bluuebunny/crossref_metadata_2025_split huggingface dataset:
Functional Status of Perilymphatic Mast Cells is Critical for Lymphatic and Immune Functions
Authors: Gashev Anatoliy A. , Dusio Giuseppina F. , Gasheva Olga Yu. , Skoog Hunter , Tobin Richard , Peddaboina Chander , Meininger Cynthia , Newell‐Rogers M. Karen , Zawieja David C. , Nizamutdinova Irina Tsoy 
Published: November 2023

Mesenteric lymphatic vessels surrounded by mast cells which inhabit in high density in close proximity to lymphatic wall. In the events of acute inflammation mast cells, upon activation, release numerous bio‐active and, in particular, vasoactive mediators that continuously modulate the mesenteric lymphatic vessels and surrounding tissues. In current study we attempted to establish mechanistic links between the functional status of mast cells and the responses of mesenteric lymphatic vessels and perilymphatic mesenteric tissues to acute inflammatory insult in adulthood and elderly. We used an in vivo model of acute peritoneal inflammation induced by lipopolysaccharide (LPS) treatment (24 hours after single IP injection of LPS in dose of 10 mg/kg body weight into adult (9‐mo) and aged (24‐mo) F‐344 rats). We analyzed contractility of isolated mesenteric lymphatic vessels, mast cell activation, activation of nuclear factor‐κB (NF‐κB) without and with stabilization of mast cells by cromolyn or blockade of all types of histamine receptors and production of 27 major pro‐inflammatory cytokines in adult and aged perilymphatic mesenteric tissues and blood. We found that the reactivity of aged contracting mesenteric lymphatic vessels to LPS‐induced acute inflammation was abolished and that activated mast cells trigger NF‐κB signaling in the mesentery through release of histamine. The aging‐associated activation of mast cells maintains chronic histamine‐dependent activation of NF‐κB signaling in aged perilymphatic mesenteric tissues and limits NF‐κB activation in aged mesentery in response to acute inflammation. These findings correlate with our previously published findings which link together chronic activation of aged mast cells with diminished recruitment/activation of MHC class II+ cells and eosinophils towards/near aged mesenteric lymphatic vessels during acute inflammation (Chatterjee V, Gashev A.A. Amer. J. Physiol., 2012, Lymphatic Research and Biology, 2014). Cumulatively our studies indicate that healthy functional status of mast cells is critical for proper performance of the lymphatic and immune functions. In healthy adults the mast cells‐derived histamine is necessary trigger of the NF‐κB‐mediated reactions of mesentery to acute inflammation. The decreased ability of the aged immune system to respond to acute inflammatory challenge in peritoneal compartment of the body appears to be generated by chronically activated mast cells.Support or Funding InformationNIH grants AG030578, DK099161, DK076169 and HL123420, research funds from Texas A&M University Health Science Center College of Medicine and Department of Medical Physiology, and from the Raleigh R. White IV endowment fund from Baylor Scott & White Foundation.
Culture Medium Fatty Acid Withdrawal Prompts Insulin‐Producing Cell Death
Authors: Rosim Mariana Papaléo , Nunes Viviane Abreu , Lensen Sigurd , Curi Rui , Azevedo‐Martins Anna Karenina 
Published: November 2023

Fatty acids (FAs) are important components of cell membranes and precursors of many bioactive molecules. The deficiency of FA in the diet has been associated with the development of human diseases (e.g. neuronal diseases) and tissue damage in experimental animal models. FAs regulate several cell functions including insulin secretion. We investigated herein the effects of FA withdrawal in the culture medium on insulin producing cell (RINm5F) metabolism, survival, and response to death cell stimuli. Cells were cultivated in a lipid‐reduced fetal bovine serum (LRS) medium for 24, 48 or 72 h. Cell proliferation was measured by BrdU incorporation into cells and glucose metabolism was analyzed by measurement of 14CO2 production from [U‐14C]‐glucose. Production of reactive oxygen species (ROS) was determined using dichlorofluorescein diacetate assay. After the culture period, cells were exposed to death inducers (either UV radiation or staurosporine). In order to evaluate the effect of FA deprivation on RINm5F cell survival and susceptibility to death induction, and the mechanism of cell death, we analyzed, by flow cytometry, loss of plasma membrane integrity, DNA fragmentation, phosphatidylserine externalization and activation of caspases 3, 8 and 9. The removal of FAs from the culture medium increased the number of dead cells as assessed by loss of plasma membrane integrity and occurrence of DNA fragmentation. Cells cultured in LRS exhibited higher cell death rates as compared to those cultivated with conventional serum. Cultivation in LRS for 48 hours increased numbers of cells with externalized phosphatidylserine, activation of caspases, raised production of ROS and glucose oxidation. In conclusion, FA deprivation induced RINm5F cell death through apoptosis and exacerbated the cell death response to staurosporine and UV radiation treatments. Cell death was associated with changes in generation of ROS and glucose oxidation.Support or Funding InformationThe authors thank the financial support of FAPESP, CAPES and CNPq
Sirtuin 1 regulation of mitophagy and function in hindlimb muscles of aging mice
Authors: Myers Matthew J. , Mohamed Junaith S. , Alway Stephen E. 
Published: November 2023

Age‐related muscle wasting, or sarcopenia, results in a functional decline in muscle strength, mobility, and overall independence of aging populations. Muscles of old animals and humans have reduced levels of repair, which contributes to sarcopenia. Aging also reduces muscle mitophagy, which results in an accumulation of dysfunctional mitochondria, but sirtuin 1 (SIRT1) appears to improve mitophagy.PurposeThe objective of this project was to determine the long‐term effects of SIRT1 on mitophagy, repair, and function of muscles in aging mice.MethodsCardiotoxin (naja mossambica) or the vector control was injected into the tibialis anterior or gastrocnemius of one leg of wild type, SIRT1 overexpresser, adult skeletal muscle‐specific knockout, and satellite cell‐specific knockout mice that were 20wk or 80wk of age. The muscles were analyzed 21 days post‐injection.ResultsThe data show that SIRT1 increased in vivo fatigue‐resistance, the percentage of type I fibers, muscle force, mitochondrial function and mitophagy in muscles of aged mice after injury.ConclusionOur results suggest that targeting activation of SIRT1 is a potential therapeutic strategy for offsetting muscle sarcopenia in aging.
Palmitic Acid Methyl Ester Regulates Proliferation of Human Mesenchymal Stem Cells
Authors: Lin Jian‐Hong , Yang Kun‐Ta 
Published: November 2023

BACKGROUNDHuman mesenchymal stem cell (hMSCs) have unique multilineage differentiation and self‐renewal capabilities that can be used for cell therapy and regenerative medicine. Although MSCs can be easily isolated from many tissues such as bone marrow, adipose tissue, and umbilical cord etc., it is still challenging to obtain sufficient numbers of cells for clinical use. In vitro environments, chemical cues such as fatty acid affect stem cell proliferation. Palmitic acid methyl ester (PAME) is released from adipose tissue and synthesized through transesterification of palmitic acid(PA) with methanol. PAME was shown to possess potent anti‐inflammatory and anti‐fibrotic. However, the effect of PAME on MSCs proliferation is unknown.PURPOSEIn this study, we investigated effects and mechanisms of PAME on proliferation of the cultured hMSCs.METHODShMSCs were cultured in αMEM for 1 day, and then treated with PAME for 2 days. We performed MTT and BrdU assays to determine the proliferate capacity of these cells, and evaluated cell cycle and mitochondrial reactive oxygen species (ROS) using flow cytometry. The action of PAME on signaling pathway was analyzed by using Western Blots and Quantitative real time‐PCR.RESULTSOur results show that PAME inhibited the proliferation of hMSCs in a dose‐dependent manner (10, 30, 50 and 100 μM) through G2/M phase arrest, which is mediated by down‐regulation of G2/M‐related mRNA and proteins (CDK1 and Cyclin B1). In addition, we found that PAME treatment (2 days) increased p53 and p21 expression, but did not affect the mitochondrial ROS level.CONCLUSIONPAME inhibits the proliferation of hMSCs through G2/M phase arrest by regulating the p53–p21 signaling pathway.
Effect of Hypoxia and Heat Inactivated Serum on Mesenchymal Stem Cell Migration
Authors: Boomsma Robert A. , Jochems Valerie K. , Spotts Anna R. 
Published: November 2023

Previous studies have shown that intravenous injection of mouse bone marrow mesenchymal stem cells (MSC) attenuate the loss of function in the heart after coronary artery occlusion (Boomsma et al, Int J Cardiol 122:17 2007) and secrete paracrine factors that may be responsible for their beneficial effect (Boomsma and Geenen, PLoSONE 7:e3685, 2012). The purpose of the current study was to determine the effect of hypoxia and heat inactivated (HI) serum on MSC migration across surfaces coated with laminin or fibronectin, conditions that better mimic the situation MSC encounter as they migrate in an ischemic heart. MSC were cultured in Mesencult + 20% serum supplement (Stem Cell Technologies) under normoxic (21% O2 + 5% CO2) conditions until confluent. The cells were lifted, placed in Mesencult containing either 20% supplement or 20% HI serum, and transferred to 12 mm Millicell cell culture inserts (PCF membranes, 3 μm pores; 600,000 cells/insert) that were either uncoated, coated with gelatin‐fibronectin, or coated with laminin. The inserts were placed in 24 well plates containing the same media and incubated to allow for cell migration at 37°C in normoxic or hypoxic (1% O2, 5% CO2, 95% N2) conditions. After 24 hours the cells on top of the membranes were removed and the cells on the underside of the membranes were stained with 0.9% crystal violet. The cells were lysed with 10% acetic acid and the absorbance at 560 nm was determined using a plate reader. The amount of migration was calculated as the % change in absorbance from the mean control value. Mean ± SE was calculated and significance was determined by Student's t‐test (p<0.05; n=9). When MSC were cultured in 20% supplement and normoxia, there was no significant change in migration on laminin or fibronectin compared to uncoated membranes. When cultured in HI serum, migration across uncoated membranes was 87% lower compared to supplement. However, migration in HI serum significantly increased 5‐fold on laminin and 9‐fold on fibronectin compared to uncoated membranes returning migration levels back to that found in supplement. Cells in HI serum were also cultured in hypoxic conditions. Hypoxia significantly reduced migration on uncoated (75%) and fibronectin coated (25%) membranes but was unaffected on laminin. These data show that in the absence of serum stimulatory factors, MSC migration is reduced on uncoated membranes, but laminin and fibronectin are able to increase migration in those conditions. Hypoxia reduces migration on fibronectin but not on laminin. Together, these data suggest that MSC migration in the ischemic conditions of the heart is best on fibronectin but is also promoted by laminin.Support or Funding InformationVanderVelde Research Scholarship from Trinity Christian College
Modeling ion channel function with murine intestinal 3D stem cell cultures
Authors: Sowick Joshua A , Shah Kalpit , Bradbury Neil A. 
Published: November 2023

Cystic Fibrosis (CF) is a persistent and progressive genetic disease, manifesting as frequent lung, gastrointestinal, and pancreatic inflammation and chronic infection. The majority of patients with CF have a mutation caused by a single amino acid deletion of phenylalanine (F508del) in the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) protein; this causes an unstable protein to form, with subsequent failure of membrane insertion or lack of function and early degradation. A lack of functional CFTR leads to the production of viscous mucus that is hard to clear from the lungs and gastrointestinal tract, providing a medium for bacterial growth and infection. While treatments for Cystic Fibrosis have made advancements, the median survival age for patients with CF is still 40.In order to utilize a mouse model for human CF disease, we developed a protocol culturing mouse intestinal crypts into three dimensional organoids. Mature organoids are subsequently dosed with Forskolin, an adenylyl cyclase activator, to elicit ion and fluid transport into the lumen of the organoid, via stimulation of CFTR channels. The unidirectional flow of ions and fluid resulted in the swelling of the organoids as fluid is moved into the lumen. We were able to measure the change in volume and surface area as well as movement of ions across the epithelium using a modified two electrode voltage clamp technique. Forskolin induced swelling was inhibited by the presence of the CFTR inhibitor cftrinh172, consistent with the role of CFTR ion fluid transport. We will present data using intestinal organoids from mice lacking transport proteins, such as LMTK2.Support or Funding InformationNIH Heart Lung and Blood
K‐Cl Cotransporter Activity Contributes to Mammalian Cancer Cell Morphology
Authors: Gagnon Kenneth B , Barati Michelle T , Palumbo Alex , Conklin Caryl A , Lederer Eleanor D 
Published: November 2023

Changes in cell shape and volume mediated by ion and water flux across cell membranes are an important component of cell migration. Previous studies have demonstrated the importance of K‐Cl cotransporters (KCCs) in a chemically‐induced rat glioblastoma cell line (C6). Inhibition of KCC3 using siRNA reduced C6 cell migration by ~50% in an in vitro scratch wound assay. Dephosphorylation of specific threonine residues within the intracellular cytoplasmic tail of the KCCs by protein phosphatase 1A (PP1A) results in cotransporter activation. The present study aimed to further characterize the expression and regulation of the KCC family of proteins in the motility of mouse melanoma (B16F10) and human breast adenocarcinoma (MDA231) cell lines. B16F10 and MDA231 cells were both grown to confluence in 10 cm diameter cell culture dishes with Dulbecco's minimum essential media including 10% fetal bovine serum and 1% penicillin/streptomycin in a humidified 37°C incubator with 95% O2/5% CO2. Crude membrane preparations were subjected to SDS‐PAGE and immunoblotted for KCC1, KCC3, KCC4, and PP1A proteins. B16F10 and MDA231 cells were also grown to 50% confluence on coverslip glass and immunofluorescently imaged by confocal microscopy for KCC1, KCC3, and KCC4 proteins with and without Calyculin A (PP1A inhibitor) treatment. For both cell lines, western blotting showed KCC3, KCC4, and PP1A expression enriched in crude membrane preparations. We also observed both membrane and intracellular immunofluorescent localization of KCC3 and KCC4 in both cell lines. No WB or immunofluorescent signal was detected for KCC1 in either cell line. Treatment with Calyculin A (5 nM) for 1 h resulted in a morphological change in cell phenotype from flattened with many filopodia to rounded and swollen without visible filopodia. This morphological change was reversible when the Calyculin A was removed and the cells were cultured in control DMEM media for 10 h. These results suggest that mammalian cancer cell lines overexpress KCC3 and KCC4 proteins as well as an important regulator of their activity, PP1A. Based on the Calyculin A studies, we hypothesize that PP1A dephosphorylation of specific K‐Cl cotransporter isoforms is a component of filipodia development and directional cell motility.Support or Funding InformationFunding for this research was provided by Robley Rex Veterans Affairs Medical Center and University of Louisville School of Medicine (to EDL).
Cardiotoxin III suppresses HGF‐ and EGF‐induced EMT and signaling network in breast cancer MDA‐MB‐231 cells
Authors: Tsai Pei‐Chien , Fu Yaw‐Syan , Chang Long‐Sen , Hsu Sodio C.N. , Lu Tzongshi 
Published: November 2023

Introduction and hypothesisMetastasis causes a poor prognosis and high mortality rate of breast cancer patients. Because the epithelial‐to‐mesenchymal transition (EMT) is the initial step for metastasis of breast cancer, suppression of EMT might improve breast therapy. Cardiotoxin III (CTX III), a basic polypeptide isolated from Naja naja atra venom, has been reported to show anticancer activity. Thus, we investigated the inhibitory activity of CTX III on the HGF‐ and EGF‐induced EMT of breast cancer MDA‐MB‐231 cells.MethodsCTX III was purified from the venom of Naja naja atra (Taiwan cobra) using chromatographies on Sephadex G‐50 and SP‐Sephadex C‐25 columns. MTT assay, transwell assay and western blotting analyses were employed to study the inhibitory activity of CTX3 on HGF‐ or EGF‐induced cell proliferation, migration, invasion and EMT‐related protein expression.ResultsCTX III treatment resulted in morphological changes of HGF‐ or EGF‐treated cells from elongated and spindle shape to rounded and epithelial‐like shape. Meanwhile, CTXIII induced E‐cadherin upregulation and down‐regulation of MMP‐9, N‐cadherin and vimentin expression, thereby inhibiting migration and invasion of HGF‐ and EGF‐treated cells. Consistently, CTX III suppressed HGF‐ or EGF‐induced expression of transcription factors that regulated the expression of EMT‐related proteins. Moreover, CTX III abrogated phosphorylation of Src, FAK, p130cas, paxillin, c‐Met and EGFR in HGF‐ or EGF‐treated cells. Thus, abolishment of HGF/c‐Met and EGF/EGFR‐mediated activation of ERK1/2 and PI3K/Akt pathways was observed after CTX III treatment.ConclusionsOur data reveal that CTX III reverses HGF‐ and EGF‐induced epithelial‐to‐mesenchymal transition of MDA‐MB‐231 cells, and suggest that it may be served as an agent for repressing the malignancy‐promoting effect of HGF and EGF on breast cancer.Support or Funding InformationNSYSUKMU‐104‐P‐001
The Mechanobiology of Cervical Remodeling: Characterization of Select Cytoskeletal and Rho‐GTPase Signaling Factors in Mice Cervical Tissue During Pregnancy
Authors: Gordon Jacob , Frank Martin , Mowa Chishimba Nathan 
Published: November 2023

During pregnancy the developing fetus exerts an increasing gravitational force as it grows in size. However, the effects and underlying molecular mechanism of this process on the cervix is not well known. Our earlier proteomics studies have provided evidence of presence and changes in levels of cytoskeletal mechano‐mediators with advancement of pregnancy. In the present study, we use real time PCR analysis to further characterize expression of select cytoskeletal genes (Filamin A, Gelsolin, Vimentin, Actinin 1, Caveolin 1, Transgelin, Keratin 1, Profilin 1 and Focal Adhesion Kinase) (day 0, day 11 and day 17) to verify findings of the earlier proteomics study. Additionally, we also investigate the expression levels of Rho‐GTPases (RhoA, RhoB and Cdc42), molecules that play cell‐signaling roles upstream of proliferation and cytoskeletal protein organization. The levels of mRNA of all the (12) genes examined increased over the course of pregnancy except 3 that decreased. We conclude that the growing fetus influences cervical remodeling and likely the timing of birth by manipulating mechano‐transduction during pregnancy.Support or Funding InformationOffice of Students Research, Appalachian State University.
H Ficolin: Polymerization and Aggregation from Blood to Cerebrospinal Fluid
Authors: Castillo‐González William , González‐Losada Cristóbal , Rodríguez‐Pérez José Alejandro , Jensenius Jens Christian , Zerr Inga , Schmitz Matthias , Dorta‐Contreras Alberto Juan 
Published: November 2023

Ficolins are one of the starters of the lectin pathway It recognizes carbohydrates over the surface of pathogen and active the complement pathway. It plays an important role in the innate immunity.H Ficolin are produced by hepatic epithelial cells, bile ducts and hepatocyte and was secreted into bile and blood.The universal validity of the molecular diffusion/cerebrospinal fluid (CSF) flow model is valid for the brain‐derived molecules, is the base for a systematic approach to determine the source of a CSF protein either derived from blood, from brain or from leptomeninges.ObjectiveTo demonstrate the dynamics of diffusion of H ficolin from blood to CSF over the base of its CSF and serum concentration and the different aggregation forms.Methods80 serum and CSF paired samples were obtained from control persons without neuroinflammation process and without oligoclonal IgG bands. H ficolin and albumin were quantified by ELISA and by an immunoturbidimetric analysis. Q albumin=CSF albumin/serum albumin and Q H Ficolin=CSF H Ficolin/serum H ficolin was calculated.In order to calculate the different aggregation forms was determinate the inflection points of the frequency distribution of H Ficolin vs Q H Ficolin.ResultsSerum and CSF H Ficolin did not follow a normal distribution according to Kolmogorov‐Smirnov test. Mean values of H Ficolin in serum was higher than its mean content in CSF. CSF H Ficolin concentration increased with the increase of Q albumin. H Ficolin is not a brain‐derived protein. Three inflection points was determined. (Table 1)ConclusionsH ficolin is a blood‐derived protein and the diffusion from blood to CSF indicates three aggregation forms.
H Ficolin aggregation forms.

G1‐H Ficolin
G2‐H Ficolin
G3‐H Ficolin

Q (Quotient ratio)
0,002
0,0002
0,00004

Molecular weight (KDa)
120
800
4500

Aggregation forms
Tetramer (original structure non aggregated)
6 aggregates
24 aggregates
Dantrolene Improves Endothelial Function in Type‐1 Diabetic Rats by Decreasing Oxidative Stress
Authors: Roman Marie , Alemar Stephanie , Zack Richard , Matias Jonathan , Morales Myrna , Torres Hector , Quidgley Jose , Crespo Maria J 
Published: November 2023

Diabetic patients have a high risk of developing cerebral vasospasms (CVSP). Experimental evidence indicates that the addition of the ryanodine receptor (RyR) blocker dantrolene to standard therapies reduces vasoconstriction in non‐diabetic patients with CVSP. Whether diabetics with CVSP also benefit from this drug, however, is unknown. In this study, we evaluated the effects of a 30‐minute incubation period with dantrolene (50 μM) on acetylcholine (ACh)–induced relaxation in phenylephrine (PHE)‐precontracted aortic rings from streptozotocin (STZ)‐induced Type‐1 diabetic rats. Age‐matched non‐diabetic rats were used as controls (CT). Concentration response curves for the ACh‐induced relaxation from 0.1 nM to 10 μM were performed on aortic rings in the presence and absence of dantrolene. The oxidative‐stress markers malondialdehyde (MDA) and 4‐hydroxyalkenal (4‐HAE) were also measured in aortic homogenates after a 30‐minute incubation period with dantrolene. The EMAX value for the ACh‐induced relaxation was reduced in diabetic rats (40.97 ± 1.92%) when compared to CT (75.74 ± 5.27%) (N=5, P<0.05) without changing the EC50 value. Acute incubation of aortic rings with 50 μM dantrolene increased the EMAX value in diabetic rats to 70.39 ± 13.84 % (N=5, P<0.05), without affecting this parameter in CT. Endothelium‐independent relaxation with 10 μM sodium nitroprusside (SNP) was not affected by dantrolene in either diabetic or CT rats. In addition, dantrolene decreased MDA +4‐HAE (μM/g protein) in the vasculature of diabetic rats from 3.19 ± 1.15 to 1.16 ± 0.10 (N=5, P<0.05). Together, our results suggest that by reducing oxidative stress and improving endothelial function, dantrolene may exert a beneficial effect in diabetic patients with CVSP by reducing arterial tone.Support or Funding InformationSupported by RCMI Grant G12‐RR03051.