DOCSLIB.ORG

( 12 ) Patent Application Publication ( 10 ) Pub . No .: US 2021/0005284 A1 Nuzhdina Et Al

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
( 12 ) Patent Application Publication ( 10 ) Pub . No .: US 2021/0005284 A1 Nuzhdina Et Al US 20210005284A1 IN ( 19 ) United States ( 12 ) Patent Application Publication ( 10 ) Pub . No .: US 2021/0005284 A1 Nuzhdina et al . ( 43 ) Pub . Date : Jan. 7 , 2021 ( 54 ) TECHNIQUES FOR NUCLEIC ACID DATA G16B 25/10 ( 2006.01 ) QUALITY CONTROL G16B 35/10 ( 2006.01 ) ( 52 ) U.S. CI . ( 71 ) Applicant: BostonGene Corporation , Waltham , CPC G16B 30/10 ( 2019.02 ) ; G16B 35/10 MA ( US ) ( 2019.02 ) ; G16B 25/10 ( 2019.02 ) ; G16B ( 72 ) Inventors : Ekaterina Nuzhdina , Moscow ( RU ); 20/20 ( 2019.02 ) Alexander Bagaev , Moscow ( RU ); Maksim Chelushkin , Moscow (RU ) ; Yaroslav Lozinsky, Moscow ( RU ); ( 57 ) ABSTRACT Natalia Miheecheva , Moscow ( RU ) ; Alexander Zaitsev , Leninskiy R - N (RU ) Described herein are various methods of collecting and processing of tumor and /or healthy tissue samples to extract ( 21 ) Appl . No .: 16 / 920,641 nucleic acid and perform nucleic acid sequencing . Also described herein are various methods of processing nucleic ( 22 ) Filed : Jul . 3 , 2020 acid sequencing data to remove bias from the nucleic acid sequencing data . Also described herein are various methods Related U.S. Application Data of evaluating the quality of nucleic acid sequence informa ( 60 ) Provisional application No. 62 / 991,570 , filed on Mar. tion . The identity and / or integrity of nucleic acid sequence 18 , 2020 , provisional application No. 62 /870,622 , data is evaluated prior to using the sequence information for filed on Jul. 3 , 2019 . subsequent analysis ( for example for diagnostic , prognostic , or clinical purposes ). The methods enable a subject, doctor, Publication Classification or user to characterize or classify various types of cancer ( 51 ) Int . Cl . precisely , and thereby determine a therapy or combination of G16B 30/10 ( 2006.01 ) therapies that may be effective to treat a cancer in a subject G16B 20/20 ( 2006.01 ) based on the precise characterization . 110 BEGIN Obtain a biological sample ( e.g. , a tumor sampte ) from a subject 111 having, suspected of having , or at risk of having cancer Extract nucleic acid from the biological sample 112 Perform one or more quality control assessments of extracted nucleic 113 acid Prepare nucleic acid library with extracted nucleic acid -114 that satisfies nucleic acid quality control Sequence DNA and / or RNA nucleic acid , -115 obtain RNA expression data Process RNA expression data to obtain bias - corrected gene 116 expression data Perform one or more quality control assessmients on gene -117 expression data Analyze gene expression data 118 Identify a cancer treatment for the subject based on 119 the gene expression data End Patent Application Publication Jan. 7 , 2021 Sheet 1 of 22 US 2021/0005284 A1 BEGIN 200100 Obtain a sample of a tumor from a subject having , suspected of 101 having , or at risk of having cancer 102 Quality control step 1 - Perform one or more quality control assessments on tumor sample Quality control step 2 - Perform one or more quality control assessments on 103 DNA , RNA , and / or libraries processes Quality control step 3 - Perform one or more quality control 104 assessments on nucleic acid sequence data End FIG . 1A Patent Application Publication Jan. 7 , 2021 Sheet 2 of 22 US 2021/0005284 A1 110 BEGIN Obtain a biological sample ( e.g., a tumor sample ) from a subject 111 having , suspected of having, or at risk of having cancer Extract nucleic acid from the biological sample 112 Perform one or more quality control assessments of extracted nucleic 113 acid Prepare nucleic acid library with extracted nucleic acid -114 that satisfies nucleic acid quality control Sequence DNA and / or RNA nucleic acid , 115 obtain RNA expression data Process RNA expression data to obtain bias - corrected gene -116 expression data Perform one or more quality control assessments on gene expression data ' 117 Analyze gene expression data -118 Identify a cancer treatment for the subject based on 119 the gene expression data End FIG . 1B Patent Application Publication Jan. 7 , 2021 Sheet 3 of 22 US 2021/0005284 A1 enrichmentpolyA FIG.2A rRNAdepletion Patent Application Publication Jan. 7 , 2021 Sheet 4 of 22 US 2021/0005284 A1 FIG.2B WX08 ) uossa Patent Application Publication Jan. 7 , 2021 Sheet 5 of 22 US 2021/0005284 A1 IILE Samples FIG . 3 Patent Application Publication Jan. 7 , 2021 Sheet 6 of 22 US 2021/0005284 A1 Histone length ( Hela ) Gene Median length , nt HIST1H4C 20,0 HISTIH1C 25,5 HIST1H2BK 71,0 HIST1H2AE 14,0 HIST1H3B 15,0 HIST3H2A 17,0 FIG . 4A Patent Application Publication Jan. 7 , 2021 Sheet 7 of 22 US 2021/0005284 A1 Polya Total FIG . 4B Patent Application Publication Jan. 7 , 2021 Sheet 8 of 22 US 2021/0005284 A1 SIKIO Polya Total FIG . 4C Patent Application Publication Jan. 7 , 2021 Sheet 9 of 22 US 2021/0005284 A1 b FIG . 5 Patent Application Publication Jan. 7. 2021 Sheet 10 of 22 US 2021/0005284 A1 BEGIN 200 Obtain a first sample of a first tumor from a -201 subject having , suspected of having , or at risk of having cancer 202 Extract RNA from the first sample of the first tumor Enrich the extracted RNA for coding RNA to obtain 203 enriched RNA Prepare a first library of CDNA fragments from the 204 enriched RNA for non - stranded RNA sequencing Perform non - stranded RNA sequencing on 205 the first library of cDNA fragments from the enriched RNA End FIG . 6A Patent Application Publication Jan. 7 , 2021 Sheet 11 of 22 US 2021/0005284 A1 BEGIN 210 Obtain RNA expression data for a subject having , suspected of 211 having , or at risk of having cancer Align the RNA expression data to a reference and annotate the RNA * 212 expression data Remove non - coding transcripts from the annotated RNA expression data to -213 obtain filtered RNA expression data Normalize the filtered RNA expression data to obtain gene expression 214 data in transcripts per million ( TPM ) Identify at least one gene that introduces bias in the gene expression data 215 Remove at least one gene from the gene expression data to obtain 216 bias - corrected gene expression data Identify a cancer treatment for the subject using the bias - corrected -217 gene expression data End FIG . 6B Patent Application Publication Jan. 7 , 2021 Sheet 12 of 22 US 2021/0005284 A1 220 BEGIN Enrich RNA for coding RNA in a sample of extracted RNA from a first 221 tumor sample from a subject having , suspected of having , or at risk of having cancer Perform non - stranded RNA sequencing on a first library of cDNA fragments prepared from the enriched RNA to obtain RNA 222 expression data Convert the RNA expression data to gene expression data 223 Identify at least one gene that introduces bias in the gene expression 224 data in transcripts per kilobase million ( TPM ) Remove at least one gene from the gene expression data to 225 obtain bias - corrected gene expression data Identify a cancer treatment for the subject using the 226 bias - corrected gene expression data End FIG . 6C Patent Application Publication Jan. 7. 2021 Sheet 13 of 22 US 2021/0005284 A1 300 308 301 309 302 . 310 303 311 304 . 312 305 313 306 314 307 315 FIG . 7 Patent Application Publication Jan. 7 , 2021 Sheet 14 of 22 US 2021/0005284 A1 800 Obtain nucleic acid data comprising : sequence data and asserted information indicating an asserted 801 source and / or an asserted integrity of the sequence data Process the nucleic acid data 802a 802b 802 Process the sequence data to Process the sequence data to obtain determined information obtain determined information indicating a determined source indicating a determined of the sequence data integrity of the sequence data 803 Match ? Yes No Process the sequence data to determine whether the sequence data is indicative of one or more disease 804 features and / or determine a therapy for the subject Perform remedial action ( s ) 805 FIG . 8 Patent Application Publication Jan. 7 , 2021 Sheet 15 of 22 US 2021/0005284 A1 500 Kiowaw 520 550 FIG9. 540 510 530 Patent Application Publication Jan. 7 , 2021 Sheet 16 of 22 US 2021/0005284 A1 640 660 600 620 670 610 FIG.10 SampleBokcal 089 680 *** 650 OD Patent Application Publication Jan. 7 , 2021 Sheet 17 of 22 US 2021/0005284 A1 301302303 Normal 60400701 8:00 C.06:02 83 WES 8,04:0103:03 0:0802,06:02 8:5301.550 FIG.11 FIG.12 RNA-Seq C.06:02,06:02 8370103 8633602 3703 103 Patent Application Publication Jan. 7. 2021 Sheet 18 of 22 US 2021/0005284 A1 FIG.13A Good *** Patent Application Publication Jan. 7. 2021 Sheet 19 of 22 US 2021/0005284 A1 FIG.13B Patent Application Publication Jan. 7. 2021 Sheet 20 of 22 US 2021/0005284 A1 Total FIG.14A Polya VAjodjejoi jo killigeqoid Patent Application Publication Jan. 7. 2021 Sheet 21 of 22 US 2021/0005284 A1 Total Polya FIG.14B 0.5 Vhod / e10l jo yliqeqoid Patent Application Publication Jan. 7 , 2021 Sheet 22 of 22 US 2021/0005284 A1 ...ilili *** 369211 260240, FIG.15 US 2021/0005284 A1 Jan. 7 , 2021 1 TECHNIQUES FOR NUCLEIC ACID DATA or a determined integrity of the sequence data ; and deter QUALITY CONTROL mining whether the determined information matches the asserted information . CROSS REFERENCE TO RELATED [ 0005 ] Some embodiments provide at least one non - tran APPLICATIONS sitory computer - readable storage medium storing processor executable instructions that, when executed by at least one [ 0001 ] This application claims the benefit under 35 U.S.C. computer hardware processor, cause the at least one com $ 119 ( e ) of U.S. Provisional Application No. 62 / 870,622 , puter hardware processor to perform a method . The method filed Jul . 3 , 2019 , entitled “ Compositions and Methods for comprising: obtaining nucleic acid data comprising : Sample Preparation and Characterization of Cancer There sequence data indicating a nucleotide sequence for at least 5 from " and U.S.
Load more

Recommended publications
  • Histone Isoform H2A1H Promotes Attainment of Distinct Physiological
    Bhattacharya et al. Epigenetics & Chromatin (2017) 10:48 DOI 10.1186/s13072-017-0155-z Epigenetics & Chromatin RESEARCH Open Access Histone isoform H2A1H promotes attainment of distinct physiological states by altering chromatin dynamics Saikat Bhattacharya1,4,6, Divya Reddy1,4, Vinod Jani5†, Nikhil Gadewal3†, Sanket Shah1,4, Raja Reddy2,4, Kakoli Bose2,4, Uddhavesh Sonavane5, Rajendra Joshi5 and Sanjay Gupta1,4* Abstract Background: The distinct functional efects of the replication-dependent histone H2A isoforms have been dem- onstrated; however, the mechanistic basis of the non-redundancy remains unclear. Here, we have investigated the specifc functional contribution of the histone H2A isoform H2A1H, which difers from another isoform H2A2A3 in the identity of only three amino acids. Results: H2A1H exhibits varied expression levels in diferent normal tissues and human cancer cell lines (H2A1C in humans). It also promotes cell proliferation in a context-dependent manner when exogenously overexpressed. To uncover the molecular basis of the non-redundancy, equilibrium unfolding of recombinant H2A1H-H2B dimer was performed. We found that the M51L alteration at the H2A–H2B dimer interface decreases the temperature of melting of H2A1H-H2B by ~ 3 °C as compared to the H2A2A3-H2B dimer. This diference in the dimer stability is also refected in the chromatin dynamics as H2A1H-containing nucleosomes are more stable owing to M51L and K99R substitu- tions. Molecular dynamic simulations suggest that these substitutions increase the number of hydrogen bonds and hydrophobic interactions of H2A1H, enabling it to form more stable nucleosomes. Conclusion: We show that the M51L and K99R substitutions, besides altering the stability of histone–histone and histone–DNA complexes, have the most prominent efect on cell proliferation, suggesting that the nucleosome sta- bility is intimately linked with the physiological efects observed.
    [Show full text]
  • University of California, San Diego
    UNIVERSITY OF CALIFORNIA, SAN DIEGO The post-terminal differentiation fate of RNAs revealed by next-generation sequencing A dissertation submitted in partial satisfaction of the requirements for the degree Doctor of Philosophy in Biomedical Sciences by Gloria Kuo Lefkowitz Committee in Charge: Professor Benjamin D. Yu, Chair Professor Richard Gallo Professor Bruce A. Hamilton Professor Miles F. Wilkinson Professor Eugene Yeo 2012 Copyright Gloria Kuo Lefkowitz, 2012 All rights reserved. The Dissertation of Gloria Kuo Lefkowitz is approved, and it is acceptable in quality and form for publication on microfilm and electronically: __________________________________________________________________ __________________________________________________________________ __________________________________________________________________ __________________________________________________________________ __________________________________________________________________ Chair University of California, San Diego 2012 iii DEDICATION Ma and Ba, for your early indulgence and support. Matt and James, for choosing more practical callings. Roy, my love, for patiently sharing the ups and downs of this journey. iv EPIGRAPH It is foolish to tear one's hair in grief, as though sorrow would be made less by baldness. ~Cicero v TABLE OF CONTENTS Signature Page .............................................................................................................. iii Dedication ....................................................................................................................
    [Show full text]
  • 1General Introduction and Outline Glycosphingolipids, Carbohydrate
    Lipophilic iminosugars : synthesis and evaluation as inhibitors of glucosylceramide metabolism Wennekes, T. Citation Wennekes, T. (2008, December 15). Lipophilic iminosugars : synthesis and evaluation as inhibitors of glucosylceramide metabolism. Retrieved from https://hdl.handle.net/1887/13372 Version: Corrected Publisher’s Version Licence agreement concerning inclusion of doctoral thesis in the License: Institutional Repository of the University of Leiden Downloaded from: https://hdl.handle.net/1887/13372 Note: To cite this publication please use the final published version (if applicable). General Introduction and Outline Glycosphingolipids, Carbohydrate- 1 processing Enzymes and Iminosugar Inhibitors General Introduction The study described in this thesis was conducted with the aim of developing lipophilic iminosugars as selective inhibitors for three enzymes involved in glucosylceramide metabolism. Glucosylceramide, a β-glycoside of the lipid ceramide and the carbohydrate d-glucose, is a key member of a class of biomolecules called the glycosphingolipids (GSLs). One enzyme, glucosylceramide synthase (GCS), is responsible for its synthesis and the two other enzymes, glucocerebrosidase (GBA1) and β-glucosidase 2 (GBA2), catalyze its degradation. Being able to influence glucosylceramide biosynthesis and degradation would greatly facilitate the study of GSL functioning in (patho)physiological processes. This chapter aims to provide background information and some history on the various subjects that were involved in this study. The chapter will start out with a brief overview of the discovery of GSLs and the evolving view of the biological role of GSLs and carbohydrate containing biomolecules in general during the last century. Next, the topology and dynamics of mammalian GSL biosynthesis and degradation will be described with special attention for the involved carbohydrate-processing enzymes.
    [Show full text]
  • Environmental Influences on Endothelial Gene Expression
    ENDOTHELIAL CELL GENE EXPRESSION John Matthew Jeff Herbert Supervisors: Prof. Roy Bicknell and Dr. Victoria Heath PhD thesis University of Birmingham August 2012 University of Birmingham Research Archive e-theses repository This unpublished thesis/dissertation is copyright of the author and/or third parties. The intellectual property rights of the author or third parties in respect of this work are as defined by The Copyright Designs and Patents Act 1988 or as modified by any successor legislation. Any use made of information contained in this thesis/dissertation must be in accordance with that legislation and must be properly acknowledged. Further distribution or reproduction in any format is prohibited without the permission of the copyright holder. ABSTRACT Tumour angiogenesis is a vital process in the pathology of tumour development and metastasis. Targeting markers of tumour endothelium provide a means of targeted destruction of a tumours oxygen and nutrient supply via destruction of tumour vasculature, which in turn ultimately leads to beneficial consequences to patients. Although current anti -angiogenic and vascular targeting strategies help patients, more potently in combination with chemo therapy, there is still a need for more tumour endothelial marker discoveries as current treatments have cardiovascular and other side effects. For the first time, the analyses of in-vivo biotinylation of an embryonic system is performed to obtain putative vascular targets. Also for the first time, deep sequencing is applied to freshly isolated tumour and normal endothelial cells from lung, colon and bladder tissues for the identification of pan-vascular-targets. Integration of the proteomic, deep sequencing, public cDNA libraries and microarrays, delivers 5,892 putative vascular targets to the science community.
    [Show full text]
  • A Computational Approach for Defining a Signature of Β-Cell Golgi Stress in Diabetes Mellitus
    Page 1 of 781 Diabetes A Computational Approach for Defining a Signature of β-Cell Golgi Stress in Diabetes Mellitus Robert N. Bone1,6,7, Olufunmilola Oyebamiji2, Sayali Talware2, Sharmila Selvaraj2, Preethi Krishnan3,6, Farooq Syed1,6,7, Huanmei Wu2, Carmella Evans-Molina 1,3,4,5,6,7,8* Departments of 1Pediatrics, 3Medicine, 4Anatomy, Cell Biology & Physiology, 5Biochemistry & Molecular Biology, the 6Center for Diabetes & Metabolic Diseases, and the 7Herman B. Wells Center for Pediatric Research, Indiana University School of Medicine, Indianapolis, IN 46202; 2Department of BioHealth Informatics, Indiana University-Purdue University Indianapolis, Indianapolis, IN, 46202; 8Roudebush VA Medical Center, Indianapolis, IN 46202. *Corresponding Author(s): Carmella Evans-Molina, MD, PhD ([email protected]) Indiana University School of Medicine, 635 Barnhill Drive, MS 2031A, Indianapolis, IN 46202, Telephone: (317) 274-4145, Fax (317) 274-4107 Running Title: Golgi Stress Response in Diabetes Word Count: 4358 Number of Figures: 6 Keywords: Golgi apparatus stress, Islets, β cell, Type 1 diabetes, Type 2 diabetes 1 Diabetes Publish Ahead of Print, published online August 20, 2020 Diabetes Page 2 of 781 ABSTRACT The Golgi apparatus (GA) is an important site of insulin processing and granule maturation, but whether GA organelle dysfunction and GA stress are present in the diabetic β-cell has not been tested. We utilized an informatics-based approach to develop a transcriptional signature of β-cell GA stress using existing RNA sequencing and microarray datasets generated using human islets from donors with diabetes and islets where type 1(T1D) and type 2 diabetes (T2D) had been modeled ex vivo. To narrow our results to GA-specific genes, we applied a filter set of 1,030 genes accepted as GA associated.
    [Show full text]
  • Focus on the Small Heat Shock Protein HSPB1 Autofagie in De Erfelij
    Faculteit Faculteit Farmaceutische, Biomedische en Diergeneeskundige wetenschappen Biochemie en Biotechnologie Autophagy in inherited peripheral neuropathies: Focus on the small heat shock protein HSPB1 Autofagie in de erfelijke perifere neuropathieën: Focus op de kleine heat shock proteïne HSPB1 Proefschrift voorgelegd tot het behalen van de graad van Doctor in de Wetenschappen: Biochemie en Biotechnologie aan de Universiteit Antwerpen. te verdedigen door Mansour HAIDAR Promotor Prof. Dr. Vincent Timmerman Antwerpen, 2018 1 2 “Haud igitur redit ad Nihilum res ulla, sed omnes Discidio redeunt in corpora materiai” Lucretius, De Rerum Natura, Book I. 250 3 4 Members of the jury Chair Prof. Dr. Wim Vanden Berghe, PhD (UA, Antwerp, Belgium) Promotor Prof. Dr. Vincent Timmerman, PhD (UA, Antwerp, Belgium) Internal jury member Prof. Dr. Wim Martinet, PhD (UA, Antwerp, Belgium) External jury members Prof. Dr. Joy Irobi (UHasselt, Hasselt, Belgium) Prof. Dr. Maurizio D’Antonio (San Raffaele Institute, Milan, Italy) Prof. Dr. Ir. Winnok De Vos (UA, Antwerp, Belgium) 5 6 Table of Contents Summary/Samenvatting 9 Rationale and Aims 13 Introduction Chapter 1 Autophagy as an emerging common pathomechanism in inherited 15 peripheral neuropathies Chapter 2 Small heat shock proteins: Their role in proteostasis 79 and neurodegeneration Results Chapter 3 HSPB1 is required for Autophagy: Insights from CMT-causing mutations 103 Chapter 4 An interactomics study of HSPB1 wild-type and mutant links it to the 129 autophagy receptor P62 Discussion 179 List of abbreviations 195 Curriculum Vitae 199 Acknowledgements 203 7 8 Summary Inherited peripheral neuropathies (IPNs) are genetically heterogeneous disorders affecting mainly the peripheral nervous system and with over 1500 mutations in more than 80 affected genes discovered so far.
    [Show full text]
  • Download Download
    Supplementary Figure S1. Results of flow cytometry analysis, performed to estimate CD34 positivity, after immunomagnetic separation in two different experiments. As monoclonal antibody for labeling the sample, the fluorescein isothiocyanate (FITC)- conjugated mouse anti-human CD34 MoAb (Mylteni) was used. Briefly, cell samples were incubated in the presence of the indicated MoAbs, at the proper dilution, in PBS containing 5% FCS and 1% Fc receptor (FcR) blocking reagent (Miltenyi) for 30 min at 4 C. Cells were then washed twice, resuspended with PBS and analyzed by a Coulter Epics XL (Coulter Electronics Inc., Hialeah, FL, USA) flow cytometer. only use Non-commercial 1 Supplementary Table S1. Complete list of the datasets used in this study and their sources. GEO Total samples Geo selected GEO accession of used Platform Reference series in series samples samples GSM142565 GSM142566 GSM142567 GSM142568 GSE6146 HG-U133A 14 8 - GSM142569 GSM142571 GSM142572 GSM142574 GSM51391 GSM51392 GSE2666 HG-U133A 36 4 1 GSM51393 GSM51394 only GSM321583 GSE12803 HG-U133A 20 3 GSM321584 2 GSM321585 use Promyelocytes_1 Promyelocytes_2 Promyelocytes_3 Promyelocytes_4 HG-U133A 8 8 3 GSE64282 Promyelocytes_5 Promyelocytes_6 Promyelocytes_7 Promyelocytes_8 Non-commercial 2 Supplementary Table S2. Chromosomal regions up-regulated in CD34+ samples as identified by the LAP procedure with the two-class statistics coded in the PREDA R package and an FDR threshold of 0.5. Functional enrichment analysis has been performed using DAVID (http://david.abcc.ncifcrf.gov/)
    [Show full text]
  • Supporting Information
    Supporting Information Edgar et al. 10.1073/pnas.1601895113 SI Methods (Actimetrics), and recordings were analyzed using LumiCycle Mice. Sample size was determined using the resource equation: Data Analysis software (Actimetrics). E (degrees of freedom in ANOVA) = (total number of exper- – Cell Cycle Analysis of Confluent Cell Monolayers. NIH 3T3, primary imental animals) (number of experimental groups), with −/− sample size adhering to the condition 10 < E < 20. For com- WT, and Bmal1 fibroblasts were sequentially transduced − − parison of MuHV-4 and HSV-1 infection in WT vs. Bmal1 / with lentiviral fluorescent ubiquitin-based cell cycle indicators mice at ZT7 (Fig. 2), the investigator did not know the genotype (FUCCI) mCherry::Cdt1 and amCyan::Geminin reporters (32). of the animals when conducting infections, bioluminescence Dual reporter-positive cells were selected by FACS (Influx Cell imaging, and quantification. For bioluminescence imaging, Sorter; BD Biosciences) and seeded onto 35-mm dishes for mice were injected intraperitoneally with endotoxin-free lucif- subsequent analysis. To confirm that expression of mCherry:: Cdt1 and amCyan::Geminin correspond to G1 (2n DNA con- erin (Promega E6552) using 2 mg total per mouse. Following < ≤ anesthesia with isofluorane, they were scanned with an IVIS tent) and S/G2 (2 n 4 DNA content) cell cycle phases, Lumina (Caliper Life Sciences), 15 min after luciferin admin- respectively, cells were stained with DNA dye DRAQ5 (abcam) and analyzed by flow cytometry (LSR-Fortessa; BD Biosci- istration. Signal intensity was quantified using Living Image ences). To examine dynamics of replicative activity under ex- software (Caliper Life Sciences), obtaining maximum radiance perimental confluent conditions, synchronized FUCCI reporter for designated regions of interest (photons per second per − − − monolayers were observed by time-lapse live cell imaging over square centimeter per Steradian: photons·s 1·cm 2·sr 1), relative 3 d (Nikon Eclipse Ti-E inverted epifluorescent microscope).
    [Show full text]
  • HIST2H3C(27Ac) Antibody Purified Mouse Monoclonal Antibody Catalog # Ao2159a
    10320 Camino Santa Fe, Suite G San Diego, CA 92121 Tel: 858.875.1900 Fax: 858.622.0609 HIST2H3C(27Ac) Antibody Purified Mouse Monoclonal Antibody Catalog # AO2159a Specification HIST2H3C(27Ac) Antibody - Product Information Application E, WB, FC, IHC Primary Accession Q71DI3 Reactivity Human Host Mouse Clonality Monoclonal Isotype IgG1 Calculated MW 15.4kDa KDa Description Histones are basic nuclear proteins that are responsible for the nucleosome structure of the chromosomal fiber in eukaryotes. This structure consists of approximately 146 bp of DNA wrapped around a nucleosome, an octamer composed of pairs of each of the four core histones (H2A, H2B, H3, and H4). The chromatin fiber is further compacted through the interaction of a linker histone, H1, with the DNA between the nucleosomes to form higher order chromatin structures. This gene is intronless and encodes a member of the histone H3 family. Transcripts from this gene lack polyA tails; instead, they contain a palindromic termination element. This gene is found in a histone cluster on chromosome 1. This gene is one of four histone genes in the cluster that are duplicated; this record represents the telomeric copy. Immunogen Synthesized peptide of human HIST2H3C (AA: ATKAARK(Ac)SAPATGGV). Formulation Purified antibody in PBS with 0.05% sodium azide HIST2H3C(27Ac) Antibody - Additional Information Gene ID 126961;333932;653604 Dilution E~~1/10000 Page 1/2 10320 Camino Santa Fe, Suite G San Diego, CA 92121 Tel: 858.875.1900 Fax: 858.622.0609 WB~~1/500 - 1/2000 FC~~1/200 - 1/400 IHC~~1/200 - 1/1000 Storage Maintain refrigerated at 2-8°C for up to 6 months.
    [Show full text]
  • Expression Analysis of Progesterone‑Regulated Mirnas in Cells Derived from Human Glioblastoma
    MOLECULAR MEDICINE REPORTS 23: 475, 2021 Expression analysis of progesterone‑regulated miRNAs in cells derived from human glioblastoma DIANA ELISA VELÁZQUEZ‑VÁZQUEZ1, AYLIN DEL MORAL‑MORALES1, JENIE MARIAN CRUZ‑BURGOS2, EDUARDO MARTÍNEZ‑MARTÍNEZ3, MAURICIO RODRÍGUEZ‑DORANTES2 and IGNACIO CAMACHO‑ARROYO1 1Unidad de Investigación en Reproducción Humana, Instituto Nacional de Perinatología‑Facultad de Química, Universidad Nacional Autónoma de México, Mexico City 04510; 2Oncogenomics Laboratory, The National Institute of Genomic Medicine; 3Laboratory of Cell Communication and Extracellular Vesicles, The National Institute of Genomic Medicine, Mexico City 14610, Mexico Received August 16, 2020; Accepted February 2, 2021 DOI: 10.3892/mmr.2021.12114 Abstract. Glioblastomas (GBMs) are the most frequent and is characterized by being highly infiltrative, angiogenic and malignant type of brain tumor. It has been reported that resistant to chemotherapy and radiotherapy. The medical progesterone (P4) regulates the progression of GBMs by modi‑ history of patients with GBM is short as few of them survive fying the expression of genes that promote cell proliferation, more than one year (1‑3). GBM is mainly diagnosed in adults migration and invasion; however, it is not fully understood >50 years old, but it can occur at any age and the incidence is how these processes are regulated. It is possible that P4 medi‑ higher in men than in women (3:2) (4). ates some of these effects through changes in the microRNA Studies have focused on the identification of new biomarkers (miRNA) expression profile in GBM cells. The present study and therapeutic agents in GBM. Of particular interest are the investigated the effects of P4 on miRNAs expression profile microRNAs (miRNAs), which are single‑stranded, short, in U‑251MG cells derived from a human GBM.
    [Show full text]
  • Genome-Wide Screen of Cell-Cycle Regulators in Normal and Tumor Cells
    bioRxiv preprint doi: https://doi.org/10.1101/060350; this version posted June 23, 2016. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. Genome-wide screen of cell-cycle regulators in normal and tumor cells identifies a differential response to nucleosome depletion Maria Sokolova1, Mikko Turunen1, Oliver Mortusewicz3, Teemu Kivioja1, Patrick Herr3, Anna Vähärautio1, Mikael Björklund1, Minna Taipale2, Thomas Helleday3 and Jussi Taipale1,2,* 1Genome-Scale Biology Program, P.O. Box 63, FI-00014 University of Helsinki, Finland. 2Science for Life laboratory, Department of Biosciences and Nutrition, Karolinska Institutet, SE- 141 83 Stockholm, Sweden. 3Science for Life laboratory, Division of Translational Medicine and Chemical Biology, Department of Medical Biochemistry and Biophysics, Karolinska Institutet, S-171 21 Stockholm, Sweden To identify cell cycle regulators that enable cancer cells to replicate DNA and divide in an unrestricted manner, we performed a parallel genome-wide RNAi screen in normal and cancer cell lines. In addition to many shared regulators, we found that tumor and normal cells are differentially sensitive to loss of the histone genes transcriptional regulator CASP8AP2. In cancer cells, loss of CASP8AP2 leads to a failure to synthesize sufficient amount of histones in the S-phase of the cell cycle, resulting in slowing of individual replication forks. Despite this, DNA replication fails to arrest, and tumor cells progress in an elongated S-phase that lasts several days, finally resulting in death of most of the affected cells.
    [Show full text]
  • HIST2H2AA3 Is Differentially Expressed in Brain Metastatic
    1 HIST2H2AA3 is differentially expressed in the lymph node and brain metastases of patients with metastatic breast cancer. 2 Shahan Mamoor, MS1 3 [email protected] 4 East Islip, NY USA 5 6 Metastasis to the brain is a clinical problem in patients with breast cancer1-3. We mined published microarray data4,5 to compare primary and metastatic tumor transcriptomes to discover genes associated 7 with brain metastasis in patients with metastatic breast cancer. We found that the histone cluster 2, H2aa3, encoded by HIST2H2AA3 was among the genes whose expression was most different in the brain 8 and lymph node metastases of patients with metastatic breast cancer as compared to primary tumors of the 9 breast and normal breast tissues, respectively. HIST2H2AA3 mRNA was present at higher quantities in brain metastatic tissues as compared to primary tumors of the breast. These data combined suggest some 10 level of common origin for metastases that reside in the lymph nodes and colonize the brain. 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 Keywords: breast cancer, metastasis, brain metastasis, central nervous system metastasis, lymph node metastasis, HIST2H2AA3, histone cluster 2, H2aa3, systems biology of breast cancer, targeted 27 therapeutics in breast cancer. 28 PAGE 1 1 One report described a 34% incidence of central nervous system metastases in patients treated with trastuzumab for breast cancer2. More recently, the NEfERT-T clinical trial6 which compared 2 administration of either neratinib or trastuzumab in conjunction with paclitaxel demonstrated that in a randomized, controlled setting, in breast cancer patients treated with neratinib, not only was the incidence 3 of central nervous system recurrence significantly lower, the time to central nervous system metastasis 4 was significantly delayed as compared to patients administered trastuzumab.
    [Show full text]