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Real-time PCR analysis of enteric pathogens from fecal samples of irritable bowel syndrome subjects

GUT PATHOGENS 2011年第2011期3卷 6-6页

作者： Rinttila, Teemu Lyra, Anna Krogius-Kurikka, Lotta Palva, Airi Univ Helsinki Fac Vet Med Dept Vet Biosci FIN-00014 Helsinki Finland Alimetrics Ltd FIN-02920 Espoo Finland Danisco Sweeteners Hlth & Nutr FIN-02460 Kantvik Finland

Background: Growing amount of scientific evidence suggests that microbes are involved in the pathophysiology of irritable bowel syndrome (IBS). The predominant fecal microbiota composition of IBS subjects has been widely studied with DNA-based techniques but less research has been focused on the intestinal pathogens in this disorder. Here, we optimized a highly sensitive panel of 12 quantitative real-time PCR (qPCR) assays to shed light on the putative presence of intestinal pathogens in IBS sufferers. The panel was used to screen fecal samples from 96 IBS subjects and 23 healthy controls. Results: Fifteen IBS samples (17%) tested positive for Staphylococcus aureus with a thermonuclease (nuc) gene-targeting qPCR assay, whereas none of the healthy controls were positive for S. aureus (p <0.05). The S. aureus-positive IBS samples were confirmed by sequencing of the PCR amplicons. Clostridium perfringens was detected from IBS and control groups with a similar frequency (13% and 17%, respectively) with a-toxin (plc) gene targeting qPCR assay while none of the samples tested positive for the Cl. perfringens enterotoxin-encoding gene (cpe). Conclusions: The qPCR panel consisting of 12 assays for an extensive set of pathogenic microorganisms provides an efficient alternative to the conventional detection of gastrointestinal pathogens and could accelerate the initiation of targeted antibiotic therapy reducing the risk of post-infectious IBS (PI-IBS). S. aureus has not been previously reported to be associated with the onset of IBS. Although we discovered significant differences in the prevalence of S. aureus between the study groups, its importance in giving rise to IBS symptoms requires further studies.

关键词： Irritable Bowel Syndrome Fecal Sample basic local alignment search tool qPCR Assay Genomic Equivalent

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Echinobase: leveraging an extant model organism database to build a knowledgebase supporting research on the genomics and biology of echinoderms

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NUCLEIC ACIDS REsearch 2022年第D1期50卷 D970-D979页

作者： Arshinoff, Bradley, I Cary, Gregory A. Karimi, Kamran Foley, Saoirse Agalakov, Sergei Delgado, Francisco Lotay, Vaneet S. Ku, Carolyn J. Pells, Troy J. Beatman, Thomas R. Kim, Eugene Cameron, R. Andrew Vize, Peter D. Telmer, Cheryl A. Croce, Jenifer C. Ettensohn, Charles A. Hinman, Veronica F. Univ Calgary Dept Biol Sci Calgary AB T2N 1N4 Canada Carnegie Mellon Univ Dept Biol Sci Pittsburgh PA 15213 USA CALTECH Div Biol & Biol Engn Pasadena CA 91125 USA Sorbonne Univ CNRS Inst Mer Villefranche IMEV Lab Biol Dev Villefranche Sur Mer LBDV Villefranche Sur Mer France Jackson Lab Bar Harbor ME 04609 USA Univ Pittsburgh Sch Med Pittsburgh PA 15213 USA Univ Calif San Francisco San Francisco CA 94143 USA

Echinobase (***) is a third generation web resource supporting genomic research on echinoderms. The new version was built by cloning the mature Xenopus model organism knowledge-base, Xenbase, refactoring data ingestion pipelines and modifying the user interface to adapt to multispecies echinoderm content. This approach leveraged over 15 years of previous database and web application development to generate a new fully featured informatics resource in a single year. In addition to the software stack, Echinobase uses the private cloud and physical hosts that support Xenbase. Echinobase currently supports six echinoderm species, focused on those used for genomics, developmental biology and gene regulatory network analyses. Over 38 000 gene pages, 18 000 publications, new improved genome assemblies, JBrowse genome browser and BLAST + services are available and supported by the development of a new echinoderm anatomical ontology, uniformly applied formal gene nomenclature, and consistent orthology predictions. A novel feature of Echinobase is integrating support for multiple, disparate species. New genomes from the diverse echinoderm phylum will be added and supported as data becomes available. The common code development design of the integrated knowledgebases ensures parallel improvements as each resource evolves. This approach is widely applicable for developing new model organism informatics resources.

关键词： cloning echinodermata genes genome genomics software user-computer interface xenopus basic local alignment search tool knowledge bases

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PseudoMLSA: a database for multigenic sequence analysis of Pseudomonas species

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BMC MICROBIOLOGY 2010年第1期10卷 118-118页

作者： Bennasar, Antoni Mulet, Magdalena Lalucat, Jorge Garcia-Valdes, Elena Univ Illes Balears Dept Biol Palma de Mallorca 07122 Spain Inst Mediterrani Estudis Avancats IMEDEA CSIC UIB Palma de Mallorca 07122 Spain Inst Univ Invest Ciencies Salut IUNICS UIB Palma de Mallorca 07122 Spain

Background: The genus Pseudomonas comprises more than 100 species of environmental, clinical, agricultural, and biotechnological interest. Although, the recommended method for discriminating bacterial species is DNA-DNA hybridisation, alternative techniques based on multigenic sequence analysis are becoming a common practice in bacterial species discrimination studies. Since there is not a general criterion for determining which genes are more useful for species resolution;the number of strains and genes analysed is increasing continuously. As a result, sequences of different genes are dispersed throughout several databases. This sequence information needs to be collected in a common database, in order to be useful for future identification-based projects. Description: The PseudoMLSA Database is a comprehensive database of multiple gene sequences from strains of Pseudomonas species. The core of the database is composed of selected gene sequences from all Pseudomonas type strains validly assigned to the genus through 2008. The database is aimed to be useful for MultiLocus Sequence Analysis (MLSA) procedures, for the identification and characterisation of any Pseudomonas bacterial isolate. The sequences are available for download via a direct connection to the National Center for Biotechnology Information (NCBI). Additionally, the database includes an online BLAST interface for flexible nucleotide queries and similarity searches with the user's datasets, and provides a user-friendly output for easily parsing, navigating, and analysing BLAST results. Conclusions: The PseudoMLSA database amasses strains and sequence information of validly described Pseudomonas species, and allows free querying of the database via a user-friendly, web-based interface available at http://***/microbiologiaBD/***. The web-based platform enables easy retrieval at strain or gene sequence information level;including references to published peer-reviewed articles, and direct ext

关键词： basic local alignment search tool ampC Database Schema basic local alignment search tool search MultiLocus Sequence Analysis

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A genomic perspective on the potential of Actinobacillus succinogenes for industrial succinate production

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BMC GENOMICS 2010年第1期11卷 680-680页

作者： McKinlay, James B. Laivenieks, Maris Schindler, Bryan D. McKinlay, Anastasia A. Siddaramappa, Shivakumara Challacombe, Jean F. Lowry, Stephen R. Clum, Alicia Lapidus, Alla L. Burkhart, Kirk B. Harkins, Victoria Vieille, Claire Michigan State Univ Dept Microbiol & Mol Genet E Lansing MI 48824 USA Univ Washington Dept Genome Sci & Med Seattle WA 98195 USA DOE Joint Genome Inst Los Alamos NM 87545 USA Los Alamos Natl Lab Los Alamos NM 87545 USA DOE Joint Genome Inst Walnut Creek CA 94598 USA Michigan State Univ Dept Biochem & Mol Biol E Lansing MI 48824 USA Univ Washington Dept Microbiol Seattle WA 98195 USA Univ Wisconsin Genet Lab Madison WI 53706 USA Michigan State Univ Dept Zool E Lansing MI 48824 USA

Background: Succinate is produced petrochemically from maleic anhydride to satisfy a small specialty chemical market. If succinate could be produced fermentatively at a price competitive with that of maleic anhydride, though, it could replace maleic anhydride as the precursor of many bulk chemicals, transforming a multi-billion dollar petrochemical market into one based on renewable resources. Actinobacillus succinogenes naturally converts sugars and CO2 into high concentrations of succinic acid as part of a mixed-acid fermentation. Efforts are ongoing to maximize carbon flux to succinate to achieve an industrial process. Results: Described here is the 2.3 Mb A. succinogenes genome sequence with emphasis on A. succinogenes's potential for genetic engineering, its metabolic attributes and capabilities, and its lack of pathogenicity. The genome sequence contains 1,690 DNA uptake signal sequence repeats and a nearly complete set of natural competence proteins, suggesting that A. succinogenes is capable of natural transformation. A. succinogenes lacks a complete tricarboxylic acid cycle as well as a glyoxylate pathway, and it appears to be able to transport and degrade about twenty different carbohydrates. The genomes of A. succinogenes and its closest known relative, Mannheimia succiniciproducens, were compared for the presence of known Pasteurellaceae virulence factors. Both species appear to lack the virulence traits of toxin production, sialic acid and choline incorporation into lipopolysaccharide, and utilization of hemoglobin and transferrin as iron sources. Perspectives are also given on the conservation of A. succinogenes genomic features in other sequenced Pasteurellaceae. Conclusions: Both A. succinogenes and M. succiniciproducens genome sequences lack many of the virulence genes used by their pathogenic Pasteurellaceae relatives. The lack of pathogenicity of these two succinogens is an exciting prospect, because comparisons with pathogenic Pasteurellaceae cou

关键词： basic local alignment search tool Cluster Regularly Interspaced Short Palindromic Repeat Succinate Production Cytolethal Distend Toxin Natural Competence

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Sample prep for proteomics of breast cancer: proteomics and gene ontology reveal dramatic differences in protein solubilization preferences of radioimmunoprecipitation assay and urea lysis buffers

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PROTEOME SCIENCE 2008年第1期6卷 30-30页

作者： Ngoka, Lambert C. M. Virginia Commonwealth Univ Dept Chem Richmond VA 23284 USA Texas Tech Univ Hlth Sci Ctr Paul L Foster Sch Med Dept Biomed Sci El Paso TX 79905 USA

Background: An important step in the proteomics of solid tumors, including breast cancer, consists of efficiently extracting most of proteins in the tumor specimen. For this purpose, Radio-Immunoprecipitation Assay (RIPA) buffer is widely employed. RIPA buffer's rapid and highly efficient cell lysis and good solubilization of a wide range of proteins is further augmented by its compatibility with protease and phosphatase inhibitors, ability to minimize non-specific protein binding leading to a lower background in immunoprecipitation, and its suitability for protein quantitation. Results: In this work, the insoluble matter left after RIPA buffer extraction of proteins from breast tumors are subjected to another extraction step, using a urea-based buffer. It is shown that RIPA and urea lysis buffers fractionate breast tissue proteins primarily on the basis of molecular weights. The average molecular weight of proteins that dissolve exclusively in urea buffer is up to 60% higher than in RIPA. Gene Ontology (GO) and Directed Acyclic Graphs (DAG) are used to map the collective biological and biophysical attributes of the RIPA and urea proteomes. The Cellular Component and Molecular Function annotations reveal protein solubilization preferences of the buffers, especially the compartmentalization and functional distributions. It is shown that nearly all extracellular matrix proteins (ECM) in the breast tumors and matched normal tissues are found, nearly exclusively, in the urea fraction, while they are mostly insoluble in RIPA buffer. Additionally, it is demonstrated that cytoskeletal and extracellular region proteins are more soluble in urea than in RIPA, whereas for nuclear, cytoplasmic and mitochondrial proteins, RIPA buffer is preferred. Extracellular matrix proteins are highly implicated in cancer, including their proteinase-mediated degradation and remodelling, tumor development, progression, adhesion and metastasis. Thus, if they are not efficiently extracted by RIP

关键词： Directed Acyclic Graph Extracellular Matrix Protein basic local alignment search tool Extracellular Region HFBA

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A novel method of differential gene expression analysis using multiple cDNA libraries applied to the identification of tumour endothelial genes

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BMC GENOMICS 2008年第1期9卷 153-153页

作者： Herbert, John M. J. Stekel, Dov Sanderson, Sharon Heath, Victoria L. Bicknell, Roy Univ Birmingham Sch Med Inst Biomed Res Canc Res UK Angiogenesis GrpMed Sch Birmingham B15 2TT W Midlands England Univ Birmingham Sch Biosci Ctr Syst Biol Birmingham B15 2TT W Midlands England Univ Oxford Canc Res UK Inst Mol Med Oxford OX3 9DS England

Background: In this study, differential gene expression analysis using complementary DNA (cDNA) libraries has been improved. Firstly by the introduction of an accurate method of assigning Expressed Sequence Tags (ESTs) to genes and secondly, by using a novel likelihood ratio statistical scoring of differential gene expression between two pools of cDNA libraries. These methods were applied to the latest available cell line and bulk tissue cDNA libraries in a two-step screen to predict novel tumour endothelial markers. Initially, endothelial cell lines were in silico subtracted from non-endothelial cell lines to identify endothelial genes. Subsequently, a second bulk tumour versus normal tissue subtraction was employed to predict tumour endothelial markers. Results: From an endothelial cDNA library analysis, 431 genes were significantly up regulated in endothelial cells with a False Discovery Rate adjusted q-value of 0.01 or less and 104 of these were expressed only in endothelial cells. Combining the cDNA library data with the latest Serial Analysis of Gene Expression (SAGE) library data derived a complete list of 459 genes preferentially expressed in endothelium. 27 genes were predicted tumour endothelial markers in multiple tissues based on the second bulk tissue screen. Conclusion: This approach represents a significant advance on earlier work in its ability to accurately assign an EST to a gene, statistically measure differential expression between two pools of cDNA libraries and predict putative tumour endothelial markers before entering the laboratory. These methods are of value and available http://***/data/*** to researchers that are interested in the analysis of transcriptomic data.

关键词： cDNA Library basic local alignment search tool Gene Assignment Unigene Cluster Endothelial Gene

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Pervasive sequence patents cover the entire human genome

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GENOME MEDICINE 2013年第3期5卷 27-27页

作者： Rosenfeld, Jeffrey Mason, Christopher E. Univ Med & Dent New Jersey IST Div High Performance & Res Comp Newark NJ 07103 USA Amer Museum Nat Hist Sackler Inst Comparat Genom New York NY 10024 USA Cornell Univ Dept Physiol & Biophys Weill Med Coll New York NY 10065 USA Cornell Univ HRH Prince Alwaleed Bin Talal Bin Abdulaziz Alsau Weill Med Coll New York NY 10065 USA Yale Univ Sch Law Informat Soc Project New Haven CT 06520 USA

The scope and eligibility of patents for genetic sequences have been debated for decades, but a critical case regarding gene patents (Association of Molecular Pathologists v. Myriad Genetics) is now reaching the US Supreme Court. Recent court rulings have supported the assertion that such patents can provide intellectual property rights on sequences as small as 15 nucleotides (15mers), but an analysis of all current US patent claims and the human genome presented here shows that 15mer sequences from all human genes match at least one other gene. The average gene matches 364 other genes as 15mers;the breast-cancer-associated gene BRCA1 has 15mers matching at least 689 other genes. Longer sequences (1,000 bp) still showed extensive cross-gene matches. Furthermore, 15mer-length claims from bovine and other animal patents could also claim as much as 84% of the genes in the human genome. In addition, when we expanded our analysis to full-length patent claims on DNA from all US patents to date, we found that 41% of the genes in the human genome have been claimed. Thus, current patents for both short and long nucleotide sequences are extraordinarily non-specific and create an uncertain, problematic liability for genomic medicine, especially in regard to targeted re-sequencing and other sequence diagnostic assays.

关键词： basic local alignment search tool Gene Patent Memorial Sloan Kettering Cancer Center Patent Claim Entire Human Genome

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Leukostasis aggravated by red blood cell transfusion in a chronic lymphocytic leukemia patient

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AMERICAN JOURNAL OF CLINICAL PATHOLOGY 2021年第Sup1期156卷 S162-S163页

作者： Jum'ah, H. Chitsaz, M. Kundrapu, S. Case Western Reserve Univ Metrohlth Med Ctr Pathol Cleveland OH 44106 USA

Introduction/Objective Leukostasis/symptomatic hyperleukocytosis is commonly seen in acute leukemias and is characterized by high blast counts and symptoms of decreased tissue perfusion with a one-week mortality of 20-40%, if left untreated. It is a rare complication in chronic lymphocytic leukemia (CLL) and is seen in CLL patients with white blood cell (WBC) counts > 500x10^9/L. Studies have shown that transfusion of blood products prior to decreasing the WBC count may lead to increased blood viscosity and worsen leukostasis. We report a CLL patient with a lower WBC count presenting with symptoms of leukostasis, further worsened by a red blood cell (RBC) transfusion prior to leukapheresis. Methods/Case Report A 73-year-old male with history of CLL for 5 years, hypertension and chronic kidney disease presented with acute dyspnea for 1 day. The WBC count was 208x10^9/L, hemoglobin was 6.5 g/dL, troponin I was 1.554 ng/mL, creatinine was 1.68 mg/dL and chest x-ray showed bilateral interstitial lung edema. The patient was diagnosed with acute hypoxic respiratory failure, acute kidney injury and myocardial infarction due to leukostasis. He received RBC transfusion, shortly after which his dyspnea worsened. There were no other signs or symptoms that could suggest a transfusion reaction. A single leukapheresis was performed following which there was a significant improvement in symptoms with a drop in WBC count to 123.1x10^9/L and creatinine to 1.3 mg/dL. Results (if a Case Study enter NA) NA Conclusion CLL may present with symptomatic hyperleukocytosis at lower leukocyte counts than has been described in the literature. Symptoms due to leukostasis can be precipitated/worsened by transfusion of blood products due to increased blood viscosity. It is critical to identify the signs and symptoms of this medical emergency as prompt diagnosis and management with leukapheresis significantly reduces the leukocyte count and blood products should be transfused slowly during or afte

关键词： myocardial infarction hypertension leukemia acute dyspnea pulmonary edema kidney failure chronic hypoxia creatinine respiratory failure acute erythrocytes hemoglobin erythrocyte transfusion leukapheresis chronic lymphocytic leukemia chronic b-cell leukemias leukocyte count leukocytes leukostasis chest x-ray blood viscosity diagnosis mortality troponin i transfusion reaction transfusion renal trauma blood products tissue perfusion leukocytosis marked basic local alignment search tool symptom aggravating factors

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Bioinformatics and wet laboratory analysis of rs1800562 to predict genetic aetiology of iron overload in Nigeria

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AMERICAN JOURNAL OF CLINICAL PATHOLOGY 2021年第Sup1期156卷 S145-S146页

作者： Bolarinwa, A. B. Onawoga, F. Lagos Univ Teaching Hosp Haematol & Blood Transfus Lagos Nigeria

Introduction/Objective The most reported single nucleotide polymorphism (SNP) of the HFE gene is rs1800562, representing the substitution of Adenine for Guanine at position 847 of the HFE gene. This has been widely implicated in hereditary haemochromatosis and other conditions like altered cholesterol balance, Alzheimer’s disease and cutaneous photosensitivity. Abnormal HFE protein resulting from the mutant HFE gene leads to formation of excess iron which has been postulated as likely mechanism for these diseases. Although there is evidence of iron overload in Africans, only few studies have explored possible genetic causes, and prevalence of rs1800562 is not known in West African population. Hence the need to determine the prevalence of rs1800562 in Nigeria using computational and wet laboratory approach. Methods/Case Report Details of rs1800562 were retrieved from Ensembl Genome Browser version 99. Severity of the consequences of this SNP on protein product was determined using bioinformatics tools including SIFT, Polyphen, Mutation Assessor, HOPE, I-mutant and MutPred2. Genotyping of rs1800562 was done In silico using restriction fragment length polymorphism (RFLP). Primer3plus was used for primer design, NCBI BLAST and SMS were used for primer validation. We used Webcutter 2.0 to determine suitable restriction enzymes. The genotyping was simulated using USCS virtual PCR and RestrictionMapper. Whole blood samples were obtained from 200 participants selected randomly from a pool of blood donors. DNA was extracted and flanking region of rs1800562 was amplified. The amplified product was digested by RSA1and fragments examined on agarose gel electrophoresis. Results (if a Case Study enter NA) The MAF was found to be 0.01 globally and 0.02 in Africa. In the two Nigerian population examined (Yoruba and Esan population), MAF was 0.00. Mutation Assessor and SIFT Polyphen consistently predicted the mutation to be of severe consequences. Analysis on HOPE, I-mutant and Mutp

关键词： alleles polymerase chain reaction hemochromatosis iron alzheimer's disease mutation cholesterol adenine africa blood donors dna electrophoresis agar gel genes genome guanine homozygote iron overload nigeria restriction fragment length polymorphism single nucleotide polymorphism enzymes genetics iron metabolism whole blood photosensitivity hfe gene genotype determination causality basic local alignment search tool bioinformatics hemochromatosis protein

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Myeloperoxidase Expression in B-Lymphoblastic Leukemia by Immunohistochemistry as a Diagnostic Confounder

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AMERICAN JOURNAL OF CLINICAL PATHOLOGY 2020年第Sup1期154卷 S1-S1页

作者： Du, Jing Moklebust, Amanda Cherian, Sindhu Edlefsen, Kerstin Fromm, Jonathan R. Li, K. David Thomason, Ron Soma, Lorinda Cherian, Sindhu Univ Washington Med Ctr Seattle Canc Care Alliance Dept Pathol Seattle WA 98195 USA Univ Washington Med Ctr Integrated Oncol Seattle WA 98195 USA

Background B-lymphoblastic leukemia (BLL) is a B lineage neoplasm that expresses typical immature B cell markers, but may also express myeloid-associated antigens. Myeloperoxidase (MPO) is a heme-containing peroxidase, which has been used as the single most specific myeloid marker when assigning lineage to acute leukemia. MPO positivity by immunohistochemistry (IHC) in BLL has been historically described in a subset of patients; however, further detailed comparison of IHC to flow cytometry and IHC methodology is described *** The University of Washington pathology database was searched for new or relapsed adult BLL from 2011–2019. Cases with blasts >30% of marrow cellularity by morphology were selected. MPO IHC was performed using the Dako, polyclonal antibody (rabbit) on a Ventana instrument with streptavidin-biotin (SB) detection method. MPO IHC SB positive cases were also stained using the same antibody and platform, but a different detection method, multimer-optiview (MO). MPO IHC was called positive when >10% of neoplastic blasts showed expression. MPO positive blast percentage, staining intensity and pattern were assessed. Intensity: 0=negative, 1=mild, 2=moderate, 3=bright/same level as myeloids. Cytoplasmic pattern: homogenous or granular. Concurrent flow cytometry results and cytogenetic and/or molecular studies were *** 35 cases were identified. Positive MPO IHC expression was present in 7/35 cases by SB method, with the percentage of MPO positive blasts ranging from 20–90% (majority >70%), all ranged from 1 - 2+ intensity and most (5/7) were homogenous pattern. 4/5 MPO SB positive cases were negative by MO detection method. MPO evaluation by flow cytometry was negative in 3 of 3 cases and myeloid associated antigens were negative or low on a subset. 2/8 BLL, BCR-ABL1 cases were MPO IHC positive by *** MPO staining by IHC in BLL is present in 17% of cases, often present in the majority of blasts, and positive using the SB d

关键词： leukemia acute immunohistochemistry b-lymphocytes flow cytometry adult antigens biotin cytogenetics cytoplasm heme leukemia peroxidase oryctolagus cuniculus streptavidin antibodies diagnosis neoplasms pathology polyclonal antibody bone marrow aspiration blast cells basic local alignment search tool

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