bluuebunny/crossref_metadata_2025_split huggingface dataset:
Maternal immune activation affects precursor cell populations and developmental processes in the rodent spinal cord in a temporally and spatially dependent manner
Authors: Mcdermott Kieran W. , Anderson Rebecca C. , Foley Tara , Radford Jay , Allardyce Joanna M. , O'Hallaron Sophie , Uzun Burak , O'Keeffe Gerard W. 
Published: June 2021

Studies suggest that maternal immune activation (MIA) during pregnancy may be implicated in development of neurological disorders with timing of immune challenge and spatial differences playing an important role. However, further research is necessary in order to elucidate fully the CNS regions, cell populations and timing of gestational vulnerability. As the development of the spinal cord (SC) is well characterised, here we investigate how MIA affects cell populations and developmental processes in this CNS region.Time‐mated dams received an intraperitoneal injection of 100μg/kg lipopolysaccharide (LPS) or saline on embryonic day (E)12 or 16. Embryos were harvested 5hr post‐injection, or delivered and sacrificed at postnatal day (P)14. Offspring were fixed, frozen whole or following SC dissection and cryosectioned. In order to determine how MIA affects oligodendrocyte precursors, microglia, and reelin expression during development, sections were immunostained with Olig2, Iba‐1 and reelin. In addition total RNA was extracted from fresh frozen SCs and prepared for microarray, RNAseq and small RNAseq.At E12, numbers of Olig2+ and Iba‐1+ cells were unchanged 5hrs post‐LPS injection but Olig2+ cells were decreased in the ventral grey matter of the rostral SC at P14. The number of Iba‐1+ cells were unchanged at P14. At E16, Olig2+ and Iba‐1+ cells were decreased in grey and white matter 5 hrs post‐LPS, however by P14 there was no difference between the LPS and saline treated groups. Reelin expression following MIA at E12 appeared unchanged at 5hrs and P14 however, at E16, reelin expression was decreased 5hrs after MIA. Total cell numbers at E16, at both the 5hr and P14 survival times, were unchanged following LPS injection.These findings suggest MIA affects development in the SC in a temporally and spatially precise manner. At E12 MIA does not appear to affect olig2+ oligodendrocyte precursors acutely but may affect their survival to P14. In contrast, at E16, MIA affects Olig2+, Iba‐1+ and reelin expression acutely. Ongoing work is characterising postnatal reelin expression following LPS at E16, and the phenotype of Iba1+ cells to understand more fully the detailed cellular perturbations resulting from MIA.Support or Funding InformationAnatomical Society, Graduate Entry Medical School, Health Research Institute, University of LimerickThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
A New Pathway of Transcription‐Coupled Repair
Authors: Myka Kamila , Washburn Robert , Kusters Kira , Gottesman Max 
Published: June 2021

Transcription‐coupled repair (TCR) of damaged DNA in E. coli can involve several different pathways. We propose that one pathway entails release of RNAP at the site of DNA damage without dissociation of the RNA:DNA hybrid. The RNA can serve as a primer for PolI, which initiates the repair process. We are investigating how TCR repairs nalidixic acid‐induced DNA damage. Nalidixic acid, like Etoposide generates a DNA double‐strand break bearing a TopA adduct at each 5′ DNA end. We show that efficient repair requires two transcription factors. The first DksA, is a coiled‐coil protein that inserts into the RNAP secondary channel and, in concert with ppGpp, regulates the activity of certain promoters. The role of DksA in transcription elongation is undefined, although it is known to suppresses replisome – RNAP clashes. DksA plays a direct role in repair, rather than blocking access of GreA to RNAP. Thus, a ΔdksA mutation is not suppressed by a greA deletion. Recent work from the Murakami laboratory reveals that DksA binding distorts the structure of RNAP, opening the pincers, thus potentially reducing the processivity of transcription elongation. DksA in association with ppGpp, however, does not affect RNAP structure. We find that repair is more efficient in strains carrying an RNAP mutation that reduces ppGpp binding to site 2, the location of DksA insertion. We have isolated a suppressor of ΔdksA based on resistance to nalidixic acid. The mutation, rpoBQ148H, phenocopies RNAP bound to DksA in the absence of ppGpp, i.e. it opens the pincers and may favor release of elongating RNAP. These findings suggest that DksA may act as a transcription termination factor that can release RNAP without removing the RNA:DNA hybrid.The second, transcription termination protein Rho, is an RNA‐dependent DNA:RNA helicase. The defects in TCR of rho and dksA mutations are additive, suggesting that they act on different substrates. Whereas Rho is thought to release RNAP by unwinding the RNA:DNA helix, rho mutants that terminate transcription without releasing RNA have been isolated.Another reaction that entails elongation of an RNA:DNA hybrid by PolI is constitutive stable DNA replication (cSDR). In cSDR, elongation of an R‐loop by PolI creates a new origin of replication. We find that cSDR is dependent on DksA. Presumably the mechanisms of nalidixic acid DNA damage and cSDR both involve allowing access of PolI to the 3′ end of an RNA:DNA hybrid.Support or Funding InformationGM037219 GM037219 NIH: GM037219 NIH: GM037219 GM037219This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Escherichia coli RarA commits cells to post‐replication repair pathways by facilitating replisome skipping
Authors: Gopal Kamya , Stanage Tyler , Cox Michael M. 
Published: June 2021

Members of the highly conserved RarA/Mgs1/WRNIP1 protein family have been implicated in the maintenance of genomic stability, but their exact functions remain unknown. Loss of one of these proteins rarely results in an altered phenotype, but a knockout mutation in genes encoding proteins of this family in tandem with the loss of other DNA metabolism functions can adversely affect cell survival. We hypothesize that the RarA protein of E. coli commits the cell to post‐replication repair pathways, namely translesion DNA synthesis (TLS) and daughter strand gap synthesis. RarA overexpression is toxic to the cell and knocking out DNA Polymerase IV and DNA Polymerase V, TLS‐competent polymerases, alleviates this toxicity. Furthermore, the presence of RarA induces sensitivity in cells lacking TLS polymerases to the DNA‐damaging agents nitrofurazone (NFZ) and 4‐nitroquinoline‐1‐oxide (4‐NQO). Deletion of RarA suppresses DNA Polymerase IV mediated sensitivity to NFZ and 4‐NQO. These data suggest that RarA can promote rapid replisome progress by committing cells to using post‐replication repair pathways and blocking access to other repair pathways when the replisome encounters certain types of lesions.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Understanding the role of viral RNA sequence in host shut‐off by SARS‐CoV
Authors: Nag Anita , Chockalingam Prem K. , Adams Jessica L. , Gonzalez Fabiola G. , Abrar Fareeha 
Published: June 2021

Nonstructural Protein 1 (nsp1), generated by Severe Acute Respiratory Syndrome Coronavirus (SARS‐CoV), is responsible for host cell mRNA degradation via endonucleolytic cleavage. While the cellular mRNA is targeted for cleavage, few viral mRNAs are resistant. These mRNAs include mRNA from SARS‐CoV, hepatitis C virus and cricket paralysis virus. Other studies concluded that a stem‐loop forming sequence in the 5′‐UTR (untranslated region) may be responsible for this cleavage resistance. We designed multiple mutations in the UTR to partially or fully disrupt the stem‐loop, or reinstate a stem‐loop with mutated sequence. These RNAs were subjected to nsp1 mediated cleavage and RNA stability was quantified using reverse transcription followed by quantitative polymerase chain reaction. We used 18S ribosomal RNA as a control since it does not undergo nsp1 mediated cleavage. Surprisingly, our results show that a partially disrupted stem‐loop leads to enhanced stability of the RNA. In order to understand any sequence driven stability that arises due to stronger binding by nsp1, we are currently investigating RNA binding properties of nsp1.Support or Funding InformationSC INBRE faculty fellows awardSCICU summer fellows awardFurman fellows awardThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
An in vivo method to study post‐transcriptional regulation in germ stem cells
Authors: Doenier Jon , Aoki Scott Takeo , Kimble Judith 
Published: June 2021

Stem cells in regenerative tissues must balance factors promoting self‐renewal with those driving differentiation. Faulty regulation of this balance can lead to tumor growth or loss of regenerative ability. The tractable germline of the nematode C. elegans has allowed the identification of a wealth of key stem cell regulatory factors and remarkably, virtually all are RNA binding proteins (RBP). Despite this insight, a mechanistic understanding of how these RBPs regulate their target mRNAs is lacking. Here, we develop an in vivo method to investigate their mechanisms. Our assay probes how a given RBP affects expression of a reporter mRNA when recruited to its 3′ untranslated region (3′UTR). To perform the assay, an RBP of interest is genetically engineered to contain a small λN peptide. The λN peptide binds a specific RNA hairpin, called boxB, with high affinity. Thus, a reporter mRNA engineered to contain boxB hairpins in its 3′UTR will bind the λN tagged RBP and either increase or decrease expression, depending on the RBP regulatory function. By imaging reporter expression in germline stem cells, we can gain insight into how the RBP regulates target transcripts in its normal biological context. The simple and rapid readout of the assay allows it to be used to probe how an RBP accomplishes its RNA regulation or screen for other factors necessary for RBP function. We have applied this assay to several of the key stem cell RBP regulators to test their functions in vivo. Our results demonstrate the tractability and ease of this method to study RBPs in the C. elegans germline as well as providing mechanistic insights into how the RBPs regulate stem cell fate decisions.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Mass Spectrometry Analysis of RBM20 Phosphorylation and Its Role in Titin Splicing
Authors: Sun Mingming , Jin Yutong , Zhu Chaoqun , Rexiati Maimaiti , Cai Hanfang , Chen Zhilong , Ge Ying , Guo Wei 
Published: June 2021

ObjectiveThe SR protein family has a characteristic domain which is rich in arginine and serine residues. They play significant roles in constitutive pre‐mRNA splicing and the phosphorylation status of them is important in the regulation of alternative splicing. RNA binding motif 20 (RBM20), a newly identified SR protein, mainly regulates splicing of a giant muscle gene titin, a major heart disease‐causing gene. However, the mechanism of RBM20 modulation on titin splicing is not well defined. We hypothesize that RBM20 phosphorylation plays an important role in titin splicing. In this study, we employed middle‐down mass spectrometry and in vitro splicing assay to explore the phosphorylation status of RBM20 and its role in titin splicing.MethodsBoth bacteria and insect cell expressed RBM20 were used for ProQ diamond staining and middle down mass spectrometry analysis. In vitro approaches with co‐transfection of mutated RBM20 and titin minigene, and a dual luciferase splice reporter assay were performed to explore the role of RBM20 phosphorylation in titin splicing.ResultsInsect cell expressed RBM20 was stained by ProQ diamond, a dye which is specifically used for phosphorylated protein staining in SDS‐PAGE gel, whereas ProQ diamond stained bands were not observed with bacteria expressed RBM20. Mass spectrometry analysis on the insect cell expressed RBM20 identified 16 phosphorylation sites with four of them located in RS domain. 16 mutated RBM20 plasmids on individual phosphorylation sites co‐transfected with titin minigene in HeLA cells, and the results indicated that only phosphorylation in RS domain affects titin splicing, and splice report assay further confirmed the results.ConclusionRBM20 is massively phosphorylated with 16 phosphorylation sites, and only RS domain phosphorylation plays a role in titin splicing. These results reveal that the phosphorylation sites on RS domain residues could be potential therapeutic targets for titin splicing‐induced heart disease.Support or Funding InformationNIFA‐USDA 1009266, NIH NIGMSP20GM103432, AHA BGIA and Faculty‐Grant‐in‐Aid from University of WyomingThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Monitoring Innate Immune Receptor Stability via Post‐Translational Modification by OGT
Authors: Drake Walter R. , Hou Ching‐Wen , Zachara Natasha E. , Grimes Catherine Leimkuhler 
Published: June 2021

The human body's first line of defense against infectious disease is the innate immune system, which is tasked with distinguishing harmful pathogens from commensal and beneficial microbes. On a molecular level, this system contains different pattern recognition receptors (PRRs) that detect microbe associated molecular patterns (MAMPs) specific to particular microorganisms, and the interaction between a PRR and its respective MAMP triggers an immune response. The Nod‐like receptors (NLRs) are one major family of innate immune receptors that contain the proteins Nod1 and Nod2, which use different bacterial cell wall peptidoglycan (PGN) fragments as their designated MAMPs. Binding between the NLR and PGN fragment is mediated by the leucine rich repeat domain (LRR), which exists at the C‐termini of Nod1 and Nod2. Upon LRR‐MAMP stimulation, NF‐kB and other signaling cascades are initiated, ending with the production of proinflammatory cytokines and chemokines. Misregulation of signaling by NLRs can lead to disease, and mutations in Nod1 and Nod2 are associated with gastric cancer and Crohn's disease, respectively. These NLRs are critically important for maintaining the relationship between the human body and the trillions of microorganisms that it hosts, yet so much about their functions and regulation in the body remain unclear. Recently, Nod2 was identified as being O‐‐GlcNAcylated, which is a post‐translational modification mediated by two enzymes that affect thousands of proteins in diverse ways. An emerging cellular thermal shift assay (CETSA) that is used for identifying protein‐ligand binding was repurposed to measure the stabilizing effect of O‐GlcNAc modification on Nod1 and Nod2. When protein O‐GlcNAc levels are increased, melting temperatures of both NLRs increase as well. These data are supported by cycloheximide half‐life experiments and NF‐kB luciferase assays, which demonstrated that the modification also led to increased NLR half‐lives and NF‐kB activity. Additionally, two Nod2 Crohn's associated variants were studied, and both G908R‐Nod2 and 1007fs‐Nod2 are O‐GlcNAcylated in the cell. While the modification led to similar increases in melting temperature and NF‐kB activity for 1007fs‐Nod2, its effects on G908R‐Nod2 are less clear. These results show that CETSA is a useful tool for measuring stability changes caused by O‐GlcnAc modification and that NLRs may be commonly post‐translationally modified to regulate stability.Support or Funding InformationMITZUTANI GLYCOSCIENCE FOUNDATION, NATIONAL SCIENCE FOUNDATION (CAREER CHE 1554967), NIH NATIONAL HEART, LUNG, AND BLOOD INSTITUTE (P01HL107153)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Palmitoylation Regulates the Functions of NHE1
Authors: Pitsch Emily , Wallert Mark , Provost Joseph 
Published: June 2021

The sodium hydrogen exchanger isoform 1 (NHE1) is a ubiquitously expressed transmembrane protein that regulates intercellular pH (pHi) and interacts with proteins involved in cell migration and adhesion. Within the extended cytosolic tail of NHE1, there are multiple sites of post‐translational modification including phosphorylation and lipidation. Our understanding of impact of reversible palmitoylation on membrane transporters is growing. 2‐bromopalmitate (2BP) irreversibly inhibits the enzyme, palmitoyl acyltransferase, that transfer palmitate to cysteines. We found NHE1 is reversibly palmitoylated and here, demonstrate the impact of the lipidation on NHE1 function. Lung fibroblasts expressing NHE1 (PSN) showed minimal decrease (8.5 ± 2.5% SEM) in cell viability after treatment with 50 μM 2BP after 72 h. Palmitoylation plays a critical role in cell adhesion and motility. Cells that were incubated in 15 μM 2BP for 18 hrs displayed a significant decrease cell adhesion in low serum conditions and cells treated with 2BP did not fully migrate into vacated space after 24 hrs in a wounding assay. Impact on cytoskeletal structure by 2BP treatment was also investigated. Lysophosphatidic acid (LPA) increased actin stress fiber formation 40% over control cells. This LPA‐induced stress fiber formation was inhibited more than two‐fold in the presence of 2BP. This is important as LPA‐induced stress fiber formation requires NHE1; therefore, palmitoylation is required for NHE1‐dependent actin stress fiber formation. To determine the effect of palmitoylation on NHE1 transport activity, PSN cells were treated with 2BP and the pHi was measured. Addition of increasing concentrations of 2BP inhibited NHE1 transport in cells with and without serum, but the effect was enhanced in low serum conditions. Interestingly, LPA stimulation of NHE1 activity was inhibited by 60%, but not to baseline. This indicates that in addition to cell adherence, motility, and cytoskeletal formation, palmitoylation is critical for NHE1 transport.Support or Funding InformationThis research was partially funded by the Beckman FoundationThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Structural comparison of the Arabidopsis thaliana family of β‐amylases
Authors: Chandrasekharan Nithesh , Monroe Jonathan , Berndsen Christopher 
Published: June 2021

The β‐amylase (BAM) family in Arabidopsis thaliana has nine members, some of which are known to hydrolyze starch to maltose. The members of this family show unique functional, regulatory, and catalytic behaviors due to changes in sequence or domain composition. However, the molecular basis for functional specificity and regulation is not always clear. Some of the inactive members of the lack the catalytic amino acids required for hydrolysis. However, this is not the case for all the enzymes such as BAM2, which appears to be allosterically regulated and requires KCl for activity. Given the lack of structural information on any member of this protein family, we homology modeled all 9 BAM proteins from Arabidopsis and performed molecular dynamics simulations to propose how these proteins may differ at a functional level. All simulations were equilibrated for at least 50 ns in explicit solvent, the equilibrated models were aligned structurally, and then analyzed to compare the structure, dynamics, and chemical properties. All 9 proteins modeled well as TIM barrel proteins with most of the structural deviations occurring in predicted loops. Comparison of the simulated dynamics shows that overall the BAM proteins show similar areas of high and low motion. Interestingly, BAM4 and BAM9 showed fluctuation profiles that were distinct from the other BAM proteins, which matches their predicted lack of catalytic activity and distinct active site sequences. BAM7 and BAM8 which also have distinct active sites however these enzymes show dynamics similar to those of the catalytically active BAM1 and BAM3 suggesting that BAM7 and BAM8 may also have activity, however the proper conditions are not known. We further modeled BAM2 as a tetramer based on recent biochemical information to identify how allostery and solution conditions may influence the activity of this protein. We identified predicted changes in the dynamics upon binding to a ligand in the starch‐binding site as well as changes in hydrogen bonding within the protein in potassium. Both findings provide testable proposals for the regulation of BAM2 activity.Support or Funding InformationThis work was supported by a National Science Foundation Research at Undergraduate Institutions Grant MCB‐1616467 and National Science Foundation Research Experience for Undergraduates Grant CHE‐1461175This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Distinct functions for the flexible loops of zinc specific solute binding proteins from Paracoccus denitrificans
Authors: NEUPANE DURGA P. , YUKL ERIK T. 
Published: June 2021

In bacteria, ATP binding cassette (ABC) transporters of cluster A‐I exist for the acquisition of the essential trace element zinc from limiting environments. These transporters are crucial for bacterial survival, and the gene knockout strains often demonstrate a pronounced growth defect in minimal media and attenuated virulence in the case of pathogens. Thus such systems are attractive targets for the development of novel antibiotics. Zinc binding with high affinity and specificity is mediated by the solute binding protein (SBP) component of ABC transporters. Zinc‐specific SBPs are characterized by the presence of a flexible loop near zinc binding pocket of the protein. P. denitrificans encodes two zinc ABC transporter operons, znuABC and aztABCD. The SBPs for these systems (AztC and ZnuA) are distinct in the length and sequence of flexible loop regions. The function of the flexible loop in each protein was investigated using fluorescence binding assays and structural methods for WT and loop mutant proteins. The flexible loop of AztC has no impact on zinc binding affinity, stoichiometry or protein structure, yet it is essential for zinc transfer from the metallochaperone AztD. Site specific mutagenesis was employed to evaluate the roles of specific residues in this process and the results showed that three His residues in the loop temporarily coordinate zinc and then convey it to the high‐affinity binding site. Consistent with this, mutation of any of these residues to Ala abrogated zinc transfer from AztD. AztD does not transfer zinc to ZnuA, despite the fact that ZnuA also contains a flexible loop rich in His residues. The ZnuA flexible loop contains 15 His, 11 Glu and 3 Asp residues, providing multiple additional zinc binding sites. In fact, P. denitrificans ZnuA binds up to 6 zinc ions, the most for any zinc SBP studied to date. The results demonstrate distinct roles for zinc SBP flexible loops depending on loop structure and context, potentially allowing for acquisition of zinc from different environments.Support or Funding InformationNational Institute of General Medical Science of the National Institutes of Health under award number 1SC2GM111170‐01.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.