bluuebunny/crossref_metadata_2025_split huggingface dataset:
Combinatorial Effects of PK11195 and 5‐Azacytidine as an Epigenetic Modulator For MPC1
Authors: Duran Daniel , O'Donnell Robert Watts 
Published: November 2023

According to the Warburg effect, cancer cells typically favor glycolysis over oxidative phosphorylation wherein only a few select molecules of pyruvate are oxidized. The mitochondrial pyruvate carrier, MPC, contributes significantly to proper cellular respiration and its under expression correlates strongly with the observed “fractional pyruvate oxidation.” The associated MPC1 gene is under expressed or even deleted across multiple tumor cell lines, contributing to their preference of glycolysis as an energy supplying mechanism. We hope to reverse the Warburg effect by enhancing MPC1 gene expression and thereby increase the potential for tumor cells to undergo oxidative phosphorylation following glycolysis. Use of the DNA methyltransferase inhibitor 5‐azacytidine may, through its role as an epigenetic modulator, contribute to enhanced expression or even re‐expression of the MPC1 gene. An additional drug, PK11195, was chosen for its role as a peripheral benzodiazepine receptor ligand to similarly observe it potential effect on MPC1 expression. The drug itself binds with nanomolar affinity to the PBR on the outer mitochondrial membrane and alters mitochondrial respiration. Through qRT‐PCR we have demonstrated an increased expression of MPC1 in the HTB‐4 cell line upon co‐treatment with PK11195 and 5‐azacytidine over either drug used alone. This suggests the cells are navigating from glycolysis to an oxidative pathway in which MPC1 is an integral part. Experiments are planned to verify whether or not the MPC‐1 gene is similarly up regulated in other tumor cell lines; furthermore, we will investigate if the treated cells show an increase in reactive oxygen species, indicating a transition to oxidative phosphorylation.Support or Funding InformationThe Geneseo Foundation and the SUNY Geneseo Biology Department.
Deciphering the Dynamics of UHRF1‐dependent DNA Methylation
Authors: Tiedemann Rochelle L. , Jones Peter A. , Rothbart Scott B. 
Published: November 2023

The epigenetic inheritance of DNA methylation patterning is a coordinated process involving UHRF1‐directed recruitment of DNMT1 to S‐phase chromatin, where the parental methylation patterns are copied on nascent DNA. As UHRF1 is now recognized as a key regulator of DNA methylation maintenance, it is considered as a promising therapeutic target for modulating the DNA methylation program in cancer and other diseases. UHRF1 itself is targeted to chromatin in a multivalent manner by the interconnected activities of three reader domains, which directs its ubiquitin ligase activity towards several lysine residues on histone H3 (a proposed docking site for DNMT1). While the mechanistic links between UHRF1 and DNA methylation maintenance are beginning to be unraveled, little is known about the dynamics of this process at epigenome‐scale resolution. To study the dynamics of UHRF1‐dependent DNA methylation, we engineered a human colorectal carcinoma cell line that expresses a doxycycline‐inducible shRNA targeting UHRF1. Importantly, this system is reversible, allowing us to model the effects of acute depletion of UHRF1 in these cells. Using this system, we are following UHRF1‐dependent DNA methylation dynamics using Illumina's Infinium Methylation EPIC Bead Array and layering this information with transcriptome, nucleosome occupancy, and histone modification dynamics. We are also querying the contributions of specific UHRF1 regulatory domains to the maintenance of epigenome integrity. Collectively, the results of this study are providing high‐resolution temporal snapshots of epigenetic programming (and reprogramming) facilitated by UHRF1 and are revealing how the epigenome responds to targeted inhibition of UHRF1 function.Support or Funding InformationNIH NCI R00 CA181343 to SBR; NIH NCI R01 CA082422, R01 CA170550 to PAJ
Epigenetic Regulation of Alternative Developmental Trajectories
Authors: Romney Amie Lynn T. , Podrabsky Jason E. 
Published: November 2023

The annual killifish, Austrofundulus limnaeus, is capable of entering into a state of metabolic and developmental dormancy during embryogenesis termed diapause. However, individuals may also follow an alternative phenotypic trajectory and instead “escape” entry into diapause and develop continuously until hatching. Early development along the two trajectories is morphologically indistinguishable, and gene regulatory mechanisms are currently unknown. Phenotype appears to be influenced by maternal provisioning based on the observation that young females produce predominately escape embryos and older females produce diapausing embryos. Additionally, the incubation temperature of embryos can override this maternal pattern and alter trajectory. Maternal provisioning is known in many vertebrates to direct early development prior to the initiation of embryonic gene transcription. Using RNA‐seq, we have generated transcriptomic profiles of messenger RNAs (mRNAs) during embryogenesis of A. limnaeus. Embryos that are destined for either the diapause or escape phenotypes have unique profiles of mRNA isoforms at fertilization; well before they are morphologically distinct. We hypothesize that maternally packaged gene products coordinate the cellular events that determine developmental trajectory in A. limnaeus. In addition, we propose the expression of environmentally responsive microRNAs during development that can override maternal provisioning. RNA‐seq data during early development, after the maternal‐to‐zygotic transition, suggest distinct profiles of microRNA expression that are phenotype specific. Our findings suggest maternal programming of diapause through the packaging of specific mRNAs as well as temperature induced microRNAs that target maternal transcripts allowing an escape from the diapause trajectory. This presents a novel example of maternal‐embryonic‐conflict in gene regulation that provides the embryo with alternatives in developmental trajectories in the face of fluctuating environmental patterns. These results will not only impact our understanding of genetic mechanisms that regulate entrance into diapause, but will also provide insight into epigenetic control of development.Support or Funding InformationNSF IOS 1354549
Differential Alternative Splicing in Clinical Strains of Cryptococcus neoformans
Authors: Merryman Mitch Carlton , McClelland Erin , Seipelt‐Thiemann Rebecca 
Published: November 2023

Alternative splicing allows eukaryotes to produce a larger number of proteins from a limited number of genes. Alternative splicing has also been associated with virulence in pathogenic eukaryotes, such as fungi like Cryptococcus neoformans. It is expected that clinical strains of C. neoformans will exhibit differing patterns of alternative splicing in virulence factor genes than less‐pathogenic laboratory strains of C. neoformans. If new isoforms of virulence factor genes could be identified in pathogenic strains, they could potentially be used as targets for new therapeutics for cryptococcosis. To more fully understand the differential patterns of alternative splicing in clinical strains and identify these potential therapeutic targets, mRNA from seven different clinical isolates and one laboratory strain, H99S, was isolated and analyzed using RNA sequencing. Initial bioinformatics analyses of the RNA sequencing data was undertaken using TopHat, Cufflinks, and Cuffdiff tools. These data will be compared to other models and tool sets including replicate multivariate analysis of transcript splicing (rMATS), Splicing Compass, Exon Quantitation Pipeline (EQP).Support or Funding InformationBiology Department, Middle Tennessee State UniversityURECA
Export of discarded, splicing intermediates provides insight into mRNA export
Authors: Zeng Yi , Staley Jonathan 
Published: November 2023

The precise removal of introns is essential for cell survival. To promote fidelity in nuclear pre‐‐mRNA splicing, the spliceosome rejects and discards suboptimal splicing substrates that have engaged the spliceosome. Although these discarded splicing substrates can be degraded in the nucleus, discarded splicing species, the 5′ exon and lariat intermediate, can be exported to the cytoplasm, as the discarded species can be translated and degraded by cytoplasmic nucleases. However, it is unknown how these suboptimal intermediates are exported.Here, we used single molecule RNA‐FISH in budding yeast to provide direct evidence that cells export suboptimal splicing intermediates, both the 5′ exon and lariat intermediate. We observed that the export of suboptimal splicing intermediates requires the nuclear basket and the general mRNA export receptor Mex67. Further, we found that two essential Mex67 adaptors, Yra1 and Nab2, are required for exporting suboptimal lariat intermediates, demonstrating that both mRNAs and lariat intermediates undergo a similar process to assemble export machinery. Surprisingly, we discovered that the export of lariat intermediates requires Mlp1, a factor implicated in retaining unspliced pre‐‐mRNA in the nucleus of budding yeast. Importantly, Mlp1 promoted the export of lariat intermediates through its interaction with Nab2. Together, our findings establish the importance of the mRNA export pathway in exporting discarded splicing intermediates and implicate a dual role for Mlp1 in not only retaining unspliced transcripts but also exporting partially spliced transcripts. We propose that this dual role of Mlp1 reflects a function in docking mRNAs at the nuclear basket during export.Support or Funding InformationNIH
Engineering of an Immunogenic pre‐Trans‐splicing RNA (iPTR) to Block Growth and Express a Glioblastoma Specific Epitope
Authors: Falotico Sarah C. , Sivetz Nicole , Nekrasov Peter , Hicks Martin J. 
Published: November 2023

The most common and lethal malignancy of the central nervous system (CNS) is glioblastoma multiforme (GBM). Due to the blood brain barrier and the relatively immunologically privileged status of the CNS, clinical strategies have not improved the standard of care. Epidermal growth factor receptor (EGFR), a type of tyrosine kinase receptor, has been found to be overexpressed in as much as 60% of GBM tumors. Upon binding of its cognate ligand, EGFR promotes tumor growth and proliferation. The cytokine, interleukin‐13 receptor alpha variant 2 (IL13Rα2) is an isoform selectively expressed in GBM. It is highly immunogenic, attracting cytotoxic T‐cells to the tumor microenvironment. Therapeutically, this cytokine has the potential reactivate the immune system toward GBM. In the current study, we have designed and cloned an immunogenic pre‐trans splicing RNA molecule (iPTR) against EGFR. The iPTR has the potential to synergistically block growth and reactivate the immune system toward the tumor microenvironment by generation of a GBM‐specific epitope. In a GBM tissue culture model, we use RT‐PCR to detect changes in IL13Rα2 peptide expression. In addition, we use western blot and ELISA to measure changes in EGFR protein localization. Genetic delivery of our highly immunogenic IL13Rα2 peptide using the iPTR has the potential to redirect the immune system to recognize and induce apoptosis in GBM cells.Support or Funding InformationMonmouth University School of Science Research, Bristol‐Meyers Squibb, Johnson and Johnson, and the Independent College Fund of New Jersey
A Genetic Interaction Between DED1 and HAT1 in Saccharomyces cerevisiae Suggests a Role for Hat1p in mRNA storage
Authors: Hilliker Angie , Kindsfather Audrey , Winters Aidan , Rothbard Nicholas , Robins Sean , Fronek Lisa 
Published: November 2023

Ded1p is an essential translation initiation factor that interacts with and is modulated by eIF4F. Ded1p also promotes the assembly and disassembly of stress granules, which contain repressed mRNAs and translation initiation factors. As Ded1p affects both mRNA storage and translation initiation, regulation of Ded1p's function may affect whether mRNAs are stored or translated. To identify regulators of Ded1p in Saccharomyces cerevisiae, we screened for over‐expression suppressors of the severe growth defect conferred by high levels of Ded1p. We found that over‐expression of HAT1, a lysine acetyltransferase that acetylates free histone H4, can suppress this DED1 growth defect. Mutations within the catalytic core of Hat1p substantially reduce this suppression, suggesting that acetyltransferase activity is required for the genetic interaction. We demonstrate that HAT1 antagonizes the accumulation of P‐bodies under short‐term stresses. Under sustained glucose deprivation during diauxic shift and stationary phase, strains lacking HAT1 form more stress granules and have a survival advantage, revealing a new function of Hat1p. Given this genetic connection between HAT1 and DED1, we screened for other lysine acetyltransferases and deacetylases that have a genetic interaction with DED1, identifying several more of these post‐translational modifiers as possible regulators of mRNA storage and/or translation. These results demonstrate new connections between lysine acetylation and the control of cytoplasmic mRNA localization.Support or Funding InformationJeffress Memorial Trust; University of Richmond
PAF53 is essential in mammalian cells: CRISPR/Cas9 fails to eliminate PAF53 expression.
Authors: Rothblum Lawrence , Chang Eugenie , Rothblum Katrina 
Published: November 2023

When mammalian cells are nutrient and/or growth factor deprived, exposed to inhibitors of protein synthesis, stressed by heat shock or grown to confluence, rDNA transcription is essentially shut off. Various mechanisms are available to accomplish this downshift in ribosome biogenesis. Muramatsu's laboratory first demonstrated that mammalian PAF53 was essential for specific rDNA transcription and that PAF53 levels were regulated in response to growth factorsWhile S. cerevisae A49, the homologue of PAF53, is not essential for viability, deletion of yA49 results in colonies that grow at 6% of the wild type rate at 25C. This included the genes encoding Rrn3, PAF49 and PAF53. Experiments described by Wang et al. identified PAF53 as a gene “essential for optimal proliferation “. However, they did not discriminate genes essential for viability. Hence, in order to resolve this question, we designed a series of experiments to determine if PAF53 was essential for cell survival. We set out to delete the gene product from mammalian cells using CRISPR/CAS9 technology.MethodsWe designed several small guide RNAs (sgRNA) for human PAF53 and cloned them into lentiCRISPR v2. Human 293 cells were transfected with the viral DNA. In some experiments, the cells were cotransfected in parallel with plasmids encoding tagged forms of mouse PAF53. After treating the transfected cells with puromycin (to select for the lentiCRISPR backbone), cells were cloned and analyzed by western blots for PAF53 expression. Genomic DNA was amplified across the “CRISPRd” exon, cloned and sequenced to identify mutated PAF53 genes.ResultsWe obtained cell lines in which the endogenous PAF53 gene was “knocked out” only when we rescued with FLAF‐PAF53. DNA sequencing demonstrated that in the absence of ectopic PAF53 expression, cells demonstrated unique means of surviving; including recombination or the utilization of alternative reading frames.Conclusions
PAF53 is an essential protein. We did not obtain any clones that did not express the protein.
PAF53 expression requires both genes. We never observed a clone in which one gene is expressed, unless there was also ectopic expression In the absence of ectopic gene expression, the gene products of both endogenous genes were expressed, irrespective of whether they were partially mutant proteins or not.
Role of cystines in the structure and function of the RNA‐binding protein LARP6
Authors: Peña Eleuterio L , Lane LaPatience F , Salas Eliseo , Castro Jose Miguel , Lewis Karen A. 
Published: November 2023

La‐related protein 6, formerly known as Acheron, is an RNA binding protein associated with the translation of mRNAs that encode type‐I collagen. This protein belongs to a superfamily of RNA‐binding proteins known as the La‐related proteins, which share a conserved La Module consisting of a La‐Motif (LaM) and RNA Recognition Motif (RRM). Although extensive research on the mechanism of binding by LARP6 is being performed, very little is known about the physiological conditions that induce the protein to bind to its ligand. A multiple sequence alignment of the LARP6 proteins from vertebrates identified a fully conserved cysteine near the RRM (position C258 in the human protein), and two semi‐conserved cysteines near the C‐terminal region (positions C378 and C490 in human LARP6). To determine if disulfide bonds were present in the protein, the reducing agent dithiothreitol (DTT), was added during purification. In the presence of DTT, LARP6 eluted from a size exclusion column at a volume that is indicative of an increase in the hydrodynamic radius of the protein. Therefore, we hypothesize that at least one pair of the conserved cysteine residues is involved in intramolecular disulfide bonding. Using the human LARP6 (HsLARP6) as a model, the location of the structurally relevant disulfide bonds is being identified using a combination of site‐directed mutagenesis, chemical modification, and mass spectrometry. By mutating each cysteine to serine, we can monitor changes in protein stability through limited proteolysis and chemical and thermal denaturation assays. Additionally, the effects of cysteine mutagenesis on the biochemical activity of LARP6 will be assessed using electrophoretic mobility shift assays against the previously identified stem‐loop structure in the 5′UTR of the COL1a1 mRNA. The identification of the existence and function of disulfide bonds within LARP6 will provide further understanding of the physiological function of this posttranscriptional regulator.Support or Funding InformationFunding provided by the Texas State University Department of Chemistry and Biochemistry; the National Science Foundation through the Texas State University CheMIE Summer Research Experience for Undergraduates (NSF 1156579 to A.K.), the National Institutes of Health through the South Texas Doctoral Bridge Program (GM102783 to J.M.C., E.S. & ELP), the Student Undergraduate Research Fund through the Texas State University Honors College (to E.L.P.), and the National Institutes of Health grant (GM119096 to K.A.L).
Sphingosine Kinase‐1 inhibitor, PF‐543 ameliorates Bronchopulmonary Dysplasia in a neonatal murine model
Authors: Harijith Anantha , Ha Alison W , Ebenezer David L , Fu Panfeng , Berdyshev Evgeny , Kanteti Prasad , Natarajan Viswanathan 
Published: November 2023

BackgroundWe have earlier demonstrated that genetic knock out of sphingosine kinase1 (SphK1) ameliorated hyperoxia (75%) induced lung injury in a neonatal murine model of bronchopulmonary dysplasia (BPD). SphK 1 catalyzes the formation of sphingosine‐1‐phosphate (S1P), which acts through G protein coupled receptors S1P1–5. Exposure of neonatal mice pups to hyperoxia enhanced SphK1 expression and S1P levels and promoted formation of reactive oxygen species (ROS) in lungs. In vitro, exposure of human lung microvascular endothelial cells (HLMVECs) to hyperoxia (3–24 h) stimulated SphK1 activity, S1P generation and ROS production via p47phox, which was blocked by PF‐543, a potent and specific SphK1 inhibitor.HypothesisPF‐543 protects neonatal lungs from hyperoxia‐induced BPD.MethodsNeonatal mice were administered PF‐543 (5 mg/kg/dose, intraperitoneal injection) starting on postnatal day 3 (PN3) and exposed to normoxia or hyperoxia (75% O2) for 7 days. Mice received three additional doses of PF‐543, which were administered on alternate days and mice were sacrificed on PN 11. Broncho alveolar lavage (BAL) fluid and lung tissue were collected, and markers of inflammation and lung histology were evaluated. Plasma S1P levels were measured to determine the efficacy of PF‐543 in inhibiting SphK1.ResultsSphK1 inhibition by PF‐543 resulted in amelioration of hyperoxia‐induced BPD as evidenced by improved alveolarization characterized by decreased mean linear intercept compared to controls. Also, neonatal pups that received PF‐543 showed decreased infiltration of inflammatory cells in alveolar space, lower levels of protein and ROS in BAL fluid compared to controls. Administration of PF543 was accompanied by reduced plasma S1P levels.ConclusionsInhibition of SphK1 activity by, PF‐543 significantly reduced hyperoxia‐induced lung inflammation and BPD in newborn mice. These results suggest that PF‐543 can serve as a potential therapeutic drug to treat BPD.Support or Funding InformationThis work was supported by the University of Illinois at Chicago (UIC) Proof of Concept grant to A.H and by the National Institutes of Health Grant P01 HL98050 to V.N.
Sphingosine‐1‐phosphate as an angiocrine mediator in type II cell mediated alveoli epithelial repair following lung injury
Authors: Chen Qian , Huang Longshuang , Fu Panfeng , Rehman Jalees , Malik Asrar , Natarajan Viswanathan , Liu Yuru 
Published: November 2023

The alveolar barrier is continuously exposed to pathogens and is highly susceptible to inflammation and injury. Hence, orderly and exquisitely regulated repair of injured alveoli is essential, but the underlying mechanisms are not fully understood. Lung alveolar epithelium is composed of Type I and Type II cells. While Type II cells occupy only 5% of the surface area, they act as progenitor cells by self‐renewal and differentiation into Type I cells to repair the injured alveolar barrier. Type I cells occupy 95% of the surface area and enable gas exchange and are juxtaposed to the lung microvascular endothelium. Lung microvascular ECs comprising up to 40% of the lung cells, may play a major (but as yet unappreciated) role in influencing the regeneration of alveolar epithelium. Using genetically modified mice in which generation of Sphingosine‐1‐phosphate (S1P) specifically in endothelial cells was blocked [(through deletion of sphingosine kinase 1 (SphK1)], we have found evidence that endothelial‐derived S1P participated in repairing of the alveolar epithelium. After acute lung injury were induced in these mutant mice by i.t. Pseudomonas aeruginosa, Type II cells were abnormal in that they showed higher proliferation and these cells failed to show reduced expression of the Type II cell marker Sp‐C during the phase of Type II to Type I transition, suggesting a failure to differentiate into Type I cells. We also found the expression of S1P receptor S1PR2, which is a G protein‐coupled receptor, was induced on Type II cells post injury and preceding alveolar repair. Our results indicate that lung microvessel ECs release S1P after injury and activate receptor S1PR2 on Type II alveolar epithelial cells to initiate Type II to Type I cell transition required for alveolar repair.Support or Funding InformationNIH National Heart, Lung, and Blood Institute Grant R01HL105947.