bluuebunny/crossref_metadata_2025_split huggingface dataset:
Electrostatic and Hydrophobic Interactions Mediate Single‐Stranded DNA Recognition and Acta2 Repression by Purine‐Rich Element Binding Protein B
Authors: Ferris Lauren , Rumora Amy , Wheeler Tamar , Kelm Robert 
Published: November 2023

Fibroblast to myofibroblast differentiation is characterized by increased expression of cytoskeletal smooth muscle α‐actin. In human and murine fibroblasts, the gene encoding smooth muscle α‐actin (Acta2) is tightly regulated by a network of transcription factors that either activate or repress the 5′ promoter in response to environmental cues signaling tissue repair and remodeling. Purine‐rich element binding protein B (Purβ) suppresses the expression of Acta2 by cooperatively interacting with the sense strand of a 5′ polypurine sequence containing an inverted muscle‐CAT (MCAT) motif required for gene activation. In this study, we evaluated the physical and chemical basis of nucleoprotein complex formation between the Purβ repressor and the purine‐rich strand of the MCAT cis‐regulatory element in the mouse Acta2 promoter. Quantitative single‐stranded DNA (ssDNA)‐binding assays conducted in the presence of increasing concentrations of monovalent salt or anionic detergent indicated that high affinity nucleoprotein complex assembly is driven by a combination of electrostatic and hydrophobic interactions. Results of pH titration analysis suggested that basic amino acid residues are likely involved in mediating the interaction of Purβ with ssDNA. Computational homology modeling was performed to identify positionally‐conserved cationic residues in each ssDNA‐binding subdomain of Purβ. Site‐directed mutagenesis confirmed that several solvent‐exposed lysine and arginine residues located in the intermolecular (R267) and intramolecular (K82, R159) subdomains are essential for the transcriptional repressor function of Purβ. In keeping with their stable expression but diminished Acta2 repressor activity in transfected fibroblasts, purified Purβ mutants R267A, R159A/R267A, and K82A/R159A/R267A were unaltered in terms of protein folding and quaternary structure but exhibited markedly reduced binding affinity for the purine‐rich strand of the Acta2 MCAT element. Moreover, the Purβ double and triple point mutants were also found to be defective in binding to the Acta2 corepressor protein, Y‐box binding protein 1. Collectively, these findings establish the repertoire of non‐covalent interactions that account for the unique structural and functional properties of the Acta2 repressor protein Purβ.Support or Funding InformationAHA 09GRNT2170060 and NIH T32 HL007594
Activation of p21 following DNA damage transcriptionally controlled by a telomeric shelterin protein TRF2 in a telomere‐length dependent fashion
Authors: Chowdhury Shantanu 
Published: November 2023

We report and unexpected finding ‐ TRF2 occupancy at the p21, as well as other promoters, depends on telomere‐length, even though the sites are as much as 62 Mb distal to the nearest telomere. And, p21 repression by TRF2 was telomere length‐dependent. Together our data are consistent with a model that involves partitioning of TRF2 between telomeres and interstitial sites, which influences gene regulation. DNA secondary structures called G‐quadruplexes were recently detected in‐vivo using antibodies. Further, it remains to be understood whether and how detected interstitial G‐quadruplexes function biologically. In support of the underlying mechanism of TRF2‐p21 regulation we find telomere repeat factor 2 (TRF2) associates with hundreds of potential interstitial G‐quadruplex sites, a significant number of which overlap with gene promoter regions. Detailed mutational and expression analysis of the cyclin‐dependent kinase CDKNIA (p21) promoter quadruplex site revealed TRF2 occupancy results in recruitment of the REST‐repressor protein complex and decreases p21 expression in response to drug induced DNA damage.Support or Funding InformationWellcome Trust ‐ Department of Biotechnology India Alliance and Council of Scientific and Industrial Research
Defining a Transcriptional Regulatory Network for Peroxisome Biogenesis
Authors: Paz Edwin A. , Gaasterland Terry , La Spada Albert 
Published: November 2023

Peroxisomes are highly dynamic organelles that are involved in lipid metabolism and hydrogen peroxide break‐down. Dysregulation of peroxisomal processes are associated with a group of neurodevelopmental disorders including Zellweger syndrome, neonatal adrenoleukodystrophy, and infantile Refsum disease. Underlying these disorders are mutations in PEX genes resulting in faulty peroxin production and function. We hypothesize that important contributors to neurodegenerative diseases are dysregulated transcriptional networks affecting peroxisome biology. Using a bioinformatics approach, we have identified potential transcription factors and their consensus transcriptional factor binding site (TFBS) motifs, and are testing if these TFBSs are coordinately regulating peroxisome biogenesis and function. Identifying novel transcriptional regulators of peroxisome biogenesis may reveal novel targets that can be exploited for therapeutic intervention.Support or Funding InformationR01 AG033082K12 GM068524
Microarray expression profile analysis of long non‐coding RNAs in pulmonary tuberculosis with Traditional Chinese Medicine
Authors: Jiang Tingting , Shi Li‐Ying , Wei Li‐Liang , Chen Zhong‐Liang , Wang Chong , Liu Chang‐Ming , Li Zhong‐Jie , Li Ji‐Cheng 
Published: November 2023

The present study aimed to explore the expression profile of long non‐coding RNAs (lncRNAs) and mRNAs in pulmonary tuberculosis (TB) patients with Traditional Chinese Medicine (TCM) by Arraystar Human LncRNA Microarray. TB patients were divided according to the TCM syndromes into pulmonary Yin deficiency (PYD) syndrome, hyperactivity of fire due to Yin deficiency (HFYD) syndrome, and deficiency of Qi and Yin (DQY) syndrome. Plasma samples were collected from 33 TB patients and 11 healthy controls. Total RNA was extracted and purified, and then hybridized with Arraystar Human LncRNA Microarray V3.0 containing 30586 lncRNAs probes. Bioinformatic analyses including gene ontology (GO), and kyoto encyclopedia of genes and genomes (KEGG) pathway were conducted to analyze the differentially expressed lncRNAs of TB with different TCM syndromes. The related clinical data was also analyzed.1523, 558, and 596 differentially expressed lncRNAs were found in PYD, HFYD, and DQY patients, respectively, compared with the healthy controls (ratios of >2, or <0.5, and P<0.05). Pathway analysis revealed that fatty acid metabolism (P=0.005067587), protein digestion and absorption (P=5.89017E‐05), and Hippo signaling pathway (P=0.000164) have higher enrichment score. The expression of 18 transcripts with zinc ion binding function, 10 transcripts related to the metabolic process and 9 transcripts involved in the apoptotic process were progressively increased from PYD→HFYD→DQY syndrome. Clinical analysis (143 controls, 90 PYD cases, 116 HFYD cases, and 85 DQY cases) showed that the lipid indexes of TB patients was abnormal, and the triglyceride concentration was significantly different among PYD, HFYD, and DQY patients (P=0.0352). Our study is the first to acquire the microarray expression profile of lncRNAs, and analyze the pathway enrichment and expression tendency of differentially expressed lncRNAs among PYD, HFYD, and DQY syndromes. Our results indicate that the analysis of expression profile of lncRNAs may be a novel method to explore the biological essence of TCM syndrome.Support or Funding InformationThis work was supported by grants from National Basic Research Program of China (No.2014CB543002), National Special Sci‐Tech Projects (No. 2012ZX10005001‐006), and National Natural Science Foundation of China (No.81273882, NO. 81573709).
Ascertaining a Role For Rnp1p in Post‐Transcriptional Gene Control in S. cerevisiae
Authors: Mackay Katherine E. , Segal Scott P. 
Published: November 2023

Translational control plays a vital role in the response to such physiological stresses, as high cell density or glucose deprivation. When physiological stress is present, translation is repressed and mRNA becomes unbound to ribosomes. A number of proteins are involved in the process of removing mRNA from the translational pool, where they are, in turn, sent to stress granules (SGs) to be stored or P‐bodies to be degraded. One of these translational repression proteins is Sbp1p, which contains two RRMs that flank a central RGG box. Sbp1p has been shown to localize to P‐bodies and SG. However, its interactions with translational initiation factors through its RGG box, suggests it may play a greater role in mRNA storage.RNP1, which is an SBP1 paralog, encodes a protein containing two RRMs but lacks the RGG box present in SBP1. Given these structural similarities, RNP1 may also play a role in translational repression and possible localization of mRNA to P‐bodies or SGs. To determine whether Rnp1p may play a role in these processes, Rnp1p was overexpressed, and assembly of P‐bodies and SGs were ascertained. Upon overexpression assembly of P‐bodies was induced in unstressed cells, whereas SGs were not. This suggested that Rnp1p plays a role in P‐body assembly directly, or through translational repression. To support this claim, physiologically stressed rnp1Δ cells had a significant reduction in P‐body assembly. Perhaps the lack of an RGG box may target Rnp1p to P‐bodies allowing it, in turn, to have a role in mRNA for degradation, rather than storage.To further ascertain whether Rnp1p has a direct role in P‐body assembly or translational repression, future experiments will be conducted to determine if Rnp1p localizes to P‐bodies under physiological stress. Additionally, translation will be monitored, and mRNA half‐lives will be measured in strains over‐expressing and lacking Rnp1p.
Biochemical Characterization of the Interaction between Innate Immune Receptor Nod2 and its Chaperones
Authors: Wastyk Hannah C. , Mohanan Vishnu , Schaefer Amy , Hou Ching‐Wen , Lauro Mackenzie , Grimes Catherine L. 
Published: November 2023

The human body houses over 10 trillion bacterial cells, of which our innate immune system must delineate between pathogenic and commensal. Disease can arise during misregulation of this bacteria, and one example is the debilitating inflammatory bowel disorder, Crohn's disease (CD). The molecular pathology of CD is known to be a mutation in Nod2, an innate immune receptor responsible for binding directly to bacterial cell wall fragment, muramyl dipeptide (MDP). MDP has recently been shown to stabilize Nod2 in a cell based assay, and after binding to MDP, Nod2 signaling proteins activate the NF‐κB pathway, a protective inflammatory response. In Crohn's variants, however, Nod2 is rapidly degraded, thereby the downstream signaling is disrupted. Our initial work identified Hsp70 as a chaperone molecule that binds to and stabilizes Nod2. The purpose of this study is to characterize this interaction and stabilization effect, such that the natural stabilization of Nod2 within the cell can be extrapolated to a possible treatment option that mimics Hsp70's activity on unstable Nod2 Crohn's variants. Hsp70 is a highly ubiquitous protein that is able to confer selective stability by tuning its specific activity via a number of different cochaperones. Our data shows that Hsp40 contributes to the chaperone complex, and through limited proteolysis, we show that both Hsp70 and Hsp40 mediate Nod2 stability in vitro, and that this stability is further enhanced with the addition of Nod2's ligand, MDP. Furthermore, we have biochemically characterized the importance of Hsp70's ATPase activity for Nod2's stability. Significance of this study extends beyond CD, as understanding Hsp70's action and determining a method of control for Nod2 could be extrapolated to stabilize a large number of disease‐relevant mutated proteins.Support or Funding InformationThe authors would like to thank the following sources of funding: the Chemistry‐Biology Interface program of NIH, the Howard Hughes Medical Institute, and the Hofmann Fund through Nucleus.
En'Cas'ing The Stress: Engineering a Human Cell Line Knockout of Heat Shock Response Coordinator Genes Using CRISPR Cas9 System
Authors: Canales Gabriela Isla‐Inez , Pani Bibhusita , Nudler Evgeny 
Published: November 2023

The Heat Shock Response (HSR) is an evolutionarily conserved response to high temperatures and other stresses that controls adaptive proteostasis, and is primarily regulated by the factor, Heat shock transcription factor 1 (HSF1). In mammalian cells, HSF1 is converted from an inactive monomeric form to an active trimer in response to heat stress. A ribonucleoprotein complex comprising of eukaryotic translation elongation factor eEF1A1, and a long noncoding RNA HSR1 are the key components of HSF1 activation. Once activated, HSF1 is recruited to Heat Shock Protein (HSP) promoter regions, upregulating chaperone activity in the cell. Along with mediating initiation of HSR, eEF1A1 is also a vital component of protein synthesis machinery. Interestingly, another isoform of eEF1A, called eEF1A2, is expressed in some specialized terminally differentiated cells of skeletal muscle, heart, pancreatic islets and motor neurons, all of which are prone to protein aggregation. The two isoforms are 92% identical and are reciprocally regulated. To better understand the role of eEF1A1 and HSF1 proteins in humans, we use a CRISPR‐Cas9 nickase system to knockout HSF1 and eEF1A1 in a human cell line. We showed that the hTERT‐immortalized, normal diploid foreskin fibroblast cell line, BJ‐5ta, produces both eEF1A isoforms. This will allow us to perform eEF1A1 knockout in these cells. We hypothesize that HSF1 knockout cell line will survive under normal conditions but express very low thermotolerance. Conjointly, we hypothesize that the elimination of eEF1A1 may be compensated by the upregulation of eEF1A2. If viable, the eEF1A1 knockout cell line will be used for screening mutants of eEF1A2 restoring activation of HSR. Both HSF1 and eEF1A1 knockout lines will also be used for future studies to improve upon the current model of the HSR pathway and potentially reveal therapeutic targets for diseases like ALS, Alzheimer's disease, Parkinson's disease, type 2 diabetes, and amyloidosis.Support or Funding InformationThis investigation was supported in part by a MARC Undergraduate Student Training in Academic Research (U‐STAR) National Research Service Award (NRSA) Institutional Research Training Grant (2 T34 GM008663) from the National Institutes of Health, National Institute for General Medical Sciences.This research was supported in part by a grant to UMBC from the Howard Hughes Medical Institute through the Precollege and Undergraduate Science Education Program.
Evaluation of ADAMTS Levels in Patients with Chronic and Aggressive Periodontitis
Authors: Demircan Kadir , Tayman Ayşe , Bayram Nezihe Aslı , Demir Berrin Tuğtağ , Karagöz Zehra Fırat , Fıstık Tuba , Kurga Şivge , Önder Canan , Günhan Meral 
Published: November 2023

IntroductionADAMTS is a 19‐membered gene family which was first discovered in colon cancer, is associated with inflammation. ADAMTS is a member of matrix metalloproteinases family and they are involved in degradation and repair of extracellular matrix. In recent studies, ADAMTSs are activated with lots of signaling pathways and regulated with lots of cytokines. They play an important role in pathogenesis of many diseases. Based on this information it has been hypothesized that ADAMTS levels would be increased in inflammatory diseases such as periodontal disease. Chronic periodontitis that is the most prevalent form of periodontitis, shows the characteristics of a slowly progressing inflammatory disease. Also, systemic and environmental factors (e.g., diabetes mellitus, smoking) may revise the host's immune response so that periodontal destruction becomes more progressive. The aim of this study is to investigate the presence of ADAMTS genes and proteins in chronic and aggressive periodontitis patients.MethodsFifteen aggressive and 15 chronic periodontitis patients that were systemically healthy were included in this study. Patients presented with chronic or aggressive periodontitis and needed tooth extractions were selected. Gingival biopsies containing epithelium and connective tissue were obtained during the extraction of periodontally involved teeth. Systemically healthy patients who need crown lengthening or gingivectomy procedure for aesthetic reasons were included to the control group. mRNA expression of ADAMTSs were evaluated by real‐time PCR method and position of the marked protein in the tissue is determined by Immunohistochemistry method.Results and ConclusionDifferences were observed between the control and disease groups. According to results of the study, it can be concluded that ADAMTS expressions have a potential to be a new marker in pathogenesis of periodontal disease. Western‐blot studies are continuing to determine the protein levels.
Comparison Between hPEP and spPEP Inhibition When Varying Size and Position of Inhibitory Peptide of Casein α s1
Authors: Vishram Avleen Kaur , Clack Beatrice A 
Published: November 2023

Eurygaster integriceps Puton (Sunn pest) is the most damaging insect pest of wheat in areas of North Africa, Eastern Europe and Western and Central Asia. Sunn pest injects an enzyme characterized as a prolyl endylprotease (spPEP) into wheat grains that digests the gluten protein (Darkoh et al., 2010). Previous studies have shown that casein peptides generated by Lactobacillus species have inhibitory effects on human (hPEP) and bacterial prolyl endoproteases (PEP) which makes casein a good natural candidate against spPEP. Developing biopesticides for food products from casein αs1 is complicated due to some allergenic amino acid sequences. Ruiter et al (2005) identifies a non‐allergenic peptide region on casein αs1 that includes a previously identified inhibitory sequence (LNENLLRFFVAPFPEVFG) to hPEP (Jeanneret, 2011). The Objective of this study was to produce 16 peptides derived from aS1 casein of varying lengths and varying size containing the hPEP inhibitory sequence. The inhibitory region is positioned at 0, 10, 20 and 30 amino acids from either N′ terminal or C‐terminal end. Each of the peptides were cloned into pET15b for expression with a His tag for purification. Peptides were incubated with either hPEP or spPEP in the presence of the substrate GPpNA. We report the inhibition of both enzymes based on size and placement of the inhibitory sequence.Support or Funding InformationFunding: Office of Research and Sponsored Projects, SFASU and Ed and Gwen Cole, Nacogdoches, TX.
Deletion of nicastrin Leads to Hypomyelination and Transcriptional Changes in Juvenile Mice
Authors: Yamato Mizuho , Zhu Yi , Dries Daniel , Yu Gang 
Published: November 2023

The γ‐secretase complex is a four‐subunit membrane‐bound protease complex that regulates a number of critical cellular functions through the cleavage of transmembrane substrate proteins. Of the four subunits, nicastrin is involved in substrate recruitment. Germline deletion of any of the four subunits leads to death during embryonic development. Previous work described a line of conditional knockout (cKO) mice in which nicastrin was knocked out of oligodendrocytes (OLs, the myelinating cells in the central nervous system). To examine the transcriptional changes that led to the pathological differences in juvenile mice, RNA‐Seq was performed. Here, we present the analysis of several genes with altered expression from the study using qPCR. Meanwhile, optic nerves were examined by electron microscopy (EM) to observe myelination during development. The results indicate that deletion of nicastrin from γ‐secretase leads to transcriptional changes and significantly affects myelination in the central nervous system of juvenile mice.Support or Funding InformationThis work was supported by the National Institute of Neurological Disorders and Stroke (R01 NS079796), the National Institute of Aging (AG029547, R01 AG023104 and F32 AG031625), the Welch Foundation (I‐1776), the Hartwell Foundation, and the Alzheimer's AssociationNational Institute of Neurological Disorders and Stroke (R01 NS079796), the National Institute of Aging (R01 AG023104 and F32 AG031625), the Welch Foundation (I‐1776), the Hartwell Foundation, and the Alzheimer's Association.
Preclinical Trials of Amblyomin‐X: a Toxicity Study in Mice
Authors: Chudzinski‐Tavassi Ana Marisa , Will Sonia Elisabete , Sciani Juliana Mozer , Bosch Rosemary , Goldfeder Mauricio B. , Maria Durvanei A. 
Published: November 2023

IntroductionAmblyomin‐X is a recombinant protein, which cause apoptosis of tumor cells, in a selective way, beside tumor regression, in in vivo tumor models. Due to the interesting effects, this protein is being developed as an antitumor drug, and toxicity studies in animals are required by regulatory agencies. Amblyomin‐X selectively induces apoptosis in tumor cells and was able to promote a reduction in tumor size in a melanoma model.AimThe aim of this study was to perform preclinical assays to evaluate the toxicity of Amblyomin‐X in mice after acute (200 mg/kg) and chronic (5 and 50 mg/kg per day) intravenous administration. During the experimental period, indications of toxicity, body weight variations and water and food consumption were evaluated. No mortality was observed following treatment with Amblyomin‐X.ResultsAmblyomin‐X is well tolerated by mice, both male and female, that were treated with a high concentration of the drug (200 times higher than the effective dose therapy) and therefore is not be expected to have any acute or long‐term adverse effects in humans. The therapeutic concentration of Amblyomin‐X, which is less than 5 mg/kg, causes tumor reduction in mouse models and is pharmacologically safe. In general, Amblyomin‐X did not cause clinical alterations, and treated animals exhibited no changes in hematological and biochemical parameters. The macroscopic and histologic analysis of organs demonstrated that the protein increased the white pulp of the spleen, caused vacuolization in hepatocytes and induced intestinal lesions, which can be considered toxic effects of Amblyomin‐X, at concentrations of 50 mg/kg to 200 mg/kg. However, these effects do not appear to alter the function of the organ or homeostasis.ConclusionAfter this study, we can conclude that the liver changes that were observed in animals treated with high doses of Amblyomin‐X are changes that can take place without jeopardizing the integrity of the organ, which is responsible for the most important metabolic processes in the body. Careful interpretation and extrapolation of the data obtained from these models are essential to reduce the current clinical costs of new anticancer therapeutic agents for humans. This study provides data regarding important safety parameters that enable the implementation of further experiments aimed at developing this molecule as an anti‐tumor drug.Support or Funding InformationSupport by FAPESP