BDX2, a BDX Rat Fibrosarcoma Cell Line (1) Was Maintained in DMEM/10%
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The Purification and Identification of Interactors to Elucidate Novel Connections in the HEK 293 Cell Line
The Purification and Identification of Interactors to Elucidate Novel Connections in the HEK 293 Cell Line Brett Hawley Biochemistry, Microbiology and Immunology Faculty of Medicine University of Ottawa © Brett Hawley, Ottawa, Canada, 2012 ABSTRACT The field of proteomics studies the structure and function of proteins in a large scale and high throughput manner. My work in the field of proteomics focuses on identifying interactions between proteins and discovering novel interactions. The identification of these interactions provides new information on metabolic and disease pathways and the working proteome of a cell. Cells are lysed and purified using antibody based affinity purification followed by digestion and identification using an HPLC coupled to a mass spectrometer. In my studies, I looked at the interaction networks of several AD related genes (Apolipoprotein E, Clusterin variant 1 and 2, Low-density lipoprotein receptor, Phosphatidylinositol binding clathrin assembly protein, Alpha- synuclein and Platelet-activating factor receptor) and an endosomal recycling pathway involved in cholesterol metabolism (Eps15 homology domain 1,2 and 4, Proprotein convertase subtilisin/kexin type 9 and Low-density lipoprotein receptor). Several novel and existing interactors were identified and these interactions were validated using co-immunopurification, which could be the basis for future research. ii ACKNOWLEDGEMENTS I would like to take this opportunity to thank my supervisor, Dr. Daniel Figeys, for his support and guidance throughout my studies in his lab. It was a great experience to work in his lab and I am very thankful I was given the chance to learn and work under him. I would also like to thank the members of my lab for all their assistance in learning new techniques and equipment in the lab. -
Dependent Traits in Mice Models of Hyperthyroidism and Hypothyroidism
Phenotypical characterization of sex- dependent traits in mice models of hyperthyroidism and hypothyroidism Inaugural-Dissertation zur Erlangung des Doktorgrades Dr. rer. nat. der Fakultät für Biologie an der Universität Duisburg-Essen vorgelegt von Helena Rakov aus St. Petropawlowsk April 2017 Die der vorliegenden Arbeit zugrunde liegenden Experimente wurden am Universitätsklinikum Essen in der Klinik für Endokrinologie und Stoffwechselerkrankungen durchgeführt. 1. Gutachter: Prof. Dr. Dr. Dagmar Führer-Sakel 2. Gutachter: Prof. Dr. Elke Cario Vorsitzender des Prüfungsausschusses: Prof. Dr. Ruth Grümmer Tag der mündlichen Prüfung: 17.07.2017 Publications Publications Engels Kathrin*, Rakov Helena *, Zwanziger Denise, Moeller Lars C., Homuth Georg, Köhrle Josef, Brix Klaudia, Fuhrer Dagmar. Differences in mouse hepatic thyroid hormone transporter expression with age and hyperthyroidism. Eur Thyroid J 2015;4(suppl 1):81–86. DOI: 10.1159/000381020. *contributed equally Zwanziger Denise*, Rakov Helena*, Engels Kathrin, Moeller Lars C., Fuhrer Dagmar. Sex-dependent claudin-1 expression in liver of eu- and hypothyroid mice. Eur Thyroid J. 2015 Sep; 4(Suppl 1): 67–73. DOI: 10.1159/000431316. *contributed equally Engels Kathrin*, Rakov Helena*, Zwanziger Denise, Hoenes Georg Sebastian, Rehders Maren, Brix Klaudia, Koehrle Josef, Moeller Lars Christian, Fuhrer Dagmar. Efficacy of protocols for induction of chronic hyperthyroidism in male and female mice. Endocrine. 2016 Oct;54(1):47-54. DOI: 10.1007/s12020-016-1020-8. Rakov Helena*, Engels Kathrin*, Hönes Georg Sebastian, Strucksberg Karl-Heinz, Moeller Lars Christian, Köhrle Josef, Zwanziger Denise, Führer Dagmar. Sex-specific phenotypes of hyperthyroidism and hypothyroidism in mice. Biol Sex Differ. 2016 Aug 24;7(1):36. DOI: 10.1186/s13293-016-0089-3. -
Anti-Inflammatory Role of Curcumin in LPS Treated A549 Cells at Global Proteome Level and on Mycobacterial Infection
Anti-inflammatory Role of Curcumin in LPS Treated A549 cells at Global Proteome level and on Mycobacterial infection. Suchita Singh1,+, Rakesh Arya2,3,+, Rhishikesh R Bargaje1, Mrinal Kumar Das2,4, Subia Akram2, Hossain Md. Faruquee2,5, Rajendra Kumar Behera3, Ranjan Kumar Nanda2,*, Anurag Agrawal1 1Center of Excellence for Translational Research in Asthma and Lung Disease, CSIR- Institute of Genomics and Integrative Biology, New Delhi, 110025, India. 2Translational Health Group, International Centre for Genetic Engineering and Biotechnology, New Delhi, 110067, India. 3School of Life Sciences, Sambalpur University, Jyoti Vihar, Sambalpur, Orissa, 768019, India. 4Department of Respiratory Sciences, #211, Maurice Shock Building, University of Leicester, LE1 9HN 5Department of Biotechnology and Genetic Engineering, Islamic University, Kushtia- 7003, Bangladesh. +Contributed equally for this work. S-1 70 G1 S 60 G2/M 50 40 30 % of cells 20 10 0 CURI LPSI LPSCUR Figure S1: Effect of curcumin and/or LPS treatment on A549 cell viability A549 cells were treated with curcumin (10 µM) and/or LPS or 1 µg/ml for the indicated times and after fixation were stained with propidium iodide and Annexin V-FITC. The DNA contents were determined by flow cytometry to calculate percentage of cells present in each phase of the cell cycle (G1, S and G2/M) using Flowing analysis software. S-2 Figure S2: Total proteins identified in all the three experiments and their distribution betwee curcumin and/or LPS treated conditions. The proteins showing differential expressions (log2 fold change≥2) in these experiments were presented in the venn diagram and certain number of proteins are common in all three experiments. -
Supplementary Material
BMJ Publishing Group Limited (BMJ) disclaims all liability and responsibility arising from any reliance Supplemental material placed on this supplemental material which has been supplied by the author(s) J Neurol Neurosurg Psychiatry Page 1 / 45 SUPPLEMENTARY MATERIAL Appendix A1: Neuropsychological protocol. Appendix A2: Description of the four cases at the transitional stage. Table A1: Clinical status and center proportion in each batch. Table A2: Complete output from EdgeR. Table A3: List of the putative target genes. Table A4: Complete output from DIANA-miRPath v.3. Table A5: Comparison of studies investigating miRNAs from brain samples. Figure A1: Stratified nested cross-validation. Figure A2: Expression heatmap of miRNA signature. Figure A3: Bootstrapped ROC AUC scores. Figure A4: ROC AUC scores with 100 different fold splits. Figure A5: Presymptomatic subjects probability scores. Figure A6: Heatmap of the level of enrichment in KEGG pathways. Kmetzsch V, et al. J Neurol Neurosurg Psychiatry 2021; 92:485–493. doi: 10.1136/jnnp-2020-324647 BMJ Publishing Group Limited (BMJ) disclaims all liability and responsibility arising from any reliance Supplemental material placed on this supplemental material which has been supplied by the author(s) J Neurol Neurosurg Psychiatry Appendix A1. Neuropsychological protocol The PREV-DEMALS cognitive evaluation included standardized neuropsychological tests to investigate all cognitive domains, and in particular frontal lobe functions. The scores were provided previously (Bertrand et al., 2018). Briefly, global cognitive efficiency was evaluated by means of Mini-Mental State Examination (MMSE) and Mattis Dementia Rating Scale (MDRS). Frontal executive functions were assessed with Frontal Assessment Battery (FAB), forward and backward digit spans, Trail Making Test part A and B (TMT-A and TMT-B), Wisconsin Card Sorting Test (WCST), and Symbol-Digit Modalities test. -
Identification of Potential Key Genes and Pathway Linked with Sporadic Creutzfeldt-Jakob Disease Based on Integrated Bioinformatics Analyses
medRxiv preprint doi: https://doi.org/10.1101/2020.12.21.20248688; this version posted December 24, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. All rights reserved. No reuse allowed without permission. Identification of potential key genes and pathway linked with sporadic Creutzfeldt-Jakob disease based on integrated bioinformatics analyses Basavaraj Vastrad1, Chanabasayya Vastrad*2 , Iranna Kotturshetti 1. Department of Biochemistry, Basaveshwar College of Pharmacy, Gadag, Karnataka 582103, India. 2. Biostatistics and Bioinformatics, Chanabasava Nilaya, Bharthinagar, Dharwad 580001, Karanataka, India. 3. Department of Ayurveda, Rajiv Gandhi Education Society`s Ayurvedic Medical College, Ron, Karnataka 562209, India. * Chanabasayya Vastrad [email protected] Ph: +919480073398 Chanabasava Nilaya, Bharthinagar, Dharwad 580001 , Karanataka, India NOTE: This preprint reports new research that has not been certified by peer review and should not be used to guide clinical practice. medRxiv preprint doi: https://doi.org/10.1101/2020.12.21.20248688; this version posted December 24, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. All rights reserved. No reuse allowed without permission. Abstract Sporadic Creutzfeldt-Jakob disease (sCJD) is neurodegenerative disease also called prion disease linked with poor prognosis. The aim of the current study was to illuminate the underlying molecular mechanisms of sCJD. The mRNA microarray dataset GSE124571 was downloaded from the Gene Expression Omnibus database. Differentially expressed genes (DEGs) were screened. -
Unintentional Genomic Changes Endow Cupriavidus Metallidurans with an Augmented Heavy-Metal Resistance
G C A T T A C G G C A T genes Article Unintentional Genomic Changes Endow Cupriavidus metallidurans with an Augmented Heavy-Metal Resistance Felipe A. Millacura 1, Paul J. Janssen 2, Pieter Monsieurs 2, Ann Janssen 2, Ann Provoost 2, Rob Van Houdt 2 and Luis A. Rojas 3,* 1 School of Biological Sciences, University of Edinburgh, Edinburgh EH9 3JQ, UK; [email protected] 2 Interdisciplinary Biosciences, Belgian Nuclear Research Centre, SCK•CEN, 2400 Mol, Belgium; [email protected] (P.J.J.); [email protected] (P.M.); [email protected] (A.J); [email protected] (A.P.); [email protected] (R.V.H.) 3 Chemistry Department, Faculty of Sciences, Universidad Católica del Norte, UCN, Antofagasta 1240000, Chile * Correspondence: [email protected]; Tel.: +56-55-235-5629 Received: 6 October 2018; Accepted: 8 November 2018; Published: 13 November 2018 Abstract: For the past three decades, Cupriavidus metallidurans has been one of the major model organisms for bacterial tolerance to heavy metals. Its type strain CH34 contains at least 24 gene clusters distributed over four replicons, allowing for intricate and multilayered metal responses. To gain organic mercury resistance in CH34, broad-spectrum mer genes were introduced in a previous work via conjugation of the IncP-1β plasmid pTP6. However, we recently noted that this CH34-derived strain, MSR33, unexpectedly showed an increased resistance to other metals (i.e., Co2+, Ni2+, and Cd2+). To thoroughly investigate this phenomenon, we resequenced the entire genome of MSR33 and compared its DNA sequence and basal gene expression profile to those of its parental strain CH34. -
Regulator of G Protein Signaling 2 Mediates Cardiac Compensation to Pressure Overload and Antihypertrophic Effects of PDE5 Inhibition in Mice
Regulator of G protein signaling 2 mediates cardiac compensation to pressure overload and antihypertrophic effects of PDE5 inhibition in mice Eiki Takimoto, … , Michael E. Mendelsohn, David A. Kass J Clin Invest. 2009;119(2):408-420. https://doi.org/10.1172/JCI35620. Research Article Cardiology The heart initially compensates for hypertension-mediated pressure overload by enhancing its contractile force and developing hypertrophy without dilation. Gq protein–coupled receptor pathways become activated and can depress function, leading to cardiac failure. Initial adaptation mechanisms to reduce cardiac damage during such stimulation remain largely unknown. Here we have shown that this initial adaptation requires regulator of G protein signaling 2 (RGS2). Mice lacking RGS2 had a normal basal cardiac phenotype, yet responded rapidly to pressure overload, with increased myocardial Gq signaling, marked cardiac hypertrophy and failure, and early mortality. Swimming exercise, which is not accompanied by Gq activation, induced a normal cardiac response, while Rgs2 deletion in Gαq- overexpressing hearts exacerbated hypertrophy and dilation. In vascular smooth muscle, RGS2 is activated by cGMP- –/– dependent protein kinase (PKG), suppressing Gq-stimulated vascular contraction. In normal mice, but not Rgs2 mice, PKG activation by the chronic inhibition of cGMP-selective phosphodiesterase 5 (PDE5) suppressed maladaptive cardiac hypertrophy, inhibiting Gq-coupled stimuli. Importantly, PKG was similarly activated by PDE5 inhibition in myocardium from both genotypes, but PKG plasma membrane translocation was more transient in Rgs2–/– myocytes than in controls and was unaffected by PDE5 inhibition. Thus, RGS2 is required for early myocardial compensation to pressure overload and mediates the initial antihypertrophic and cardioprotective effects of PDE5 inhibitors. -
Early Growth Response 1 Regulates Hematopoietic Support and Proliferation in Human Primary Bone Marrow Stromal Cells
Hematopoiesis SUPPLEMENTARY APPENDIX Early growth response 1 regulates hematopoietic support and proliferation in human primary bone marrow stromal cells Hongzhe Li, 1,2 Hooi-Ching Lim, 1,2 Dimitra Zacharaki, 1,2 Xiaojie Xian, 2,3 Keane J.G. Kenswil, 4 Sandro Bräunig, 1,2 Marc H.G.P. Raaijmakers, 4 Niels-Bjarne Woods, 2,3 Jenny Hansson, 1,2 and Stefan Scheding 1,2,5 1Division of Molecular Hematology, Department of Laboratory Medicine, Lund University, Lund, Sweden; 2Lund Stem Cell Center, Depart - ment of Laboratory Medicine, Lund University, Lund, Sweden; 3Division of Molecular Medicine and Gene Therapy, Department of Labora - tory Medicine, Lund University, Lund, Sweden; 4Department of Hematology, Erasmus MC Cancer Institute, Rotterdam, the Netherlands and 5Department of Hematology, Skåne University Hospital Lund, Skåne, Sweden ©2020 Ferrata Storti Foundation. This is an open-access paper. doi:10.3324/haematol. 2019.216648 Received: January 14, 2019. Accepted: July 19, 2019. Pre-published: August 1, 2019. Correspondence: STEFAN SCHEDING - [email protected] Li et al.: Supplemental data 1. Supplemental Materials and Methods BM-MNC isolation Bone marrow mononuclear cells (BM-MNC) from BM aspiration samples were isolated by density gradient centrifugation (LSM 1077 Lymphocyte, PAA, Pasching, Austria) either with or without prior incubation with RosetteSep Human Mesenchymal Stem Cell Enrichment Cocktail (STEMCELL Technologies, Vancouver, Canada) for lineage depletion (CD3, CD14, CD19, CD38, CD66b, glycophorin A). BM-MNCs from fetal long bones and adult hip bones were isolated as reported previously 1 by gently crushing bones (femora, tibiae, fibulae, humeri, radii and ulna) in PBS+0.5% FCS subsequent passing of the cell suspension through a 40-µm filter. -
The Microbiota-Produced N-Formyl Peptide Fmlf Promotes Obesity-Induced Glucose
Page 1 of 230 Diabetes Title: The microbiota-produced N-formyl peptide fMLF promotes obesity-induced glucose intolerance Joshua Wollam1, Matthew Riopel1, Yong-Jiang Xu1,2, Andrew M. F. Johnson1, Jachelle M. Ofrecio1, Wei Ying1, Dalila El Ouarrat1, Luisa S. Chan3, Andrew W. Han3, Nadir A. Mahmood3, Caitlin N. Ryan3, Yun Sok Lee1, Jeramie D. Watrous1,2, Mahendra D. Chordia4, Dongfeng Pan4, Mohit Jain1,2, Jerrold M. Olefsky1 * Affiliations: 1 Division of Endocrinology & Metabolism, Department of Medicine, University of California, San Diego, La Jolla, California, USA. 2 Department of Pharmacology, University of California, San Diego, La Jolla, California, USA. 3 Second Genome, Inc., South San Francisco, California, USA. 4 Department of Radiology and Medical Imaging, University of Virginia, Charlottesville, VA, USA. * Correspondence to: 858-534-2230, [email protected] Word Count: 4749 Figures: 6 Supplemental Figures: 11 Supplemental Tables: 5 1 Diabetes Publish Ahead of Print, published online April 22, 2019 Diabetes Page 2 of 230 ABSTRACT The composition of the gastrointestinal (GI) microbiota and associated metabolites changes dramatically with diet and the development of obesity. Although many correlations have been described, specific mechanistic links between these changes and glucose homeostasis remain to be defined. Here we show that blood and intestinal levels of the microbiota-produced N-formyl peptide, formyl-methionyl-leucyl-phenylalanine (fMLF), are elevated in high fat diet (HFD)- induced obese mice. Genetic or pharmacological inhibition of the N-formyl peptide receptor Fpr1 leads to increased insulin levels and improved glucose tolerance, dependent upon glucagon- like peptide-1 (GLP-1). Obese Fpr1-knockout (Fpr1-KO) mice also display an altered microbiome, exemplifying the dynamic relationship between host metabolism and microbiota. -
Supplementary Information
Supplementary information (a) (b) Figure S1. Resistant (a) and sensitive (b) gene scores plotted against subsystems involved in cell regulation. The small circles represent the individual hits and the large circles represent the mean of each subsystem. Each individual score signifies the mean of 12 trials – three biological and four technical. The p-value was calculated as a two-tailed t-test and significance was determined using the Benjamini-Hochberg procedure; false discovery rate was selected to be 0.1. Plots constructed using Pathway Tools, Omics Dashboard. Figure S2. Connectivity map displaying the predicted functional associations between the silver-resistant gene hits; disconnected gene hits not shown. The thicknesses of the lines indicate the degree of confidence prediction for the given interaction, based on fusion, co-occurrence, experimental and co-expression data. Figure produced using STRING (version 10.5) and a medium confidence score (approximate probability) of 0.4. Figure S3. Connectivity map displaying the predicted functional associations between the silver-sensitive gene hits; disconnected gene hits not shown. The thicknesses of the lines indicate the degree of confidence prediction for the given interaction, based on fusion, co-occurrence, experimental and co-expression data. Figure produced using STRING (version 10.5) and a medium confidence score (approximate probability) of 0.4. Figure S4. Metabolic overview of the pathways in Escherichia coli. The pathways involved in silver-resistance are coloured according to respective normalized score. Each individual score represents the mean of 12 trials – three biological and four technical. Amino acid – upward pointing triangle, carbohydrate – square, proteins – diamond, purines – vertical ellipse, cofactor – downward pointing triangle, tRNA – tee, and other – circle. -
ATP2A2 Gene Atpase Sarcoplasmic/Endoplasmic Reticulum Ca2+ Transporting 2
ATP2A2 gene ATPase sarcoplasmic/endoplasmic reticulum Ca2+ transporting 2 Normal Function The ATP2A2 gene provides instructions for making an enzyme called sarco(endo) plasmic reticulum calcium-ATPase 2 (SERCA2). This enzyme belongs to a family of ATPase enzymes that helps control the level of positively charged calcium atoms ( calcium ions) inside cells. Within the cell, SERCA2 is found in the endoplasmic reticulum and a related structure in muscle cells called the sarcoplasmic reticulum. The endoplasmic reticulum is a structure inside the cell that is involved in protein processing and transport. The sarcoplasmic reticulum assists with muscle contraction and relaxation by releasing and storing calcium ions. Calcium ions act as signals for a large number of activities that are important for the normal development and function of cells. SERCA2 allows calcium ions to pass into and out of the cell in response to cell signals. Health Conditions Related to Genetic Changes Darier disease More than 130 mutations in the ATP2A2 gene have been found to cause Darier disease. Most of these mutations change a single protein building block (amino acid) in the SERCA2 enzyme. All mutations cause the production of a nonfunctional SERCA2 enzyme or cause no SERCA2 to be produced from one copy of the gene. Cells with only one functional copy of the ATP2A2 gene produce half the normal amount of SERCA2 protein. It is thought that insufficient amounts of SERCA2 combined with outside factors such as heat and minor injury cause the signs and symptoms of Darier -
Supplementary Informations SI2. Supplementary Table 1
Supplementary Informations SI2. Supplementary Table 1. M9, soil, and rhizosphere media composition. LB in Compound Name Exchange Reaction LB in soil LBin M9 rhizosphere H2O EX_cpd00001_e0 -15 -15 -10 O2 EX_cpd00007_e0 -15 -15 -10 Phosphate EX_cpd00009_e0 -15 -15 -10 CO2 EX_cpd00011_e0 -15 -15 0 Ammonia EX_cpd00013_e0 -7.5 -7.5 -10 L-glutamate EX_cpd00023_e0 0 -0.0283302 0 D-glucose EX_cpd00027_e0 -0.61972444 -0.04098397 0 Mn2 EX_cpd00030_e0 -15 -15 -10 Glycine EX_cpd00033_e0 -0.0068175 -0.00693094 0 Zn2 EX_cpd00034_e0 -15 -15 -10 L-alanine EX_cpd00035_e0 -0.02780553 -0.00823049 0 Succinate EX_cpd00036_e0 -0.0056245 -0.12240603 0 L-lysine EX_cpd00039_e0 0 -10 0 L-aspartate EX_cpd00041_e0 0 -0.03205557 0 Sulfate EX_cpd00048_e0 -15 -15 -10 L-arginine EX_cpd00051_e0 -0.0068175 -0.00948672 0 L-serine EX_cpd00054_e0 0 -0.01004986 0 Cu2+ EX_cpd00058_e0 -15 -15 -10 Ca2+ EX_cpd00063_e0 -15 -100 -10 L-ornithine EX_cpd00064_e0 -0.0068175 -0.00831712 0 H+ EX_cpd00067_e0 -15 -15 -10 L-tyrosine EX_cpd00069_e0 -0.0068175 -0.00233919 0 Sucrose EX_cpd00076_e0 0 -0.02049199 0 L-cysteine EX_cpd00084_e0 -0.0068175 0 0 Cl- EX_cpd00099_e0 -15 -15 -10 Glycerol EX_cpd00100_e0 0 0 -10 Biotin EX_cpd00104_e0 -15 -15 0 D-ribose EX_cpd00105_e0 -0.01862144 0 0 L-leucine EX_cpd00107_e0 -0.03596182 -0.00303228 0 D-galactose EX_cpd00108_e0 -0.25290619 -0.18317325 0 L-histidine EX_cpd00119_e0 -0.0068175 -0.00506825 0 L-proline EX_cpd00129_e0 -0.01102953 0 0 L-malate EX_cpd00130_e0 -0.03649016 -0.79413596 0 D-mannose EX_cpd00138_e0 -0.2540567 -0.05436649 0 Co2 EX_cpd00149_e0