bluuebunny/crossref_metadata_2025_split huggingface dataset:
2‐Amino Muramyl Dipeptide Derivatives: Chemical probes to assay the stability and activation of NOD2
Authors: Brown Ashley R , Ukaegbu Ophelia , Grimes Catherine Leimkuhler 
Published: June 2021

Mutations of the cytosolic protein nucleotide‐binding oligomerization domain‐containing protein 2 (NOD2), which detects the synthetic bacterial cell wall fragment muramyl dipeptide (MDP), are implicated in making individuals more susceptible to Crohn's disease (CD). However, the mode by which these mutations affect the pathogenesis of this disease is not fully understood. Interestingly, studies suggest NOD2 positively and negatively regulates the expression of toll‐like receptors (TLRs) and the consequent production of inflammatory molecules upon activation. Like NOD2, TLRs can detect bacterial fragments and produce an inflammatory response via the NF‐kB pathway, though through distinct mechanisms. Stabilizing the NOD2 Crohn's mutants through protein‐ligand binding can be a potential therapeutic target for CD. A library of MDP derivatives with altering functionality at the 2‐amino position were synthesized in order to investigate the stabilization imparted to NOD2 upon binding. A simplified 7‐step synthetic route to prepare 2‐amino MDP and subsequent functionalization of the molecule with mild NHS coupling or acetylating conditions is utilized. Compounds are then tested for stabilization with the cellular thermal shift assay (CESTA). In this cell‐based assay, binding of a ligand to a protein stabilizes a protein and protects it against thermal degradation at increased temperatures, resulting in a thermal shift (ΔTm) when compared to the unbound protein. The activation of the NF‐kB pathway by these molecules is tested concurrently, using a luciferase reporter assay to determine if changes in functionalization also yield a modulated immune response.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 Mammalian UDP‐Galactose 4′‐Epimerase (GalE) Is Required for Cell Surface Glycome Structure and Function
Authors: Florwick Alyssa T , Broussard Alex , Desbiens Chelsea , Nischan Nicole , Robertson Corrina , Guan Ziqiang , Kohler Jennifer , Wells Lance , Boyce Michael 
Published: June 2021

Nucleotide‐sugars are required for glycosylation reactions in mammals, but it is largely unknown how their levels are dynamically regulated in response to cellular signals. The UDP‐galactose 4’‐epimerase (GalE) has recently emerged as a potential regulator of nucleotide‐sugar flux in response to stimuli such as feeding, ischemia and endoplasmic reticulum (ER) stress. The importance of this enzyme is underscored by human patients with hypomorphic GalE alleles, constituting a disease termed type III galactosemia. Critically, the molecular underpinnings of galactosemic pathophysiology are unclear.Because it controls the levels of multiple substrates in several signaling contexts, GalE is an ideal candidate for studying the importance of nucleotide‐sugar regulation in both normal and disease physiology. In this work, we generated GalE−/− human cell lines using CRISPR‐Cas9 mutagenesis. We characterized the effects of GalE loss on nucleotide‐sugar metabolism, protein glycosylation, and death receptor function. Our results demonstrate that loss of the GalE enzyme effects dramatic dysfunction in nucleotide‐sugar metabolism along with protein and lipid glycosylation. We have observed mis‐glycosylation of various cell‐surface proteins. GalE−/− human cells exhibit dysregulated cellular signaling, which we hypothesize is due to mis‐glycosylation of cell‐surface receptors.Support or Funding InformationResearch in the Boyce Lab has been supported by NIH grants R01GM118847 and R01GM117473 and the Rita Allen Foundation Scholars programThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Targeting the O‐GlcNAc Transferase to Specific Proteins Using RNA Aptamers
Authors: Zhu Yi , Hart Gerald 
Published: June 2021

O‐linked β‐D‐N‐acetylglucosamine (O‐GlcNAc) is an abundant post‐translational modification (PTM) of thousands of proteins that regulates signaling, transcription and cytoskeletal functions. A major roadblock toward elucidating O‐GlcNAc's functions is our inability to alter the levels of O‐GlcNAcylation on a single protein at a time. Herein, we describe a novel method to alter the O‐GlcNAcylation of a specific protein. We designed series of modulated RNA aptamers that are able to induce the proximity between β‐catenin and O‐GlcNAc transferase (OGT). These aptamers are able to increase O‐GlcNAcylation of β‐catenin by ~6‐fold in HEK293T cells without affecting the O‐GlcNAcylation of other proteins. Increased O‐GlcNAcylation of β‐catenin concomitantly altered the phosphorylation state at the degradation‐related sites (Ser33, Ser37, Thr41 and Ser45), indicating a cross‐talk between O‐GlcNAcylation and phosphorylation in regulating the stability and transcriptional activity of β‐catenin. Detailed analyses of a series of similar modulated aptamers indicate that the distance and spatial alignment between OGT and β‐catenin in the induced proximity are critical considerations when designing the modulated aptamers. This modulated aptamer design is also able to increase O‐GlcNAcylation on some other cellular proteins. Taken together, our research provides a novel tool to study how O‐GlcNAcylation specifically regulates individual proteins, establishes a novel method to induce proximity between cellular components, as well as providing insights on how O‐GlcNAcylation regulates the function of β‐catenin.Support or Funding InformationSupported by NIH P01HL107153, & R01GM116891, the Georgia Research Alliance and the Univ. of Georgia. Dr. Hart receives a share of royalty received by the university on sales of the CTD 110.6 antibody, which are managed by JHU.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
MicroRNA Sequencing of Discoid Lupus Erythematosus Skin
Authors: Calderon Irene , Mina Rina 
Published: June 2021

BackgroundDiscoid lupus erythematous (DLE) is an autoimmune skin condition that causes profound disfigurement. Environmental effects are linked to increased risk and worsening disease off activity in DLE. MicroRNA (miRNA) are increasingly found to have important roles in the environmental modulation of inflammation and cellular differentiation in autoimmune diseases.ObjectiveThe goal of the pilot study was to comprehensively identify the miRNA transcriptome in DLE.MethodsThe specimens were from African American women (DLE=4, normal=4) and were age‐matched [median age=55 years (range=50–61), median DLE duration=18 years]. Subjects with DLE did not have concomitant systemic lupus and were not on systemic immunosuppression during skin biopsy. Skin specimens were composed of keratinocytes, epidermal appendages, vasculature, dermal fibroblasts and skin homing immune cell populations. We extracted total RNA using a method that preserved small RNAs (Ambion). RNA was prepared for sequencing with the Small RNA Sample Prep Kit,□□ □□ adapters were ligated to approximately 1 microgram of total RNA. Each library was loaded on a single Illumina lane at 20 picogram and subjected to 86 cycles of sequencing on HiSeq. We generated 52.4 million raw and 15.3 million qualified reads. We mapped qualified reads to miRNA precursors, functional non‐coding RNAs and build 37 (hg19) of the human genome. The adapter‐trimmed, qualified reads were distributed around an average length of 21 nucleotides. The number of reads and mapping proportions in the DLE group approximate that in the normal skin groups. We detected mature miRNAs derived from 1240 known miRNA precursor families. These were represented by at least one read in the cumulative data set. We analyzed the data from our next generation sequencing based on relative comparisons of normalized read counts between DLE and normal skin to detect relative changes. Standardized fold changes of miRNAs were calculated as mean differences in read counts divided by pooled standard deviations in the log scale.ResultsWe identified 18 known miRNAs that were significantly over or under expressed in active DLE lesions versus normal skin (p‐values < 0.05). Among these are mir‐146a, mir‐223, mir‐21, mir‐23b. These miRNAs are linked to IFN‐regulation, hypomethylation, ultraviolet exposure, differentiation of the keratinocytes, epidermal dendritic cells, and T cells, and estrogen response.ConclusionFrom this discovery‐based study, we have identified several miRNAs that may play an important role in epigenetic regulation in DLE that ultimately affects disease severity.Support or Funding InformationCTSA of University of CincinnatiThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Src and cortactin are involved in pemphigus skin blistering
Authors: Kugelmann Daniela , Rötzer Vera , Walter Elias , Egu Desalegn Tadesse , Vielmuth Franziska , Vargas‐Robles Hilda , Schnoor Michael , Hertl Michael , Rottner Klemens , Spindler Volker , Waschke Jens 
Published: June 2021

Autoantibodies against desmoglein (Dsg) 1 and Dsg3 primarily cause blister formation in the autoimmune disease pemphigus vulgaris (PV). Src was proposed to contribute to loss of keratinocyte cohesion. However, the role and underlying mechanisms are unclear. In keratinocytes, cell cohesion in response to autoantibodies was reduced in a Src‐dependent manner by two patient‐derived PV‐IgG fractions as well as by AK23, but not by a third PV‐IgG fraction, although Src was activated by all autoantibodies. Loss of cell cohesion was progredient and AK23 similar to PV‐IgG interfered with reconstitution of cell cohesion after Ca2+‐switch, indicating that the autoantibodies also interfered with desmosome assembly. Dsg3 co‐localized along cell contacts and interacted with the Src substrate cortactin. Concomitantly, cell adhesion was impaired in keratinocytes isolated from cortactin‐deficient mice in comparison to keratinocytes isolated from wildtype (wt). AK23‐induced loss of cell cohesion was Src‐dependent only when cortactin was expressed. Similarly, AK23 impaired reconstitution of desmosomal adhesion in Src‐dependent fashion only in the presence of cortactin and AK23‐induced skin blistering was abolished by Src inhibition in wt but not cortactin‐deficient mice. However, in human epidermis, PV‐IgG‐induced skin blistering and ultrastructural alterations of desmosomes were not affected by Src inhibition. Our data suggest that Src and cortactin are involved in pemphigus skin blistering but the contribution of Src is variable.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Novel Junctional and Nuclear Roles of the Mitochondrial Protein Mitofusin 2 During Inflammation
Authors: Kim Young‐Mee , Krantz Sarah , Toth Peter T. , Rehman Jalees 
Published: June 2021

BackgroundSevere inflammation can cause widespread damage to the endothelium and result in fatal illnesses such as acute lung injury (ALI) or acute respiratory distress syndrome (ARDS). Vascular endothelial barrier dysfunction is the major vascular complication in ALI. One of the key mechanisms of barrier disruption is the disassembly of adherens junction (AJ) complexes consisting of VE‐cadherin as well as accessory proteins such as β‐catenin and p120‐catenin. We observed that Mfn2, which has previously been primarily studied as a mitochondrial protein, is also located at the endothelial plasma membrane and co‐localizes with VE‐cadherin as well as translocates into nucleus under inflammatory condition, suggesting novel roles in AJs.Methods and ResultsBy using three‐dimensional structured illumination microscopy (3D‐SIM) which provides high spatial resolution (□130 nm), we studied Mfn2 localization in human lung microvascular ECs (HLMVECs). Mfn2 predominantly co‐localizes with the mitochondrial membrane protein Tom20 but it also co‐localizes at the plasma membrane with VE‐cadherin in resting ECs. However, upon pro‐inflammatory stimulation with TNF‐α, Mfn2 is dissociated from AJs and no longer co‐localizes with VE‐cadherin. Mechanistically, Mfn2 directly interacts with VE‐cadherin and β‐catenin in resting ECs as assessed by immunoprecipitation assay and a proximity ligation assay (PLA). To determine whether Mfn2 affects endothelial barrier integrity and function, Mfn2 was depleted in HLMVECs with a doxycycline inducible lentiviral shMfn2 RNA. Mfn2 silencing disrupted AJ junctions as visualized by VE‐cadherin and β‐catenin immunostaining. Mfn2 depletion significantly increased transendothelial permeability of ECs as assessed by a FITC‐conjugated albumin tracer assay and transendothelial resistance (TER). We also identified novel binding partners of Mfn2 using the high affinity tandem mass spectrometry (LC‐MS/MS). The analysis of gene ontology indicates that Mfn2 interacts with cell‐cell interaction proteins including cadherin binding and nuclear factor binding proteins with >1.5‐fold increase. Finally, the complex of Mfn2 and β‐catenin translocates into the nucleus upon TNF‐α stimulation.ConclusionThese data suggest that Mfn2 may directly regulate AJ stability in ECs by interacting with the AJ complex proteins VE‐cadherin and β‐catenin. Mfn2 may act as a co‐regulator with β‐catenin, a key transcription factor in inflammatory condition. These studies enhance our understanding of how the “mitochondrial” protein Mfn2 regulates inflammatory activation via novel “non‐mitochondrial” functions.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Aerobic glycolysis is essential to remodel the epigenetic landscape and initiate transcription during T cell activation
Authors: Coffer Paul , Russo Laura , Vastert Bas , Mokry Michal , Burgering Boudewijn , Mocholi Enric 
Published: June 2021

Recent work has started to provide evidence that cellular metabolism is critical in the regulation of immune cell differentiation and function, thereby influencing the final outcome of both the adaptive and innate immune response. Upon engagement of the T Cell Receptor (TCR) after antigen recognition, T cells rapidly upregulate aerobic glycolysis, a process often termed the Warburg effect. It has become clear that this switch to aerobic glycolysis is essential for T cell proliferation, differentiation, and function. However, glycolysis is extremely inefficient for ATP production, suggesting that likely has alternative roles during T cells activation.Using a combination of RNAseq, ChIP‐seq, and metabolomics, our results show for the first time that after TCR engagement, pyruvate produced by induction of glycolysis is essential to remodel the human CD4+ T cell epigenome. Pyruvate is translocated to the nucleus where it is converted to acetyl‐CoA and can act as a co‐factor for histone acetyltransferases (HATs) resulting in chromatin remodeling and subsequent expression of T cell activation markers. Preventing TCR‐induced pyruvate generation severely impacts T cell activation and transcriptional reprogramming. We further show that inflammatory microenvironment can reprogram CD4+ T cell metabolism and through epigenetic reprogramming can thereby trigger aberrant, pathogenic gene expression.Taken together our findings demonstrate a direct link between cellular metabolism, chromatin remodeling, and T cell function/fate. Understanding the influence of metabolic alterations in inflammatory environments and there impact on chromatin remodeling may result in the design of novel interventions to specifically target these pathways and alter T cell function.Support or Funding InformationThis work was supported by a grant from the Dutch Arthritis Society (ReumaNederland)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Can enhancing the expression and activity of the Cystic Fibrosis Transmembrane Regulator improve efficacy of gene therapy for Cystic Fibrosis?
Authors: Woodall Maximillian , Tarran Robert , Baines Deborah 
Published: June 2021

Cystic Fibrosis, an autosomal recessive genetic disease, is the most common cause of respiratory disease in children of the Caucasian population. Due to the monogenetic nature of the disease and the accessibility of the airways to nebulizers, it is a desirable candidate for gene therapy/editing. It has been suggested that ~10% of endogenous cystic fibrosis transmembrane regulator (CFTR) expression within target cells in vivo is sufficient for prevention of lung disease. With gene editing technology gaining traction it is important for CF researchers to understand how much functional CFTR is required to prevent the development of lung disease and how to make this more achievable.To answer this question we used two models: cultures of mixed CF:non‐CF primary epithelial cells (a model for gene correction) and cultures of CF cells transduced with CFTR under expression of a high activity promoter (a model for gene therapy). Cultures were grown on semipermeable membranes and differentiated at airway liquid interface. Ussing chamber, Airway surface liquid height and immunohistochemistry were performed as functional readouts. It was observed that essential CFTR functions increased in a linear fashion in CF:WT primary epithelial mixes (CFTRinhIsc μA/cm2: R2=0.91, n=28) whilst only ~16% transduction of a CF culture with CFTR fully restored ion channel activity and airway surface liquid height (n=6).Our data shows that overexpression of CFTR is a more effective method for restoring function in a CF culture. Therefore, we have since developed an enhanced CFTR gene with ~7 fold greater processing in to protein and dramatically increased anion transport activity as shown by a YFP quenching assay (n=3). It is our hope that this enhanced gene could be used as a tool for a more effective gene therapy.Support or Funding InformationCystic Fibrosis Trust UKSt. George's University LondonUniversity of North Carolina, Chapel HillThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Rb S249 as an Emerging Biomarker in the Prognosis of Prostate Cancer
Authors: Camacho‐Hernández Elaine , Feliciano‐Rivera Maylee , Rodriguez‐Lopez Xavier , Perez‐Morales Jaileene , Chardon‐Colon Camille , Caban‐Rivera Jennifer , Santiago‐Cardona Pedro 
Published: June 2021

Biomarkers have become a potential tool in the diagnosis of cancer patients, allowing a more accurate diagnosis and treatment. Prostate cancer is the second most common type of cancer in men worldwide. In prostate cancer, it is important to identify which patients would benefit from early treatment. Here we report the combination of phosphorylation of the retinoblastoma protein (Rb) on serine 249 (Rb S249) wit with p39 expression as biomarkers for predicting prostate cancer. We performed an immunohistochemical analysis of Rb S249 and p39 expression. Our results indicate that there is an inverse relationship between Rb S249 and prostate adenocarcinoma. Hence, Rb S249 could help in determining a more accurate prognosis for prostate cancer patients.Support or Funding InformationFunded by NIH‐NIGMS #2R25GM096955This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Investigating the Expression of Wnt5a Isoforms in Urothelial Carcinoma
Authors: Mehlman Amy N , Malgor Ramiro , Coschigano Karen T 
Published: June 2021

It is projected that there will be over 81,000 new bladder cancer diagnoses in 2018 in the United States alone. In fact, 1 in 27 men will develop bladder cancer in their lifetime. Despite its prevalence, little is known about this cancer's molecular mechanism. Research performed both at Ohio University and by other scientists has linked a protein, Wnt5a, to urothelial carcinoma (UC), which is the most common form of bladder cancer. This protein has been shown to be involved in an array of diseases, including several types of cancer. Recent research has suggested that two Wnt5a isoforms, Wnt5a‐Short (S) and Wnt5a‐Long (L), may play differing roles in a variety of cancers, with Wnt5a‐S promoting cellular proliferation and Wnt5a‐L suppressing cellular proliferation. Based on this and the research linking Wnt5a to UC, we want to investigate the roles of the Wnt5a isoforms in UC cell lines and human UC biopsies. We extracted RNA from human UC cell lines derived from low and high grade tumors to quantify the amount of each Wnt5a isoform and total Wnt5a expression using real‐time reverse transcription / polymerase chain reaction (RT‐RT/PCR). Trends in expression for Wnt5a‐L and Wnt5a‐Total were extremely similar, suggesting that Wnt5a‐L is the predominantly expressed isoform in urothelial cells. Wnt5a‐L RNA expression was highest in a cell line derived from a high grade tumor but also lowest in a cell line derived from a second high grade tumor. In contrast, expression of Wnt5a‐S RNA was highest in cell lines derived from high grade tumors in comparison to low grade tumors. Linking this to previous research published by our lab, the pattern of Wnt5a‐S expression was similar to that of vimentin, a marker of epithelial mesenchymal transition (EMT) linked to metastasis. We are currently performing immunohistochemistry (IHC) and laser microdissection (LMD) to visualize the location of Wnt5a isoform expression in human UC biopsies from both low grade and high grade tumors. Our hypothesis is that high grade tumors will express higher levels of the Wnt5a‐S isoform than low grade tumors; in contrast, high grade tumors will express lower levels of Wnt5a‐L than low grade tumors. An affirmative outcome of our hypothesis would suggest that the Wnt5a‐S isoform promotes oncogenic properties in UC, while the Wnt5a‐L isoform promotes tumor suppressive properties. Future experiments will be aimed at determining the molecular pathway(s) through which each isoform acts in order to advance diagnostic, prognostic, and therapeutic approaches to UC.Support or Funding InformationFunding and support were provided in part by Ohio University Heritage College of Osteopathic Medicine and its Research and Scholarly Advancement Fellowship (RSAF), the Diabetes Institute at Ohio University, as well as by an Ohio University Student Enhancement Award (SEA).This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.