bluuebunny/crossref_metadata_2025_split huggingface dataset:
Replisome Disassembly At The Termination Of Eukaryotic Replication Forks
Authors: Gambus Agnieszka 
Published: November 2023

To ensure duplication of the whole genome DNA replication initiates from thousands of origins of replication. The pre‐loaded inactive helicase (Mcm2‐7 double hexameric complexes) becomes activated during replication initiation by loading of additional components: Cdc45 and GINS in a carefully regulated process. The progressing replication fork moves through the chromatin until it encounters fork from the neighboring origin. During the termination of replication forks the replisomes disassemble and topisomerase II resolves the daughter DNA molecules. If not resolved properly, the terminating forks are at risk of stalling and fork reversal, leading to DNA damage and genomic instability.Using the Xenopus laevis egg extract system, we have shown (Priego Moreno et al. Science 2014) that blocking polyubiquitylation results in the prolonged association of the active helicase with replicating chromatin. It was accompanied by a defect in replication forks termination leading to conclusion that polyubiquitylation is essential for the disassembly of the active helicase at the termination of replication forks. The Mcm7 subunit of the Mcm2‐7 complex was the only component of the active helicase that we found polyubiquitylated during S‐phase and its ubiquitylation was blocked when forks could not terminate. Finally, disassembly of the helicase was dependent on p97/VCP/Cdc48 segregase activity.This process of replisome dissolution is very well conserved through evolution (Maric et al. Science 2014), however, the ubiquitin ligase driving ubiquitylation of replicative helicase is not. I will present, therefore, our recent findings on the identity of the enzyme ubiquitylating Mcm7 in higher eukaryotes.Support or Funding InformationThis work was funded by MRC CDA Fellowship and Lister Institute Research Award for A.G.
Adiponectin Inhibits LPS‐Induced HMGB1 Release through Heme Oxygenase‐1 Dependent Pathway in RAW 264 Cells
Authors: Elfeky Mohamed , Yoneshiro Takeshi , Okamatsu‐Ogura Yuko , Kimura Kazuhiro 
Published: November 2023

IntroductionHigh mobility group protein B1 (HMGB1) is an important late inflammatory mediator in sepsis. Adiponectin, an adipokine predominantly secreted from adipose tissue, has potent anti‐inflammatory properties. However, the possible anti‐inflammatory effects of adiponectin on lipopolysaccharides (LPS)‐induced HMGB1 release are unknown. The aim of this study was to investigate whether adiponectin could inhibit HMGB1 release in LPS‐stimulated RAW 264 cells.Materials and methodsMurine macrophage RAW 264 cells were treated with or without LPS (200 ng/ml) in the presence or absence of adiponectin, interleukin (IL)‐10, HO‐1 inhibitor (ZnPP) and p38 MAPK inhibitor (SB203580). HMGB1 translocation and release were measured by immunofluorescence staining of the cells and western blotting analysis of the medium, respectively. Expression levels of mRNA for toll like receptor (TLR) 4, TLR2, myeloid differentiation factor‐2 (MD‐2), HMGB1, IL‐10, heme oxygenase‐1(HO‐1) and nuclear factor erythoid‐derived 2 related factor 2 (Nrf2) mRNA were assessed by real‐time PCR.Results and discussionTreatment of the cells with LPS induced significant HMGB1 release and obvious translocation from nucleus to cytosol. However, prior treatment with either adiponectin or IL‐10 suppressed the LPS‐induced HMGB1 release and translocation. Adiponectin treatment decreased TLR4 mRNA and induced expression of HO‐1 mRNA, but not IL‐10 mRNA, while IL‐10 treatment decreased TLR2 and HMGB1 mRNA and enhanced both IL‐10 and HO‐1 gene expressions. Treatment of RAW 264 cells with ZnPP before adiponectin and IL‐10 treatment obviated the suppression of HMGB1 release by adiponectin completely, but partially by IL‐10 while treatment with SB203580 didn't had a significant effect. In conclusion, our results indicate that adiponectin suppresses HMGB1 release by LPS through a HO‐1‐dependent while neither HO‐1 nor p38 MAPK was involved in IL‐10 anti‐inflammatory effect. Therefore, both adiponectin and IL‐10 may play a role as an anti‐inflammatory molecule in the late inflammatory process by preventing release of a septic exaggeration factor, HMGB1.
Interlukin‐17A Promotes Aortic Endothelial Activation Via Transcriptionally and Post Transcriptionally Activating p38 MAPK Pathway
Authors: Mai Jietang , Nanayakkara Gayani , Lopez‐Pastrana Jahaira , Li Xinyuan , Li Ya‐Feng , Wang Xin , Virtue Anthony , Shao Ying , Shan Huimin , Liu Fang , Autieri Michael , Iwakura Yoichiro , Jiang Xiaohua , Wang Hong , Yang Xiao‐Feng 
Published: November 2023

Interleukin‐17A (IL‐17) is a potent mediator of various inflammatory and autoimmune diseases, and is known to be induced by hyperlipidemia. However, the roles of IL‐17A in atherosclerosis and aortic endothelial cell activation is not clearly defined and remains controversial. Nevertheless, its role as a pro‐atherogenic stimuli prevails, thus we formulated the hypothesis that IL‐17A promotes endothelial cell activation under hyperlipidemic stress. The objective of this study was to elucidate the mechanism of how IL‐17A promote endothelial activation that leads to atherosclerosis under hyperlipidemia. We utilized biochemical, immunological, microarray, flow cytometry, intra‐vital microscopy, data mining analysis techniques and ApoE−/−/ IL‐17A−/− null mice to test our hypothesis. Our data revealed that hyperlipidemic stimuli can induce IL‐17A receptor expression in human/ mouse aortic endothelial cells (HAEC/ MAEC). In addition, IL‐17A treatment significantly induced monocyte adhesion to HAEC. Also, by gene expression studies, we implicated that IL‐17A induces pro‐inflammatory chemokines C‐X‐C motif ligand 1 and 2 (CXCL 1 and 2) expression in HAEC. Interestingly, our mouse aortic microarray data revealed that IL‐17A upregulates the endothelial cell adhesion molecules and the p38 component of the mitogen activated protein kinase (MAPK) pathway. We have demonstrated that the pharmacological inhibition of p38 MAPK can significantly impair IL‐17A induced endothelial adhesion molecules and CXCL1/ 2 expression, suggesting that p38 MAPK is a mediator of IL‐17A induced endothelial cell activation. Moreover, we observed that IL‐17A depletion in ApoE mice had significantly decreased levels of phosphorylated p38 MAPK relative to controls. We further consolidated these finding in‐vivo by utilizing intra‐vital microscopy to demonstrate that IL‐17A knockout in ApoE−/− mice reduced leukocyte rolling and adhesion to the endothelium. Based on our findings, we conclude that IL‐17A mediates the early phase of hyperlipidemia induced endothelial cell activation by activating the p38 MAPK pathway.Support or Funding InformationNIH R01, AHA predoctoral and postdoctoral grants
HSP70 and HSP90 Inhibitors Repress Expression of TSC1 in T24 Bladder Cancer Cells and UOK139, UOK 140 Kidney Cancer Cells
Authors: Andolino Chaylen , Bresticker Julia , Berg Adrian , Cox Avery , Williams Heinric , Chernin Mitchell 
Published: November 2023

Normal cells are able to survive in stressful conditions by increased expression of heat shock protein (HSP) genes. Both HSP90 and HSP70 are up regulated in cancers of the bladder and kidney. Next generation sequencing studies have identified pathways involved in kidney and bladder cancer. The objective of this study was to elucidate the impact of HSP70 and HSP90 inhibition on the signal transduction pathways driving their tumorigenic process. The TSC1‐TSC2 complex has emerged as a central signal‐integrating node within the cell.MethodsWe analyzed the expression of key mediators of the TSC1‐TSC2 pathway when treated with the HSP90 inhibitor (STA9090) or HSP70 inhibitors (VER155008, MAL3‐101) in bladder cancer (T24) and kidney cancer (UOK139 and UOK140) cell lines. The impact of treatment on cell growth was analyzed using a MTT assay and on signal protein expression by Western blot analyses.ResultsGrowth inhibition was seen in T24 bladder cancer cells when treated with 1 μM STA9090 (STA) and 50 μM VER155008 (VER) but not with 30 μM MAL3‐101 (MAL) for 72 h. Synergistic growth inhibition was seen with combination STA+VER, STA+MAL and MAL+VER in T24 cell lines. We tested the effect of these inhibitors on the expression of TSC pathway mediators in both bladder and kidney cancer cell lysates and noted that TSC1 was down regulated in all cell lines tested in the presence STA and VER. In T24 cells, only the combination VER + MAL led to TSC1 down regulation. Neither STA, VER nor MAL had any impact on S6 kinase (a downstream mediator) expression in T24 cells. Rather, only STA and not VER or MAL downregulated the expression of the active form of S6 kinase (pS6kinase).ConclusionThe inhibition of HSP70 and HSP90 appears to cause a down regulation of TSC1. TSC1/2 functions as a GTPase‐activating protein (GAP) for the small Ras‐related GTPase Rheb. The active, GTP‐bound form of Rheb directly interacts with mTORC1 to stimulate its activity. mTORC1 promotes protein synthesis by phosphorylating S6 kinase. Together, these data suggest a role for HSP90 and HSP70 inhibitors in kidney and bladder cancer management.Support or Funding InformationBucknell‐Geisinger Research Initiative
Comparative Analysis of ELISA and MALDI‐TOF Mass Spectrometry Methods for Microcystins in Freshwater Samples
Authors: Kadlec Kimberly Michele , Turczyn Andrew , Grant Jennifer 
Published: November 2023

Health risks associated with freshwater cyanobacterial blooms have led to the development of a variety of analytical methods to detect the presence of microcystins, which are monocyclic heptapeptides that can cause acute and chronic hepatotoxicity when ingested by animals and humans. In this study of soluble microcystin levels in lake water, the sensitivity of detection of Matrix‐Assisted Laser Desorption/Ionization Time‐of‐Flight mass spectrometry (MALDI‐TOF MS) was compared to that of Enzyme Linked Immunosorbent Assay (ELISA) methods.Lake water samples were collected monthly during the first week of each month, May through August in 2012, 2013 and 2014 and samples were frozen at −20 °C. Prior to analysis, lake water was centrifuged (5 min @ 10,000 × g) to remove suspended solids. ELISA was performed using Microcystins (ADDA)‐DM ELISA Microtiter Plate, following the manufacturer's protocol (Abraxis Inc.). Absorbance was measured at 450 nm on a BioTek Epoch Microplate spectrophotometer. MALDI‐TOF MS was performed by applying a small volume of sample to a stainless steel MALDI plate, followed by an equal volume of α‐cyano‐4‐hydroxycinnamic acid, using the dried droplet method. Relative quantitation of microcystin content was performed using angiotensin II to create standards ranging from 0.06 μM to 5.0 μM in concentration. Data were acquired using a Bruker Microflex Linear Time of Flight MALDI‐TOF Mass Spectrometer. Spectra were acquired over the mass range of 0.6 to 4 kDa using 50 laser shots in the positive ion mode at a laser power of 25%. Calibration of the mass spectra was achieved using a calibration solution comprised of 1 μM bradykinin and 1 μM neurotensin.Semi‐quantitation with the ELISA kit showed seasonal fluctuations of microcystins peaking in July or August with concentrations up to 10‐fold greater than detected using the MALDI‐TOF MS method. The ELISA kit indicated microcystin levels less than 1.0 μg/L, the maximum concentration for drinking water set by the WHO, in 50% of the samples. MALDI‐TOF MS analysis of the same samples detected seasonal fluctuations of microcystins and indicated the presence of 18 different congeners of microcystin over the course of the entire study. The congener that was visualized most consistently was MC‐LAba (m/z 923), recorded at both locations at concentrations ranging from 0.25 – 0.47 μg/L in 70% of the samples. The congener present in the greatest concentration was found to be MC‐RR (m/z 1037), at 3.42 μg/L in August 2013.Our findings showed general agreement between the ELISA and MALDI‐TOF MS data. The primary drawback in the ELISA methodology is that ELISA cross‐reacts with non‐microcystin targets (e.g. nodularins, N‐hemi‐ADDA and ADDA), often causing an overestimation of the actual concentration of the target microcystins in the sample. MALDI‐TOF MS, in addition to requiring far less sample preparation, identified and quantified specific congeners.
Pneumolysin induces cellular senescence in microglial cells: involvement of MAPK and NF‐κB pathways
Authors: Kwon Ii‐Seul , Pyo Suhkneung 
Published: November 2023

Senescence, which is an irreversible proliferation arrest, is induced by various stress stimuli such as genotoxic stress and oxidative stress. Pneumolysin (Ply) that is important pathogenicity factor unique to S. pneumoniae plays a pathogenic role in pneumococcal‐induced meningitis. Brain cells exposed to PLY undergo cell‐cycle arrest or apoptosis. However, the cell fate response to toxin is mechanistically unclear. In the present study, we investigated the effect of PLY on cellular senescence in BV‐2 microglial cells. Exposure to PLY resulted in changes in the expression of genes associated with senescence and increased senescence‐associated β‐galactosidase (SA‐β‐gal) activity. PLY‐treatment also showed a concentration‐ and time‐dependent increase in PAI‐1 expression and G1/G0 phase arrest. PLY induced NF‐κB activation through the suppression of SIRT‐1 expression and phosphorylation of ERK, JNK and p38 MAP kinase. Additionally, PLY increased the production of reactive oxygen species. Overall, our results suggest that PLY induces cellular senescence by enhancing ROS production, activation of MAPK and NF‐κB, and the inhibition of the SIRT‐1 expression, resulting in regulation of cellular senescence in microglial cells.Senescence, which is an irreversible proliferation arrest, is induced by various stress stimuli such as genotoxic stress and oxidative stress. Pneumolysin (Ply) that is important pathogenicity factor unique to S. pneumoniae plays a pathogenic role in pneumococcal‐induced meningitis. Brain cells exposed to PLY undergo cell‐cycle arrest or apoptosis. However, the cell fate response to toxin is mechanistically unclear. In the present study, we investigated the effect of PLY on cellular senescence in BV‐2 microglial cells. Exposure to PLY resulted in changes in the expression of genes associated with senescence and increased senescence‐associated β‐galactosidase (SA‐β‐gal) activity. PLY‐treatment also showed a concentration‐ and time‐dependent increase in PAI‐1 expression and G1/G0 phase arrest. PLY induced NF‐κB activation through the suppression of SIRT‐1 expression and phosphorylation of ERK, JNK and p38 MAP kinase. Additionally, PLY increased the production of reactive oxygen species. Overall, our results suggest that PLY induces cellular senescence by enhancing ROS production, activation of MAPK and NF‐κB, and the inhibition of the SIRT‐1 expression, resulting in regulation of cellular senescence in microglial cells.
Obesity intensifies hepatotoxicity by asparaginase in mice deleted for GCN2 but not ATF4
Authors: Nikonorova Inna A. , Mirek Emily T. , Wang Yongping , Dixon Joseph L. , Anthony Tracy G. 
Published: November 2023

Liver dysfunction is a serious adverse effect of asparaginase treatment in patients with acute lymphoblastic leukemia, reducing overall survival. Asparaginase depletes asparagine and glutamine levels in the blood causing rapid tumor regression. Depletion of these amino acids also reduces liver protein synthesis via phosphorylation of eukaryotic initiation factor 2 (eIF2) by the GCN2 (aka eIF2AK4) kinase. Phosphorylation of eIF2 promotes synthesis of activating transcription factor 4 (ATF4) which reconfigures the cellular transcriptome to alleviate the amino acid stress. Failure of this integrated stress response (ISR) leads to severe liver dysfunction. A potent clinical predictor of hepatotoxicity by asparaginase is pre‐existing obesity. Molecular mechanisms of this clinical observation remain to be determined. The focus of our study was to examine the role of the ISR pathway on the background of obesity in the development of hepatotoxicity by asparaginase. Wild type, Gcn2−/− and liver specific‐Atf4−/−(ls‐Atf4−/−) mice of C57Bl/6J background were fed a high fat diet for 12 weeks. The onset of obesity was monitored by assessing body composition and glucose metabolism. Mice were randomly assigned to receive daily injections of Elspar L‐asparaginase at 0 or 3 IU per gram body weight for 8 days. Liver tissues were collected and analyzed by immunoblotting for activation of the ISR pathway and its crosstalk to usual suspects of liver dysfunction, mTORC1 and unfolded protein response (UPR) pathways. Our data showed that asparaginase treatment of obese mice resulted in hepatic steatosis that intensified significantly in Gcn2−/− but not ls‐Atf4−/− mice. Liver pathology in Gcn2−/− mice corresponded with induction of the UPR pathway that was assessed by PERK (aka eIF2AK3) phosphorylation. PERK phosphorylation was moderate in the livers of asparaginase‐treated wild type and ls‐ATF4−/− mice (5‐fold) and very intense in the livers of Gcn2−/− mice (20‐fold). Asparaginase treatment corresponded with reduced mTORC1 activity (3‐fold reduction in p70S6K phosphorylation and 2‐fold reduction in 4EBP1 phosphorylation) in wild type and ls‐Atf4−/− livers, whereas Gcn2−/− group demonstrated strong activation of mTORC1 pathway (2‐fold increase in mTOR‐Ser2448 and 4EBP1 phosphorylation, 3‐fold increase in p70S6K phosphorylation). Downregulation of mTORC1 pathway in the livers of asparaginase‐treated wild type mice and the absence of such downregulation in asparaginase‐treated Gcn2−/− mice suggest that GCN2 signals to mTORC1 pathway. Furthermore, downregulation of mTORC1 in asparaginase‐treated ls‐Atf4−/− livers suggest that the point of coordination of GCN2 pathway to mTORC1 pathway is not through ATF4. Overall our study provides important molecular insights into the mechanism of acute liver damage by asparaginase and identifies a novel role for the ISR in coordinating mTORC1 signal transduction.Support or Funding InformationNIH HD070487
Hepatic ATGL manipulation regulates lipid metabolism through altered autophagy
Authors: Sathyanarayan Aishwarya , Mashek Mara T , Mashek Douglas G 
Published: November 2023

Adipose triglyceride lipase (ATGL) catalyzes the rate‐limiting step of triacylglycerol (TAG) hydrolysis in multiple tissues. Our laboratory has previously shown that hepatic ATGL preferentially channels hydrolyzed fatty acids (FAs) to β‐oxidation with a concomitant induction of peroxisome proliferator activated receptor‐α (PPAR‐α), independent of a FA carrier. Additionally, our work describes a novel signaling axis involving cAMP/PKA, ATGL‐catalyzed lipolysis and sirtuin1 (SIRT1) activation that governs transcriptional regulation of oxidative metabolism and mitochondrial biogenesis. Recent studies have also shown SIRT1 promotes autophagy, which also contributes to lipid droplet catabolism (lipophagy). Thus, the objective of these studies is to determine the effects of ATGL on autophagy in the liver. Hepatic ATGL overexpression promotes the expression of autophagy genes, whereas ATGL knockdown reduces the expression of the same genes. ATGL regulates autophagosome biogenesis and lysosomal levels through alterations in the microtubule‐associated protein 1A/1B‐light chain 3 (LC3), sequestome1 (p62) and lysosomal‐associated membrane protein 1 (LAMP1). Use of a chemical inhibitor of ATGL, ATGListatin (Astat) in hepatocytes shows a marked suppression of autophagy, similar to the effects observed with ATGL knockdown in vivo. Finally, ATGL requires SIRT1 to govern its effects on autophagy as evidenced by studies showing that ATGL overexpression in liver‐specific SIRT1 KO mice is unable to induce autophagy. Thus, our findings identify ATGL‐mediated lipolysis as an important and potent regulator of autophagy that could have important implications for NAFLD etiology and its comorbidities.Support or Funding InformationFunding sources include the NIDDK (DK085008 to DGM) and the Minnesota Obesity Center (DK050456).
Intact Autophagy is Requisite for Shear‐Stress‐Induced Nitric Oxide Generation by Endothelial Cells via a Novel Glucose‐Dependent Purinergic Signaling Pathway
Authors: Bharath Leena P , Ruan Ting , Babu PVA , Boudina Sihem , Graham Timothy E , Symons J David 
Published: November 2023

Impaired intracellular autophagy has been implicated as a cause of vascular dysfunction in aging human arteries. Earlier we reported that shear‐stress (180‐min × 20 dyne•cm2) induces autophagy that is concurrent with phosphorylation of endothelial nitric oxide (NO) synthase (eNOS) at serine (S) 1177 (p‐eNOSS1177) and NO generation in bovine aortic endothelial cells (ECs). Genetic inhibition of autophagy via Atg3 or Atg5 siRNA suppresses shear‐induced p‐eNOSS1177 and NO generation, increases reactive oxygen species generation, and unleashes inflammatory/adhesion responses, suggesting strongly that autophagy is critical for maintaining EC function. Here we report one mechanism through which autophagy regulates shear‐induced p‐eNOSS1177 and NO production in ECs. Tumor suppressor and metabolism regulator, p53, is strongly upregulated by Atg3 siRNA, causing reduced shear‐induced Glut‐1 expression, glucose transport, glycolysis, and ATP production in ECs. As expected, AMPK is activated by the energy deficit, but does not restore p‐eNOSS1177 and NO generation in this setting. However, shear‐evoked p‐eNOSS1177 and NO generation can be restored in autophagy‐impaired ECs by (i) adding extracellular ADP or by (ii) overexpressing Glut‐1. These findings prompted a further investigation of purinergic signaling to eNOS in the context of suppressed EC autophagy. As predicted, P2Y1 receptor blockade (MRS2179) and P2Y1 siRNA can recapitulate the Atg3 phenotype i.e., shear‐induced p‐eNOSS1177 and NO generation are prevented. Furthermore, shear‐induced ATP/ADP release increases p‐PKCδThr505, a downstream effector of P2Y1 and activator of p‐eNOSS1177. As would be anticipated by these findings, shear‐induced p‐eNOSS1177 and NO generation can be restored in autophagy‐impaired ECs by (i) the PKCδ activator bryostatin or by (ii) PKCδ overexpression, whereas shear‐induced p‐eNOSS1177 and NO generation are prevented when autophagy‐intact ECs are transfected with PKCδ siRNA. Ongoing experiments are determining the extent to which this mechanism is responsible for impaired endothelial function that we have observed in arteries from old mice, that is concurrent with lower Atg3 protein expression, compromised autophagy, and attenuated p‐eNOSS1177 : total eNOS, vs. arteries from adult mice. Collectively, targeted activation of purinergic signaling and/or PKCδ may present a new strategy for treating arterial dysfunction that exists with impaired EC autophagy in the context of healthy aging.Support or Funding InformationUU Diabetes and Metabolism Center; UU Center on Aging; UU College of Health