is responsible for macrophage activation
Authors: Ching John C.H. , Loewen Matthew E 
Published: November 2023

The CLCA gene family produces both secreted and membrane‐associated proteins that modulate ion‐channel function, drive mucus production and have a poorly understood pleiotropic effect on airway inflammation. The mechanism of how hCLCA1 regulates airway inflammation remains unclear. However, hCLCA1 is highly expressed in airway diseases in which alveolar macrophages’ are central to disease progression. Thus, it is possible that hCLCA1's role in airway inflammation is to regulate the immune response of macrophages. Previously, we showed that secreted hCLCA1 is able to activate macrophages, inducing them to express cytokines and to undertake a pivotal role in airway inflammation. In this study, we have purified the functional domains of hCLCA1, and we activated macrophages with these domains. There are three functional domains in hCLCA1 (hydrolase domain, von Willebrand type A domain, and fibronectin type III domain), and we found that one of these hCLCA1's domains significantly induced the expression of pro‐inflammatory cytokines in macrophages. To further investigate this activation, we have also found the cell signaling pathways that are involved in this activationSupport or Funding InformationNatural Sciences and Engineering Research Council (NSERC) 371364/2010 to MEL
Absence of PKCα Alters the Renal Immune Response in Angiotensin II‐dependent Hypertension
Authors: Borchart Matthew R. , Pulous Fadi E. , Wang Yirong , Sun Michael A. , Himmel Nathaniel J. , Vanderford Thomas H. , Blount Mitsi A. 
Published: November 2023

Angiotensin II (Ang II) stimulation of T‐cell proliferation and accumulation plays an important role in the development of hypertension and end‐stage kidney damage. Unfortunately, the signaling mechanisms behind the immune response within the kidney remains underexplored. One pathway that is important to both Ang II and T‐cell response is mediated by PKCα. To determine the role of PKCα in facilitating T‐cell infiltration in the kidney during Ang II‐dependent hypertension, PKCα null (KO) mice were treated with either saline or Ang II and compared to PKCα control (WT) mice treated similarly. After 14 days, systolic blood pressure, water intake and urine output were increased in all Ang II‐treated mice. Kidney damage and collagen deposition is elevated with ANG II treatment and appears more extensive in KO mice. Ang II‐treatment significantly increased spleen weight in WT mice only; suggesting that the absence of PKCα attenuates increased immune cell production. Histological examination of immune cell infiltration of the renal cortex revealed that Ang II‐treatment increased CD4+ and CD8+ T‐cells but not macrophage invasion in both WT and KO mice and suggests that CD4+ T‐cell recruitment is higher in Ang II‐treated WT compared to treated KO mice. Immune cell infiltration and cytokine production was also measured via flow cytometry and results suggest that PKCα is important for Ang II‐meditated cytokine release in renal tissues. Collectively, these data suggest that the absence of PKCα decreases inflammatory response to Ang II‐induced hypertension in the kidney.Support or Funding InformationNIDDK R25‐DK101390
Screening Bioactivity of Virus Surface Glycoprotein Peptides using C. elegans Electropharyngiogram
Authors: Hincks Joshua B , Monrean Billyann B , Lu Brianna , Taylor Barbara E , Hueffer Karsten , Harris Michael B 
Published: November 2023

The rabies virus surface glycoprotein contains an amino acid sequence similar to neurotoxin peptides known to bind to and inhibit the function of nicotinic acetylcholine receptors (nAChR). In preliminary experiments, we have shown that a peptide fraction of the neurotoxin‐like‐domain of the rabies virus inhibits signaling of central nervous system nAChRs. It has been shown, in the nematode Caenorhabditis elegans, that defined motor neuron MC acts as a neurogenic pacemaker for pharyngeal pumping and that nAChR are critical to excitation of the pharynx. We propose to use pharyngeal pumping in C. elegans as a screening assay for bioactivity of variants of virus glycoprotein peptides. In the present study we either administered a 29 amino acid peptide fraction (or a scrambled peptide) by microinjection through the cuticle proximal to the pharynx, or incubated animals for 90 minutes in concentrated peptide (5 or 2.5 mM) or M9 buffer solution. Following treatments, we assessed pharyngeal pumping frequency during 2‐minute observation periods using a non‐invasive electropharyngeogram (NemaMetrix Katalyst 100). Pharyngeal pumping was absent in 5 of 6 animals receiving active peptide injection, while pharyngeal pumping in those receiving scrambled peptide was identical to untreated animals. Pharyngeal pumping was similarly absent or greatly attenuated in animals incubated in active peptide compared to controls. Results indicate that internal and external exposure to a peptide proposed to inhibit nAChR attenuate pharyngeal pumping in C. elegans in a manner expected by nAChR antagonism. Results support the bioactivity of this virus peptide fraction and the utility of C. elegans electropharyngeogram as a screen for bioactivity of variants of this peptide.Support or Funding InformationWork reported in this publication was supported by the National Heart Lung and Blood Institute and National Institute of General Medical Sciences of the National Institutes of Health under awards 1R15HL126105, 1SC2GM112570 and three linked awards number RL5GM118990, TL4GM118992 and 1UL1GM118991. The work is solely the responsibility of the authors and does not necessarily represent the official view of the National Institutes of Health or any other funding body.