bluuebunny/crossref_metadata_2025_split huggingface dataset:
NFATc1 mediates Pak1 activation in the modulation of chemokine‐induced human aortic smooth muscle cell F‐actin stress fiber formation, migration, and proliferation and injury‐induced vascular wall remodeling (1158.5)
Authors: Singh Nikhlesh , Kundumani‐Sridharan Venkatesh , Kumar Sanjay , Gadepalli Ravishekhar , Rao Gadiparthi 
Published: June 2021

In addition to their role in cell proliferation, cyclin‐dependent kinases (CDKs) mediate cell migration; however the underlying mechanisms are not clear. Therefore, the objective of this study is to test whether cyclin/CDKs activate Pak1, an effector of Rac1, whose involvement in the modulation of cell migration and proliferation is well established. Monocyte chemotactic protein 1 (MCP1) induced Pak1 phosphorylation/activation in human aortic smooth muscle cells (HASMCs) in a time‐dependent but delayed manner. MCP1 also stimulated F‐actin stress fiber formation as well as the migration and proliferation of these cells and inhibition of Pak1 reversed these effects. MCP1 also induced cyclin D1 expression and CDK6/4 activities, and these effects were dependent on NFATc1 activation. Depletion of NFATc1, cyclin D1, CDK6, or CDK4 levels attenuated MCP1‐induced Pak1 phosphorylation/activation resulting in decreased HASMC F‐actin stress fiber formation, migration and proliferation. CDK4, which appeared to be activated downstream of CDK6, formed a complex with Pak1 in response to MCP1. Depletion/inhibition of Rac1 levels/activation abrogated MCP1‐induced NFATc1‐cyclin D1‐CDK6‐CDK4‐Pak1 signaling, and thereby, decreased HASMC F‐actin stress fiber formation, migration, and proliferation. In addition, smooth muscle‐specific deletion of NFATc1 led to decreased cyclin D1 expression and CDK6, CDK4, and Pak1 activities resulting in reduced neointima formation in response to injury. Thus, these observations reveal that Pak1 is a downstream effector of CDK4 and Rac1‐dependent NFATc1‐mediated cyclin D1 expression and CDK6 activity mediate this effect in the modulation of MCP1‐induced HASMC migration and proliferation in vitro and vascular wall remodeling in vivo.Grant Funding Source: NHLBI
Maternal deposition of ApoA‐I‐mCherry transgene indicates an important role in early zebrafish development (1161.1)
Authors: Reece Takisha , Otis Jessica , Farber Steven 
Published: June 2021

Our goal is to investigate the function of high‐density lipoprotein (HDL) particles of hepatic origin through in vivo visualization in the optically clear larval zebrafish (Danio rerio). We generated zebrafish stably expressing fluorescently tagged human apolipoprotein A‐I (ApoA‐I), a marker of HDL, through a liver‐specific promoter (LFABP:ApoA‐I‐mCherry; 6 lines). At 6‐days post fertilization (dpf), transgenic larvae show variable ApoA‐I‐mCherry localization: a subset of larvae show fluorescence in the liver, circulation, pronephric kidney and yolk syncytial layer, as expected; a second subset shows fluorescence in all of these regions except the liver. We hypothesized that variable ApoA‐I‐mCherry expression results from the first subset of larvae containing LFABP:ApoA‐I‐mCherry in their genome and that the second subset of larvae lack the transgene and are displaying maternally deposited ApoA‐I‐mCherry. Maternal RNA products deposited in embryos are degraded within hours, but the duration of maternal protein expression is less characterized. We pairwise crossed wild type and transgenic LFABP:ApoA‐I‐mCherry zebrafish and characterized ApoA‐I‐mCherry expression and localization. Only the progeny of LFABP:ApoA‐I‐mCherry females showed absence of ApoA‐I‐mCherry in the liver (n=12 experiments in 5 lines), supporting the conclusion that maternal deposition of ApoA‐I‐mCherry in non‐transgenic larvae created this phenotype. On‐going studies include Western Blot and PCR analyses to confirm and investigate the function of maternally deposited ApoA‐I in early development.Grant Funding Source: APS 2013 Frontiers in Physiology Research Teacher Fellowship
Changes in macrophage phenotype and induction of epithelial‐to‐mesenchymal transition genes following acute Achilles tenotomy and repair (1166.3)
Authors: Sugg Kristoffer , Gumucio Jonathan , Lubardic Jovan , Mendias Christopher 
Published: June 2021

Tendon injuries occur frequently in physically active individuals, but the clinical outcomes for these injuries can be poor. In many injured tissues the repair process is orchestrated by two types of cells, macrophages and fibroblasts. Macrophages, which have both proinflammatory (M1) and antiinflammatory (M2) phenotypes, can directly participate in tissue remodeling and direct the response of other cells through the secretion of cytokines and growth factors. Fibroblasts have a well‐described role in synthesizing and remodeling extracellular matrix (ECM) after tissue injury. In many organ systems, epithelial cells can transdifferentiate into fibroblasts, which can then regenerate damaged ECM. This process is triggered via activation of epithelial‐to‐mesenchymal transition (EMT) signaling programs. Most tendons are surrounded by sheets of epithelial cells, and these tissue layers could provide a source of fibroblasts to repair injured tendons. To gain greater insight into the biology of tendon regeneration, we performed a tenotomy and repair in Achilles tendons of adult rats and determined changes in macrophage phenotype, and ECM‐ and EMT‐related genes over a four week time course. The results from this study suggest that changes in macrophage phenotype and activation of canonical EMT programs likely contribute to the degradation and subsequent repair of injured tendon tissue.Grant Funding Source: NIH grants R01‐AR063649, T32‐GM008616 and T32‐GM008322
Calcium signaling and glucose response is different in adult compared to neonatal rat astrocytes (1177.7)
Authors: Rarick Kevin , Eisenhauer Jessica , Harder David 
Published: June 2021

Much of our mechanistic understanding of astrocytes comes from neonatal cells which may not represent the mature glial phenotype. We sought to determine if neonatal and adult astrocytes respond similarly when challenged with high glucose. Neonatal and adult rat astrocytes were cultured in 5.5 mM low glucose DMEM (LG) and then maintained in LG or switched to 25 mM high glucose DMEM (HG) for two weeks. A developmental decrease was observed in expression of the inositol trisphosphate receptor (IP3R) and the metabotropic glutamate 5 receptor (mGluR5). The L‐type calcium channel (CaV1.2) was only expressed in adult astrocytes. Adult and neonatal astrocytes both responded to HG with increased mGluR5 and decreased IP3R protein expression. Adult astrocytes also increased CaV1.2 expression following HG. Neonatal astrocytes had a greater magnitude and rate of rise of [Ca2+]i in response to ATP and glutamate compared to adult astrocytes. The [Ca2+]i responses to ATP and glutamate were similarly affected by HG, although adult astrocytes demonstrated minimal response to glutamate in all conditions. Cell viability decreased only in adult astrocytes following HG. These results suggest HG alters calcium signaling in astrocytes and developmental differences may exist in downstream pro‐survival signaling. The response of adult glia in specific disease paradigms may not be accurately reflected when using neonatal cells.Grant Funding Source: Supported by T32 HL007792, R01 HL033833, R01 HL 092105, and R01 HL105997
The influence of different carbon sources on the structural characteristics of the bacterial cellulose membrane (1180.15)
Authors: Bravo Mariangela , Lazarini Silmara , de Carvalho Renata , Corbi Fabiana , Corbi Pedro , Lustri Wilton , Amaral André 
Published: June 2021

Recent research shows that bacterial cellulose membranes (BCm), which represent a promising biomaterial for the use in regenerative medicine, can be used as biological support and/or as a system of extended release of bioactive compounds. The objective of this work was to characterize the influence of different carbon sources (CS1 and CS2), obtained from the processing of sugar cane, on the physical and structural characteristics of the BCm. To develop this work, bacterial strains of Gluconacetobacter hansenii (ATCC 23769 ) were cultivated in media containing nitrogen source (5,6g/L) and increasing concentrations of CS1 (2.5, 5 , 7.5, 10, 12.5 and 15%) and CS2 (5, 10, 15, 20, 25 and 30%). The static culture was maintained at room temperature for 14 days. The influence of the carbon sources and the different concentrations was established by analysis of dry matter yield, tumescence capacity and ultra‐structural analysis. The best yield was obtained with the mass concentration of 10% CS1 (0.71 g/L) and 15% CS2 (0.84 g/L) and percentage of tumescence of 70,7% and 71,6 % respectively. The ultra‐structural analysis of the BCm best yield in the two sources showed a difference among the reticular structures. It was concluded that carbon sources obtained from sugar cane provided, in the concentrations mentioned above, favorable conditions for the biosynthesis of BCm by G. hansenii, with potential for the use in regenerative medicine.Grant Funding Source: Supported by: Conselho Nacional de Desenvolvimento Científico e Tecnológico ‐ CNPq
Polarized mechanotransduction in osteocytes: role of αVβ3 integrin on the cell processes (1180.9)
Authors: Thi Mia , Suadicani Sylvia , Schaffler Mitchell , Weinbaum Sheldon , Spray David 
Published: June 2021

Osteocyte processes attach to the bony canalicular wall at discrete integrin sites and loading‐induced fluid flow in the lacunar‐canalicular system results in focally high membrane strains around these attachments, but not at the cell body. While the presence of integrin in the attachment sites to the canalicular wall suggests its participation in osteocyte mechano‐transduction, such a role still remains to be demonstrated. The goal of this study was to address these points by using the novel Stokesian fluid stimulus probe (SFSP) to achieve the spatial resolution required to stimulate MLO‐Y4 osteocyte processes and cell body with physiologically relevant hydrodynamic forces at pN levels. Ca2+ imaging studies demonstrated that osteocyte processes are uniquely mechanosensitive. 1‐5 pN level forces applied to the cell processes triggered Ca2+ signaling while comparable stimulation of cell bodies did not. Inhibition of αVβ3 integrin attachment sites with IntegriSense probe blocked the Ca2+ response to SFSP stimulation. Furthermore, we found that ATP is a major autocrine/paracrine signaling messenger that amplifies spread of Ca2+ signals from the mechanically stimulated process to cell body and from one osteocyte to another in the network. We thus validated the essential role of αVβ3 integrin on osteocyte processes in focal mechanosensing and transduction.Grant Funding Source: Supported by AR057139, DK091466
Phosphorylation regulates clathrin‐mediated endocytosis of the thiazide‐sensitive cotransporter NCC (1181.12)
Authors: Rosenbaek Lena , Kortenoeven Marleen , Aroankins Takwa , Fenton Robert 
Published: June 2021

Our recent work suggests that AQP2 endocytosis rates can be regulated via phosphorylation. As NCC is phosphorylated at conserved residues in its intracellular NH2 terminus, we hypothesized that a similar mechanism may regulate NCC plasma membrane abundance. Novel MDCKI cell line models stably expressing NCC were generated to examine trafficking pathways of NCC and the role of phosphorylation in regulation of NCC internalization. In MDCKI‐NCC cells, NCC was observed as a highly glycosylated monomer and dimer. NCC constitutively recycled to/from the apical plasma membrane of MDCKI‐NCC cells, with 20‐30% of the apical membrane pool of NCC being internalized within 30 min. Using specific inhibitors of either clathrin‐dependent or ‐independent endocytosis we determined that NCC is internalized via a clathrin‐mediated pathway and reduction of endocytosis results in greater steady‐state NCC levels in the plasma membrane. Inducing phosphorylation of NCC via low chloride treatment or mimicking phosphorylation by replacing Thr53, Thr58, and Ser71 residues with Asp, resulted in increased apical membrane abundance and reduced rates of NCC internalization. Our data support a model where NCC is constitutively cycled to the plasma membrane, where upon stimulation it can be phosphorylated resulting in increased NCC activity and decreased NCC endocytosis, which together increase NaCl transport in the DCT.Grant Funding Source: Supported by Lundbeck Foundation and Danish Medical Research Foundation
High‐fat diet induced expansion of colon crypt epithelial proliferative zone towards lumen correlates with elevated innate inflammatory markers in the human‐relevant porcine model (123.2)
Authors: Vanamala Jairam , Radhakrishnan Sridhar , Eriksson Elisabeth , Charepalli Venkata , Kim Sung , Reddivari Lavanya 
Published: June 2021

Recent evidence shows that high‐fat diet (HFD) alone causes colon cancer in mice; however, underlying mechanisms are not fully elucidated. An expansion of proliferative zone towards the crypt lumen is a biomarker for colon cancer in animal models and humans. We hypothesized that HFD induced expansion of colonic proliferative zone and elevation of proliferating index correlates with innate inflammatory markers in the porcine model that has similar gastrointestinal function/physiology as humans. Pigs (n = 8/group) were provided either standard diet (SD; 5% fat) or HFD (25% fat) for 13 wk. HFD elevated proliferative zone (Ki‐67; immunofluorescence; p = 0.02) and proliferative index (p = 0.06). HFD numerically increased (p = 0.15) distal colon expression of pattern recognition receptors TLRs 2/4 and TLR‐4 responsive genes NF‐κB, COX‐2 and TNF‐α (qPCR) compared to SD group. However, HFD elevated proliferative zone correlated with TLR‐4, NF‐κB, COX‐2 and TNF‐α expression (r = 0.77, 0.71, 0.78 and 0.62, respectively, p = 0.02). HFD group had greater distal colon macrophage infiltration (mac387; immunohistochemistry; p = 0.1) that correlated with TLR‐4 expression (r = 0.52, p = 0.05). As proliferating colonocytes are highly susceptible to damage due to oxidative stress/inflammation, expanded proliferative zone may partly explain how chronic HFD consumption causes colon cancer.Grant Funding Source: Supported by USDA‐NIFA NRI Grant 2009‐55200‐05197
PINCH‐Rsu‐1 complex in regulating liver size and tumorigenesis (144.6)
Authors: Donthamsetty Shashi , Bhave Vishakha , Mars Wendy , Michalopoulos George 
Published: June 2021

PINCH protein is part of the ternary complex known as the IPP (Integrin linked kinase(ILK)‐PINCH‐Parvinα) complex. PINCH itself binds to ILK and to another protein known as Rsu1 (Ras suppressor 1). We generated PINCH 1 and PINCH 2 Double knockout mice (referred as PINCH DKO mice). PINCH2 elimination was systemic whereas PINCH1 elimination was targeted to hepatocytes. The genetically modified mice were born normal. The mice were sacrificed at different ages after birth. Soon after birth, they developed abnormal hepatic histology characterized by disorderly hepatic plates, increased proliferation of hepatocytes and biliary cells and increased deposition of extracellular matrix. After a sustained and prolonged proliferation of all epithelial components, proliferation subsided and final liver weight by the end of 30 weeks in livers with PINCH DKO deficient hepatocytes was 40% larger than the control mice. The livers of the PINCH DKO mice were also very stiff due to increased ECM deposition throughout the liver, with no observed nodularity. Mice developed liver cancer by one year. These mice regenerated normally when subjected to 70% partial hepatectomy and did not show any termination defect. Ras suppressor 1 (Rsu‐1) protein, the binding partner of PINCH is frequently deleted in human liver cancers. Rsu‐1 expression is dramatically decreased in PINCH DKO mouse livers. Increased expression of Rsu‐1 suppressed hepatocyte proliferation and migration in HCC cell lines. These changes were brought about not by affecting activation of Ras (as its name suggests) but by suppression of Ras downstream signaling via RhoGTPase proteins. In conclusion, our studies suggest that removal of PINCH results in enlargement of liver and tumorigenesis. Decreased levels of Rsu‐1, a partner for PINCH and a protein often deleted in human liver cancer, may play an important role in the development of the observed phenotype..Grant Funding Source: (5R01CA035373 and 5R01CA103958
Modulating myeloperoxidase‐induced endothelial damage by a carbon monoxide‐releasing molecule, CORM‐3 (146.9)
Authors: Patterson Eric , Capretta Alfredo , Fraser Douglas , Potter Richard , Cepinskas Gediminas 
Published: June 2021

Previous work has demonstrated that carbon monoxide releasing molecules (CORMs) suppress inflammation, however, the mechanisms are not well understood. Neutrophil (PMN)‐derived myeloperoxidase (MPO) is known to play a significant role in the pathophysiology of numerous inflammatory disorders through: 1) direct peroxidation and 2) producing hypohalous acids, (e.g. hypochlorous acid). Thus, an overwhelming MPO‐dependent production of oxidants in the sub‐endothelial space after PMN degranulation, contributes to tissue damage and propagating inflammation.We hypothesized that CORM‐3 interferes with MPO’s catalytic activity, and thereby mitigates endothelial inflammatory damage.In vitro assays were used to study MPO catalytic activity inhibition. Further, live cell in vitro assays were used to investigate endothelial damage through measuring reactive oxygen species (ROS) and cellular permeability.Our results indicate that CORM‐3 inhibits total MPO activity as well as MPO’s peroxidation and halogenation cycles. Cellular assays determined that ROS production and cellular permeability are decreased when extracellular MPO is treated with CORM‐3. In all assays inactivated CORM‐3 (iCORM‐3) was significantly less effective than CORM‐3.We conclude that CORM‐3 may mitigate inflammatory damage partly through inhibiting MPO activity.Grant Funding Source: Supported by Heart and Stroke Foundation, HSFO‐393 and LHRI IRF‐025‐09
Regulation of intestinal epithelial apoptosis by protease‐activated receptor 2 involves EGFR‐transactivation and is associated with Mcl‐1 upregualtion (151.1)
Authors: Iablokov Vadim , MacNaughton Wallace 
Published: June 2021

Colonic biopsies from inflamed colon have higher levels of epithelial apoptosis than biopsies from healthy individuals. Therapies that inhibit intestinal epithelial apoptosis may decrease the inflammation observed in inflammatory bowel disease. We previously showed that signaling by PAR2, a GPCR activated by serine proteases, delayed IFN‐γ &amp; TNF‐α‐induced apoptosis in colonic epithelial (HT‐29) cells. The observed decrease in the cleavage of caspase‐8, 9, 3 and PARP following the activation of PAR2 by 2‐furoyl‐LIGRLO (2fLI) was shown to be dependent on the activities of PI3K and ERK1/2. In this study we found that selective inhibition of EGFR with AG1478 blocked the phosphorylation of PI3K and ERK1/2. Pre‐treatment with AG1478 prior to the addition of IFN‐γ &amp; TNF‐α also blocked 2fLI‐induced intestinal epithelial survival, as determined by PARP cleavage. Modulation of BCL‐2 family members was then investigated due to their known function in regulating mitochondrial permeability and apoptosis. Protein expression of pro‐apoptotic BAK, BAX and BIM were not altered by either cytokine or 2fLI treatment. Likewise, no change in expression of anti‐apoptotic BCL‐XL was observed. Treatment with 2fLI stimulated phosphorylation and inactivation of the apoptotic BH3‐only protein, BAD. Knockdown of BAD with siRNA, however, did not alter 2fLI‐stimulated intestinal survival. Finally, 2fLI stimulated a rapid increase in anti‐apoptotic MCL‐1 expression. PAR2 signaling reduces epithelial apoptosis in response to inflammatory cytokines via EGFR transactivation and possibly by regulation of MCL‐1 expression. Our findings may represent a mechanism whereby proteases facilitate epithelial cell survival and colonic healing after inflammation.Grant Funding Source: Supported by the Crohn’s and Colitis Foundation of Canada
High MUC2 production in goblet cells causes increased susceptibility to ER stress and apoptosis (151.4)
Authors: Tawiah Adelaide , Moreau France , Chadee Kris 
Published: June 2021

MUC2, a large glycoprotein produced by goblet cells in the colon, forms a protective mucus blanket over the epithelium as the first line of host defense. During pathological conditions such as inflammatory bowel diseases, there is accelerated biosynthesis and secretion of MUC2. Surprisingly, little information is known on how MUC2 production is regulated and whether goblet cells undergo increased stress in response to high MUC2 biosynthesis and secretion. In this study we investigated the role of MUC2 in goblet cell stress using a high mucin producing cell line, HT29‐H, and a clone of HT29‐H (HT29‐L) in which MUC2 has been silenced using lentivirus shRNA. Cells were treated with tunicamycin and MG132, and markers for endoplasmic reticulum (ER) stress and apoptosis were quantified. Compared to HT29‐L cells, HT29‐H cells exhibited a greater dose‐and time‐dependent increase in ER stress markers GRP78, ATF4, CHOP, sXBP1 and AGR2 in response to both tunicamycin and MG132. ER stress also caused a decrease in phosphorylation of Akt and increased apoptosis in HT29‐H cells 6h after treatment. Phospho‐Akt in HT29‐L cells was however sustained, decreasing slightly only after 24h with less apoptosis compared to HT29‐H cells. Our findings indicate that MUC2 induces goblet cell stress and apoptosis which could subsequently lead to diminished mucus barrier function.Grant Funding Source: Supported by CIHR
The mini ultrasound institute: does incorporating ultrasound in the anatomy curriculum increase students’ understanding of living anatomy and comfort with new technology? (211.4)
Authors: Smither Fantley , Bhagra Anjali , Lachman Nirusha , Pawlina Wojciech 
Published: June 2021

Despite increase in residency programs including ultrasound training, few medical schools have incorporated it into their curricula. The Gross Anatomy course at Mayo Medical School has introduced ultrasound in the curriculum. Cadaver dissection teaches students static anatomical relationships, but ultrasound offers dynamic display of how those relationships can change with movement. Ultrasound curriculum consists of four 1 hour didactic sessions and five 30 minute hands‐on modules, covering Carpal tunnel, Heart, Abdominal viscera, and Doppler imaging of blood flow. Each module is guided by a checklist of techniques and structures. Students are graded using ARS system, and ultrasound objectives are incorporated into the final exam.This study aimed to assess effectiveness of ultrasound curriculum in a 7 week anatomy course. Students were asked to complete pre‐ and post‐ test surveys that assessed whether ultrasound sessions allowed them to better appreciate living anatomy, learn the basics of operating a portable ultrasound machine, and become more comfortable with medical technology. Pre and post surveys showed that ultrasound helped students appreciate living anatomy, and that they were comfortable with technology. There was an increase in students’ perception of their ability to interpret ultrasound images with a p‐value of 0.000026. Ultrasound was a successful addition to the anatomy curriculum.