bluuebunny/crossref_metadata_2025_split huggingface dataset:
Discrete histone marks are translated into degenerate readouts by reader proteins (739.11)
Authors: Wang Wei 
Published: June 2021

The current paradigm to evaluate the combinatorial regulatory effect of histone modifications on gene transcription is to consider individual histone sites as the primary/basic functional units which are distinct from one another. However, numerous observations in chromatin biology cannot be explained by this paradigm. We propose a new framework to reconcile the evidences conflicting with the current paradigm: the individual histone sites can be functionally degenerate; thus, when considering combinatorial histone modifications, combination of these degenerate modifications but not that of the individual sites should be considered. This framework is based on our observation of significant degenerate binding between chromodomains and histone peptides, i.e. different histone sites can be recognized by the same set of chromodomains, and different chromodomains can share similar binding profiles to individual histone sites. Such degenerate binding is not dictated by amino acid sequence or PTM motif but rather rooted in the physiochemical properties defined by the PTMs on the histone peptides. This molecular mechanism is confirmed by the accurate prediction of the binding specificity by a computational model that captures the structural and energetic patterns of the domain‐peptide interaction. Our work represents the first but a crucial step towards elucidating the fundamental mechanism by which histone modifications correlate with their biological outputs.Grant Funding Source: NIH
Phosphorylation of threonine 45 of histone H3 and the effects of alanine and glutamic acid mutations (739.5)
Authors: Yates Saiah , Campos Eric , Reinberg Danny 
Published: June 2021

Epigenetics is the study of non‐genetic components that alter gene expression. One of the most notable examples is the chemical posttranslational modification of the DNA‐associated histone proteins. For my project, we focused on the effects of phosphorylation of threonine 45 on histone H3 by the STK38 kinase. In order to do this, we carried out a number of techniques: Western blotting of extracts from synchronized cells showed that the phosphorylation of H3 threonine 45 peaks at the G1/S transition of the cell cycle. Using Polymerase Chain Reaction (PCR)‐based Site‐directed Mutagenesis, we mutated a plasmid encoding wild‐type H3 into plasmids with Alanine (pET‐H3 T45A) and Glutamic Acid (pET‐H3 T45D) substitutions at threonine 45. Alanine eliminates phosphorylation while the negative charge on glutamic acid mimics the effects of threonine 45 when it is phosphorylated. We will now use these mutants to determine whether STK38 phosphorylates additional sites on histone H3 in vitro, and to immunoprecipitate histone H3 in its non‐phosphorylated and phospho‐mimic forms in vivo. This will allow us to determine whether phosphorylation at threonine 45 alters histone H3 binding proteins. From, current results we hypothesize that phosphorylation of threonine 45 plays an important role in the cell cycle, possibly having impact in the choice of whether or not the cell divides. In completing this study more will be known about how epigenetics can affect the cell cycle and more knowledge can be accessed about how histones and their modifications affect nucleosomes.Grant Funding Source: Supported by Howard Hughes Medical Institute
Investigating the transcription initiation mechanism of E. coli RNAP by FRET (743.1)
Authors: Zhang Yurun , Sreenivasan Raashi , Heitkamp Sara , Record M. 
Published: June 2021

E. coli RNA Polymerase (RNAP) recognizes the ‐10 and ‐35 upstream elements of the promoter DNA, which bends and wraps around RNAP on the back of the β subunit of the RNAP core. The recognition between RNAP and DNA promoter initiates the melting of about 13bp of DNA duplex surrounding the transcription start site in the active site of RNAP to form a promoter‐RNAP open complex (RPo). Even though transcription of DNA plays a critical role in gene expression and regulation, many important questions about the transcription initiation mechanism remains unanswered. To study the wrapping of DNA around RNAP, we have performed equilibrium FRET experiments on a strong promoter, λPr with Cy3 and Cy5 fluorophores on the upstream and downstream ends of the promoter DNA. We find that wrapping is present in closed complexes, as seen from complexes at 2°C and it persists in open complexes at 19°C. We further confirmed that the fluorophore‐labeled DNA complexed with RNAP is transcriptionally competent. Single‐labeled constructs also show permanganate reactivity, proving that fluorophore‐labeled promoter‐RNAP complexes are open. Further experiments are underway to study the kinetics of wrapping of promoter DNA around RNAP.Grant Funding Source: NIH GM103061
Cataloging and comparing long noncoding RNAs generated at enhancers (eRNAs) in prostate, breast, and ovarian cancer cells (744.2)
Authors: Konovalov Sergiy , Mackintosh Carlos , Garcia‐Bassets Ivan 
Published: June 2021

An important purpose of sequencing the human genome was to determine the number of human genes and to catalog them for future study. The final gene count matched the most conservative predictions, with gene sequences representing only 1.5% of the entire human genome. However, more recent transcriptomic analyses have revealed that the cumulative coverage of transcribed regions is at least 75‐85% of the human genome. Moreover, since these analyses did not investigate the process of transcription itself, but only the accumulation of RNAs, the range of cumulative transcribed genome is likely close to the full human genome. The question is how much of the human transcriptome (i.e. the transcribed genome) is functional and how much of the human transcriptome is transcriptional noise. Thus, we return to the question of how many genes (i.e. sequences coding for functional transcripts) are there in the human genome, and to the challenge of cataloging them.In this study, we catalog the full repertoire of long noncoding (lnc)RNAs (length >200 nucleotides) generated at enhancers, also known as enhancer (e)RNAs, in prostate (LNCaP), breast (MCF7), and ovarian (A2780) cancer cells. We use profiles of intergenic DNaseI hypersensitive sites (DNase‐seq) and H3K4me1/3 and H3K27ac regions (ChIP‐seq) to identify enhancers, combined with global run‐on next‐generation sequencing (GRO‐seq) and RNA‐seq to identify, characterize, and compare the eRNA transcriptomes in these three cancer lines. We expect that our analyses will facilitate the study of the functionality of the eRNA transcriptome, and will contribute to distinguishing the fraction of the human transcriptome that is functional from the fraction that could be regarded as noise.
Characterization of eukaryotic translation initiation factor 5A isoform A (752.13)
Authors: Tamborlin Letícia , Pereira Karina , Almeida Isadora , Meneguello Letícia , Cambiaghi Tavane , Luchessi Augusto 
Published: June 2021

The eukaryotic translation initiation factor 5A (EIF5A) protein is highly conserved in eukaryotes. Some studies shown that eIF5A has important roles in cell proliferation control and HIV‐1 replication. EIF5A possesses a particular amino acid residue named hypusine that is not found in others proteins. The hypusine is essential for its activity and is produced by a post‐translational modification using spermidine as substrate. The EIF5A protein has been involved with the protein synthesis process and recently it was shown that it acts on translation of polyproline motifs. In humans, four transcript variants for EIF5A are known (variants A, B, C and D), derived from alternative splicing associated with the first exon. The variants B, C and D encode for the canonical isoform B, the main studied isoform to date. It is predicted that the variant A is responsible for the establishment of an upstream start codon in frame to the start codon of the others variants. In HeLa cells, we confirmed that variant A encodes an isoform containing an additional sequence of 30 amino acids at the N‐terminus of EIF5A (isoform A). The subcellular localizations of EIF5A isoforms were also compared. In addition, it was observed that the start codon of isoform A has a weak Kozak consensus, showing that the variant A encodes for both isoforms. The isoform A is not predicted for mice, however it was detected in different tissues. The results obtained so far contribute to the characterization of EIF5A isoform A in mammals.Grant Funding Source: Supported by FAPESP 2010/18095‐0
ATP signaling complex is altered in muscular dystrophy and was partly recovered after nifedipine treatment (762.3)
Authors: Valladares Denisse , Altamirano Francisco , Henríquez‐Olguín Carlos , Díaz‐Vegas Alexis , Intriago Giomar , Contreras‐Ferrat Ariel , Buvinic Sonja , López José , Allen Paul , Jaimovich Enrique 
Published: June 2021

Duchenne muscular dystrophy (DMD) is a common genetic disorder characterized by a severe muscle wasting caused by the absence of dystrophin. In mdx muscle fibers, we have shown that basal ATP release is increased and that high extracellular ATP is a pro‐apoptotic stimuli. We also have shown that Dihydropyridine receptors (DHPR) are needed for ATP release through pannexin‐1(Pnx1) channels. The aim of this work was to study the potential therapeutic effect of DHPR blockage by nifedipine in DMD. We used muscle fibers isolated from six‐week‐old normal and mdx mice that were treated with daily intraperitoneal injections of 1mg/Kg nifedipine for 1‐week. We studied differences in the interaction between DHPR and Pnx1by proximity ligation assay. In mdx fibers there was an increase in the number and volume of positive particles but not in the intensity of the positive reaction when compared to normal fibers. After the treatment with nifedipine, both number and volume of the positive reaction particles was normalized. This result correlates well with a decrease in both ATP release and intracellular resting Ca2+ concentration in mdx fibers after the treatment. Moreover, ATP signaling was no longer a pro‐apoptotic stimuli. Finally, nifedipine treatment increased muscle strength assessed by both the inverted grip‐hanging and forced swimming test. These data suggest that dihydropyridines can be used as a therapeutical tool to reduce muscle damage observed in dystrophic muscles.Grant Funding Source: Fondecyt 1110467, ACT1111, AFM14562, AT‐24110211
PGC1α‐1 nucleosome position in skeletal muscle of overweight individuals may determine mRNA splicing and be predictive of adiposity and hyperlipidemia (763.1)
Authors: Henagan Tara , Stewart Laura , Johannsen Neil , Forney Laura , Sparks Lauren , Church Timothy 
Published: June 2021

Epigenetic alterations, leading to aberrant metabolism and dyslipidemia, partially determine obesity and associated CVD. PGC1α is a transcriptional coactivator whose expression in skeletal muscle is decreased during obesity and CVD. Several splice variants of PGC1α are known to have similar effects on metabolism as the full length mRNA isoform (FL) and may compensate for decreased FL expression. Here, we employed scanning PCR to determine if FL and N‐truncated PGC1α (NT) mRNA splice variant expression may be epigenetically regulated through ‐1 nucleosome positioning (‐1N) in the skeletal muscle of overweight/obese, diabetic individuals. The ‐1N tended to determine splice variant mRNA expression, with a ‐1N upstream (‐1NU) in the promoter (N=9) tending to have higher FL (P=0.175) and those with a ‐1N further downstream (‐1ND; N=4) exhibited higher NT (P=0.032). The ‐1ND group had a significantly higher body fat percentage (P=0.046) and cholesterol (P=0.025) and a higher trend in LDL cholesterol (P=0.055) compared to the ‐1NU group. Importantly, ‐1N occurs upstream of changes in mRNA expression and may occur before changes in gene expression or lipids are observed. These preliminary data include a small sample size yet reveal that PGC1α ‐1N may determine mRNA splicing and serve as a predictive biomarker for the onset of obesity and adverse cardiometabolic outcomes.Grant Funding Source: Supported by NIH 5T32‐DK064584‐09, Cola‐cola Company and NIDDK R01‐068298.
Knockdown of UDP‐glucose dehydrogenase in INS‐1 832/13 cells inhibits insulin release (767.12)
Authors: Perez Jaritza , MacDonald Michael 
Published: June 2021

Introduction: Our unpublished mass spectrometry studies of pancreatic islet insulin‐producing cells, as well as a published study by others, showed that several nucleotide‐sugar compounds increased with glucose stimulation. These compounds included ADP‐glucose (not known to be present in mammals), UDP‐glucose and GDP‐mannose. That these compounds increased with glucose stimulation suggests they might play a role in insulin secretion. We focused our study on two enzymes that use UDP‐glucose as a substrate: UDP‐glucose Dehydrogenase (UGDH) and UDP‐glucose glycoprotein glucosyltransferase (UGGT1).Results: Transcripts of both genes were detected in human pancreatic islets and in the glucose‐responsive INS‐1 832/13 cell line. Human and rat islets showed six‐fold more UGDH mRNA than liver. Enzyme activity of UGDH was detected in rat liver, but was below the level of detection in the INS‐1 832/13 cell line. Two shRNAs targeting the UGDH gene significantly knocked down the mRNA of the gene in INS‐1 832/13‐derived cell lines. Knockdown of the UGDH mRNA was associated with 38% and 22% decrease in glucose‐stimulated insulin release in two UGDH shRNA / INS‐1 832/13‐derived cell lines. Cell lines with knockdown of UGGT1 are currently being generated.Conclusions: Based on enzyme activity assay, the concentration of UGDH in INS‐1 832/13 cells is very low. However an inhibition of insulin release was observed in two separate UGDH knockdown cell lines, and it is unlikely that the inhibition of insulin release was due to an off target shRNA effect. Further studies are needed to establish whether UGDH and UGGT1 play a role in insulin release in INS‐1 832/13 cells.Acknowledgements: NIH / NIDDK STEP‐UP Program award to JMP and NIH Grant DK28348 to MJM.Grant Funding Source: Supported by NIH / NIDDK STEP‐UP Program award to JMP and NIH Grant DK28348 to MJM
Effect of ketones on ROS, NADPH oxidases, and monocyte adhesion in endothelial cells (767.8)
Authors: Kanikarla Marie Preeti , Jain Sushil 
Published: June 2021

Cardiovascular disease (CVD) incidence is higher in Type 1 diabetic (T1D) patients. Along with hyperglycemia, T1D is associated with hyperketonemia, a condition with elevated blood levels of acetoacetate (AA) and 3‐β‐hydroxybutyrate (BHB). It is not known whether hyperketonemia per se contributes to the excess CVD in T1D. This study investigates the hypothesis that ketones upregulate NADPH oxidases (NOX) and ROS and therby increase monocyte‐endothelial adhesion. HUVEC were treated with AA or BHB (0‐8 mM) ± high‐glucose (25 mM) for 24 h. Results show that AA, but not BHB, increased ROS production, NOX activity, and NOX4 expression in HUVEC. Pretreatment with DPI (20 μM , inhibitor of NOX) abolished the effect of AA on ROS production. This suggests that AA effect on ROS production is mediated through the NOX. To further examine the role of NOX, we determined the effect of ketones in NOX4 knockdown (KD) HUVEC. There were significantly lower levels of ROS (65% vs. 20%), ICAM‐1 (42% vs. 23%), and monocyte adhesion (22% vs. 1%) in AA (4 mM) treated NOX4 KD cells compared with control cells. Effect of AA on ROS and ICAM‐1 was higher in cells knocked down for GCLC (that induces GSH deficiency). This suggests that ketone‐induced increase in ICAM‐1 and adhesion is mediated by the upregulation of NOX and ROS production. This study provides evidence for a biochemical mechanism for the role of hyperketonemia in the development of CVD in T1D.Grant Funding Source: Supported by NIH and Malcolm Feist Chair in Diabetes
Determination of hydrolases, oxidases and synthases in hypertensive patients (771.1)
Authors: Osman Mohamed Yousry , Osman Hassan , Moharem Dalia , Elsaiedy Ahmed 
Published: June 2021

Hypertension (HT) affects about 20 percent of the adult population placing a high burden on health care systems. Cholinestrase is responsible of the hydrolysis of the vasodilator acetylcholine. Monoamine oxidase (MAO) is responsible for degradation of the vasoconstrictor biogenic amines. Nitric oxide synthase (eNOS) is responsible for the production of the vasodilator nitric oxide (NO). The study aimed to find the relationship between these enzymes and essential hypertension in recently diagnosed patients. The results indicated a significant decrease in the activity of pseudo ChE in HT patients compared to control resulting in increase of ACh content and also indicated a significant decrease in plasma MAO‐A in HT patients saving the biogenic amines. Similarly; there is a significant decrease in the plasma nitrites + nitrates [NOx] of HT patients due to decrease in NOS activity. Eventually; it could be concluded that the investigated enzymes played a role in the regulation of essential HT. As the study showed; increase of 5‐hydroxytryptamine and adrenaline (due to decrease in MAO) that exceeded the decrease of pseudo ChE (increasing ACh content) and NOS (decreasing NO) resulting in vasoconstriction exceeding vasodilatation and the blood pressure consequently rises. Reversal of the previous mechanism could be helpful in treating essential HT.Grant Funding Source: Alexandria University
Lipoprotein‐associated phospholipase A2 structure and function (774.3)
Authors: Gonzalez Tara , Bahnson Brian 
Published: June 2021

Lipoprotein‐associated phospholipase A2 (LpPLA2) or plasma platelet‐activating factor acetylhydrolase (pPAFAH) is a serine hydrolase that acts on platelet‐activating factor (PAF) and oxidized phospholipids. The enzyme has a classic α/β hydrolase fold and predicted membrane binding regions. Plasma PAFAH binds readily to both high density lipoproteins (HDL) and low density lipoproteins (LDL) and is believed to play a pro‐atherosclerotic role. The purpose of this study is to further elucidate the structure and function of LpPLA2 through the use of specific inhibitors. It was necessary to develop and refine an expression method in E. coli to produce high quantities of protein of high purity for a truncated form of LpPLA2. Successful expression and purification of this LpPLA2 construct have been achieved and initial trials of crystallization were positive. The next step in this study is to obtain the crystal structure of LpPLA2 along with its selective inhibitors.Grant Funding Source: Supported by NIHNHLBI grant #RO1HL084366
Genetic variability in CRHR1 and its association with participant response to glucocorticoid receptor‐mediated signaling (776.2)
Authors: Knox Rachel , Biette Kelly , Fox Olivia , Murphy Patrick 
Published: June 2021

The goal of this study was to identify correlations between interpersonal genetic variability and alterations in the intracellular glucocorticoid receptor (GR)‐mediated signaling pathway. Salivary cortisol concentrations were measured in healthy volunteers (n=66) in order to determine differences in circulating cortisol concentrations and cortisol response to HPA stimulation. Statistical analysis indicated inter‐participant cortisol variability was not correlated to participant demographics, health history, or SNPs in genes encoding the GR (NR3C1) or the essential GR chaperone protein hsp90 (HSP90AA1). Two participants with highly similar demographic profiles and opposing responses to HPA stimulation were selected for matched exome sequencing. Filtering analysis identified candidate SNPs in approximately 30 genes involved in mediating the cortisol response and GR signaling. Interestingly, corticotropin releasing hormone receptor 1 (CRHR1) showed a difference of over 20 SNPs between the matched exome pair. CRHR1 regions were sequenced from the larger participant group, and participants with average cortisol concentrations that slightly decreased following stimulation most commonly exhibited a unique SNP profile. We speculate participants with SNP profiles associated with altered cortisol response may share a corresponding sensitivity to corticosteroid‐based drug therapies.Grant Funding Source: Supported by NIH AREA grant GM086822
Regulated axonal transport of APP and alcadein by kinesin‐1 (783.1)
Authors: Suzuki Toshiharu , Chiba Kyoko , Okumura Yuka , Shiraki Yuzuha , Sobu Yuriko , Kinjo Masataka , Hata Saori , Taru Hidenori 
Published: June 2021

Alzheimer’s β‐amyloid precursor protein (APP) and Alcadein (Alc) are cargo of kinesin‐1. APP associates to kinesin‐1 by JIP1b mediation, while Alc directly binds to kinesin‐1. APP is subject to axonal transport faster than Alc in independent cargos, even if both APP and Alc are transported kinesin‐1 in axon. To understand molecular mechanisms how both cargos are transported in different velocities, we revealed molecular interactions biochemically among APP, JIP1b and kinesin light chain (KLC), and between Alc and KLC.Methods: Detailed interaction between Alc and KLC, and JIP1b and KLC were analyzed biochemically by co‐immunoprecipitaion and GST pull‐down assays. We generated many deletion mutants for Alcα (one of Alcadein family) and JIP1b (JIP1 family which can bind APP strongly), and analyzed the interaction with KLC1 (neuron‐specific isoform of KLC) mutants. Function was investigated with analysis of APP‐GFP and Alcα‐GFP transport in neural axon with TRIF microscopy.Results and Discussion: Direct binding between Alcα and KLC shows a simple but strong affinity, while JIP1b‐mediated interaction between APP and KLC is more complex. The mediation of JIP1b may regulate fast velocity of anterograde cargo transport by kinesin‐1.Grant Funding Source: Supported by Grants‐in‐Aid for Scientific Research from JSPS, Japan.
investigating the mechanisms involved in aberrant neurosphere migration in schizophrenia (784.2)
Authors: Patruno Anna , Tran Ngoc , Abdelrahim Mohammed , Brennand Kristen 
Published: June 2021

Schizophrenia (SCZD) is a chronic, debilitating disorder with a prevalence of 1% and typically appears in the late adolescent and early adulthood years. Postmortem studies have been pivotal in understanding the neuropathology of schizophrenic brains by revealing characteristics such as reduced cell size, brain volume and neuronal connections; however, the cellular and molecular mechanisms involved in the disease as well as its development remain unclear. To investigate disease progression, we modeled genetic predisposition to SCZD by reprogramming fibroblasts from SCZD patients into human induced pluripotent stems cells (hiPSCs) and differentiating these cells into neural progenitor cells (NPCs) and neurons. Using a neurosphere migration assay, we observed aberrant migration of SCZD NPCs in vitro. To investigate the mechanism responsible for this aberrant migration, we manipulated the RHO‐ROCK (Rho‐associated kinase) signaling pathway in SCZD NPCs. In addition, we investigated whether treatment with antipsychotics such as Loxapine and Clozapine was sufficient to ameliorate aberrant migration. Neurosphere migration assays are a useful model to study molecular mechanisms in SCZD as well as disease predisposition, which may contribute in developing potential treatments.Grant Funding Source: Supported by NYSCF and NIH/NIMH, R01 MH101464