bluuebunny/crossref_metadata_2025_split huggingface dataset:
Development of a Specialized Yeast Strain to Monitor Recruitment Patterns of Saw1 Mediated Rad1‐Rad10 Recruitment to DNA Damage Sites through Single‐Strand Annealing
Authors: Fregoso Fred E , Fischhaber Paula L. 
Published: November 2023

Damage to the structural integrity of DNA by exposure to ionizing radiation is the cause of many mutations that accumulate in the human genome, which can lead to cancer and aging. The DNA double‐strand break repair (DSBR) pathways limit this accumulation by continuously repairing such damage as it forms. Single‐strand annealing (SSA) is one mode of DSB repair that occurs in regions that have tandem arrays of DNA repeats. Upon DSB induction, the processing of broken ends exposes complementary single‐stranded regions that anneal and produce 3′ overhanging flaps deriving from the sequence originally situated between the repeats. This DNA flap structure recruits Rad1‐Rad10 endonuclease to cleave the overhanging 3′ DNA. Although recent literature reported that the Saw1 protein mediates recruitment of Rad1‐Rad10 when flaps are 500 deoxynucleotides long but not ~10, the minimum flap length for Saw1 to recruit Rad1‐Rad10 in vivo is unknown.The ultimate aim of this project is to determine whether Saw1 is required to recruit Rad1‐Rad10 to SSA sites when 3′ overhanging flaps are slightly longer (~20 deoxynucleotides). To address this important question, we have prepared a specific yeast strain containing an inducible DSB site flanked by DNA repeats that will yield 20 bp overhang flaps upon DSB induction and repair by SSA. The strain will additionally contain a fluorescently labeled Rad10 gene (Rad10‐YFP) and DSB site (DSB‐RFP) to monitor Rad10 localization to DSB sites by fluorescence microscopy in the presence or absence of Saw1.Cloning of the 20 bp flap yeast strain was achieved by constructing two DNA plasmids that each contained the regions required to derive the desired substrate. The two plasmids were linearized and transformed into a progenitor yeast strain. Resulting transformants were screened by PCR to confirm integration of the recombinant DNA cassettes; an ISce‐I restriction site situated between two homologous HIS3 DNA repeats however spaced away from each other by 10 nonhomologous bp. DNA sequencing and ISce‐I restriction digest further supported proper insertion of the DNA cassettes and functionality of the inducible DSB site necessary for microscopy. This specialized yeast strain will be able to be used as an SSA substrate in consequent fluorescent microscopy experiments and will determine the necessity of Saw1 in recruiting Rad1‐Rad10 to SSA sites bearing ~20 deoxynucleotide flaps.Support or Funding InformationThe authors thank NIH grant (SC3GM093858) for funding.
Acetylpyrazine Thiosemicarbazone Inhibiting Topoisomerase II
Authors: Ngo Lana C , Stults Garrett A. , Hill Jessica , Bowman Sarah B. , Lisic Edward C. , Jiang Xiaohua 
Published: November 2023

We have previously showed that Cu(II) acetylpyrazine methylthiosemicarbazone chloride ([Cu(APZ‐MTSC)Cl]) inhibits the ATP hydrolysis of human topoisomerase (Topo II) as catalytic inhibitor and share some common features as covalent poisons such as increasing TopoII‐mediate DNA cleavage which can be abolished by the presence of DTT. To further test if acteylpyrazine thiosemicarbazone metal complexes all have the similar characteristics, we synthesized a series of acetylpyrazine thiosemicarbazone metal (copper and palladium) complexes with different side chain substitutions including methyl, ethyl, turt‐butyl, benzyl, phenyl and dimethyl functional groups on the terminal nitrogen. We examined their effect on TopoII‐mediated DNA relaxation and DNA cleavage assays. Our data suggest that all metal acetylpyrazine thiosemicarbazones display similar characteristics as [Cu(APZ‐MTSC)Cl], but metal ions play a very important role in inhibiting TopoII.Support or Funding InformationThis project is funded by Faculty Development Research Grant from Tennessee Board of Regents and Faculty Research Grant from Tennessee Technological University.
Differential Gene Expression and Variant Analysis of Clinical Strains of Cryptococcus neoformans
Authors: Seipelt‐Thiemann Rebecca , McClelland Erin 
Published: November 2023

Cryptococcus neoformans is a fungal opportunistic pathogen that kills more than 500,000 people each year. To investigate the relationships of gene expression, genetic variant, and pathogenesis characteristics, RNA sequencing was performed on seven different clinical isolates and one laboratory strain H99S. TopHat, Cufflinks, and Cuffdiff bioinformatics tools were utilized to analyze the data. As expected, all strains showed differential expression of some genes (≤2 or ≥2 fold) compared to the laboratory strain. About 1700 up regulation differences and about 2300 down regulation differences were found compared to wildtype. When the clinical strains were compared to each other, more than 6200 up regulation differences and 7500 down regulation differences were observed in total. These data are currently being analyzed to determine common patterns of gene expression among the clinical strains, discover common biological pathways using gene ontology and gene enrichment analyses, and investigate relationships between gene expression and clinical patient data. Further, variant analyses will be undertaken to investigate genetic variation in the expressed genes of the clinical strains.Support or Funding InformationURECA Grant at MTSU
Transgene Expression Is Increased When Cells Are Cultured on Defined Biophysical Cues as Detected by a Novel Fluorescence Quantification Method
Authors: Veen Sarah C , Youssef Amanda J , Liu Claire , Gasiorowski Joshua Z 
Published: November 2023

Studies have shown that the interaction between cells and their extracellular matrix (ECM) is critical in directing migration, development, differentiation, and gene expression. Gene array studies have shown that cells seeded onto topographically‐patterned substrates that mimic biophysical ECM cues demonstrate a significantly different expression profile than those seeded onto a flat control. This led us to believe that biophysical cues may also have a significant effect on the expression level of transgenes for transfections and gene therapy experiments. However, quantifying transgene fluorescence for our experiments with commonly used techniques to measure live‐cell gene expression were limiting. Employing microscopes to measure fluorescent reporter expression are useful in observing single cell transgene expression, but macro‐scale cell populations cannot be observed as a whole unless time‐consuming stitch image acquisition is performed. This method of observation and quantification can become strenuous, inaccurate, and subjected to user bias. To address this limitation, we developed a non‐invasive method to acquire sensitive, quantitative, visual data over time of whole cell populations for transgene expression analysis. Through the use of a macro‐scale fluorescence photo‐imager with appropriate light sources and filters, we investigated the influence of surface topography on transgene expression. Mesenchymal and epithelial cell lines were transfected with a plasmid encoding GFP and seeded onto either a range of nano‐, submicro‐, and micro‐scale topographic surfaces with repeating patterns of ridges and grooves, or on a flat control. An increase in transgene expression was observed in cell populations plated onto the topographic surfaces when compared to the flat control, at multiple time points over a 48 hour time course. Cells plated onto the submicron sized patterns consistently displayed the highest amount of transgene expression. With similar trends observed in several cell types, our results suggest that defined biophysical ECM cues have a direct effect on the level of transgene expression. Additionally, our macro‐scale fluorescence imaging method is a non‐invasive, efficient way to observe and quantify transgene expression in large cell populations over extended time courses.Support or Funding InformationThe research presented here was supported by funds from Midwestern University's College of Health Sciences and Department of Biomedical Sciences.
Nutrient regulation of TATA‐Box binding protein by O‐GlcNAcylation
Authors: Hardiville Stephan , Han Guanghui , Ma Junfeng , Hu Ping , Banerjee Partha S. , Hart Gerald W. 
Published: November 2023

O‐GlcNAcylation is a dynamic post‐translational modification that is responsive to nutrient avabilality via the hexosamine biosynthetic pathway and its endproduct UDP‐GlcNAc. O‐GlcNAcylation serves as a nutrient sensor to regulate the activities of many proteins involved in nearly all biological processes. Interestingly, literature points out a regulation of the basal transcription machinery by O‐GlcNAcylation. TATA‐box binding protein (TBP) is a key player in regulating transcription since it is required for the activity of all three RNA polymerases. DNA‐bound TBP acts as a dock for recruitment of basal transcription factors and the RNA polymerase II to formation of a functional pre‐initiation complex. DNA‐bound TBP can also be recognized by NC2 and BTAF1. NC2 stabilizes the chromatin interaction of TBP, whereas the association of TBP with BTAF1 promotes its release upon hydrolysis of ATP. Here, we have shown that O‐GlcNAcylated TBP is bound to DNA, and that the sugar inhibits the interaction with BTAF1. Three O‐GlcNAc sites on the N‐terminus domain of TBP have been revealed by mass spectrometry. Site‐specific O‐GlcNAc mutants have been used to point out the T114 as the main regulator of TBP/BTAF1 interaction. Owing to the fact that BTAF1 dynamically regulates the chromatin interaction of TBP, we investigated the dynamic behavior of TBP and TBP O‐GlcNAc by Strip‐FRAP and ChIP assays. Collectively, our results indicate that O‐GlcNAcylation at T114 regulates TBP dynamics on gene promoters.Support or Funding InformationThe authors are supported by R01DK61671 and P01HL107153. G.W.H. receives a share of royalties received by Johns Hopkins University on sales of the CTD110.6 antibody, which is managed by JHU.
Cell penetrating peptide‐mediated nuclear delivery of Cas9 to enhance the utility of CRISPR/Cas genome editing
Authors: Axford David S. , Morris Daniel P. , McMurry Jonathan L 
Published: November 2023

CRISPR/Cas9 is a transformative technology that allows genome editing in a variety of eukaryotic cells. However, many cell types and tissues are resistant to transfection or other delivery of the CRISPR/Cas9 complex, which has limited its utility. Our novel cell‐penetrating peptide (CPP) adaptor, TAT‐CaM, allows cytoplasmic delivery and release of a wide variety of biomolecular cargos. The integration of CRISPR/Cas9 with CPP‐mediated delivery could make CRISPR/Cas9 much more widely utile. Recombinant Cas9 containing a calmodulin binding site (CBS‐Cas9) was expressed in Escherichia coli, from which it was purified using affinity chromatography on a calmodulin sepharose column. TAT‐CaM/CBS‐Cas9 complexes were assayed for cell penetrating capabilities and subcellular localization using confocal microscopy. Several other CPPs and adaptor proteins, e.g. SAP and CALL3, and model cargos with subcellular localization signals were assayed in an attempt to develop CPP‐adaptor deliveries with altered kinetics and destinations. After cell penetrating capability and subcellular localization were assessed, a nuclear localization sequence was added to the gene encoding Cas9. Enhanced nuclear localization of Cas9 would make it more effective in potentially editing genes. Future work will integrate the present results into an effective CRISPR/Cas9 delivery and gene editing system.Support or Funding InformationThis work was supported by NIH grant R15GM120691
Postmortem Samples from Aviation Accident Victims Maintain Tissue‐specific mRNA Expression Profiles
Authors: Burian Dennis , Hutchings David C , Uyhelji Hilary A , Williams Daniel L , White Vicky L 
Published: November 2023

The translation of marker panels for aerospace medical factors from discovery phase to a forensic setting requires that RNA stability and the factors that affect it be assessed in post‐accident samples. Increased understanding of these parameters will allow investigators to optimize assay design and conditions to compensate for expected post‐accident sample degradation.To assess the utility of samples from accident victims for gene expression analysis, total RNA was purified from accident victim blood and tissue samples. Purified RNA was characterized for integrity, yield, purity and the origin of ribosomal RNA. Data was gathered regarding accident conditions and postmortem interval. Gene expression data was gathered by qRT‐PCR for panels of genes on victim RNA. Live subject data was obtained from microarray data available in public databases. Correlation analyses were performed to determine mRNA expression patterns within victim samples and tissue‐specific expression patterns between live and victim samples. Primers specific for bacterial rRNA were characterized for sensitivity and specificity on purified E. coli RNA, then used on purified accident victim RNAs exhibiting either prokaryotic or eukaryotic rRNA alone, both rRNAs, or no rRNA peaks.RNA integrity was highly variable between victim samples but there was no correlation between RNA integrity and yield, postmortem interval, trauma level, or accident condition parameters. Across 35 genes in 87 victim samples, mRNA levels were generally very highly correlated suggesting that mRNA degradation is not dependent on accident‐related factors and, for these genes, is mechanistically similar. In a comparison of victim samples to live subject expression levels with 44 genes, blood, brain, and muscle relative expression levels were moderately well correlated. The assay for bacterial rRNA was found to be sensitive for very low levels of purified bacterial RNA and highly specific for bacterial rRNA when challenged with mixtures of bacterial and human RNA but had lower Cts than expected in accident victim samples without prokaryotic rRNA peaks.In conclusion, aviation accident victims exhibit a wide range of degradation but very few failed to yield qRT‐PCR data of sufficient quality that relative expression values between target genes were not preserved. That victim samples have similar expression patterns to live subject data demonstrates that marker panels developed in human subject studies can be translated to accident investigation and will allow additional information to be incorporated into accident investigation.Support or Funding InformationFAA Office of Aviation Medicine, FAA Office of Accident Investigation and Prevention
Functional Characterization of a Cysteine‐Less Isoform of the Human GLUT2 Hexose Transporter
Authors: Reyes Alejandro Mauricio , Pérez Alejandra Andrea , Vargas‐Uribe Mauricio , Elgueta Coral , Cuevas Alexei , Ojeda Lorena , Arce Robinson , Salas Monica 
Published: November 2023

The GLUT2 carrier is the major glucose transporter isoform expressed in hepatocytes, insulin‐secreting pancreatic beta cells, and absorptive epithelial cells of the intestinal mucosa and kidney. GLUT2 is suggested to play a critical role in maintaining glucose homeostasis because it functions as a low affinity, high‐turnover transport machinery. However, little is known regarding the mechanism of transport at the structural level. Here, we designed a cysteine‐less isoform of GLUT2 (C‐less GLUT2) to serve as platform for functional and structural studies, i.e. cysteine‐scanning mutagenesis in combination with thiol chemical modification. The kinetic properties of the C‐less GLUT2 expressed into Xenopus oocytes were like those of the native GLUT2, except that the overall activity was lower than that of the wild‐type transporter. Like the native protein, the C‐less GLUT2 transported methylglucose and deoxyglucose, and the activity was reduced in the presence of other sugars such as fructose, sorbitol, 3‐O‐methyl‐D‐glucose, galactose and ribose. Similarly, the activity of the C‐less isoform was inhibited by the classical GLUT2 inhibitors cytochalasin B, quercetin and resveratrol. This C‐less protein was refractory to MTSET, a membrane‐impermeable thiol‐alkylating reagent that obliterated the activity of native GLUT2. Our data suggest that the C‐less isoform of GLUT2 retains native‐like function and therefore is a suitable platform to perform structure‐function analysis by cysteine‐scanning mutagenesis.Support or Funding InformationSupported by grants from FONDECYT 1130386, and FONDEF D11I1131
ALS and Ubiquilin‐2: Effects of ALS Mutations on Ubiquilin‐2 Structure and Function
Authors: Castaneda Carlos , Dao Thuy 
Published: November 2023

Amyotrophic lateral sclerosis (ALS) and other neurodegenerative diseases are caused by malfunctions in ubiquitin‐mediated protein degradation pathways. Ubiquilin‐2 is a 624‐amino acid multi‐domain adaptor protein critical for maintaining protein homeostasis through the ubiquitin‐proteasome system, the endoplasmic reticulum‐associated protein degradation (ERAD) pathway and autophagy. Mutations in ubiquilin‐2 have recently been shown to cause dominant x‐linked inheritance of ALS and ALS/dementia. Moreover, wild‐type ubiquilin‐2 is present in protein inclusions found in patients diagnosed with either familial or sporadic ALS, further linking ubiquilin‐2 to the pathogenesis of ALS. Interestingly, most ALS‐linked mutations are localized to the proline‐rich repeat (Pxx) region that is unique to ubiquilin‐2 and not present in the other members of the ubiquilin protein family. The consequences of these mutations (specifically to residues P497, P506, P509, P525 and P533) on ubiquilin‐2's structure and function remain unknown. Towards that end, we are using biomolecular NMR spectroscopy and other biophysical techniques to characterize the Pxx region of ubiquilin‐2. 13C‐detect NMR experiments have allowed for the direct and necessary observation of the prolines as well as aided in resonance assignment for a significant portion of this ubiquilin‐2 region. Significantly, even though the Pxx region is intrinisically disordered, we observed sequence‐distant perturbations in several ALS‐linked mutants. Serendipitously, similar to some other ALS‐linked proteins such as TDP‐43 and hnRNPA1, our ubiquilin‐2 constructs exhibit salt and temperature dependent liquid‐like phase‐separation, a behavior that is also affected by ALS mutations. Our data suggest that the C‐terminal region of ubiquilin‐2 is important for modulating phase separation behavior. We are working towards modeling the structure of ubiquilin‐2 containing the Pxx region, as well as identifying protein‐binding surfaces involving the Pxx region.Support or Funding InformationALS Association Starter Grant to C.A.C.
Modulatory Role of Anti‐Tau Antibodies on Phosphorylation of Tau‐441 by MARK4
Authors: Ziu Iva , Martic Sanela 
Published: November 2023

Tau protein stabilizes microtubules in neurons. The detachment of tau from microtubules, induced by post‐translational modifications, leads to tau aggregation, neuronal cell death, and cytotoxicity. The hyperphosphorylation of tau is one of the main causes of tau pathology in neurodegeneration. The current immunotherapies that target tau protein have shown clearance of tau pathology in animal models. However, it is still unclear how antibody‐based inhibition of tauopathies takes place. We have shown that anti‐tau antibody, targeting R1 epitope of tau, inhibited in vitro aggregation of non‐phosphorylated tau‐441. We also reported that phosphorylation of tau‐441 at S199 may be inhibited by antibodies to phosphorylated tau. Here, we report on the effects of anti‐tau antibodies to non‐phosphorylated tau (epitope at R4 repeat domain) on the phosphorylation outcomes of tau‐441 when MARK4 was used as a protein kinase. Using Dot blots and Western blots we evaluated the phosphorylation at S199, T231 and S262 residues of tau‐441. MARK4‐catalyzed phosphorylation of tau‐441 indicated a successful phosphorylation at S262 and S199, but not at T231. However, in the presence of anti‐tau antibodies (binding to tau R4 epitope) the phosphorylation at T231 was turned on, while phosphorylation at S199 and S262 remained unchanged. These findings suggest that phosphorylation of tau at specific sites may be modulated by using antibodies specific tau epitopes.Support or Funding InformationNIH/NIGMS R15 GM11905301
Effect of Rad 23 selectivity to polyubiquitin chain linkage on Far‐1 degradation in yeast
Authors: Liu Yixuan , Cooper Eric 
Published: November 2023

Ubiquitin is a small, 76‐amino acid polypeptide that can be covalently attached to other proteins to regulate their activities. The role of ubiquitin in tagging proteins for proteasome‐mediated degradation is its most well‐known function, but ubiquitin regulates non‐proteolytic processes as well. Ubiquitin's versatility is due, in part, by its ability to be linked into different types of polymers, depending on which of its seven lysine connect the individual ubiquitin subunits together.Polymers assembled through ubiquitin's lysine 48 are generally considered to be the primary “flag” targeting substrates to the proteasome for degradation. Although ubiquitin can be recognized by the proteasome directly, there is abundant evidence indicating certain ubiquitinated substrates requiring a “shuttle” protein to deliver them to the proteasome. For example, in yeast, the cyclin‐dependent kinase inhibitor Far1 is only efficiently degraded by the proteasome in cells expressing a wild type version of a shuttle protein called Rad23. We are performing an in vivo experiment to test the hypothesis that Lys48‐linked polyubiquitin is, indeed, the primary degradation in cells. By switching the ubiquitin polymer‐binding selectivity of Rad23 from Lys48‐selective to Lys63‐selective, we will determine whether Far1 is still efficiently degraded in these cells.
Proteasome Assembly and the Formation of Non‐canonical Complexes in Vivo
Authors: Kusmierczyk Andrew R , Hammack Lindsay J 
Published: November 2023

The 26S proteasome is the heart of the ubiquitin‐proteasome system (UPS) in eukaryotes with a central role in the degradation of proteins and the maintenance of protein homeostasis. The 26S proteasome is comprised of a compartmentalized protease, the 20S proteasome or core particle (CP), whose activity is modulated by its interaction with one or more copies of the 19S regulatory particle (RP). The quaternary structure of the CP, found in all domains of life, is absolutely conserved and consists of four heptameric rings stacked coaxially. The rings are made of structurally related α and β subunits. In eukaryotes, assembly factors chaperone the seven different α and seven different β subunits to form a structure with α(1‐7)β(1‐7) β(1‐7) α(1‐7) stoichiometry. Here we demonstrate that CP subunits can assemble into structures other than the canonical CP in vivo. Specifically, the yeast α4 subunit forms high molecular weight complexes (α4 HMWCs) whose abundance increases when proteasome function is compromised. Results from a disulfide crosslinking approach are consistent with these complexes being ring‐shaped. Though several eukaryotic α subunits can form rings when expressed recombinantly in bacteria, this is the first evidence that such non‐canonical complexes exist in vivo. Our results place the α4 subunit at an important crossroads in the assembly of the CP. In addition to being present in the canonical CP, and these newly discovered α4 HMWCs, the α4 subunit also assembles into an alternate CP isoform called an α4–α4 proteasome, whose formation is evolutionarily conserved from yeast to humans. How α4 is distributed among these 3 possible complexes in vivo is not known. We present evidence suggesting that relative levels of the Rpn4 transcription factor might determine the extent to which α4 populates assembly pathways that lead to the formation of α4 HMWCs and α4–α4 proteasomes.Support or Funding InformationThis work was supported in part by a Research Support Funds Grant from Indiana University‐Purdue University Indianapolis (to A.R.K.).