bluuebunny/crossref_metadata_2025_split huggingface dataset:
Investigation of the DNA strand separation step by DNA methyltransferase Caulobacter Crescentus
Authors: Konttinen Olivia Rae , Reich Norbert O. , Carmody Jason , Kurnik Martin 
Published: April 2020

Epigenetic DNA methylation by CcrM C. crescentus is an important regulator of gene expression. CcrM methylates adenine bases on hemimethylated double stranded DNA substrates containing 5′‐GANTC‐3′ sites to regulate gene expression. The recent cocrystal structure shows that CcrM uses a unique DNA recognition mechanism by which four of the five basepairs are completely disrupted. The target strand interacts in base‐specific ways whereas the non‐target strand is largely unperturbed, and does not involve any base‐specific interactions. The dimeric enzyme’s methyltransferase domain and c‐terminal domain interact with the DNA target strand and non‐target strand respectively. Here we present a novel assay that tracks the DNA strand separation step by using a fluorescent probe (Pyrollo‐dC). Placement of Pyrollo‐dC within double stranded DNA at the N position of the CcrM recognition site results in a dramatic increase in fluorescence when CcrM binds; this only occurs when the analog is positioned within the target strand, with much smaller effects involving the non‐target strand. Serine 315 and Histidine 317, which reside in the highly conserved 80‐residue C‐terminal tail, interact with phosphates within the non‐target strand and mutation to alanine results in significant losses of activity and a loss of strand separation, as determine by the Pyrollo‐dC assay. Residue G305A does not interact with the phosphate backbone of the non‐target strand and does show fluorescent signal, suggesting that this mutant is able to strand separate. This mechanism appears to be widespread since other enzymes (e.g., HinfI and an ortholog of CcrM, BabI) exhibit fluorescent signals similar to CcrM.Support or Funding InformationNIH Grant
Expression and purification of human Neuronal PAS domain protein 2 (hNPAS2) for structural and functional studies
Authors: Chen Shawnan , Moreno Brenda , Moriel Karla , Xiao Chuan 
Published: April 2020

The circadian rhythm, a roughly 24‐hour biological clock, is found in most living organisms. The oscillation of the rhythm is controlled by a molecular mechanism called the transcriptional‐translational feedback loop (TTFL). In mammals, the circadian rhythm is responsible for a variety of biological and physiological processes such as hormone regulation, digestion, and body temperature. Human neuronal PAS domain protein 2 (hNPAS2) has been proven to work as a transcription factor important in regulating the circadian rhythm. This project aims to determine the structure of hNPAS2 in order to elucidate its function in the circadian rhythm. In this study, full length hNPAS2 gene was cloned into a bacterial expression vector. To obtain high yields of the soluble protein, the expression was optimized. Recombinant hNPAS2 proteins were purified by affinity and size exclusion chromatography. The high concentration of purified hNPAS2 protein will be used in structural and functional studies. The results will deepen our knowledge about hNPAS2 and its role in the circadian rhythm.Support or Funding InformationMentored Experiences in Research, Inquiry, and Creativity for Undergraduate Scholors (MERITUS), Campus Office of Undergraduate Research Initiatives (COURI), Office of the Provost and Vice President for Student Affairs, BUILDing SCHOLARS, National Research of General Medical Sciences of NIH
Growing and Sustaining a Nationwide CURE: Genomics Education Partnership Enhances Research Opportunities for Students and Faculty at Diverse Institutions
Authors: Goodman Anya L. , Van Stry Melanie , Lopatto David , Leung Wilson , Reed Laura K. , Community GEP Faculty 
Published: April 2020

The Genomics Education Partnership (GEP) is a community of practice focused on the integration of genomics research experiences into undergraduate curriculum and supporting faculty in this endeavor. Founded in 2006, GEP has grown to 100+ participating institutions and introduced thousands of students to eukaryotic gene structure, comparative genomics, and genome evolution. Recently, GEP has transitioned to a collaborative leadership structure. GEP members contribute their expertise and enthusiasm to the community through four committees: science and IT, curriculum development, assessment, and professional development and mentoring. Digital communication tools facilitate collaboration in this geographically distributed community and allow faculty with similar interests to work together. We seek to grow our network of faculty and to increase the diversity of our community by making a variety of training opportunities available, including online mentoring via the QUBES platform and regional node workshops. We are also expanding our repertoire of scientific projects. GEP has partnered with Galaxy to develop G‐OnRamp, an open‐source platform for constructing UCSC Assembly Hubs and JBrowse/Apollo genome browsers to facilitate collaborative annotation of eukaryotic genomes. These tools allow us to expand GEP projects, for example, to include investigations of the evolution of venom in parasitoid wasps and an investigation of the evolution of insulin pathway genes across 27 Drosophila genomes. Our education research is focused on student experiences, attitudes, and learning gains in the Course‐based Undergraduate Research Experience (CURE) setting. An important dimension of CUREs that distinguishes them from the traditional laboratory classroom is iteration: students have opportunities to make mistakes and redo (Auchincloss et al, 2014). The scaling of undergraduate research from an apprentice model to the classroom, however, poses a major challenge for faculty, creating the need to provide feedback to a large number of inexperienced student researchers. We find that custom bioinformatics tools provide rapid feedback to GEP students and allow for iterative revisions of student projects. Student surveys and focus groups reveal that bioinformatics CUREs foster “formative frustration,” whereby students can safely fail in their original analysis, adjust, recover, and succeed; students value this experience. We have created a supportive community of faculty and welcome new colleagues (no experience necessary!) eager for the challenge of bringing genomics research to all undergraduate students (freshmen through seniors). Faculty interested in joining the GEP can contact us at http://gep.wustl.edu/contact_us.Support or Funding InformationSupported by NSF IUSE‐1915544 and NIH IPERT‐1R25GM130517‐01 to LKR.
ROLE OF DNA METHYLAION IN THE PROMOTER REGION OF CLOCK GENES IN THE REGULATION OF LIVER DISEASE
Authors: Aljobaily Nouf , Han Yuyan 
Published: April 2020

Background and aimObesity is becoming a major global health concern, as it is associated with many diseases, such as Non‐Alcoholic Fatty Liver Disease (NAFLD). NAFLD is associated with diabetes, metabolic syndrome and, and is known to be multifactorial. Meaning that it is affected by multiple external and environmental factors. The progression of NAFLD to Non‐alcoholic Steatohepatitis (NASH), liver carcinoma and further to liver failure, makes it very crucial that we pay attention to the disease at its earlier stages. Further, NAFLD is affected by the circadian rhythm, which is a 24‐hour cycle that controls biological activities of all cells. In the liver, this rhythmic cycle is called liver clock and is very sensitive to nutritional intake, physical activity and sleeping patterns, and it is controlled by Clock genes. The expression of clock genes can be controlled by DNA methylation, which is a modification that acts as a switch to turn genes on and off. Therefore, this study aimed to study the methylation status of clock genes and clock‐controlled genes at the promoter region.MethodsC57BL/6 mice were treated with high‐fat diet (HFD) or control (CTR) for 8 wk. Mouse livers were harvested for further analysis. Liver function was evaluated by serum chemistry, fibrosis was measured by IHC and PCR and lipid deposition were evaluated by Oil red O staining. Genomic DNA and total RNA were obtained for measuring the DNA methylation of core clock genes and RNA expression as described above. Methylation‐Specific PCR (MSP) and Sanger sequencing were used to evaluate the change in the gene expression of genes of interest; CLOCK, BMAL‐1 and PPAR‐a in a high‐fat diet group and control‐diet group.ResultsWe found increased steatosis and hepatic fibrosis in mice fed a high‐fat diet compared to control. Expression of fibrotic markers (a‐SMA, FN1 and collagen I) was upregulated in mice fed high‐fat diet compared to control. Differential DNA methylation status in promoter regions was observed in different core clock genes in high‐fat‐fed mice.ConclusionDNA methylation of core clock genes and clock‐controlled genes may lead to dysregulation of the circadian rhythm during consumption of a high fat diet, thus leading to increased steatosis and liver fibrosis.
Maternal Immune Activation Alters Placental and Fetal Brain Amino Acid Transport in Rats
Authors: McColl Eliza R. , Piquette-Miller Micheline 
Published: April 2020

ProblemMaternal immune activation (MIA) during pregnancy is an environmental risk factor for neurodevelopmental disorders such as autism and schizophrenia. However, the exact mechanism through which this occurs is unknown. Due to the important roles that amino acids play in neurodevelopment, altered transport of amino acids across the placenta and throughout the developing fetal brain could play a role in neurodevelopmental disorders. Our objectives were to determine whether virally mediated MIA alters amino acid transporter expression in the placenta and fetal brain, and to explore the underlying mechanism(s) associated with these changes.MethodsPregnant rats received poly(I:C) on gestational day 14 and placental and fetal tissues were collected 6, 24, and 48 hours later. Amino acid transporter expression in the placenta and fetal brain was measured using qPCR and immunoblotting. Free amino acid concentrations in the fetal brain were quantified using HPLC. To explore a possible mechanism, the expression and phosphorylation of components of the placental mTOR and AMPK signaling pathways were investigated using immunoblotting.ResultsPoly(I:C) induced the mRNA expression of multiple amino acid transporters in the placenta and fetal brain over all three timepoints. Conversely, poly(I:C) significantly decreased protein expression of ASCT1 and EAAT2 in the placenta, and SNAT5, EAAT1, and GLYT1 in the fetal brain. Changes in transporter expression were accompanied by functional changes in the concentrations of numerous amino acids in the fetal brain. We observed activation of AMPK and inhibition of mTOR signaling in the placentas of poly(I:C)‐treated dams.ConclusionsMIA induced by poly(I:C) significantly alters placental and fetal brain amino acid transporter expression as well as amino acid concentrations in the fetal brain. Due to the importance of amino acids in neurodevelopment, changes in amino acid transporters could contribute to the development of a neurodevelopmental disorder. AMPK and mTOR have been previously shown to regulate amino acid transporter expression. Since poly(I:C) altered the activation of placental and AMPK and mTOR, these could provide novel treatment targets to alleviate MIA‐induced changes in amino acid transport and investigate the implications of these changes in neurodevelopment.Support or Funding InformationThis work was supported by an operating grant from the Canadian Institutes of Health Research (CIHR) awarded to Dr. Piquette‐Miller [MOP 13346]. Eliza McColl was a recipient of a CIHR Canadian Graduate Scholarship (Master’s) and an Ontario Graduate Scholarship, and is currently funded by a CIHR Banting and Best Canadian Graduate Scholarship (Doctoral) [GSD‐164238].
Recapitulating GSK3β and Tau interaction in brain injury dynamic model
Authors: Cheng Pei-Wen , Lin Yu-Te , Wu Yi-Chung , Wong Tzyy-Yue 
Published: April 2020

IntroductionLiving systems experience various form of mechanical stimulations in daily life. Some mechanical stimulation are required for cell survival, others are capable of causing injuries. In vivo, the neuronal system is linked closely to the muscular system. As a result, we respond to pain immediately after physical injury. Proliferating neuron cells can be used to study the effect of mechanical stimulation on neurons. However, cell models developed from neurons have been lacking due to the fact that neuron cells do not or are difficult to proliferate by in vitro culture. In this study, we investigated the effect of stretching on the SH‐SY5Y cells, neuroblastoma cells that proliferate in vitro. The cells retain the dopamine‐β‐hydroxylase activity; thus, it is suitable for neuron mechanistic study.MethodThe culture conditions contain non‐stretched (control) and stretched groups. SH‐SY5Y cells were cultured on stretchable membranes, subjected to 6, 24h of cyclic stretching, 25% elongation at 1Hz.ResultsAfter 6 and 24h stretching, cells showed decreased red fluorescence for JC‐1 probe which stains the mitochondrial membrane, indicating that cells experienced oxidative stress. Results show that stretching increased b‐amyloid precursor, total Tau protein by western blot. Co‐immunoprecipitation showed that Tau formed complex with GSK3b after 6h stretching.SummaryThe observations of change in mitochondrial potential, and formation of Tau complex imply that the neuron cells were under a stress. The stress may be a kind of injury suffered from physical stimulation.Support or Funding Information108‐2320‐B‐075B‐002 ‐
Filament Formation Induces a Shape Change and Activation of the Nuclease SgrAI
Authors: Horton Nancy C. 
Published: April 2020

SgrAI is allosteric and is both activated as well as undergoes a change in substrate specificity (cleaving a secondary class of DNA sequences) upon binding to its primary recognition sequence in DNA. We have investigated the allosteric activation of SgrAI and this unusual expansion of DNA sequence specificity using structural, biophysical, and enzyme kinetic methods.
1–
2A variety of biophysical techniques (native gel electrophoresis, analytical ultracentrifugation, ion mobility native mass spectrometry, x‐ray crystallography and electron microscopy) to investigate complexes formed by SgrAI. Single turnover DNA cleavage assays were used to investigate the reaction mechanism of SgrAI, and global modeling to create a computation model of the full reaction pathway including rate constants for each important step. The biophysical characterizations revealed that SgrAI forms run‐on oligomers or filaments when activated, and that these filaments are induced when SgrAI binds to its primary recognition sequence. SgrAI binds tightly to secondary site sequences, which differ from primary by a single base pair, but fails to cleave them and does not induce filament formation. However, SgrAI bound to the secondary site sequences will join filaments formed from SgrAI bound to primary site sequences. Protein‐protein and protein‐DNA contacts between SgrAI/DNA complexes in a filament stabilize a different conformation of SgrAI, hypothesized to be activated due to a reorganization of critical residues in the active site. DNA cleavage kinetics reveal activation of DNA cleavage activity by SgrAI of 200–1000 fold for primary and secondary site sequences upon filament formation. Full global kinetic analysis reveals a slow second order association step of SgrAI addition to a filament, followed by rapid DNA cleavage, then dissociation of SgrAI from the filament, and finally rapid product (i.e. cleaved DNA) release. The slow association step controls SgrAI activity to form filaments preferentially on DNA contiguous with primary site sequences, and incorporation of secondary site sequences into the filament increases the number of DNA cleavages on the contiguous DNA. Hence, this system allows for rapid cleavage of invading phage DNA (with unprotected primary sites).The SgrAI system uses formation of a filamentous form to stabilize an activated conformation. DNA sequence and slow second order association kinetics control where and when this filament is formed in a way that is tuned for optimal performance of its biological role.Support or Funding InformationThis work was supported by the National Science Foundation under Grant No. MCB‐1410355 (to N.H.).
Glycolysis enzymes coalesce in G bodies in hypoxia nucleated by RNA
Authors: Fuller Gregory , Freeberg Mallory , Han Ting , Kim John 
Published: April 2020

Cells deprived of oxygen rely on glycolysis alone for energy production, generating far less energy per molecule of glucose than aerobic respiration. While modulating levels of glycolysis enzymes and their activity can increase the rate of glycolysis, spatial reorganization of pathway components may also contribute to enhancing rates of glycolysis. We have demonstrated that in S. cerevisiae, glycolysis enzymes, including the rate limiting phosphofructokinase (PFK), coalesce into single non‐membrane‐bound cytoplasmic foci called “glycolytic (G) bodies
1” in hypoxia. Cells lacking the AMP activated protein kinase, SNF1, fail to form single foci and are enriched for upstream glycolysis metabolites, while being depleted for the downstream metabolite, acetyl CoA. Furthermore, cells preconditioned in hypoxia (thereby forming G bodies) deplete glucose from media at a faster rate than those cultured in normoxia (lacking G bodies). Thus, the presence of G bodies is correlated with greater rates of glycolysis. However, we know very little about how G bodies form. Many glycolysis enzymes moonlight as RNA binding proteins despite lacking known RNA binding domains, and proteomic analysis of purified G bodies revealed an enrichment of RNA binding proteins, suggesting that G bodies are ribonucleoprotein (RNP) granules. Tethering PFK to nonspecific endoribonucleases to degrade RNA at sites of G body formation resulted in complete loss of G body formation, indicating that RNA nucleates nascent G bodies. Induction of PFK‐RNase in cells with existing G bodies leads to fragmentation into multiple foci, indicating that RNA is required to maintain G body integrity. Consistent with RNA nucleating G body formation, RNAs bound to glycolysis enzymes in normoxic conditions identified by PAR‐CLIP strongly overlapped with RNAs that copurify with G bodies in hypoxia. Many RNP granules form via phase separation of protein and RNA. Although G bodies are distinct from stress granules, G bodies exhibit similar physical properties to stress granules, which are thought to form via phase separation. Proteomic analysis revealed similar protein components as stress granules including HSP70 chaperones. In addition, we have shown that G bodies in mating yeast cells have the ability to fuse and do so during their biogenesis. Furthermore, FRAP revealed that G body components exchange with the cytoplasm. G body fusion, however, takes multiple minutes to occur and FRAP recovery rates occur with half times of greater than 10 minutes, indicating that G bodies behave more akin to gels than a liquid‐liquid phase condensate. Taken together, we propose that G bodies are novel RNP granules that function to enhance rates of glycolysis in response to energy limiting hypoxic stress. Future studies will clarify the mechanisms of G body function and further test their physiological roles in cells.Support or Funding InformationThis work was funded by NIH grant (GM129301‐01).
CD248 plays important role in profibrotic phenotype switching of macrophages and tissue fibrosis
Authors: Pai Chen-Hsueh P. , Lin Shu-Rung , Liu Chia-Hao , Pan Szu-Yu , Hsu Hao , Chen Yi-Ting , Tsai Yu-Fei , Yu I-Shing , Lin Shuei-Liong , Lin Shu-Wha 
Published: April 2020

In human chronic kidney disease, upregulated CD248 in myofibroblasts is linked to poor renal survival. Although the role of macrophages and myofibroblasts in organ fibrosis have been extensively studied, how macrophages and myofibroblasts crosstalk to promote organ fibrosis is not clear. In this study we showed that Cd248 was upregulated in myofibroblasts in murine models of renal and peritoneal fibrosis. Cd248 knockout (Cd248−/−) could attenuate both renal and peritoneal fibrosis. By parabiosis of both the GFP reporter mice and Cd248−/− mice, we showed that decrease of fibrosis was related to a decrease in macrophage infiltration. Moreover, decreases of chemokine (C‐C motif) ligand 17 and Ccl22 was also found in Cd248−/− or galectin‐3‐deficient macrophages demonstrated that CD248 and galectin‐3 played a similar role in profibrotic phenotype switching of macrophages in injured organs. We also showed that CD248 interacted specifically with macrophage galectin‐3 and promoted myofibroblasts collagen synthesis. Mice with DNA vaccination targeting CD248 showed decreased fibrosis. We propose a novel interaction between CD248 on myofibroblasts and galectin‐3 on macrophages is a key step in mediating kidney fibrosis.Support or Funding InformationThis study is supported by Taiwan MOST (108‐2319‐B‐002‐002 and 108‐2320‐B‐002‐051‐MY3), National Core Facility for Biopharmaceuticals, and the Mrs. Hsiu‐Chin Lee Kidney Research Foundation.
Is Hyperoxic Hyperventilation Caused by Reduced Carbon Dioxide Washout or Disturbed Brain Redox Homeostasis?
Authors: Fernandes Igor A. , Mattos João D. , Monique Campos O. , Rocha Marcos P. , Mansur Daniel E. , Rocha Helena M. , Garcia Vinícius P. , Alvares Thiago S. , Secher Niels H. , Nóbrega Antonio C. L. 
Published: April 2020

Under oxygen‐enriched conditions, humans present a biphasic ventilatory response that is initially characterized by reduced ventilation (V̇E) as hyperoxia (HX) inhibits the peripheral chemoreceptors. This short‐lived response precedes a paradoxical increase in V̇E that has been termed hyperoxic hyperventilation. Proposed but untested mechanisms for this paradoxical response include central chemoreceptor stimulation via either an increased cerebral partial pressure of carbon dioxide (CO2), which would result from reductions in cerebral blood flow (CBF) and brain CO2–[H+] washout, or (2) an HX‐induced exaggerated production of reactive oxygen species (ROS). To determine the mechanism behind the paradoxical increase in V̇E during hyperoxemia, we submitted 8 healthy young men (24±1 yrs) to 10‐min trials of poikilocapnic HX under (1) saline or (2) ascorbic acid (AA) infusion. V̇E, arterial (a) and internal right jugular venous (Jv) PO2, PCO2, pH, oxidant (8‐isoprostane), and antioxidant (AA) markers, as well as volumetric CBF (Doppler ultrasound) responses, were quantified at the last minute of each trial. Under saline infusion, HX evoked an increase in PaO2 followed by a hyperventilatory response (+3.0 ± 1.2 L/min, p = 0.003) and arterial hypocapnia (−2.4 ± 0.9 mmHg, p = 0.03). HX also evoked a small but significant reduction in CBF (−219.6 ± 36.8 mL/min, p = 0.001) that was followed by unchanged PjvCO2 (−0.1 ± 0.7 mmHg, p = 0.619) and pH (0.001 ± 0.005, p = 0.957). HX under saline infusion also provoked an increase in arterial 8‐isoprostane (18.6 ± 7.5 pg/mL, p = 0.04). On the other hand, intravenous AA augmented arterial antioxidant markers (+584.1 ± 64.6, p = 0.0001 vs. saline) and blunted not only the increase in arterial 8‐isoprostane (−10.7 ± 9.5 pg/mL, p = 0.204) but also the HX‐induced hyperventilation (+0.87 ± 0.64 L/min, p = 0.232) and the arterial hypocapnia (−1.7 ± 0.5 mmHg, p = 0.04 vs. saline) observed during infusion of saline. HX under AA infusion also blunted CBF hypoperfusion (−41.8 ± 26.3 mL/min, p = 0.001 vs. saline) that, in turn, was followed by increases in PjvO2 (+5.3 ± 0.7 mmHg, p = 0.006 vs. saline), PjvCO2 (+1.1 ± 0.50 mmHg, p = 0.03 vs. saline) and a reduction in pH (−0.012 ± 0.004, p = 0.01) that may be explained by the Haldane effect. These findings suggest that hyperoxic hyperventilation is not a consequence of an increased cerebral CO2 partial pressure and a reduced brain CO2–[H+] washout but mediated by central chemoreceptor stimulation likely evoked by excessive production of ROS.Support or Funding InformationSupport: FaperRJ, CNPq, CAPES