bluuebunny/crossref_metadata_2025_split huggingface dataset:
Development of Antigen Binding Protein Monobodies as a Tool for Antigen Specific Treatment for Autoimmune Kidney Disease
Authors: Stocks Omar , Goldman Liam , Stoddard Shana Victoria 
Published: June 2021

Autoimmune disease (AD) is a disease where antibodies incorrectly attack healthy self cells by binding to antigens. As a result of this self attacking patients experience long term sickness or even death. Current treatment methods include the use of non‐specific immunosuppressive medicines, which are somewhat effective at diminishing the effects of the disease. While effective, these therapies leave the patient with a significantly weakened immune system and decreased ability to fight off simple pathogens. This emphasizes the need for a more targeted treatment for AD. The work here focuses on the disease primary membranous nephropathy (PMN), which targets three domains of the phospholipase A2 receptor (PLA2R): the ricin domain and C‐type lectin domains numbers 1 and 7 (CTLD1 and CTLD7). The goal of this project is design of antigen specific treatment for PMN, which involves preventing autoantibodies from binding to the PLA2R antigen, through design of protein monobodies. In this work, the epitope regions on CTLD7 were predicted using the programs Epitopia and EPCES to define target sites for the epitope caps. The data suggest that there are three distinct epitope locations on CTLD7 that could be targeted by PMN autoantibodies. Currently, 26 epitope caps have been designed using the templates 3UYO and 5A40. The results show that in silico mutagenesis of protein monobodies enhanced binding of epitope caps to epitope sites on the CTLD7 domain from −4.94 Rosetta Energy Units (REU) on the 3UYO template to −5.094 REU, and −5.081 in 3UYO‐004‐OS and 3UYO‐022‐OS, respectively. In silico mutagenesis to the 5A40 template, producing the 5A40‐001‐OS cap, improved binding by approximately 2 REUs: improving the REU score from −5.618 to −7.596 to the CTLD7 domain. Results indicate that in the 5A40‐001‐OS cap, introduction of a negatively charged aspartate created an electrostatic interaction with CTLD7, which improved the potency of binding. The 5A40‐001‐OS cap along with 3UYO‐004‐OS and 3UYO‐022‐OS caps are being expressed in E. coli for further evaluation through ELISA assays. The results of this work could lead to a much safer and more antigen specific treatment for autoimmune disease.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Proteomic and Phosphoproteomic Signatures of Severe Alcoholic Hepatitis
Authors: Hardesty Josiah , Warner Jeffrey C. , Warner Dennis C. , Jacobs Jon , McClain Craig C. , Kirpich Irina A 
Published: June 2021

BackgroundThe increasing prevalence of alcoholic liver disease (ALD) presents a large global healthcare burden. Currently, no therapeutics are available and the mechanisms underlying ALD development and progression are not well understood. Alcoholic hepatitis (AH) is a severe stage of ALD with a high mortality. Thus, identifying new signaling pathways involved in AH development, as well as novel biomarkers of AH progression are of a great clinical importance. The current study was aimed to determine hepatic proteomic and phosphoproteomic signatures and impaired metabolic pathways in severe AH.MethodsLiquid chromatography and tandem mass spectrometry analysis were performed to examine proteomic and phosphoproteomic changes in explanted liver tissues obtained from AH patients (n=6) and patients without ALD (controls, n=12). A Student's two‐tailed unpaired t‐test was used to determine statistical significance between the two groups. MetaCore ontology analyses (false‐discovery rate < 0.05) were performed to identify proteomic changes, transcription factors and metabolic/signaling pathways altered in AH patients.Results3,288 peptides and 5,923 phospho‐peptides were significantly changed in AH liver samples compared to controls. The most diminished transcription factors in AH patients were hepatocyte nuclear factor 4‐alpha (HNF4a), hepatocyte nuclear factor 1‐alpha (HNF1a), and thyroid hormone receptor‐beta (THRb) as well as their target genes (45, 26, and 17 genes decreased in the networks, respectively). In contrast, the expression of transcription factors related to inflammation such as nuclear factor kappa B subunit 1 and 2 (NFkB1, NFkB2), as well as specificity protein 3 (SP3), were increased as were their target genes (9, 16, and 32 genes increased in each network, respectively). Further, the pathway involved in phagocytic bacterial clearance was significantly diminished in AH patients compared to control (p=4.2e‐8), while the neutrophil extracellular trap pathway was elevated (p=1.6e‐8). In addition, proteins involved in hepatocyte maturation (p=1.4e‐6) and glutathione metabolism (p=5.1e‐5) pathways were downregulated in AH patients.ConclusionsLiver proteomic and phosphoproteomic analysis identified multiple alterations in transcriptional networks in AH patients characterized by diminished expression of transcription factors such as HNF4a, HNF1a, and THRb and elevation of NFkB1/2, and SP3. Further investigations are required to uncover new mechanisms in AH development and progression, as well as to identify novel biomarkers and therapeutic targets.Support or Funding InformationNIH NIAAA RO1 AA024102NIH NIGMS P20 GM113226This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
The Association of Fibroblast Growth Factor 5 (FGF5), Fibroblast Growth Factor Receptor 1 (FGFR1), and Prolactin (PRL) with Hair Length in Scottish Highland Cattle
Authors: Li Haoju , Lipsett Koren A 
Published: June 2021

Investigating genetic disorders in large mammals is important because large mammals mimic humans due to their size, lifespan and physiology. Therefore, genetic mutations attributed to mammal disease can be correlated to similar human genetic diseases. Fibroblast growth factor family (FGF) and its receptor (FGFR1) are responsible for cell growth and wound healing. Prolactin (PRL) and its receptor (PRLR) are also in charge of the growth hormone and responsible for milk production in mammals. Previous research has shown that mutations in the FGF5 and FGFR1 genes result in abnormal hair growth in mice, dogs, sheep and donkeys. In addition, deficiency in dairy production and abnormally long hair are the result of mutations in PRL and PRLR. By defining a mutation associated with abnormal hair growth, and by extension, unregulated cell growth, abnormal growth of cancer cells can be further understood. However, to date no studies have found mutations in these genes in cattle. The Scottish Highland cattle is the only cattle breed with long hair (year‐round). In this study, FGF5, FGFR1, PRL were sequenced using our experimental cattle and control cattle. Our results showed that single nucleotide polymorphisms (SNPs) exist between the long‐hair cattle and control cattle, however, the mutations are silent mutations.To further investigate, we expanded our research to the intronic regions and other genes through whole genome (WGS) sequencing by Illumina. We processed the raw mega‐data utilizing Amazon Web Service (AWS) including EC2, S3 coupled with Dragen by Edico Genome to map, align, and complete variant calling of the WGS from Scottish Highland cattle. This work generated BAM and VCF files that are being used for further variant analysis. Here, we report our progress leading to the identification of variants that may be responsible for long hair growth in Scottish Highland cattle.Support or Funding InformationProvost's Senior Research Fund, X‐SIG Fund, & Albaugh Fund, Gettysburg College, Gettysburg, PA 17325This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Transforming Growth Factor Beta Induces Fibroblasts to Express and Release the Immunomodulatory Protein PD‐L1 into Extracellular Vesicles
Authors: Kang Jeong‐han , Jung Mi‐Yeon , Leof Edward B 
Published: June 2021

Transforming growth factor beta (TGF‐β) is an enigmatic protein with various roles in normal tissue homeostasis/development as well as the development and/or progression of cancer, wound healing, fibrotic disorders, and immune modulation, to name a few. As TGF‐β is causal to various fibroproliferative disorders featuring localized or systemic tissue/organ fibrosis as well as the activated stroma observed in numerous malignancies, characterizing the pathways and players mediating its action is fundamental. To that end, in the current study we found that (i) TGF‐β induces the expression of the immunoinhibitory molecule Programmed Death‐Ligand 1 (PD‐L1) in human and murine fibroblasts in a Smad2/3‐dependent manner; (ii) profibrotic TGF‐β signaling is dependent upon PD‐L1 expression; and (iii) following treatment with TGF‐β, human lung fibroblasts secrete PD‐L1 into extracellular vesicles (EVs) capable of inhibiting T‐cell proliferation in response to T cell receptor stimulation. These findings provide new insights and potential targets for a variety of fibrotic and/or malignant diseases.Support or Funding InformationThis work was supported by Public Health Service grants GM‐55816 and GM‐54200 from the National Institute of General Medical Sciences.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
The Impact of Cancer‐Associated Mutations in Thyroid Hormone Receptor (TR) on TR Localization
Authors: Malapati Sri Harshini , Punzalan Paige , Allison Lizabeth 
Published: June 2021

The thyroid hormone receptor (TR)—a nuclear receptor that is essential for the proper regulation of metabolism and development, as it regulates gene expression in response to thyroid hormone—contains nuclear localization signals (NLSs) and nuclear export signals (NESs) that allow for TR transport into and out of the nucleus, respectively. Previous research has shown that nuclear import, nuclear retention, and nuclear export of TR are associated with modulation of gene expression, the alteration of which can contribute to various diseases, including Resistance to Thyroid Hormone (RTH) Syndrome, type II diabetes mellitus, and several types of cancer. There are three main subtypes of TR: TRα1, TRβ1, and TRβ2. Here, through mammalian cell transfection of expression plasmids for green fluorescent protein (GFP)‐tagged TR and fluorescence microscopy, we examined particular groups of TRα1 mutations that were observed in patients with hepatocellular carcinoma and renal carcinoma and are associated with NLSs and NESs of TRα1. More specifically, we calculated the nuclear‐to‐cytoplasmic ratios of TRα1 variants to determine the impact of TRα1 mutations on receptor localization within HeLa cells. We found that, while wild‐type TR has a primarily nuclear localization with a smooth distribution in human cells grown in culture, the TRα1 mutations S40T, K136R, L251P, V390A and A225G, D246N, G350K lead to significantly decreased nuclear‐to‐cytoplasmic TRα1 ratios (greater cytoplasmic distribution) and formation of TRα1 aggregates. We observed that the distribution of a TRα1 mutant with just one of the four mutations (L251P) is very similar to the distribution of the TRα1 mutant with all four mutations (S40T, K136R, L251P, V390A), suggesting that L251P plays a major role in the mislocalization of TRα1. In addition, we found that TRα1 with a G24E mutation had a significantly greater cytoplasmic distribution, compared to wild‐type TRα1. In conclusion, our findings suggest that certain TRα1 mutations that are associated with carcinoma lead to TRα1 mislocalization, which may alter the expression of thyroid hormone‐dependent genes. These results contribute to an improved understanding of TR structure and function, which could lead to novel strategies for discovering therapies for TR‐related illnesses.Support or Funding InformationNIH #2R15DK058028‐04This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Ouabain Regulated Phosphoproteome Reveals Molecular Mechanisms Behind Na, K‐ATPase Control of Cell Adhesion, Proliferation and Survival
Authors: Panizza Elena , Svensson Daniel , Zhang Liang , Fontana Jacopo Maria , Akkuratov Evgeny , Scott Lena , Brismar Hjalmar , Lehtiö Janne , Aperia Anita 
Published: June 2021

Ouabain is a cardiotonic steroid with a history of medical uses. It acts as a ligand to the ubiquitous ion pump Na+,K+‐ATPase which it inhibits at high concentrations. At lower concentrations it triggers calcium oscillations and activation of MAP kinases, stimulates cell proliferation as well as adhesion and acts anti‐apoptotic. In light of this, it has been proposed that the pump may have a dual function as a signaling transducer. To investigate this, we have analyzed the phosphoproteome of COS‐7 kidney cells after treatment with sub‐saturating levels (100 nM) of ouabain, which were high enough to induce Ca2+ oscillations but below those needed to modulate the ion pumping function of Na+,K+‐ATPase. Gene ontology analysis was performed to find distinct cellular processes regulated by ouabain after 10 and 20 min of treatment in the phopshoproteomic analysis. The analysis revealed 2580 ouabain‐regulated phosphorylation sites, many of which were associated with cell adhesion and proliferation. Two of the regulated phospho‐proteins were the inositol triphosphoate receptor (InsP3) and stromal interaction molecule (STIM), both of which are essential for ouabain‐induced calcium oscillations. We used siRNA and Western blotting for target confirmation. One confirmed target, calcium/calmodulin‐dependent protein kinase II gamma (CAMK2γ), were shown to be involved in the anti‐apoptotic effect of ouabain; siRNA silencing of CaMK2γ in primary renal cells eliminates the protective effect of ouabain against apoptosis induced by either serum starvation or glucose. In conclusion, our findings support a role for Na+,K+‐ATPase as a signal transducer that serves to protect cell and tissue integrity.Support or Funding InformationThis study was supported by the Swedish Research Council and Erling‐Persson Family Foundation. D.S. is supported by a Novo Nordisk postdoctoral fellowship run in partnership with Karolinska Institutet.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Nuclear Receptor Corepressor 1 Regulates Thyroid Hormone Receptor Nuclear Retention
Authors: Sun Xiaopeng , Allison Lizabeth 
Published: June 2021

The thyroid hormone receptor (TR) mediates gene transcription in response to thyroid hormone (T3) and significantly affects cell growth, development and metabolism. Although TR shuttles rapidly between the nucleus and cytoplasm, it is primarily nuclear localized at steady state. A shift in the balance between export and import leading to TR mislocalization may correlate with certain cancers or endocrine disorders. Previously, we have identified multiple nuclear localization signals and nuclear export signal within TR that interact with importins and exportins, respectively, to mediate shuttling. Given that TR contains multiple, competing import and export signals, the question arises: what factors are involved in nuclear retention of TR? In addition to DNA‐binding sites, the lab's prior research indicated that nuclear coactivator MED1 facilitates TR nuclear localization. Thus, we hypothesize that certain transcriptional coregulators, including Nuclear Receptor Corepressor 1 (NCoR1), contribute to TR nuclear retention. Using transient transfection assays and fluorescence microscopy, we measured the fluorescent intensity of GFP or mCherry‐tagged TR in the nucleus (N) and cytoplasm (C) of HeLa cells, and then calculated the N/C ratio as an indicator of intracellular localization patterns. We discovered that when NCoR1 is knocked down by shRNA, the amount of TR in the nucleus decreased significantly (lower N/C ratio). Inversely, when NCoR1 is over‐expressed, the amount of TR in the nucleus significantly increased (higher N/C ratio). An even more dramatic cytosolic shift was observed for a TR mutant that cannot bind to NCoR1 (AHT‐TR). Taken together, our results suggest that NCoR1 is a key regulator of TR nuclear retention. This finding will contribute to the development of a dynamic mathematical model that aims to predict TR's N/C ratio during the interaction with different complexes and TR localization's effect on transcriptional output of T3‐dependent genes.Support or Funding InformationNIH 2R15DK058028This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Assembly and Annotation of the Starfish Egg Transcriptome: The Search for the Elusive Molecular Controllers of Fertilization
Authors: Bates Lauren , Wiseman Emily , Kitson Jamie , Dunn Leah , Baghban Mehrasa , Carroll David 
Published: June 2021

Fertilization is the process of sperm‐egg interaction leading to a permanent block to polyspermy and re‐initiation of the cell cycle. Release of Ca2+ from the endoplasmic reticulum is the integral molecular mechanism responsible for activation of fertilization; however, the molecular players involved in the initiation and regulation of the Ca2+ release are not well‐established in any organism. Research in the fertilization field has been hampered due to difficulty in studying the sperm‐egg cell system. Eggs and sperm of the starfish Patiria miniata present an attractive model to study the molecular aspects of fertilization due to the ease of obtaining millions of viable sperm and eggs, as well as, their amenability to in vitro fertilization and biochemical assays. However, the lack of specific antibodies for the identification of proteins in the starfish model system has delayed discovery. To address this deficiency, we produced the first P. miniata egg transcriptome by de novo RNA sequencing, which will allow us to predict which proteins could be present and function in controlling the fertilization signaling pathway. The goal of this project is to annotate the starfish egg transcriptome and categorize the transcripts into known protein families. We present evidence for the presence of all major signaling pathways and confirm biologically‐relevant RNAs via RT‐PCR against freshly isolated starfish egg RNA. This work will be used to establish a potential proteome for the identification of proteins that may function in the fertilization signaling pathway.Support or Funding InformationNational Institutes of Health R15This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Identification of GDP‐dependent Interactions Between TCL and Potential Effector Proteins
Authors: Tader Brooke R , Hamann Michael J 
Published: June 2021

TCL/RhoJ belongs to the Rho family of GTPases and has been shown to contribute to tumor‐associated angiogenesis as well as metastatic melanoma. While the role of TCL in these pathological events is becoming clearer, less is known about its biochemistry and molecular/cellular function. To better understand the cellular events controlled by TCL, experiments in our lab have focused on where TCL localizes to in cells based upon its nucleotide loading status and prenyl modifications. Using constitutively active (CA) and dominant negative (DN) mutants of TCL, we have shown that GTP‐loaded TCL localizes to the plasma membrane while GDP‐loaded TCL is more vesicular in appearance. Clearly, localization of CA TCL to the plasma membrane is also dependent on prenylation of the TCL CAAX motif as mutation of this signal sequence results in diffuse cytoplasmic localization. However, the same CAAX mutation in the context of the DN mutation lead to a marked vesicular localization. Together, our results have led us to hypothesize that TCL is specifically retained to vesicular membranes through an interaction with an unidentified binding partner, and current experiments using biotin‐ligase tags of CA and DN TCL and BioID procedures are aimed at identifying a vesicle‐bound partner protein. We anticipate that a more complete understanding of how TCL functions at vesicular and plasma membranes will help identify its potential role in vesicular trafficking in pathology‐related processes.Support or Funding InformationThis work was supported by Bemidji State University Biology Department and the College of Business, Mathematics, and Science. Support was also provided through the Neilson Foundation, Bemidji, MN and Regenerative Medicine Minnesota (RMM‐2017‐EP‐04).This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Interplay of Soluble, Mechanical and Redox Signaling in Airway Remodeling During Asthma
Authors: Al‐Azzam Nosayba Zakariya , Gombedza Farai , Snyderman Matthew , Ghebreigziabher Samarawit , Covington Kayla , Thodeti Charles , Paruchuri Sailaja 
Published: June 2021

NADPH oxidase 4 (NOX4) is a member of the NADPH oxidase family, encoded by the NOX4 gene, and has been implicated in TGF‐β1‐induced human cardiac fibroblast to myofibroblast differentiation1. NOX4 has been shown to potentiate lung fibroblast differentiation in vitro and its transcript level is enhanced in fibroblasts isolated from patients with idiopathic pulmonary fibrosis (IPF) compared to normal fibroblasts2. Our lab has previously identified that Transient Receptor Potential Vanilloid 4 (TRPV4), a mechano‐sensitive ion channel, is involved in D. farinae‐induced airway remodeling during asthma in vivo and myofibroblast differentiation in vitro. However, the role of NOX4 in asthma, its interaction with TRPV4, and the mechanism of action still remains elusive. In the present study, we show that NOX4 is up‐regulated during lung remodeling in asthma and fibroblast differentiation in a TRPV4‐dependent pathway. TGF‐β1 treatment up‐regulated NOX4 gene expression and function. Further, employing antioxidants and NOX4 siRNA, our results reveal that NOX4 is involved in TGF‐β1‐induced α smooth muscle actin (αSMA), SM22, fibronectin (FN), myocardin‐related transcription factor‐A (MRTF‐A) and plasminogen activator inhibitor ‐1 (PAI‐1) expression in human lung fibroblasts (hLF). Interestingly, TRPV4 inhibition led to attenuation in TGF‐β1‐mediated NOX4 expression and fibroblast differentiation. Furthermore, NOX4 transcripts were up‐regulated in lung tissues in response to D. farinae in an asthma model supporting our in vitro findings. More importantly, TRPV4−/− mice were protected from D. farinae‐induced asthma and exhibited lower levels of NOX4 gene expression in response to D. farinae, further suggesting that TRPV4 promotes airway remodeling through NOX4.Support or Funding InformationJames Foght research supportThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.