bluuebunny/crossref_metadata_2025_split huggingface dataset:
The ERG1 K+ Channel Protein Is More Abundant in the Denervated Skeletal Muscle of Rodents
Authors: Cobb Brittan A , Anderson Luke B. , Latour Sawyer M , Mosole Simone , Davie Judith K. , Cheatwood Joesph , Carraro Ugo , Zampieri Sandra , Pond Amber L. 
Published: November 2023

Skeletal muscle atrophy is the loss of muscle mass and strength that occurs with normal aging, neural and muscle injuries, starvation and diseases such as diabetes, neuropathies, muscular dystrophy, cancer and sepsis cachexia, etc. The atrophy of skeletal muscle can be incapacitating and is related to increased disability, morbidity and mortality in people. We have shown that the ERG1a alternative splice variant of the ERG1 K+ channel is up‐regulated in the skeletal muscle of mice experiencing atrophy as a consequence of cancer cachexia and disuse (i.e., hind limb unweighting, a condition in which muscle action potentials are reduced). In fact, the ERG1 protein is up‐regulated after 4 days of hind limb unweighting, which is prior to a significant loss in muscle cross sectional area, strongly suggesting ERG1 is involved in the onset of muscle loss. Further, we have shown that the ERG1a channel contributes to protein breakdown in muscle by increasing both ubiquitin proteasome proteolysis (UPP) and intracellular calcium concentration with a subsequent increase in calpain activity. Because skeletal muscle atrophy occurs rapidly in denervated skeletal muscle, we hypothesized that ERG1 would also increase in denervated muscles. To test this hypothesis, we performed sciatic nerve transection on mice and rats and collected gastrocnemius and soleus muscles at 0, 4, 7 and 14 days after surgery. Decreased muscle weight and myofiber cross sectional area demonstrate that the muscles were successfully denervated and underwent atrophy. Immunoblot with anti‐ERG1 antibodies demonstrates that indeed the ERG1 protein increases post denervation in mice. Further, immunohistochemistry demonstrates that ERG1 protein also increases in denervated soleus of rats. Interestingly, confocal microscopy analysis of these tissues shows that the ERG1 protein is localized in the sarcolemma and t‐tubules of denervated rat skeletal muscle as it is in heart. The t‐tubule location places it in the vicinity of the calcium release apparatus where it can affect calcium release. We conclude that ERG1 protein is upregulated in the t‐tubules and sarcolemma of denervated muscle and, because it is up‐regulated in other atrophy models, ERG1 is a consistent marker of skeletal muscle atrophy.Support or Funding InformationThis research was supported by the National Institute of Arthritis and Musculoskeletal and Skin Diseases of the National Institutes of Health under Award Number NIH NIAMS 1R03AR053706‐01A2 to ALP. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.
RECQ1 interacts with rDNA and promotes pre‐rRNA transcription
Authors: Parvathaneni Swetha , Lu Xing , Sharma Sudha 
Published: November 2023

Ribosomal DNA (rDNA) is organized in tandem repeats and presents a significant barrier to replication fork progression. RECQ1 helicase is a major player in maintaining replication fork progression under stress. Nascent rRNAs generated during RNA Polymerase (Pol) I‐mediated rDNA transcription have a tendency to re‐associate with template rDNA and form rRNA:rDNA hybrids that can inhibit rRNA transcription, impede replication fork and facilitate rDNA recombination. Here, we show that RECQ1 is enriched in the nucleolus, the factory of ribosome biogenesis. RECQ1‐depletion in HeLa and U2OS cells significantly reduces pre‐rRNA levels. Genetic rescue experiments indicate a helicase activity‐dependent function of RECQ1 in rRNA biogenesis. RECQ1 binds to the rDNA promoter, enhances RNA pol I recruitment to rDNA promoter and enhances rDNA promoter activity. Depletion of RECQ1 causes ribosomal stress, dysregulated mRNA translation and reduced global translation. Consistent with this disruption of ribosome biogenesis, RECQ1‐depleted cells display impaired growth and proliferation. Collectively, these results identify rDNA as a novel target of RECQ1 helicase to maintain rRNA transcription and ribogenesis.Support or Funding InformationThis work was funded by the NIGMS/NIH grant SC1GM093999 to Sudha Sharma. We also acknowledge support from the NIMHD/NIH award number G12MD007597 and NIA/NIH award number 1R25 AG047843‐02. We thank Drs. Ritu Chaudhary, Xiaoling Li and Ashish Lal (NCI) for their help and support.
Characterization of the Interaction between Mps3 and Cdc5 in Saccharomyces cerevisiae
Authors: Antoniacci Lisa , Breymeier Corinne 
Published: November 2023

Mps3 is a nuclear envelope protein identified in Saccharomyces cerevisiae and is involved in several chromosome functions such as sister chromatid cohesion, DNA damage repair, and telomere clustering. Mps3 is hypothesized to be involved in many of these processes through its association with several different chromatin associated proteins (Elg1, Htz1, Ctf18, Eco1). Recently our lab identified a physical interaction between Mps3 and the polo‐like kinase Cdc5. Cdc5 functions in regulation of many essential cell pathways including several that Mps3 also functions. Here we characterize the physical, genetic, and functional interaction of Mps3 and Cdc5.Support or Funding InformationSchierling Endowed Chair Marywood University
Development of Novel Nickase Enzymes for Targeted Gene Modification in Higher Eukaryotes
Authors: Klipp Alexa , Antoine Michael , Kaplan Emily , Robins Lori , Turner Betsy , Hillers Kenneth 
Published: November 2023

In the developing field of genomic engineering, several strategies for targeted, specific genetic modification have recently been developed. Many current methods, such as CRISPR, utilize double strand breaks (DSBs) in DNA to initiate an innate repair process called homology directed repair (HDR). DSBs can also trigger another repair pathway, non‐homologous end joining (NHEJ). However, NHEJ has a lower fidelity and can lead to harmful mutations or translocations. Single strand breaks (SSBs) in DNA stimulate HDR while avoiding the NHEJ pathway. Nickases are enzymes that are capable of inducing SSBs in DNA via a single cut, or nick, in one strand of DNA. Therefore, nickases are ideal for developing targeted and specific genetic modifications.LAGLIDLAG homing endonuclease (LHEs) are monomeric enzymes with long recognition sequences; these properties make then good candidates for conversion to nickases. In collaboration with Dr. Lori Robins (University of Washington Bothell), we are carrying out a structure‐guided mutagenesis of various LHEs with the goal of converting them into functional nickases. Two LHEs, LtrWI and SmaMI, have been chosen to mutate into functional nickases. Site‐directed mutagenesis of these LHE genes is aimed at altering single amino acids that are believed to catalyze DNA hydrolysis in one of the active sites. By inactivating one of the active sites in the monomer and allowing the other active site to remain intact, this produces a semi‐functional enzyme capable of nicking DNA at specific sequences.Through PCR‐directed mutagenesis of specific active‐site lysine residues, we have successfully produced LtrWI and SmaMI mutants with predominately nicking activity. Current efforts seek to extend these observations to additional LHEs, through identification and mutagenesis of homologous lysine residues in the cognate active sites.Support or Funding InformationThank you to the Cal Poly Student based fee and Chevron Applied Biotechnology Research Program for funding; Ellen Calcagno for administrative assistance and Michael Black for technical assistance; members of the Hillers/Turner lab for helpful discussion and support; Conor Beck for diagrammatic interpretation of data.
Differential Requirements for Nonhomologous End‐joining (NHEJ) Pathway Genes in DNA Repair and Telomere Stability
Authors: Rodriguez Nestor Daniel , Lewis Kevin 
Published: November 2023

Deoxyribonucleic acid (DNA) is constantly being damaged due to environmental chemicals, free radicals, and radiation. These damaging agents cause varying effects from phosphate, sugar, or base modification to double‐stranded breaks (DSBs) in the DNA. If DSBs are left untreated in a cell, the consequences can be grave, potentially causing loss of function, mutations, and cell death. In order to repair DSBs, two independent pathways are used, homologous recombination (HR) and nonhomologous end‐joining (NHEJ). NHEJ is the preferred pathway in humans but secondary in the yeast Saccharomyces cerevisiae. The NHEJ pathway involves three major complexes in yeast cells: the Ku complex, Mrx complex, and DNA ligase IV complex. Each complex has a specific function in repairing DSBs.The Ku complex is a heterodimer found in both humans and yeast (Ku70 and Ku80 for humans, Yku70 and Yku80 in yeast), which is responsible for first binding to the broken ends of the DNA and protecting the ends from degradation. A mutation in the Ku70/Ku80 complex genes causes severe combined immunodeficiency (SCID) in humans and animals. The Mrx complex is composed of Mre11, Rad50, and Xrs2 in yeast and binds to the DNA ends to form an end‐bridging complex. The last complex is the DNA ligase IV complex that is composed of Dnl4 and Lif1. This complex performs the last step in NHEJ whose function is to ligate the two DNA ends together.Many past studies have studied NHEJ repair using plasmid end‐joining assays. In this method, cells are transformed with a plasmid containing a DSB that cannot be repaired by HR and the number of transformants with plasmids recircularized by NHEJ is determined. Previous reports observed that repair in yku, mrx and dnl4 mutants is reduced by 10–30 fold and suggested that the three major complexes are approximately equally important in the pathway. Using high efficiency transformation methods and early stationary phase cell cultures, we have been able to detect strong differences in the requirements for each complex. These results and additional experiments suggest that cell cycle phase distribution within cell cultures affects repair complex requirements.
Unstable Telomere Cap Structures in Saccharomyces cerevisiae yku70 Mutants Cause Altered Cell Cycle Phase Distributions
Authors: Posey Tressie , Lewis L Kevin 
Published: November 2023

Eukaryotic cells keep their DNA compartmentalized within nuclei, but this does not guard the DNA from damage. There are a number of environmental and intracellular factors such as ionizing radiation, e.g., X‐rays and gamma rays, UV radiation, nucleases, DNA modifying agents, and free radicals that can damage the DNA. One of the most detrimental lesions that can occur in the DNA is a double‐strand break (DSB), where both strands of the double helix are fractured. This damage is the most difficult to repair and holds a number of consequences for the cell's viability. In humans, the main repair pathway for DSBs is NHEJ, or the Nonhomologous End‐Joining pathway. This repair pathway primarily uses three protein complexes to directly rejoin the two broken ends. In Saccharomyces cerevisiae (budding yeast) these three complexes are Mrx (Mre11, Rad50, and Xrs2), DNA ligase IV (Dnl4 and Lif1), and the Ku complex (Yku70 and Yku80). The Ku complex binds to the broken ends first, protecting them from further degradation. Mrx then bridges the gap between the broken ends and DNA ligase IV covalently links the ends together.In this project, yeast yku70 cells, which exhibit a growth defect when grown at 37° C, were analyzed microscopically after growth at multiple temperatures (20° C, 30° C, and 37° C). Normally, yeast cells spend approximately the same proportion of time in G1, S, and G 2/M during log phase growth, but yku70 cells exhibit an increased number of G2/M cells. This phenomenon was not seen in other NHEJ mutants such as dnl4 cells. Further experiments involving the quantitation of cells with different nuclear morphologies and testing the role of DNA damage checkpoints will be presented.
DNA double‐strand break repair deficiency is associated with changes in cell cycling and cell morphology in Saccharomyces cerevisiae
Authors: Weis Monica , Lewis L Kevin 
Published: November 2023

Chromosomal DNA experiences a variety of different types of damage often and naturally. Considered potentially the most lethal of DNA lesions, double‐strand breaks (DSBs) involve a complete severance of both strands of DNA at some point in the chromosome. If not repaired, DSBs can lead to an increase in DNA mutations, chromosome loss events and ultimately loss in cell viability. In eukaryotes, DNA DSBs are repaired almost exclusively by two mechanisms, nonhomologous end‐joining (NHEJ) and homologous recombination (HR). NHEJ is the simpler of the two pathways, involving the direct rejoining of broken chromosome ends, while HR involves the use of a region of a homologous chromosome as a template for repair. RAD52 group proteins (Rad51, Rad52, Rad54, etc.) play an important role in the HR pathway. In Saccharomyces cerevisiae homologous recombination is considered the primary pathway for repair. Checkpoint proteins also play an important role in DSB repair by making sure that damage is repaired before a cell completes the cell cycle. If there is significant damage that requires attention, such as DSBs induced by chemicals or radiation, growth arrest is induced and the cells accumulate in G2 phase.Yeast rad52 mutants cannot repair DSBs by HR and have elevated levels of unrepaired DNA lesions. In the current project, we have observed that rad52 cell cultures have high levels of enlarged large‐budded cells, and that log phase cells spend 64 minutes of each 128 minute cell cycle in G2 phase. Similar results were seen with rad51, rad54, rad55, rad57, and rad59 mutants, but not rdh54 cells. Inactivation of 7 critical DNA damage checkpoint genes (RAD9, RAD17, RAD24, CHK1, DDC1, MRC1, and MEC3) in rad52 cells, creating double mutants, abolished the high large‐budded cell phenotype. It is likely that rad52 cells have elevated levels of unrepaired DNA lesions leading to constitutively activated damage‐responsive cell cycle checkpoints. Additional experiments involving analysis of changes in nuclear and cellular morphology will be presented.
Exploring the Role of ATP Hydrolysis in the RecA Protein via Stalled DNA Intermediates
Authors: Sowin Laura , Cox Michael M 
Published: November 2023

A central participant in the recombinational DNA‐repair system is the recombinase protein, RecA, which acts as a DNA pairing enzyme that is responsible for promoting an exchange of homologous DNA strands. Physical studies have revealed three DNA binding sites buried within the interior of the RecA filament. This current understanding of RecA is incomplete as it fails to explain how a four‐strand DNA exchange occurs between two duplex DNA fragments.The bacterial RecA homologue is unique in that it couples ATP‐hydrolysis with DNA strand exchange. ATP hydrolysis occurrs uniformly throughout the RecA filament, with only a small fraction of ATP used for filament disassembly. The mechanism by which the RecA filament utilizes the rest of ATP hydrolysis to perform DNA strand exchange reactions is not fully understood. My aim is to explore the hypothesis that the majority of ATP utilized by the reaction is hydrolyzed at binding sites located longitudinally along both the interior and exterior of the filament, providing the energy needed to rotate one segment of DNA around another.This will be explored by performing RecA mediated strand‐exchange reactions with a variety of linear, double stranded DNA substrates containing heterologous regions. When the RecA protein is run into a heterologous barrier, a stalled DNA‐protein intermediate molecule is the product. Formation of these intermediates has been obtained. Imaging with Electron Microscopy shows topological changes that offer fundamental insight into the universal mechanics of RecA‐mediated strand exchanges. Furthermore, evidence shows that introduction of the protein Uup modifies the structure of the intermediates, providing insight into Uup's function.
Effects of DNA Damage on Natural Transformation in Bacillus subtilis
Authors: Korry Benjamin Joseph , Belenky Peter A 
Published: November 2023

Horizontal gene transfer is an important mechanism that promotes rapid evolution in microbial communities. Understanding how environmental conditions impact horizontal gene transfer in microorganisms can identify factors that lead to the transfer of virulence and antibiotic resistance genes. Natural transformation is a form of horizontal gene transfer that allows bacteria to take up and incorporate extracellular double stranded DNA. Competence machinery utilized for the import of DNA is typically induced by cellular state or external stimuli. Following import, DNA is recombined into the genome in a RecA‐dependent fashion. Cellular stress has been shown to modulate natural transformation in various bacterial species by targeting either the competence or recombination machinery. Here we investigate the links between genotoxic stress and changes in the rate of natural transformation in naturally competent B. subtilis. We find that genotoxic agents have divergent impacts likely related to their effects on DNA replication and processing in bacteria. Traditional high‐throughput methods are applied to analyze transcriptional responses induced by genotoxic agents in order to link known mechanisms of natural transformation to phenotypic outcomes.Support or Funding Information“Monitoring the Impact of Climate Change Stressors on Horizontal Gene Transfer in Marine Microbial Populations” 2016. Rhode Island Research Alliance Collaborative Research Grant, Rhode Island Science & Technology Advisory Council
Stress Response and Natural Transformation in Vibrio cholerae
Authors: Rowan Aislinn D , Belenky Peter A 
Published: November 2023

Antimicrobial resistance is a major threat facing modern medicine, which is accelerated by various forms of horizontal gene transfer (HGT). A contributing problem is that antibiotics themselves, especially those inducing DNA damage and the SOS response, can actually increase the rates of transfer of resistance genes through conjugation and transduction. Evidence from other species indicates that DNA‐damaging stress may also have effects on natural transformation, a form of HGT in which extracellular DNA is promiscuously taken up and recombined into the genome and which may contribute to the spread of resistance genes. We are studying the effects of DNA‐damaging stress in Vibrio cholerae, an important human pathogen capable of both natural transformation and the SOS response. Here, we demonstrate that genotoxic agents can alter the rates of natural transformation in V. cholerae and explore the mechanisms by which these changes occur.Support or Funding Information“Monitoring the Impact of Climate Change Stressors on Horizontal Gene Transfer in Marine Microbial Populations.” 2016. Rhode Island Research Alliance Collaborative Research Grant, Rhode Island Science and Technology Advisory Council (STAC).Brown University Presidential Fellowship
Down‐Regulation of Ubiquitin‐Specific Protease 24 Regulates DNA Repair by Homologous Recombination in Lung Cancer Cells
Authors: Wang Shao‐An , Wang Yi‐Chang , Hung Jan‐Jong 
Published: November 2023

Homologous recombination is a type of genetic recombination in which nucleotide sequences are exchanged between two similar or identical molecules of DNA. It is most widely used by cells to accurately repair harmful breaks that occur on both strands of DNA, known as double‐strand breaks. Ubiquitin‐specific peptidases (USPs) belong to a large family of cysteine proteases and are deubiquitinating enzymes that specifically recognize and remove ubiquitin from proteins. USP disorders have been reported to be involved in many human diseases, such as neurodegenerative disorders, inflammation, and cancer progression. In this study, we want to elucidate the role of a novel deubiquitinating enzyme, ubiquitin‐specific peptidase 24 (USP24) in DNA damage repair. Our recent study showed that downregulation of USP24 increases cell cycle progression by enhancing the G1‐S transition. The molecular mechanism involves a decrease in the USP24 level, which reduces the expression of E2F4 and thus increases the G1/S transition. In this study, we found that USP24 knockdown enabled the cells to overcome CPT‐induced cell death was also reversed when E2F4 overexpression was performed after USP24 knockdown. Furthermore, RAD51 involved in DNA damage repair was also dramatically up‐regulated after USP24 knockdown and USP24 knockdown A549 cells formed more colonies than scramble knockdown group after DNA damaging agent CPT treatment. E2F4 overexpression can inhibit USP24 knockdown induced RAD51 promoter activity. Loss of transcription repressive effect of E2F4, RAD51 was up‐regulated and increased CPT resistance through promoting homologous recombination. Based on these results, E2F4 is one of the critical factors in USP24 knockdown induced genes upregulation, and E2F4 overexpression can partially reverse USP24 knockdown induced proliferation and CPT resistance. Taken together, we have proofed that USP24 plays important roles in DNA damage repair and drug resistance.Support or Funding InformationMOST‐103‐2320‐B‐006‐035‐MY3
Regulation of Interleukin 33 by Transforming Growth Factor‐β in Epithelia Cells, Fibroblasts and Keratinocytes
Authors: Woelfel Jennifer M , DenHaese Jason P , Gervasi Megan E , Ruvio Stacy 
Published: November 2023

Interleukin 33 (IL‐33), a member of the IL‐1 family of cytokines, plays a key role in inflammatory responses functioning as an alarmin, alerting the immune system to tissue damage. IL‐33 signaling molecules following damage, IL‐33 coordinates recruitment and activation of immune cells leading to the release of mediators such as VEGF, which drives angiogenesis and skin remodeling.I L‐33 is expressed in epithelial cells, stromal cells, and endothelial cells. High levels of IL‐33 as has been implicated in diseases such as asthma, inflammatory skin and rheumatological diseases. Preliminary studies have shown that the pluripotent cytokine Transforming Growth Factor‐β (TGF‐β) represses expression of IL‐33 in mouse and human mammary epithelial cells, human kidney and lung epithelial cells and human primary fibroblast in vitro. TGF‐β is a cytokine involved in a number of cellular responses including cell differentiation, growth inhibition, extracellular matrix sti mulation, immunosuppression, and immunomodulation. Our current studies revealed that repression of IL‐33 was dependent on the TGF‐β canonical pathway of signaling. Continuing studies will utilize mast cells to determine the biological implications of repression of IL‐33 by TGF‐β.Support or Funding InformationWork supported by the Department of Biology and Mathematics, D'Youville College.