bluuebunny/crossref_metadata_2025_split huggingface dataset:
Aberrant Activation of the Kynurenine Pathway in Gliomas Increases Polymerase Kappa Expression and Promotes Genomic Instability and Chemoresistance
Authors: Bostian April , Maddukuri Leena , Reed Megan , Savenka Tatsiana , Hartman Jessica , Davis Lauren , Pouncey Dakota , Miller Grover , Eoff Robert 
Published: November 2023

Glioblastoma multiforme (GBM) is the most malignant primary brain tumor and has a median survival time of 12 months, due in large part to poor responses to chemo‐ and radio‐therapy. These tumors exhibit high levels of DNA damage response activation before treatment and this likely plays a role in the inherent chemo‐ and radio‐resistance of GBM. Y‐family polymerases aid cells in bypassing genotoxic damage, but they are mis‐regulated in many cancers. Over‐expression of the Y‐family enzyme human polymerase kappa (hpol κ), which is up‐regulated in GBM patients, has been linked to an increase in genomic instability. The factors promoting over‐expression of hpol κ in GBM are unknown, but previous work implicates the aryl hydrocarbon receptor (AhR) in the regulation of hpol k levels. Up‐regulation of the AhR pathway through aberrant production of the endogenous ligand kynurenine is associated with malignant progression and shorter survival in glioma patients. We tested the hypothesis that the AhR signaling pathway transcriptionally regulates hpol κ levels in gliomas using in vitro cell culture models. Exposure to the AhR agonist 3‐methylcholanthrene increases hpol κ protein and mRNA levels 2–3‐fold in GBM cells. Furthermore, when the enzyme tryptophan 2,3‐dioxygenase (TDO), which is responsible for aberrant kynurenine production in gliomas, is inhibited by the small molecule 680C91, hpol κ expression decreased 20–60% in three GBM cell lines. We then examined the idea that stimulation of hpol k expression by the AhR pathway contributes to the inherent genomic instability seen in gliomas. Supporting our hypothesis, TDO inhibition leads to a decrease in spontaneous micronuclei formation in three GBM cell lines (33.6% DMSO to 12.9% 10 μM 680C91 in T98G cells; 12.3% DMSO to 8.7% 10 μM 680C91 in U87MG; and 25.3% DMSO to 20.1% 10 μM 680C91 in A172 cells). Additionally, treatment with an AhR antagonist also decreases micronuclei formation in T98G cells (33.6% DMSO to 17.2% 10 μM CH‐223191), as does knock down of hpol κ expression (25.5% for scr siRNA to 19.7% hpol κ). Furthermore, we performed epistasis assays which supported our hypothesis that TDO activity, AhR signaling, and hpol κ expression act in the same pathway to promote genomic instability in gliomas. Moreover, we found that TDO inhibition also significantly increases sensitivity of the T98G cells to various concentrations (10 – 100 μM) of cisplatin. Our study reveals how activation of AhR signaling could impact replication dynamics in gliomas and may provide insight into the mechanisms leading to the genomic instability and chemoresistance often inherent to this disease.Support or Funding InformationThis work was supported by NIH grant CA 183895 (to R.L.E.).
Identifying Y‐family DNA Polymerase Inhibitors to Improve Anti‐Cancer Drugs
Authors: Maddukuri Leena , Zafar Maroof Khan , Penthala Narsimha , Crooks Peter A , Eoff Robert L 
Published: November 2023

Replication stress is a hallmark of cancer and many of the current chemo‐ and radio‐therapies act to limit tumor growth primarily though the induction of DNA damage in order to block replication. Translesion DNA polymerases (TLS pols), such as the Y‐family members pols h, k, i and Rev1, can attenuate the effects of genotoxic anti‐cancer drugs by facilitating bypass of otherwise cytotoxic DNA damage. Increased levels of hpol h expression has been implicated in platinum‐based drug resistance in ovarian cancer stem cells, as well as in patients suffering from metastatic stomach cancer. We have screened a series of N‐substituted indole thiobarbituric acid (ITBA) derivatives for inhibition of pol h activity using a fluorescence‐based activity assay. From these assays, we have identified several ITBA molecules that inhibit hpol h with low micromolar IC50 values. Ovarian cancer cells were co‐treated with the most potent hpol h inhibitors and cisplatin (CDDP) to determine if the combination treatment produced a synergistic effect on cell viability. Treating cells with a combination of PNR‐7‐02 and CDDP decreased OVCAR3 ovarian cancer cell survival in a synergistic fashion (combination index value of 0.40). As expected, transient knock‐down of hpol h sensitizes OVCAR3 cells to CDDP relative to control siRNA. Combining hpol h knock‐down with PNR‐7‐02 does not further sensitize OVCAR3 cells to CDDP, which indicates that both treatments act through the same pathway to influence cell viability in response to CDDP. This suggests that PNR‐7‐02 may specifically inhibit hpol h in cells. In addition, flow cytometry analysis of cells treated with CDDP and PNR‐7‐02 revealed enrichment of cells in G2/M phase compared to cells treated with CDDP alone. From this data, it appears that combining TLS pol inhibitors with CDDP has the potential to impede ovarian cancer cell growth by inhibiting hpol h‐catalyzed bypass of CDDP‐induced DNA damage and blocking exit from the G2/M phase of the cell cycle.Support or Funding Information(Supported by NIH grants CA183895 to RLE and CA140409 to PAC. This research was also supported by a grant from the Arkansas Breast Cancer Research Program, the UAMS CTSA and a Seeds of Science Award from the Winthrop P. Rockefeller Cancer Institute).
Effect of Inflammatory Cytokines on DNA Methylation and Demethylation
Authors: Winfield Jacquay , Esbitt Alexis , Seutter Sara F , Desai Biren , Abdo Mohamed , Vasconez Megan , Laidlaw William , Green Kevan , Shamseddin Seyed M , Borghaei Ruth C 
Published: November 2023

ObjectiveTo determine the effects of inflammatory cytokines and mediators on expression of DNMT and TET enzymes in human gingival fibroblasts isolated from patients with periodontitis as well as normal and transformed cell lines.MethodsExpression levels of DNA methylatransferase (DNMT1 and 3a) and demethylase (TET1) were examined by RT‐PCR in cells treated for up to 24 hours with IL‐1, IL‐6 or prostaglandin E2. The role of PGE2 in IL‐1 suppression of DNMT3a expression was assessed by treating cells with IL‐1 in the presence and absence of NS‐398. Global levels of DNA methylation following treatment with IL‐1 were measured in an ELISA‐like assay.ResultsTreatment of HGF with IL‐1 caused a three‐fold increase in levels of DNMT1 mRNA over 24 hours, but a two‐fold decrease in DNMT3a mRNA in the same samples. Global levels of 5mC were increased in HGF treated with IL‐1 for 72 hours relative to untreated controls. PGE2 caused a dose‐dependent decrease in levels of DNMT3a similar to IL‐1, but also slightly decreased levels of DNMT1.ConclusionThese data are consistent with the hypothesis that exposure to inflammatory cytokines or PGE2 alters levels and/or activity of enzymes involved in DNA methylation, resulting in global and possibly gene‐specific changes which can lead to changes in cellular characteristics over time. Further experiments will be aimed at testing the effects of these cytokines on expression of DNMT and TET enzymes in normal and transformed cell lines, and at elucidating mechanisms involved in cytokine regulation of these enzymes.Support or Funding InformationThis work has been supported by the Philadelphia College of Osteopathic Medicine Division of Research.
Sphingosine kinase 2 Generated Nuclear S1P is an Epigenetic Co‐Regulator of Pseudomonas aeruginosa‐Induced Lung Inflammation
Authors: Ebenezer David Lenin , Fu Panfeng , Berdyshev Evgeny , Natarajan Viswanathan 
Published: November 2023

Pseudomonas aeruginosa (PA), a gram‐negative, opportunistic, aerobic bacilli is associated with rapid deterioration of the cystic fibrotic lung and is the significant cause of pneumonia in immunocompromised and mechanical ventilated patients. A distinctive feature of P. aeruginosa lung infection is the incessant inflammation‐driven respiratory failure that contributes to increased morbidity and mortality. Our research aims to define the epigenetic role of Sphingolipids in regulating immunological events in P. aeruginosa‐induced lung inflammation that has remained undefined so far. Sphingosine‐1‐Phosphate (S1P), is a bioactive sphingolipid that plays a key role in regulating signaling pathways modulating inflammatory responses in cells. Sphingosine Kinase (SphK) 1 & 2 generate intracellular S1P and Sphingosine‐1‐Phosphate Lyase (S1PL) and S1P Phosphatase (SPP) catalyze its degradation. Our murine model studies, as evident from decreased levels of inflammatory cytokines, proteins and cell counts in bronchoalveolar lavage and neutrophil migration to the alveolar space, suggest that knocking out SphK2, but not SphK1, offers protection against PA‐induced lung inflammation. PA infection also induced phosphorylation of SphK2 and subsequent translocation to the nucleus in mouse lung tissues and mouse lung epithelial cell line (MLE‐12). PA‐induced SphK2 phosphorylation resulted in increased acetylation of Histone H3 and H4 that correlated with increased IL‐6 gene expression at mRNA and protein levels. Moreover, PA infection decreased Histone Deacetylase (HDAC) activity in a time dependent manner in mouse alveolar type II cells and we validated SphK2 association with HDAC 1 & 2 by co‐immuno precipitation with nuclear HDAC repressor complex proteins. Nuclear fractions isolated from MLE‐12 cells exhibited significant levels of sphingosine and ceramide and stimulation with PA enhanced nuclear S1P in the presence of S1P lyase inhibitor, 4‐deoxypyridoxine. Interaction of sphingosine and S1P with HDAC1 and 2 in MLE‐12 nuclear fraction was confirmed by bio‐conjugate technique using biotin tagged sphingosine and S1P. Furthermore, using MLE‐12 nuclear fraction, we were able to demonstrate, that inhibition of nuclear HDAC activity by S1P is as potent as the inhibition obtained using Trichostatin A (TSA), a HDAC inhibitor. To examine the therapeutic potential of targeting SphK2‐S1P signaling axis in the treatment of PA‐induced lung inflammation, we evaluated ABC 294640, a specific SphK2 inhibitor in mice and observed reduction in PA‐induced inflammation. Taken together, these results emphasize the novel role of nuclear S1P in PA‐induced lung inflammation and targeting SphK2 could be a therapeutic option to treat bacterial infection of the lung.Support or Funding InformationThis work is supported by NIH/HLBI P01 HL98050 to VN.
Characterization of MarR family transcriptional regulator (BTH_I0021) from Burkholderia thailandensis
Authors: SABRIN AFSANA , Gupta Ashish , Grove Anne 
Published: November 2023

Burkholderia thailandensis, a soil dwelling bacterium, is a Gram‐negative bacillus, which is structurally similar to pathogenic species such as Burkholderia pseudomallei but a much less virulent species. B. thailandensis encodes nine Multiple antibiotic resistance Regulator (MarR) homologs, which act as transcriptional regulators of multiple genes, some of which may be involved in virulence and pathogenesis. The genomic locus of one of the MarR homologs, BTH_I0021 (BtMarR), predicts that it regulates the expression of both an EmrB/QacA family drug resistance transporter (BTH_I0022) and a Resistance Nodulation Division (RND) efflux pump (BTH_I0019). Increased expression of EmrB/QacA transporter and RND efflux systems have been associated with enhanced resistance to a wide variety of antimicrobials. Using Electrophoretic Mobility Shift Assays we have shown that BtMarR binds to the intergenic DNA of its own gene and a divergently oriented gene encoding a conserved hypothetical protein (BTH_I0020) of unknown subcellular localization. Upon treatment with inorganic and organic oxidants, SDS PAGE revealed the formation of reversible oligomeric species of BtMarR. DNA binding is attenuated by oxidized protein suggesting structural changes due to disulfide bond formation between redox‐active cysteines. PAR (4‐(2‐Pyridylazo) resorcinol) assay illustrated that this protein binds divalent metals such as zinc. This raises the possibility that BtMarR controls gene expression in response to oxidative stress via cysteine oxidation and by metal binding. Attenuation of DNA binding in presence of oxidants may lead to increased expression of efflux systems. Since oxidative stress is an important host defense mechanism, this study should shed light on the understanding of adaptive responses of Burkholderia strains towards the oxidative burst that characterizes the initial host response to bacterial infection.Support or Funding InformationThis work was supported by National Institutes of Health grant 1R15GM107825 (to Anne Grove)
Antitumor DNA‐Damaging C‐1748 is a New Inhibitor of Autophagy that Triggers Apoptosis in Human Pancreatic Cancer Cell Lines
Authors: Borowa‐Mazgaj Barbara , Skwarska Anna , Augustin Ewa , Radomińska‐Pandya Anna , Mazerska Zofia 
Published: November 2023

Despite the enormous progress that has been made over the past decades in diagnosis, treatment and prevention of many types of tumors, survival rates in pancreatic cancer still remain poor. Pancreatic cancer is one of the most malignant and chemoresistant tumors and the profound mechanism supporting these phenomena is the constitutively activated prosurvival autophagy. The antitumor 1‐nitroacridine derivative C‐1748 (9‐(2′‐hydroxyethylamino)‐4‐methyl‐1‐nitroacridine) belongs to a new set of DNA‐damaging agents developed in our laboratory and is currently ready for phase I clinical trials. Here, we have uncovered a new role for C‐1748 as a potent inhibitor of autophagy in four tested pancreatic cancer cell lines: Panc‐1, MiaPaCa‐2, BxPC‐3 and AsPC‐1. C‐1748 treatment at 0.1 – 1 μM significantly reduced the typical hallmarks of autophagy, such as conversion of soluble LC3‐I protein to its lipid‐bound LC3‐II isoform or development of acidic vesicular organelles (AVOs), and importantly shifted pancreatic cancer cells into apoptotic cell death. Co‐treatment with C‐1748 and wortmannin, an early‐stage autophagy inhibitor, had a synergistic effect on autophagy inhibition and potentiated C‐1748‐induced apoptosis. In particular, compared with exposure to C‐1748 alone, the combination treatment increased time‐ and dose‐dependent cytotoxic activity of C‐1748 with the most profound effect observed in BxPC‐3 cells. Moreover, along with decrease in autophagy, co‐treatment with wortmannin significantly elevated dysfunction of mitochondria, as manifested by a drop in mitochondrial membrane potential (ΔΨm), increased caspase‐3 activation, PARP cleavage, DNA fragmentation into apoptotic bodies and accumulation of lipid droplets arising due to the interruption of autophagy. In all studied cell lines, the level of apoptotic cells after dual exposure with C‐1748 and wortmannin increased by about 20% as compared to C‐1748 alone. Surprisingly, simultaneous exposure of pancreatic cancer cells to C‐1748 in combination with the late‐stage autophagy inhibitor, chloroquine, neither significantly increased the cytotoxic activity of C‐1748 nor ameliorated its apoptosis‐inducing potential. The synergistic effect of C‐1748 and wortmannin on the inhibition of autophagy and lack of such effect when C‐1748 was co‐administered with chlroquine, suggests that C‐1748, in addition to its DNA‐damaging properties may also affects early stages of autophagy. Further studies are undergoing to uncover the role of C‐1748 as a new inhibitor of autophagy, which is especially effective against pancreatic cancer.Support or Funding InformationResearch Grant from the Polish Ministry of Science and Higher Education NR 13‐0133‐10/2011 to BB‐M, AS, EA, ZM; DoD‐W81XWH1110795 to AR‐P and UAMS TRI ABCRP funds to AR‐P
Cholesterol‐Mediated Degradation of 7‐Dehydrocholesterol Reductase Switches the Balance from Cholesterol to Vitamin D Synthesis
Authors: Sharpe Laura Jane , Prabhu Anika Vinayak , Luu Winnie , Brown Andrew John 
Published: November 2023

Cholesterol is essential for normal embryogenesis, but in excess, it can be detrimental to human health. To achieve balanced cholesterol levels, enzymes involved in its synthesis possess many layers of regulation. Here, we investigated the post‐translational regulation of 7‐dehydrocholesterol reductase (DHCR7), a terminal enzyme of cholesterol synthesis, converting 7‐dehydrocholesterol to cholesterol. In the absence of functional DHCR7, accumulation of 7‐dehydrocholesterol and a lack of cholesterol production leads to the devastating developmental disorder, Smith‐Lemli‐Opitz Syndrome (SLOS). We found that statin treatment can ameliorate the low DHCR7 expression observed with common SLOS mutants. Furthermore, we show that DHCR7 protein is rapidly turned over and this degradation is induced by the cholesterol product itself, resulting in decreased DHCR7 protein levels and activity. The loss of enzymatic activity results in the accumulation of the substrate 7‐dehydrocholesterol, which in turn leads to an increased production of vitamin D. Thus, DHCR7 is an important enzymatic switch between cholesterol and vitamin D synthesis.Support or Funding InformationNational Health and Medical Research Council Grant (1060515), UNSW Goldstar Award, UNSW Career Advancement Fund
Development of Methionyl‐tRNA Synthetase from Pseudomonas aeruginosa as a Platform to Screen for Inhibitors of Protein Synthesis
Authors: Robles Sara , Hu Yanmei , Bullard James M 
Published: November 2023

IntroductionPseudomonas aeruginosa is an opportunistic pathogen and a common cause of nosocomial infections. Aminoacyl‐tRNA synthetases (aaRSs) are a class of enzymes that catalyze the covalent attachment of amino acids to their cognate tRNAs during protein biosynthesis. We describe here the enzymatic characterization and development of a screening platform based on methionyl tRNA synthetase (MetRS) from P. aeruginosa.ResultsMetRS from P. aeruginosa was cloned and expressed in E. coli and purified to greater than 98% homogeneity. Sequence analysis shows that this protein contains the characteristic motifs of class I aminoacyl‐tRNA synthetases. The kinetic parameters for the interaction of P. aeruginosa MetRS with its three substrates (tRNA, ATP, and methionine) were determined. Initial velocities were determined for charging of tRNA using tRNAMet concentrations between 0.5 and 5 μM. The KM and Vmax was determined to be 29 μM and 3.0 μM min−1, respectively, resulting in an observed kcat of 1.9 sec−1. This resulted in a kcat/KM value of 0.067 s−1μM−1. The ATP:PPi exchange reaction was used to monitor interaction with ATP and methionine (Met). The KM, Vmax, observed kcat, and kcat/KM for interaction with ATP and Met was 515, 145, 24.2, 0.05 and 35.5, 223, 37.2, 1.05, all respectively. Scintillation proximity assay (SPA) technology was adapted to the aminoacylation assay and then used to screen for inhibitors of activity of P. aeruginosa MetRS in a high throughput format. Using this assay, a natural product (800 compounds) and a synthetic compound (890 compounds) library was screened. Two compounds were identified which inhibit greater than 50% of enzymatic activity. Compounds BM01F04 and BM01C11 inhibit the activity of P. aeruginosa MetRS with IC50 values of 26 and 70 μM, respectively and both of these compounds exhibited promising MICs against Gram+ bacteria and mutant forms of Gram‐ bacteria.ConclusionMetRS identified in P. aeruginosa was cloned, expressed characterized and developed into a screening platform to identify compounds that have the potential for development as an antibacterial agent against pathogenic organisms.Support or Funding InformationResearch was funded by NIH grant 1SC3GM098173‐01A1. Partial student support was from a Departmental Grant from the Robert A. Welch Foundation (Grant No. BG‐0017) and the Howard Hughes Medical Institute (HHMI) Precollege and Undergraduate Science Education Program grant #52007568.
Assembly of human translational elongation factor 1B is based on the interaction of the glutathione transferase‐like domains from alpha and gamma subunits
Authors: Choi Yoon Seo , Lee Hyun Joo , Kang Beom Sik 
Published: November 2023

In the process of human translational elongation, eEF1A·GDP is exchanged to eEF1A·GTP by eEF1B, which consists of three subunits, α, β, and γ. Among them, α and β subunits have guanidine exchange function while eEF1Bγ can bind to both α and β subunits. The N‐terminal GST domain of eEF1Bγ is responsible for the interaction to the N‐terminal regions of eEF1Bα and β. We obtained the 2:2 complex of the N‐terminal eEF1Bα and γ domains, and determined the crystal structure of the complex at the resolution of 2.1 Å. eEF1Bγ has a canonical GST domain consisted GST‐N containing a four stranded β‐sheet and helical GST‐C subdomains while the N‐terminal region of the eEF1Bα forms a GST‐C subdomain. The tetrameric complex has an order of α‐γ‐γ‐α subunits. The interaction between γ and γ subunits is similar to that of canonical GST dimer. however, the interaction between α and γ subunits is using their GST‐C domains. Binding assay with α and γ subunits containing mutations at the binding interfaces confirmed the interfaces for α‐γ and γ‐γ subunit interactions. Although the N‐terminal domains of eEF1Bα and eEF1Bγ form a heterotetramer, gel filtration analysis of the full‐length complex indicate their 4:4 complex suggesting another interactions through other regions than the N‐terminal domains. Furthermore, these two subunits could form even a bigger complex (8:8:8) in the presence of eEF1Bβ subunit.Support or Funding InformationKNU Creative BioResearch Group, School of Life Sciences and Biotechnology, Kyungpook National University, Daegu 702‐701, Korea