bluuebunny/crossref_metadata_2025_split huggingface dataset:
Computational studies on HdeA and its pH‐Dependent Activation
Authors: Pacheco Sayuri , Ravinder Abrol 
Published: June 2021

HdeA is a chaperone protein found in pathogenic gram‐negative bacteria such as E. coli that forms homodimers, provides bacterial stress response and acid resistance. HdeA is inactive in its dimeric form and becomes activated in low pH environments such as the mammalian stomach. In low pH environments it binds to other periplasmic proteins protecting them from aggregation and denaturation. Computational biophysical methods such as constant pH molecular dynamics in explicit and implicit solvent are utilized to couple protein dynamics and protonation states in order to reveal a plausible unfolding mechanism for HdeA. Our computational models are providing insight into the unfolding mechanisms for HdeA. The structural models have shown important interactions, some of which have not yet been observed in experimental structures, which can potentially stabilize the monomers at neutral pH while destabilizing and triggering partial unfolding in acidic pH conditions.Support or Funding InformationBuilding Infrastructure Leading to Diversity (BUILD) # 5TL4GM118977This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Ghrelin sensitises colonic myenteric neurons to the neurostimulatory effects of glucagonlike peptide‐1 in Sprague Dawley and Wistar Kyoto rats.
Authors: O'Brien Rebecca , Buckley Maria M. , O'Malley Dervla 
Published: June 2021

Irritable Bowel Syndrome (IBS) is a common functional bowel disorder affecting approximately 10–20% of the population with many experiencing post‐prandial exacerbation of symptoms. Although IBS pathophysiology remains elusive, dysfunctional endocrine signalling has been implicated. Ghrelin is an orexigenic hormone which peaks prior to food ingestion. Glucagon‐like peptide‐1 (GLP‐1) is secreted by L‐cells in the mucosa of the small and large intestine in response to the arrival of nutrients. Previous studies have shown that ghrelin directly primes L‐cells to secrete GLP‐1 and increases GLP‐1 release (Gagnon et al., 2014). The aims of this study were to investigate if ghrelin sensitises colonic enteric neurons for GLP‐1 mediated signalling in two animal models; Sprague Dawley (control) and Wistar Kyoto (stress model of IBS).Immunofluorescence was used to stain for ghrelin and GLP‐1 receptors. Colonic motility was assessed in organ baths, using SD and WKY colonic segments, in response to a GLP‐1 mimetic before and after ghrelin stimulation.Ghrelin and GLP‐1 receptors were expressed in discrete co‐localised clusters in myenteric neurons. GLP‐1 reduced contractile activity in both SD and WKY rat colons. However, this response was potentiated by prior exposure to ghrelin in both circular and longitudinal muscle in Sprague Dawley ((p=0.0116), (p=0.0093), respectively) and Wistar Kyoto ((p= 0.0129), (p=0.0006), respectively) rat colonic segments.Ghrelin sensitises the colon to GLP‐1‐evoked contractile activity. Although similar mechanisms of action were evidence in both species, the baseline contractile activity was aberrant in WKY rats.Support or Funding InformationFunded by UCC TRAP funding and the Department of Physiology, University College of CorkThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Molecular Basis of Hypoxia‐Induced Excessive Erythrocytosis of High Altitude
Authors: Azad Priti , Haddad Gabriel 
Published: June 2021

RationaleSince polycythemia is a predominant trait in some high altitude dwellers (Chronic Mountain Sickness, CMS, or Monge's disease) but not others living at the same altitude in the Andes, we took advantage of this human “experiment in nature” and studied both populations (with CMS and without, non‐CMS).MethodsIn order to understand the molecular basis for polycythemia of high altitude, we generated a disease in‐the dish‐model by re‐programming fibroblasts from CMS and non‐CMS subjects. In the past, we have discovered that there are selective sweeps that seem to be statistically different between CMS and non‐CMS and hence contain candidate genes. In this study, by manipulating expression of these candidate genes (Eg. SENP1, ARID1B) in our in‐vitro model system, we delineate the molecular mechanism(s) of the CMS polycythemia..ResultsAs compared to sea level controls who responded to hypoxia by increasing their RBCs modestly, CMS cells increased theirs remarkably (up to 60 fold) with a dose‐dependent response to graded hypoxia (1.5, 5, 10% O2). Non‐CMS cells showed a blunted increase in RBC production under hypoxia. In order to understand the genetic basis of these phenotypes, we knocked down SENP1 (a desumoylase) in CMS iPS cells using lentiviral constructs and observed a striking reduction (>90%) of the CMS excessive erythropoietic response to low O2. Similarly, by knocking down ARID1B in non‐CMS we observed that RBC formation increased significantly under hypoxia.ConclusionOur results demonstrate the presence of a regulatory system (SENP1 and ARID1B) that is critical in erythropoiesis and that is out of balance in the CMS subjects.Further, by combining iPS technology and genomics, we believe that we will be able to determine a comprehensive model of the basis for hypoxia‐induced polycythemia.Support or Funding InformationThis study is funded by National Institutes of Health (NIH) grants (1P01HL098053 and 5P01HD32573) to G.G. HaddadThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Rhinovirus infection alters cellular architecture of highly differentiated airway epithelial cells
Authors: Michi Aubrey , Proud David 
Published: June 2021

RATIONALEHuman rhinoviruses (HRV) are responsible for over 50% of viral respiratory tract infections, and pose a significant health challenge for sufferers of chronic inflammatory airway diseases. Much of our current knowledge of the HRV lifecycle is based on other picornavirus models using cell lines of non‐airway origin (e.g. HeLa cells). However, the human airway epithelial cell (HAE) is the natural host cell that is subject to HRV infection and replication. Using highly‐differentiated HAE, we seek to elucidate the mechanisms that HRV uses to remodel the cellular architecture of HAE during replication, and to temporally relate these alterations to transient intercellular junctional changes.METHODSHighly differentiated air‐liquid interface (ALI) cultures of HAE, isolated from normal human lungs, were treated with three different HRV serotypes: HRV‐16, HRV‐1A, and HRV‐C15 for 24, 48, 72, 96, 120, 144 hours, and the junctional protein, ZO‐1, was examined. Studies of the architecture of the cytoskeleton were conducted using HAEs grown in monolayer submersion culture. Other stimuli include replication‐deficient HRV, poly(I:C) (a mimic of the double‐stranded RNA produced during HRV replication), cytochalasin D (actin disruptor), and paclitaxel (microtubule stabilizer). Cells were fixed for immunofluorescence using a resonance scanning confocal microscope. HRV replication kinetics were determined by serially washing ALI cultures every 24 hours to determine viral release by RT‐qPCR. Barrier function studies in Ussing chambers used FITC‐dextran (permeability) and measured real‐time transepithelial electrical resistance (TEER).RESULTSHRV‐1A and HRV‐C15, but not HRV‐16 induce dramatic remodeling of cellular architecture in highly differentiated HAE. These changes included the co‐localization of nuclei with apically expressed ZO‐1. This response depends upon the replication cycle of HRV, as replication‐deficient HRV does not induce these changes. Additionally, the cells associated with remodeled architecture are positive for HRV infection, as assessed by expression of replication intermediates. Preliminary data suggests that cytoskeletal inhibitors diminish the ability of HRV replication to occur, indicating a critical role for the actin cytoskeleton in HRV replication.CONCLUSIONWe conclude that some, but not all, HRV serotypes induce altered cellular architecture in HAE that is associated with disruption of the actin cytoskeleton. Future studies will seek to temporally relate altered cytoskeletal architecture with HRV replication sites.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
P311 Modulates Adipocyte Development
Authors: Nunez Sha'Kayla , Kotha Peddanna , Young Corey , Adebayo Olayinka , Badri Kameswara Rao 
Published: June 2021

Adipogenesis is a complex process of adipocytes formed from progenitor cells that involves changes in gene expression, cell morphology and hormone sensitivity. The differentiation of adipocytes is regulated by a well‐orchestrated network of transcriptional factors and associated cell signaling proteins responsible for adipocyte development and maturation. An in‐depth understanding of adipogenesis is essential to fight against metabolic disorders including obesity. Our lab investigates the P311 protein that plays a key role in adipocyte development. P311 has been shown to play an essential role in blood pressure regulation and vascular contractility (Badri et al., J Clin Inv 2013). In this study, we evaluate the adipogenic function of P311 using NIH3T3‐L1 cell culture model and genetically modified animal models. We show that P311 expression, initiated with the induction of adipogenesis in 3T3‐L1 cells, increased during the adipogenesis process. Additionally, siRNA mediated P311 knockdown inhibited adipogenic differentiation in 3T3‐L1 cells. The increase in P311 expression during adipogenesis correlated with the increase in the expression of key adipogenic transcriptional factors PPARγ2 and C/EBPα. Further, we observed nuclear localization of P311 in immunofluorescence studies indicating potential transcriptional regulatory/co‐regulatory function/s of P311. To verify the transcriptional regulatory/co‐regulatory functions of P311, we conducted chromatin immunoprecipitation (ChIP) assays using adipogenesis induced 3T3‐L1 cells transfected with pCMV‐P311 or an empty vector, pCMV. P311 overexpressing adipocytes demonstrated the recruitment of P311 to the PPARγ2 promoter regions (~800 fold) compared to empty vector transfected adipocytes. However, P311 immunocomplexes failed to amplify C/EBPα promoter. Further, to verify the involvement of P311 in adipose tissue development, we conducted animal studies and did not find any differences in the white fat weights (gonadal and subcutaneous fat) between wild type mice (WT) and P311 knockout mice (KO) (18–21 weeks). However, we observed an increase in the numbers of LipidTox stained adipocytes as detected in fluorescenceactivated cell sorting (FACS) analysis in white fat of WT mice than P311 KO mice. Our current studies demonstrate the critical role of P311 in adipocyte development through the transcriptional regulation of PPARγ2.Support or Funding InformationThis study is supported by grant funds from NIMHD/NIH (U54MD008621 to Hampton University and S21MD000101).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Chronic Pain Induced Inflammation in the Hypothalamus of Rats
Authors: DeForest Elizabeth , Semke Lori , Duric Vanja , Clayton Sarah C 
Published: June 2021

Chronic pain has been shown in humans to be associated with cardiovascular disease and hypertension but the mechanism of that association is unclear. Our overall hypothesis of this study is that chronic pain has a sensitizing effect on the central nervous system (CNS) that increases sympathetic nerve activity and predisposes to hypertension. Because inflammation can influence neuronal activation, in this study, we hypothesized that chronic pain generates a pro‐inflammatory phenotype in the cardiovascular control centers in the brain (e.g. the paraventricular nucleus of the hypothalamus). A chronic inflammatory pain model was generated by CFA injection into the left hind paw. Animals were then sacrificed 24 hours after the injection (acute phase) or 14 days following the injection (Chronic phase). RNA was isolated from the samples and expression of the pro‐ and anti‐inflammatory markers were assessed by RT‐qPCR. The expression of the pro‐inflammatory marker interleukin ‐ 1β (IL‐1β) was elevated in the hypothalamus 24 hours after CFA injection (2.3‐fold vs sham) and remained elevated at 14 days (2.5‐fold vs. sham) (P= .002). The expression of an anti‐inflammatory marker, transforming growth factor‐β 1 (TGF‐β 1), was decreased in the hypothalamus 24 hours after CFA injection (.8‐fold vs. sham) and remained decrease at 14 days (.63‐fold vs. sham) (P=.05) This data suggests that chronic pain induces a rapid and sustained pro‐inflammatory phenotype in the hypothalamus. This could lead to inappropriate activation of the sympathetic nervous system that could contribute to the development of hypertension.Support or Funding InformationSC IOERThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
The Functional Organization and Integration of Purkinje Cells in the Cerebellum
Authors: Heck Daniel Glenn , Person Abigail , Fukushima Sawako 
Published: June 2021

The overall object of this project is to create a 3D model of the cerebellum in order to illustrate the anatomical organization of Purkinje neurons, and to provide a better understanding of how they are integrated into other locations of the brain. Because the communication between the cerebrum and cerebellum is a closed system of polysynaptic circuits, understanding the underlying anatomy and physiology is a significant challenge. Classic staining techniques do not have the resolution nor the sophistication to capture the complete organization of the cerebellar cortex, let alone the entire cerebrocerebellar circuit. To overcome this challenge, a more precise staining technique has been developed to understand the complex functional organization of the cerebellum. Using a genetically modified variant of the rabies virus, neural connectivity can be imaged with extreme detail. The rabies is modified so that the glycoprotein required for the trans‐synaptic jump is deleted and replaced with a GFP protein, thus creating the ability to accurately label the connections to a single neuron. As a result of this staining technique, the Purkinje neurons can be traced using the cutting edge tracing software Neurolucida. The traced Purkinje cell somas and their axons can then be rendered in 3D and used for analysis and quantification. Having the ability to visualize the origin of the axons of specific cell types that communicate with the deep cerebellar nuclei in high 3D resolution may begin to provide an understanding of how the input of Purkinje cells is integrated with other excitatory inputs from elsewhere in the brain. Moreover, a 3D model of the cerebellum could also provide a more complete explanation of how the cerebellum quickly and accurately coordinates the inputs and outputs into movement, and is able to predict body movements in space.Support or Funding Informationhttps://www.sciencedirect.com/science/article/pii/0306452284901933This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
The anti‐apoptotic activity of protein S worsens liver fibrosis by activating the Akt pathway
Authors: Totoki Toshiaki , D'Alessandro‐Gabazza Corina N. , Toda Masaaki , Takeshita Atsuro , Yasuma Taro , Nishihama Kota , Horiki Noriyuki , Takei Yoshiyuki , Gabazza Esteban C 
Published: June 2021

Protein S is a vitamin K‐dependent glycoprotein produced mainly in the liver with anticoagulant, anti‐inflammatory, immune‐modulatory and anti‐apoptotic properties. The anti‐apoptotic activity of protein S can ameliorate or worsen a disease state depending on the organ involved. For example, protein S attenuates pulmonary fibrosis by inhibiting apoptosis of lung alveolar epithelial cells or improve diabetes mellitus by inhibiting apoptosis of pancreatic islet b‐cells. On the contrary, exogenous protein S exerts detrimental effects during acute alcoholic hepatitis by its ability to activate and prolong the survival of liver natural killer T cells. In the present study, we investigated whether human protein S can affect chronic liver injury and fibrosis. Liver injury/fibrosis was induced by carbon tetrachloride injection in mice overexpressing human protein S and in wild type mice and the degree of liver injury and fibrotic changes were evaluated. Human protein S transgenic mice receiving carbon tetrachloride showed significantly higher circulating levels of liver transaminases, increased liver expression of inflammatory cytokines and chemokines, significantly much extended liver fatty degeneration and fibrosis and increased areas with DNA breakage after chronic injury compared to wild‐type mice. Liver injury was also exacerbated in mice after one bolus injection of carbon tetrachloride. Wild‐type mice infused with exogenous human protein S exhibited exacerbated liver injury, had significantly less apoptosis associated with increased number of hepatic stellate cells compared to untreated mice. An in vitro study using primary human hepatic stellate cells demonstrated that protein S inhibits apoptosis and increases the degree of Akt pathway phosphorylation in these liver cells. Western analysis showed significantly less cleavage of caspase‐3 in liver tissue from protein S transgenic mice compared to wild type mice. In conclusion, the results of this investigation showed that the anti‐apoptotic activity of protein S may play a role in chronic liver injury and subsequent liver fibrosis.Support or Funding InformationI have no financial relationships to disclose.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Lack of a‐CGRP Prevents High Fat Diet Induced Biliary Proliferation and Hepatic Steatosis
Authors: O'Brien April , Kyritsi Tina , Chen Lixian , Glaser Shannon , Alpini Gianfranco 
Published: June 2021

Nonalcoholic fatty liver disease (NAFLD) has become a prevalent problem in westernized societies where incidents of obesity lead to a reduction in blood flow and a deposition of fat into the liver increasing incidents of maladies ranging from minor nonclinical hepatic steatosis (NASH) to severe cirrhosis and cholangiocarcinoma (CCA). We have previously shown that cholangiocytes participate in a sub epithelial fibrotic process that is the hallmark of hepatobiliary injury. Employing a well‐established mouse model of mechanical liver injury through bile duct ligation (BDL), we have shown that cholangiocytes are regulated, in part, through sensory innervation, and manipulation of the innervation pathway through a‐calcitonin gene‐related peptide (a‐CGRP) signaling results in a reduction in cholangiocyte proliferation as well as liver fibrosis. Due to the fibrotic nature of fatty liver disease and our previous research demonstrating the interplay between cholangiocytes, neuropeptides, and sensory receptors, we AIM to evaluate the role a‐CGRP plays in the biliary progression from hepatic steatosis to the profibrotic phenotype found in the damaged biliary epithelium.METHODSStudies were performed in C57 (WT) and CGRP KO mice (4 wk of age). Mice were treated with Control and High Fat Diet (0.2% cholesterol, 45% fat calories, with 30% of the fat in the form of partially hydrogenated vegetable oil) with 45% fructose, 55% glucose (42g/L) drinking water for 20 wks. Fat deposition was visualized via Oil Red O staining. Ductular reaction (IBDM), CGRP accessory protein and collagen deposition was evaluated by IHC for CK‐19, RAMP1 and Sirius Red, respectively, in total liver sections. a‐CGRP was evaluated via protein expression in mouse total liver and mRNA in human tissue by IHC and qPCR. mRNA data for CLR, CALCRL, RAMP1, and CGRP was obtained via qPCR in pure cholangiocytes in WT CD and WT HFD.RESULTSThere was a significant decrease in the fat deposition in the a‐CGRP KO HFD compared to the WT HFD. There was a significant decrease in IBDM and collagen deposition in CGRP KO HFD mice compared to WT HFD. Both mouse HFD and human steatotic livers showed significant increases in a‐CGRP when compared to their WT counterpart. Additionally, IHC demonstrated a significant increase in RAMP1 in the WT HFD when compared to CD. Cholangiocytes had significant increase in the expression of mRNA in the CGRP receptor (CLR), the receptor component (CGRPRC), its accessory, RAMP1, and the protein CGRP in HFD when compared to the WT CD.CONCLUSIONa‐CGRP plays a substantial role in proliferation and hepatic fibrosis. Modulation of the a‐CGRP axis may represent a novel therapeutic approach for the management of NAFLD.Support or Funding InformationNIH R01 DK76898This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Spatial Organization of a cAMP/Ca2+‐Regulated Signaling Complex: A Solution Structural Small‐Angle X‐ray and Neutron Scattering Study of an AKAP79‐Scaffolded Complex Containing Type IIβ PKA and Calcineurin
Authors: Hall James , Copps Jeffery , Zhang Ping , Heck Albert , Blumenthal Donald , Taylor Susan 
Published: June 2021

cAMP‐dependent protein kinase (PKA) regulates many cell processes including development, differentiation, and memory. In its resting state, PKA is a tetrameric holoenzyme consisting of a regulatory (R) subunit dimer and two catalytic (C) subunits. Four isoforms of the R‐subunit exist (RIα, RIβ, RIIα, and RIIβ). Although each isoform varies in sequence and quaternary structure, all share a common domain organization. The strength, duration, and specificity of PKA activity within the cell are mostly dependent upon isoform specific subcellular localization. The D/D domain interface of the R‐subunits serves as a docking site for A Kinase Anchoring Proteins (AKAPs) that target PKA to specific cellular locations. In cardiac myocytes and neurons, PKA is localized to Cav1.2 L‐type calcium channels by AKAP79 and up‐regulates calcium influx by phosphorylating the channel's cytosolic C‐terminal tail. AKAP79 also binds the Ca2+/calmodulin‐dependent protein phosphatase type 2B (PP2B, or calcineurin) at a site very close to that of PKA binding. Given the close proximity of the calcineurin and PKA binding sites on AKAP79, calcineurin has been suggested to play roles in regulation of calcium channels, along with regulation of PKA activity itself. Using a variety of biochemical and biophysical techniques, we have isolated and characterized a stable complex of the C‐terminal tail of AKAP79, RIIβ holoenzyme, and calcuneurin (“ARC” for short). We show a stoichiometry of 1:1:1 (AKAP79: RIIβ2C2:CN) using native mass spectroscopy, and SAXS/SANS techniques confirm that this is a compact complex, suggesting a close interaction between PKA and calcineurin when bound to AKAP79. Altogether, we suggest beyond its function of localizing PKA and CN in close spatial proximity to the Cav1.2 L‐type calcium channel, the ARC signalosome fosters extended protein‐protein interactions and regulations that suggest tight spatiotemporal control.Support or Funding InformationJames Hall is a San Diego IRACDA Fellow supported by National Institutes of Health (NIH)/NIGMS K12 GM06852 Award. Support for this work was provided through NIH GM034921.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.