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Sample records for human isolates final

  1. Isolation of cDNAs from the human X chromosome and derivation of related STSs. Final progress report, April 1992--March 1995

    Energy Technology Data Exchange (ETDEWEB)

    Nelson, D.L.

    1995-09-01

    Over the course of this funding period, the number of genes assigned to the human X chromosome has approximately tripled from less than one hundred to nearly three hundred characterized, cloned genes assigned to it. The aims of this project were to develop methods for gene identification and to identify and characterize expressed sequences from the X chromosome. The rapidly changing environment of the human genome project provided abundant resources for gene characterization, and since methods for gene identification became rather robust over this period, these aims were de-emphasized during the project. Among the methods developed was a local one (reciprocal probing) that was developed by Drs. Cheng Chi Lee and C. Thomas Caskey, with emphasis on the human X chromosome. The development of this method offered significant expressed sequence resources for this project, particularly when coupled with the efforts to identify cosmid clones from specific X chromosome locations, as the reciprocal probing process results in paired genomic (cosmid) and cDNA materials. Attention, then has been paid to characterization of genes rather than to their identification.

  2. Isolation, cultivation and transfection of human keratinocytes.

    Science.gov (United States)

    Zare, Sona; Zarei, Mohammad Ali; Ghadimi, Tayyeb; Fathi, Fardin; Jalili, Ali; Hakhamaneshi, Mohammad Saeed

    2014-04-01

    Human keratinocytes could be used in the repair of damaged skin, in tissue engineering applications, gene therapy and recently, the generation of iPS cells. We isolated human keratinocytes from foreskin and subsequently cultured them on fibronectin, collagen type I, gelatin and laminin-coated dishes that contained three different types of serum-free medium (epilife, KSM or CnT). We developed improved conditions for efficient transfection of these human keratinocytes by testing three common transfection methods and a GFP plasmid vector. The isolated cells showed typical keratinocyte morphology and expressed the epithelial cell specific antigen, cytokeratin 14. Collagen type 1, epilife medium and lipofectamin 2000 gave the best results for isolation and transfection of human keratinocytes. Our protocol can be used as a reproducible, simple and efficient method for isolation, cultivation and genetic manipulation of human keratinocytes, which may be useful in cell and gene therapy applications.

  3. Genetic differences between avian and human isolates of Candida dubliniensis.

    LENUS (Irish Health Repository)

    McManus, Brenda A

    2009-09-01

    When Candida dubliniensis isolates obtained from seabird excrement and from humans in Ireland were compared by using multilocus sequence typing, 13 of 14 avian isolates were genetically distinct from human isolates. The remaining avian isolate was indistinguishable from a human isolate, suggesting that transmission may occur between humans and birds.

  4. Final environmental impact statement. Waste Isolation Pilot Plant

    Energy Technology Data Exchange (ETDEWEB)

    1980-10-01

    This volume contains the appendices for the Final Environmental Impact Statement for the Waste Isolation Pilot Plant (WIPP). Alternative geologic environs are considered. Salt, crystalline rock, argillaceous rock, and tuff are discussed. Studies on alternate geologic regions for the siting of WIPP are reviewed. President Carter's message to Congress on the management of radioactive wastes and the findings and recommendations of the interagency review group on nuclear waste management are included. Selection criteria for the WIPP site including geologic, hydrologic, tectonic, physicochemical compatability, and socio-economic factors are presented. A description of the waste types and the waste processing procedures are given. Methods used to calculate radiation doses from radionuclide releases during operation are presented. A complete description of the Los Medanos site, including archaeological and historic aspects is included. Environmental monitoring programs and long-term safety analysis program are described. (DMC)

  5. Total excitation of the isolated human heart

    NARCIS (Netherlands)

    Durrer, D.; Dam, R.Th. van; Freud, G.E.; Janse, M.J.; Meijler, F.L.; Arzbaecher, R.C.

    1970-01-01

    To obtain information conceming the time course and instantaneous distribution of the excitatory process of the normal human healt, studies were made on isolated human hearts from seven individuals who died from various cerebral conditions, but who had no history of cardiac disease. Measurements wer

  6. Isolation of Cancer Stem Cells From Human Prostate Cancer Samples

    Science.gov (United States)

    Vidal, Samuel J.; Quinn, S. Aidan; de la Iglesia-Vicente, Janis; Bonal, Dennis M.; Rodriguez-Bravo, Veronica; Firpo-Betancourt, Adolfo; Cordon-Cardo, Carlos; Domingo-Domenech, Josep

    2014-01-01

    The cancer stem cell (CSC) model has been considerably revisited over the last two decades. During this time CSCs have been identified and directly isolated from human tissues and serially propagated in immunodeficient mice, typically through antibody labeling of subpopulations of cells and fractionation by flow cytometry. However, the unique clinical features of prostate cancer have considerably limited the study of prostate CSCs from fresh human tumor samples. We recently reported the isolation of prostate CSCs directly from human tissues by virtue of their HLA class I (HLAI)-negative phenotype. Prostate cancer cells are harvested from surgical specimens and mechanically dissociated. A cell suspension is generated and labeled with fluorescently conjugated HLAI and stromal antibodies. Subpopulations of HLAI-negative cells are finally isolated using a flow cytometer. The principal limitation of this protocol is the frequently microscopic and multifocal nature of primary cancer in prostatectomy specimens. Nonetheless, isolated live prostate CSCs are suitable for molecular characterization and functional validation by transplantation in immunodeficient mice. PMID:24686446

  7. Final Reclamation Report: Basalt Waste Isolation Project exploratory shaft site

    Energy Technology Data Exchange (ETDEWEB)

    Brandt, C.A.; Rickard, W.H. Jr.

    1990-06-01

    The restoration of areas disturbed by activities of the Basalt Waste Isolation Project (BWIP) constitutes a unique operation at the US Department of Energy's (DOE) Hanford Site, both from the standpoint of restoration objectives and the time frame for accomplishing these objectives. The BWIP reclamation program comprises three separate projects: borehole reclamation, Near Surface Test Facility (NSTF) reclamation, and Exploratory Shaft Facility (ESF) reclamation. The main focus of this report is on determining the success of the revegetation effort 1 year after work was completed. This report also provides a brief overview of the ESF reclamation program. 21 refs., 7 figs., 14 tabs.

  8. Final environmental impact statement. Waste Isolation Pilot Plant

    Energy Technology Data Exchange (ETDEWEB)

    1980-10-01

    In accordance with the National Environmental Policy Act (NEPA) of 1969, the US Department of Energy (DOE) has prepared this document as environmental input to future decisions regarding the Waste Isolation Pilot Plant (WIPP), which would include the disposal of transuranic waste, as currently authorized. The alternatives covered in this document are the following: (1) Continue storing transuranic (TRU) waste at the Idaho National Engineering Laboratory (INEL) as it is now or with improved confinement. (2) Proceed with WIPP at the Los Medanos site in southeastern New Mexico, as currently authorized. (3) Dispose of TRU waste in the first available repository for high-level waste. The Los Medanos site would be investigated for its potential suitability as a candidate site. This is administration policy and is the alternative preferred by the DOE. (4) Delay the WIPP to allow other candidate sites to be evaluated for TRU-waste disposal. This environmental impact statement is arranged in the following manner: Chapter 1 is an overall summary of the analysis contained in the document. Chapters 2 and 4 set forth the objectives of the national waste-management program and analyze the full spectrum of reasonable alternatives for meeting these objectives, including the WIPP. Chapter 5 presents the interim waste-acceptance criteria and waste-form alternatives for the WIPP. Chapters 6 through 13 provide a detailed description and environmental analysis of the WIPP repository and its site. Chapter 14 describes the permits and approvals necessary for the WIPP and the interactions that have taken place with Federal, State, and local authorities, and with the general public in connection with the repository. Chapter 15 analyzes the many comments received on the DEIS and tells what has been done in this FEIS in response. The appendices contain data and discussions in support of the material in the text.

  9. MLVA genotyping of human Brucella isolates from Peru

    NARCIS (Netherlands)

    H.L. Smits; B. Espinosa; R. Castillo; E. Hall; A. Guillen; M. Zevaleta; R.H. Gilman; P. Melendez; C. Guerra; A. Draeger; A. Broglia; K. Nöckler

    2009-01-01

    Recent human Brucella melitensis isolates from Peru were genotyped by multiple locus variable number repeat analysis. All 24 isolates originated from hospitalized patients living in the central part of Peru and consisted of six genomic groups comprising two to four isolates and nine unique genotypes

  10. Isolation and characterization of human spermatogonial stem cells

    Directory of Open Access Journals (Sweden)

    Liu Shixue

    2011-10-01

    Full Text Available Abstract Background To isolate and characterization of human spermatogonial stem cells from stem spermatogonium. Methods The disassociation of spermatogonial stem cells (SSCs were performed using enzymatic digestion of type I collagenase and trypsin. The SSCs were isolated by using Percoll density gradient centrifugation, followed by differential surface-attachment method. Octamer-4(OCT4-positive SSC cells were further identified using immunofluorescence staining and flow cytometry technques. The purity of the human SSCs was also determined, and a co-culture system for SSCs and Sertoli cells was established. Results The cell viability was 91.07% for the suspension of human spermatogonial stem cells dissociated using a two-step enzymatic digestion process. The cells isolated from Percoll density gradient coupled with differential surface-attachement purification were OCT4 positive, indicating the cells were human spermatogonial stem cells. The purity of isolated human spermatogonial stem cells was 86.7% as assessed by flow cytometry. The isolated SSCs were shown to form stable human spermatogonial stem cell colonies on the feeder layer of the Sertoli cells. Conclusions The two-step enzyme digestion (by type I collagenase and trypsin process is an economical, simple and reproducible technique for isolating human spermatogonial stem cells. With little contamination and less cell damage, this method facilitates isolated human spermatogonial stem cells to form a stable cell colony on the supporting cell layer.

  11. Thermolysin in human cultured keratinocyte isolation

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    A. Gragnani

    Full Text Available BACKGROUND: When treating extensively burned patients using cultured epidermal sheets, the main problem is the time required for its production. Conventional keratinocyte isolation is usually done using Trypsin. We used a modification of the conventional isolation method in order to improve this process and increase the number of colonies from the isolated epidermal cell population. PURPOSE: To compare the action of trypsin and thermolysin in the keratinocyte isolation using newborn foreskin. METHODS: This method used thermolysin as it selectively digests the dermo-epidermal junction. After dermis separation, the epidermis was digested by trypsin in order to obtain a cell suspension. RESULTS: Compared to the conventional procedure, these experiments demonstrated that in the thermolysin group, the epidermis was easily detached from the dermis, there was no fibroblast contamination and there were a larger number of keratinocyte colonies which had a significant statistical difference. CONCLUSION: The number of colonies in the thermolysin group was significantly greater than in the trypsin group.

  12. Isolation of Campylobacter from human stool samples

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    S M Salim

    2014-01-01

    Full Text Available Context: Campylobacter is an undetected cause of diarrhoea especially under 5 years of age in most of the countries. Isolation of this organism is difficult, expensive and cumbersome. Aims: Our objective of this study was to isolate this pathogen from the stool specimens on routinely available blood containing laboratory media using the candle jar for creating the microaerophilic atmosphere in our setup. Settings and Designs: A descriptive study. Materials and Methods: A total of 50 stool samples were inoculated onto selective and non-selective media with and without filtration using a 0.45 μm membrane. The inoculated media were simultaneously incubated in microaerophilic conditions using the Anoxomat as well as in candle jars at temperatures 37°C and 42°C. The culture isolates were confirmed by standard phenotypic tests. A simplex polymerase chain reaction (PCR targeting the 16S ribosomal deoxyribonucleic acid of Campylobacter was performed on the deoxyribonucleic acid (DNA of the culture isolates as well as on the DNA extracted from the stool filtrates. Statistical Analysis: Data was expressed as a proportion. Results: Campylobacter could be isolated in 5 out of 50 stool samples using both the Anoxomat as well as the candle jar. Furthermore, we did not find any difference between the isolation using the selective and blood containing media as well as the different incubation temperatures. All the five were confirmed phenotypically and genotypically to be Campylobacter jejuni. The PCR results corroborated with that of the culture. Conclusions: Isolation by culture was as sensitive as that of the PCR.

  13. Anaerococcus nagyae sp. nov., isolated from human clinical specimens

    NARCIS (Netherlands)

    Veloo, A C M; Vries , de E. D.; Jean-Pierre, H; van Winkelhoff, A J

    We describe a new Anaerococcus species isolated from human clinical specimens. Analyses of 16S rRNA gene sequences of three strains showed <98% similarity with its closest relative Anaerococcus octavius. Phylogenetically the isolated strains form a cluster and can be differentiated from other

  14. Anaerococcus degenerii sp nov., isolated from human clinical specimens

    NARCIS (Netherlands)

    Veloo, A. C. M.; Elgersma, P. E.; van Winkelhoff, A. J.

    Four clinical isolates of gram-positive strict anaerobic cocci were isolated from four different human mixed anaerobic infections. The taxonomical status of the four strains could not be established using standard identification techniques. The biochemical features of the strains were established

  15. Mechanical design of a pre-isolator for the CLIC final focusing magnets

    CERN Document Server

    Gaddi, A; Ramos, F; Siegrist, N

    2012-01-01

    Due to the very small vertical beam sizes, the final focusing elements at the future CLIC linear collider need to be stable against vibrations to below 0.15 nanometres at frequencies above about 4 Hz. One of the key elements in the strategy to achieve such a stable environment is a passive, heavy pre-isolator. In this report, the results from the dynamic finite element analyses of the proposed design for such a passive preisolator are summarized. Furthermore, the results from a low frequency, heavy mass passive vibration isolation test set-up used to validate the calculations are shown.

  16. First isolation of Streptococcus downei from human dental plaques.

    Science.gov (United States)

    Yoo, So Young; Kim, Kwan-Joong; Lim, Seong-Hoon; Kim, Kwang-Won; Hwang, Ho-Keel; Min, Byung-Moo; Choe, Son-Jin; Kook, Joong-Ki

    2005-08-15

    In this study, we isolated four bacterial strains grown on mitis-salivarius sucrose bacitracin agar. The strains had similar biochemical characteristics to biotypes I or II of mutans streptococci. The four isolates were identified as Streptococcus downei by 16S rDNA and dextranase gene (dex) sequencing as well as polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) targeting dex. To our knowledge, this is the first report of the isolation and identification of S. downei from dental plaque in humans. The results suggest that S. downei can inhabit the human oral cavity.

  17. Comparison of Staphylococcus aureus isolates associated with food intoxication with isolates from human nasal carriers and human infections.

    Science.gov (United States)

    Wattinger, L; Stephan, R; Layer, F; Johler, S

    2012-04-01

    Staphylococcus aureus represents an organism of striking versatility. While asymptomatic nasal colonization is widespread, it can also cause serious infections, toxinoses and life-threatening illnesses in humans and animals. Staphylococcal food poisoning (SFP), one of the most prevalent causes of foodborne intoxication worldwide, results from oral intake of staphylococcal enterotoxins leading to violent vomiting, diarrhea and cramps shortly upon ingestion. The aim of the present study was to compare isolates associated with SFP to isolates collected from cases of human nasal colonization and clinical infections in order to investigate the role of S. aureus colonizing and infecting humans as a possible source of SFP. Spa typing and DNA microarray profiling were used to characterize a total of 120 isolates, comprising 50 isolates collected from the anterior nares of healthy donors, 50 isolates obtained from cases of clinical infections in humans and 20 isolates related to outbreaks of staphylococcal food poisoning. Several common spa types were found among isolates of all three sources (t015, t018, t056, t084). DNA microarray results showed highly similar virulence gene profiles for isolates from all tested sources. These results suggest contamination of foodstuff with S. aureus colonizing and infecting food handlers to represent a source of SFP.

  18. Romboutsia timonensis, a new species isolated from human gut

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    D. Ricaboni

    2016-07-01

    Full Text Available The exploration of the human microbiome was recently revolutionized by microbial culturomics and taxonogenomics. Thanks to this approach, we report here the main characteristics of Romboutsia timonensis strain Marseille-P326, a new bacterium isolated from the right human colon by colonoscopy in a 63-year-old French man with severe anaemia with melaena.

  19. Isolation and characterization of human rhinovirus antigenic variants

    Energy Technology Data Exchange (ETDEWEB)

    Watson, D.G.

    1985-01-01

    Isolation of antigenic variants of human rhinovirus types 2, 14, and 17 was attempted by plaquing untreated virus (P-isolates), selecting variants in the presence of homologous antiserum (C-isolates), and by selecting variants in the presence of antibody following 5-fluorouracil mutagenesis (M-isolates). All viruses were triple-plaque purified and purity neutralization tested prior to isolate selection. Based on a fourfold reduction in neutralizing antibody titer to homologous antiserum, no antigenic variation was found in P-isolates from the three serotypes examined. Antigenic variants of all three serotypes could be isolated by the antiserum selection method (C-isolates). However, antigenic variants of RV17 were isolated at a much higher frequency and showed a larger degree of variation than those of RV2 and RV14. At least two of the variants selected, RV17 (C301) and RV2 (M803), failed to be neutralized by the known 89 rhinovirus antiserum. SDS-polyacrylamide gel electrophoresis of (/sup 35/S) methionine-labelled virion polypeptides revealed that each serotype had a characteristic pattern and that selected RV2 and RV17 isolates had patterns identical to those of the prototype strains. By isoelectric focusing an antigenic variant of RV2 was shown to contain altered virion polypeptides VP1 and VP2 whereas two RV17 antigenic variants demonstrated alterations only in the VP1 polypeptide.

  20. Isolation of human apolipoprotein E by chromatofocusing.

    Science.gov (United States)

    Weisweiler, P; Schwandt, P

    1982-09-01

    Human prolipoprotein E is implicated in the transport of serum cholesterol and the binding of lipoproteins to cell receptors. Further investigations on this apolipoprotein would be facilitated by improved purification methods. We prepared human apo E by the combination of high performance gel filtration and chromatofocusing from serum very low density lipoproteins. Chromatofocusing was performed with a pH gradient from 7 to 4. Apo E contained all isoforms, but was homogeneous in SDS-polyacrylamide gel electrophoresis and in double immunodiffusion against a monospecific antiserum. The reported purification method allows a rapid and simple preparation of large amounts of apo E.

  1. Hawthorn extract inhibits human isolated neutrophil functions.

    Science.gov (United States)

    Dalli, Ernesto; Milara, Javier; Cortijo, Julio; Morcillo, Esteban J; Cosín-Sales, Juan; Sotillo, José Francisco

    2008-06-01

    Hawthorn extract is a popular herbal medicine given as adjunctive treatment for chronic heart failure. In contrast to the cardiac properties of hawthorn extract, its anti-inflammatory effect has been scarcely investigated. This study examines the effects of a dry extract of leaves and flowers of Crataegus laevigata on various functional outputs of human neutrophils in vitro. Incubation of human neutrophils obtained from peripheral blood of healthy donors with C. laevigata extract (0.75-250 microg/ml) inhibited N-formyl-Met-Leu-Phe (FMLP)-induced superoxide anion generation, elastase release and chemotactic migration with potency values of 43.6, 21.9, and 31.6 microg/ml, respectively. By contrast, serum-opsonized zymosan-induced phagocytosis was unaltered by plant extract. C. laevigata extract (125 microg/ml) reduced FMLP-induced leukotriene B(4) production and lipopolysaccharide-induced generation of tumour necrosis factor-alpha and interleukin-8. Extract inhibited FMLP-induced intracellular calcium signal with potency of 17.4 microg/ml. Extract also markedly inhibited the extracellular calcium entry into calcium-depleted neutrophils, and the thapsigargin-induced intracellular calcium response. In conclusion, C. laevigata extract inhibited various functional outputs of activated human neutrophils which may be relevant to the pathophysiology of cardiac failure.

  2. Anaerococcus nagyae sp. nov., isolated from human clinical specimens.

    Science.gov (United States)

    Veloo, A C M; de Vries, E D; Jean-Pierre, H; van Winkelhoff, A J

    2016-04-01

    We describe a new Anaerococcus species isolated from human clinical specimens. Analyses of 16S rRNA gene sequences of three strains showed octavius. Phylogenetically the isolated strains form a cluster and can be differentiated from other species of the genus Anaerococcus based on its phenotypic characteristics and its MALDI-TOF MS profile. We propose the name Anaerococcus nagyae, with A. nagyae DSM101193 (accession number KU043522) as the type strain. Copyright © 2015 Elsevier Ltd. All rights reserved.

  3. Distinct replicative and cytopathic characteristics of human immunodeficiency virus isolates.

    Science.gov (United States)

    Fenyö, E M; Morfeldt-Månson, L; Chiodi, F; Lind, B; von Gegerfelt, A; Albert, J; Olausson, E; Asjö, B

    1988-01-01

    According to their capacity to replicate in vitro, human immunodeficiency virus (HIV) isolates can be divided into two major groups, rapid/high and slow/low. Rapid/high viruses can easily be transmitted to a variety of cell lines of T-lymphoid (CEM, H9, and Jurkat) and monocytoid (U937) origin. In contrast, slow/low viruses replicate transiently, if at all, in these cell lines. Except for a few isolates, the great majority of slow/low viruses replicate in peripheral blood mononuclear cells and Jurkat-tatIII cells constitutively expressing the tatIII gene of HIV-1. The viruses able to replicate efficiently cause syncytium formation and are regularly isolated from immunodeficient patients. Poorly replicating HIV isolates, often obtained from individuals with no or mild disease, show syncytium formation and single-cell killing simultaneously or, with some isolates, cell killing only. Images PMID:2459416

  4. Phylogenetic analysis of Escherichia coli strains isolated from human samples

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    Abdollah Derakhshandeh

    2013-12-01

    Full Text Available Escherichia coli (E. coli is a normal inhabitant of the gastrointestinal tract of vertebrates, including humans. Phylogenetic analysis has shown that E. coli is composed of four main phylogenetic groups (A, B1, B2 and D. Group A and B1 are generally associated with commensals, whereas group B2 is associated with extra-intestinal pathotypes. Most enteropathogenic isolates, however, are assigned to group D. In the present study, a total of 102 E. coli strains, isolated from human samples, were used. Phylogenetic grouping was done based on the Clermont triplex PCR method using primers targeted at three genetic markers, chuA, yjaA and TspE4.C2. Group A contained the majority of the collected isolates (69 isolates, 67.64%, followed by group B2 (18 isolates, 17.64% and D (15 isolates, 14.7% and no strains were found to belong to group B1. The distribution of phylogenetic groups in our study suggests that although the majority of strains were commensals, the prevalence of enteropathogenic and extra-intestinal pathotypes was noteworthy. Therefore, the role of E. coli in human infections including diarrhea, urinary tract infections and meningitis should be considered.

  5. Blastocystis phylogeny among various isolates from humans to insects.

    Science.gov (United States)

    Yoshikawa, Hisao; Koyama, Yukiko; Tsuchiya, Erika; Takami, Kazutoshi

    2016-12-01

    Blastocystis is a common unicellular eukaryotic parasite found not only in humans, but also in various kinds of animal species worldwide. Since Blastocystis isolates are morphologically indistinguishable, many molecular biological approaches have been applied to classify these isolates. The complete or partial sequences of the small subunit rRNA gene (SSU rDNA) are mainly used for comparisons and phylogenetic analyses among Blastocystis isolates. However, various lengths of the partial SSU rDNA sequence have been used for phylogenetic inference among genetically different isolates. Based on the complete SSU rDNA sequences, consensus terminology of nine subtypes (STs) of Blastocystis sp. that were supported by phylogenetically monophyletic nine clades was proposed in 2007. Thereafter, eight additional kinds of STs comprising non-human mammalian Blastocystis isolates have been reported based on the phylogeny of SSU rDNA sequences, while STs 11 and 12 were only proposed on the base of partial sequences. Although many sequence data from mammalian and avian Blastocystis are registered in GenBank, only limited data on SSU rDNA are available for poikilotherm-derived Blastocystis isolates. Therefore, the phylogenetic positions of the reptilian/amphibian Blastocystis clades are unstable. The phylogenetic inference of various STs comprising mammalian and/or avian Blastocystis isolates was verified herein based on comparisons between partial and complete SSU rDNA sequences, and the phylogenetic positions of reptilian and amphibian Blastocystis isolates were also investigated using 14 new Blastocystis isolates from reptiles with all known isolates from other reptilians, amphibians, and insects registered in GenBank. Copyright © 2016. Published by Elsevier Ireland Ltd.

  6. Isolation of functionally active and highly purified neuronal mitochondria from human cortex.

    Science.gov (United States)

    Khattar, Nicolas K; Yablonska, Svitlana; Baranov, Sergei V; Baranova, Oxana V; Kretz, Eric S; Larkin, Timothy M; Carlisle, Diane L; Richardson, R Mark; Friedlander, Robert M

    2016-04-01

    Functional and structural properties of mitochondria are highly tissue and cell dependent, but isolation of highly purified human neuronal mitochondria is not currently available. We developed and validated a procedure to isolate purified neuronal mitochondria from brain tissue. The method combines Percoll gradient centrifugation to obtain synaptosomal fraction with nitrogen cavitation mediated synaptosome disruption and extraction of mitochondria using anti mitochondrial outer membrane protein antibodies conjugated to magnetic beads. The final products of isolation are non-synaptosomal mitochondria, which are a mixture of mitochondria isolated from different brain cells (i.e. neurons, astrocytes, oligodendrocytes, microglia) and synaptic mitochondria, which are of neuronal origin. This method is well suited for preparing functional mitochondria from human cortex tissue that is surgically extracted. The procedure produces mitochondria with minimal cytoplasmic contaminations that are functionally active based on measurements of mitochondrial respiration as well as mitochondrial protein import. The procedure requires approximately four hours for the isolation of human neuronal mitochondria and can also be used to isolate mitochondria from mouse/rat/monkey brains. This method will allow researchers to study highly enriched neuronal mitochondria without the confounding effect of cellular and organelle contaminants. Copyright © 2016 Elsevier B.V. All rights reserved.

  7. [Human social activity under conditions of relative social isolation].

    Science.gov (United States)

    Prokhvatilov, A Iu

    1992-01-01

    The differences in using a "social isolation" concept in the psychological literature are presented. The term of "relative social isolation" is clarified. A relationship between human adaptation to the relative social isolation environments and the development of his social qualities and social activities is presented. The "social context", dictating motivation attitudes of a man to the isolation situation, emotional experiences, self-appraisal of activity is of crucial importance for evaluating the real environments of relative social isolations. Social activity of a personality is studied as the relations of a man with the conditions of his activity. The results of studying the dynamics of the psychic state of a man during individual and group isolation are compared. It is concluded that social activity of man and his functional state are interrelated. The particular manifestations and direction of the changes in the social activity of the subject depend on the duration of isolation and are determined first of all by social significance and meaningful and balanced work for a person as well as by the amount and frequency of direct and mediated social contacts under specific conditions of relative social isolation.

  8. The human genome: Some assembly required. Final report

    Energy Technology Data Exchange (ETDEWEB)

    NONE

    1994-12-31

    The Human Genome Project promises to be one of the most rewarding endeavors in modern biology. The cost and the ethical and social implications, however, have made this project the source of considerable debate both in the scientific community and in the public at large. The 1994 Graduate Student Symposium addresses the scientific merits of the project, the technical issues involved in accomplishing the task, as well as the medical and social issues which stem from the wealth of knowledge which the Human Genome Project will help create. To this end, speakers were brought together who represent the diverse areas of expertise characteristic of this multidisciplinary project. The keynote speaker addresses the project`s motivations and goals in the larger context of biological and medical sciences. The first two sessions address relevant technical issues, data collection with a focus on high-throughput sequencing methods and data analysis with an emphasis on identification of coding sequences. The third session explores recent advances in the understanding of genetic diseases and possible routes to treatment. Finally, the last session addresses some of the ethical, social and legal issues which will undoubtedly arise from having a detailed knowledge of the human genome.

  9. Detection and prevalence of antimicrobial resistance genes in Campylobacter spp. isolated from chickens and humans

    Directory of Open Access Journals (Sweden)

    Samantha Reddy

    2017-01-01

    Full Text Available Campylobacter spp. are common pathogenic bacteria in both veterinary and human medicine. Infections caused by Campylobacter spp. are usually treated using antibiotics. However, the injudicious use of antibiotics has been proven to spearhead the emergence of antibiotic resistance. The purpose of this study was to detect the prevalence of antibiotic resistance genes in Campylobacter spp. isolated from chickens and human clinical cases in South Africa. One hundred and sixty one isolates of Campylobacter jejuni and Campylobacter coli were collected from chickens and human clinical cases and then screened for the presence of antimicrobial resistance genes. We observed a wide distribution of the tetO gene, which confers resistance to tetracycline. The gyrA genes that are responsible quinolone resistance were also detected. Finally, our study also detected the presence of the blaOXA-61, which is associated with ampicillin resistance. There was a higher (p < 0.05 prevalence of the studied antimicrobial resistance genes in chicken faeces compared with human clinical isolates. The tetO gene was the most prevalent gene detected, which was isolated at 64% and 68% from human and chicken isolates, respectively. The presence of gyrA genes was significantly (p < 0.05 associated with quinolone resistance. In conclusion, this study demonstrated the presence of gyrA (235 bp, gyrA (270 bp, blaOXA-61 and tetO antimicrobial resistance genes in C. jejuni and C. coli isolated from chickens and human clinical cases. This indicates that Campylobacter spp. have the potential of resistance to a number of antibiotic classes.

  10. Human Streptococcus agalactiae isolate in Nile tilapia (Oreochromis niloticus)

    Science.gov (United States)

    Streptococcus agalactiae, the Lancefield group B Streptococcus (GBS), long recognized as a mammalian pathogen, is an emerging pathogen to fish. We show that a GBS serotype Ia, multilocus sequence type ST-7 isolate from a human neonatal meningitis clinical case causes disease signs and mortality in N...

  11. Human Streptococcus agalactiae Isolate in Nile Tilapia (Oreochromis niloticus)

    OpenAIRE

    Evans, Joyce J.; Phillip H. Klesius; Pasnik, David J.; Bohnsack, John F.

    2009-01-01

    Streptococcus agalactiae, the Lancefield group B streptococcus (GBS) long recognized as a mammalian pathogen, is an emerging concern with regard to fish. We show that a GBS serotype Ia multilocus sequence type ST-7 isolate from a clinical case of human neonatal meningitis caused disease and death in Nile tilapia (Oreochromis niloticus).

  12. Francisella tularensis subsp. novicida isolated from a human in Arizona

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    Birdsell Dawn N

    2009-11-01

    Full Text Available Abstract Background Francisella tularensis is the etiologic agent of tularemia and is classified as a select agent by the Centers for Disease Control and Prevention. Currently four known subspecies of F. tularensis that differ in virulence and geographical distribution are recognized:tularensis (type A, holarctica (type B, mediasiatica, and novicida. Because of the Select Agent status and differences in virulence and geographical location, the molecular analysis of any clinical case of tularemia is of particular interest. We analyzed an unusual Francisella clinical isolate from a human infection in Arizona using multiple DNA-based approaches. Findings We report that the isolate is F. tularensis subsp. novicida, a subspecies that is rarely isolated. Conclusion The rarity of this novicida subspecies in clinical settings makes each case study important for our understanding of its role in disease and its genetic relationship with other F. tularensis subspecies.

  13. Isolation of human liver angiotensin-converting enzyme by chromatofocusing.

    Science.gov (United States)

    Sakharov IYu; Danilov, S M; Sukhova, N V

    1987-10-01

    Angiotensin-converting enzyme (EC 3.4.15.1) has been isolated from human liver by chromatofocusing. The isolation procedure permitted us to obtain a 9000-fold purified enzyme with a 22% yield. Specific activity of the angiotensin-converting enzyme was 10 units/mg of protein. The molecular mass of enzyme determined by polyacrylamide gel electrophoresis under denaturing conditions was 150,000. The isoelectric point (4.2-4.3) was also determined by chromatofocusing. The Km values of the enzyme for hippuryl-L-histidyl-L-leucine and N-benzyloxycarbonyl-L-phenylalanyl-L-histidyl-L-leucine are 5000 and 125 microM, respectively. The human liver angiotensin-converting enzyme is inhibited by bradykinin-potentiating factor SQ 20881 (IC50 = 18 nM).

  14. Color-Removal by Microorganisms Isolated from Human Hands

    Directory of Open Access Journals (Sweden)

    Tsukasa Ito

    2013-08-01

    Full Text Available Microorganisms are essential for human life. Microorganisms decompose the carbon compounds in dead animals and plants and convert them into carbon dioxide. Intestinal bacteria assist in food digestion. Some vitamins are produced by bacteria that live in the intestines. Sewage and industrial wastewater are treated by activated sludge composed of microbial communities. All of these are due to the ability of microbes to produce many enzymes that can degrade chemicals. How do teachers make students understand that microorganisms are always associated with humans, and that microorganisms have the ability to degrade chemicals? The presence of microorganisms on humans can be shown by incubating agar plates after they are touched by the hands of students. The ability of microorganisms to degrade chemicals can be shown by an analytical measurement of the degradation of chemicals. When the chemicals are dyes (colorants in water, microbial activity on degradation of dyes can be demonstrated by observing a decreasing degree of color as a result of the enzymatic activity (e.g., azoreductase. Dyes are widely used in the textile, food, and cosmetic industries. They are generally resistant to conventional biological wastewater treatment systems such as the activated sludge process (4. The discharge of wastewater containing dye pollutes surface water. The ability of microorganisms to decolorize and degrade dyes has been widely investigated to use for bioremediation purposes (5. The goal of this tip is to understand the presence of bacteria on human skin and the ability of bacteria to degrade colorant chemicals (decolorization. In this tip, students first cultivate and isolate bacteria on their hands, and then examine potential decolorization activity of each bacterium by observing the degree of color of the liquid in tubes in which bacteria isolated from students’ hands were inoculated. Decolorization activity of bacterial isolates from human skin has been

  15. Isolation and Quantification of Glycosaminoglycans from Human Hair Shaft

    Science.gov (United States)

    Bonovas, Stefanos; Sitaras, Nikolaos

    2016-01-01

    Background There is evidence that glycosaminoglycans (GAGs) are present in the hair shaft within the follicle but there are no studies regarding GAGs isolation and measurement in the human hair shaft over the scalp surface, it means, in the free hair shaft. Objective The purpose of our research was to isolate and measure the total GAGs from human free hair shaft. Methods Seventy-five healthy individuals participated in the study, 58 adults, men and women over the age of 50 and 17 children (aged 4~9). GAGs in hair samples, received from the parietal and the occipital areas, were isolated with 4 M guanidine HCl and measured by the uronic acid-carbazole reaction assay. Results GAGs concentration was significantly higher in the occipital area than in the parietal area, in all study groups. GAG levels from both areas were significantly higher in children than in adults. GAG levels were not associated with gender, hair color or type. Conclusion We report the presence of GAGs in the human free hair shaft and the correlation of hair GAG levels with the scalp area and participants' age. PMID:27746630

  16. Isolated deficiency of IgE in humans: update

    Directory of Open Access Journals (Sweden)

    D.V. Maltsev

    2017-04-01

    Full Text Available Isolated deficiency IgE is one of the most common primary immunodeficiency diseases in human with prevalence of 1 case per 30 people of total population. Genetic basis of this immunodeficiency are polymorphisms of 5923A/G and 7888C/T in AICDA gene of B-lymphocytes. Recently several new studies have been conducted that expand the current understanding of the nature of an isolated IgE deficiency in human. Several recent epidemiological studies specified immunodeficiency frequency in different cohorts of patients and confirmed the relationship of immunological and clinical phenotypes, including recurrent infections, autoimmunity and oncology. The results of a number of clinical cases have expanded the knowledge of the heterogeneity of clinical symptoms of the di­sease. Several studies investigated the complications of an isolated deficiency IgE, including chronic gastritis and peptic stomach ulcer associated with H. pylori, and atherosclerosis and related vascular accidents. The results of comparative clinical studies demonstrate high efficiency of base immunotherapy with normal human immunoglobulin preparations for the intramuscular and intravenous usage.

  17. Detecting Genetic Isolation in Human Populations: A Study of European Language Minorities

    Science.gov (United States)

    Capocasa, Marco; Battaggia, Cinzia; Anagnostou, Paolo; Montinaro, Francesco; Boschi, Ilaria; Ferri, Gianmarco; Alù, Milena; Coia, Valentina; Crivellaro, Federica; Bisol, Giovanni Destro

    2013-01-01

    The identification of isolation signatures is fundamental to better understand the genetic structure of human populations and to test the relations between cultural factors and genetic variation. However, with current approaches, it is not possible to distinguish between the consequences of long-term isolation and the effects of reduced sample size, selection and differential gene flow. To overcome these limitations, we have integrated the analysis of classical genetic diversity measures with a Bayesian method to estimate gene flow and have carried out simulations based on the coalescent. Combining these approaches, we first tested whether the relatively short history of cultural and geographical isolation of four “linguistic islands” of the Eastern Alps (Lessinia, Sauris, Sappada and Timau) had left detectable signatures in their genetic structure. We then compared our findings to previous studies of European population isolates. Finally, we explored the importance of demographic and cultural factors in shaping genetic diversity among the groups under study. A combination of small initial effective size and continued genetic isolation from surrounding populations seems to provide a coherent explanation for the diversity observed among Sauris, Sappada and Timau, which was found to be substantially greater than in other groups of European isolated populations. Simulations of micro-evolutionary scenarios indicate that ethnicity might have been important in increasing genetic diversity among these culturally related and spatially close populations. PMID:23418562

  18. Isolation, identification and differentiation of human embryonic cartilage stem cells.

    Science.gov (United States)

    Fu, Changhao; Yan, Zi; Xu, Hao; Zhang, Chen; Zhang, Qi; Wei, Anhui; Yang, Xi; Wang, Yi

    2015-07-01

    We isolated human embryonic cartilage stem cells (hECSCs), a novel stem cell population, from the articular cartilage of eight-week-old human embryos. These stem cells demonstrated a marker expression pattern and differentiation potential intermediate to those of human embryonic stem cells (hESCs) and human adult stem cells (hASCs). hECSCs expressed markers associated with both hESCs (OCT4, NANOG, SOX2, SSEA-3 and SSEA-4) and human adult stem cells (hASCs) (CD29, CD44, CD90, CD73 and CD10). These cells also differentiated into adipocytes, osteoblasts, chondrocytes, neurons and islet-like cells under specific inducing conditions. We identified N(6), 2'-O-dibutyryl cyclic adenosine 3':5'-monophosphate (Bt2cAMP) as an inducer of chondrogenic differentiation in hECSCs. Similar results using N(6), 2'-O-dibutyryl cyclic adenosine 3':5'-monophosphate (Bt2cAMP) were obtained for two other types of human embryonic tissue-derived stem cells, human embryonic hepatic stem cells (hEHSCs) and human embryonic amniotic fluid stem cells (hEASCs), both of which exhibited a marker expression pattern similar to that of hECSCs. The isolation of hECSCs and the discovery that N(6), 2'-O-dibutyryl cyclic adenosine 3':5'-monophosphate (Bt2cAMP) induces chondrogenic differentiation in different stem cell populations might aid the development of strategies in tissue engineering and cartilage repair.

  19. Epidemiological relationship of human and swine Streptococcus suis isolates.

    Science.gov (United States)

    Tarradas, C; Luque, I; de Andrés, D; Abdel-Aziz Shahein, Y E; Pons, P; González, F; Borge, C; Perea, A

    2001-06-01

    Two cases of meningitis due to Streptococcus suis in humans are reported here. A butcher and an abattoir worker were referred to a health centre in Castellón (Spain) with fever and symptoms of meningitis. After adequate treatment, a slight hipoacusia persisted as sequelae in both cases. Colonies of S. suis group R, serotype 2 and phenotype MRP+EF+ were isolated from cerebroespinal fluid. Epidemiological studies showed that both workers had in common the handling of pork meat of slaughtered healthy pigs from three closed farms. A study of the tonsils from apparently healthy, slaughtered pigs was carried out. A total of 234 tonsillar samples were obtained and 81 strains of S. suis were isolated from them. Serotype 2 appeared to be the most frequent (50.6%), and the analysis for phenotype showed a high percentage of tonsillar strains with the phenotype MRP+EF+ (35.9%). The humans and 28 tonsillar swine strains showed a similar profile (S. suis group R, serotype 2 and phenotype MRP+EF+). A total of 26 of the swine isolates were analysed by ribotyping using EcoRI. The human strains showed the same six-band hybridization pattern that shared five bands with the pattern most frequently shown by most of the tonsillar N. suis group R, serotype 2 and phenotype MRP+EF+ strains, differing only in the lightest, faintest band which was slightly less anodical in human (> or = 1.8 kb) than in swine (approximately 1.8 kb). From these results, both groups of strains, humans and porcine, showed differences; how can these differences in the pattern of ribotyping be explained if they should have the same origin? Is it possible that they have undergone an adaptation to the new host or perhaps the modification is due to other unknown causes? Further studies in this area are required in order to answer these questions.

  20. Sterols of Pneumocystis carinii hominis Organisms Isolated from Human Lungs

    Science.gov (United States)

    Kaneshiro, Edna S.; Amit, Zunika; Chandra, Jyotsna; Baughman, Robert P.; Contini, Carlo; Lundgren, Bettina

    1999-01-01

    The opportunistic pathogen Pneumocystis carinii causes pneumonia (P. carinii pneumonia, or PCP) in immunocompromised individuals such as AIDS patients. Rat-derived P. carinii carinii organisms have distinct sterols which are not synthesized by mammals and not found in other microbes infecting mammalian lungs. The dominant sterol present in the organism is cholesterol (which is believed to be scavenged from the host), but other sterols in P. carinii carinii have an alkyl group at C-24 of the sterol side chain (C28 and C29 24-alkylsterols) and a double bond at C-7 of the nucleus. Recently, pneumocysterol (C32), which is essentially lanosterol with a C-24 ethylidene group, was detected in lipids extracted from a formalin-fixed human P. carinii-infected lung, and its structures were elucidated by gas-liquid chromatography, mass spectrometry, and nuclear magnetic resonance spectrometry in conjunction with analyses of chemically synthesized authentic standards. The sterol composition of isolated P. carinii hominis organisms has yet to be reported. If P. carinii from animal models is to be used for identifying potential drug targets and for developing chemotherapeutic approaches to clear human infections, it is important to determine whether the 24-alkylsterols of organisms found in rats are also present in organisms in humans. In the present study, sterol analyses of P. carinii hominis organisms isolated from cryopreserved human P. carinii-infected lungs and from bronchoalveolar lavage fluid were performed. Several of the same distinct sterols (e.g., fungisterol and methylcholest-7-ene-3β-ol) previously identified in P. carinii carinii were also present in organisms isolated from human specimens. Pneumocysterol was detected in only some of the samples. PMID:10548595

  1. Innate immune functions of microglia isolated from human glioma patients

    Directory of Open Access Journals (Sweden)

    Grimm Elizabeth

    2006-03-01

    Full Text Available Abstract Background Innate immunity is considered the first line of host defense and microglia presumably play a critical role in mediating potent innate immune responses to traumatic and infectious challenges in the human brain. Fundamental impairments of the adaptive immune system in glioma patients have been investigated; however, it is unknown whether microglia are capable of innate immunity and subsequent adaptive anti-tumor immune responses within the immunosuppressive tumor micro-environment of human glioma patients. We therefore undertook a novel characterization of the innate immune phenotype and function of freshly isolated human glioma-infiltrating microglia (GIM. Methods GIM were isolated by sequential Percoll purification from patient tumors immediately after surgical resection. Flow cytometry, phagocytosis and tumor cytotoxicity assays were used to analyze the phenotype and function of these cells. Results GIM expressed significant levels of Toll-like receptors (TLRs, however they do not secrete any of the cytokines (IL-1β, IL-6, TNF-α critical in developing effective innate immune responses. Similar to innate macrophage functions, GIM can mediate phagocytosis and non-MHC restricted cytotoxicity. However, they were statistically less able to mediate tumor cytotoxicity compared to microglia isolated from normal brain. In addition, the expression of Fas ligand (FasL was low to absent, indicating that apoptosis of the incoming lymphocyte population may not be a predominant mode of immunosuppression by microglia. Conclusion We show for the first time that despite the immunosuppressive environment of human gliomas, GIM are capable of innate immune responses such as phagocytosis, cytotoxicity and TLR expression but yet are not competent in secreting key cytokines. Further understanding of these innate immune functions could play a critical role in understanding and developing effective immunotherapies to malignant human gliomas.

  2. Isolation and characterization of the human MRE11 homologue

    Energy Technology Data Exchange (ETDEWEB)

    Petrini, J.H.J.; Walsh, M.E.; DiMare, C. [Harvard Medical School, Boston, MA (United States)] [and others

    1995-09-01

    Mutation of the Saccharomyces cerevisiae RAD52 epistasis group gene, MRE11, blocks meiotic recombination, confers profound sensitivity to double-strand break damage, and has a hyperrecombinational phenotype in mitotic cells. We isolated a highly conserved human MRE11 homologue using a two-hybrid screen for DNA ligase I-interacting proteins. Human MRE11 shares approximately 50% identity with its yeast counterpart over the N-terminal half of the protein. MRE11 is expressed at the highest levels in proliferating tissues, but is also observed in other tissues. The MRE11 locus maps to human chromosome 11q21 in a region frequently associated with cancer-related chromosomal abnormalities. A MRE11-related locus was found on chromosome 7q11.2-q11.3. 60 refs., 4 figs.

  3. Genotyping of Giardia lamblia isolates from human in southern Iran.

    Science.gov (United States)

    Sarkari, B; Ashrafmansori, A; Hatam, G R; Motazedian, M H; Asgari, Q; Mohammadpour, I

    2012-09-01

    Giardia lamblia cysts isolated from human faeces in South of Iran were analyzed with PCR-restriction fragment length polymorphism (RFLP) assay, based on the detection of glutamate dehydrogenase (gdh) genes. Among 205 faecal samples from microscopically diagnosed giardiasis patients, the gdh gene was amplified from 172 cases with a semi nested PCR assay and typed by RFLP analysis. Of the 172 positive samples, 128 (74.41%) were typed as assemblage AII, 30 (17.44%) assemblage BIII, 6 (3.49%) assemblage BIV and in 8 (4.66%) isolates, mixed assemblages AII and BIV were detected. Clinical features were available for 52 successfully typed cases and the possible correlation of Giardia assemblages and clinical symptoms was evaluated. Both assemblages caused similar illness, but assemblage AII was significantly more frequently associated with abdominal pain, nausea and vomiting. Since these isolates, A and B, are of human origin, anthroponotic transmission of Giardia can be suggested for the route of infection in this region.

  4. Measurements of atmospheric trace gases by means of matrix-isolation-spectroscopy. Final report. Atmosphaerische Spurengasmessungen mittels Matrix-Isolations-Spektroskopie. Abschlussbericht

    Energy Technology Data Exchange (ETDEWEB)

    Wilson, S.; Griffith, D.; Schuster, G.; Helas, G.

    1988-12-01

    The report describes the results and general experience of project No. 0743188 0 (ATP 88). The method of matrix isolation spectroscopy is described, as are the flights funded during this project. This is followed by tables of the samples taken, by trace elements. Finally, the data measured during all flights are summarized. (orig.).

  5. Isolation, Culture, and Imaging of Human Fetal Pancreatic Cell Clusters

    Science.gov (United States)

    Lopez, Ana D.; Kayali, Ayse G.; Hayek, Alberto; King, Charles C.

    2014-01-01

    For almost 30 years, scientists have demonstrated that human fetal ICCs transplanted under the kidney capsule of nude mice matured into functioning endocrine cells, as evidenced by a significant increase in circulating human C-peptide following glucose stimulation1-9. However in vitro, genesis of insulin producing cells from human fetal ICCs is low10; results reminiscent of recent experiments performed with human embryonic stem cells (hESC), a renewable source of cells that hold great promise as a potential therapeutic treatment for type 1 diabetes. Like ICCs, transplantation of partially differentiated hESC generate glucose responsive, insulin producing cells, but in vitro genesis of insulin producing cells from hESC is much less robust11-17. A complete understanding of the factors that influence the growth and differentiation of endocrine precursor cells will likely require data generated from both ICCs and hESC. While a number of protocols exist to generate insulin producing cells from hESC in vitro11-22, far fewer exist for ICCs10,23,24. Part of that discrepancy likely comes from the difficulty of working with human fetal pancreas. Towards that end, we have continued to build upon existing methods to isolate fetal islets from human pancreases with gestational ages ranging from 12 to 23 weeks, grow the cells as a monolayer or in suspension, and image for cell proliferation, pancreatic markers and human hormones including glucagon and C-peptide. ICCs generated by the protocol described below result in C-peptide release after transplantation under the kidney capsule of nude mice that are similar to C-peptide levels obtained by transplantation of fresh tissue6. Although the examples presented here focus upon the pancreatic endoderm proliferation and β cell genesis, the protocol can be employed to study other aspects of pancreatic development, including exocrine, ductal, and other hormone producing cells. PMID:24895054

  6. Isolation, culture, and imaging of human fetal pancreatic cell clusters.

    Science.gov (United States)

    Lopez, Ana D; Kayali, Ayse G; Hayek, Alberto; King, Charles C

    2014-05-18

    For almost 30 years, scientists have demonstrated that human fetal ICCs transplanted under the kidney capsule of nude mice matured into functioning endocrine cells, as evidenced by a significant increase in circulating human C-peptide following glucose stimulation(1-9). However in vitro, genesis of insulin producing cells from human fetal ICCs is low(10); results reminiscent of recent experiments performed with human embryonic stem cells (hESC), a renewable source of cells that hold great promise as a potential therapeutic treatment for type 1 diabetes. Like ICCs, transplantation of partially differentiated hESC generate glucose responsive, insulin producing cells, but in vitro genesis of insulin producing cells from hESC is much less robust(11-17). A complete understanding of the factors that influence the growth and differentiation of endocrine precursor cells will likely require data generated from both ICCs and hESC. While a number of protocols exist to generate insulin producing cells from hESC in vitro(11-22), far fewer exist for ICCs(10,23,24). Part of that discrepancy likely comes from the difficulty of working with human fetal pancreas. Towards that end, we have continued to build upon existing methods to isolate fetal islets from human pancreases with gestational ages ranging from 12 to 23 weeks, grow the cells as a monolayer or in suspension, and image for cell proliferation, pancreatic markers and human hormones including glucagon and C-peptide. ICCs generated by the protocol described below result in C-peptide release after transplantation under the kidney capsule of nude mice that are similar to C-peptide levels obtained by transplantation of fresh tissue(6). Although the examples presented here focus upon the pancreatic endoderm proliferation and β cell genesis, the protocol can be employed to study other aspects of pancreatic development, including exocrine, ductal, and other hormone producing cells.

  7. Isolation and functional characterization of the human 90K promoter

    DEFF Research Database (Denmark)

    Brakebusch, C; Sures, I; Jallal, B;

    1999-01-01

    90K is a secreted protein thought to be involved in the body's defense against pathogens and cancer. To elucidate its transcriptional regulation, the promoter of human 90K (HGMW-approved symbol LGAL S3BP) was isolated and characterized. Analysis of the 3. 3-kb 5'-flanking region revealed that it ......90K is a secreted protein thought to be involved in the body's defense against pathogens and cancer. To elucidate its transcriptional regulation, the promoter of human 90K (HGMW-approved symbol LGAL S3BP) was isolated and characterized. Analysis of the 3. 3-kb 5'-flanking region revealed...... that it is a TATA-less promoter, but neither GC-rich nor dependent on SP1 sites. RNase protection assays detected one major transcription start site (+1) and several minor transcription start sites upstream and downstream. Deletion studies defined a minimal promoter (-103 --> -49) and indirectly suggested positive...... synergism between different elements within it. Consistent with the proposed function of 90K, its promoter activity could be stimulated by poly(I). poly(C), mimicking viral infection. Two regions mediating induction by poly(I). poly(C) (-171 --> -112, -32 --> 46) were identified by deletion mutants. A small...

  8. Neuron enriched nuclear proteome isolated from human brain.

    Science.gov (United States)

    Dammer, Eric B; Duong, Duc M; Diner, Ian; Gearing, Marla; Feng, Yue; Lah, James J; Levey, Allan I; Seyfried, Nicholas T

    2013-07-05

    The brain consists of diverse cell types including neurons, astrocytes, oligodendrocytes, and microglia. The isolation of nuclei from these distinct cell populations provides an opportunity to identify cell-type-specific nuclear proteins, histone modifications, and regulation networks that are altered with normal brain aging or neurodegenerative disease. In this study, we used a method by which intact neuronal and non-neuronal nuclei were purified from human post-mortem brain employing a modification of fluorescence activated cell sorting (FACS) termed fluorescence activated nuclei sorting (FANS). An antibody against NeuN, a neuron specific splicing factor, was used to isolate neuronal nuclei. Utilizing mass spectrometry (MS) based label-free quantitative proteomics, we identified 1755 proteins from sorted NeuN-positive and negative nuclear extracts. Approximately 20% of these proteins were significantly enriched or depleted in neuronal versus non-neuronal populations. Immunoblots of primary cultured rat neuron, astrocyte, and oligodendrocyte extracts confirmed that distinct members of the major nucleocytoplasmic structural linkage complex (LINC), nesprin-1 and nesprin-3, were differentially enriched in neurons and astrocytes, respectively. These comparative proteomic data sets also reveal a number of transcription and splicing factors that are selectively enriched in a cell-type-specific manner in human brain.

  9. Human dental pulp mesenchymal stem cells isolation and osteoblast differentiation

    Directory of Open Access Journals (Sweden)

    Moustafa Alkhalil

    2015-02-01

    Full Text Available Aim This study was focused on the isolation and characterization of mesenchymal stem cells (MSCs from human dental pulp (DPSC. Methods The study was performed in the Department for Oral and Cranio-Maxillo- Facial Surgey Hamad Medical Corporation, Doha, Qatar and Weill Cornell Medical Colleague Doha, Qatar, in period 2010-2011. Dental pulp was extracted from premolars and third molars of 19 healthy patients. The pulp was digested in a solution of 3 mg/mL collagenase type I and 4 mg/mL dispase for 1 hour at 37C. After filtration, cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM Low Glucoses with 20% Fetal Bovine Serum (FBS, 2mM L-glutamine and antibiotics (100 U/mL penicillin, 100 ug/mL streptomycin at 37 °C under 5% CO2. Cultures were treated with osteoinductive medium for differentiation MSC in to the osteoblast cell line. Staining with Alizarin red were used for the detection of the osteoblast production and calcification new formed tissue. Results On the total of three out of 19 patients it was possible to isolate DPMSCs after 2 to 3 weeks: in one patient it was not possible to expand MSCs because of infection, and in other two patients positive Alizarin red staining reaction showed osteogenic differentiation capability and strong mineralization in vitro. Conclusion The main advantage of using DPSC is absence of morbidity. MSCs could be isolated noninvasively from teeth, routinely extracted in the clinic and discarded as medical waste. Standardization of clinical and laboratory protocols for DPMSCs isolation and team work coordination could lead to significantly improved result.

  10. Antigenic Relationships among Human Pathogenic Orientia tsutsugamushi Isolates from Thailand

    Science.gov (United States)

    Nawtaisong, Pruksa; Tanganuchitcharnchai, Ampai; Smith, Derek J.; Day, Nicholas P. J.; Paris, Daniel H.

    2016-01-01

    Background Scrub typhus is a common cause of undiagnosed febrile illness in certain tropical regions, but can be easily treated with antibiotics. The causative agent, Orientia tsutsugamushi, is antigenically variable which complicates diagnosis and efforts towards vaccine development. Methodology/Principal Findings This study aimed to dissect the antigenic and genetic relatedness of O. tsutsugamushi strains and investigate sero-diagnostic reactivities by titrating individual patient sera against their O. tsutsugamushi isolates (whole-cell antigen preparation), in homologous and heterologous serum-isolate pairs from the same endemic region in NE Thailand. The indirect immunofluorescence assay was used to titrate Orientia tsutsugamushi isolates and human sera, and a mathematical technique, antigenic cartography, was applied to these data to visualise the antigenic differences and cross-reactivity between strains and sera. No functional or antigen-specific analyses were performed. The antigenic variation found in clinical isolates was much less pronounced than the genetic differences found in the 56kDa type-specific antigen genes. The Karp-like sera were more broadly reactive than the Gilliam-like sera. Conclusions/Significance Antigenic cartography worked well with scrub typhus indirect immunofluorescence titres. The data from humoral responses suggest that a Karp-like strain would provide broader antibody cross-reactivity than a Gilliam-like strain. Although previous exposure to O. tsutsugamushi could not be ruled out, scrub typhus patient serum antibody responses were characterised by strong homologous, but weak heterologous antibody titres, with little evidence for cross-reactivity by Gilliam-like sera, but a broader response from some Karp-like sera. This work highlights the importance of antigenic variation in O. tsutsugamushi diagnosis and determination of new serotypes. PMID:27248711

  11. Antigenic Relationships among Human Pathogenic Orientia tsutsugamushi Isolates from Thailand.

    Directory of Open Access Journals (Sweden)

    Sarah L James

    2016-06-01

    Full Text Available Scrub typhus is a common cause of undiagnosed febrile illness in certain tropical regions, but can be easily treated with antibiotics. The causative agent, Orientia tsutsugamushi, is antigenically variable which complicates diagnosis and efforts towards vaccine development.This study aimed to dissect the antigenic and genetic relatedness of O. tsutsugamushi strains and investigate sero-diagnostic reactivities by titrating individual patient sera against their O. tsutsugamushi isolates (whole-cell antigen preparation, in homologous and heterologous serum-isolate pairs from the same endemic region in NE Thailand. The indirect immunofluorescence assay was used to titrate Orientia tsutsugamushi isolates and human sera, and a mathematical technique, antigenic cartography, was applied to these data to visualise the antigenic differences and cross-reactivity between strains and sera. No functional or antigen-specific analyses were performed. The antigenic variation found in clinical isolates was much less pronounced than the genetic differences found in the 56kDa type-specific antigen genes. The Karp-like sera were more broadly reactive than the Gilliam-like sera.Antigenic cartography worked well with scrub typhus indirect immunofluorescence titres. The data from humoral responses suggest that a Karp-like strain would provide broader antibody cross-reactivity than a Gilliam-like strain. Although previous exposure to O. tsutsugamushi could not be ruled out, scrub typhus patient serum antibody responses were characterised by strong homologous, but weak heterologous antibody titres, with little evidence for cross-reactivity by Gilliam-like sera, but a broader response from some Karp-like sera. This work highlights the importance of antigenic variation in O. tsutsugamushi diagnosis and determination of new serotypes.

  12. Isolation of biologically-active exosomes from human plasma.

    Science.gov (United States)

    Muller, Laurent; Hong, Chang-Sook; Stolz, Donna B; Watkins, Simon C; Whiteside, Theresa L

    2014-09-01

    Effects of exosomes present in human plasma on immune cells have not been examined in detail. Immunological studies with plasma-derived exosomes require their isolation by procedures involving ultracentrifugation. These procedures were largely developed using supernatants of cultured cells. To test biologic activities of plasma-derived exosomes, methods are necessary that ensure adequate recovery of exosome fractions free of contaminating larger vesicles, cell fragments and protein/nucleic acid aggregates. Here, an optimized method for exosome isolation from human plasma/serum specimens of normal controls (NC) or cancer patients and its advantages and pitfalls are described. To remove undesirable plasma-contaminating components, ultrafiltration of differentially-centrifuged plasma/serum followed by size-exclusion chromatography prior to ultracentrifugation facilitated the removal of contaminants. Plasma or serum was equally acceptable as a source of exosomes based on the recovered protein levels (in μg protein/mL plasma) and TEM image quality. Centrifugation on sucrose density gradients led to large exosome losses. Fresh plasma was the best source of morphologically-intact exosomes, while the use of frozen/thawed plasma decreased exosome purity but not their biologic activity. Treatments of frozen plasma with DNAse, RNAse or hyaluronidase did not improve exosome purity and are not recommended. Cancer patients' plasma consistently yielded more isolated exosomes than did NCs' plasma. Cancer patients' exosomes also mediated higher immune suppression as evidenced by decreased CD69 expression on responder CD4+ T effector cells. Thus, the described procedure yields biologically-active, morphologically-intact exosomes that have reasonably good purity without large protein losses and can be used for immunological, biomarker and other studies.

  13. Final report of the cooperative study on seismic isolation design. The second stage

    Energy Technology Data Exchange (ETDEWEB)

    Uryu, Mitsuru; Terada, Syuji; Shioya, Tsutomu (and others)

    1999-05-01

    The applicability of the seismic isolation design onto the nuclear fuel facilities, which must clear severe criteria of integrity, has been examined. Following the first stage of the cooperative study, conducted from 1988 to 1991, the second stage included critical vibration testing, seismic observation of seismic isolation building and founded buildings of non-isolation, with the objectives of clarifying the policies on critical design of seismic isolation building. Integrity of the seismic isolation piping system was tested by means of static deformation test, with variable inner water pressure and relative deformation. (Yamamoto, A.)

  14. Characterization of Staphylococcus caprae Clinical Isolates Involved in Human Bone and Joint Infections, Compared with Goat Mastitis Isolates.

    Science.gov (United States)

    d'Ersu, J; Aubin, G G; Mercier, P; Nicollet, P; Bémer, P; Corvec, S

    2016-01-01

    Staphylococcus caprae is an emerging microorganism in human bone and joint infections (BJI). The aim of this study is to describe the features of S. caprae isolates involved in BJI (H for human) compared with those of isolates recovered in goat mastitis (A for animal). Fourteen isolates of each origin were included. Identifications were performed using a Vitek 2 GP ID card, tuf gene sequencing, and matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) Vitek MS. Molecular typing was carried out using pulsed-field gel electrophoresis (PFGE) and DiversiLab technology. The crystal violet method was used to determine biofilm-forming ability. Virulence factors were searched by PCR. Vitek MS technology provides an accurate identification for the two types of isolates compared to that of gold-standard sequencing (sensitivity, 96.4%), whereas the Vitek 2 GP ID card was more effective for H isolates. Molecular typing methods revealed two distinct lineages corresponding to the origin despite few overlaps: H and A. In our experimental conditions, no significant difference was observed in biofilm production ability between H and A isolates. Nine isolates (5 H isolates and 4 A isolates) behaved as weak producers while one A isolate was a strong producer. Concerning virulence factors, the autolysin atlC and the serine aspartate adhesin (sdrZ) genes were detected in 24 isolates (86%), whereas the lipase gene was always detected, except in one H isolate (96%). The ica operon was present in 23 isolates (82%). Fibrinogen-binding (fbe) or collagen-binding (cna) genes were not detected by using primers designed for Staphylococcus aureus or Staphylococcus epidermidis, even in low stringency conditions. Although S. caprae probably remains underestimated in human infections, further studies are needed to better understand the evolution and the adaptation of this species to its host.

  15. Buried waste integrated demonstration human engineered control station. Final report

    Energy Technology Data Exchange (ETDEWEB)

    1994-09-01

    This document describes the Human Engineered Control Station (HECS) project activities including the conceptual designs. The purpose of the HECS is to enhance the effectiveness and efficiency of remote retrieval by providing an integrated remote control station. The HECS integrates human capabilities, limitations, and expectations into the design to reduce the potential for human error, provides an easy system to learn and operate, provides an increased productivity, and reduces the ultimate investment in training. The overall HECS consists of the technology interface stations, supporting engineering aids, platform (trailer), communications network (broadband system), and collision avoidance system.

  16. Candida nivariensis isolated from an Indonesian human immunodeficiency virus-infected patient suffering from oropharyngeal candidiasis

    NARCIS (Netherlands)

    Wahyuningsih, Retno; SahBandar, Ivo N.; Theelen, Bart; Hagen, Ferry; Poot, Ge; Meis, Jacques F.; Rozalyani, Anna; Sjam, Ridhawati; Widodo, Djoko; Djauzi, Samsuridjal; Boekhout, Teun

    2008-01-01

    Candida nivariensis was isolated from an Indonesian human immunodeficiency virus-infected patient who suffered from oropharyngeal candidiasis and was identified with molecular tools. Our isolate demonstrated low MICs to amphotericin B, flucytosine, posaconazole, caspofungin, and isavueonazole and wa

  17. Isolation of Mesenchymal Stem Cells from Human Deciduous Teeth Pulp

    Directory of Open Access Journals (Sweden)

    Aileen I. Tsai

    2017-01-01

    Full Text Available This study aimed to identify predictors of success rate of mesenchymal stem cell (MSC isolation from human deciduous teeth pulp. A total of 161 deciduous teeth were extracted at the dental clinic of Chang Gung Memorial Hospital. The MSCs were isolated from dental pulps using a standard protocol. In total, 128 colonies of MSCs were obtained and the success rate was 79.5%. Compared to teeth not yielding MSCs successfully, those successfully yielding MSCs were found to have less severe dental caries (no/mild-to-moderate/severe: 63.3/24.2/12.5% versus 12.5/42.4/42.4%, P<0.001 and less frequent pulpitis (no/yes: 95.3/4.7% versus 51.5/48.5%, P<0.001. In a multivariate regression model, it was confirmed that the absence of dental caries (OR = 4.741, 95% CI = 1.564–14.371, P=0.006 and pulpitis (OR = 9.111, 95% CI = 2.921–28.420, P<0.001 was significant determinants of the successful procurement of MSCs. MSCs derived from pulps with pulpitis expressed longer colony doubling time than pulps without pulpitis. Furthermore, there were higher expressions of proinflammatory cytokines, interleukin- (IL- 6 and monocyte chemoattractant protein- (MCP- 1, P<0.01, and innate immune response [toll-like receptor 1 (TLR1 and TLR8, P<0.05; TLR2, TLR3, and TLR6, P<0.01] in the inflamed than noninflamed pulps. Therefore, a carious deciduous tooth or tooth with pulpitis was relatively unsuitable for MSC processing and isolation.

  18. Isolation of Mesenchymal Stem Cells from Human Deciduous Teeth Pulp

    Science.gov (United States)

    Tsai, Aileen I.; Hong, Hsiang-Hsi; Fu, Jen-Fen; Chang, Chih-Chun; Wang, I-Kuan; Huang, Wen-Hung; Weng, Cheng-Hao; Hsu, Ching-Wei

    2017-01-01

    This study aimed to identify predictors of success rate of mesenchymal stem cell (MSC) isolation from human deciduous teeth pulp. A total of 161 deciduous teeth were extracted at the dental clinic of Chang Gung Memorial Hospital. The MSCs were isolated from dental pulps using a standard protocol. In total, 128 colonies of MSCs were obtained and the success rate was 79.5%. Compared to teeth not yielding MSCs successfully, those successfully yielding MSCs were found to have less severe dental caries (no/mild-to-moderate/severe: 63.3/24.2/12.5% versus 12.5/42.4/42.4%, P < 0.001) and less frequent pulpitis (no/yes: 95.3/4.7% versus 51.5/48.5%, P < 0.001). In a multivariate regression model, it was confirmed that the absence of dental caries (OR = 4.741, 95% CI = 1.564–14.371, P = 0.006) and pulpitis (OR = 9.111, 95% CI = 2.921–28.420, P < 0.001) was significant determinants of the successful procurement of MSCs. MSCs derived from pulps with pulpitis expressed longer colony doubling time than pulps without pulpitis. Furthermore, there were higher expressions of proinflammatory cytokines, interleukin- (IL-) 6 and monocyte chemoattractant protein- (MCP-) 1, P < 0.01, and innate immune response [toll-like receptor 1 (TLR1) and TLR8, P < 0.05; TLR2, TLR3, and TLR6, P < 0.01] in the inflamed than noninflamed pulps. Therefore, a carious deciduous tooth or tooth with pulpitis was relatively unsuitable for MSC processing and isolation. PMID:28377925

  19. Fourth international workshop on human chromosome 5. Final progress report

    Energy Technology Data Exchange (ETDEWEB)

    McPherson, J.D.

    1996-12-31

    The Fourth International Workshop on Human Chromosome 5 was held in Manchester, UK on November 9--10, 1996 and was hosted by the University of Manchester. The major goals of the workshop were: (1) to collate the various genetic, cytogenetic and physical maps of human chromosome 5; (2) to integrate these maps and identify/correct discrepancies between them wherever possible; (3) to catalogue the sequence-ready contigs of the chromosome; (4) to co-ordinate the various sequencing efforts to avoid future duplication; (5) to establish the first (to the author`s knowledge) web site for the human chromosome 5 community which contains the above information in a readily accessible form.

  20. Trichophyton onychocola sp. nov. isolated from human nail.

    Science.gov (United States)

    Hubka, Vit; Cmokova, Adela; Skorepova, Magdalena; Mikula, Peter; Kolarik, Miroslav

    2014-04-01

    A previously undescribed Trichophyton species was isolated from the nail of a 33-year-old man with a history of probable distal lateral subungual onychomycosis (without confirmation by mycological examination). The infection occurred for the first time five years earlier (in 2006) and affected the right great toenail, with complete clinical remission after treatment with ciclopirox olamine. This undescribed species was isolated during probable relapse in 2011, but its etiological significance was not confirmed, that is, direct microscopy was negative and additional clinical samples were not collected. The species is probably geophilic based on phylogenetic analysis (internal transcribed spacer [ITS] rDNA) and is most closely related to the anamorphic T. thuringiense, homothallic Arthroderma ciferrii (anamorph T. georgiae), and heterothallic A. melis. The new species is characterized by yellowish colonies, red reverse on several media, positive urease test, negative hair-perforation test, absence of growth at 34°C, absence of macroconidia, formation of one-celled clavate microconidia, and spiral hyphae. The species grows well on sterilized human hairs placed on agar medium without any additional nutrients and forms gymnothecium-like structures covered by peridial hyphae. The combination of unique micro- and macromorphological features and physiological and sequence data from four unlinked loci (ITS, benA, RPB2, and act1 gene) justified the proposal of a new species T. onychocola sp. nov.

  1. Streptococcus rubneri sp. nov., isolated from the human throat.

    Science.gov (United States)

    Huch, Melanie; De Bruyne, Katrien; Cleenwerck, Ilse; Bub, Achim; Cho, Gyu-Sung; Watzl, Bernhard; Snauwaert, Isabel; Franz, Charles M A P; Vandamme, Peter

    2013-11-01

    The novel, Gram-stain-positive, ovoid, lactic acid bacterial isolates LMG 27205, LMG 27206, LMG 27207(T) and MRI-F 18 were obtained from throat samples of healthy humans. 16S rRNA gene sequence analyses indicated that these isolates belong to the genus Streptococcus, specifically the Streptococcus mitis group, with Streptococcus australis and Streptococcus mitis as the nearest neighbours (99.45 and 98.56 % 16S rRNA gene sequence similarity to the respective type strains). Genotypic fingerprinting by fluorescent amplified fragment length polymorphism (FAFLP) and pulsed-field gel electrophoresis (PFGE), DNA-DNA hybridizations, comparative sequence analysis of pheS, rpoA and atpA and physiological and biochemical tests revealed that these bacteria formed a taxon well separated from its nearest neighbours and other species of the genus Streptococcus with validly published names and, therefore, represent a novel species, for which the name Streptococcus rubneri sp. nov. is proposed, with LMG 27207(T) ( = DSM 26920(T)) as the type strain.

  2. Biofilm formation in Hafnia alvei HUMV-5920, a human isolate

    Directory of Open Access Journals (Sweden)

    Itziar Chapartegui-González

    2016-11-01

    Full Text Available Hafnia alvei is a Gram-negative, rodshaped, facultative anaerobic bacterium of the family Enterobacteriaceae that has been isolated from various mammals, fish, insects and birds. In humans, case reports of Hafnia-associated enteric infections have been chiefly reported in Spain. Although H. alvei shares some virulence mechanisms with other Gram-negative enteropathogens little is known about the factors that contribute to its pathogenesis or virulence factors and regulatory circuits that may enhance the establishment and survival of H. alvei in the environment. The goal of the present study was to analyze the capacity of a H. alvei clinical isolate (strain HUMV-5920 to form biofilms. Biofilm formation by this strain increases during growth at 28 °C compared to 37 °C. Investigation of multicellular behavior by confocal microscopy, crystal violet and calcofluor staining in this strain showed biofilm formation associated with the production of cellulose. Importantly, several genes related to cellulose production including bcsABZC and yhjQ are present in the H. alvei HUMV-5920 chromosome. The ability of H. alvei to adhere to abiotic surfaces and to form biofilms likely contributes to its persistence in the hospital environment or food processing environments, increasing the probability of causing infections. Therefore, a better understanding of the adherence properties of this species will provide greater insights into the diseases it causes.

  3. The third international workshop of human chromosome 5. Final report

    Energy Technology Data Exchange (ETDEWEB)

    NONE

    1994-12-31

    The Third International Workshop on Human Chromosome 5 was held in Laguna Beach, California, March 5-8, 1994. The pace at which new mapping information has been published in the last year make almost any report outdated before publication. Much of the information in this report and the most recent data from the Human chromosome 5 Genome Center at U.C. Irvine on the physical map of chromosome 5 are accessible via a WWW server. For most loci referred to in this report that can be detected by Polymerase Chain Reaction, the sequences of the oligonucleotide primers are available and some primer sequences are provided in this report.

  4. Sterols of Pneumocystis carinii hominis organisms isolated from human lungs

    DEFF Research Database (Denmark)

    Kaneshiro, E S; Amit, Z; Chandra, Jan Suresh

    1999-01-01

    The opportunistic pathogen Pneumocystis carinii causes pneumonia (P. carinii pneumonia, or PCP) in immunocompromised individuals such as AIDS patients. Rat-derived P. carinii carinii organisms have distinct sterols which are not synthesized by mammals and not found in other microbes infecting...... mammalian lungs. The dominant sterol present in the organism is cholesterol (which is believed to be scavenged from the host), but other sterols in P. carinii carinii have an alkyl group at C-24 of the sterol side chain (C(28) and C(29) 24-alkylsterols) and a double bond at C-7 of the nucleus. Recently...... in conjunction with analyses of chemically synthesized authentic standards. The sterol composition of isolated P. carinii hominis organisms has yet to be reported. If P. carinii from animal models is to be used for identifying potential drug targets and for developing chemotherapeutic approaches to clear human...

  5. [Isolation and physico-chemical characteristics of human cancerocerebral antigen].

    Science.gov (United States)

    Prokopenko, P G; Borisenko, S A; Tatarinov, Iu S

    1984-01-01

    During gel filtration on Sephadex G-200 human cancerocerebral antigen (CCA) was eluted as two protein fractions with molecular mass of 135,000 and 270.000 daltons. Only one band of protein with molecular mass of about 15,000 daltons was noted after electrophoresis in 10% polyacrylamide gel containing SDS. As characteristic properties of CCA were recognized an electrophoretic polymorphism and a distinct trend to polymerization and isomeria. The antigen was not stained with dyes designed for staining base proteins, lipo-,glyco- and ferroproteins; CCA was thermostable (5 min at 80 degrees), it was inactivated by trypsin and protease but was resistant to pronase, hexokinase, alpha-amylase and beta-glucuronidase. A procedure was developed for isolation of CCA from brain, including fractionation with ammonium sulfate, ion exchange chromatography on DEAE-Sephadex A-50. The procedure enabled to obtain the CCA preparations suitable for radioimmunological, immunobiological assays and amino acid analyses.

  6. PIXE analysis of human spermatozoa isolated from seminal plasma

    Science.gov (United States)

    Maeda, K.; Sasa, Y.; Kusuyama, H.; Yoshida, K.; Uda, M.

    1990-04-01

    PIXE has been applied to the multielemental and microanalysis of human spermatozoa. This is the first attempt to determine the chemical compositions of the motile spermatozoa free from contaminations of seminal plasma without loss of component elements during washing. The spermatozoa were isolated from semen by letting them swim into a kind of physiological saline, Tyrode's solution. Relative concentrations of P, K, Ca, Ti, Fe, Zn and Br in motile spermatozoa were determined by the use of the chlorine K X-ray peak intensity for evaluating the amount of Tyrode's solution contained in the sample targets. The concentrations of calcium and iron in spermatozoa were considerably higher than in seminal plasma. The concentrations of P, K, Zn and Br in spermatozoa were not so different from those in seminal plasma.

  7. Isolation and characterization of DNA probes for human chromosome 21.

    Science.gov (United States)

    Watkins, P C

    1990-01-01

    A coordinated effort to map and sequence the human genome has recently become a national priority. Chromosome 21, the smallest human chromosome accounting for less than 2% of the human genome, is an attractive model system for developing and evaluating genome mapping technology. Several strategies are currently being explored including the development of chromosome 21 libraries from somatic cell hybrids as reported here, the cloning of chromosome 21 in yeast artificial chromosomes (McCormick et al., 1989b), and the construction of chromosome 21 libraries using chromosome flow-sorting techniques (Fuscoe et al., 1989). This report describes the approaches used to identify DNA probes that are useful for mapping chromosome 21. Probes were successfully isolated from both phage and cosmid libraries made from two somatic cell hybrids that contain human chromosome 21 as the only human chromosome. The 15 cosmid clones from the WA17 library, reduced to cloned DNA sequences of an average size of 3 kb, total 525 kb of DNA which is approximately 1% of chromosome 21. From these clones, a set of polymorphic DNA markers that span the length of the long arm of chromosome 21 has been generated. All of the probes thus far analyzed from the WA17 libraries have been mapped to chromosome 21 both by physical and genetic mapping methods. It is therefore likely that the WA17 hybrid cell line contains human chromosome 21 as the only human component, in agreement with cytogenetic observation. The 153E7b cosmid libraries will provide an alternative source of cloned chromosome 21 DNA. Library screening techniques can be employed to obtain cloned DNA sequences from the same genetic loci of the two different chromosome 21s. Comparative analysis will allow direct estimation of DNA sequence variation for different regions of chromosome 21. Mapped DNA probes make possible the molecular analysis of chromosome 21 at a level of resolution not achievable by classical cytogenetic techniques (Graw et al

  8. Advisory Committee on human radiation experiments final report

    Energy Technology Data Exchange (ETDEWEB)

    NONE

    1995-10-01

    When the Advisory Committee began work in April 1994 we were charged with determining whether the radiation experiments design and administration adequately met the ethical and scientific standards, including standards of informed consent, that prevailed at the time of the experiments and that exist today and also to determine the ethical and scientific standards and criteria by which it shall evaluate human radiation experiments. Although this charge seems straightforward, it is in fact difficult to determine what the appropriate standards should be for evaluating the conduct and policies of thirty or fifty years ago. First, we needed to determine the extent to which the standards of that time are similar to the standards of today. To the extent that there were differences we needed to determine the relative roles of each in making moral evaluations. In Chapter 1 we report what we have been able to reconstruct about government rules and policies in the 1940s and 1950s regarding human experiments. We focus primarily on the Atomic Energy Commission and the Department of Defense. In Chapter 2 we turn from a consideration of government standards to an exploration of the norms and practices of physicians and medical scientists who conducted research with human subjects during this period. Using the results of our Ethics Oral History Project, and other sources, we also examine how scientists of the time viewed their moral responsibilities to human subjects as well as how this translated into the manner in which they conducted their research.

  9. Sanitary Transportation of Human and Animal Food. Final rule.

    Science.gov (United States)

    2016-04-06

    The Food and Drug Administration (FDA or we) is issuing a final rule to establish requirements for shippers, loaders, carriers by motor vehicle and rail vehicle, and receivers engaged in the transportation of food, including food for animals, to use sanitary transportation practices to ensure the safety of the food they transport. This action is part of our larger effort to focus on prevention of food safety problems throughout the food chain and is part of our implementation of the Sanitary Food Transportation Act of 2005 (2005 SFTA) and the Food Safety Modernization Act of 2011 (FSMA).

  10. Isolation of 24 novel cDNA fragments from microdis—sected human chromosome band

    Institute of Scientific and Technical Information of China (English)

    ZHANGMIN; LONGYU; 等

    1998-01-01

    The strategy of isolating the band0specific expression fragments from a probe pool generated by human chromosome microdissection was reported.A chromosome 14q 24.3 band-specific single copy DNA pool was constructed based on this probe pool.Using total DNA of the pool as probe to hybridize the human marrow cDNA library,68 primary positive clones were selected from 5×105 cDNA clones.Among these primary clones,32 secondary clones were obtained after second-round screening and designed as cFD14-1-32.Finally,24 band-specific expression fragments were identified from these 32 positive clones by DNA hybridization.Those band-specific clones can hybridize to both 14q24.3 DNA and human genomic DNA but cann't hybridize to 17q11-12 DNA,Partial sequences of 13 fragments of them were sequenced and idenfified as novel cDNA sequences,and these sequences were proved to have some homology with known genes in NCBI database.Analysis of expression spectrum of cFD 14-1 suggested that the cDNA fragments thus obtained should be used to isolate the genes can not been cloned in 14q24.3 region.

  11. Prevalence of Salmonella serotypes isolated in Spain from human and non human sources (1983-1987).

    Science.gov (United States)

    Echeita, M A; Usera, M A

    1989-09-01

    Salmonella serotypes over a five year period were studied in order to know their prevalence in Spain. The Salmonella Reference Centre received a total of 17,612 strains from 1983-1987. The majority (16,133) were of human origin and only 1,479 strains were isolated from non-human sources. The serotyping yielded 100 different serotypes, Salmonella enterica serotype Enteritidis (8) being the commonest in both groups, 61.18% of human origin and 31.91% of non-human origin. Salmonella enterica serotype Typhimurium the commonest serotype in many countries, occupies second place in our results with the following percentages 11.87% and 9.67% respectively. Among the strains of human origin Salmonella enterica serotype Typhi occupies fourth place (3.24%). This is very low compared with the high number of clinically diagnosed typhoid fever cases declared in the country: over 5,000 cases per year.

  12. Rhodococcus equi human clinical isolates enter and survive within human alveolar epithelial cells.

    Science.gov (United States)

    Ramos-Vivas, J; Pilares-Ortega, L; Remuzgo-Martínez, S; Padilla, D; Gutiérrez-Díaz, J L; Navas-Méndez, J

    2011-05-01

    Rhodococcus equi is an emerging opportunistic human pathogen associated with immunosuppressed people, especially those infected with the human immunodeficiency virus (HIV). This pathogen resides primarily within lung macrophages of infected patients, which may explain in part its ability to escape normal pulmonary defense mechanisms. Despite numerous studies as a pulmonary pathogen in foals, where a plasmid seems to play an important role in virulence, information on the pathogenesis of this pathogen in humans is still scarce. In this study, fluorescence microscopy and vancomycin protection assays were used to investigate the ability of R. equi human isolates to adhere to and to invade the human alveolar epithelial cell line A549. Our findings indicate that some R. equi clinical strains are capable of adhering, entering and surviving within the alveolar cell line, which may contribute to the pathogen persistence in lung tissues. Copyright © 2011 Institut Pasteur. Published by Elsevier SAS. All rights reserved.

  13. Final Report: Contractor Readiness Assessment (CRA) for TREAT Fuel Movement and Control Rod Drives Isolation

    Energy Technology Data Exchange (ETDEWEB)

    Rowsell, David Leon [Idaho National Laboratory (INL), Idaho Falls, ID (United States)

    2015-06-01

    This report documents the Contractor Readiness Assessment (CRA) for TREAT Fuel Movement and Control Rod Drives Isolation. The review followed the approved Plan of Action (POA) and Implementation Plan (IP) using the identified core requirements. The activity was limited scope focusing on the control rod drives functional isolation and fuel element movement. The purpose of this review is to ensure the facility's readiness to move fuel elements thus supporting inspection and functionally isolate the control rod drives to maintain the required shutdown margin.

  14. A summary of the sources of input parameter values for the Waste Isolation Pilot Plant final porosity surface calculations

    Energy Technology Data Exchange (ETDEWEB)

    Butcher, B.M.

    1997-08-01

    A summary of the input parameter values used in final predictions of closure and waste densification in the Waste Isolation Pilot Plant disposal room is presented, along with supporting references. These predictions are referred to as the final porosity surface data and will be used for WIPP performance calculations supporting the Compliance Certification Application to be submitted to the U.S. Environmental Protection Agency. The report includes tables and list all of the input parameter values, references citing their source, and in some cases references to more complete descriptions of considerations leading to the selection of values.

  15. Isolation & molecular characterization of human parainfluenza virus in Chennai, India

    Directory of Open Access Journals (Sweden)

    C P Indumathi

    2015-01-01

    Full Text Available Background & objectives: Human parainfluenza virus (HPIV accounts for a significant proportion of lower respiratory tract infections in children as well as adults. This study was done to detect the presence of different subtypes of HPIV from patients having influenza like illness (ILI. Methods: Throat and nasal swabs from 232 patients with ILI who were negative for influenza viruses were tested by multiplex reverse transcription polymerase chain reaction(mRT-PCR for the detection of human parainfluenza virus. All samples were inoculated in rhesus monkey kidney (LLC-MK2 cell line. Results: Of the 232 samples, 26(11.2% were positive by mRT-PCR and nine (34.6% showed cytopathic effect with syncytium formation for HPIV and all were HPIV-3 serotype, other serotypes like 1,2,4 were negative. The HPIV-3 strains (HN gene were sequenced and analysed. Two novel mutations were identified at amino acid residues 295 and 297. Interpretation & conclusions: The mRT-PCR assay offers a rapid, sensitive and accurate diagnostic method for detection of HPIV which enables early detection and control. In our study there was a predominance of HPIV among 1-5 yr age group and the school going age group was less affected. Further studies need to be done to characterize HPIV isolated from different parts of the country.

  16. Regional aerosol deposition in human upper airways. Final report

    Energy Technology Data Exchange (ETDEWEB)

    Swift, D.L.

    1997-11-01

    During the award period, a number of studies have been carried out related to the overall objective of the project which is to elucidate important factors which influence the upper airway deposition and dose of particles in the size range 0.5 nm - 10 {mu}m, such as particle size, breathing conditions, age, airway geometry, and mode of breathing. These studies are listed below. (1) A high voltage electrospray system was constructed to generate polydispersed 1-10 {mu}m diameter di-ethylhexyl sebacate aerosol for particle deposition studies in nasal casts and in human subjects. (2) The effect of nostril dimensions, nasal passage geometry, and nasal resistance on particle deposition efficiency in forty healthy, nonsmoking adults at a constant flowrate were studied. (3) The effect of nostril dimensions, nasal passage dimensions and nasal resistance on the percentage of particle deposition in the anterior 3 cm of the nasal passage of spontaneously breathing humans were studied. (4) The region of deposition of monodispersed aerosols were studied using replicate casts. (5) Ultrafine aerosol deposition using simulated breath holding path and natural path was compared. (6) An experimental technique was proposed and tested to measure the oral deposition of inhaled ultrafine particles. (7) We have calculated the total deposition fraction of ultrafine aerosols from 5 to 200 n in the extrathoracic airways and in the lung. (8) The deposition fraction of radon progeny in the head airways was studied using several head airway models.

  17. Aerosol deposition in the human respiratory system. Final report

    Energy Technology Data Exchange (ETDEWEB)

    Yu, C.P.

    1988-01-01

    Attempts were made to develop mathematical models for the deposition of aerosols in the human respiratory system. Expressions were obtained for the mean deposition efficiency for nasal inspiration, nasal expiration, and mouth inspiration. A determination was made of statistical properties associated with each deposition efficiency due to intersubject and intrasubject variabilities. Expressions were then derived for head deposition with combined nose and mouth breathing. In the lung, deposition is a result primarily of impaction, sedimentation, and diffusion. While there was no adequate model for impaction, several deposition formulae for sedimentation were derived as well as ones for diffusion. Studies were also made of the particle charge effect, as the electrostatic image force on a particle contributes to its deposition. There is, however, a threshold charge per particle below which the particle charge has no effect on deposition. Deposition data on ultrafine particles is scarce due to the difficulties in conducting proper experiments.

  18. Efficient Generation of Glucose-Responsive Beta Cells from Isolated GP2+ Human Pancreatic Progenitors

    Directory of Open Access Journals (Sweden)

    Jacqueline Ameri

    2017-04-01

    Full Text Available Stem cell-based therapy for type 1 diabetes would benefit from implementation of a cell purification step at the pancreatic endoderm stage. This would increase the safety of the final cell product, allow the establishment of an intermediate-stage stem cell bank, and provide a means for upscaling β cell manufacturing. Comparative gene expression analysis revealed glycoprotein 2 (GP2 as a specific cell surface marker for isolating pancreatic endoderm cells (PECs from differentiated hESCs and human fetal pancreas. Isolated GP2+ PECs efficiently differentiated into glucose responsive insulin-producing cells in vitro. We found that in vitro PEC proliferation declines due to enhanced expression of the cyclin-dependent kinase (CDK inhibitors CDKN1A and CDKN2A. However, we identified a time window when reducing CDKN1A or CDKN2A expression increased proliferation and yield of GP2+ PECs. Altogether, our results contribute tools and concepts toward the isolation and use of PECs as a source for the safe production of hPSC-derived β cells.

  19. Metabolically Derived Human Ventilation Rates: A Revised Approach Based Upon Oxygen Consumption Rates (Final Report, 2009)

    Science.gov (United States)

    EPA announced the availability of the final report, Metabolically Derived Human Ventilation Rates: A Revised Approach Based Upon Oxygen Consumption Rates. This report provides a revised approach for calculating an individual's ventilation rate directly from their oxygen c...

  20. Vertically integrated analysis of human DNA. Final technical report

    Energy Technology Data Exchange (ETDEWEB)

    Olson, M.

    1997-10-01

    This project has been oriented toward improving the vertical integration of the sequential steps associated with the large-scale analysis of human DNA. The central focus has been on an approach to the preparation of {open_quotes}sequence-ready{close_quotes} maps, which is referred to as multiple-complete-digest (MCD) mapping, primarily directed at cosmid clones. MCD mapping relies on simple experimental steps, supported by advanced image-analysis and map-assembly software, to produce extremely accurate restriction-site and clone-overlap maps. We believe that MCD mapping is one of the few high-resolution mapping systems that has the potential for high-level automation. Successful automation of this process would be a landmark event in genome analysis. Once other higher organisms, paving the way for cost-effective sequencing of these genomes. Critically, MCD mapping has the potential to provide built-in quality control for sequencing accuracy and to make possible a highly integrated end product even if there are large numbers of discontinuities in the actual sequence.

  1. The human genetic history of the Americas: the final frontier.

    Science.gov (United States)

    O'Rourke, Dennis H; Raff, Jennifer A

    2010-02-23

    The Americas, the last continents to be entered by modern humans, were colonized during the late Pleistocene via a land bridge across what is now the Bering strait. However, the timing and nature of the initial colonization events remain contentious. The Asian origin of the earliest Americans has been amply established by numerous classical marker studies of the mid-twentieth century. More recently, mtDNA sequences, Y-chromosome and autosomal marker studies have provided a higher level of resolution in confirming the Asian origin of indigenous Americans and provided more precise time estimates for the emergence of Native Americans. But these data raise many additional questions regarding source populations, number and size of colonizing groups and the points of entry to the Americas. Rapidly accumulating molecular data from populations throughout the Americas, increased use of demographic models to test alternative colonization scenarios, and evaluation of the concordance of archaeological, paleoenvironmental and genetic data provide optimism for a fuller understanding of the initial colonization of the Americas.

  2. Comparative Proteomic Analysis of Human Liver Tissue and Isolated Hepatocytes with a Focus on Proteins Determining Drug Exposure.

    Science.gov (United States)

    Vildhede, Anna; Wiśniewski, Jacek R; Norén, Agneta; Karlgren, Maria; Artursson, Per

    2015-08-07

    Freshly isolated human hepatocytes are considered the gold standard for in vitro studies of liver functions, including drug transport, metabolism, and toxicity. For accurate predictions of the in vivo outcome, the isolated hepatocytes should reflect the phenotype of their in vivo counterpart, i.e., hepatocytes in human liver tissue. Here, we quantified and compared the membrane proteomes of freshly isolated hepatocytes and human liver tissue using a label-free shotgun proteomics approach. A total of 5144 unique proteins were identified, spanning over 6 orders of magnitude in abundance. There was a good global correlation in protein abundance. However, the expression of many plasma membrane proteins was lower in the isolated hepatocytes than in the liver tissue. This included transport proteins that determine hepatocyte exposure to many drugs and endogenous compounds. Pathway analysis of the differentially expressed proteins confirmed that hepatocytes are exposed to oxidative stress during isolation and suggested that plasma membrane proteins were degraded via the protein ubiquitination pathway. Finally, using pitavastatin as an example, we show how protein quantifications can improve in vitro predictions of in vivo liver clearance. We tentatively conclude that our data set will be a useful resource for improved hepatocyte predictions of the in vivo outcome.

  3. Epidemiological relationships of Campylobacter jejuni strains isolated from humans and chickens in South Korea.

    Science.gov (United States)

    Oh, Jae-Young; Kwon, Yong-Kuk; Wei, Bai; Jang, Hyung-Kwan; Lim, Suk-Kyung; Kim, Cheon-Hyeon; Jung, Suk-Chan; Kang, Min-Su

    2017-01-01

    Thirty-nine human isolates of Campylobacter jejuni obtained from a national university hospital during 2007-2010 and 38 chicken isolates of C. jejuni were collected from poultry farms during 2009-2010 in South Korea were used in this study. Campylobacter genomic species and virulence-associated genes were identified by PCR. Pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST) were performed to compare their genetic relationships. All isolates were highly resistant to ciprofloxacin, nalidixic acid, and tetracycline. Of all isolates tested, over 94% contained seven virulence associated genes (flaA, cadF, racR, dnaJ, cdtA, cdtB, and cdtC). All isolates were classified into 39 types by PFGE clustering with 90% similarity. Some chicken isolates were incorporated into some PFGE types of human isolates. MLST analysis for the 39 human isolates and 38 chicken isolates resulted in 14 and 23 sequence types (STs), respectively, of which 10 STs were new. STs overlapped in both chicken and human isolates included ST-21, ST-48, ST-50, ST-51, and ST-354, of which ST-21 was the predominant ST in both human and chicken isolates. Through combined analysis of PFGE types and STs, three chicken isolates were clonally related to the three human isolates associated with food poisoning (VII-ST-48, XXII-ST-354, and XXVIII-ST-51). They were derived from geographically same or distinct districts. Remarkably, clonal spread of food poisoning pathogens between animals and humans was confirmed by population genetic analysis. Consequently, contamination of campylobacters with quinolone resistance and potential virulence genes in poultry production and consumption may increase the risk of infections in humans.

  4. Prevotella colorans sp. nov., isolated from a human wound.

    Science.gov (United States)

    Buhl, Michael; Willmann, Matthias; Liese, Jan; Autenrieth, Ingo B; Marschal, Matthias

    2016-08-01

    A strain of obligately anaerobic, Gram-stain-negative and non-spore-forming rod-shaped bacterium was isolated from a human wound and characterized both phenotypically and genotypically. The strain was moderately saccharolytic and proteolytic. Phylogenetic analysis was based on full-length 16S rRNA gene sequence analysis and revealed the strain to represent a member of the genus Prevotella, but to be different from the described species, with the closest relationship to Prevotella bergensis and Prevotella multisaccharivorax. The genomic DNA G+C content was 43.2 mol%. The most abundant cellular long-chain fatty acids were 3-OH iso-C17 : 0, anteiso-C15 : 0 and iso-C15 : 0. In view of phenotypical and biochemical characteristics as well as gene sequencing, strain A1336T is considered to represent a novel species within the genus Prevotella, for which the name Prevotella colorans sp. nov. is proposed. The type strain is A1336T (=DSM 100333T =CCUG 67421T =CCOS 902T).

  5. Human attribute concepts: relative ubiquity across twelve mutually isolated languages.

    Science.gov (United States)

    Saucier, Gerard; Thalmayer, Amber Gayle; Bel-Bahar, Tarik S

    2014-07-01

    It has been unclear which human-attribute concepts are most universal across languages. To identify common-denominator concepts, we used dictionaries for 12 mutually isolated languages-Maasai, Supyire Senoufo, Khoekhoe, Afar, Mara Chin, Hmong, Wik-Mungkan, Enga, Fijian, Inuktitut, Hopi, and Kuna-representing diverse cultural characteristics and language families, from multiple continents. A composite list of every person-descriptive term in each lexicon was closely examined to determine the content (in terms of English translation) most ubiquitous across languages. Study 1 identified 28 single-word concepts used to describe persons in all 12 languages, as well as 41 additional terms found in 11 of 12. Results indicated that attribute concepts related to morality and competence appear to be as cross-culturally ubiquitous as basic-emotion concepts. Formulations of universal-attribute concepts from Osgood and Wierzbicka were well-supported. Study 2 compared lexically based personality models on the relative ubiquity of key associated terms, finding that 1- and 2-dimensional models draw on markedly more ubiquitous terms than do 5- or 6-factor models. We suggest that ubiquitous attributes reflect common cultural as well as common biological processes.

  6. The Use of Isolated Human Lymphocytes in Mycotoxin Cytotoxicity Testing

    Directory of Open Access Journals (Sweden)

    Mike F. Dutton

    2008-08-01

    Full Text Available The cytotoxicity of selected mycotoxins against isolated human lymphocytes was investigated, as a means of detecting mycotoxins in extracts derived from cereal samples. The methodology was based on the ability of viable cells to reduce methyl tetrazolium bromide to a purple formazan dye that could be quantitated by spectrophometric means and hence give a measure of the cytotoxicity of added substances. The results showed that there was good correlation with the occurrence of identified mycotoxins with only a minimum of false positives. For example, of the 13 samples of barley or barley derivatives that were positive for the mycotoxins, fumonisin B1 (FB1 deoxynivalenol (DON and ochratoxin A (OTA, all gave positive cytotoxicity responses. Two samples negative for mycotoxins gave no cytotoxicity responses. There was little variation between the results for lymphocytes drawn from the same healthy volunteer on three different occasions. Furthermore, for two of the mycotoxins tested (FB1 and DON it was possible to correlate general levels of mycotoxins present to the cytotoxic response of the lymphocytes but not for OTA, where it was concluded that interfering substances prevented direct correlation. It was concluded that this method was suited for general application as it could handle relatively high number of samples in a short period of time.

  7. Description of Campylobacter fetus subsp. testudinum subsp. nov., isolated from humans and reptiles

    Science.gov (United States)

    A polyphasic study was undertaken to determine the taxonomic position of 13 Campylobacter fetus-like isolates from humans (n=8) and reptiles (n=5). Phenotypic characterization, Genusgenus-specific and sap insertion-PCR initially identified all human isolates as type A Campylobacter fetus. Phylogenet...

  8. Comparison of virulence factors and capsular types of Streptococcus agalactiae isolated from human and bovine infections.

    Science.gov (United States)

    Emaneini, Mohammad; Khoramian, Babak; Jabalameli, Fereshteh; Abani, Samira; Dabiri, Hossein; Beigverdi, Reza

    2016-02-01

    Streptococcus agalactiae is a leading cause of human and bovine infections. A total of 194 S. agalactiae isolates, 55 isolates from bovines and 139 from humans, were analyzed for capsular types, virulence genes (scpB, hly, rib, bca and bac) and mobile genetic elements (IS1548 and GBSi1) using polymerase chain reaction (PCR) and multiplex PCR. Capsular type III was predominant (61%), followed by types V, II, Ib, and IV. The scpB, hly, bca and bac virulence genes were only found among human isolates. Twelve and 2 distinct virulence gene profiles were identified among human and bovine isolates respectively. The virulence gene profiles scpB- hly- IS1548- rib-bca (51%) and scpB- hly- IS1548- bca (19%) were only predominant among human isolates. The rib gene was the most common virulence gene in both human and bovine isolates. The study showed a high prevalence of virulence genes in S. agalactiae strains isolated from human infections, these result can support the idea that S. agalactiae isolated from humans and bovines are generally unrelated and probably belonged to separate populations.

  9. Multilocus sequence types of Finnish bovine Campylobacter jejuni isolates and their attribution to human infections

    Directory of Open Access Journals (Sweden)

    Corander Jukka

    2010-07-01

    Full Text Available Abstract Background Campylobacter jejuni is the most common bacterial cause of human gastroenteritis worldwide. Due to the sporadic nature of infection, sources often remain unknown. Multilocus sequence typing (MLST has been successfully applied to population genetics of Campylobacter jejuni and mathematical modelling can be applied to the sequence data. Here, we analysed the population structure of a total of 250 Finnish C. jejuni isolates from bovines, poultry meat and humans collected in 2003 using a combination of Bayesian clustering (BAPS software and phylogenetic analysis. Results In the first phase we analysed sequence types (STs of 102 Finnish bovine C. jejuni isolates by MLST and found a high diversity totalling 50 STs of which nearly half were novel. In the second phase we included MLST data from domestic human isolates as well as poultry C. jejuni isolates from the same time period. Between the human and bovine isolates we found an overlap of 72.2%, while 69% of the human isolates were overlapping with the chicken isolates. In the BAPS analysis 44.3% of the human isolates were found in bovine-associated BAPS clusters and 45.4% of the human isolates were found in the poultry-associated BAPS cluster. BAPS reflected the phylogeny of our data very well. Conclusions These findings suggest that bovines and poultry were equally important as reservoirs for human C. jejuni infections in Finland in 2003. Our results differ from those obtained in other countries where poultry has been identified as the most important source for human infections. The low prevalence of C. jejuni in poultry flocks in Finland could explain the lower attribution of human infection to poultry. Of the human isolates 10.3% were found in clusters not associated with any host which warrants further investigation, with particular focus on waterborne transmission routes and companion animals.

  10. Detection of CC17 Enterococcus faecium in dogs and a comparison with human isolates.

    Science.gov (United States)

    Kwon, K H; Moon, B Y; Hwang, S Y; Park, Y H

    2012-09-01

    Enterococcus faecium strains of clonal complex (CC) 17 were isolated from domestic dogs. The strains were more prevalent in infectious isolates than in colonized isolates, suggesting that strains of the CC17 lineage may have an advantage in causing infections in dogs. The pulsed field gel electrophoresis patterns of some dog and human isolates were over 90% similar. However, antimicrobial resistance patterns and virulence factors were not identical, which might reflect different use of antimicrobials in veterinary medicine or in host specificity.

  11. Antimicrobial drug resistance of Salmonella isolates from meat and humans, Denmark

    DEFF Research Database (Denmark)

    Skov, Marianne Nielsine; Andersen, Jens Strodl; Aabo, Søren

    2007-01-01

    We compared 8,144 Salmonella isolates collected from meat imported to or produced in Denmark, as well as from Danish patients. Isolates from imported meat showed a higher rate of antimicrobial drug resistance, including multidrug resistance, than did isolates from domestic meat. Isolates from...... humans showed resistance rates lower than those found in imported meat but higher than in domestic meat. These findings indicate that programs for controlling resistant Salmonella spp. are a global issue...

  12. Myeloperoxidase impairs the contractile function in isolated human cardiomyocytes.

    Science.gov (United States)

    Kalász, Judit; Pásztor, Enikő T; Fagyas, Miklós; Balogh, Ágnes; Tóth, Attila; Csató, Viktória; Édes, István; Papp, Zoltán; Borbély, Attila

    2015-07-01

    We set out to characterize the mechanical effects of myeloperoxidase (MPO) in isolated left-ventricular human cardiomyocytes. Oxidative myofilament protein modifications (sulfhydryl (SH)-group oxidation and carbonylation) induced by the peroxidase and chlorinating activities of MPO were additionally identified. The specificity of the MPO-evoked functional alterations was tested with an MPO inhibitor (MPO-I) and the antioxidant amino acid Met. The combined application of MPO and its substrate, hydrogen peroxide (H2O2), largely reduced the active force (Factive), increased the passive force (Fpassive), and decreased the Ca(2+) sensitivity of force production (pCa50) in permeabilized cardiomyocytes. H2O2 alone had significantly smaller effects on Factive and Fpassive and did not alter pCa50. The MPO-I blocked both the peroxidase and the chlorinating activities, whereas Met selectively inhibited the chlorinating activity of MPO. All of the MPO-induced functional effects could be prevented by the MPO-I and Met. Both H2O2 alone and MPO + H2O2 reduced the SH content of actin and increased the carbonylation of actin and myosin-binding protein C to the same extent. Neither the SH oxidation nor the carbonylation of the giant sarcomeric protein titin was affected by these treatments. MPO activation induces a cardiomyocyte dysfunction by affecting Ca(2+)-regulated active and Ca(2+)-independent passive force production and myofilament Ca(2+) sensitivity, independent of protein SH oxidation and carbonylation. The MPO-induced deleterious functional alterations can be prevented by the MPO-I and Met. Inhibition of MPO may be a promising therapeutic target to limit myocardial contractile dysfunction during inflammation.

  13. Isolating nasal olfactory stem cells from rodents or humans.

    Science.gov (United States)

    Girard, Stéphane D; Devéze, Arnaud; Nivet, Emmanuel; Gepner, Bruno; Roman, François S; Féron, François

    2011-08-22

    The olfactory mucosa, located in the nasal cavity, is in charge of detecting odours. It is also the only nervous tissue that is exposed to the external environment and easily accessible in every living individual. As a result, this tissue is unique for anyone aiming to identify molecular anomalies in the pathological brain or isolate adult stem cells for cell therapy. Molecular abnormalities in brain diseases are often studied using nervous tissue samples collected post-mortem. However, this material has numerous limitations. In contrast, the olfactory mucosa is readily accessible and can be biopsied safely without any loss of sense of smell(1). Accordingly, the olfactory mucosa provides an "open window" in the adult human through which one can study developmental (e.g. autism, schizophrenia)(2-4) or neurodegenerative (e.g. Parkinson, Alzheimer) diseases(4,5). Olfactory mucosa can be used for either comparative molecular studies(4,6) or in vitro experiments on neurogenesis(3,7). The olfactory epithelium is also a nervous tissue that produces new neurons every day to replace those that are damaged by pollution, bacterial of viral infections. This permanent neurogenesis is sustained by progenitors but also stem cells residing within both compartments of the mucosa, namely the neuroepithelium and the underlying lamina propria(8-10). We recently developed a method to purify the adult stem cells located in the lamina propria and, after having demonstrated that they are closely related to bone marrow mesenchymal stem cells (BM-MSC), we named them olfactory ecto-mesenchymal stem cells (OE-MSC)(11). Interestingly, when compared to BM-MSCs, OE-MSCs display a high proliferation rate, an elevated clonogenicity and an inclination to differentiate into neural cells. We took advantage of these characteristics to perform studies dedicated to unveil new candidate genes in schizophrenia and Parkinson's disease(4). We and others have also shown that OE-MSCs are promising candidates

  14. Isolation of Human Intestinal Bacteria Capable of Producing the Bioactive Metabolite Isourolithin A from Ellagic Acid

    Directory of Open Access Journals (Sweden)

    María V. Selma

    2017-08-01

    Full Text Available Urolithins are intestinal microbial metabolites produced from ellagitannin- and ellagic acid-containing foods such as walnuts, strawberries, and pomegranates. These metabolites, better absorbed than their precursors, can contribute significantly to the beneficial properties attributed to the polyphenols ellagitannins and ellagic acid (EA. However, both the ability of producing the final metabolites in this catabolism (urolithins A, B and isourolithin A and the health benefits associated with ellagitannin consumption differ considerably among individuals depending on their gut microbiota composition. Three human urolithin metabotypes have been previously described, i.e., metabotype 0 (urolithin non-producers, metabotype A (production of urolithin A as unique final urolithin and metabotype B (urolithin B and/or isourolithin A are produced besides urolithin A. Although production of some intermediary urolithins has been recently attributed to intestinal species from Eggerthellaceae family named Gordonibacter urolithinfaciens and Gordonibacter pamelaeae, the identification of the microorganisms responsible for the complete transformation of EA into the final urolithins, especially those related to metabotype B, are still unknown. In the present research we illustrate the isolation of urolithin-producing strains from human feces of a healthy adult and their ability to transform EA into different urolithin metabolites, including isourolithin A. The isolates belong to a new genus from Eggerthellaceae family. EA transformation and urolithin production arisen during the stationary phase of the growth of the bacteria under anaerobic conditions. The HPLC-DAD-MS analyses demonstrated the sequential appearance of 3,8,9,10-tetrahydroxy-urolithin (urolithin M6, 3,8,9-trihydroxy-urolithin (urolithin C and 3,9-dihydroxy-urolithin (isourolithin A while 3,8-dihydroxy-urolithin (urolithin A and 3-hydroxy-urolithin (urolithin B were not detected. For the first time

  15. Molecular typing of Brucella species isolates from livestock and human.

    Science.gov (United States)

    Nagalingam, Mohandoss; Shome, Rajeswari; Balamurugan, Vinayagamurthy; Shome, Bibek Ranjan; NarayanaRao, Krishnamsetty; Vivekananda; Isloor, Shrikrishna; Prabhudas, Krishnamsetty

    2012-01-01

    Although host specificity has been observed in different species of Brucella, crossing the animal host boundary is likely to occur at any time. In this study, Bruce ladder PCR and abortus-melitensis-ovis-suis (AMOS) PCR assays were used to characterize 47 Brucella isolates from Indian origin in order to know exact species for understanding epidemiology of brucellosis. Out of them, 28, 14, and 5 isolates were found to be Brucella abortus, Brucella melitensis, and Brucella suis, respectively. Further analysis by AMOS PCR has identified that all the B. abortus isolates belong to any one of the biovar 1, 2, or 4; of the five B. suis isolates, three belong to biovar 1 and two belong to any one of the biovar 2, 3, 4, or 5. Although this multiplex Bruce ladder PCR is useful in differentiating all Brucella species, elaborate study is required to further characterize the isolates at exact biovar level.

  16. Final Scientific/Technical Report for "Nanite" for Better Well-Bore Integrity and Zonal Isolation

    Energy Technology Data Exchange (ETDEWEB)

    Veedu, Vinod [Oceanit Laboratories, Inc., Honolulu, HI (United States); Hadmack, Michael [Oceanit Laboratories, Inc., Honolulu, HI (United States); Pollock, Jacob [Oceanit Laboratories, Inc., Honolulu, HI (United States); Pernambuco-Wise, Paul [Oceanit Laboratories, Inc., Honolulu, HI (United States); Ah Yo, Derek [Oceanit Laboratories, Inc., Honolulu, HI (United States)

    2017-05-30

    Nanite™ is a cementitious material that contains a proprietary formulation of functionalized nanomaterial additive to transform conventional cement into a smart material responsive to pressure (or stress), temperature, and any intrinsic changes in composition. This project has identified optimal sensing modalities of smart well cement and demonstrated how real-time sensing of Nanite™ can improve long-term wellbore integrity and zonal isolation in shale gas and applicable oil and gas operations. Oceanit has explored Nanite’s electrical sensing properties in depth and has advanced the technology from laboratory proof-of-concept to sub-scale testing in preparation for field trials.

  17. Characterization of clinical and environmental Mycobacterium avium spp. isolates and their interaction with human macrophages.

    Directory of Open Access Journals (Sweden)

    Evelyn Guirado

    Full Text Available Members of the Mycobacterium avium complex (MAC are naturally occurring bacteria in the environment. A link has been suggested between M. avium strains in drinking water and clinical isolates from infected individuals. There is a need to develop new screening methodologies that can identify specific virulence properties of M. avium isolates found in water that predict a level of risk to exposed individuals. In this work we have characterized 15 clinical and environmental M. avium spp. isolates provided by the US Environmental Protection Agency (EPA to improve our understanding of the key processes involved in the binding, uptake and survival of these isolates in primary human macrophages. M. avium serovar 8 was predominant among the isolates studied. Different amounts and exposure of mannose-capped lipoarabinomannan (ManLAM and glycopeptidolipids (GPLs, both major mycobacterial virulence factors, were found among the isolates studied. Reference clinical isolate 104 serovar 1 and clinical isolates 11 and 14 serovar 8 showed an increased association with macrophages. Serum opsonization increased the cell association and survival at 2 h post infection for all isolates. However, only the clinical isolates 104 and 3 among those tested showed an increased growth in primary human macrophages. The other isolates varied in their survival in these cells. Thus we conclude that the amounts of cell envelope ManLAM and GPL, as well as GPL serovar specificity are not the only important bacterial factors for dictating the early interactions of M. avium with human macrophages.

  18. Isolation of single-base genome-edited human iPS cells without antibiotic selection.

    Science.gov (United States)

    Miyaoka, Yuichiro; Chan, Amanda H; Judge, Luke M; Yoo, Jennie; Huang, Miller; Nguyen, Trieu D; Lizarraga, Paweena P; So, Po-Lin; Conklin, Bruce R

    2014-03-01

    Precise editing of human genomes in pluripotent stem cells by homology-driven repair of targeted nuclease-induced cleavage has been hindered by the difficulty of isolating rare clones. We developed an efficient method to capture rare mutational events, enabling isolation of mutant lines with single-base substitutions without antibiotic selection. This method facilitates efficient induction or reversion of mutations associated with human disease in isogenic human induced pluripotent stem cells.

  19. Characterization of temperate phages infecting Clostridium difficile isolates of human and animal origins.

    Science.gov (United States)

    Sekulovic, Ognjen; Garneau, Julian R; Néron, Audrey; Fortier, Louis-Charles

    2014-04-01

    Clostridium difficile is a Gram-positive pathogen infecting humans and animals. Recent studies suggest that animals could represent potential reservoirs of C. difficile that could then transfer to humans. Temperate phages contribute to the evolution of most bacteria, for example, by promoting the transduction of virulence, fitness, and antibiotic resistance genes. In C. difficile, little is known about their role, mainly because suitable propagating hosts and conditions are lacking. Here we report the isolation, propagation, and preliminary characterization of nine temperate phages from animal and human C. difficile isolates. Prophages were induced by UV light from 58 C. difficile isolates of animal and human origins. Using soft agar overlays with 27 different C. difficile test strains, we isolated and further propagated nine temperate phages: two from horse isolates (ΦCD481-1 and ΦCD481-2), three from dog isolates (ΦCD505, ΦCD506, and ΦCD508), and four from human isolates (ΦCD24-2, ΦCD111, ΦCD146, and ΦCD526). Two phages are members of the Siphoviridae family (ΦCD111 and ΦCD146), while the others are Myoviridae phages. Pulsed-field gel electrophoresis and restriction enzyme analyses showed that all of the phages had unique double-stranded DNA genomes of 30 to 60 kb. Phages induced from human C. difficile isolates, especially the members of the Siphoviridae family, had a broader host range than phages from animal C. difficile isolates. Nevertheless, most of the phages could infect both human and animal strains. Phage transduction of antibiotic resistance was recently reported in C. difficile. Our findings therefore call for further investigation of the potential risk of transduction between animal and human C. difficile isolates.

  20. Effect of cAMP on short-circuit current in isolated human ciliary body

    Institute of Scientific and Technical Information of China (English)

    WU Ren-yi; MA Ning; HU Qian-qian

    2013-01-01

    Background Cyclic adenosine monophosphate (cAMP) could activate chloride channels in bovine ciliary body and trigger an increase in the ionic current (short-circuit current,Isc) across the ciliary processes in pigs.The purpose of this study was to investigate how cAMP modulates Isc in isolated human ciliary processes and the possible involvement of chloride transport across the tissue in cAMP-induced Isc change.Methods In an Ussing-type chamber system,the Isc changes induced by the cAMP analogue 8-bromo-cAMP and an adenylyl cyclase activator forskolin in isolated human ciliary processes were assessed.The involvement of Cl-component in the bath solution was investigated.The effect of Cl-channel (10 μmol/L niflumic acid and 1 mmol/L 4,4′-diisothiocyanostilbene-2,2′-disulfonic acid (DIDS)),K+ channel (10 mmol/L tetraethylammonium chloride (TEA)),or Na+ channel blockers (1 mmol/L amiloride) on 8-bromo-cAMP-induced Isc change was also studied.Results Dose-dependently,8-bromo-cAMP (10 nmol/L-30 μmol/L) or forskolin (10 nmol/L-3 μmol/L) increased Isc across the ciliary processes with an increase in negative potential difference on the non-pigmented epithelium (NPE) side of the tissue.Isc increase induced by 8-bromo-cAMP was more pronounced when the drug was applied on the NPE side than on the pigmented epithelium side.When the tissue was bathed in low Cl-solutions,the Isc increase was significantly inhibited.Finally,niflumic acid and DIDS,but not TEA or amiloride,significantly prevented the Isc increase induced by 8-bromo-cAMP.Conclusions cAMP stimulates stroma-to-aqueous anionic transport in isolated human ciliary processes.Chloride is likely to be among the ions,the transportation of which across the tissue is triggered by cAMP,suggesting the potential role of cAMP in the process of aqueous humor formation in human eyes.

  1. Microbial Gas Generation Under Expected Waste Isolation Pilot Plant Repository Conditions: Final Report

    Energy Technology Data Exchange (ETDEWEB)

    Gillow, J.B.; Francis, A.

    2011-07-01

    Gas generation from the microbial degradation of the organic constituents of transuranic (TRU) waste under conditions expected in the Waste Isolation Pilot Plant (WIPP) was investigated. The biodegradation of mixed cellulosic materials and electron-beam irradiated plastic and rubber materials (polyethylene, polyvinylchloride, hypalon, leaded hypalon, and neoprene) was examined. We evaluated the effects of environmental variables such as initial atmosphere (air or nitrogen), water content (humid ({approx}70% relative humidity, RH) and brine inundated), and nutrient amendments (nitogen phosphate, yeast extract, and excess nitrate) on microbial gas generation. Total gas production was determined by pressure measurement and carbon dioxide (CO{sub 2}) and methane (CH{sub 4}) were analyzed by gas chromatography; cellulose degradation products in solution were analyzed by high-performance liquid chromatography. Microbial populations in the samples were determined by direct microscopy and molecular analysis. The results of this work are summarized.

  2. Hydraulic Testing of Salado Formation Evaporites at the Waste Isolation Pilot Plant Site: Final Report

    Energy Technology Data Exchange (ETDEWEB)

    Beauheim, Richard L.; Domski, Paul S.; Roberts, Randall M.

    1999-07-01

    This report presents interpretations of hydraulic tests conducted in bedded evaporates of the Salado Formation from May 1992 through May 1995 at the Waste Isolation Pilot Plant (WIPP) site in southeastern New Mexico. The WIPP is a US Department of Energy research and development facility designed to demonstrate safe disposal of transuranic wastes from the nation's defense programs. The WIPP disposal horizon is located in the lower portion of the Permian Salado Formation. The hydraulic tests discussed in this report were performed in the WIPP underground facility by INTERA inc. (now Duke Engineering and Services, Inc.), Austin, Texas, following the Field Operations Plan and Addendum prepared by Saulnier (1988, 1991 ) under the technical direction of Sandia National Laboratories, Albuquerque, New Mexico.

  3. Pantoea intestinalis sp. nov., isolated from the human gut.

    Science.gov (United States)

    Prakash, Om; Nimonkar, Yogesh; Vaishampayan, Ankita; Mishra, Mrinal; Kumbhare, Shreyas; Josef, Neetha; Shouche, Yogesh S

    2015-10-01

    A novel bacterial strain, 29Y89BT, was isolated from a faecal sample of a healthy human subject. Cells were Gram-stain-negative, motile, non-spore-forming and rod-shaped. Strain 29Y89BT formed cream-coloured colonies 2 mm in diameter on trypticase soy agar and showed optimum growth at 35 °C. Strain 29Y89BT showed highest 16S rRNA gene sequence similarity to Pantoea gaviniae A18/07T (98.4 %) followed by Pantoea calida 1400/07T (97.2 %). Multi-locus sequence analysis using atpD (ATP synthase β subunit), gyrB (DNA gyrase), infB (initiation translation factor 2) and rpoB (RNA polymerase β subunit) genes also supported the result of 16S rRNA gene sequence based phylogeny. Strain 29Y89BT showed 62 and 40.7 % DNA-DNA relatedness with P. calida DSM 22759T and P. gaviniae DSM 22758T. Strain 29Y89BT contained C17  : 0 cyclo, C19  : 0 cyclo ω8c, C16 : 0, C14 : 0 and C12 : 0 as predominant fatty acids. In addition, strain 29Y89BT showed physiological and phenotypic differences from its closest relatives P. gaviniae DSM 22758T and P. calida DSM 22759T. The polar lipid profile mainly comprised phospholipids. The DNA G+C content was 59.1 mol%. Thus, based on the findings of the current study, strain 29Y89BT showed clear delineations from its closest relatives P. gaviniae DSM 22758T and P. calida DSM 22759T, and is thus considered to represent a novel species of the genus Pantoea, for which the name Pantoea intestinalis sp. nov. is proposed. The type strain is 29Y89BT ( = DSM 28113T = MCC 2554T).

  4. Receptor binding properties of human and animal H1 influenza virus isolates.

    Science.gov (United States)

    Rogers, G N; D'Souza, B L

    1989-11-01

    It has been previously reported that several human H1 influenza viruses isolated prior to 1956, in contrast to human H3 isolates which are quite specific for SA alpha 2,6Gal sequences, apparently recognize both SA alpha 2,3Gal and SA alpha 2,6Gal sequences (Rogers, G.N., and Paulson, J.C., Virology 127, 361-373, 1983). In this report human H1 isolates representative of two epidemic periods, from 1934 to 1957 and from 1977 to 1986, and H1 influenza isolated from pigs, ducks, and turkeys were compared for their ability to utilize sialyloligosaccharide structures containing terminal SA alpha 2,3Gal or SA alpha 2,6Gal sequences as receptor determinants. Five of the eight human isolates from the first epidemic period recognize both SA alpha 2,3Gal and SA alpha 2,6Gal linkages, in agreement with our previous results. Of the remaining three strains, all isolated towards the end of the first epidemic, two appear to prefer SA alpha 2,6Gal sequences while the third preferentially binds SA alpha 2,3Gal sequences. In contrast to the early isolates, 11 of 13 human strains isolated during the second epidemic period preferentially bind SA alpha 2,6Gal containing oligosaccharides. On the basis of changes in receptor binding associated with continued passage in the laboratory for some of these later strains, it seems likely that human H1 isolates preferentially bind SA alpha 2,6Gal sequences in nature, and that acquisition of SA alpha 2,3Gal-binding is associated with laboratory passage. Influenza H1 viruses isolated from pigs were predominantly SA alpha 2,6Gal-specific while those isolated from ducks were primarily SA alpha 2,3Gal-specific. Thus, as has been previously reported for H3 influenza isolates, receptor specificity for influenza H1 viruses appears to be influenced by the species from which they were isolated, human isolates binding preferentially to SA alpha 2,6Gal-containing oligosaccharides while those isolated from ducks prefer SA alpha 2,3Gal

  5. Isolation and full-genome sequences of Japanese encephalitis virus genotype I strains from Cambodian human patients, mosquitoes and pigs.

    Science.gov (United States)

    Duong, Veasna; Choeung, Rithy; Gorman, Christopher; Laurent, Denis; Crabol, Yoann; Mey, Channa; Peng, Borin; Di Francesco, Juliette; Hul, Vibol; Sothy, Heng; Santy, Ky; Richner, Beat; Pommier, Jean-David; Sorn, San; Chevalier, Véronique; Buchy, Philippe; de Lamballerie, Xavier; Cappelle, Julien; Horwood, Paul Francis; Dussart, Philippe

    2017-09-01

    Japanese encephalitis remains the most important cause of viral encephalitis in humans in several southeast Asian countries, including Cambodia, causing at least 65 000 cases of encephalitis per year. This vector-borne viral zoonosis - caused by Japanese encephalitis virus (JEV) - is considered to be a rural disease and is transmitted by mosquitoes, with birds and pigs being the natural reservoirs, while humans are accidental hosts. In this study we report the first two JEV isolations in Cambodia from human encephalitis cases from two studies on the aetiology of central nervous system disease, conducted at the two major paediatric hospitals in the country. We also report JEV isolation from Culextritaeniorhynchus mosquitoes and from pig samples collected in two farms, located in peri-urban and rural areas. Out of 11 reverse-transcription polymerase chain reaction-positive original samples, we generated full-genome sequences from 5 JEV isolates. Five additional partial sequences of the JEV NS3 gene from viruses detected in five pigs and one complete coding sequence of the envelope gene of a strain identified in a pig were generated. Phylogenetic analyses revealed that JEV detected in Cambodia belonged to genotype I and clustered in two clades: genotype I-a, mainly comprising strains from Thailand, and genotype I-b, comprising strains from Vietnam that dispersed northwards to China. Finally, in this study, we provide proof that the sequenced JEV strains circulate between pigs, Culex tritaeniorhynchus and humans in the Phnom Penh vicinity.

  6. Isolation and cryopreservation of human peripheral blood monocytes.

    Science.gov (United States)

    Tsvetkov, T; Nickolov, C; Buckureshtliev, A; Mincheff, M; Tsoney, L; Terziev, R; Popov, D

    1986-12-01

    A modification of the Freundlich and Avdalovic method (J. Immunol. Methods 62, 31 (1983] is reported. Buffy coats, separated and pooled together, are used for isolation of monocytes (70% yield, 100% purity). Cell density of working suspension is increased up to 0.65 X 10(9) cells/75 cm2 surface by multiplication of the active fibronectin sites. For the purpose, cryoprecipitate is used instead of plasma for coating the glass-gelatin surface. Monocytes, isolated by that procedure, could be successfully cryopreserved with dimethyl sulfoxide cryoprotective solution.

  7. Search for Gluinos using Final States with One Isolated Lepton in the LHC- ATLAS Experiment

    CERN Document Server

    Chen, Shion

    This thesis presents the updated search for gluinos via proton-proton collisions with the center- of-mass energy of √s = 13 TeV at LHC, by focusing on the final state with exactly one lepton. With respect to the past searches, the sensitivity to heavier gluino is gained using the improved analysis technique and updated data statistics (36.1 fb−1 of integrated luminosity) collected in the ATLAS detector. No significant data excess is found in the unblinded dataset, and the exclusion limits are set on various targeted gluino decay scenarios. As a general conclusion, it is confirmed that up to 1.7 TeV ∼ 2.0 TeV in gluino mass and up to ∼ 1 TeV in the lightest neutralino mass is excluded for typical mass spectra, while the limit extends up to 1.5 TeV ∼ 1.9 TeV in gluino mass for the mass spectra motivated by the well-tempered neutralino scenario.

  8. Draft Genome of Debaryomyces fabryi CBS 789T, Isolated from a Human Interdigital Mycotic Lesion.

    Science.gov (United States)

    Tafer, Hakim; Sterflinger, Katja; Lopandic, Ksenija

    2016-02-04

    The yeast genus Debaryomyces comprises species isolated from various natural habitats, man-made environments, and clinical materials. Here, the draft genome of D. fabryi CBS 789(T), isolated from a human interdigital mycotic lesion, is presented. Copyright © 2016 Tafer et al.

  9. Paenibacillus spp. isolated from human and environmental samples in Spain: detection of 11 new species

    Directory of Open Access Journals (Sweden)

    J.A. Sáez-Nieto

    2017-09-01

    Full Text Available One hundred thirty-six isolates, 88 human and 48 environmental, that met the requirements to belong to the genus Paenibacillus were identified using a polyphasic taxonomic approach known as 16S rRNA plus phenotypic traits. Thirty-seven Paenibacillus species were identified; some had not been previously reported from clinical samples. The main species were P. pabuli (13 isolates, P. provencensis (11, P. phoenicis (9 and P. lautus (8. P. pabuli (11/13 and P. provencensis (8/11 were mainly environmental isolates, while P. phoenicis (9/9 and P. lautus (6/8 were mainly human isolates. Despite the difficulties in assigning to human Paenibacillus isolates a role as a pathogen or contaminant, here 25% of the isolates were involved in true infections, especially in those cases that affected abscesses, wound exudates, ocular infections and diverse fluids. In addition, 15 isolates were identified as 11 ‘Candidatus’ to a new species, all of them from human specimens except one that was obtained from laboratory air. The antimicrobial susceptibility testing showed 95.6% of isolates were resistant to ampicillin, 44% were resistant to cotrimoxazole, 20 to 30% were resistant to cefotaxime and vancomycin and 13% were resistant to rifampicin and erythromycin.

  10. Relaxant mechanisms of 3, 5, 7, 30, 40-pentamethoxyflavone on isolated human cavernosum

    DEFF Research Database (Denmark)

    Jansakul, Chaweewan; Tachanaparuksa, Kuldej; Mulvany, Michael J.

    2012-01-01

    We have investigated effects and mechanisms responsible for the activity of 3, 5, 7, 30, 40-pentamethoxyflavone (PMF) on isolated human cavernosum. PMF is the major flavone isolated from Kaempferia parviflora claimed to act as an aphrodisiac. PMF caused relaxation of phenylephrine precontracted h...

  11. Molecular typing of Brucella species isolates from Human and livestock bloods in Isfahan province

    Directory of Open Access Journals (Sweden)

    Ebtehaj Pishva

    2015-01-01

    Conclusion: Our findings confirm abundance of B. melitensis, particularly biovar 1 in human and sheep are identical but B. abortus biovar 3 as the etiological agent of cattle brucellosis most frequently isolated in the Isfahan area.

  12. Hydrogen generation by metal corrosion in simulated Waste Isolation Pilot Plant environments. Final report

    Energy Technology Data Exchange (ETDEWEB)

    Telander, M.R.; Westerman, R.E. [Battelle Pacific Northwest Lab., Richland, WA (United States)

    1997-03-01

    The corrosion and gas-generation characteristics of four material types: low-carbon steel (the current waste packaging material for the Waste Isolation Pilot Plant), Cu-base and Ti-base (alternative packaging) materials, and Al-base (simulated waste) materials were determined in both the liquid and vapor phase of Brine A, a brine representative of an intergranular Salado Formation brine. Test environments consisted primarily of anoxic brine with overpressures of N{sub 2}, CO{sub 2}, H{sub 2}S, and H{sub 2}. Limited tests of low-carbon steel were also performed in simulated-backfill environments and in brine environments with pH values ranging from 3 to 11. Low-carbon steel reacted at a slow, measurable rate with anoxic brine, liberating H{sub 2} on an equimolar basis with Fe reacted. Presence of CO{sub 2} caused the initial reaction to proceed more rapidly, but CO{sub 2}-induced passivation stopped the reaction if the CO{sub 2} were present in sufficient quantities. Addition of H{sub 2}S to a CO{sub 2}-passivated system caused reversal of the passivation. Low-carbon steel immersed in brine with H{sub 2}S showed no reaction, apparently because of passivation of the steel by formation of FeS. Addition of CO{sub 2} to an H{sub 2}S-passivated system did not reverse the passivation. Cu- and Ti-base materials showed essentially no corrosion when exposed to brine and overpressures of N{sub 2}, CO{sub 2}, and H{sub 2}S except for the rapid and complete reaction between Cu-base materials and H{sub 2}S. The Al-base materials reacted at approximately the same rate as low-carbon steel when immersed in anoxic Brine A; considerably more rapidly in the presence of CO{sub 2} or H{sub 2}S; and much more rapidly when iron was present in the system as a brine contaminant. High-purity Al was much more susceptible to corrosion than the 6061 alloy. No significant reaction took place on any material in any environment in the vapor-phase exposures.

  13. The isolated perfused human skin flap model: A missing link in skin penetration studies?

    OpenAIRE

    Ternullo, Selenia; de Weerd, Louis; Flaten, Gøril Eide; Holsæter, Ann Mari; Skalko-Basnet, Natasa

    2016-01-01

    Development of effective (trans)dermal drug delivery systems requires reliable skinmodels to evaluate skin drug penetration. The isolated perfused human skin flap remainsmetabolically active tissue for up to 6 h during in vitro perfusion. We introduce the isolated perfused human skin flap as a close-to-in vivo skin penetration model. To validate the model's ability to evaluate skin drug penetration the solutions of a hydrophilic (calcein) and a lipophilic (rhodamine) fluorescence ...

  14. Comparative expression analysis of isolated human adipocytes and the human adipose cell lines LiSa-2 and PAZ6

    NARCIS (Netherlands)

    Beek, van E.A.; Bakker, A.H.; Kruyt, P.M.; Vink, C.; Saris, W.H.; Keijer, J.

    2008-01-01

    Objective: To obtain insight in the extent to which the human cell lines LiSa-2 and PAZ6 resemble isolated primary human adipocytes. Design: A combination of cDNA subtraction (representative difference analysis; RDA) and cDNA microarray analysis was used to select adipose specific genes to compare i

  15. Lipooligosaccharide locus classes and putative virulence genes among chicken and human Campylobacter jejuni isolates.

    Science.gov (United States)

    Ellström, Patrik; Hansson, Ingrid; Nilsson, Anna; Rautelin, Hilpi; Olsson Engvall, Eva

    2016-11-21

    Campylobacter cause morbidity and considerable economic loss due to hospitalization and post infectious sequelae such as reactive arthritis, Guillain Barré- and Miller Fischer syndromes. Such sequelae have been linked to C. jejuni harboring sialic acid structures in their lipooligosaccharide (LOS) layer of the cell wall. Poultry is an important source of human Campylobacter infections but little is known about the prevalence of sialylated C. jejuni isolates and the extent of transmission of such isolates to humans. Genotypes of C. jejuni isolates from enteritis patients were compared with those of broiler chicken with pulsed-field gel electrophoresis (PFGE), to study the patterns of LOS biosynthesis genes and other virulence associated genes and to what extent these occur among Campylobacter genotypes found both in humans and chickens. Chicken and human isolates generally had similar distributions of the putative virulence genes and LOS locus classes studied. However, there were significant differences regarding LOS locus class of PFGE types that were overlapping between chicken and human isolates and those that were distinct to each source. The study highlights the prevalence of virulence associated genes among Campylobacter isolates from humans and chickens and suggests possible patterns of transmission between the two species.

  16. Genetic diversity and antibiotic resistance profiles of Campylobacter jejuni isolates from poultry and humans in Turkey.

    Science.gov (United States)

    Abay, Secil; Kayman, Tuba; Otlu, Baris; Hizlisoy, Harun; Aydin, Fuat; Ertas, Nurhan

    2014-05-16

    In this study, the investigation of clonal relations between human and poultry Campylobacter jejuni isolates and the determination of susceptibilities of isolates to various antibiotics were aimed. A total of 200 C. jejuni isolates concurrently obtained from 100 chicken carcasses and 100 humans were genotyped by the Pulsed-Field Gel Electrophoresis (PFGE) and automated Repetitive Extragenic Palindromic PCR (Rep-PCR, DiversiLab system) methods and were tested for their susceptibility to six antibiotics with disk diffusion method. The minimum inhibitory concentration (MIC) values of ciprofloxacin (CI), enrofloxacin (EF) and erythromycin (EM) were evaluated by E-test. By using PFGE 174 of (87.0%) the isolates were able to be typed. The clonally related strains were placed in 35 different clusters and 115 different genotypes were obtained. All of the two hundred isolates could be typed by using Rep-PCR and were divided into 133 different genotypes. One hundred and fourteen clonally related isolates (57.0%) were included in 47 clusters. In disk diffusion test, while the susceptibility rates of AMC and S to human and chicken derived C. jejuni isolates were 84.0%-96.0% and 96.0%-98.0%, respectively, all isolates were susceptible to gentamicin. The resistance rates of human isolates to AMP, NA and TE were detected as 44.0%, 84.0% and 38.0% of the resistances of chicken isolates to these antibiotics were 34.0%, 95.0% and 56.0%, respectively. The MIC values of human and chicken isolates to CI, EF and EM were detected as 81.0-93.0%, 85.0-88.0% and 6.0-7.0%, respectively. The clonal proximity rates were detected between human and poultry origin C. jejuni isolates. The discriminatory power of PFGE and Rep-PCR was similar, with Simpson's diversity indexes of 0.993 and 0.995, respectively. Concordance of the two methods as determined by Adjusted Rand coefficient was 0.198 which showed the low congruence between Rep-PCR and PFGE. High rates of quinolone resistance were detected in

  17. Nisin-Producing Lactococcus lactis Strains Isolated from Human Milk

    OpenAIRE

    Beasley, Shea S.; Saris, Per E.J.

    2004-01-01

    Characterization by partial 16S rRNA gene sequencing, ribotyping, and green fluorescent protein-based nisin bioassay revealed that 6 of 20 human milk samples contained nisin-producing Lactococcus lactis bacteria. This suggests that the history of humans consuming nisin is older than the tradition of consuming fermented milk products.

  18. Comparison of epidemiologically linked Campylobacter jejuni isolated from human and poultry sources.

    Science.gov (United States)

    Lajhar, S A; Jennison, A V; Patel, B; Duffy, L L

    2015-12-01

    Campylobacter jejuni is responsible for most foodborne bacterial infections worldwide including Australia. The aim of this study was to investigate a combination of typing methods in the characterization of C. jejuni isolated from clinical diarrhoeal samples (n = 20) and chicken meat (n = 26) in order to identify the source of infection and rank isolates based on their relative risk to humans. Sequencing of the flaA short variable region demonstrated that 86% of clinical isolates had genotypes that were also found in chicken meat. A polymerase chain reaction binary typing system identified 27 different codes based on the presence or absence of genes that have been reported to be associated with various aspects of C. jejuni pathogenicity, indicating that not all isolates may be of equal risk to human health. The lipooligosaccharide (LOS) of the C. jejuni isolates was classified into six classes (A, B, C, E, F, H) with 10·4% remaining unclassified. The majority (72·7%) of clinical isolates possessed sialylated LOS classes. Sialylated LOS classes were also detected in chicken isolates (80·7%). Antimicrobial tests indicated a low level of resistance, with no phenotypic resistance found to most antibiotics tested. A combination of typing approaches was useful to assign isolates to a source of infection and assess their risk to humans.

  19. Genotyping of Pseudomonas aeruginosa isolated from cockroaches and human urine.

    Science.gov (United States)

    Saitou, Keiko; Furuhata, Katsunori; Fukuyama, Masafumi

    2010-10-01

    Molecular-epidemiological analysis of Pseudomonas aeruginosa isolated from cockroaches captured in hospitals and from patient urine was performed, employing randomly amplified polymorphic DNA (RAPD) analysis to investigate the usefulness of RAPD analysis. Four specific bands at positions of 993, 875, 521, and 402 bp were commonly detected using primer 272 in 16 of 45 cockroach-derived strains (35.6%), but not in 21 urine-derived strains. On analysis using primer 208, 4 specific bands at positions of 1,235, 1,138, 1,068, and 303 bp were commonly detected in 15 of the 45 cockroach-derived (33.3%) and 10 of the 21 patient urine-derived (47.6%) strains, in a total of 25 of 66 strains (37.8%). On cluster analysis, 12 (48.5%) and 16 (66.7%) clusters were grouped based on a homology of 89% or greater, using primer 272 and primer 208, respectively, showing that primer 208 was suitable for the confirmation of diversity. Seven patterns were clustered based on 100% homology using either primer, and 6 of these consisted of only cockroach-derived strains. In the individual groups with 100% homology, all strains in the group were isolated at an identical site during the same period. P. aeruginosa isolated from cockroaches showed diverse genotypes suggesting several sources of contamination, indicating the necessity for investigating infection control targeting cockroaches inhabiting hospitals.

  20. Monochromosomal hybrids for the analysis of the human genome. Final technical report

    Energy Technology Data Exchange (ETDEWEB)

    Athwal, R.S. [Temple Univ. School of Medicine, Philadelphia, PA (United States). Fels Inst. for Cancer Research and Molecular Biology

    1994-09-01

    The objective of this research project is to produce panels of mouse/human and/or Chinese hamster/human hybrid cell lines each harboring a single different human chromosome. The human chromosome present in rodent cell will be marked with a dominant selectable marker and maintained by selection. In these experiments human chromosomes first ``tagged`` with a selectable marker in human cells are subsequently transferred to rodent cells by microcell fusion method. Several different experimental schemes have been developed to ``tag`` human chromosomes with a selectable marker. Amphotropic retroviral vectors provide a highly efficient system to introduce selectable markers into normal diploid human cells. The integration of retroviral vector into the cell genome occurs at random by recombination at a defined nucleotide sequence in the LTRs and only a single copy of the vector integrates in a cell. This property of retroviral vectors allows to isolate a segment of the chromosomal DNA flanking the vector integration site by PCR amplification. In these studies the amphotropic retroviral vector pZIPgpt that carries a dominant selectable marker gpt, is used to tag the human chromosomes in normal diploid cells. Human DNA flanking the integrated vector is rescued by PCR amplification and cloned into a plasmid vector. Cloned human DNA is then used to probe Southern blots of DNAs from a panel of hybrid cell lines to identify the chromosome of its origin. This allows them to identify clonal human cell lines, each carrying the marker integrated into a different chromosome. Marked chromosomes are then transferred to rodent cells by MMCT.

  1. Social isolation disrupts hippocampal neurogenesis in young non-human primates

    Directory of Open Access Journals (Sweden)

    Simone M Cinini

    2014-03-01

    Full Text Available Social relationships are crucial for the development and maintenance of normal behavior in non-human primates. Animals that are raised in isolation develop abnormal patterns of behavior that persist even when they are later reunited with their parents. In rodents, social isolation is a stressful event and is associated with a decrease in hippocampal neurogenesis but considerably less is known about the effects of social isolation in non-human primates during the transition from adolescence to adulthood. To investigate how social isolation affects young marmosets, these were isolated from other members of the colony for one or three weeks and evaluated for alterations in their behavior and hippocampal cell proliferation. We found that anxiety-related behaviors like scent-marking and locomotor activity increased after social isolation when compared to baseline levels. In agreement, grooming - an indicative of attenuation of tension - was reduced among isolated marmosets. These results were consistent with increased cortisol levels after one and three weeks of isolation. After social isolation (one or three weeks, reduced proliferation of neural cells in the subgranular zone of dentate granule cell layer was identified and a smaller proportion of BrdU-positive cells underwent neuronal fate (doublecortin labeling. Our data is consistent with the notion that social deprivation during the transition from adolescence to adulthood leads to stress and produces anxiety-like behaviors that in turn might affect neurogenesis and contribute to the deleterious consequences of prolonged stressful conditions.

  2. Isolation and culture of human umbilical vein endothelial cells (HUVEC).

    Science.gov (United States)

    Cheung, Ambrose L

    2007-02-01

    Human-derived endothelial cells can now be routinely harvested from human umbilical veins. Studies with human umbilical vein endothelial cells (HUVEC) have been conducted with cells from passage 2 to 5. It is now also possible to cryopreserve primary and early-passaged HUVEC for future propagation and for forwarding to an end user by express courier. Stored HUVEC have been stably retrieved even after several years. These retrieval techniques have facilitated the deployment of HUVEC for many studies, including those for homeostasis, inflammatory disorders, atherosclerosis, cancer, and microbial adhesion and invasion. In this unit, we will delineate the procedure for harvesting, propagation, and storage of HUVEC.

  3. ERIC-PCR Genotyping of Some Campylobacter jejuni Isolates of Chicken and Human Origin in Egypt.

    Science.gov (United States)

    Ahmed, Heba A; El Hofy, Fatma I; Ammar, Ahmed M; Abd El Tawab, Ashraf A; Hefny, Ahmed A

    2015-12-01

    The public health importance of the genus Campylobacter is attributed to several species causing diarrhea in consumers. Poultry and their meat are considered the most important sources of human campylobacteriosis. In this study, 287 samples from chicken (131 cloacal swabs, 39 chicken skin, 78 chicken meat, and 39 cecal parts) obtained from retail outlets as well as 246 stool swabs from gastroenteritis patients were examined. A representative number of the biochemically identified Campylobacter jejuni isolates were identified by real-time PCR, confirming the identification of the isolates as C. jejuni. Genotyping of the examined isolates (n = 31) by enterobacterial repetitive intergenic consensus PCR (ERIC-PCR) revealed a high discriminatory index of ERIC-PCR (D = 0.948), dividing C. jejuni isolates of chicken and human origins into 18 profiles and four clusters. The 18 profiles obtained indicated the heterogeneity of C. jejuni. Dendrogram analysis showed that four clusters were generated; all human isolates fell into clusters I and III. These observations further support the existence of a genetic relationship between human and poultry isolates examined in the present study. In conclusion, the results obtained support the speculation that poultry and poultry meat have an important role as sources of infection in the acquisition of Campylobacter infection in humans.

  4. Comparison of Hemagglutination and Hemolytic Activity of Various Bacterial Clinical Isolates Against Different Human Blood Groups

    Science.gov (United States)

    HRV, Rajkumar; Devaki, Ramakrishna

    2016-01-01

    Among the various pathogenic determinants shown by microorganisms hemagglutination and hemolysin production assume greater significance in terms of laboratory identification. This study evaluated the hemagglutination and hemolytic activity of various bacterial isolates against different blood groups. One hundred and fifty bacterial strains, isolated from clinical specimens like urine, pus, blood, and other body fluids were tested for their hemagglutinating and hemolytic activity against human A, B, AB, and O group red blood cells. Among the 150 isolates 81 were Escherichia coli, 18 were Klebsiella pneumoniae, 19 were Pseudomonas aeruginosa, 10 were Pseudomonas spp, six were Proteus mirabilis, and the rest 16 were Staphylococcus aureus. Nearly 85% of the isolates agglutinated A group cells followed by B and AB group (59.3% and 60.6% respectively). Least number of isolates agglutinated O group cells (38.0%). When the hemolytic activity was tested, out of these 150 isolates 79 (52.6%) hemolyzed A group cells, 61 (40.6%) hemolyzed AB group cells, 46 (30.6%) hemolyzed B group cells, and 57 (38.6%) isolates hemolyzed O group cells. Forty-six percent of the isolates exhibited both hemagglutinating and hemolytic property against A group cells, followed by B and AB group cells (28.6% and 21.3% respectively). Least number of isolates i.e., 32 (21.3%) showed both the properties against O group cells. The isolates showed wide variation in their hemagglutination and hemolytic properties against different combinations of human blood group cells. The study highlights the importance of selection of the type of cells especially when human RBCs are used for studying the hemagglutination and hemolytic activity of bacterial isolates because these two properties are considered as characteristic of pathogenic strains. PMID:27014523

  5. MLVA16 typing of Portuguese human and animal Brucella melitensis and Brucella abortus isolates.

    Science.gov (United States)

    Ferreira, Ana Cristina; Chambel, Lélia; Tenreiro, Tania; Cardoso, Regina; Flor, Lídia; Dias, Isabel Travassos; Pacheco, Teresa; Garin-Bastuji, Bruno; Le Flèche, Philippe; Vergnaud, Gilles; Tenreiro, Rogério; de Sá, Maria Inácia Corrêa

    2012-01-01

    To investigate the epidemiological relationship of isolates from different Portuguese geographical regions and to assess the diversity among isolates, the MLVA16(Orsay) assay (panels 1, 2A and 2B) was performed with a collection of 126 Brucella melitensis (46 human and 80 animal isolates) and 157 B. abortus field isolates, seven vaccine strains and the representative reference strains of each species. The MLVA16(Orsay) showed a similar high discriminatory power (HGDI 0.972 and 0.902) for both species but panel 1 and 2A markers displayed higher diversity (HGDI 0.693) in B. abortus compared to B. melitensis isolates (HGDI 0.342). The B. melitensis population belong to the "Americas" (17%) and "East Mediterranean" (83%) groups. No isolate belonged to the "West Mediterranean" group. Eighty-five percent of the human isolates (39 in 46) fit in the "East-Mediterranean" group where a single lineage known as MLVA11 genotype 116 is responsible for the vast majority of Brucella infections in humans. B. abortus isolates formed a consistent group with bv1 and bv3 isolates in different clusters. Four MLVA11 genotypes were observed for the first time in isolates from S. Jorge and Terceira islands from Azores. From the collection of isolates analysed in this study we conclude that MLVA16(Orsay) provided a clear view of Brucella spp. population, confirming epidemiological linkage in outbreak investigations. In particular, it suggests recent and ongoing colonisation of Portugal with one B. melitensis lineage usually associated with East Mediterranean countries.

  6. MLVA16 typing of Portuguese human and animal Brucella melitensis and Brucella abortus isolates.

    Directory of Open Access Journals (Sweden)

    Ana Cristina Ferreira

    Full Text Available To investigate the epidemiological relationship of isolates from different Portuguese geographical regions and to assess the diversity among isolates, the MLVA16(Orsay assay (panels 1, 2A and 2B was performed with a collection of 126 Brucella melitensis (46 human and 80 animal isolates and 157 B. abortus field isolates, seven vaccine strains and the representative reference strains of each species. The MLVA16(Orsay showed a similar high discriminatory power (HGDI 0.972 and 0.902 for both species but panel 1 and 2A markers displayed higher diversity (HGDI 0.693 in B. abortus compared to B. melitensis isolates (HGDI 0.342. The B. melitensis population belong to the "Americas" (17% and "East Mediterranean" (83% groups. No isolate belonged to the "West Mediterranean" group. Eighty-five percent of the human isolates (39 in 46 fit in the "East-Mediterranean" group where a single lineage known as MLVA11 genotype 116 is responsible for the vast majority of Brucella infections in humans. B. abortus isolates formed a consistent group with bv1 and bv3 isolates in different clusters. Four MLVA11 genotypes were observed for the first time in isolates from S. Jorge and Terceira islands from Azores. From the collection of isolates analysed in this study we conclude that MLVA16(Orsay provided a clear view of Brucella spp. population, confirming epidemiological linkage in outbreak investigations. In particular, it suggests recent and ongoing colonisation of Portugal with one B. melitensis lineage usually associated with East Mediterranean countries.

  7. Long Chain N-acyl Homoserine Lactone Production by Enterobacter sp. Isolated from Human Tongue Surfaces

    Directory of Open Access Journals (Sweden)

    Kok-Gan Chan

    2012-10-01

    Full Text Available We report the isolation of N-acyl homoserine lactone-producing Enterobacter sp. isolate T1-1 from the posterior dorsal surfaces of the tongue of a healthy individual. Spent supernatants extract from Enterobacter sp. isolate T1-1 activated the biosensor Agrobacterium tumefaciens NTL4(pZLR4, suggesting production of long chain AHLs by these isolates. High resolution mass spectrometry analysis of these extracts confirmed that Enterobacter sp. isolate T1-1 produced a long chain N-acyl homoserine lactone, namely N-dodecanoyl-homoserine lactone (C12-HSL. To the best of our knowledge, this is the first isolation of Enterobacter sp., strain T1-1 from the posterior dorsal surface of the human tongue and N-acyl homoserine lactones production by this bacterium.

  8. The Isolation and Characterization of Human Prostate Cancer Stem Cells

    Science.gov (United States)

    2015-05-01

    IGF1, SOX15, BMPR1B, TGFBR1, etc), which fall into distinct GO categories including SC, development, stress response, and wound healing (unpublished...prostate cancer through the elucidation of the role of cancer stem cells in the pathogenesis of the disease. During the past year, we have made the...studies, ii) in vitro co-culture of human prostate cancer cells (established cell lines and primary patient samples) with human prostate fibroblasts

  9. Comparative Analysis of Human and Canine Campylobacter upsaliensis Isolates by Amplified Fragment Length Polymorphism

    DEFF Research Database (Denmark)

    Damborg, Peter; Guardabassi, Luca; Pedersen, Karl

    2008-01-01

    Human (n = 33) and canine (n = 53) Campylobacter upsaliensis isolates from seven countries were genotyped by a new amplified fragment length polymorphism method. We observed 100% typeability and high overall diversity. The majority of human strains (23/33) clustered separately from canine strains...

  10. Complete genome sequence of Streptococcus salivarius PS4, a strain isolated from human milk.

    Science.gov (United States)

    Martín, Virginia; Maldonado-Barragán, Antonio; Jiménez, Esther; Ruas-Madiedo, Patricia; Fernández, Leónides; Rodríguez, Juan M

    2012-08-01

    Streptococcus salivarius is a commensal species commonly found in the human oropharyngeal tract. Some strains of this species have been developed for use as oral probiotics, while others have been associated with a variety of opportunistic human infections. Here, we report the complete sequence of strain PS4, which was isolated from breast milk of a healthy woman.

  11. Draft Genome Sequence of Herpotrichiellaceae sp. UM 238 Isolated from Human Skin Scraping.

    Science.gov (United States)

    Ng, Kee Peng; Yew, Su Mei; Chan, Chai Ling; Tan, Ruixin; Soo-Hoo, Tuck Soon; Na, Shiang Ling; Hassan, Hamimah; Ngeow, Yun Fong; Hoh, Chee-Choong; Lee, Kok Wei; Yee, Wai-Yan

    2013-01-01

    Herpotrichiellaceae spp. are known to be opportunistic human pathogens. Here, we report the ~28.46-Mb draft genome of Herpotrichiellaceae sp. UM 238, isolated from human skin scraping. The UM 238 genome was found to contain many classes of protective genes that are responsible for fungal adaptation under adverse environmental conditions.

  12. Draft Genome Sequence of Ochroconis constricta UM 578, Isolated from Human Skin Scraping.

    Science.gov (United States)

    Chan, Chai Ling; Yew, Su Mei; Na, Shiang Ling; Tan, Yung-Chie; Lee, Kok Wei; Yee, Wai-Yan; Ngeow, Yun Fong; Ng, Kee Peng

    2014-04-17

    Ochroconis constricta is a soilborne dematiaceous fungus that has never been reported to be associated with human infection. Here we report the first draft genome sequence of strain UM 578, isolated from human skin scraping. The genomic information revealed will contribute to a better understanding of this species.

  13. Antimicrobial resistance of Listeria monocytogenes isolated from seafood and humans in Iran.

    Science.gov (United States)

    Abdollahzadeh, Esmail; Ojagh, Seyed Mahdi; Hosseini, Hedayat; Ghaemi, Ezzat Allah; Irajian, Gholamreza; Naghizadeh Heidarlo, Masoud

    2016-11-01

    Fourteen Listeria monocytogenes isolates previously collected from seafood (n = 7) and human patients (n = 7) were studied for their antimicrobial susceptibility against eight common antimicrobials (ampicillin, penicillin, gentamicin, streptomycin, tetracycline, trimethoprim-sulfamethoxazole, chloramphenicol, and cefotaxime). A high resistance level to ampicillin, cefotaxime (100%), and pencillin (57% in seafood isolates and 71.4% in clinical isolates) was observed in this study. However, all of the isolates were susceptible to trimethoprim-sulfamethoxazole, chloramphenicol, and tetracycline. Simultaneous resistance was identified in 4 clinical isolates (57.1%). Genotypic characterization of fish isolates (isolated from three fish species) was performed by pulsed-field gel electrophoresis (PFGE). A high diversity among fish isolates was observed. PFGE analyses distinguished the 4 isolates into 4 reproducible pulsotypes. There was no correlation between the antibiograms with pulsotypes. In conclusion, the resistance of seafood isolates to the antibiotics commonly used to treat listeriosis could be a potential health hazard for consumers. Copyright © 2016 Elsevier Ltd. All rights reserved.

  14. Isolation, growth, and characterization of human renal epithelial cells using traditional and 3D methods.

    Science.gov (United States)

    Gildea, John J; McGrath, Helen E; Van Sciver, Robert E; Wang, Dora Bigler; Felder, Robin A

    2013-01-01

    The kidney is a highly heterogeneous organ that is responsible for fluid and electrolyte balance. Much interest is focused on determining the function of specific renal epithelial cells in humans, which can only be accomplished through the isolation and growth of nephron segment-specific epithelial cells. However, human renal epithelial cells are notoriously difficult to maintain in culture. This chapter describes the isolation, growth, immortalization, and characterization of the human renal proximal tubule cell. In addition, we describe new paradigms in 3D cell culture which allow the cells to maintain more in vivo-like morphology and function.

  15. CRISPR Diversity in E. coli Isolates from Australian Animals, Humans and Environmental Waters.

    Directory of Open Access Journals (Sweden)

    Maxim S Sheludchenko

    Full Text Available Seventy four SNP genotypes and 54 E. coli genomes from kangaroo, Tasmanian devil, reptile, cattle, dog, horse, duck, bird, fish, rodent, human and environmental water sources were screened for the presence of the CRISPR 2.1 loci flanked by cas2 and iap genes. CRISPR 2.1 regions were found in 49% of the strains analysed. The majority of human E. coli isolates lacked the CRISPR 2.1 locus. We described 76 CRISPR 2.1 positive isolates originating from Australian animals and humans, which contained a total of 764 spacer sequences. CRISPR arrays demonstrated a long history of phage attacks especially in isolates from birds (up to 40 spacers. The most prevalent spacer (1.6% was an ancient spacer found mainly in human, horse, duck, rodent, reptile and environmental water sources. The sequence of this spacer matched the intestinal P7 phage and the pO111 plasmid of E. coli.

  16. Pellet-free isolation of human and bovine milk extracellular vesicles by size-exclusion chromatography

    Science.gov (United States)

    Blans, Kristine; Hansen, Maria S.; Sørensen, Laila V.; Hvam, Michael L.; Howard, Kenneth A.; Möller, Arne; Wiking, Lars; Larsen, Lotte B.; Rasmussen, Jan T.

    2017-01-01

    ABSTRACT Studies have suggested that nanoscale extracellular vesicles (EV) in human and bovine milk carry immune modulatory properties which could provide beneficial health effects to infants. In order to assess the possible health effects of milk EV, it is essential to use isolates of high purity from other more abundant milk structures with well-documented bioactive properties. Furthermore, gentle isolation procedures are important for reducing the risk of generating vesicle artefacts, particularly when EV subpopulations are investigated. In this study, we present two isolation approaches accomplished in three steps based on size-exclusion chromatography (SEC) resulting in effective and reproducible EV isolation from raw milk. The approaches do not require any EV pelleting and can be applied to both human and bovine milk. We show that SEC effectively separates phospholipid membrane vesicles from the primary casein and whey protein components in two differently obtained casein reduced milk fractions, with one of the fractions obtained without the use of ultracentrifugation. Milk EV isolates were enriched in lactadherin, CD9, CD63 and CD81 compared to minimal levels of the EV-marker proteins in other relevant milk fractions such as milk fat globules. Nanoparticle tracking analysis and electron microscopy reveals the presence of heterogeneous sized vesicle structures in milk EV isolates. Lipid analysis by thin layer chromatography shows that EV isolates are devoid of triacylglycerides and presents a phospholipid profile differing from milk fat globules surrounded by epithelial cell plasma membrane. Moreover, the milk EV fractions are enriched in RNA with distinct and diverging profiles from milk fat globules. Collectively, our data supports that successful milk EV isolation can be accomplished in few steps without the use of ultracentrifugation, as the presented isolation approaches based on SEC effectively isolates EV in both human and bovine milk. PMID:28386391

  17. Contribution of Avian Salmonella enterica Isolates to Human Salmonellosis Cases in Constantine (Algeria

    Directory of Open Access Journals (Sweden)

    Rachid Elgroud

    2015-01-01

    Full Text Available An epidemiological investigation was carried out on one hundred Salmonella isolates from broiler farms, slaughterhouses, and human patients in the Constantine region of Algeria, in order to explore the contribution of avian strains to human salmonellosis cases in this region over the same period of time. The isolates were characterized by phenotypic as well as genotypic methods. A large variety of antimicrobial resistance profiles was found among human isolates, while only seven profiles were found among avian isolates. Enterobacterial Repetitive Intergenic Consensus-PCR (ERIC-PCR, Insertion Sequence 200-PCR (IS200-PCR, and Pulsed Field Gel Electrophoresis (PFGE resulted in the allocation of the isolates to 16, 20, and 34 different profiles, respectively. The 3 genotyping methods led to complementary results by underlining the clonality of some serovars with the diffusion and persistence of a single clone in the Constantine area as well as stressing the polymorphism present in isolates belonging to other serovars, indicating the diversity of potential reservoirs of nontyphoidal Salmonella. Altogether, our results seem to indicate that nontyphoidal avian Salmonella may play an important role in human salmonellosis in the Constantine region.

  18. Contribution of Avian Salmonella enterica Isolates to Human Salmonellosis Cases in Constantine (Algeria)

    Science.gov (United States)

    Elgroud, Rachid; Granier, Sophie A.; Marault, Muriel; Kerouanton, Annaëlle; Lezzar, Abdesslem; Bouzitouna-Bentchouala, Chafia; Brisabois, Anne; Millemann, Yves

    2015-01-01

    An epidemiological investigation was carried out on one hundred Salmonella isolates from broiler farms, slaughterhouses, and human patients in the Constantine region of Algeria, in order to explore the contribution of avian strains to human salmonellosis cases in this region over the same period of time. The isolates were characterized by phenotypic as well as genotypic methods. A large variety of antimicrobial resistance profiles was found among human isolates, while only seven profiles were found among avian isolates. Enterobacterial Repetitive Intergenic Consensus-PCR (ERIC-PCR), Insertion Sequence 200-PCR (IS200-PCR), and Pulsed Field Gel Electrophoresis (PFGE) resulted in the allocation of the isolates to 16, 20, and 34 different profiles, respectively. The 3 genotyping methods led to complementary results by underlining the clonality of some serovars with the diffusion and persistence of a single clone in the Constantine area as well as stressing the polymorphism present in isolates belonging to other serovars, indicating the diversity of potential reservoirs of nontyphoidal Salmonella. Altogether, our results seem to indicate that nontyphoidal avian Salmonella may play an important role in human salmonellosis in the Constantine region. PMID:26543858

  19. Isolation and In vitro characterization of anti-Gardnerellavaginalisbacteriocin producing Lactobacillus fermentum HV6b isolated from human vaginal ecosystem

    Directory of Open Access Journals (Sweden)

    Baljinder Kaur

    2012-09-01

    Full Text Available Bacteriocin producing strains of lactic acid bacteria were isolated from vaginal swabs of healthy andfecund females and evaluated for their antimicrobial activity against pathogens causing important humandiseases such as gastrointestinal infections, nosocomial and skin diseases. Vaginal isolate HV6b is anagent that could be used to combat growing prevalence of sexually transmitted microbial infections andviral diseases. Therapeutic application of this probiotic strain to protect against gastrointestinal infectionsmay be of great importance for future medicinal use. Bacteriocin HV6b shows maximum inhibitionagainst bacterial vaginosis causing G. vaginalis. It was identified as Lactobacillus fermentum on the basisof biochemical testing and 16S rDNA sequencing. Based on the antibiotic sensitivity profiles vaginalLABs, HV6b was suggested as a strain for formulating topical personal care therapeutics aimed atprevention and treatment of many human diseases.

  20. Human intestinal mucus proteins isolated by transanal irrigation and proctosigmoidoscopy

    Directory of Open Access Journals (Sweden)

    Paola Andrea Gómez Buitrago

    2015-10-01

    Full Text Available Human intestinal mucus essentially consistsof a network of Mucin2 glycoproteinsembedded in many lower molecularweight proteins. This paper contributes tothe proteomic study of human intestinalmucus by comparing two sample collectionmethods (transanal irrigation and brushcytology during proctosigmoidoscopy andanalysis techniques (electrophoresis anddigestion in solution. The entire samplecollection and treatment process is explained,including protein extraction, digestion anddesalination and peptide characterisationusing a nanoAcquity UPLC chromatographcoupled to an HDMS spectrometer equippedwith a nanoESI source. Collecting mucus viatransanal irrigation provided a larger samplevolume and protein concentration from asingle patient. The proctosigmoidoscopysample could be analysed via digestion insolution after depleting albumin. The analysisindicates that a simple mucus lysis methodcan evaluate the electrophoresis and digestionin solution techniques. Studying humanintestinal mucus complexes is importantbecause they perform two essential survivalfunctions for humans as the first biochemicaland physical defences for the gastrointestinaltract and a habitat for intestinal microbiota,which are primarily hosted in the colon andexceeds the human genetic information andcell number 100- and 10-fold (1.

  1. Failure to infect laboratory rodent hosts with human isolates of Rodentolepis(= Hymenolepis) nana

    OpenAIRE

    MacNish, M.G.; Morgan, U. M.; Behnke, Jerzy M.; Thompson, R. C. A.

    2002-01-01

    Confusion exists over the species status and host-specificity of the tapeworm Rodentolepis (= Hymenolepis) nana. It has been described as one species, R. nana, found in both humans and rodents. Others have identified a subspecies; R. nana var. fraterna, describing it as morphologically identical to the human form but only found in rodents. The species present in Australian communities has never been identified with certainty. Fifty one human isolates of Rodentolepis (= Hymenolepis) nana were ...

  2. Mycobacterium bovis in Burkina Faso: epidemiologic and genetic links between human and cattle isolates.

    Directory of Open Access Journals (Sweden)

    Adama Sanou

    2014-10-01

    Full Text Available In sub-Saharan Africa, bovine tuberculosis (bTB is a potential hazard for animals and humans health. The goal of this study was to improve our understanding of bTB epidemiology in Burkina Faso and especially Mycobacterium bovis transmission within and between the bovine and human populations.Twenty six M. bovis strains were isolated from 101 cattle carcasses with suspected bTB lesions during routine meat inspections at the Bobo Dioulasso and Ouagadougou slaughterhouses. In addition, 7 M. bovis strains were isolated from 576 patients with pulmonary tuberculosis. Spoligotyping, RDAf1 deletion and MIRU-VNTR typing were used for strains genotyping. The isolation of M. bovis strains was confirmed by spoligotyping and 12 spoligotype signatures were detected. Together, the spoligotyping and MIRU-VNTR data allowed grouping the 33 M. bovis isolates in seven clusters including isolates exclusively from cattle (5 or humans (1 or from both (1. Moreover, these data (genetic analyses and phenetic tree showed that the M. bovis isolates belonged to the African 1 (Af1 clonal complex (81.8% and the putative African 5 (Af5 clonal complex (18.2%, in agreement with the results of RDAf1 deletion typing.This is the first detailed molecular characterization of M. bovis strains from humans and cattle in Burkina Faso. The distribution of the two Af1 and putative Af5 clonal complexes is comparable to what has been reported in neighbouring countries. Furthermore, the strain genetic profiles suggest that M. bovis circulates across the borders and that the Burkina Faso strains originate from different countries, but have a country-specific evolution. The genetic characterization suggests that, currently, M. bovis transmission occurs mainly between cattle, occasionally between cattle and humans and potentially between humans. This study emphasizes the bTB risk in cattle but also in humans and the difficulty to set up proper disease control strategies in Burkina Faso.

  3. Comparative transcriptional analysis of human macrophages exposed to animal and human isolates of Mycobacterium avium subspecies paratuberculosis with diverse genotypes.

    Science.gov (United States)

    Motiwala, Alifiya S; Janagama, Harish K; Paustian, Michael L; Zhu, Xiaochun; Bannantine, John P; Kapur, Vivek; Sreevatsan, Srinand

    2006-11-01

    Mycobacterium avium subsp. paratuberculosis is the causative agent of Johne's disease in animals and has been hypothesized to be associated with Crohn's disease in humans. Recently, M. avium subsp. paratuberculosis isolates recovered from Crohn's disease patients were shown to have limited diversity, implying the existence of human disease-associated genotypes and strain sharing with animals (A. H. Ghadiali et al., J. Clin. Microbiol. 42:5345-5348, 2004). To explore whether these genotypic differences or similarities among human and animal isolates translated to functionally significant attributes such as variance in host preference and/or difference in magnitude of infections, we performed a global scale analysis of M. avium subsp. paratuberculosis isolates that were representative of different genotypes and host species using DNA microarrays. Genome-wide characterization of the transcriptional changes was carried out using a human monocytic cell line (THP-1 cells) in response to different genotypes of M. avium subsp. paratuberculosis isolates recovered from various hosts. We identified several differentially expressed genes during early intracellular infection, including those involved in common canonical pathways such as NF-kappaB, interleukin-6 (IL-6), mitogen-activated protein kinase/extracellular signal-regulated kinase, and Jun N-terminal protein kinase signaling, as well as genes involved in T helper type 1 (Th1) responses (such as CCL5 ligand) and those that encode several proinflammatory cytokines and chemokine receptors. The cattle and human isolates of M. avium subsp. paratuberculosis, regardless of their short sequence repeat (SSR) genotype, induced similar global gene expression patterns in THP-1 cells. They differentially regulated genes necessary for cell survival without causing major alterations in proinflammatory genes. In contrast, the sheep isolates representing diverse SSR genotypes closely resembled the global gene expression pattern of an M

  4. Mycobacterium abscessus isolated from municipal water - a potential source of human infection

    OpenAIRE

    Thomson, Rachel; Tolson, Carla; Sidjabat, Hanna; Huygens, Flavia; Hargreaves, Megan

    2013-01-01

    Background Mycobacterium abscessus is a rapidly growing mycobacterium responsible for progressive pulmonary disease, soft tissue and wound infections. The incidence of disease due to M. abscessus has been increasing in Queensland. In a study of Brisbane drinking water, M. abscessus was isolated from ten different locations. The aim of this study was to compare genotypically the M. abscessus isolates obtained from water to those obtained from human clinical specimens. Methods Between 2007 and ...

  5. Human cultured cells are capable to incorporate isolated plant mitochondria loaded with exogenous DNA

    Directory of Open Access Journals (Sweden)

    Laktionov P. P.

    2012-07-01

    Full Text Available Aim. To investigate the possibility of human cultured cells to incorporate isolated mitochondria together with exogenous DNA introduced into organelles. Methods. Two approaches were used for this purpose, fluorescent labelling of mitochondria and/or DNA with subsequent analysis of the cells subjected to incubation by microscopy or by quantitative PCR. Results. We have shown that human cultured cells lines, HeLa and HUVEC, are capable to uptake isolated plant mitochondria and that this process depends on the incubation time and concentration of organelles present in medium. The incorporated mitochondria can serve as vehicles to deliver exogenous DNA into human cells, this DNA is then distributed in different cell compartments. Conclusions. These results are preliminary and need further investigations, including testing the possibility of human cells to incorporate the mitochondria of human or animal origin and creating genetic construction which could provide certain selectivity or stability of the transferred exogenous DNA upon cell uptake of the mitochondria as vectors.

  6. Pellet-free isolation of human and bovine milk extracellular vesicles by size-exclusion chromatography

    DEFF Research Database (Denmark)

    Blans, Kristine Ingrid Marie; Hansen, Maria Stenum; Sørensen, Laila V.

    2017-01-01

    -marker proteins in other relevant milk fractions such as milk fat globules. Nanoparticle tracking analysis and electron microscopy reveals the presence of heterogeneous sized vesicle structures in milk EV isolates. Lipid analysis by thin layer chromatography shows that EV isolates are devoid of triacylglycerides...... accomplished in three steps based on size-exclusion chromatography (SEC) resulting in effective and reproducible EV isolation from raw milk. The approaches do not require any EV pelleting and can be applied to both human and bovine milk. We show that SEC effectively separates phospholipid membrane vesicles...

  7. Isolation of Human Amnion Epithelial Cells According to Current Good Manufacturing Procedures.

    Science.gov (United States)

    Gramignoli, Roberto; Srinivasan, Raghuraman C; Kannisto, Kristina; Strom, Stephen C

    2016-05-12

    Different cell types can be isolated from human placental tissues, and some have been reported to retain phenotypic plasticity and characteristics that make them a promising source of cells for regenerative medicine. Among these are human amnion epithelial cells (hAECs). Adoption of current good manufacturing practices (cGMP) and enhanced quality control is essential when isolating hAECs in order to deliver a safe and effective cellular product for clinical purposes. This unit describes a detailed protocol for selective isolation of hAECs from human term placenta with little to no contamination by other cell types. A method for characterizing the heterogeneity of the hAEC suspension is also provided. The resulting cell product will be useful for clinical as well as basic research applications. © 2016 by John Wiley & Sons, Inc.

  8. Isolation of Bacteroides from fish and human fecal samples for identification of unique molecular markers.

    Science.gov (United States)

    Kabiri, Leila; Alum, Absar; Rock, Channah; McLain, Jean E; Abbaszadegan, Morteza

    2013-12-01

    Bacteroides molecular markers have been used to identify human fecal contamination in natural waters, but recent work in our laboratory confirmed cross-amplification of several human-specific Bacteroides spp. assays with fecal DNA from fish. For identification of unique molecular markers, Bacteroides from human (n = 4) and fish (n = 7) fecal samples were cultured and their identities were further confirmed using Rapid ID 32A API strips. The 16S rDNA from multiple isolates from each sample was PCR amplified, cloned, and sequenced to identify unique markers for development of more stringent human-specific assays. In human feces, Bacteroides vulgatus was the dominant species (75% of isolates), whereas in tilapia feces, Bacteroides eggerthii was dominant (66%). Bacteroides from grass carp, channel catfish, and blue catfish may include Bacteroides uniformis, Bacteroides ovatus, or Bacteroides stercoris. Phylogenic analyses of the 16S rRNA gene sequences showed distinct Bacteroides groupings from each fish species, while human sequences clustered with known B. vulgatus. None of the fish isolates showed significant similarity to Bacteroides sequences currently deposited in NCBI (National Center for Biotechnology Information). This study expands the current sequence database of cultured fish Bacteroides. Such data are essential for identification of unique molecular markers in human Bacteroides that can be utilized in differentiating fish and human fecal contamination in water samples.

  9. Isolation and characterization of human membrane carboxypeptidase (HMCP)

    Energy Technology Data Exchange (ETDEWEB)

    Skidgel, R.A.; Davis, R.M.

    1986-03-05

    The authors detected a membrane-bound carboxypeptidase in human placenta and other tissues which cleaves C-terminal Lys or Arg of peptides such as Lys/sup 6/-Met/sup 5/-enkephalin. The enzyme was solubilized from placental microvilli with 0.8% CHAPS and purified 427-fold by ion-exchange chromatography, Sepharose-arginine affinity chromatography, chromatofocusing and gel filtration on HPLC. HMCP had a mol. wt. of 67,000 in SDS-PAGE and 65,300 in gel filtration and a pH optimum of 7.0. HMCP cleaved Bz-Gly-argininic acid the fastest (90 ..mu..mol/min/mg) followed by Bz-Ala-Lys (41), Bz-Phe-Lys (26), Bz-Gly-Arg (1.7) and Bz-Gly-Lys (1.6). Activity was stimulated by CoCl/sub 2/ and inhibited by cadmium acet., o-phenanthroline and 2-mercaptomethyl-3-guanidinoethylthiopropanoic acid but not by phenylmethylsulfonyl fluoride, aprotinin or p-chloromercuriphenylsulfonate. The enzyme was stable for 1 hr at room temp. at pH 4.25, but lost 31% activity at pH 4.0. HMCP did not react with antiserum to human plasma carboxypeptidase N in Western blotting. This study shows that human placental microvilli contain a membrane carboxypeptidase, that differs from other carboxypeptidases, and cleaves C-terminal basic amino acids from peptides. This enzyme could be involved in regulating the level of peptide hormones in the placenta and other tissues.

  10. Epidemiology and public health significance of Cryptosporidium isolated from cattle, buffaloes, and humans in Egypt.

    Science.gov (United States)

    Ibrahim, M A; Abdel-Ghany, A E; Abdel-Latef, G K; Abdel-Aziz, S A; Aboelhadid, S M

    2016-06-01

    The epidemiology and public health significance of Cryptosporidium species and genotypes were investigated in Beni-Suef Governorate, Egypt. A total of 610 animal fecal samples (480 from cattle and 130 from buffaloes) beside 290 stool samples from humans were collected in the period between January and December 2014. Based on the microscopic examination, the overall estimated prevalence of Cryptosporidium spp. in cattle, buffaloes, and humans was 10.2, 12.3, and 19 %, respectively. The highest detection rates were in calves less than 2 months of age (17.1 %) and diarrheic animals (13.0 %). Likewise in humans, the highest prevalence of Cryptosporidium was in infants (31.3 %) and diarrheic individuals (21.1 %). The gender distribution in humans denoted that Cryptosporidium was reported more frequently in males (21.7 %) than females (14.5 %). Based on the molecular characterization of Cryptosporidium, Cryptosporidium oocyst wall protein (COWP) and gp60 genes were successfully amplified in 36 out of 50 samples subjected to genotyping. Restriction fragment length polymorphism (RFLP) analysis of the COWP fragments revealed that Cryptosporidium parvum was the only species detected in cattle (12 isolates) and buffaloes (4 isolates), while in humans, the detected species were Cryptosporidium hominis (15 isolates) and C. parvum (5 isolates). Sequence analysis of the gp60 gene identified the subtype IIdA20G1 within C. parvum isolated from both animals and humans. The common occurrence of zoonotic subtypes of C. parvum in cattle and buffaloes highlights the potential role of these animals as significant reservoirs of infection to humans. Also, the presence of C. hominis and C. parvum in humans indicates that both anthroponotic and zoonotic pathways are expected.

  11. Advisory Committee on human radiation experiments. Supplemental Volume 2a, Sources and documentation appendices. Final report

    Energy Technology Data Exchange (ETDEWEB)

    NONE

    1995-01-01

    This large document provides a catalog of the location of large numbers of reports pertaining to the charge of the Presidential Advisory Committee on Human Radiation Research and is arranged as a series of appendices. Titles of the appendices are Appendix A- Records at the Washington National Records Center Reviewed in Whole or Part by DoD Personnel or Advisory Committee Staff; Appendix B- Brief Descriptions of Records Accessions in the Advisory Committee on Human Radiation Experiments (ACHRE) Research Document Collection; Appendix C- Bibliography of Secondary Sources Used by ACHRE; Appendix D- Brief Descriptions of Human Radiation Experiments Identified by ACHRE, and Indexes; Appendix E- Documents Cited in the ACHRE Final Report and other Separately Described Materials from the ACHRE Document Collection; Appendix F- Schedule of Advisory Committee Meetings and Meeting Documentation; and Appendix G- Technology Note.

  12. Comparison of Campylobacter jejuni pulsotypes isolated from humans and poultry in Split and Dalmatia County, Croatia.

    Science.gov (United States)

    Kovačić, Ana; Carev, Merica; Tripković, Ingrid; Srečec, Siniša; Siško-Kraljević, Katarina

    2015-01-01

    Consumption of poultry is considered to be an important source of human infection with Campylobacter. In the period from 2008 to 2010, 50 isolates of Campylobacter jejuni from human faeces were analysed and compared with 61 isolates from poultry by pulsed field gel electrophoresis using SmaI and KpnI. Based on the analysis of SmaI macrorestriction profiles, 86 isolates (77.5 %) were assigned to 15 S clusters: 31 (62 %) from humans and 55 from poultry (90.2 %). Altogether 21 isolates (19 %) exhibited macrorestriction profiles common to both humans and poultry after restriction with SmaI and KpnI. A total of five identical pulsotypes were isolated from both poultry and patients and one of them appeared in eight different locations in the time interval of one year. These results indicate that poultry could be an important source of Campylobacter infection in Split and Dalmatia County which is the biggest County in Croatia and the most important tourist destination.

  13. Antibiotics and heavy metals resistance patterns of Enterococcus faecalis and faecium bacteria isolated from the human and the livestock sources

    Directory of Open Access Journals (Sweden)

    Yaser Sharifi

    2015-12-01

    Full Text Available Background: Enterococci have emerged as a major cause of nosocomial infections and within this group, Enterococcus faecalis and Enterococcus faecium cause the majority of human and livestock enterococcal infections. In this article, we tried to determine antibiotics and metals resistance patterns of E. faecalis and E. faecium strains. Methods: One hundred sixty different strains of E. faecalis and E. faecium were collected from livestock sewage and the human fecal waste during 15 months. Then bacterial antibiotics sensitivity tests were carried out using the Agar disc diffusion method. Results: Generally, 100% of E. faecalis strains separated from human and livestock sources (i.e. sheep showed penicillin (P/ kanamycin (K/ nitrofurantoin (N/ loracarbef (L/ Ciprofloxacin (Cc/ ampicillin (AN/ nalidixic acid (NA/ sulfamethoxazole (S antibiotics resistance patterns. In addition, 55% of isolated E. faecium showed P/S/AN/NA antibiotics resistance patterns. Each strain showed a resistance to at least two aminoglycoside antibiotics. However, E. faecalis strains from human and the livestock sources showed 94% and 100% of resistance to nitrofurantoin, respectively. The effects of different metal concentrations was evaluated in both strains. The agar dilution method was applied in this stage. Hg at 0.05 mmol/L of minimum inhibitory concentration (MIC showed toxicity to both the human and livestock Enterococcus strains. Cadmium at 1 mmol/L and 0.5 mmol/L concentrations had the most toxicity to E. faecalis and E. faecium strains, respectively. Obviously, toxicity to bacteria is less than other metals. As a result, Zn/Ni/Cu/Co resistance pattern is suggested for both strains. Finally, antibiotics and heavy metals resistance patterns were monitored simultaneously. Conclusion: Almost all E. faecalis strains isolated from humans and livestock showed antibiotics and heavy metals resistance patterns of P/K/L/Cc/S/AN/NA/Zn/Cu/Co simultaneously. Moreover, 55% of E

  14. Isolation

    DEFF Research Database (Denmark)

    Agerholm, Frank Juul

    2011-01-01

    Næringsstoffet har i dette nummer sat fokus på ”velvære i vinterkulden”, ”indendørsaktiviteter” og ”fedtafgift”. I klummen vises det, at disse tre fokusområder, der for en umiddelbar betragtning måske nok synes noget uensartede, falder sammen i ét tema: Isolation!......Næringsstoffet har i dette nummer sat fokus på ”velvære i vinterkulden”, ”indendørsaktiviteter” og ”fedtafgift”. I klummen vises det, at disse tre fokusområder, der for en umiddelbar betragtning måske nok synes noget uensartede, falder sammen i ét tema: Isolation!...

  15. Isolation and genetic characteristics of human genotype 1 Japanese encephalitis virus, China, 2009.

    Directory of Open Access Journals (Sweden)

    Jiu-Song Zhang

    Full Text Available BACKGROUND: Several studies have shown that the predominant genotype of Chinese Japanese encephalitis virus (JEV is evolving from genotype 3 to genotype 1. However, in recent years, almost all genotype 1 isolates were from mosquitoes, and genotype 1 has been less associated with human disease than genotype 3. This study reports the isolation of human genotype 1 JEV and its genetic characteristics to provide additional insights into human JE pathogens that are currently circulating in China. METHODS AND RESULTS: In 2009, 31 cerebrospinal fluid samples were collected from patients living in Yunnan and Shanxi provinces and were used to inoculate Aedes albopictus C6/36 cells for virus isolation. The JEV strains were identified using immunofluorescent assays and the reverse transcription-polymerase chain reaction. Phylogenetic analyses based on the partial capsid/pre-membrane and full envelope (E sequences were performed using Clustalx 1.8 software. Three JEV isolates were obtained from a 4-year-old girl and a 2-year-old boy living in Yunnan and an 82-year-old woman in Shanxi. The boy had been immunized with one dose of JE live attenuated vaccine. New isolates were grouped into genotype 1. Amino acid sequence for the viral E protein indicated 95% to 100% identity with each other and with other JEV strains. When compared with a consensus sequence of E protein, two amino acid substitutions were found: Ser(E-123-Asn in the two Yunnan isolates and Lys(E-166-Arg in the Shanxi isolate. CONCLUSIONS: Our findings indicate that the genotype 1 of JEV is causing human infections in China. Our observation of a previously vaccinated boy developing JE from genotype 1 virus infection also calls for more detailed studies, both in vitro and in vivo neutralization tests as well as active surveillance, to examine the possibility of a lack of complete protection conferred by the live attenuated JE vaccine against genotype 1 virus.

  16. Characterization of RNA isolated from eighteen different human tissues: results from a rapid human autopsy program.

    Science.gov (United States)

    Walker, Douglas G; Whetzel, Alexis M; Serrano, Geidy; Sue, Lucia I; Lue, Lih-Fen; Beach, Thomas G

    2016-09-01

    Many factors affect the integrity of messenger RNA from human autopsy tissues including postmortem interval (PMI) between death and tissue preservation and the pre-mortem agonal and disease states. In this communication, we describe RNA isolation and characterization of 389 samples from 18 different tissues from elderly donors who were participants in a rapid whole-body autopsy program located in Sun City, Arizona ( www.brainandbodydonationprogram.org ). Most tissues were collected within a PMI of 2-6 h (median 3.15 h; N = 455), but for this study, tissue from cases with longer PMIs (1.25-29.25 h) were included. RNA quality was assessed by RNA integrity number (RIN) and total yield (ng RNA/mg tissue). RIN correlated with PMI for heart (r = -0.531, p = 0.009) and liver (r = -558, p = 0.0017), while RNA yield correlated with PMI for colon (r = -485, p = 0.016) and skin (r = -0.460, p = 0.031). RNAs with the lowest integrity were from skin and cervix where 22.7 and 31.4 % of samples respectively failed to produce intact RNA; by contrast all samples from esophagus, lymph node, jejunum, lung, stomach, submandibular gland and kidney produced RNA with measurable RINs. Expression levels in heart RNA of 4 common housekeeping normalization genes showed significant correlations of Ct values with RIN, but only one gene, glyceraldehyde-3 phosphate dehydrogenase, showed a correlation of Ct with PMI. There were no correlations between RIN values obtained for liver, adrenal, cervix, esophagus and lymph node and those obtained from corresponding brain samples. We show that high quality RNA can be produced from most human autopsy tissues, though with significant differences between tissues and donors. The RNA stability and yield did not depend solely on PMI; other undetermined factors are involved, but these do not include the age of the donor.

  17. Improved methodology for the affinity isolation of human protein complexes expressed at near endogenous levels

    DEFF Research Database (Denmark)

    Domanski, Michal; Molloy, Kelly; Jiang, Hua;

    2012-01-01

    An efficient and reliable procedure for the capture of affinity-tagged proteins and associated complexes from human cell lines is reported. Through multiple optimizations, high yield and low background affinity-purifications are achieved from modest quantities of human cells expressing endogenous......-level tagged proteins. Isolations of triple-FLAG and GFP-tagged fusion proteins involved in RNA metabolism are presented.......An efficient and reliable procedure for the capture of affinity-tagged proteins and associated complexes from human cell lines is reported. Through multiple optimizations, high yield and low background affinity-purifications are achieved from modest quantities of human cells expressing endogenous...

  18. [Microbiological characterisation of Listeria monocytogenes isolates from human cases in Andalusia].

    Science.gov (United States)

    Lepe, José A; Torres, María José; Liró, Julia; Luque, Rafael; Aznar, Javier

    2012-12-01

    The aim of this study was to perform a retrospective study by genotyping 154 isolates from human listeriosis cases occurred in the region of Andalusia (southern Spain) in the period 2005-2009. Serotyping was performed for 1 and 4 somatic antigens using commercial Listeria antisera, and by multiplex-PCR serogrouping according to the method described by Doumith et al. (2004). The antimicrobial susceptibility was performed by Epsilon test and interpreted by CLSI criteria. PFGE was performed according to the PulseNet protocol with the ApaI enzyme. The similarity of PFGE profiles was evaluated using the Bionumerics software. The multiplex PCR protocol described by Chen and Knabel (2007) was used for the identification of isolates belonging to L. monocytogenes ECI, ECII, and ECIII epidemic clones. The 154 isolates were grouped into four serotypes: 4b [94 (61%)] strains, 1/2b [30 (19%)] strains, 1/2a [27 (18%)] strains, and 1/2c [3 (2%)] strains, with 100% of susceptibility to ampicillin and cotrimoxazole. A further sixty-two ApaI distinct pulsotypes were recognized. Thirty-seven isolates (24%) showed unique ApaI pulsotypes, and the remaining 117 strains (76%) were assigned to 25 ApaI clusters (60% in clusters of more than two isolates). The EC markers were found in 62 (40.3%) of the L. monocytogenes isolates tested. The ECI marker was present in 43 (46.2%) 4b serotype isolates, ECII in 10 (10.7%) 4b serotype isolates, and ECIII in 9 (33,3%) 1/2a serotype isolates. A large proportion of the human listeriosis cases under investigation could be grouped into molecular subtype clusters, and our cases could be related to international food-borne outbreaks. Copyright © 2011 Elsevier España, S.L. All rights reserved.

  19. Antibiotic susceptibility profiles of some Vibrio strains isolated from wastewater final effluents in a rural community of the Eastern Cape Province of South Africa

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    Igbinosa Etinosa O

    2010-05-01

    Full Text Available Abstract Background To evaluate the antibiogram and antibiotic resistance genes of some Vibrio strains isolated from wastewater final effluents in a rural community of South Africa. V. vulnificus (18, V. metschnikovii (3, V. fluvialis (19 and V. parahaemolyticus (12 strains were isolated from final effluents of a wastewater treatment plant (WWTP located in a rural community of South Africa. The disk diffusion method was used for the characterization of the antibiogram of the isolates. Polymerase chain reaction (PCR was employed to evaluate the presence of established antibiotic resistance genes using specific primer sets. Results The Vibrio strains showed the typical multidrug-resistance phenotype of an SXT element. They were resistant to sulfamethoxazole (Sul, trimethoprim (Tmp, cotrimoxazole (Cot, chloramphenicol (Chl, streptomycin (Str, ampicillin (Amp, tetracycline (Tet nalidixic acid (Nal, and gentamicin (Gen. The antibiotic resistance genes detected includes dfr18 and dfrA1 for trimethoprim; floR, tetA, strB, sul2 for chloramphenicol, tetracycline, streptomycin and sulfamethoxazole respectively. Some of these genes were only recently described from clinical isolates, demonstrating genetic exchange between clinical and environmental Vibrio species. Conclusions These results demonstrate that final effluents from wastewater treatment plants are potential reservoirs of various antibiotics resistance genes. Moreover, detection of resistance genes in Vibrio strains obtained from the wastewater final effluents suggests that these resistance determinants might be further disseminated in habitats downstream of the sewage plant, thus constituting a serious health risk to the communities reliant on the receiving waterbodies.

  20. Antibacterial susceptibility of enterobacteriaceae isolated from raw horsemeat isolated for human consumption (basashi).

    Science.gov (United States)

    Furuhata, Katsunori; Ishizaki, Naoto; Fukuyama, Masafumi

    2015-01-01

    Drug susceptibility testing was carried out using 14 antibiotics in order to identify trends in the antibiotic tolerance of 142 strains of Enterobacteriaceae isolated from horsemeat commercially available for raw consumption (basashi). A comparison of the sensitivity to the 14 antibiotics using the 90% MIC (minimum inhibitory concentration) values (MIC90) showed the strongest tolerance to ampicillin (ABPC) at a concentration of > 128 μg/mL, followed by that to fosfomycin (FOM) at a concentration of 128 μg/mL. When the sensitivity to these antibiotics was examined for each individual genus of tested bacteria, Hafnia spp. exhibited relative tolerance to ceftazidime (CAZ) and ceftriaxone (CTRX) at a concentration of 4 μg/mL and 2 μg/mL, respectively, which was high in comparison to that observed for the other strains. Furthermore, Raoultella spp. and Serratia spp. were found to be highly resistant to tetracycline (TC) at a concentration of 128 μg/mL and 64 μg/mL, respectively. Of the 142 strains of test bacteria, 140 (98.6%) demonstrated resistance to ABPC, with the exception of Hafnia alvei and Klebsiella pneumonia. In addition, a total of eight strains (5.6%), seven Serratia marcescens strains and one Raoultella terrigena strain, were found to be resistant to TC. Furthermore, one strain of Citrobacter freundii exhibited resistance to nalidixic acid (NA), while another displayed resistance to ofloxacin (OFLX) (0.7% each), and one strain (0.7%) each of Enterobacter cloacae, Serratia marcescens and Citrobacter youngae demonstrated resistance to fosfomycin (FOM), streptomycin (SM) and kanamycin (KM), respectively. A single strain of C. freundii was found to be resistant to three antibiotics, ABPC, NA and OFLX. Resistance to two antibiotics was confirmed in 11 strains, including seven strains of S. marcescens and one strain of R. terrigena (a total of eight strains) resistant to ABPC and TC, and one strain each of C. youngae, S. marcescens and E. cloacae resistant

  1. Genetic diversity of human blastocystis isolates in khorramabad, central iran.

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    Ebrahim Badparva

    2014-03-01

    Full Text Available There are some genetic differences in Blastocystis that show the existence of species or genotypes. One of these genes that help in identifying Blastocystis is SSUrRNA. The aim of this study was assessment of genetic diversity of Blastocystis by PCR with seven pairs of STS primers.This study was done on 511 stool samples collected from patients referred to the health care centers of Khorramabad, Central Iran, in 2012. Genomic DNA was extracted and in order to determine the Blastocystis subtype in contaminated samples, seven pairs of primers STS (subtype specific sequence-tagged site were used.Out of 511 samples, 33 (6.5% samples were infected with Blastocystis. Subtype (ST of 30 samples was identified and three subtypes 2, 3 and 4 were determined. Mix infection was reported 10% which 3.33% of the infection was for the mixture of ST 3 and ST5 and 6.67% was for the mixture of ST 2 and ST 3.The predominant subtype was ST3 that is the main human subtype. The dominance of ST2 and 5 are important in this study. This superiority has been reported in some of the studies in ST 2 which is different from the studies in other countries, because they have announced priorities of the ST1 and ST6 after ST3.

  2. Virulence-associated gene pattern of porcine and human Yersinia enterocolitica biotype 4 isolates.

    Science.gov (United States)

    Schneeberger, M; Brodard, I; Overesch, G

    2015-04-02

    Yersinia enterocolitica 4/O:3 is the most important human pathogenic bioserotype in Europe and the predominant pathogenic bioserotype in slaughter pigs. Although many studies on the virulence of Y. enterocolitica strains have showed a broad spectrum of detectable factors in pigs and humans, an analysis based on a strict comparative approach and serving to verify the virulence capability of porcine Y. enterocolitica as a source for human yersiniosis is lacking. Therefore, in the present study, strains of biotype (BT) 4 isolated from Swiss slaughter pig tonsils and feces and isolates from human clinical cases were compared in terms of their spectrum of virulence-associated genes (yadA, virF, ail, inv, rovA, ymoA, ystA, ystB and myfA). An analysis of the associated antimicrobial susceptibility pattern completed the characterization. All analyzed BT 4 strains showed a nearly similar pattern, comprising the known fundamental virulence-associated genes yadA, virF, ail, inv, rovA, ymoA, ystA and myfA. Only ystB was not detectable among all analyzed isolates. Importantly, neither the source of the isolates (porcine tonsils and feces, humans) nor the serotype (ST) had any influence on the gene pattern. From these findings, it can be concluded that the presence of the full complement of virulence genes necessary for human infection is common among porcine BT 4 strains. Swiss porcine BT 4 strains not only showed antimicrobial susceptibility to chloramphenicol, cefotaxime, ceftazidime, ciprofloxacin, colistin, florfenicol, gentamicin, kanamycin, nalidixic acid, sulfamethoxazole, streptomycin, tetracycline and trimethoprim but also showed 100% antibiotic resistance to ampicillin. The human BT 4 strains revealed comparable results. However, in addition to 100% antibiotic resistance to ampicillin, 2 strains were resistant to chloramphenicol and nalidixic acid. Additionally, 1 of these strains was resistant to sulfamethoxazole. The results demonstrated that Y. enterocolitica BT 4

  3. Association Between Antimicrobial Resistance in Escherichia coli Isolates from Food Animals and Blood Stream Isolates from Humans in Europe: An Ecological Study

    DEFF Research Database (Denmark)

    Vieira, Antonio; Collignon, Peter; Aarestrup, Frank Møller

    2011-01-01

    Background: In addition to medical antimicrobial usage, the use of antimicrobials in food animals contributes to the occurrence of resistance among some bacterial species isolated from infections in humans. Recently, several studies have indicated that a large proportion of Escherichia coli causing...... infections in humans, especially those resistant to antimicrobials, have an animal origin.Methods: We analyzed the correlation between the prevalence of antimicrobial resistance in E. coli isolates from blood stream infections in humans and in E. coli isolates from poultry, pigs, and cattle between 2005...... and 2008 for 11 countries, using available surveillance data. We also assessed the correlation between human antimicrobial usage and the occurrence of resistance in E. coli isolates from blood stream infections.Results: Strong and significant correlations between prevalences of resistance to ampicillin (r...

  4. Detection and quantification of human adenovirus genomes in Acanthamoeba isolated from swimming pools

    Directory of Open Access Journals (Sweden)

    RODRIGO STAGGEMEIER

    2016-01-01

    Full Text Available ABSTRACT Acanthamoeba is the most common free-living environmental amoeba, it may serve as an important vehicle for various microorganisms living in the same environment, such as viruses, being pathogenic to humans. This study aimed to detect and quantify human adenoviruses (HAdV in Acanthamoebas isolated from water samples collected from swimming pools in the city of Porto Alegre, Southern Brazil. Free-living amoebae of the genus Acanthamoeba were isolated from water samples, and isolates (n=16 were used to investigate the occurrence of HAdVs. HAdV detection was performed by quantitative real-time polymerase chain reaction (qPCR. HAdVs were detected in 62.5% (10/16 of Acanthamoeba isolates, ranging from 3.24x103 to 5.14x105 DNA copies per milliliter of isolate. HAdV viral loads found in this study are not negligible, especially because HAdV infections are associated with several human diseases, including gastroenteritis, respiratory distress, and ocular diseases. These findings reinforce the concept that Acanthamoeba may act as a reservoir and promote HAdV transmission through water.

  5. Frequency of resistance to methicillin and other antimicrobial agents among Staphylococcus aureus strains isolated from pigs and their human handlers in Trinidad

    Directory of Open Access Journals (Sweden)

    Annika Gordon

    2014-04-01

    Full Text Available Background: Methicillin-resistant Staphylococcus aureus (MRSA has emerged recently worldwide in production animals, particularly pigs and veal calves, which act as reservoirs for MRSA strains for human infection. The study determined the prevalence of MRSA and other resistant strains of S. aureus isolated from the anterior nares of pigs and human handlers on pig farms in Trinidad. Methods: Isolation of S. aureus was done by concurrently inoculating Baird-Parker agar (BPA and Chromagar MRSA (CHROM with swab samples and isolates were identified using standard methods. Suspect MRSA isolates from Chromagar and BPA were subjected to confirmatory test using Oxoid PBP2 latex agglutination test. The disc diffusion method was used to determine resistance to antimicrobial agents. Results: The frequency of isolation of MRSA was 2.1% (15 of 723 for pigs but 0.0% (0 of 72 for humans. Generally, for isolates of S. aureus from humans there was a high frequency of resistance compared with those from pigs, which had moderate resistance to the following antimicrobials: penicillin G (54.5%, 51.5%, ampicillin (59.1%, 49.5%, and streptomycin (59.1%, 37.1%, respectively. There was moderate resistance to tetracycline (36.4%, 41.2% and gentamycin (27.2%, 23.7% for human and pig S. aureus isolates, respectively, and low resistance to sulfamethoxazole-trimethoprim (4.5%, 6.2% and norfloxacin (9.1%, 12.4%, respectively. The frequency of resistance to oxacillin by the disc method was 36.4 and 34.0% from S. aureus isolates from humans and pigs, respectively. Out of a total of 78 isolates of S. aureus from both human and pig sources that were resistant to oxacillin by the disc diffusion method, only 15 (19.2% were confirmed as MRSA by the PBP'2 latex test kit. Conclusions: The detection of MRSA strains in pigs, albeit at a low frequency, coupled with a high frequency of resistance to commonly used antimicrobial agents in pig and humans could have zoonotic and therapeutic

  6. Molecular typing of Leptospira spp. strains isolated from field mice confirms a link to human leptospirosis.

    Science.gov (United States)

    Li, S J; Wang, D M; Zhang, C C; Li, X W; Yang, H M; Tian, K C; Wei, X Y; Liu, Y; Tang, G P; Jiang, X G; Yan, J

    2013-11-01

    In recent years, human leptospirosis has been reported in Jinping and Liping counties, Guizhou province, but the leptospires have never been isolated. To track the source of infection and understand the aetiological characteristics, we performed surveillance for field mice carriage of leptospirosis in 2011. Four strains of leptospire were isolated from Apodemus agrarius. PCR confirmed the four isolates as pathogenic. Multiple-locus variable-number tandem repeat analysis (MLVA) showed that the four strains were closely related to serovar Lai strain 56601 belonging to serogroup Icterohaemorrhagiae, which is consistent with the antibody detection results from local patients. Furthermore, the diversity of leptospiral isolates from different hosts and regions was demonstrated with MLVA. Our results suggest that A. agrarius may be the main carrier of Leptospira in Jinping and Liping counties, and the serogroup Icterohaemorrhagiae serovar may be the epidemic serogroup of Leptospira. This will contribute to the control and prevention of leptospirosis in these localities.

  7. Identification of species and genetic variation in Taenia isolates from human and swine of North India.

    Science.gov (United States)

    Singh, Satyendra K; Prasad, Kashi N; Singh, Aloukick K; Gupta, Kamlesh K; Chauhan, Ranjeet S; Singh, Amrita; Singh, Avinash; Rai, Ravi P; Pati, Binod K

    2016-10-01

    Taenia solium is the major cause of taeniasis and cysticercosis/neurocysticercosis (NCC) in the developing countries including India, but the existence of other Taenia species and genetic variation have not been studied in India. So, we studied the existence of different Taenia species, and sequence variation in Taenia isolates from human (proglottids and cysticerci) and swine (cysticerci) in North India. Amplification of cytochrome c oxidase subunit 1 gene (cox1) was done by polymerase chain reaction (PCR) followed by sequencing and phylogenetic analysis. We identified two species of Taenia i.e. T. solium and Taenia asiatica in our isolates. T. solium isolates showed similarity with Asian genotype and nucleotide variations from 0.25 to 1.01 %, whereas T. asiatica displayed nucleotide variations ranged from 0.25 to 0.5 %. These findings displayed the minimal genetic variations in North Indian isolates of T. solium and T. asiatica.

  8. Isolation and culture of human hematopoietic progenitors for studies of dendritic cell biology.

    Science.gov (United States)

    Svensson, Mattias

    2009-01-01

    Understanding the regulation of distinct dendritic cell (DC) function and differentiation pathways is important in many physiological and pathophysiological processes. This includes infectious and neoplastic diseases, vaccination and immunotherapy, allograft rejection, and the pathogenesis of autoimmune diseases. Isolation and culture of human hematopoietic progenitor cells provide a valuable model for studies on DC biology and may help uncover new means to manipulate DC differentiation and function in therapeutic settings. Here, a detailed protocol for the isolation of CD34+ hematopoietic progenitor cells from human cord blood is described. The isolated cell population consists of approximately 85% CD34+ CD45+ hematopoietic progenitor cells that in response to granulocyte-macrophage colony-stimulating factor (GM-CSF) plus tumor necrosis factor (TNF) expand and differentiate into CD11c+ HLA-DR+ DC-expressing CD1a.

  9. Rapid isolation of antibody from a synthetic human antibody library by repeated fluorescence-activated cell sorting (FACS.

    Directory of Open Access Journals (Sweden)

    Sung Sun Yim

    Full Text Available Antibodies and their derivatives are the most important agents in therapeutics and diagnostics. Even after the significant progress in the technology for antibody screening from huge libraries, it takes a long time to isolate an antibody, which prevents a prompt action against the spread of a disease. Here, we report a new strategy for isolating desired antibodies from a combinatorial library in one day by repeated fluorescence-activated cell sorting (FACS. First, we constructed a library of synthetic human antibody in which single-chain variable fragment (scFv was expressed in the periplasm of Escherichia coli. After labeling the cells with fluorescent antigen probes, the highly fluorescent cells were sorted by using a high-speed cell sorter, and these cells were reused without regeneration in the next round of sorting. After repeating this sorting, the positive clones were completely enriched in several hours. Thus, we screened the library against three viral antigens, including the H1N1 influenza virus, Hepatitis B virus, and Foot-and-mouth disease virus. Finally, the potential antibody candidates, which show K(D values between 10 and 100 nM against the target antigens, could be successfully isolated even though the library was relatively small (∼ 10(6. These results show that repeated FACS screening without regeneration of the sorted cells can be a powerful method when a rapid response to a spreading disease is required.

  10. Molecular Characterisation of Salmonella enterica Serovar Typhi Isolated from Typhoidial Humans

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    Arunava Das

    2012-09-01

    Full Text Available Aims: Salmonella enterica serovar Typhi is the major causative agent for typhoidial fever around the globe among human population reported till date. Present research work was carried out for detection and molecular characterisation of Salmonella enterica serovar Typhi isolated from humans with Typhoidial fever by biochemical, phenotypical and virulence gene based polymerase chain reaction (PCR techniques. The isolated strains were also investigated for antibiotic susceptibility patterns as a control measure. Methodology and Results: A total of 16 clinical samples were collected from the same numbers of patients (7 males and 9 females from Coimbatore, Erode and Salem districts of Tamil Nadu and were processed via broth enrichment methods for isolation and identification of the causative agent S. enterica serovar Typhi. Microbiological and biochemical investigations revealed the presence of S. Typhi from 16 samples. The biotyping of the isolates showed that all the isolates belonged to biotype IV. The PCR analysis confirmed the presence of invA (Invasion gene, 244bp, tyv (Tyveloseepimerase gene, 615 bp, fliC-d (Phage-1 flagellin gene for d-antigen, 750 bp and viaB (Vi antigen gene, 439bp in all 16 clinical samples. The antibiotic susceptibility test that was carried out among the isolates against 12 antimicrobial agents, showed 100 % resistance to only ampicillin and 100 % sensitivity to carbenicillin, chloramphenicol, clindamycin, gentamycin, kanamycin and tetracycline.Conclusion, significance and impact of study: This study confirmed the association of virulent strains of S. enterica serovar Typhi from Typhoidial fever among human population and suggested that PCR based diagnostic could be very useful for the rapid detection of S. Typhi isolates. Present study emphasized the use of antibiotic like chloramphenicol or in combination with other antibiotics for the effective control of S. Typhi.

  11. SEQUENCE VARIABILITY OF HUMAN CYTOMEGALOVIRUS UL144 OPEN READING FRAME IN LOW-PASSAGE CLINICAL ISOLATES

    Institute of Scientific and Technical Information of China (English)

    Rong He; Yao-hua Ji; Qiang Ruan; Chang Xia; Lan-qing Liu; Sheng-min Lü; Ying Lu; Ying Qi; Yan-ping Ma; Qing Liu

    2004-01-01

    Objective To explore the relationship between human cytomegalovirus (HCMV) UL144 sequence variability and clinical disease.Methods HCMV UL144 open reading frame (ORF) was amplified by PCR assay in 72 lowpassage isolates [65 congenitally infective children and 7 healthy children who were HCMV-DNA positive by quantitative PCR (qPCR)]. All positive PCR products were analyzed by heteroduplex mobility assay and single-stranded conformation polymorphism (HMA-SSCP) and 32 of them were sequenced.Resuits Fifty-five patient isolates and five healthy children isolates were HCMV-UL144 positive by PCR. Sequencing and HMA-SSCP analysis showed that significant strain-specific variability was present in the UL144 ORF. Phylogenetic analysis indicated that the nucleotide sequences could be separated into 3 major genotypes. Comparing between UL144 sequences and the corresponding symptoms showed that genotype 2 did not exist in megacolon isolates. And genotype 1 and 3 were the major types among microcephaly and jaundice isolates respectively.Conclusions HCMV-UL144 existed in most of low passage isolates and sequences were hypervariable. The UL144ORF and its predicted product with the high level of sequence variability in different kinds of isolates suggest that UL144ORF might play a role in HCMV infectivity and subsequent diseases.

  12. Antibiotic susceptibility profile of bacilli isolated from the skin of healthy humans.

    Science.gov (United States)

    Tarale, Prashant; Gawande, Sonali; Jambhulkar, Vinay

    2015-01-01

    In the present work, twelve bacilli were isolated from four different regions of human skin from Bela population of Nagpur district, India. The isolated bacilli were identified by their morphological, cultural and biochemical characteristics. Seven isolates were Gram negative rods, out of which five were belong to genus Pseudomonas. Three among the five Gram positive isolates were identified as Dermabactor and the remaining two Bacillus. Their antimicrobial susceptibility profile was determined by Kirby-Bauer disc diffusion method. The isolates showed resistance to several currently used broad-spectrum antibiotics. The Dermabactor genus was resistant to vancomycin, although it was earlier reported to be susceptible. Imipenem was found to be the most effective antibiotic for Pseudomonas while nalidixic acid, ampicillin and tetracycline were ineffective. Isolates of Bacillus displayed resistance to the extended spectrum antibiotics cephalosporin and ceftazidime. Imipenem, carbenicillin and ticarcillin were found to be the most effective antibiotics as all the investigated isolates were susceptible to them. Antibiotic resistance may be due to the overuse or misuse of antibiotics during the treatment, or following constant exposure to antibiotic-containing cosmetic formulations.

  13. Isolation and clinical sample typing of human leptospirosis cases in Argentina.

    Science.gov (United States)

    Chiani, Yosena; Jacob, Paulina; Varni, Vanina; Landolt, Noelia; Schmeling, María Fernanda; Pujato, Nazarena; Caimi, Karina; Vanasco, Bibiana

    2016-01-01

    Leptospira typing is carried out using isolated strains. Because of difficulties in obtaining them, direct identification of infective Leptospira in clinical samples is a high priority. Multilocus sequence typing (MLST) proved highly discriminatory for seven pathogenic species of Leptospira, allowing isolate characterization and robust assignment to species, in addition to phylogenetic evidence for the relatedness between species. In this study we characterized Leptospira strains circulating in Argentina, using typing methods applied to human clinical samples and isolates. Phylogenetic studies based on 16S ribosomal RNA gene sequences enabled typing of 8 isolates (6 Leptospira interrogans, one Leptospira wolffii and one Leptospira broomii) and 58 out of 85 (68.2%) clinical samples (55 L. interrogans, 2 Leptospira meyeri, and one Leptospira kirschneri). MLST results for the L. interrogans isolates indicated that five were probably Canicola serogroup (ST37) and one was probably Icterohaemorrhagiae serogroup (ST17). Eleven clinical samples (21.6%), provided MLST interpretable data: five were probably Pyrogenes serogroup (ST13), four Sejroe (ST20), one Autumnalis (ST22) and one Canicola (ST37). To the best of our knowledge this study is the first report of the use of an MLST typing scheme with seven loci to identify Leptospira directly from clinical samples in Argentina. The use of clinical samples presents the advantage of the possibility of knowing the infecting strain without resorting to isolates. This study also allowed, for the first time, the characterization of isolates of intermediate pathogenicity species (L. wolffii and L. broomii) from symptomatic patients.

  14. Draft Genome Sequence of Lactobacillus plantarum CMPG5300, a Human Vaginal Isolate

    NARCIS (Netherlands)

    Malik, S.; Siezen, R.J.; Renckens, B.; Vaneechoutte, M.; Vanderleyden, J.; Lebeer, S.

    2014-01-01

    The draft genome of a highly auto-aggregating Lactobacillus plantarum strain isolated from a human vagina is reported. The peculiar phenotype also provides an adhesive and co-aggregative potential with various pathogens, which could be of significance in the vaginal niche. Detailed genome analysis c

  15. Molecular analysis of Tn1546 in Enterococcus faecium isolated from animals and humans

    DEFF Research Database (Denmark)

    Jensen, Lars Bogø; Ahrens, Peter; Dons, L.

    1998-01-01

    origins from Europe and the United States, Only minor variations in the coding regions within Tn1546 were found, suggesting high genetic stability, The isolates originated from broilers (n = 5), a chicken (n = 1), a duck (n = 1), a turkey (n = 1), pigs (n = 8), a pony (n = 1), and humans (n = 23). A total...

  16. Evaluation of phenotypic and genotypic methods for subtyping Campylobacter jejuni isolates from humans, poultry, and cattle

    DEFF Research Database (Denmark)

    Nielsen, Eva Møller; Engberg, J.; Fussing, V.

    2000-01-01

    Six methods for subtyping of Campylobacter jejuni were compared and evaluated with a collection of 90 isolates from poultry, cattle, and sporadic human clinical cases as well as from a waterborne outbreak. The applied methods were Penner heat-stable serotyping; automated ribotyping (Ribo...

  17. Draft Genome Sequence of Lactobacillus plantarum CMPG5300, a Human Vaginal Isolate

    NARCIS (Netherlands)

    Malik, S.; Siezen, R.J.; Renckens, B.; Vaneechoutte, M.; Vanderleyden, J.; Lebeer, S.

    2014-01-01

    The draft genome of a highly auto-aggregating Lactobacillus plantarum strain isolated from a human vagina is reported. The peculiar phenotype also provides an adhesive and co-aggregative potential with various pathogens, which could be of significance in the vaginal niche. Detailed genome analysis c

  18. Draft Genome Sequence of Lactobacillus plantarum CMPG5300, a Human Vaginal Isolate

    NARCIS (Netherlands)

    Malik, S.; Siezen, R.J.; Renckens, B.; Vaneechoutte, M.; Vanderleyden, J.; Lebeer, S.

    2014-01-01

    The draft genome of a highly auto-aggregating Lactobacillus plantarum strain isolated from a human vagina is reported. The peculiar phenotype also provides an adhesive and co-aggregative potential with various pathogens, which could be of significance in the vaginal niche. Detailed genome analysis

  19. Body temperature predicts the direction of internal desynchronization in humans isolated from time cues

    NARCIS (Netherlands)

    Daan, Serge; Honma, Sato; Honma, Ken-ichi

    2013-01-01

    This publication presents a new analysis of experiments that were carried out in human subjects in isolation from time cues, under supervision of Jurgen Aschoff and Rutger Wever at the Max Planck Institute for Behavioural Physiology (Erling-Andechs, Germany, 1964-1974). Mean rectal temperatures

  20. Isolation of DNA from bacterial samples of the human gastrointestinal tract

    NARCIS (Netherlands)

    Zoetendal, E.G.; Heilig, G.H.J.; Klaassens, E.S.; Booijink, C.C.G.M.; Kleerebezem, M.; Smidt, H.; Vos, de W.M.

    2006-01-01

    The human gastrointestinal (GI) tract contains a complex microbial community that develops in time and space. The most widely used approaches to study microbial diversity and activity are all based on the analysis of nucleic acids, DNA, rRNA and mRNA. Here, we present a DNA isolation protocol that i

  1. [Isolation of viable human tumor cells and their characteristics during culturing in diffusion chambers].

    Science.gov (United States)

    Semenova-Kobzar', R A; Iakhimovich, L V; Liul'kin, V D; Konovalenko, V F; Palivets, A Iu

    1987-01-01

    Methodical approaches for obtaining the viable tumour cells from solid human tumours are developed. Combination of the short-term enzymatic treatment of the tumour tissue pieces and their gradual rubbing through the metal sieve with the decreasing pore sizes permitted obtaining a large number of isolated tumour cells with the high percentage of viability.

  2. Complete Genome Sequences of Four Different Bordetella sp. Isolates Causing Human Respiratory Infections

    Science.gov (United States)

    Peng, Yanhui; Loparev, Vladimir; Batra, Dhwani; Bowden, Katherine E.; Cassiday, Pamela K.; Davis, Jamie K.; Johnson, Taccara; Juieng, Phalasy; Miner, Christine E.; Rowe, Lori; Sheth, Mili; Tondella, M. Lucia; Williams, Margaret M.

    2016-01-01

    Species of the genus Bordetella associate with various animal hosts, frequently causing respiratory disease. Bordetella pertussis is the primary agent of whooping cough and other Bordetella species can cause similar cough illness. Here, we report four complete genome sequences from isolates of different Bordetella species recovered from human respiratory infections.

  3. Effect of human liver source on the functionality of isolated hepatocytes and liver slices

    NARCIS (Netherlands)

    Olinga, Peter; Hof, I.H; de Jong, Kurt; Slooff, M.JH; Meijer, D.K F; Groothuis, Geny; Merema, M.T.

    1998-01-01

    In vitro experiments using human liver tissue to study drug metabolism and transport are usually performed and interpreted without real consideration of the differences in procurement of the tissue, if it is obtained from different sources. Therefore, in this study the functionality of isolated hepa

  4. Body temperature predicts the direction of internal desynchronization in humans isolated from time cues

    NARCIS (Netherlands)

    Daan, Serge; Honma, Sato; Honma, Ken-ichi

    2013-01-01

    This publication presents a new analysis of experiments that were carried out in human subjects in isolation from time cues, under supervision of Jurgen Aschoff and Rutger Wever at the Max Planck Institute for Behavioural Physiology (Erling-Andechs, Germany, 1964-1974). Mean rectal temperatures (t(b

  5. Isolation and characterization of human articular chondrocytes from surgical waste after total knee arthroplasty (TKA)

    Science.gov (United States)

    Gradišnik, Lidija; Gorenjak, Mario; Vogrin, Matjaž

    2017-01-01

    Background Cartilage tissue engineering is a fast-evolving field of biomedical engineering, in which the chondrocytes represent the most commonly used cell type. Since research in tissue engineering always consumes a lot of cells, simple and cheap isolation methods could form a powerful basis to boost such studies and enable their faster progress to the clinics. Isolated chondrocytes can be used for autologous chondrocyte implantation in cartilage repair, and are the base for valuable models to investigate cartilage phenotype preservation, as well as enable studies of molecular features, nature and scales of cellular responses to alterations in the cartilage tissue. Methods Isolation and consequent cultivation of primary human adult articular chondrocytes from the surgical waste obtained during total knee arthroplasty (TKA) was performed. To evaluate the chondrogenic potential of the isolated cells, gene expression of collagen type 2 (COL2), collagen 1 (COL1) and aggrecan (ACAN) was evaluated. Immunocytochemical staining of all mentioned proteins was performed to evaluate chondrocyte specific production. Results Cartilage specific gene expression of COL2 and ACAN has been shown that the proposed protocol leads to isolation of cells with a high chondrogenic potential, possibly even specific phenotype preservation up to the second passage. COL1 expression has confirmed the tendency of the isolated cells dedifferentiation into a fibroblast-like phenotype already in the second passage, which confirms previous findings that higher passages should be used with care in cartilage tissue engineering. To evaluate the effectiveness of our approach, immunocytochemical staining of the evaluated chondrocyte specific products was performed as well. Discussion In this study, we developed a protocol for isolation and consequent cultivation of primary human adult articular chondrocytes with the desired phenotype from the surgical waste obtained during TKA. TKA is a common and very

  6. Isolation and characterization of human articular chondrocytes from surgical waste after total knee arthroplasty (TKA

    Directory of Open Access Journals (Sweden)

    Jakob Naranda

    2017-03-01

    Full Text Available Background Cartilage tissue engineering is a fast-evolving field of biomedical engineering, in which the chondrocytes represent the most commonly used cell type. Since research in tissue engineering always consumes a lot of cells, simple and cheap isolation methods could form a powerful basis to boost such studies and enable their faster progress to the clinics. Isolated chondrocytes can be used for autologous chondrocyte implantation in cartilage repair, and are the base for valuable models to investigate cartilage phenotype preservation, as well as enable studies of molecular features, nature and scales of cellular responses to alterations in the cartilage tissue. Methods Isolation and consequent cultivation of primary human adult articular chondrocytes from the surgical waste obtained during total knee arthroplasty (TKA was performed. To evaluate the chondrogenic potential of the isolated cells, gene expression of collagen type 2 (COL2, collagen 1 (COL1 and aggrecan (ACAN was evaluated. Immunocytochemical staining of all mentioned proteins was performed to evaluate chondrocyte specific production. Results Cartilage specific gene expression of COL2 and ACAN has been shown that the proposed protocol leads to isolation of cells with a high chondrogenic potential, possibly even specific phenotype preservation up to the second passage. COL1 expression has confirmed the tendency of the isolated cells dedifferentiation into a fibroblast-like phenotype already in the second passage, which confirms previous findings that higher passages should be used with care in cartilage tissue engineering. To evaluate the effectiveness of our approach, immunocytochemical staining of the evaluated chondrocyte specific products was performed as well. Discussion In this study, we developed a protocol for isolation and consequent cultivation of primary human adult articular chondrocytes with the desired phenotype from the surgical waste obtained during TKA. TKA is a

  7. Executive summary and guide to final report: Advisory committee on human radiation experiments

    Energy Technology Data Exchange (ETDEWEB)

    NONE

    1995-01-01

    On January 15, 1994, President Clinton appointed the Advisory Committee on Human Radiation Experiments to investigate reports of possibly unethical experiments funded by the government decades ago. The Committee was directed to uncover the history of human radiation experiments during the period 1944 through 1974 and to examine cases in which the government had intentionally released radiation into the environment for research purposes. The Committee was further charged with identifying the ethical and scientific standards for evaluating these events, and with making recommendations to ensure that whatever wrongdoing may have ocurred in the past cannot be repeated. The Committee undertook three projects: A review of how each agency of the federal government that currently conducts or funds research involving human subjects regulates this activity or oversees it; An examination of the documents and consent forms of research projects that are today sponsored by the federal government in order to develop insight into the current status of protections for the rights and interests of human subjects; and, Interviews of nearly 1,900 patients receiving out-patient medical care in private hospitals and federal facilities throughout the country. This booklet provides an overview of the Final Report, summarizing each chapter.

  8. Positive inotropy mediated via CGRP receptors in isolated human myocardial trabeculae

    DEFF Research Database (Denmark)

    Saetrum Opgaard, O; Hasbak, P; de Vries, R;

    2000-01-01

    Isometric contractile force were studied on isolated human myocardial trabeculae that were paced at 1.0 Hz in tissue baths. Alpha calcitonin gene-related peptide (alpha-CGRP) had a potent positive inotropic effect in most trabeculae from both the right atrium and left ventricle, and this effect...... reaction (PCR) mRNAs encoding the human calcitonin receptor-like receptor and the receptor associated modifying proteins (RAMPs) RAMP1, RAMP2, and RAMP3 were detected in human myocardial trabeculae from both the right atrium and left ventricle. In conclusion, functional CGRP(1) and CGRP(2) receptors may...

  9. Degradation of Marine Algae-Derived Carbohydrates by Bacteroidetes Isolated from Human Gut Microbiota.

    Science.gov (United States)

    Li, Miaomiao; Shang, Qingsen; Li, Guangsheng; Wang, Xin; Yu, Guangli

    2017-03-24

    Carrageenan, agarose, and alginate are algae-derived undigested polysaccharides that have been used as food additives for hundreds of years. Fermentation of dietary carbohydrates of our food in the lower gut of humans is a critical process for the function and integrity of both the bacterial community and host cells. However, little is known about the fermentation of these three kinds of seaweed carbohydrates by human gut microbiota. Here, the degradation characteristics of carrageenan, agarose, alginate, and their oligosaccharides, by Bacteroides xylanisolvens, Bacteroides ovatus, and Bacteroides uniforms, isolated from human gut microbiota, are studied.

  10. Prevalence of plant beneficial and human pathogenic bacteria isolated from salad vegetables in India.

    Science.gov (United States)

    Nithya, Angamuthu; Babu, Subramanian

    2017-03-14

    The study aimed at enumerating, identifying and categorizing the endophytic cultivable bacterial community in selected salad vegetables (carrot, cucumber, tomato and onion). Vegetable samples were collected from markets of two vegetable hot spot growing areas, during two different crop harvest seasons. Crude and diluted vegetable extracts were plated and the population of endophytic bacteria was assessed based on morphologically distinguishable colonies. The bacterial isolates were identified by growth in selective media, biochemical tests and 16S rRNA gene sequencing. The endophytic population was found to be comparably higher in cucumber and tomato in both of the sampling locations, whereas lower in carrot and onion. Bacterial isolates belonged to 5 classes covering 46 distinct species belonging to 19 genera. Human opportunistic pathogens were predominant in carrot and onion, whereas plant beneficial bacteria dominated in cucumber and tomato. Out of the 104 isolates, 16.25% are human pathogens and 26.5% are human opportunistic pathogens. Existence of a high population of plant beneficial bacteria was found to have suppressed the population of plant and human pathogens. There is a greater potential to study the native endophytic plant beneficial bacteria for developing them as biocontrol agents against human pathogens that are harboured by plants.

  11. Isolation and identification of tyramine-producing enterococci from human fecal samples.

    Science.gov (United States)

    Ladero, Victor; Fernández, María; Alvarez, Miguel A

    2009-02-01

    Lactic acid bacteria (LAB) are recognized as a group of important microorganisms because of their crucial role in food fermentation and their contribution to the maintenance of health homeostasis, as natural inhabitants of the human mucosa. However, the metabolic activities of some strains, such as the ability to synthesize biogenic amines (BAs), can be detrimental to human health. BAs are low molecular weight compounds synthesized by the enzymatic decarboxylation of amino acids. Tyramine, one of the most biologically active BAs, is produced by certain strains of LAB related to food fermentations. Since no data are available as to whether tyramine originates exclusively from food intake, or, like polyamines, could be formed by gut bacteria, this study was focused on the isolation of tyramine-producing LAB from human feces. Different strains of Enterococcus faecium and Enterococcus faecalis able to produce tyramine in culture conditions were isolated.

  12. Relaxant mechanisms of 3, 5, 7, 30, 40-pentamethoxyflavone on isolated human cavernosum

    DEFF Research Database (Denmark)

    Jansakul, Chaweewan; Tachanaparuksa, Kuldej; Mulvany, Michael J.

    2012-01-01

    We have investigated effects and mechanisms responsible for the activity of 3, 5, 7, 30, 40-pentamethoxyflavone (PMF) on isolated human cavernosum. PMF is the major flavone isolated from Kaempferia parviflora claimed to act as an aphrodisiac. PMF caused relaxation of phenylephrine precontracted...... did not significantly inhibit the relaxant activity of glyceryltrinitrate or acetylcholine on human cavernosal strips precontracted with phenylephrine. In contrast, sildenafil (phosphodiesterase inhibitor) potentiated the relaxant activity of glyceryl trinitrate but not of acetylcholine. In normal...... of nitric oxide, and had no effect as a KATP- or KCa channel opener, a phosphodiesterase inhibitor, a store-operated Ca2þ channel blocker or a Rho-kinase inhibitor. Therefore, these studies suggest that PMF causes relaxation of human cavernosum through voltagedependent Ca2þ channels and other mechanisms...

  13. Explant culture: An advantageous method for isolation of mesenchymal stem cells from human tissues.

    Science.gov (United States)

    Hendijani, Fatemeh

    2017-04-01

    Mesenchymal stem cell (MSC) research progressively moves towards clinical phases. Accordingly, a wide range of different procedures were presented in the literature for MSC isolation from human tissues; however, there is not yet any close focus on the details to offer precise information for best method selection. Choosing a proper isolation method is a critical step in obtaining cells with optimal quality and yield in companion with clinical and economical considerations. In this concern, current review widely discusses advantages of omitting proteolysis step in isolation process and presence of tissue pieces in primary culture of MSCs, including removal of lytic stress on cells, reduction of in vivo to in vitro transition stress for migrated/isolated cells, reduction of price, processing time and labour, removal of viral contamination risk, and addition of supporting functions of extracellular matrix and released growth factors from tissue explant. In next sections, it provides an overall report of technical highlights and molecular events of explant culture method for isolation of MSCs from human tissues including adipose tissue, bone marrow, dental pulp, hair follicle, cornea, umbilical cord and placenta. Focusing on informative collection of molecular and methodological data about explant methods can make it easy for researchers to choose an optimal method for their experiments/clinical studies and also stimulate them to investigate and optimize more efficient procedures according to clinical and economical benefits. © 2017 John Wiley & Sons Ltd.

  14. Antigenic typing of brazilian rabies virus samples isolated from animals and humans, 1989-2000

    Directory of Open Access Journals (Sweden)

    FAVORETTO Silvana Regina

    2002-01-01

    Full Text Available Animal and human rabies samples isolated between 1989 and 2000 were typified by means of a monoclonal antibody panel against the viral nucleoprotein. The panel had been previously established to study the molecular epidemiology of rabies virus in the Americas. Samples were isolated in the Diagnostic Laboratory of the Pasteur Institute and in other rabies diagnostic centers in Brazil. In addition to the fixed virus samples CVS-31/96-IP, preserved in mouse brain, and PV-BHK/97, preserved in cell culture, a total of 330 rabies virus samples were isolated from dogs, cats, cattle, horses, bats, sheep, goat, swine, foxes, marmosets, coati and humans. Six antigenic variants that were compatible with the pre-established monoclonal antibodies panel were defined: numbers 2 (dog, 3 (Desmodus rotundus, 4 (Tadarida brasiliensis, 5 (vampire bat from Venezuela, 6 (Lasiurus cinereus and Lab (reacted to all used antibodies. Six unknown profiles, not compatible with the panel, were also found. Samples isolated from insectivore bats showed the greatest variability and the most commonly isolated variant was variant-3 (Desmodus rotundus. These findings may be related to the existence of multiple independent transmission cycles, involving different bat species.

  15. Genotypic diversity, pathogenic potential and the resistance profile of Salmonella Typhimurium strains isolated from humans and food from 1983 to 2013 in Brazil.

    Science.gov (United States)

    Almeida, Fernanda; Medeiros, Marta Inês Cazentini; Rodrigues, Dália dos Prazeres; Falcão, Juliana Pfrimer

    2015-11-01

    Salmonella enterica subsp. enterica serovar Typhimurium is one of the leading serovars that causes salmonellosis worldwide. However, few studies have molecularly characterized S. Typhimurium strains in Brazil. In this study, we genotyped 92 S. Typhimurium strains isolated from humans (43) and food (49) between 1983 and 2013 in Brazil using PFGE, multiple-locus variable number of tandem repeats analysis (MLVA) and enterobacterial repetitive intergenic consensus PCR (ERIC-PCR). Moreover, we assessed the frequency of 12 virulence markers by PCR and the resistance profile against 12 antimicrobials. More than 85.8% of the strains studied carried 11 of the virulence markers or more. Thirty-three strains (25%) were multidrug resistant (MDR). The 92 S. Typhimurium studied were grouped by PFGE as PFGE-A, PFGE-B1 and PFGE-B2; by MLVA as MLVA-A, MLVA-B1 and MLVA-B2; and, finally, by ERIC-PCR as ERIC-A and ERIC-B. The strains isolated from humans before the mid-1990s were allocated to all clusters. The strains isolated from humans after the mid-1990s were distributed in the PFGE-B1, MLVA-B1, MLVA-B2 and ERIC-A clusters. The strains isolated from food were distributed in all clusters, except in PFGE-B2. All typing results suggested that the S. Typhimurium strains of human clinical origin isolated before the mid-1990s were genetically more diverse, which might indicate the selection of a more adapted S. Typhimurium subtype after Salmonella Enteritidis became the most prevalent serovar in Brazil. Regarding strains isolated from food, the results suggest the current circulation of more than one subtype. Furthermore, the high frequency of virulence genes and the presence of MDR strains reinforces their potential hazard for humans and the risk of their presence in foods in Brazil.

  16. Aeromonas hydrophila isolated from food and drinking water: hemagglutination, hemolysis, and cytotoxicity for a human intestinal cell line (HT-29).

    Science.gov (United States)

    Handfield, M; Simard, P; Couillard, M; Letarte, R

    1996-09-01

    Aeromonas hydrophila isolated from food and drinking water was tested for pathogenicity by studying its hemolysis, hemagglutination, and cytotoxicity. Hemolysis, tested on erythrocytes from six different species, was more frequently seen with water isolates (64%) than with food isolates (48%). Hemagglutination was more frequently encountered with food isolates (92%) than with water isolates (73%). Cytotoxicity, evaluated on seven cell lines, was frequently observed with food isolates (92%) and with water isolates (73%). Heat treatment (56 degrees C for 10 min) of culture supernatant fluids inhibited the toxicity of some but not all toxin-producing isolates. Our results suggest that the human intestinal cell line HT-29 could be a useful complement for testing A. hydrophila exotoxins and for studying the enteropathogenicity of this species for humans.

  17. Key features of mcr-1-bearing plasmids from Escherichia coli isolated from humans and food.

    Science.gov (United States)

    Zurfluh, Katrin; Nüesch-Inderbinen, Magdalena; Klumpp, Jochen; Poirel, Laurent; Nordmann, Patrice; Stephan, Roger

    2017-01-01

    Mcr-1-harboring Enterobacteriaceae are reported worldwide since their first discovery in 2015. However, a limited number of studies are available that compared full-length plasmid sequences of human and animal origins. In this study, mcr-1-bearing plasmids from seven Escherichia coli isolates recovered from patients (n = 3), poultry meat (n = 2) and turkey meat (n = 2) in Switzerland were further analyzed and compared. Isolates were characterized by multilocus sequence typing (MLST). The mcr-1-bearing plasmids were transferred by transformation into reference strain E. coli DH5α and MCR-1-producing transformants were selected on LB-agar supplemented with 2 mg/L colistin. Purified plasmids were then sequenced and compared. MLST revealed six distinct STs, illustrating the high clonal diversity among mcr-1-positive E. coli isolates of different origins. Two different mcr-1-positive plasmids were identified from a single E. coli ST48 human isolate. All other isolates possessed a single mcr-1 harboring plasmid. Transferable IncI2 (size ca. 60-61 kb) and IncX4 (size ca. 33-35 kb) type plasmids each bearing mcr-1 were found associated with human and food isolates. None of the mcr-1-positive IncI2 and IncX4 plasmids possessed any additional resistance determinants. Surprisingly, all but one of the sequenced mcr-1-positive plasmids lacked the ISApl1 element, which is a key element mediating acquisition of mcr-1 into various plasmid backbones. There is strong evidence that the food chain may be an important transmission route for mcr-1-bearing plasmids. Our data suggest that some "epidemic" plasmids rather than specific E. coli clones might be responsible for the spread of the mcr-1 gene along the food chain.

  18. Phage types of Salmonella enterica ssp. enterica serovar Typhimurium isolated from production animals and humans in Denmark

    DEFF Research Database (Denmark)

    Baggesen, Dorte Lau; Wegener, Henrik Caspar

    1994-01-01

    S. Typhimurium is one of the 2 most common salmonella serotypes causing human salmonellosis in Denmark. In order to illustrate the significance of different production animals as a source of infection, 1461 isolates were characterized by phage typing. The isolates originated from human patients...

  19. Relatedness of Mycobacterium avium subspecies hominissuis clinical isolates of human and porcine origins assessed by MLVA.

    Science.gov (United States)

    Leão, Célia; Canto, Ana; Machado, Diana; Sanches, Ilda Santos; Couto, Isabel; Viveiros, Miguel; Inácio, João; Botelho, Ana

    2014-09-17

    Mycobacterium avium subsp. hominissuis (MAH) is an important opportunistic pathogen, infecting humans and animals, notably pigs. Several methods have been used to characterize MAH strains. RFLP and PFGE typing techniques have been used as standard methods but are technically demanding. In contrast, the analysis of VNTR loci is a simpler, affordable and highly reliable PCR-based technique, allowing a numerical and reproductive digitalization of typing data. In this study, the analysis of Mycobacterium avium tandem repeats (MATRs) loci was adapted to evaluate the genetic diversity of epidemiological unrelated MAH clinical strains of human (n=28) and porcine (n=69) origins, collected from diverse geographical regions across mainland Portugal. These MAH isolates were found to be genetically diverse and genotypes are randomly distributed across the country. Some of the human strains shared identical VNTR profiles with porcine isolates. Our study shows that the VNTR genotyping using selected MATR loci is a useful analysis technique for assessing the genetic diversity of MAH isolates from Portugal. This typing method could be successfully applied in other countries toward the implementation of a worldwide open-access database of MATR-VNTR profiles of MAH isolates, allowing a better assessment of the global epidemiology traits of this important pathogenic species.

  20. Genotypic and phenotypic variation among Staphylococcus saprophyticus from human and animal isolates.

    Science.gov (United States)

    Kleine, Britta; Gatermann, Sören; Sakinc, Türkan

    2010-06-10

    The main aim of this study was to examine the genotypic and phenotypic diversity of Staphylococcus saprophyticus isolates from human and animal origin. In total, 236 clinical isolates and 15 animal isolates of S. saprophyticus were characterized in respect of the occurrence of 9 potential virulence genes and four surface properties. All strains were PCR positive for the regulatory genes agr, sar >it>A and rot as well as for the surface proteins UafA and Aas. Nearly 90% of the clinical isolates were found to possess the gene for the surface-associated lipase Ssp and 10% for the collagen binding MSCRAMM SdrI. All animal isolates were negative forsdrI. Lipolytic activity could be detected in 66% of the clinical and 46% of the animal isolates. Adherence to collagen type I was shown of 20% of the clinical strains and 6% of the strains of animal origin. Most S. saprophyticus strains showed hydrophobic properties and only few could agglutinate sheep erythrocytes. We described a broad analysis of animal and human S. saprophyticus isolates regarding virulence genes and phenotypic properties such as lipase activity, hydrophobicity, and adherence. While S. saprophyticus strains from animal sources have prerequisites for colonization of the urinary tract like the D-serine-deaminase, out findings suggested that they need to acquire new genes e.g. MSCRAMMS for adherence like sdrI and to modulate their existing properties e.g. increasing the lipase activity or reducing hydrophobicity. These apparently important new genes or properties for virulence have to be further analyzed.

  1. Multilocus sequence typing analysis of human and animal Clostridium difficile isolates of various toxigenic types.

    Science.gov (United States)

    Lemee, Ludovic; Dhalluin, Anne; Pestel-Caron, Martine; Lemeland, Jean-François; Pons, Jean-Louis

    2004-06-01

    A multilocus sequence typing (MLST) scheme was developed to study the genetic relationships and population structure of 72 Clostridium difficile isolates from various hosts, geographic sources, PCR ribotypes, and toxigenic types (determined by PCR targeting tcdA and tcdB genes). MLST was performed by DNA sequence analysis of seven housekeeping genes (aroE, ddl, dutA, tpi, recA, gmk, and sodA). The number of alleles ranged from five (dutA and ddl) to eleven (recA). Allelic profiles allowed the definition of 34 different sequence types (STs). These STs lacked correlation with geographic source but were well correlated to toxigenic type. The dendrogram generated from a matrix of pairwise genetic distances showed that animal isolates did not constitute a distinct lineage from human isolates and that there was no hypervirulent lineage within the population of toxigenic human isolates (isolates recovered from pseudomembranous colitis and antibiotic-associated diarrhea did not cluster in distinct lineages). However, A(-) B(+) variant isolates shared the same ST that appeared as a divergent lineage in the population studied, indicating a single evolutionary origin. The population structure was further examined by analysis of allelic polymorphism. The dendrogram generated from composite sequence-based analysis revealed a homogeneous population associated with three divergent lineages, one of which was restricted to A(-) B(+) variant isolates. C. difficile exhibited a clonal population structure, as revealed by the estimation of linkage disequilibrium (Ia) between loci. The analysis of alleles within clonal complexes estimated that point mutation generated new alleles at a frequency eightfold higher than recombinational exchange, and the congruence of the dendrograms generated from separate housekeeping loci confirmed the mutational evolution of this species.

  2. Genotypic and phenotypic variation among Staphylococcus saprophyticus from human and animal isolates

    Directory of Open Access Journals (Sweden)

    Sakinc Türkan

    2010-06-01

    Full Text Available Abstract Background The main aim of this study was to examine the genotypic and phenotypic diversity of Staphylococcus saprophyticus isolates from human and animal origin. Findings In total, 236 clinical isolates and 15 animal isolates of S. saprophyticus were characterized in respect of the occurrence of 9 potential virulence genes and four surface properties. All strains were PCR positive for the regulatory genes agr, sar>it>A and rot as well as for the surface proteins UafA and Aas. Nearly 90% of the clinical isolates were found to possess the gene for the surface-associated lipase Ssp and 10% for the collagen binding MSCRAMM SdrI. All animal isolates were negative forsdrI. Lipolytic activity could be detected in 66% of the clinical and 46% of the animal isolates. Adherence to collagen type I was shown of 20% of the clinical strains and 6% of the strains of animal origin. Most S. saprophyticus strains showed hydrophobic properties and only few could agglutinate sheep erythrocytes. Conclusions We described a broad analysis of animal and human S. saprophyticus isolates regarding virulence genes and phenotypic properties such as lipase activity, hydrophobicity, and adherence. While S. saprophyticus strains from animal sources have prerequisites for colonization of the urinary tract like the D-serine-deaminase, out findings suggested that they need to acquire new genes e.g. MSCRAMMS for adherence like sdrI and to modulate their existing properties e.g. increasing the lipase activity or reducing hydrophobicity. These apparently important new genes or properties for virulence have to be further analyzed.

  3. Genomic Variability of Serial Human Isolates of Salmonella enterica Serovar Typhimurium Associated with Prolonged Carriage.

    Science.gov (United States)

    Octavia, Sophie; Wang, Qinning; Tanaka, Mark M; Sintchenko, Vitali; Lan, Ruiting

    2015-11-01

    Salmonella enterica serovar Typhimurium is an important foodborne human pathogen that often causes self-limiting but severe gastroenteritis. Prolonged excretion of S. Typhimurium after the infection can lead to secondary transmissions. However, little is known about within-host genomic variation in bacteria associated with asymptomatic shedding. Genomes of 35 longitudinal isolates of S. Typhimurium recovered from 11 patients (children and adults) with culture-confirmed gastroenteritis were sequenced. There were three or four isolates obtained from each patient. Single nucleotide polymorphisms (SNPs) were analyzed in these isolates, which were recovered between 1 and 279 days after the initial diagnosis. Limited genomic variation (5 SNPs or fewer) was associated with short- and long-term carriage of S. Typhimurium. None of the isolates was shown to be due to reinfection. SNPs occurred randomly, and the majority of the SNPs were nonsynonymous. Two nonsense mutations were observed. A nonsense mutation in flhC rendered the isolate nonmotile, whereas the significance of a nonsense mutation in yihV is unknown. The estimated mutation rate is 1.49 × 10(-6) substitution per site per year. S. Typhimurium isolates excreted in stools following acute gastroenteritis in children and adults demonstrated limited genomic variability over time, regardless of the duration of carriage. These findings have important implications for the detection of possible transmission events suspected by public health genomic surveillance of S. Typhimurium infections.

  4. Identification, using isoenzyme electrophoresis and monoclonal antibodies, of Leishmania isolated from humans and wild animals of Ecuador.

    Science.gov (United States)

    Mimori, T; Grimaldi, G; Kreutzer, R D; Gomez, E A; McMahon-Pratt, D; Tesh, R B; Hashiguchi, Y

    1989-02-01

    Six strains of Leishmania isolated from wild mammals and humans on the Pacific Coast of Ecuador were identified by isoenzyme electrophoresis and by their reactivity patterns to a cross-panel of specific monoclonal antibodies using a radioimmune binding assay. Single isolates from Sciurus vulgaris, Potos flavus, and Tamandua tetradactyla were identified as Leishmania amazonensis. Three other strains, isolated from cutaneous lesions of humans, were identified as Leishmania panamensis.

  5. First isolation and molecular characterization of Toxoplasma gondii from a human placenta in Argentina.

    Science.gov (United States)

    Pardini, Lais; Carral, Liliana A; Bernstein, Mariana; Gos, María L; Olejnik, Patricia; Unzaga, Juan M; Kaufer, Federico J; Freuler, Cristina B; Durlach, Ricardo A; Venturini, María C

    2014-04-01

    Blood sample and placenta were taken from a 37-week pregnant woman; serologic results indicated acute toxoplasmosis. Placenta was inoculated into mice. Seropositive mice were sacrificed and tissue cysts from brain were inoculated into new mice. Specific DNA was detected by PCR, and the isolate was characterized as Type II by nPCR-RFLP for nSAG2, SAG3, BTUB, GRA6, c29-2, c22-8, L358, PK1 and Apico markers. This is the first isolation and molecular characterization of Toxoplasma gondii from humans in Argentina. © 2013.

  6. Generation of Alveolar Epithelial Spheroids via Isolated Progenitor Cells from Human Pluripotent Stem Cells

    Directory of Open Access Journals (Sweden)

    Shimpei Gotoh

    2014-09-01

    Full Text Available No methods for isolating induced alveolar epithelial progenitor cells (AEPCs from human embryonic stem cells (hESCs and induced pluripotent stem cells (hiPSCs have been reported. Based on a study of the stepwise induction of alveolar epithelial cells (AECs, we identified carboxypeptidase M (CPM as a surface marker of NKX2-1+ “ventralized” anterior foregut endoderm cells (VAFECs in vitro and in fetal human and murine lungs. Using SFTPC-GFP reporter hPSCs and a 3D coculture system with fetal human lung fibroblasts, we showed that CPM+ cells isolated from VAFECs differentiate into AECs, demonstrating that CPM is a marker of AEPCs. Moreover, 3D coculture differentiation of CPM+ cells formed spheroids with lamellar-body-like structures and an increased expression of surfactant proteins compared with 2D differentiation. Methods to induce and isolate AEPCs using CPM and consequently generate alveolar epithelial spheroids would aid human pulmonary disease modeling and regenerative medicine.

  7. Genotypic characterisation and cluster analysis of Campylobacter jejuni isolates from domestic pets, human clinical cases and retail food

    Directory of Open Access Journals (Sweden)

    Acke Els

    2011-03-01

    Full Text Available Abstract The genetic similarity of Campylobacter jejuni isolates from pets, compared to human clinical cases and retail food isolates collected in Ireland over 2001-2006 was investigated by cluster analysis of pulsed-field gel electrophoresis (PFGE fingerprinting profiles. Comparison of the PFGE profiles of 60 pet isolates and 109 human isolates revealed that seven (4.1% profiles were grouped in clusters including at least one human and one pet C. jejuni isolate. In total six (1.6% of 60 pet and 310 food profiles were in clusters with at least one food and one pet C. jejuni isolate. The detection of only a small number of genetically indistinguishable isolates by PFGE profile cluster analysis from pets and from humans with enteritis in this study suggests that pets are unlikely to be an important reservoir for human campylobacteriosis in Ireland. However, genetically indistinguishable isolates were detected and C. jejuni from pets may circulate and may contribute to clinical infections in humans. In addition, contaminated food fed to pets may be a potential source of Campylobacter infection in pets, which may subsequently pose a risk to humans.

  8. Flow-Cytometric Isolation of Human Antibodies from a Nonimmune Saccharomyces cerevisiae Surface Display Library

    Energy Technology Data Exchange (ETDEWEB)

    Feldhaus, Michael (BATTELLE (PACIFIC NW LAB)); Siegel, Robert W.(BATTELLE (PACIFIC NW LAB)); Opresko, Lee (BATTELLE (PACIFIC NW LAB)); Coleman, James R.(BATTELLE (PACIFIC NW LAB)); Feldhaus, Jane M.(BATTELLE (PACIFIC NW LAB)); Yeung, Yik A.(Massachusetts Institute Of Tec); Cochran, Jennifer R.(Massachusetts Institute Of Tec); Heinzelman, Peter (Massachusetts Institute Of Tec); Colby, David (Massachusetts Institute Of Tec); Swers, Jeffrey (Massachusetts Institute Of Tec); Graff, Christilyn (Massachusetts Institute Of Tec); Wiley, H Steven (BATTELLE (PACIFIC NW LAB)); Wittrup, K D.(Massachusetts Institute Of Tec)

    2003-02-28

    A nonimmune library of 109 human antibody scFv fragments has been cloned and expressed on the surface of yeast, and nanomolar-affinity scFvs routinely obtained by magnetic bead screening and flow cytometric sorting. The yeast library can be amplified 1010-fold without measurable loss of clonal diversity, enabling effectively indefinite expansion of the library. The expression, stability, and antigen binding properties of more than 50 isolated scFv clones were assessed directly on the yeast cell surface by immunofluorescent labeling and flow cytometry, obviating separate subcloning, expression, and purification steps and thereby expediting the isolation of novel affinity reagents. The ability to use multiplex library screening demonstrates the utility of this approach for high throughput antibody isolation for proteomics applications.

  9. Efficient Generation of Glucose-Responsive Beta Cells from Isolated GP2+ Human Pancreatic Progenitors

    DEFF Research Database (Denmark)

    Ameri, Jacqueline; Borup, Rehannah; Prawiro, Christy

    2017-01-01

    cell manufacturing. Comparative gene expression analysis revealed glycoprotein 2 (GP2) as a specific cell surface marker for isolating pancreatic endoderm cells (PECs) from differentiated hESCs and human fetal pancreas. Isolated GP2+ PECs efficiently differentiated into glucose responsive insulin......-producing cells in vitro. We found that in vitro PEC proliferation declines due to enhanced expression of the cyclin-dependent kinase (CDK) inhibitors CDKN1A and CDKN2A. However, we identified a time window when reducing CDKN1A or CDKN2A expression increased proliferation and yield of GP2+ PECs. Altogether, our...... results contribute tools and concepts toward the isolation and use of PECs as a source for the safe production of hPSC-derived β cells....

  10. Molecular identification of nocardia isolates from clinical samples and an overview of human nocardiosis in Brazil.

    Directory of Open Access Journals (Sweden)

    Paulo Victor Pereira Baio

    Full Text Available BACKGROUND: Nocardia sp. causes a variety of clinical presentations. The incidence of nocardiosis varies geographically according to several factors, such as the prevalence of HIV infections, transplants, neoplastic and rheumatic diseases, as well as climate, socio-economic conditions and laboratory procedures for Nocardia detection and identification. In Brazil the paucity of clinical reports of Nocardia infections suggests that this genus may be underestimated as a cause of human diseases and/or either neglected or misidentified in laboratory specimens. Accurate identification of Nocardia species has become increasingly important for clinical and epidemiological investigations. In this study, seven clinical Nocardia isolates were identified by multilocus sequence analysis (MLSA and their antimicrobial susceptibility was also determined. Most Nocardia isolates were associated to pulmonary disease. METHODOLOGY/PRINCIPAL FINDINGS: The majority of Brazilian human isolates in cases reported in literature were identified as Nocardia sp. Molecular characterization was used for species identification of Nocardia nova, Nocardia cyriacigeorgica, Nocardia asiatica and Nocardia exalbida/gamkensis. Data indicated that molecular analysis provided a different Nocardia speciation than the initial biochemical identification for most Brazilian isolates. All Nocardia isolates showed susceptibility to trimethoprim-sulfamethoxazole, the antimicrobial of choice in the treatment nocardiosis. N. nova isolated from different clinical specimens from one patient showed identical antimicrobial susceptibility patterns and two distinct clones. CONCLUSIONS/SIGNIFICANCE: Although Brazil is the world's fifth-largest country in terms of land mass and population, pulmonary, extrapulmonary and systemic forms of nocardiosis were reported in only 6 of the 26 Brazilian states from 1970 to 2013. A least 33.8% of these 46 cases of nocardiosis proved fatal. Interestingly, coinfection

  11. Advisory Committee on human radiation experiments. Final report, Supplemental Volume 2. Sources and documentation

    Energy Technology Data Exchange (ETDEWEB)

    NONE

    1995-01-01

    This volume and its appendixes supplement the Advisory Committee`s final report by reporting how we went about looking for information concerning human radiation experiments and intentional releases, a description of what we found and where we found it, and a finding aid for the information that we collected. This volume begins with an overview of federal records, including general descriptions of the types of records that have been useful and how the federal government handles these records. This is followed by an agency-by-agency account of the discovery process and descriptions of the records reviewed, together with instructions on how to obtain further information from those agencies. There is also a description of other sources of information that have been important, including institutional records, print resources, and nonprint media and interviews. The third part contains brief accounts of ACHRE`s two major contemporary survey projects (these are described in greater detail in the final report and another supplemental volume) and other research activities. The final section describes how the ACHRE information-nation collections were managed and the records that ACHRE created in the course of its work; this constitutes a general finding aid for the materials deposited with the National Archives. The appendices provide brief references to federal records reviewed, descriptions of the accessions that comprise the ACHRE Research Document Collection, and descriptions of the documents selected for individual treatment. Also included are an account of the documentation available for ACHRE meetings, brief abstracts of the almost 4,000 experiments individually described by ACHRE staff, a full bibliography of secondary sources used, and other information.

  12. Characterization of quinolone resistance in Salmonella spp. isolates from food products and human samples in Brazil

    Science.gov (United States)

    Pribul, Bruno Rocha; Festivo, Marcia Lima; de Souza, Miliane Moreira Soares; dos Prazeres Rodrigues, Dalia

    2016-01-01

    Non-typhoidal salmonellosis is an important zoonotic disease caused by Salmonella enterica. The aim of this study was to investigate the prevalence of plasmid-mediated quinolone resistance in Salmonella spp. and its association with fluoroquinolone susceptibility in Brazil. A total of 129 NTS isolates (samples from human origin, food from animal origin, environmental, and animal) grouped as from animal (n = 62) and human (n = 67) food were evaluated between 2009 and 2013. These isolates were investigated through serotyping, antimicrobial susceptibility testing, and the presence of plasmid-mediated quinolone resistance (PMQR) genes (qnr, aac(6′)-Ib) and associated integron genes (integrase, and conserved integron region). Resistance to quinolones and/or fluoroquinolones, from first to third generations, was observed. Fifteen isolates were positive for the presence of qnr genes (8 qnrS, 6 qnrB, and 1 qnrD) and twenty three of aac(6′)-Ib. The conserved integron region was detected in 67 isolates as variable regions, from ±600 to >1000 pb. The spread of NTS involving PMQR carriers is of serious concern and should be carefully monitored. PMID:26887245

  13. Characterization of quinolone resistance in Salmonella spp. isolates from food products and human samples in Brazil.

    Science.gov (United States)

    Pribul, Bruno Rocha; Festivo, Marcia Lima; de Souza, Miliane Moreira Soares; Rodrigues, Dalia dos Prazeres

    2016-01-01

    Non-typhoidal salmonellosis is an important zoonotic disease caused by Salmonella enterica. The aim of this study was to investigate the prevalence of plasmid-mediated quinolone resistance in Salmonella spp. and its association with fluoroquinolone susceptibility in Brazil. A total of 129 NTS isolates (samples from human origin, food from animal origin, environmental, and animal) grouped as from animal (n=62) and human (n=67) food were evaluated between 2009 and 2013. These isolates were investigated through serotyping, antimicrobial susceptibility testing, and the presence of plasmid-mediated quinolone resistance (PMQR) genes (qnr, aac(6')-Ib) and associated integron genes (integrase, and conserved integron region). Resistance to quinolones and/or fluoroquinolones, from first to third generations, was observed. Fifteen isolates were positive for the presence of qnr genes (8 qnrS, 6 qnrB, and 1 qnrD) and twenty three of aac(6')-Ib. The conserved integron region was detected in 67 isolates as variable regions, from ±600 to >1000pb. The spread of NTS involving PMQR carriers is of serious concern and should be carefully monitored.

  14. Partial Sequence Analysis of the Genome of Human Herpesvirus 7 YY5 Isolated from Saliva Samples

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    Objective To isolate and identify Nanjing local strains of Human Herpesvirus 7 (HH-V-7), and to analyze their partia l genome characteristic. Methods The saliva specimens were collected from 2 healthy adults and 5 children with kidney disease. After treatment with antibiotics and filtering. they were inoculated on to the phytohemagglutin stimulated umbilical cord blood mononuclear cells ( CBMCs). When the infected cells presented the typical ballooning and polykaryotic cytopathic effects (CPE), we identified them by transvnission electron microscopy and polymerase chain reaction.PCR product was also sequenced. Results Four strains were isolated from the seven saliva specimens. The 186-base-pair fragment of the isolated strain YY5 PCR products was sequenced, which encoded part of the HHV-7 U10 gene. The DNA sequence revealed an identity of 57. 5% and 36.0%, respectively with HHV-6 and human cytomegalovirus ( HCMV). At the amino acid level, the similarity of 51.6% was found between HHV-7 and HHV-6, and that of 25.8% between HHV-7and HCMV. Conclusion The isolated viruses were HHV-7, and 186 bp fragments revealed an identity with HHV-7 RK and Jl of 100%.

  15. ISOLATION AND PURIFICATION OF A KRINGLE 5 FROM HUMAN PLASMINOGEN USING AH-SEPHAROSE

    Directory of Open Access Journals (Sweden)

    Kapustianenko L. G.

    2014-08-01

    Full Text Available Our aim was to develop a method for isolation of human plasminogen kringle 5 possessing functional activity. The proposed method includes the following steps: hydrolysis of plasminogen with elastase, separation of mini-plasminogen from kringle fragments 1–3 and 4 on Lys-Sepharose, mini-plasminogen hydrolysis with pepsin, affinity chromatography on AH-Sepharose and polyacrilamide gel electrophoresis. We obtained the electrophoretically pure fragment of human plasminogen kringle 5 showing functional activity towards the ligands with high and low molecular mass. Weight yield was 3.8% that corresponds to 25.3% of the theoretically possible. It was established that affinity chromatography on AH-Sepharose was the sufficient step to isolate kringle 5 from mini-plasminogen hydrolysate with pepsin. This approach does not require additional purification steps while the ability of kringle 5 to bind specifically to AH-Sepharose demonstrates the functional activity of the kringle.

  16. Antimicrobial susceptibility of Clostridium difficile isolated from animals and humans in Brazil

    Directory of Open Access Journals (Sweden)

    Rodrigo Otávio Silveira Silva

    2014-05-01

    Full Text Available The objective of this study was to evaluate antimicrobial susceptibility in Clostridium difficile strains isolated from animals and humans in Brazil. The 54 C. difficile strains used were isolated from stool samples from piglets (n=16, dogs (n=13, humans (n=13, foals (n=8 calves (n=2, an ocelot (n=1 and a maned wolf (n=1. Antimicrobial susceptibility was determined using the serial plate agar dilution method for penicillin, florfenicol, oxytetracycline, erythromycin, vancomycin, metronidazole and tylosin. The C. difficile strains assessed were susceptible to metronidazole and vancomycin. Florfenicol resistance was rarely observed; 52 (96.4% strains were sensitive to this antimicrobial. Five (9.3%, five (9.3%, 14 (25.9% and 20 (37.0% strains were resistant to oxytetracycline, penicillin, tylosin and erythromycin respectively.

  17. A comparative study of two methods for the isolation of human leucocytes for DNA extraction.

    Science.gov (United States)

    Lim, L H; Ton, S H; Cheong, S K

    1990-06-01

    The 'Dextran' and the 'Buffy-coat' methods for isolation of human leucocytes for DNA extraction were compared on the basis of DNA yield from the same amounts (10 ml) of blood. Human leucocytes from a total of 11 samples were isolated using both methods for each sample after which DNA was extracted. Extracted DNA samples were treated with ribonucleases and proteinase K after which the yields were quantitated by measuring absorbance at 260 nm. The 'Buffy-coat' method yielded a mean concentration of DNA of 476.7 micrograms/ml (range: 212 to 700 micrograms/ml) while the 'Dextran' method yielded 188.4 micrograms/ml (range: 64 to 340 micrograms/ml). The difference was confirmed by subjecting the extracted DNA samples to agarose gel electrophoresis.

  18. Isolation and characterization of novel phosphate-containing sialyloligosaccharides from normal human urine.

    Science.gov (United States)

    Parkkinen, J; Finne, J

    1984-04-16

    Three phosphate-containing sialyloligosaccharides were isolated from normal human urine using charcoal adsorption, gel-filtration chromatography, ion-exchange chromatography and paper chromatography. Studies including gas-liquid chromatography of monosaccharide and disaccharide derivatives, methylation analysis, phosphate determination, ion-exchange chromatography and glycosidase and phosphatase treatments indicated the following three structures for the compounds isolated: NeuAc(alpha 2-6)Gal(beta 1-4)GlcNAc(alpha)-P; NeuAc(alpha 2-3)Gal(beta 1-4)GlcNAc(alpha)-P; NeuAc(alpha 2-3)Gal(beta 1-3)GalNAc(alpha)-P. These sialyloligosaccharide 1-phosphates represent a novel class of oligosaccharides. Their oligosaccharide chains are identical with the common sialyloligosaccharide end groups of glycoproteins and glycolipids. The excretion of these compounds in normal human urine may indicate the existence of a novel, as yet unrevealed pathway in the metabolism of complex carbohydrates.

  19. Prevalence and pathogenic potential of campylobacter isolates from free-living, human-commensal american crows.

    Science.gov (United States)

    Weis, Allison M; Miller, Woutrina A; Byrne, Barbara A; Chouicha, Nadira; Boyce, Walter M; Townsend, Andrea K

    2014-03-01

    Recent studies have suggested a potential role for wild birds in zoonotic transmission of Campylobacter jejuni, the leading cause of gastroenteritis in humans worldwide. In this study, we detected Campylobacter spp. in 66.9% (85/127) of free-ranging American crows (Corvus brachyrhyncos) sampled in the Sacramento Valley of California in 2012 and 2013. Biochemical testing and sequence analysis of 16S rRNA revealed that 93% of isolates (n = 70) were C. jejuni, with cytolethal distending toxin (CDT) and flagellin A genes detected by PCR in 20% and 46% of the C. jejuni isolates (n = 59), respectively. The high prevalence of C. jejuni, coupled with the occurrence of known virulence markers CDT and flagellin A, demonstrates that crows shed Campylobacter spp. in their feces that are potentially pathogenic to humans. Crows are abundant in urban, suburban, and agricultural settings, and thus further study to determine their role in zoonotic transmission of Campylobacter will inform public health.

  20. EPIDEMIOLOGICAL RELATIONSHIPS AMONG STRAINS OF Salmonella enterica subsp. enterica, ISOLATED FROM HUMANS, POULTRY AND FOOD

    Directory of Open Access Journals (Sweden)

    I. Méndez

    2006-06-01

    Full Text Available Human gastro-enteritis caused by Salmonella enterica is a major health problem in developing countries such as Colombia. In some parts of Colombia, the disease is endemic, and its incidence appears to be increasing, with outbreaks and sporadic cases of diarrhea becoming more frequent. At this time, it is not very clear if either poultry or food is responsible for human salmonellosis contamination in Colombia. The objectives of the present study were to analyze the Pulsed-field gel electrophoresis profiles (PFGEPs of Salmonella enterica from human patients, poultry and food found in Colombia and to determine the epidemiologic associations between these strains. Twenty-nine isolates of Salmonella enterica subsp. enterica were isolated from: 10 pediatric patients in Bogotá, D.C., 10 different types of food and 9 chickens. All isolates were analyzed by means of the molecular technique XbaI PFGE. Eleven different patterns were observed. These patterns consisted of 12-17 restriction fragments, each with a molecular size of 30-800 kb. The results suggested that Salmonella enterica was transmitted from poultry and food to humans. Surprisingly, among the strains investigated it was impossible to find a direct linkage between poultry and food, indicating, either that Salmonella was incorporated into the food during food processing by handlers, or that foods other than poultry products were the source of human infection. This study about the molecular epidemiology of Salmonella enterica in Colombia provided new information about possible means of human contamination, and should permit health institutions to take adequate measures to avoid sporadic cases and outbreaks of salmonellosis.

  1. Antimicrobial susceptibility profiles of human Campylobacter jejuni isolates and association with phylogenetic lineages

    Directory of Open Access Journals (Sweden)

    Wonhee eCha

    2016-04-01

    Full Text Available Campylobacter jejuni is a zoonotic pathogen and the most common bacterial cause of human gastroenteritis worldwide. With the increase of antibiotic resistance to fluoroquinolones and macrolides, the drugs of choice for treatment, C. jejuni was recently classified as a serious antimicrobial resistant threat. Here, we characterized 94 C. jejuni isolates collected from patients at four Michigan hospitals in 2011 and 2012 to determine the frequency of resistance and association with phylogenetic lineages. The prevalence of resistance to fluoroquinolones (19.1% and macrolides (2.1% in this subset of C. jejuni isolates from Michigan was similar to national reports. High frequencies of fluoroquinolone-resistant C. jejuni isolates, however, were recovered from patients with a history of foreign travel. A high proportion of these resistant isolates were classified as multilocus sequence type (ST-464, a fluoroquinolone-resistant lineage that recently emerged in Europe. A significantly higher prevalence of tetracycline-resistant C. jejuni was also found in Michigan and resistant isolates were more likely to represent ST-982, which has been previously recovered from ruminants and the environment in the U.S. Notably, patients with tetracycline-resistant C. jejuni infections were more likely to have contact with cattle. These outcomes prompt the need to monitor the dissemination and diversification of imported fluoroquinolone-resistant C. jejuni strains and to investigate the molecular epidemiology of C. jejuni recovered from cattle and farm environments to guide mitigation strategies.

  2. Cryptolepine, isolated from Sida acuta, sensitizes human gastric adenocarcinoma cells to TRAIL-induced apoptosis.

    Science.gov (United States)

    Ahmed, Firoj; Toume, Kazufumi; Ohtsuki, Takashi; Rahman, Mahmudur; Sadhu, Samir Kumar; Ishibashi, Masami

    2011-01-01

    Bioassay guided separation of Sida acuta whole plants led to the isolation of an alkaloid, cryptolepine (1), along with two kaempferol glycosides (2-3). Compound 1 showed strong activity in overcoming TRAIL-resistance in human gastric adenocarcinoma (AGS) cells at 1.25, 2.5 and 5 μm. Combined treatment of 1 and TRAIL sensitized AGS cells to TRAIL-induced apoptosis at the aforementioned concentrations. Copyright © 2010 John Wiley & Sons, Ltd.

  3. Evaluation of Existing Methods for Human Blood mRNA Isolation and Analysis for Large Studies

    OpenAIRE

    Meyer, Anke; Paroni, Federico; Günther, Kathrin; Dharmadhikari, Gitanjali; Ahrens, Wolfgang; Kelm, Sørge; Maedler, Kathrin

    2016-01-01

    Aims Prior to implementing gene expression analyses from blood to a larger cohort study, an evaluation to set up a reliable and reproducible method is mandatory but challenging due to the specific characteristics of the samples as well as their collection methods. In this pilot study we optimized a combination of blood sampling and RNA isolation methods and present reproducible gene expression results from human blood samples. Methods The established PAXgeneTM blood collection method (Qiagen)...

  4. Isolation and characterization of non tuberculous mycobacteria from humans and animals in Namwala District of Zambia

    OpenAIRE

    Malama, Sydney; Munyeme, Musso; Mwanza, Sydney; Muma, John Bwalya

    2014-01-01

    Background The genus Mycobacterium contains more than 100 species, most of which are classified as non-tuberculous mycobacteria (NTM). In Zambia, the NTM are slowly becoming recognized as pathogens of major public health significance with the advent of Human Immunodeficiency Virus (HIV) and Acquired Immunodeficiency Syndrome (AIDS). This study aimed at reporting the isolated NTM and ascertains their zoonotic potential and diagnostic significance in Zambia. Method A total of 100 sputum samples...

  5. Subtyping and antimicrobial susceptibility of Clostridium difficile PCR ribotype 078/126 isolates of human and animal origin.

    Science.gov (United States)

    Álvarez-Pérez, Sergio; Blanco, José L; Harmanus, Celine; Kuijper, Ed; García, Marta E

    2017-02-01

    The Clostridium difficile PCR ribotype complex 078/126 (RT078/126) is often involved in human disease and is also frequently isolated from diverse animal species. The high genetic relatedness between human and animal RT078/126 isolates found in different regions has encouraged discussion about the zoonotic potential of this lineage. We compared for the first time the genetic diversity and antimicrobial susceptibility profiles of human and animal C. difficile RT078/126 isolates from Spain. A collection of 96 isolates (50 of human and 46 of animal origin; 63 and 33 of ribotypes 078 and 126, respectively) was subtyped by an improved amplified fragment length polymorphism (AFLP) fingerprinting method and tested for in vitro antimicrobial susceptibility. A total of 67 genotypes were distinguished, three of which grouped together isolates of human and animal origin. Furthermore, two main groups of isolates that mostly correlated with PCR ribotypes could be distinguished in the AFLP dendrogram. Human origin was significantly associated with resistance to ertapenem, erythromycin and moxifloxacin; resistance to clindamycin and erythromycin was associated with RT126 and AFLP group 1. Twenty-nine isolates (30.2% of total) displayed heteroresistance to metronidazole. Substantial differences were observed in the susceptibility profiles of isolates belonging to a same genotype. Altogether, these results provide a valuable baseline for future studies on the epidemiology of C. difficile RT078/126. Copyright © 2016 Elsevier B.V. All rights reserved.

  6. Affinity-purified human interleukin I is cytotoxic to isolated islets of Langerhans

    DEFF Research Database (Denmark)

    Mandrup-Poulsen, T; Bendtzen, K; Nerup, J

    1986-01-01

    . These effects were dose-dependent and reproducible when using three different Interleukin-1 preparations. Highly purified human Interleukin-2, Lymphotoxin, Leucocyte Migration Inhibitory Factor and Macrophage Migration Inhibitory Factor were ineffective. These findings suggest that Interleukin-1 may play......Addition of highly purified human Interleukin-1 to the culture medium of isolated rat islets of Langerhans for 6 days led to 88% inhibition of glucose-induced insulin-release, reduction of islet contents of insulin and glucagon to 31% and 8% respectively, and disintegration of the islets...

  7. Establishing a cell biology platform: isolation and preservation of human blood products

    OpenAIRE

    2014-01-01

    Dissertação para obtenção do Grau de Mestre em Genética Molecular e Biomedicina The use of human primary cells provide researchers in different areas with irrefutable more biologically relevant data than using cell lines or animal blood cells. The work was performed in the scope of the Cell Biology Services @ CEDOC, aiming to provide viable and trustful human primary cells and products. We had three main objectives: protocol optimizations for blood cell isolation, culture and cryopre...

  8. Isolation and characterization of a new mutant human cell line unresponsive to alpha and beta interferons.

    OpenAIRE

    John, J.; McKendry, R; Pellegrini, S; Flavell, D; Kerr, I M; Stark, G R

    1991-01-01

    Previously we described human cell line 2fTGH, in which expression of guanine phosphoribosyltransferase is tightly controlled by the upstream region of interferon (IFN)-stimulated human gene 6-16. After mutagenesis of 2fTGH and selection with 6-thioguanine and IFN-alpha, we isolated 11.1, a recessive mutant that does not respond to IFN-alpha. We now describe U2, a second recessive mutant, selected similarly, that complements 11.1. U2 had no response to IFN-alpha or IFN-beta, and its response ...

  9. Supravital dithizone staining in the isolation of human and rat pancreatic islets

    DEFF Research Database (Denmark)

    Hansen, W A; Christie, M R; Kahn, R

    1989-01-01

    Dithizone, a zinc chelating agent, is known to selectively stain the islets of Langerhans in the pancreas. In the present study, we have used this stain to aid the identification of islets in material obtained by collagenase digestion of human pancreas. Islets were shown to rapidly and reversibly...... techniques for the large scale isolation of functionally intact human islets.......Dithizone, a zinc chelating agent, is known to selectively stain the islets of Langerhans in the pancreas. In the present study, we have used this stain to aid the identification of islets in material obtained by collagenase digestion of human pancreas. Islets were shown to rapidly and reversibly...... no effect on insulin release in tissue culture, on acute responses to stimulatory glucose concentrations or on the insulin content of cells. These results suggest that dithizone staining can assist in the identification of islets from the human pancreas and may prove to be a useful tool in developing...

  10. Distribution of intimin subtypes among Escherichia coli isolates from ruminant and human sources.

    Science.gov (United States)

    Ramachandran, Vidiya; Brett, Kim; Hornitzky, Michael A; Dowton, Mark; Bettelheim, Karl A; Walker, Mark J; Djordjevic, Steven P

    2003-11-01

    The intimin gene eae, located within the locus of enterocyte effacement pathogenicity island, distinguishes enteropathogenic Escherichia coli (EPEC) and some Shiga toxin-producing E. coli (STEC) strains from all other pathotypes of diarrheagenic E. coli. EPEC is a leading cause of infantile diarrhea in developing countries, and intimin-positive STEC isolates are typically associated with life-threatening diseases such as hemolytic-uremic syndrome and hemorrhagic colitis. Here we describe the development of a PCR-restriction fragment length polymorphism (RFLP) assay that reliably differentiates all 11 known intimin types (alpha1, alpha2, beta, gamma, kappa, epsilon, eta, iota, lambda, theta, and zeta) and three new intimin genes that show less than 95% nucleotide sequence identity with existing intimin types. We designated these new intimin genes Int- micro, Int-nu, and Int-xi. The PCR-RFLP assay was used to screen 213 eae-positive E. coli isolates derived from ovine, bovine, and human sources comprising 60 serotypes. Of these, 82 were STEC isolates, 89 were stx-negative (stx(-)) and ehxA-positive (ehxA(+)) isolates, and 42 were stx(-) and ehxA-negative isolates. Int-beta, the most commonly identified eae subtype (82 of 213 [38.5%] isolates), was associated with 21 serotypes, followed by Int-zeta (39 of 213 [18.3%] isolates; 11 serotypes), Int-theta (25 of 213 [11.7%] isolates; 15 serotypes), Int-gamma (19 of 213 [8.9%] isolates; 9 serotypes), and Int-epsilon (21 of 213 [9.9%] isolates; 5 serotypes). Intimin subtypes alpha1, alpha2, kappa, lambda, xi, micro, nu, and iota were infrequently identified; and Int-eta was not detected. Phylogenetic analyses with the Phylip package of programs clustered the intimin subtypes into nine distinct families (alpha, beta-xi, gamma, kappa, epsilon-eta-nu, iota- micro, lambda, theta, and zeta). Our data confirm that ruminants are an important source of serologically and genetically diverse intimin-containing E. coli strains.

  11. Failure to infect laboratory rodent hosts with human isolates of Rodentolepis (= Hymenolepis) nana.

    Science.gov (United States)

    Macnish, M G; Morgan, U M; Behnke, J M; Thompson, R C A

    2002-03-01

    Confusion exists over the species status and host-specificity of the tapeworm Rodentolepis (= Hymenolepis) nana. It has been described as one species, R. nana, found in both humans and rodents. Others have identified a subspecies; R. nana var. fraterna, describing it as morphologically identical to the human form but only found in rodents. The species present in Australian communities has never been identified with certainty. Fifty one human isolates of Rodentolepis (= Hymenolepis) nana were orally inoculated into Swiss Q, BALB/c, A/J, CBA/ CAH and nude (hypothymic) BALB/c mice, Fischer 344 and Wistar rats and specific pathogen free (SPF) hamsters. Twenty four human isolates of R. nana were cross-tested in flour beetles, Tribolium confusum. No adult worms were obtained from mice, rats or hamsters, even when immunosuppressed with cortisone acetate. Only one of the 24 samples developed to the cysticercoid stage in T. confusum; however, when inoculated into laboratory mice the cysticercoids failed to develop into adult worms. The large sample size used in this study, and the range of techniques employed for extraction and preparation of eggs provide a comprehensive test of the hypothesis that the human strain of R. nana is essentially non-infective to rodents.

  12. Data on isolating mesenchymal stromal cells from human adipose tissue using a collagenase-free method

    Directory of Open Access Journals (Sweden)

    Wassim Shebaby

    2016-03-01

    Full Text Available The present dataset describes a detailed protocol to isolate mesenchymal cells from human fat without the use of collagenase. Human fat specimen, surgically cleaned from non-fat tissues (e.g., blood vessels and reduced into smaller fat pieces of around 1–3 mm size, is incubated in complete culture media for five to seven days. Then, cells started to spread out from the fat explants and to grow in cultures according to an exponential pattern. Our data showed that primary mesenchymal cells presenting heterogeneous morphology start to acquire more homogenous fibroblastic-like shape when cultured for longer duration or when subcultured into new flasks. Cell isolation efficiency as well as cell doubling time were also calculated throughout the culturing experimentations and illustrated in a separate figure thereafter. This paper contains data previously considered as an alternative protocol to isolate adipose-derived mesenchymal stem cell published in “Proliferation and differentiation of human adipose-derived mesenchymal stem cells (ASCs into osteoblastic lineage are passage dependent” [1].

  13. Investigation of dental pulp stem cells isolated from discarded human teeth extracted due to aggressive periodontitis.

    Science.gov (United States)

    Sun, Hai-Hua; Chen, Bo; Zhu, Qing-Lin; Kong, Hui; Li, Qi-Hong; Gao, Li-Na; Xiao, Min; Chen, Fa-Ming; Yu, Qing

    2014-11-01

    Recently, human dental pulp stem cells (DPSCs) isolated from inflamed dental pulp tissue have been demonstrated to retain some of their pluripotency and regenerative potential. However, the effects of periodontal inflammation due to periodontitis and its progression on the properties of DPSCs within periodontally compromised teeth remain unknown. In this study, DPSCs were isolated from discarded human teeth that were extracted due to aggressive periodontitis (AgP) and divided into three experimental groups (Groups A, B and C) based on the degree of inflammation-induced bone resorption approaching the apex of the tooth root before tooth extraction. DPSCs derived from impacted or non-functional third molars of matched patients were used as a control. Mesenchymal stem cell (MSC)-like characteristics, including colony-forming ability, proliferation, cell cycle, cell surface antigens, multi-lineage differentiation capability and in vivo tissue regeneration potential, were all evaluated in a patient-matched comparison. It was found that STRO-1- and CD146-positive DPSCs can be isolated from human teeth, even in very severe cases of AgP. Periodontal inflammation and its progression had an obvious impact on the characteristics of DPSCs isolated from periodontally affected teeth. Although all the isolated DPSCs in Groups A, B and C showed decreased colony-forming ability and proliferation rate (P biomaterials were transplanted directly into an ectopic transplantation model. However, when cell-seeded scaffolds were placed in the root fragments of human teeth, all the cells formed significant dentin- and pulp-like tissues. The ability of DPSCs to generate dental tissues decreased when the cells were isolated from periodontally compromised teeth (P < 0.05). Again, increased periodontal destruction was not necessarily followed by a decrease in the amount of dentin- and pulp-like tissue formed. These findings provide preliminary evidence that periodontally compromised teeth might

  14. The molecular structure and lubricating activity of lubricin isolated from bovine and human synovial fluids.

    Science.gov (United States)

    Swann, D A; Silver, F H; Slayter, H S; Stafford, W; Shore, E

    1985-01-01

    Lubricin was isolated from bovine ankle, metacarpophalangeal and knee and human knee synovial fluids. The lubricins isolated from the bovine joint fluids had the same amino acid and carbohydrate compositions, but differences were observed in the relative molecular masses. The Mr values of bovine metacarpophalangeal and ankle lubricin determined by light-scattering measurements were about 200 000, whereas values of 132 000 and 143 000 were obtained for the bovine knee lubricin. The human knee lubricin had a similar carbohydrate composition to bovine knee lubricin except for the higher glucosamine content, and the amino acid composition differed slightly. The human sample had a lower glutamic acid content and a leucine/isoleucine ratio of 2:1 compared with 1:1 in the bovine. The Mr value of the human knee lubricin (166 000) was also lower than that of the bovine metacarpophalangeal and ankle samples. The Mr value of the bovine knee lubricin determined by sedimentation-equilibrium measurements was 171 000. The length measurements determined by electron microscopy and also the sedimentation measurements showed considerable polydispersity and indicate that the degree of extension of lubricin molecules can vary. Friction measurements showed that the human knee synovial-fluid lubricin had equivalent lubricating ability in a test system in vitro to that observed for lubricin isolated from normal bovine synovial fluids. The lubricating ability of lubricin was concentration-dependent, and each lubricin sample was able to act as a lubricant in vitro in an equivalent manner to whole synovial fluid at concentrations that are thought to occur in vivo. PMID:3977823

  15. VTEC O157 subtypes associated with the most severe clinical symptoms in humans constitute a minor part of VTEC 0157 isolates from Danish Cattle

    DEFF Research Database (Denmark)

    Roldgaard, Bemt Bjørn; Scheutz, Flemming; Boel, Jeppe;

    2004-01-01

    -positive VTEC 0 157 isolates (63 of bovine origin and 86 from human clinical cases) isolated between 1987 and 2001. All were analysed by vtx-PCR-RFLP and phage typing. The vtx-PCR-RFLP showed that isolates carrying the vtx2 gene was more than four times as prevalent among the human clinical isolates (55...

  16. Efficient Induction and Isolation of Human Primordial Germ Cell-Like Cells from Competent Human Pluripotent Stem Cells.

    Science.gov (United States)

    Irie, Naoko; Surani, M Azim

    2017-01-01

    We recently reported a robust and defined culture system for the specification of human primordial germ cell-like cells (hPGCLCs) from human pluripotent stem cells (hPSCs), both embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) in vitro (Irie et al. Cell 160: 253-268, 2015). Similar attempts previously produced hPGCLCs from hPSCs at a very low efficiency, and the resulting cells were not fully characterized. A key step, which facilitated efficient hPGCLC specification from hPSCs, was the induction of a "competent" state for PGC fate via the medium containing a cocktail of four inhibitors. The competency of hPSCs can be maintained indefinitely and interchangeably with the conventional/low-competent hPSCs. Specification of hPGCLC occurs following sequential expression of key germ cell fate regulators, notably SOX17 and BLIMP1, as well as initiation of epigenetic resetting over 5 days. The hPGCLCs can be isolated using specific cell surface markers without the need for generating germ cell-specific reporter hPSC lines. This powerful method for the induction and isolation of hPGCLCs can be applied to both hESCs and iPSCs, which can be used for advances in human germ line biology.

  17. Relaxant mechanisms of 3, 5, 7, 3', 4'-pentamethoxyflavone on isolated human cavernosum.

    Science.gov (United States)

    Jansakul, Chaweewan; Tachanaparuksa, Kuldej; Mulvany, Michael J; Sukpondma, Youwapa

    2012-09-15

    We have investigated effects and mechanisms responsible for the activity of 3, 5, 7, 3', 4'-pentamethoxyflavone (PMF) on isolated human cavernosum. PMF is the major flavone isolated from Kaempferia parviflora claimed to act as an aphrodisiac. PMF caused relaxation of phenylephrine precontracted human cavernosal strips, and this effect was slightly inhibited by N(G)-nitro-l-arginine, a nitric oxide synthase inhibitor, but not by ODQ (soluble guanylate cyclase inhibitor), TEA (tetraethylammonium, blocker of voltage-dependent K(+) channels) or glybenclamide (blocker of ATP-dependent K(+) channels). PMF did not significantly inhibit the relaxant activity of glyceryltrinitrate or acetylcholine on human cavernosal strips precontracted with phenylephrine. In contrast, sildenafil (phosphodiesterase inhibitor) potentiated the relaxant activity of glyceryl trinitrate but not of acetylcholine. In normal Krebs solution with nifedipine (blocker of l-type Ca(2+) channels), or in Ca(2+)-free Krebs solution, PMF caused a further inhibition of human cavernosum contracted with phenylephrine. In human cavernosum treated with thapsigargin (inhibitor of sarcoplasmic reticulum Ca(2+)-ATPase) in Ca(2+)-free medium, PMF suppressed the concentration-response curve of human cavernosum to phenylephrine and a further suppression was found when SKF-96365 (a blocker of store-operated Ca(2+) channels and Y-27632 (inhibitor of Rho-kinase)), but not nifedipine, were added sequentially. Thus, PMF had only a weak effect on the release of nitric oxide, and had no effect as a K(ATP)- or K(Ca) channel opener, a phosphodiesterase inhibitor, a store-operated Ca(2+) channel blocker or a Rho-kinase inhibitor. Therefore, these studies suggest that PMF causes relaxation of human cavernosum through voltage-dependent Ca(2+) channels and other mechanisms associated with calcium mobilization. Copyright © 2012 Elsevier B.V. All rights reserved.

  18. Species identification and molecular typing of human Brucella isolates from Kuwait.

    Science.gov (United States)

    Mustafa, Abu S; Habibi, Nazima; Osman, Amr; Shaheed, Faraz; Khan, Mohd W

    2017-01-01

    Brucellosis is a zoonotic disease of major concern in Kuwait and the Middle East. Human brucellosis can be caused by several Brucella species with varying degree of pathogenesis, and relapses are common after apparently successful therapy. The classical biochemical methods for identification of Brucella are time-consuming, cumbersome, and provide information limited to the species level only. In contrast, molecular methods are rapid and provide differentiation at intra-species level. In this study, four molecular methods [16S rRNA gene sequencing, real-time PCR, enterobacterial repetitive intergenic consensus (ERIC)-PCR and multilocus variable-number tandem-repeat analysis (MLVA)-8, MLVA-11 and MLVA-16 were evaluated for the identification and typing of 75 strains of Brucella isolated in Kuwait. 16S rRNA gene sequencing of all isolates showed 90-99% sequence identity with B. melitensis and real-time PCR with genus- and species- specific primers identified all isolates as B. melitensis. The results of ERIC-PCR suggested the existence of 75 ERIC genotypes of B. melitensis with a discriminatory index of 0.997. Cluster classification of these genotypes divided them into two clusters, A and B, diverging at ~25%. The maximum number of genotypes (n = 51) were found in cluster B5. MLVA-8 analysis identified all isolates as B. melitensis, and MLVA-8, MLVA-11 and MLVA-16 typing divided the isolates into 10, 32 and 71 MLVA types, respectively. Furthermore, the combined minimum spanning tree analysis demonstrated that, compared to MLVA types discovered all over the world, the Kuwaiti isolates were a distinct group of MLVA-11 and MLVA-16 types in the East Mediterranean Region.

  19. HIGH VARIABILITY OF HUMAN CYTOMEGALOVIRUS UL150 OPEN READING FRAME IN LOW-PASSAGED CLINICAL ISOLATES

    Institute of Scientific and Technical Information of China (English)

    2006-01-01

    Objective To investigate the polymorphism of human cytomegalovirus (HCMV) UL150 open reading frame(ORF) in low-passaged clinical isolates, and to study the relationship between the polymorphism and different pathogenesis of congenital HCMV infection.Methods PCR was performed to amplify the entire HCMV UL150 ORF region of 29 clinical isolates, which hadbeen proven containing detectable HCMV-DNA using fluorescence quantitative PCR. PCR amplifcation products weresequenced directly, and the data were analyzed.Results Totally 25 among 29 isolates were amplified, and 18 isolates were sequenced successfully. HCMVUL150 ORF sequences derived from congenitally infected infants were high variability. The UL150 ORF in all 18 clinical isolates shifted backward by 8 nucleotides leading to frame-shift, and contained a single nucleotide deletion at nucleotide position 226 compared with that of Toledo strain. The nucleotide diversity was 0. 1% to 6. 8% and the amino acid diversity was 0. 2% to 19. 2% related to Toledo strain. However, the nucleotide diversity was 0. 1% to 6.4% and amino acid diversity was 0. 2% to 8.3% by compared with Merlin strain. Compared with Toledo, 4 new cysteine residues and 13 additional posttranslational modification sites were observed in UL150 putative proteins of clinical isolates. Moreover, the UL150 putative protein contained an additional transmembrane helix at position of 4-17 amino acid related to Toledo.Conclusion HCMV UL150 ORF and deduced amino acid sequences of clinical strains are hypervariability. No obvious linkage between the polymorphism and different pathogenesis of congenital HCMV infection is found.

  20. Isolation and characterization of sweat gland myoepithelial cells from human skin.

    Science.gov (United States)

    Kurata, Ryuichiro; Futaki, Sugiko; Nakano, Itsuko; Tanemura, Atsushi; Murota, Hiroyuki; Katayama, Ichiro; Sekiguchi, Kiyotoshi

    2014-01-01

    Stem cells routinely maintain the main epidermal components, i.e. the interfollicular epidermis, hair follicles, and sweat glands. Human sweat glands present throughout the body are glandular exocrine organs that mainly play a role in thermoregulation by sweating. Emerging evidence points to the presence of stem cells in sweat glands, but it remains unclear whether such stem cells exist in human sweat glands. Here, we attempted to gather evidence for stem cells in human sweat glands, which would be characterized by self-renewal ability and multipotency. First, we explored human sweat gland cells for expression of stem cell markers. CD29 and Notch, epidermal stem cell markers, were found to reside among α-smooth muscle actin-positive myoepithelial cells in human sweat glands. Next, sweat gland myoepithelial cells were isolated from human skin as a CD29(hi)CD49f (hi) subpopulation. The myoepithelial cell-enriched CD29(hi)CD49f (hi) subpopulation possessed the ability to differentiate into sweat gland luminal cells in sphere-forming assays. Furthermore, CD29(hi)CD49f (hi) subpopulation-derived sphere-forming cells exhibited long-term proliferative potential upon multiple passaging, indicating that the CD29(hi)CD49f (hi) myoepithelial subpopulation includes stem cells with self-renewal ability. These findings provide evidence that human sweat gland myoepithelial cells contain stem cells that possess both self-renewal ability and multipotency to differentiate into sweat glands.

  1. Isolation of Human Antibodies Against Hepatitis E From Phage Display Library by Metal Affinity Chromatography

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    Objective To isolate human antibodies against hepatitis E virus from phage display library by a new method of panning phage antibody library based on immobilized metal affinity chromatography (IMAC). Methods Phage antibody library was allowed to mix with hex-His tagged expressed HEV specific antigen, NE2, in solution for adequate binding before affinity resin for hex-His was added. The non-specific phage antibodies were removed by extensive washing and the specific bound phage antibodies could then be eluted to infect TG1 or repeat the binding process for subsequent rounds of purification. The specificity of the selected human antibodies were tested by antigen competitive ELISA, human sera blocking ELISA, scFv expression, and sequence analysis. Results His-NE2 specific recombinant phages were successfully enriched after panning procedure. Two individual phage clones, 126 and 138, showed 50% inhibition in NE2 antigen competition ELISA and obvious blocking effect by HEV positive serum in blocking ELISA. Soluble scFv of 126, 138 bound to NE2 specifically. Conclusion Two specific human phage antibodies against hepatitis E virus (HEV) from phage display library were isolated by immobilized metal affinity chromatography. The immobilized metal affinity chromatography applied to phage antibody selection was a helpful supplement to the selection in solution.

  2. Human isolates of Bartonella tamiae induce pathology in experimentally inoculated immunocompetent mice

    Directory of Open Access Journals (Sweden)

    Kosoy Michael Y

    2010-07-01

    Full Text Available Abstract Background Bartonella tamiae, a newly described bacterial species, was isolated from the blood of three hospitalized patients in Thailand. These patients presented with headache, myalgia, anemia, and mild liver function abnormalities. Since B. tamiae was presumed to be the cause of their illness, these isolates were inoculated into immunocompetent mice to determine their relative pathogenicity in inducing manifestations of disease and pathology similar to that observed in humans. Methods Three groups of four Swiss Webster female mice aged 15-18 months were each inoculated with 106-7 colony forming units of one of three B. tamiae isolates [Th239, Th307, and Th339]. A mouse from each experimental group was sampled at 3, 4, 5 and 6 weeks post-inoculation. Two saline inoculated age-matched controls were included in the study. Samples collected at necropsy were evaluated for the presence of B. tamiae DNA, and tissues were formalin-fixed, stained with hematoxylin and eosin, and examined for histopathology. Results Following inoculation with B. tamiae, mice developed ulcerative skin lesions and subcutaneous masses on the lateral thorax, as well as axillary and inguinal lymphadenopathy. B. tamiae DNA was found in subcutaneous masses, lymph node, and liver of inoculated mice. Histopathological changes were observed in tissues of inoculated mice, and severity of lesions correlated with the isolate inoculated, with the most severe pathology induced by B. tamiae Th239. Mice inoculated with Th239 and Th339 demonstrated myocarditis, lymphadenitis with associated vascular necrosis, and granulomatous hepatitis and nephritis with associated hepatocellular and renal necrosis. Mice inoculated with Th307 developed a deep dermatitis and granulomas within the kidneys. Conclusions The three isolates of B. tamiae evaluated in this study induce disease in immunocompetent Swiss Webster mice up to 6 weeks after inoculation. The human patients from whom these

  3. Comparison of human and soil Candida tropicalis isolates with reduced susceptibility to fluconazole.

    Directory of Open Access Journals (Sweden)

    Yun-Liang Yang

    Full Text Available Infections caused by treatment-resistant non-albicans Candida species, such as C. tropicalis, has increased, which is an emerging challenge in the management of fungal infections. Genetically related diploid sequence type (DST strains of C. tropicalis exhibiting reduced susceptibility to fluconazole circulated widely in Taiwan. To identify the potential source of these wildly distributed DST strains, we investigated the possibility of the presence in soil of such C. tropicalis strains by pulsed field gel electrophoresis (PFGE and DST typing methods. A total of 56 C. tropicalis isolates were recovered from 26 out of 477 soil samples. Among the 18 isolates with reduced susceptibility to fluconazole, 9 belonged to DST149 and 3 belonged to DST140. Both DSTs have been recovered from our previous studies on clinical isolates from the Taiwan Surveillance of Antimicrobial Resistance of Yeasts (TSARY program. Furthermore, these isolates were more resistant to agricultural azoles. We have found genetically related C. tropicalis exhibiting reduced susceptibility to fluconazole from the human hosts and environmental samples. Therefore, to prevent patients from acquiring C. tropicalis with reduced susceptibility to azoles, prudent use of azoles in both clinical and agricultural settings is advocated.

  4. Haematospirillum jordaniae gen. nov., sp. nov., isolated from human blood samples.

    Science.gov (United States)

    Humrighouse, B W; Emery, B D; Kelly, A J; Metcalfe, M G; Mbizo, J; McQuiston, J R

    2016-04-01

    A Gram-negative, aerobic, motile, spiral-shaped bacterium, strain H5569(T), was isolated from a human blood sample. Phenotypic and molecular characteristics of the isolate were investigated. Optimal growth was found to occur at 35 °C under aerobic conditions on Heart Infusion Agar supplemented with 5 % rabbit blood. The major fatty acids present in the cells were identified as C16:0, C16:1ω7c and C18:1ω7c. The predominant respiratory quinone was found to be ubiquinone-Q10. The G+C content of genomic DNA for strain H5569(T) was found to be 49.9 %. Based on 16S rRNA gene sequence analysis results, 13 additional isolates were also analysed in this study. Phylogenetic analysis based on 16S rRNA gene sequences revealed that the organism, represented by strain H5569(T), forms a distinct lineage within the family Rhodospirillaceae, closely related to two Novispirillum itersonii subspecies (93.9-94.1 %) and two Caenispirillum sp. (91.2-91.6 %). Based on these results, the isolate H5569(T) is concluded to represent a new genus and species for which the name Haematospirillum jordaniae gen. nov., sp. nov. is proposed. The type strain is H5569(T) (=DSM(T) 28903 = CCUG 66838(T)).

  5. Genetic characterization of St. Louis encephalitis virus isolated from human in São Paulo, Brazil

    Directory of Open Access Journals (Sweden)

    Cecília Luiza Simões dos Santos

    2006-02-01

    Full Text Available The molecular characterization of SPH253157, a new strain of St. Louis encephalitis virus (SLEV, isolated in 2004 from the first case of human infection recognized in the state of São Paulo, Brazil, is reported. The patient, presenting a febrile illness without neurological involvement, was hospitalized as a probable case of dengue fever. Genomic RNA was isolated from the supernatant of C6/36 cells infected with acute phase-serum specimen of the patient and the envelope gene was amplified by reverse-transcription-polymerase chain reaction. The complete nucleotide sequence of the envelope gene of this isolate was directly sequenced from the amplified products and compared with other Brazilian and American SLEV strains. Phylogenetic analyses were carried out under maximum likelihood criterion with outgroups both included and excluded. Outgroups comprised four flavivirus of the Japanese encephalitis group. Phylogeny also included Bayesian analysis. The results indicated that the new SLEV isolate belongs to lineage III, being closely related to an Argentinean strain recovered from Culex sp. in 1979. It is concluded that there are at least 3 lineages of SLEV in Brazil.

  6. Molecular characterization and phylogenetic analysis of human parainfluenza virus type 3 isolated from Saudi Arabia.

    Science.gov (United States)

    Almajhdi, Fahad N; Alshaman, Mohamed S; Amer, Haitham M

    2012-08-01

    Human parainfluenza virus 3 (HPIV-3) is a leading cause of respiratory disease in children worldwide. Previous sequence analyses of the entire virus genome, among different HPIV-3 strains, demonstrated that HN is the most variable gene. There is a dearth of data on HPIV-3 strains circulating in Saudi Arabia. In this report, HPIV-3 was screened in nasopharyngeal aspirates collected from hospitalized children with acute respiratory disease during two successive seasons (2007/08 and 2008/09) using nested RT-PCR. Out of 73 samples collected during 2007/08, seven (9.59%) were positive; while 3 out of 107 samples collected during 2008/09 (2.8%) were positive. Virus isolation in cell culture was successful using HEp2, but not Vero cells. The identity of the isolated viruses was confirmed using immunofluorescence and neutralization assays. To elucidate the genetic characteristics and phylogeny of Saudi HPIV-3 strains, the complete HN gene sequence of two selected Saudi strains was analyzed in comparison to 20 strains isolated by others from different countries worldwide. Both strains showed the highest degree of sequence homology with Indian strains, followed by Chinese and most Japanese strains. Phylogenetic analysis confirmed that these strains fell into a distinct Asian lineage. This study is the first in Saudi Arabia to recover HPIV-3 isolates of confirmed identity, and to generate sequence data that may help in understanding virus diversity and evolution.

  7. Isolation of labile Fcgamma-receptors from human peripheral blood lymphocytes and production of an antiserum.

    Science.gov (United States)

    Sandilands, G P; Peel, M G; Froebel, K S; Belch, J J; MacSween, R N

    1985-05-01

    In this study, we have isolated membranelabile Fcgamma-receptors (i.e. FcgammaR I) from normal human peripheral blood lymphocytes and have produced a rabbit antiserum to this protein. Using this antiserum, we have shown that membrane-labile and membrane-stable (i.e. FcgammaR II) Fcgamma-receptors are antigenically distinct and that these two forms of the receptors probably coexist on the same lymphocyte subpopulation. Moreover, it was apparent that lymphocyte FcgammaR Is are distinct from FcgammaRs expressed on other cell types (e.g. monocytes, polymorphs and spermatozoa). Preliminary evidence does suggest, however, that human platelets express an FcgammaR which is antigenically similar to human lymphocyte FcgammaR I.

  8. Isolation, cryopreservation and culture of human amnion epithelial cells for clinical applications.

    Science.gov (United States)

    Murphy, Sean V; Kidyoor, Amritha; Reid, Tanya; Atala, Anthony; Wallace, Euan M; Lim, Rebecca

    2014-12-21

    Human amnion epithelial cells (hAECs) derived from term or pre-term amnion membranes have attracted attention from researchers and clinicians as a potential source of cells for regenerative medicine. The reason for this interest is evidence that these cells have highly multipotent differentiation ability, low immunogenicity, and anti-inflammatory functions. These properties have prompted researchers to investigate the potential of hAECs to be used to treat a variety of diseases and disorders in pre-clinical animal studies with much success. hAECs have found widespread application for the treatment of a range of diseases and disorders. Potential clinical applications of hAECs include the treatment of stroke, multiple sclerosis, liver disease, diabetes and chronic and acute lung diseases. Progressing from pre-clinical animal studies into clinical trials requires a higher standard of quality control and safety for cell therapy products. For safety and quality control considerations, it is preferred that cell isolation protocols use animal product-free reagents. We have developed protocols to allow researchers to isolate, cryopreserve and culture hAECs using animal product-free reagents. The advantage of this method is that these cells can be isolated, characterized, cryopreserved and cultured without the risk of delivering potentially harmful animal pathogens to humans, while maintaining suitable cell yields, viabilities and growth potential. For researchers moving from pre-clinical animal studies to clinical trials, these methodologies will greatly accelerate regulatory approval, decrease risks and improve the quality of their therapeutic cell population.

  9. Isolation and functional interrogation of adult human prostate epithelial stem cells at single cell resolution

    Directory of Open Access Journals (Sweden)

    Wen-Yang Hu

    2017-08-01

    Full Text Available Using primary cultures of normal human prostate epithelial cells, we developed a novel prostasphere-based, label-retention assay that permits identification and isolation of stem cells at a single cell level. Their bona fide stem cell nature was corroborated using in vitro and in vivo regenerative assays and documentation of symmetric/asymmetric division. Robust WNT10B and KRT13 levels without E-cadherin or KRT14 staining distinguished individual stem cells from daughter progenitors in spheroids. Following FACS to isolate label-retaining stem cells from label-free progenitors, RNA-seq identified unique gene signatures for the separate populations which may serve as useful biomarkers. Knockdown of KRT13 or PRAC1 reduced sphere formation and symmetric self-renewal highlighting their role in stem cell maintenance. Pathways analysis identified ribosome biogenesis and membrane estrogen-receptor signaling enriched in stem cells with NF-ĸB signaling enriched in progenitors; activities that were biologically confirmed. Further, bioassays identified heightened autophagy flux and reduced metabolism in stem cells relative to progenitors. These approaches similarly identified stem-like cells from prostate cancer specimens and prostate, breast and colon cancer cell lines suggesting wide applicability. Together, the present studies isolate and identify unique characteristics of normal human prostate stem cells and uncover processes that maintain stem cell homeostasis in the prostate gland.

  10. N-Acyl Homoserine Lactone Production by Klebsiella pneumoniae Isolated from Human Tongue Surface

    Directory of Open Access Journals (Sweden)

    Kok-Gan Chan

    2012-03-01

    Full Text Available Bacteria communicate by producing quorum sensing molecules called autoinducers, which include autoinducer-1, an N-hexanoyl homoserine lactone (AHL, and autoinducer-2. Bacteria present in the human oral cavity have been shown to produce autoinducer-2, but not AHL. Here, we report the isolation of two AHL-producing Klebsiella pneumoniae strains from the posterior dorsal surface of the tongue of a healthy individual. Spent culture supernatant extracts from K. pneumoniae activated the biosensors Agrobacterium tumefaciens NTL4(pZLR4 and Escherichia coli [pSB401], suggesting the presence of both long and short chain AHLs. High resolution mass spectrometry analyses of these extracts confirmed that both K. pneumoniae isolates produced N-octanoylhomoserine lactone and N-3-dodecanoyl-L-homoserine lactone. To the best of our knowledge, this is the first report of the isolation of K. pneumoniae from the posterior dorsal surface of the human tongue and the production of these AHLs by this bacterium.

  11. Demonstration of transplacental transmission of a human isolate of Anaplasma phagocytophilum in an experimentally infected sheep.

    Science.gov (United States)

    Reppert, E; Galindo, R C; Breshears, M A; Kocan, K M; Blouin, E F; de la Fuente, J

    2013-11-01

    Anaplasma phagocytophilum, first identified as a pathogen of sheep in Europe, more recently has been recognized as an emerging tick-borne pathogen of humans in the U.S. and Europe. Transmission of A. phagocytophilum is reported to be by ticks, primarily of the genus Ixodes. While mechanical and transplacental transmission of the type genus organism, A. marginale, occur in addition to tick transmission, these modes of transmission have not been considered for A. phagocytophilum. Recently, we developed a sheep model for studying host-tick-pathogen interactions of the human NY-18 A. phagocytophilum isolate. Sheep were susceptible to infection with this human isolate and served as a source of infection for I. scapularis ticks, but they did not display clinical signs of disease, and the pathogen was not apparent in stained blood smears. In the course of these experiments, one sheep unexpectedly gave birth to a lamb 5 weeks after being experimentally infected by inoculation with the pathogen propagated in HL-60 cells. The lamb was depressed and not feeding and was subsequently euthanized 18 h after birth. Tissues were collected at necropsy for microscopic examination and PCR to confirm A. phagocytophilum infection. At necropsy, the stomach contained colostrum, the spleen was moderately enlarged and thickened with conspicuous lymphoid follicles, and mesenteric lymph nodes were mildly enlarged and contained moderate infiltrates of eosinophils and neutrophils. Blood, spleen, heart, skin and cervical and mesenteric lymph nodes tested positive for A. phagocytophilum by PCR, and sequence analysis confirmed that the lamb was infected with the NY-18 isolate. Transplacental transmission should therefore be considered as a means of A. phagocytophilum transmission and may likely contribute to the epidemiology of tick-borne fever in sheep and other mammals, including humans.

  12. Revisions to Exceptions Applicable to Certain Human Cells, Tissues, and Cellular and Tissue-Based Products. Final rule.

    Science.gov (United States)

    2016-06-22

    : The Food and Drug Administration (FDA or Agency or we) is issuing this final rule to amend certain regulations regarding donor eligibility, including the screening and testing of donors of particular human cells, tissues, and cellular and tissue-based products (HCT/Ps), and related labeling. This final rule is in response to our enhanced understanding in this area and in response to comments from stakeholders regarding the importance of embryos to individuals and couples seeking access to donated embryos.

  13. Genetic variability of vancomycin-resistant Enterococcus faecium and Enterococcus faecalis isolates from humans, chickens, and pigs in Malaysia.

    Science.gov (United States)

    Getachew, Yitbarek; Hassan, Latiffah; Zakaria, Zunita; Abdul Aziz, Saleha

    2013-08-01

    Vancomycin-resistant enterococci (VRE) have been reported to be present in humans, chickens, and pigs in Malaysia. In the present study, representative samples of VRE isolated from these populations were examined for similarities and differences by using the multilocus sequence typing (MLST) method. Housekeeping genes of Enterococcus faecium (n = 14) and Enterococcus faecalis (n = 11) isolates were sequenced and analyzed using the MLST databases eBURST and goeBURST. We found five sequence types (STs) of E. faecium and six STs of E. faecalis existing in Malaysia. Enterococcus faecium isolates belonging to ST203, ST17, ST55, ST79, and ST29 were identified, and E. faecium ST203 was the most common among humans. The MLST profiles of E. faecium from humans in this study were similar to the globally reported nosocomial-related strain lineage belonging to clonal complex 17 (CC17). Isolates from chickens and pigs have few similarities to those from humans, except for one isolate from a chicken, which was identified as ST203. E. faecalis isolates were more diverse and were identified as ST4, ST6, ST87, ST108, ST274, and ST244, which were grouped as specific to the three hosts. E. faecalis, belonging to the high-risk CC2 and CC87, were detected among isolates from humans. In conclusion, even though one isolate from a chicken was found clonal to that of humans, the MLST analysis of E. faecium and E. faecalis supports the findings of others who suggest VRE to be predominantly host specific and that clinically important strains are found mainly among humans. The infrequent detection of a human VRE clone in a chicken may in fact suggest a reverse transmission of VRE from humans to animals.

  14. Toxigenic Corynebacterium ulcerans isolated from a wild bird (ural owl) and its feed (shrew-moles): comparison of molecular types with human isolates.

    Science.gov (United States)

    Katsukawa, Chihiro; Umeda, Kaoru; Inamori, Ikuko; Kosono, Yuka; Tanigawa, Tomokazu; Komiya, Takako; Iwaki, Masaaki; Yamamoto, Akihiko; Nakatsu, Susumu

    2016-03-22

    Corynebacterium ulcerans is a pathogen causing diphtheria-like illness to humans. In contrast to diphtheria by Corynebacterium diphtheriae circulating mostly among humans, C. ulcerans infection is zoonotic. The present study aimed to clarify how a zoonotic pathogen C. ulcerans circulates among wild birds and animals. By screening 380 birds, a single strain of toxigenic C. ulcerans was isolated from a carnivorous bird, ural owl (Strix uralensis). The bacterium was also isolated from two individuals of Japanese shrew-mole (Urotrichus talpoides), a food preference of the owl. Analysis by ribotyping showed that the owl and mole isolates were classified in a group, suggesting that C. ulcerans can be transmissible among wild birds and their prey animals. Moreover, our isolates were found to belong to a group of previously reported C. ulcerans isolates from dogs and a cat, which are known to serve as sources for human infection. The findings suggest that the shrew-mole may be a potential reservoir of a zoonotic pathogen C. ulcerans.

  15. Complete Genome Sequences of Two Methicillin-Sensitive Staphylococcus aureus Isolates Representing a Population Subset Highly Prevalent in Human Colonization

    Science.gov (United States)

    Weber, Robert E.; Layer, Franziska; Fuchs, Stephan; Bender, Jennifer K.; Fiedler, Stefan; Werner, Guido

    2016-01-01

    Here, we report the high-quality draft genome sequences of two methicillin-susceptible Staphylococcus aureus isolates, 08-02119 and 08-02300. Belonging to sequence type 582 (ST582) and ST7, both isolates are representatives of clonal lineages often associated with asymptomatic colonization of humans. PMID:27469954

  16. Frederiksenia canicola gen. nov., sp. nov. isolated from dogs and human dog-bite wounds

    DEFF Research Database (Denmark)

    Korczak, Bożena M.; Bisgaard, Magne; Christensen, Henrik

    2014-01-01

    Polyphasic analysis was done on 24 strains of Bisgaard taxon 16 from five European countries and mainly isolated from dogs and human dog-bite wounds. The isolates represented a phenotypically and genetically homogenous group within the family Pasteurellaceae. Their phenotypic profile was similar...

  17. Multiple-locus variable-number tandem-repeat analysis (MLVA) genotyping of human Brucella isolates in Malaysia.

    Science.gov (United States)

    Tay, Bee Yong; Ahmad, Norazah; Hashim, Rohaidah; Mohamed Zahidi, Jama'ayah; Thong, Kwai Lin; Koh, Xiu Pei; Mohd Noor, Azura

    2015-06-02

    Brucellosis is one of the most common zoonotic diseases worldwide. It can cause acute febrile illness in human and is a major health problem. Studies in human brucellosis in Malaysia is limited and so far no genotyping studies has been done on Brucella isolates. The aim of the study was to determine the genetic diversity among Brucella species isolated from human brucellosis, obtained over a 6-year period (2009-2014). In this study, the genotypic characteristics of 43 human Brucella melitensis isolates were analysed using multiple-locus variable-number tandem-repeat analysis (MLVA) which consisted of eight minisatellite loci (panel 1) and eight microsatellite loci; panels 2A (3 microsatellite loci) and panel 2B (5 microsatellite loci). Two human Brucella suis isolates were also investigated using the MLVA assay. Using panel 1 (MLVA8), two genotypes namely genotype 43 and 44 were obtained from the 43 B. melitensis isolates. Using the combination of panels 1 and 2A loci (MLVA11), two genotypes were obtained while using the complete panels 1, 2A and 2B, nine genotypes were obtained. The polymorphisms in using the complete panels (MLVA16) were observed in three loci from panel 2B, which showed a diversity index higher than 0.17. All B. melitensis isolates were closely related to the East Mediterranean group. For B. suis isolates, only genotype 6 and genotype 33 were obtained using panel 1 and MLVA11 respectively. In conclusion, the results of the present study showed a low genetic diversity among B. melitensis and B. suis isolates from human patients. Based on the MLVA16 assay, B. melitensis belonging to the East Mediterranean group is responsible for the vast majority of Brucella infections in our Malaysian patients. To our knowledge, this is the first genotyping study of human Brucella isolates in Malaysia.

  18. Molecular typing and genetic characterization of Mycobacterium avium subsp. hominissuis isolates from humans and swine in Japan.

    Science.gov (United States)

    Adachi, Takashi; Ichikawa, Kazuya; Inagaki, Takayuki; Moriyama, Makoto; Nakagawa, Taku; Ogawa, Kenji; Hasegawa, Yoshinori; Yagi, Tetsuya

    2016-11-01

    Mycobacterium avium subsp. hominissuis (MAH) causes disease in both humans and swine; however, the genetic variations in MAH isolates are unclear. The aim of this study was to elucidate the genetic variations in MAH isolates from humans and swine in Japan. We analysed the 16S-23S rDNA internal transcribed spacer (ITS) sequence and variable number of tandem repeats (VNTRs) using the Mycobacterium avium tandem repeat loci, prevalence of ISMav6 and clarithromycin resistance for MAH isolates from patients with pulmonary MAC (pMAC) disease (n=69), and HIV-seropositive and blood culture-positive (HIV-MAC) patients (n=28) and swine (n=23). In the minimum spanning tree based on VNTR analysis, swine MAC isolates belonged to a cluster distinguishable from that of human pMAC isolates. Isolates from HIV-MAC were scattered throughout both clusters. The three major distinct sequevars, Mav-A, Mav-B and Mav-F, were determined according to 16S-23S rDNA ITS sequence analysis in addition to three new sequevars, Mav-Q, Mav-R and Mav-S. Mav-A and Mav-F comprised the majority of human pMAC strains; in contrast, Mav-B predominated in swine isolates. Distribution of ITS sequevars in the minimum spanning tree based on VNTR analysis showed similar clusters of isolates from different origins, i.e. human pMAC, HIV-MAC and swine. These results, together with ISMav6 possession and clarithromycin resistance, revealed the genetic diversity of MAH strains recovered from humans and swine. Molecular epidemiology and genetic characterization in the present study showed the distinctive genetic evolutionary lineage of MAH strains isolated from human pMAC diseases and swine.

  19. Full genome sequences and molecular characterization of tick-borne encephalitis virus strains isolated from human patients.

    Science.gov (United States)

    Formanová, Petra; Černý, Jiří; Bolfíková, Barbora Černá; Valdés, James J; Kozlova, Irina; Dzhioev, Yuri; Růžek, Daniel

    2015-02-01

    Tick-borne encephalitis virus (TBEV) causes tick-borne encephalitis (TBE), one of the most important human neuroinfections across Eurasia. Up to date, only three full genome sequences of human European TBEV isolates are available, mostly due to difficulties with isolation of the virus from human patients. Here we present full genome characterization of an additional five low-passage TBEV strains isolated from human patients with severe forms of TBE. These strains were isolated in 1953 within Central Bohemia in the former Czechoslovakia, and belong to the historically oldest human TBEV isolates in Europe. We demonstrate here that all analyzed isolates are distantly phylogenetically related, indicating that the emergence of TBE in Central Europe was not caused by one predominant strain, but rather a pool of distantly related TBEV strains. Nucleotide identity between individual sequenced TBEV strains ranged from 97.5% to 99.6% and all strains shared large deletions in the 3' non-coding region, which has been recently suggested to be an important determinant of virulence. The number of unique amino acid substitutions varied from 3 to 9 in individual isolates, but no characteristic amino acid substitution typical exclusively for all human TBEV isolates was identified when compared to the isolates from ticks. We did, however, correlate that the exploration of the TBEV envelope glycoprotein by specific antibodies were in close proximity to these unique amino acid substitutions. Taken together, we report here the largest number of patient-derived European TBEV full genome sequences to date and provide a platform for further studies on evolution of TBEV since the first emergence of human TBE in Europe.

  20. MOLECULAR CHARACTERIZATION BY USING RANDOM AMPLIFIED POLYMORPHIC DNA (RAPD ANALYSIS OF SALMONELLA ENTERITIDIS ISOLATES RECOVERED FROM AVIAN AND HUMAN SOURCES

    Directory of Open Access Journals (Sweden)

    E. YAQOOB, I. HUSSAIN AND S. U. RAHMAN

    2007-04-01

    Full Text Available Random amplified polymorphic DNA (RAPD analysis was applied for molecular characterization of five Salmonella enteritidis strains from different avian sources and human cases of infection. A total of 16 primers were used and only five primers showed good discriminatory power for all five isolates. Dendrogram showed a common lineage among all five isolates. There was a close genetic relationship among isolates of eggs and human sources, while there was less pronounced homology among isolates of broiler meat and human sources. On the basis of results we have found that an endemic strain of S. enteritidis is prevalent between the poultry derived food and humans which gives us an insight to genetic diversity of S. enteritidis from these sources.

  1. Application of fluorescent amplified fragment length polymorphism for comparison of human and animal isolates of Yersinia enterocolitica

    DEFF Research Database (Denmark)

    Fearnley, C.; On, S.L.W.; Kokotovic, Branko

    2005-01-01

    An amplified fragment length polymorphism (AFLP) method, developed to genotype Yersinia enterocolitica, has been used to investigate 70 representative strains isolated from humans, pigs, sheep, and cattle in the United Kingdom. AFLP primarily distinguished Y enterocolitica strains according...

  2. Isolation and characterization of cytotoxic effector cells and antibody producing cells from human intestine.

    Science.gov (United States)

    MacDermott, R P

    1985-01-01

    We have examined the ability of intestinal and peripheral blood mononuclear cells isolated from patients with inflammatory bowel disease to mediate killing against cell line targets in spontaneous, antibody-dependent, lectin-induced, and interferon-induced cell-mediated cytotoxicity assays, as well as responsiveness in the allogeneic mixed leukocyte reaction, and effector capabilities in cell-mediated lympholysis. IMC were poor mediators of spontaneous or antibody-dependent cellular cytotoxicity with cell line cells as targets (in comparison to normal PBMC, but were capable of killing antibody coated chicken red blood cells. Although IMC were capable of responding to allogeneic cell surface antigens in the mixed leukocyte reaction, they did not exhibit effector function in cell-mediated lympholysis. Mitogenic lectins induced cell-mediated cytotoxicity by isolated intestinal mononuclear cells from controls and patients. HFIF induces cytotoxicity by control but not inflammatory bowel disease intestinal cells. Pokeweed mitogen was the lectin which induced the greatest amount of killing against human cell line targets. We therefore speculate that exogenous agents, or endogenous factors released during viral infection, could play a role in inducing cell mediated cytotoxic damage to the intestine in inflammatory bowel disease patients. In addition, the functional differences between IMC and PBMC indicate that intestinal MNC may have unique cell capabilities which must be better understood prior to the delineation of immunopathologic events in solid organ tissues. We have also examined the secretion of IgA, IgM, and IgG by isolated human IMC, human bone marrow MNC from rib specimens, and PBMC from patients with CD, UC, SLE, or Henoch-Schoenlein purpura (HSP). Control IMC exhibited high spontaneous secretion of IgA, while intestinal MNC from UC and CD patients exhibited only modest increases in IgA secretion. PBMC from patients with CD, UC, SLE, or HSP exhibited markedly

  3. Characterization of Naegleria fowleri strains isolated from human cases of primary amoebic meningoencephalitis in Mexico.

    Science.gov (United States)

    Cervantes-Sandoval, Isaac; de Serrano-Luna, José Jesús; Tapia-Malagón, José Luis; Pacheco-Yépez, Judith; Silva-Olivares, Angélica; Galindo-Gómez, Silvia; Tsutsumi, Victor; Shibayama, Mineko

    2007-01-01

    The protozoon Naegleria fowleri (N. fowleri) is a free-living amoeba that produces primary amoebic meningoencephalitis (PAM), which is an acute and frequently fatal infection of the central nervous system. We characterized the strains of N. fowleri isolated from the cerebrospinal fluid (CSF) of two cases presented in northwestern Mexico. The strains were isolated and cultured in 2% bactocasitone medium. Enflagellation assays, ultrastructural analysis, protein and protease electrophoresis patterns, and PCR were performed as confirmatory tests. Virulence tests were done using in Balb/c mice. Light microscopy analysis of brain tissue showed amoebae with abundant inflammatory reaction and extensive necrotic and hemorrhagic areas. The enflagellation assay was positive and the electron microscopy showed trophozoites with morphologic features typical of the genus. Protein and protease profiles of the isolated strains were identical to the reference strain. Finally, a 1500-bp PCR product was found in all three strains. Based on all the analyses performed, we concluded that the etiologic agent of both PAM cases was N. fowleri. The need for better epidemiological information and educational programs about basic clinical and pathological aspects of free-living amoebae provided by the health authorities are emphasized.

  4. Salmonella L-forms: formation in human bile in vitro and isolation culture from patients' gallbladder samples by a non-high osmotic isolation technique.

    Science.gov (United States)

    Wang, D N; Wu, W J; Wang, T; Pan, Y Z; Tang, K L; She, X L; Ding, W J; Wang, H

    2015-05-01

    Bacterial L-forms have always been considered as osmotic-pressure-sensitive cell-wall-deficient bacteria and isolation culture of L-forms must use media with high osmotic pressure. However, isolation culture of stable L-forms formed in humans and animals is very difficult because they have adapted to the physiological osmotic pressure condition of the host. We use a non-high osmotic isolation technique to isolate stable L-forms of Salmonella Typhi and Salmonella Paratyphi A from bile-inducer cultures in vitro and from patients' gallbladder specimens. Multiplex PCR assay for Salmonella-specific genes and nucleotide sequencing are used to identify the Salmonella L-forms in stable L-form isolates. Using this method, we confirmed that Salmonella Paratyphi A and Salmonella Typhi cannot be isolated from bile-inducer cultures cultured for 6 h or 48 h, but the L-forms can be isolated from 1 h to 45 days. In the 524 gallbladder samples, the positive rate for bacterial forms was 19.7% and the positive rate for Salmonella spp. was 0.6% by routine bacteriological methods. The positive rate for bacterial L-forms was 75.4% using non-high osmotic isolation culture. In the L-form isolates, the positive rate of Salmonella invA gene was 3.1%. In these invA-positive L-form isolates, four were positive for the invA and flic-d genes of Salmonella Typhi, and ten were positive for the invA and flic-a genes of Salmonella Paratyphi A.

  5. Isolation of Klebsiella terrigena from human feces: biochemical reactions, capsule types, and antibiotic sensitivity.

    Science.gov (United States)

    Podschun, R

    1991-04-01

    Colonization of the human intestinal tract by a newly proposed species, K. terrigena, was investigated. 5377 different stool specimens from healthy persons (food handlers) yielded 50 isolates (0.9%). Biochemically, low frequencies in the degradation of urea, dulcitol, and utilization of citrate at 37 degrees C were found when compared to K. pneumoniae. At 30 degrees C, urea hydrolysis was observed twice as often as at 37 degrees C. Apart from ampicillin, K. terrigena was susceptible to 12 other antimicrobial drugs tested. Multiple drug resistance was rare, few isolates being resistant against 2-4 antibiotic agents. Capsule typing revealed 30 different serotypes, K 70 and K 14 were the most frequent. Six strains expressed capsule types K 2 and K 5, which have been reported to be associated with virulence in K. pneumoniae. A possible pathogenic role of K. terrigena is discussed.

  6. Obtaining freshly isolated and cultured mesenchymal stem cells from human adipose tissue.

    Science.gov (United States)

    Boquest, Andrew C; Collas, Philippe

    2012-01-01

    The stromal compartment of adipose tissue harbors mesenchymal stem cells (MSCs) (also called stromal stem cells) that display extensive proliferative capacity and multilineage differentiation potential. Such cells offer a practical avenue of generating patient-matched tissue for use in regenerative medicine. It is relatively easy to isolate these cells from adipose tissue in large enough quantities (tens of millions) to allow for their clinical use in a native, uncultured form. Alternatively, MSCs from adipose tissue can be expanded and differentiated into the desired tissue type in vitro using straightforward cell culture techniques. In this chapter, we outline procedures for isolating large numbers of highly purified MSCs from human adipose tissue in their native, uncultured form and methods for their subsequent expansion and differentiation in vitro.

  7. Isolation and characterization of human cerebellum cDNAs containing polymorphic CAG trinucleotide repeats

    Energy Technology Data Exchange (ETDEWEB)

    Igarashi, S.; Onodera, O.; Tanaka, H. [Niigata Univ. (Japan)] [and others

    1994-09-01

    It has been discovered that neurologic diseases such as X linked spinal and bulbar muscular atrophy, Huntington`s disease, spinocerebellar ataxia type 1 (SCA1), and dentatorubral-pallidoluysian atrophy (DRPLA) are caused by unstable expansions of CAG repeats, which shed a light on a new mechanism of human hereditary diseases. The genetic anticipation, a common genetic feature in these diseases, can be explained by the trinucleotide repeat expansions, and an inverse correlation between the ages of onset and the numbers of trinucleotide repeats is demonstrated in these diseases. Furthermore, there have been diseases such as spinocerebellar ataxia 2 (SCA2) and Machado-Joseph disease showing similar genetic anticipation, which suggests that their causative mutations are unstable expansions of trinucleotide repeats. To identify candidate genes for neurodegenerative diseases which are expressed in human cerebellum and contain CAG repeats, we screened a human cerebellum cDNA library with an oligonucleotide (CAG){sub 10}, labelled with [{gamma}{sup 32}P]ATP. Out of 78 clones we have isolated, 43 clones were partially sequenced and 31 clones were shown to contain CAG or CTG tinucleotide repeats. From homology searches, 12 of the 59 clones were identified to contain known sequences including human MAR/SAR DNA binding protein, human glial fibrillary acidic protein, human myelin transcription factor 1, human neuronal growth protein 43 and human myocyte-specific enhancer 2. From 6 clones out of the 43 novel genes, we were able to develop primer pairs flanking CAG repeats and determined chromosomal localizations with human and rodent hybrid mapping panels. These CAG repeats were shown to be polymorphic and mapped to 1, 15, 17 and 18. These novel cDNAs will be useful as candidate genes for hereditary neurologic diseases showing genetic anticipation.

  8. Isolation of cellular lipid droplets: two purification techniques starting from yeast cells and human placentas.

    Science.gov (United States)

    Mannik, Jaana; Meyers, Alex; Dalhaimer, Paul

    2014-04-01

    Lipid droplets are dynamic organelles that can be found in most eukaryotic and certain prokaryotic cells. Structurally, the droplets consist of a core of neutral lipids surrounded by a phospholipid monolayer. One of the most useful techniques in determining the cellular roles of droplets has been proteomic identification of bound proteins, which can be isolated along with the droplets. Here, two methods are described to isolate lipid droplets and their bound proteins from two wide-ranging eukaryotes: fission yeast and human placental villous cells. Although both techniques have differences, the main method-- density gradient centrifugation--is shared by both preparations. This shows the wide applicability of the presented droplet isolation techniques. In the first protocol, yeast cells are converted into spheroplasts by enzymatic digestion of their cell walls. The resulting spheroplasts are then gently lysed in a loose-fitting homogenizer. Ficoll is added to the lysate to provide a density gradient, and the mixture is centrifuged three times. After the first spin, the lipid droplets are localized to the white-colored floating layer of the centrifuge tubes along with the endoplasmic reticulum (ER), the plasma membrane, and vacuoles. Two subsequent spins are used to remove these other three organelles. The result is a layer that has only droplets and bound proteins. In the second protocol, placental villous cells are isolated from human term placentas by enzymatic digestion with trypsin and DNase I. The cells are homogenized in a loose-fitting homogenizer. Low-speed and medium-speed centrifugation steps are used to remove unbroken cells, cellular debris, nuclei, and mitochondria. Sucrose is added to the homogenate to provide a density gradient and the mixture is centrifuged to separate the lipid droplets from the other cellular fractions. The purity of the lipid droplets in both protocols is confirmed by Western Blot analysis. The droplet fractions from both preps

  9. Semisolid liver infusion tryptose supplemented with human urine allows growth and isolation of Trypanosoma cruzi and Trypanosoma rangeli clonal lineages

    OpenAIRE

    2016-01-01

    Abstract: INTRODUCTION This work shows that 3% (v/v) human urine (HU) in semisolid Liver Infusion Tryptose (SSL) medium favors the growth of Trypanosoma cruzi and T. rangeli. METHODS Parasites were plated as individual or mixed strains on SSL medium and on SSL medium with 3% human urine (SSL-HU). Isolate DNA was analyzed using polymerase chain reaction (PCR) and pulsed-field gel electrophoresis (PFGE). RESULTS SSL-HU medium improved clone isolation. PCR revealed that T. cruzi strains pr...

  10. The isolated perfused human skin flap model: A missing link in skin penetration studies?

    Science.gov (United States)

    Ternullo, Selenia; de Weerd, Louis; Flaten, Gøril Eide; Holsæter, Ann Mari; Škalko-Basnet, Nataša

    2017-01-01

    Development of effective (trans)dermal drug delivery systems requires reliable skin models to evaluate skin drug penetration. The isolated perfused human skin flap remains metabolically active tissue for up to 6h during in vitro perfusion. We introduce the isolated perfused human skin flap as a close-to-in vivo skin penetration model. To validate the model's ability to evaluate skin drug penetration the solutions of a hydrophilic (calcein) and a lipophilic (rhodamine) fluorescence marker were applied. The skin flaps were perfused with modified Krebs-Henseleit buffer (pH7.4). Infrared technology was used to monitor perfusion and to select a well-perfused skin area for administration of the markers. Flap perfusion and physiological parameters were maintained constant during the 6h experiments and the amount of markers in the perfusate was determined. Calcein was detected in the perfusate, whereas rhodamine was not detectable. Confocal images of skin cross-sections shoved that calcein was uniformly distributed through the skin, whereas rhodamine accumulated in the stratum corneum. For comparison, the penetration of both markers was evaluated on ex vivo human skin, pig skin and cellophane membrane. The proposed perfused flap model enabled us to distinguish between the penetrations of the two markers and could be a promising close-to-in vivo tool in skin penetration studies and optimization of formulations destined for skin administration.

  11. Human Enteroviruses isolated during acute flaccid paralysis surveillance in Ghana: implications for the post eradication era.

    Science.gov (United States)

    Odoom, John Kofi; Obodai, Evangeline; Barnor, Jacob Samson; Ashun, Miriam; Arthur-Quarm, Jacob; Osei-Kwasi, Mubarak

    2012-01-01

    Surveillance of acute flaccid surveillance (AFP) has been used world-wide to monitor the control and eradication of circulating wild polioviruses. The Polio Laboratory since its accreditation in 1996 has supported the Disease Surveillance Department for AFP surveillance. This study aims to isolate and characterize human enteroviruses from patients with AFP in Ghana. Stool suspension was prepared from 308 samples received in 2009 from the surveillance activities throughout the country and inoculated on both RD and L20B cell lines. Isolates that showed growth on L20B were selected for real-time RT-PCR using degenerate and non-degenerate primers and probes. RD isolates were however characterized by microneutralisation technique with antisera pools from RIVM, The Netherlands and viruses that were untypable subjected to neutralization assay using antibodies specific for E71. Of the 308 samples processed, 17 (5.5%) grew on both L20B and RD cells while 32 (10.4%) grew on RD only. All 28 isolates from L20B were characterized by rRT-PCR as Sabin-like polioviruses. No wild poliovirus or VDPV was found. However from the microneutralisation assay, six different enteroviruses were characterized. Among these, Coxsackie B viruses were most predominant followed by Echovirus. Three children from whom non-polio enteroviruses were isolated had residual paralysis while one child with VAPP found. The non-polio enteroviruses circulated throughout the country with the majority (20.7%) from Ashanti region. This study showed the absence of wild or vaccine-derived poliovirus circulation in the country. However, the detection of three non-polio enteroviruses and one Sabin-like poliovirus with residual paralysis call for continuous surveillance even in the post polio eradication era.

  12. High-Level Ciprofloxacin-Resistant Campylobacter jejuni Isolates Circulating in Humans and Animals in Incheon, Republic of Korea.

    Science.gov (United States)

    Kim, J S; Lee, M Y; Kim, S J; Jeon, S-E; Cha, I; Hong, S; Chung, G T; Huh, M-J; Kang, Y-H; Yoo, C-K; Kim, J

    2016-11-01

    Campylobacter jejuni is one of the major causative pathogens of outbreaks or sporadic cases of diarrhoeal diseases worldwide. In this study, we compared the phenotypic and genetic characteristics of C. jejuni isolates of human and food-producing animal origins in Korea and examined the genetic relatedness between these two groups of isolates. Regardless of isolation source, all C. jejuni isolates harboured four virulence genes, cadF, cdtB, ciaB and racR, whereas the wlaN and virB11 genes were more frequently observed in human isolates. Antimicrobial susceptibility testing showed that the majority of C. jejuni isolates displayed high-level resistance to fluoroquinolone (95.2%) or tetracycline (76.2%) antibiotics, and 12.4% of isolates exhibited multidrug resistance (more than three classes of antibiotics tested). Pulsed-field gel electrophoresis (PFGE) of all Campylobacter isolates revealed 51 different SmaI-PFGE patterns and six major clusters containing both human and animal isolates. These results indicate that genetically diverse strains of C. jejuni with antimicrobial drug-resistance and virulence properties have prevailed in Incheon. Nevertheless, some particular populations continue to circulate within the community, providing the evidence for an epidemiological link of C. jejuni infections between humans and food-producing animals. Therefore, the continued monitoring and surveillance of C. jejuni isolates of human and food-producing animal origins are required for public health and food safety. © 2016 Blackwell Verlag GmbH.

  13. Featured Article: Isolation, characterization, and cultivation of human hepatocytes and non-parenchymal liver cells

    Science.gov (United States)

    Pfeiffer, Elisa; Kegel, Victoria; Zeilinger, Katrin; Hengstler, Jan G; Nüssler, Andreas K; Seehofer, Daniel

    2015-01-01

    Primary human hepatocytes (PHH) are considered to be the gold standard for in vitro testing of xenobiotic metabolism and hepatotoxicity. However, PHH cultivation in 2D mono-cultures leads to dedifferentiation and a loss of function. It is well known that hepatic non-parenchymal cells (NPC), such as Kupffer cells (KC), liver endothelial cells (LEC), and hepatic stellate cells (HSC), play a central role in the maintenance of PHH functions. The aims of the present study were to establish a protocol for the simultaneous isolation of human PHH and NPC from the same tissue specimen and to test their suitability for in vitro co-culture. Human PHH and NPC were isolated from tissue obtained by partial liver resection by a two-step EDTA/collagenase perfusion technique. The obtained cell fractions were purified by Percoll density gradient centrifugation. KC, LEC, and HSC contained in the NPC fraction were separated using specific adherence properties and magnetic activated cell sorting (MACS®). Identified NPC revealed a yield of 1.9 × 106 KC, 2.7 × 105 LEC and 4.7 × 105 HSC per gram liver tissue, showing viabilities >90%. Characterization of these NPC showed that all populations went through an activation process, which influenced the cell fate. The activation of KC strongly depended on the tissue quality and donor anamnesis. KC became activated in culture in association with a loss of viability within 4–5 days. LEC lost specific features during culture, while HSC went through a transformation process into myofibroblasts. The testing of different culture conditions for HSC demonstrated that they can attenuate, but not prevent dedifferentiation in vitro. In conclusion, the method described allows the isolation and separation of PHH and NPC in high quality and quantity from the same donor. PMID:25394621

  14. Isolation and transcriptome analyses of human erythroid progenitors: BFU-E and CFU-E.

    Science.gov (United States)

    Li, Jie; Hale, John; Bhagia, Pooja; Xue, Fumin; Chen, Lixiang; Jaffray, Julie; Yan, Hongxia; Lane, Joseph; Gallagher, Patrick G; Mohandas, Narla; Liu, Jing; An, Xiuli

    2014-12-04

    Burst-forming unit-erythroid (BFU-E) and colony-forming unit-erythroid (CFU-E) cells are erythroid progenitors traditionally defined by colony assays. We developed a flow cytometry-based strategy for isolating human BFU-E and CFU-E cells based on the changes in expression of cell surface markers during in vitro erythroid cell culture. BFU-E and CFU-E are characterized by CD45(+)GPA(-)IL-3R(-)CD34(+)CD36(-)CD71(low) and CD45(+)GPA(-)IL-3R(-)CD34(-)CD36(+)CD71(high) phenotypes, respectively. Colony assays validated phenotypic assignment giving rise to BFU-E and CFU-E colonies, both at a purity of ∼90%. The BFU-E colony forming ability of CD45(+)GPA(-)IL-3R(-)CD34(+)CD36(-)CD71(low) cells required stem cell factor and erythropoietin, while the CFU-E colony forming ability of CD45(+)GPA(-)IL-3R(-)CD34(-)CD36(+)CD71(high) cells required only erythropoietin. Bioinformatic analysis of the RNA-sequencing data revealed unique transcriptomes at each differentiation stage. The sorting strategy was validated in uncultured primary cells isolated from bone marrow, cord blood, and peripheral blood, indicating that marker expression is not an artifact of in vitro cell culture, but represents an in vivo characteristic of erythroid progenitor populations. The ability to isolate highly pure human BFU-E and CFU-E progenitors will enable detailed cellular and molecular characterization of these distinct progenitor populations and define their contribution to disordered erythropoiesis in inherited and acquired hematologic disease. Our data provides an important resource for future studies of human erythropoiesis.

  15. Clonal relationship between human and avian ciprofloxacin-resistant Escherichia coli isolates in North-Eastern Algeria.

    Science.gov (United States)

    Agabou, A; Lezzar, N; Ouchenane, Z; Khemissi, S; Satta, D; Sotto, A; Lavigne, J-P; Pantel, A

    2016-02-01

    The objectives of this study were to determine rates, patterns, and mechanisms of antibiotic resistance, and to assess connections between chicken commensal, human commensal, and pathogenic ciprofloxacin-resistant Escherichia coli isolates. All E. coli isolates collected from chickens, their farmers, and patients in the Constantine region (North-east Algeria) were analyzed for bla and plasmid-mediated quinolone resistance (PMQR) gene contents, phylogroups, Rep-PCR profiles, and multilocus sequence types. A high prevalence of resistance to fluoroquinolones (51.4 % to ciprofloxacin) was recorded in avian isolates. Of these, 22.2 % carried the aac(6')-Ib-cr gene, whereas lower resistance levels to these antibiotics were recorded in chicken farmers' isolates. None of the commensal isolates harbored the qnr, qepA, or oqxAB genes. One human pathogenic isolate was ertapenem-resistant and harbored the bla OXA-48 gene, 84 showed an extended-spectrum β-lactamase phenotype, with bla CTX-M-15 gene prevalent in 87.2 % of them. Seventy isolates were resistant to fluoroquinolones, with aac(6')-Ib-cr present in 72.8 %, qnrB in 5.7 %, and qnrS in 10 %. Three Rep-PCR profiles were common to chicken commensal and human pathogenic isolates (phylogroups D and B1; ST21, ST48, and ST471 respectively); one was found in both chicken and chicken-farmer commensal strains (D; ST108), while another profile was identified in a chicken-farmer commensal strain and a human pathogenic one (B1; ST19). These findings suggest clonal and epidemiologic links between chicken and human ciprofloxacin-resistant E. coli isolates and the important role that poultry may play in the epidemiology of human E. coli infections in the Constantine region.

  16. W-Tagging/B-Veto in SUSY search for squarks and gluinos in final states with missing transverse energy, jets, and one isolated lepton at ATLAS

    CERN Document Server

    Reyer, Max

    2015-01-01

    This report recapitulates my stay at CERN as a Summer Student in a 13-week period from June 29 to September 25 in 2015. During that time, I was part of the CERN ATLAS Supersymmetry working group and working on a project exploiting W-tagging and B-veto techniques for the search for squarks and gluinos in final states with missing transverse energy, jets, and one isolated lepton. In this report, I will summarize my project and present results and conclusions. Apart from that, I will also write about my experience with the Summer Student lectures, the social aspects and the CERN Summer School in general.

  17. Searches for squarks and gluinos in final states with an isolated charged lepton, jets, and missing transverse momentum with the ATLAS detector

    CERN Document Server

    Chen, Shion; The ATLAS collaboration

    2017-01-01

    Draft slides for the conference Epiphany 2017 (8.Jan- 12. Jan Cracow Poland). Expected slot is 10min+2min. Abstract for the talk is as follow: Despite the absence of experimental evidence, weak scale supersymmetry remains one of the best motivated and studied Standard Model extensions. This talk discusses recent ATLAS results on searches for supersymmetric squarks and gluinos in final states with an isolated electron or muon, multiple jets and large missing transverse momentum using proton—proton data collected by the ATLAS detector at a centre-of-mass energy of 13 TeV.

  18. Isolation, expression, and chromosomal localization of the human mitochondrial capsule selenoprotein gene (MCSP)

    Energy Technology Data Exchange (ETDEWEB)

    Aho, Hanne; Schwemmer, M.; Tessmann, D.; Murphy, D. [Institut fuer Humangenetik der Universitaet, Goettingen (Germany)] [and others

    1996-03-01

    The mitochondrial capsule selenoprotein (MCS) (HGMW-approved symbol MCSP) is one of three proteins that are important for the maintenance and stabilization of the crescent structure of the sperm mitochondria. We describe here the isolation of a cDNA, the exon-intron organization, the expression, and the chromosomal localization of the human MCS gene. Nucleotide sequence analysis of the human and mouse MCS cDNAs reveals that the 5{prime}- and 3{prime}-untranslated sequences are more conserved (71%) than the coding sequences (59%). The open reading frame encodes a 116-amino-acid protein and lacks the UGA codons, which have been reported to encode the selenocysteines in the N-terminal of the deduced mouse protein. The deduced human protein shows a low degree of amino acid sequence identity to the mouse protein. The deduced human protein shows a low degree of amino acid sequence identity to the mouse protein (39%). The most striking homology lies in the dicysteine motifs. Northern and Southern zooblot analyses reveal that the MCS gene in human, baboon, and bovine is more conserved than its counterparts in mouse and rat. The single intron in the human MCS gene is approximately 6 kb and interrupts the 5{prime}-untranslated region at a position equivalent to that in the mouse and rat genes. Northern blot and in situ hybridization experiments demonstrate that the expression of the human MCS gene is restricted to haploid spermatids. The human gene was assigned to q21 of chromosome 1. 30 refs., 9 figs.

  19. A new sialyloligosaccharide from human milk: isolation and characterization using anti-oligosaccharide antibodies.

    Science.gov (United States)

    Prieto, P A; Smith, D F

    1984-03-01

    A previously undescribed sialyloligosaccharide has been isolated from human milk using a specific anti-sialyloligosaccharide antibody. Structural studies of the radiolabeled oligosaccharide by enzyme degradation and binding by specific anti-oligosaccharide sera are consistent with the following structure: (sequence in text) The oligosaccharide is present only in milk from donors who secrete A, B, or H blood group substances; this is consistent with the requirement of at least one copy of the Se (Secretor) gene necessary for the synthesis of oligosaccharides with Fuc alpha 1-2Gal . . . linkages.

  20. A new sialyloligosaccharide from human milk: isolation and characterization using anti-oligosaccharide antibodies

    Energy Technology Data Exchange (ETDEWEB)

    Prieto, P.A.; Smith, D.F.

    1984-03-01

    A previously undescribed sialyloligosaccharide has been isolated from human milk using a specific anti-sialyloligosaccharide antibody. Structural studies of the radiolabeled oligosaccharide by enzyme degradation and binding by specific anti-oligosaccharide sera are consistent with the following structure: (sequence in text) The oligosaccharide is present only in milk from donors who secrete A, B, or H blood group substances; this is consistent with the requirement of at least one copy of the Se (Secretor) gene necessary for the synthesis of oligosaccharides with Fuc alpha 1-2Gal . . . linkages.

  1. Carbapenem resistance in a human clinical isolate identified to be closely related to Acinetobacter indicus.

    Science.gov (United States)

    Bonnin, Rémy A; Poirel, Laurent; van der Reijden, Tanny J K; Dijkshoorn, Lenie; Lescat, Mathilde; Nordmann, Patrice

    2014-10-01

    Here we report a case of carbapenem resistance in a human clinical isolate that was found to be closely related to the newly described environmental species Acinetobacter indicus. This strain harboured the blaOXA-23 carbapenemase gene located on a conjugative plasmid. Partial sequencing of 16S rDNA and rpoB genes, together with matrix-assisted laser desorption/ionisation time-of-flight (MALDI-TOF) analysis, showed that this strain was distantly related to the Acinetobacter baumannii-calcoaceticus complex and was closely related to A. indicus.

  2. Isolation of a new herpes virus from human CD4 sup + T cells

    Energy Technology Data Exchange (ETDEWEB)

    Frenkel, N.; Schirmer, E.C.; Wyatt, L.S.; Katsafanas, G.; Roffman, E.; Danovich, R.M. (National Institutes of Health, Rockville, MD (USA)); June, C.H. (Naval Medical Research Institute, Bethesda, MD (USA))

    1990-01-01

    A new human herpes virus has been isolated from CD4{sup +} T cells purified from peripheral blood mononuclear cells of a healthy individual (RK), following incubation of the cells under conditions promoting T-cell activation. The virus could not be recovered from nonactivated cells. Cultures of lymphocytes infected with the RK virus exhibited a cytopathic effect, and electron microscopic analyses revealed a characteristic herpes virus structure. RK virus DNA did not hybridize with large probes derived from herpes simplex virus, Epstein-Barr virus, varicella-zoster virus, and human cytomegalovirus. The genetic relatedness of the RK virus to the recently identified T-lymphotropic human herpes virus 6 (HHV-6) was investigated by restriction enzyme analyses using 21 different enzymes and by blot hydridization analyses using 11 probes derived from two strains of HHV-6 (Z29 and U1102). Whereas the two HHV-6 strains exhibited only limited restriction enzyme polymorphism, cleavage of the RK virus DNA yielded distinct patterns. Of the 11 HHV-6 DNA probes tested, only 6 cross-hybridized with DNA fragments derived from the RK virus. Taken together, the maximal homology amounted to 31 kilobases of the 75 kilobases tested. The authors conclude that the RK virus is distinct from previously characterized human herpesviruses. The authors propose to designate it as the prototype of a new herpes virus, the seventh human herpes virus identified to date.

  3. Occurrence of putative virulence genes in arcobacter species isolated from humans and animals.

    Science.gov (United States)

    Douidah, Laid; de Zutter, Lieven; Baré, Julie; De Vos, Paul; Vandamme, Peter; Vandenberg, Olivier; Van den Abeele, Anne-Marie; Houf, Kurt

    2012-03-01

    Interest in arcobacters in veterinary and human public health has increased since the first report of the isolation of arcobacters from food of animal origin. Since then, studies worldwide have reported the occurrence of arcobacters on food and in food production animals and have highlighted possible transmission, especially of Arcobacter butzleri, to the human population. In humans, arcobacters are associated with enteritis and septicemia. To assess their clinical relevance for humans and animals, evaluation of potential virulence factors is required. However, up to now, little has been known about the mechanisms of pathogenicity. Because of their close phylogenetic affiliation to the food-borne pathogen Campylobacter and their similar clinical manifestations, the presence of nine putative Campylobacter virulence genes (cadF, ciaB, cj1349, hecA, hecB, irgA, mviN, pldA, and tlyA) previously identified in the recent Arcobacter butzleri ATCC 49616 genome sequence was determined in a large set of human and animal Arcobacter butzleri, Arcobacter cryaerophilus, and Arcobacter skirrowii strains after the development of rapid and accurate PCR assays and confirmed by sequencing and dot blot hybridization.

  4. Final report for CCQM-K107: total elements and selenomethionine in human serum

    Science.gov (United States)

    Goenaga Infante, Heidi

    2016-01-01

    Routine tests that measure the concentration of electrolytes in serum are needed for diagnosis and management of renal, endocrine, acid-base, water balance and other conditions such as screening D- and A-vitamin disorders, kidney insufficiency, bone diseases and leukaemia. The diagnostic concentration ranges for many such markers are narrow, requiring reference methods with small uncertainty. Serum concentration of total selenium (Se) is important in health studies but there is increasing interest in the speciation of selenium compounds in clinical samples such as serum and individual Se- Species are bio-indicators of Se status. The last CCQM IAWG key comparison for elements in the clinical area (CCQM-K14: Ca in human serum) was organized in 2003 and the previous key comparison (CCQM-K60) for Se and Se species used a wheat flour sample. Therefore, the CCQM IAWG agreed that CCQM-K107 and a parallel pilot study CCQM-P146 should be carried out. The candidate human serum sample used for both CCQM-K107 and P146 is of high complexity and contains approximately 1000-fold lower concentrations of selenium methionine (SeMet) than those encountered in the CCQM-K60 wheat flour. This significantly broadens the scope and degree of difficulty of earlier measurements in this field. A total of eleven institutes participated in CCQM-K107 (11 participants for total elements and 7 for SeMet). The performance of the majority of the K107 participants for all the measurands was very good, illustrating their ability to obtain accurate results for analytes such as electrolytes at mg kg-1 level, essential elements at µg kg-1 level and selenium species at µg kg-1 level in a complex biological fluid. The range of agreement between participants was within the interval of ± 0.1% for Ca and up to ± 1.8% for Fe. CMC claims based on total elements in this study may include other elements with similar core competencies (e.g. Se, Cu, Zn) in a wide range of biological materials (including liquids

  5. Multiple-locus variable-number tandem-repeat analysis genotyping of human Brucella isolates from Turkey.

    Science.gov (United States)

    Kiliç, Selçuk; Ivanov, Ivan N; Durmaz, Riza; Bayraktar, Mehmet Refik; Ayaslioglu, Ergin; Uyanik, M Hamidullah; Aliskan, Hikmet; Yasar, Ekrem; Bayramoglu, Gülçin; Arslantürk, Ahmet; Vergnaud, Gilles; Kantardjiev, Todor V

    2011-09-01

    A multiple-locus variable-number tandem-repeat analysis (MLVA) was applied to investigate the epidemiological relationship and genetic diversity among 162 human Brucella isolates collected from all geographic regions of Turkey in an 8-year period (2001 to 2008). The isolates were genotyped by using an MLVA assay developed in Orsay, France (MLVA-16(Orsay)) including eight minisatellite (panel 1) and eight microsatellite (panel 2, subdivided into 2A and 2B) markers. Panels 1 and 2A distinguish 14 genotypes; two of these represented 85% of the strains. Panel 2B displayed a very high discriminatory power. Three loci from panel 2B had diversity index values higher than 0.74. MLVA-16(Orsay) yielded 105 genotypes; 73 were represented by a unique isolate, and 32 included two to eight isolates. The isolates from different patients within the same outbreak or from the same patient before first-line therapy and after relapse showed identical genotypes. A number of MLVA genotypes appeared to be partially restricted to some geographic areas and displayed no annual variation, possibly reflecting persistence of genotypes in certain areas for a time span of at least a decade. This study, representing the first molecular typing results of human Brucella isolates from Turkey, indicated that Turkish human Brucella melitensis isolates were most closely related to the neighboring countries' isolates included in the East Mediterranean group.

  6. Multiple-Locus Variable-Number Tandem-Repeat Analysis Genotyping of Human Brucella Isolates from Turkey▿†

    Science.gov (United States)

    Kılıç, Selçuk; Ivanov, Ivan N.; Durmaz, Rıza; Bayraktar, Mehmet Refik; Ayaşlıoğlu, Ergin; Uyanık, M. Hamidullah; Alışkan, Hikmet; Yaşar, Ekrem; Bayramoğlu, Gülçin; Arslantürk, Ahmet; Vergnaud, Gilles; Kantardjiev, Todor V.

    2011-01-01

    A multiple-locus variable-number tandem-repeat analysis (MLVA) was applied to investigate the epidemiological relationship and genetic diversity among 162 human Brucella isolates collected from all geographic regions of Turkey in an 8-year period (2001 to 2008). The isolates were genotyped by using an MLVA assay developed in Orsay, France (MLVA-16Orsay) including eight minisatellite (panel 1) and eight microsatellite (panel 2, subdivided into 2A and 2B) markers. Panels 1 and 2A distinguish 14 genotypes; two of these represented 85% of the strains. Panel 2B displayed a very high discriminatory power. Three loci from panel 2B had diversity index values higher than 0.74. MLVA-16Orsay yielded 105 genotypes; 73 were represented by a unique isolate, and 32 included two to eight isolates. The isolates from different patients within the same outbreak or from the same patient before first-line therapy and after relapse showed identical genotypes. A number of MLVA genotypes appeared to be partially restricted to some geographic areas and displayed no annual variation, possibly reflecting persistence of genotypes in certain areas for a time span of at least a decade. This study, representing the first molecular typing results of human Brucella isolates from Turkey, indicated that Turkish human Brucella melitensis isolates were most closely related to the neighboring countries' isolates included in the East Mediterranean group. PMID:21795514

  7. Multilocus sequence typing of Campylobacter jejuni and Campylobacter coli isolates from poultry, cattle and humans in Nigeria.

    Science.gov (United States)

    Ngulukun, S; Oboegbulem, S; Klein, G

    2016-08-01

    To determine the genetic diversity of Campylobacter jejuni and Campylobacter coli isolates from Nigeria and to identify the association between multilocus sequence types and hosts (poultry, cattle and humans). Isolates were identified using multiplex PCR assays. Multilocus sequence typing (MLST) was used to determine the genetic diversity of 36 Camp. jejuni and 24 Camp. coli strains isolated from poultry, cattle and humans. Of the 36 Camp. jejuni genotyped, 21 sequence types (ST) were found, 9 (43%) were new while of the 24 Camp. coli isolates genotyped, 22 STs were identified with 14 (64%) being new. The most prevalent sequence type was ST1932 followed by ST1036 and ST607 while the prevalent clonal complexes were CC-828, CC-460 and CC-353. Campylobacter isolates from Nigeria were found to be diverse with novel genotypes. There was overlap of CC-828, CC-460 and CC-353 between the poultry, cattle and human isolates. Genetic exchange was also detected in two of the Camp. coli isolates. This study highlights the genetic diversity of Campylobacter strains in Nigeria, demonstrating that Camp. jejuni and Camp. coli isolates are diverse and have both local and global strains. The predominant sequence types and clonal complexes found in this study differ from other countries; this exemplifies that different predominant Campylobacter populations exist between countries. © 2016 The Society for Applied Microbiology.

  8. Characterization of a 2016 Clinical Isolate of Zika Virus in Non-human Primates

    Directory of Open Access Journals (Sweden)

    Xiao-Feng Li

    2016-10-01

    Full Text Available Animal models are critical to understand disease and to develop countermeasures for the ongoing epidemics of Zika virus (ZIKV. Here we report a non-human primate model using a 2016 contemporary clinical isolate of ZIKV. Upon subcutaneous inoculation, rhesus macaques developed fever and viremia, with robust excretion of ZIKV RNA in urine, saliva, and lacrimal fluid. Necropsy of two infected animals revealed that systematic infections involving central nervous system and visceral organs were established at the acute phrase. ZIKV initially targeted the intestinal tracts, spleen, and parotid glands, and retained in spleen and lymph nodes till 10 days post infection. ZIKV-specific immune responses were readily induced in all inoculated animals. The non-human primate model described here provides a valuable platform to study ZIKV pathogenesis and to evaluate vaccine and therapeutics.

  9. Modification of Isolation and Culture of Human Retinal Pigment Epithelial Cells

    Institute of Scientific and Technical Information of China (English)

    ZhengJL; GuoY

    1999-01-01

    Purpose:To modify the isolation of human retinal pigment pithelial(RPE)cells and to increase the purification and production of cultured RPE cells.Methods:The human eyecups were fixed on a fubber holder.After digestion by trypsin,RPE cells were collected,then cultured and identified by morphology,immunohistochemistry and electron microscopy.Results:The cultured RPE cells grew actively in the early stage with transparent nucleus and abundant melanin particles in cytoplasm.These cells were positive in DOPA oxidase reaction and in anti-pancytokeratin antibody staining.Cellular microvilli and tight junctions could be seen through transmission electrom microscopy.Conclusion:We developed a rubber holder to fix the eyecup.Using this holder,more and purer cultured RPE cells can be obtained.These cultured REP cells are similar to those in vivo in morphology and immunohistochemical staining.

  10. Isolation and comparative mapping of a human chromosome 20-specific alpha-satellite DNA clone.

    Science.gov (United States)

    Baldini, A; Archidiacono, N; Carbone, R; Bolino, A; Shridhar, V; Miller, O J; Miller, D A; Ward, D C; Rocchi, M

    1992-01-01

    We have isolated and characterized a human genomic DNA clone (PZ20, locus D20Z2) that identifies, under high-stringency hybridization conditions, an alphoid DNA subset specific for chromosome 20. The specificity was determined using fluorescence in situ hybridization. Sequence analysis confirmed our previously reported data on the great similarity between the chromosome 20 and chromosome 2 alphoid subsets. Comparative mapping of pZ20 on chimpanzee and gorilla chromosomes, also performed under high-stringency conditions, indicates that the alphoid subset has ancestral sequences on chimpanzee chromosome 11 and gorilla chromosome 19. However, no hybridization was observed to chromosomes 21 in the great apes, the homolog of human chromosome 20.

  11. Characterization of a 2016 Clinical Isolate of Zika Virus in Non-human Primates.

    Science.gov (United States)

    Li, Xiao-Feng; Dong, Hao-Long; Huang, Xing-Yao; Qiu, Ye-Feng; Wang, Hong-Jiang; Deng, Yong-Qiang; Zhang, Na-Na; Ye, Qing; Zhao, Hui; Liu, Zhong-Yu; Fan, Hang; An, Xiao-Ping; Sun, Shi-Hui; Gao, Bo; Fa, Yun-Zhi; Tong, Yi-Gang; Zhang, Fu-Chun; Gao, George F; Cao, Wu-Chun; Shi, Pei-Yong; Qin, Cheng-Feng

    2016-10-01

    Animal models are critical to understand disease and to develop countermeasures for the ongoing epidemics of Zika virus (ZIKV). Here we report a non-human primate model using a 2016 contemporary clinical isolate of ZIKV. Upon subcutaneous inoculation, rhesus macaques developed fever and viremia, with robust excretion of ZIKV RNA in urine, saliva, and lacrimal fluid. Necropsy of two infected animals revealed that systematic infections involving central nervous system and visceral organs were established at the acute phrase. ZIKV initially targeted the intestinal tracts, spleen, and parotid glands, and retained in spleen and lymph nodes till 10days post infection. ZIKV-specific immune responses were readily induced in all inoculated animals. The non-human primate model described here provides a valuable platform to study ZIKV pathogenesis and to evaluate vaccine and therapeutics.

  12. Aerobic metabolism of human quadriceps muscle: in vivo data parallel measurements on isolated mitochondria

    DEFF Research Database (Denmark)

    Rasmussen, Ulla Fugmann; Rasmussen, Hans N.; Krustrup, Peter

    2001-01-01

    The aim of the present study was to examine whether parameters of isolated mitochondria could account for the in vivo maximum oxygen uptake ( O2 max) of human skeletal muscle. O2 max and work performance of the quadriceps muscle of six volunteers were measured in the knee extensor model (range 10......-18 mmol O2 · min 1 · kg 1 at work rates of 22-32 W/kg). Mitochondria were isolated from the same muscle at rest. Strong correlations were obtained between O2 max and a number of mitochondrial parameters (mitochondrial protein, cytochrome aa3, citrate synthase, and respiratory activities). The activities...... of citrate synthase, succinate dehydrogenase, and pyruvate dehydrogenase, measured in isolated mitochondria, corresponded to, respectively, 15, 3, and 1.1 times the rates calculated from O2 max. The respiratory chain activity also appeared sufficient. Fully coupled in vitro respiration, which is limited...

  13. Taxonomic status and ecologic function of methanogenic bacteria isolated from the oral cavity of humans

    Energy Technology Data Exchange (ETDEWEB)

    Kemp, C.W.

    1985-01-01

    The detection of methane gas in samples of dental plaque and media inoculated with dental plaque was attributed to the presence of methane-producing bacteria in the plaque microbiota. The results of a taxonomic analysis of the 12 methanogenic isolates obtained from human dental plaque, (ABK1-ABK12), placed the organisms in the genus Methanobrevibacter. A DNA-DNA hybridization survey established three distinct genetic groups of oral methanogens based on percent homology values. The groups exhibited less than 32% homology between themselves and less than 17% homology with the three known members of the genus methanobrevibacter. The ecological role of the oral methanogens was established using mixed cultures of selected methanogenic isolates (ABK1, ABK4, ABK6, or ABK7) with oral heterotrophic bacteria. Binary cultures of either Streptococcus mutans, Streptococcus sanguis, Veillonella rodentium, Lactobacillus casei, or Peptostreptococcus anaerobius together with either methanogenic isolates ABK6 or ABK7 were grown to determine the effect of the methanogens on the distribution of carbon end products produced by the heterotrophs. Binary cultures of S. mutans and ABK7 exhibited a 27% decrease in lactic acid formation when compared to pure culture of S. mutans. The decrease in lactic acid production was attributed to the removal of formate by the methanogen, (ABK7), which caused an alteration in the distribution of carbon end products by S. mutans.

  14. Schistosoma mansoni Sambon, 1907: morphometric differences between adult worms from sympatric rodent and human isolates

    Directory of Open Access Journals (Sweden)

    Neves Renata Heisler

    1998-01-01

    Full Text Available A computer software for image analysis (IMAGE PRO PLUS, MEDIA CYBERNETICS was utilized in male and females adult worms, aiming the morphological characterization of Schistosoma mansoni samples isolated from a slyvatic rodent, Nectomys squamipes, and humans in Sumidouro, Rio de Janeiro, Brazil and recovered from Mus musculus C3H/He. The following characters for males's testicular lobes were analyzed: number, area, density, larger and smaller diameter, longer and shorter axis and perimeter and extension; for females: area, longer and shorter axis, larger and smaller diameter and perimeter of the eggs and spine; oral and ventral suckers area and distance between them in both sex were determined. By the analysis of variance (one way ANOVA significant differences (p<0.05 were observed in all studied characters, except for the density of testicular lobes. Significant differences (p<0.05 were detected for all characters in the female worms. Data ratify that sympatric isolates present phenotypic differences and the adult female characters are useful for the proper identification of S. mansoni isolates.

  15. Prospective Isolation of Murine and Human Bone Marrow Mesenchymal Stem Cells Based on Surface Markers

    Directory of Open Access Journals (Sweden)

    Yo Mabuchi

    2013-01-01

    Full Text Available Mesenchymal stem cells (MSCs are currently defined as multipotent stromal cells that undergo sustained in vitro growth and can give rise to cells of multiple mesenchymal lineages, such as adipocytes, chondrocytes, and osteoblasts. The regenerative and immunosuppressive properties of MSCs have led to numerous clinical trials exploring their utility for the treatment of a variety of diseases (e.g., acute graft-versus-host disease, Crohn’s disease, multiple sclerosis, osteoarthritis, and cardiovascular diseases including heart failure and myocardial infarction. On the other hand, conventionally cultured MSCs reflect heterogeneous populations that often contain contaminating cells due to the significant variability in isolation methods and the lack of specific MSC markers. This review article focuses on recent developments in the MSC research field, with a special emphasis on the identification of novel surface markers for the in vivo localization and prospective isolation of murine and human MSCs. Furthermore, we discuss the physiological importance of MSC subtypes in vivo with specific reference to data supporting their contribution to HSC niche homeostasis. The isolation of MSCs using selective markers (combination of PDGFRα and Sca-1 is crucial to address the many unanswered questions pertaining to these cells and has the potential to enhance their therapeutic potential enormously.

  16. Isolation and characterization of cloned cDNAs as encoding human liver chlordecone reductase

    Energy Technology Data Exchange (ETDEWEB)

    Winters, C.J.; Molowa, D.T.; Guzelian, P.S. (Medical College of Virginia, Richmond (USA))

    1990-01-30

    Chlordecone (Kepone), a toxic organochlorine pesticide, undergoes bioreduction to chlorodecone alcohol in human liver. This reaction is controlled by a cytosolic enzyme, chlordecone reductase (CDR), which may be of the aldo-keto reductase family of xenobiotic metabolizing enzymes. To further investigate the primary structure and expression of CDR, the authors screened a library of human liver cDNAs cloned in the expression vector {lambda}gt11 and isolated an 800 bp cDNA that directed synthesis of a fusion protein recognized by polyclonal anti-CDR antibodies. Using this cDNA as a probe, they screened two human liver cDNA libraries and found several 1.2-kb cDNAs which would code for polypeptide with 308 residues (35.8 kDa). However, a similar full-length cDNA, possibly the transcript of a pseudogene, contained an in-frame nonsense codon. The deduced protein sequence of CDR showed 65% similarity to the primary structure of human liver aldehyde reductase and 66% similarity to the inferred protein sequence of rat lens aldose reductase. A search of GenBank revealed significant nucleotide similarity to a cDNA coding for bovine lung prostaglandin f synthase and to a partial cDNA coding for frog lens {rho}-crystallin. RNA from adult but not fetal human liver, and from the human hepatoma cell-line Hep G2, contained major (1.6 kb) and minor (2.8 kb) species hybridizable to a CDR cDNA. The relative amounts of these RNAs varied markedly among nine subjects. From this initial description of the nucleotide sequence for a human carbonyl reductase, they conclude that CDR and several related enzymes are part of a novel multigene family involved in the metabolism of such xenobiotics as chlordecone and possibly endogenous substrates.

  17. Prevalence of virulence and antimicrobial resistance genes in Salmonella spp. isolated from commercial chickens and human clinical isolates from South Africa and Brazil

    Directory of Open Access Journals (Sweden)

    Oliver T. Zishiri

    2016-03-01

    Full Text Available Salmonellosis is a significant public health concern around the world. The injudicious use of antimicrobial agents in poultry production for treatment, growth promotion and prophylaxis has resulted in the emergence of drug resistant strains of Salmonella. The current study was conducted to investigate the prevalence of virulence and antimicrobial resistance genes from Salmonella isolated from South African and Brazilian broiler chickens as well as human clinical isolates. Out of a total of 200 chicken samples that were collected from South Africa 102 (51% tested positive for Salmonella using the InvA gene. Of the overall 146 Salmonella positive samples that were screened for the iroB gene most of them were confirmed to be Salmonella enterica with the following prevalence rates: 85% of human clinical samples, 68.6% of South African chicken isolates and 70.8% of Brazilian chicken samples. All Salmonella isolates obtained were subjected to antimicrobial susceptibility testing with 10 antibiotics. Salmonella isolates from South African chickens exhibited resistance to almost all antimicrobial agents used, such as tetracycline (93%, trimethoprim-sulfamthoxazole (84%, trimethoprim (78.4%, kanamycin (74%, gentamicin (48%, ampicillin (47%, amoxicillin (31%, chloramphenicol (31%, erythromycin (18% and streptomycin (12%. All samples were further subjected to PCR in order to screen some common antimicrobial and virulence genes of interest namely spiC, pipD, misL, orfL, pse-1, tet A, tet B, ant (3"-la, sul 1 and sul. All Salmonella positive isolates exhibited resistance to at least one antimicrobial agent; however, antimicrobial resistance patterns demonstrated that multiple drug resistance was prevalent. The findings provide evidence that broiler chickens are colonised by pathogenic Salmonella harbouring antimicrobial resistance genes. Therefore, it is evident that there is a need for prudent use of antimicrobial agents in poultry production systems in

  18. Characterization of Staphylococcus aureus from Humans and a Comparison with Isolates of Animal Origin, in North Dakota, United States.

    Directory of Open Access Journals (Sweden)

    Valeria Velasco

    Full Text Available Different clones of methicillin-susceptible (MSSA and methicillin-resistant (MRSA Staphylococcus aureus have been found in humans as well as in animals and retail meat. However, more information about the genetic characteristics and similarities between strains is needed. The aim of this study was to identify and characterize Staphylococcus aureus from humans, and to compare their characteristics with isolates of animal origin. A total of 550 nasal swabs were taken from healthy humans, and S. aureus was isolated and identified. Positive S. aureus isolates were subjected to molecular typing and susceptibility testing. In addition, 108 MRSA isolates recovered from clinical patients in the state of North Dakota and 133 S. aureus isolates from animals and meat previously analyzed were included. The nasal carriage of S. aureus in healthy people was 7.6% and, in general, clones were genetically diverse. None of the S. aureus strains obtained from healthy people were mecA- or PVL-positive. A total of 105 (97.2% MRSA isolates from clinical cases harbored the mecA gene and 11 (10.2% isolated from blood stream infections harbored the PVL gene. The most common resistance profile among S. aureus from healthy people was penicillin, and from clinical cases were erythromycin-penicillin-ciprofloxacin. The rate of multidrug resistance (MDR was 70% in humans. Most of the S. aureus harboring mecA and PVL genes were identified as ST5 and ST8, and exhibited MDR. However, S. aureus isolates of animal origin used for comparison exhibited a lower rate of MDR. The most common resistance profiles in isolates of animal origin were penicillin-tetracycline and penicillin-tetracycline-erythromycin, in animals and raw meat, respectively. The ST5 was also found in animals and meat, with ST9 and ST398 being the major clones. The genetic similarity between clones from humans and meat suggests the risk of spread of S. aureus in the food chain.

  19. Methods of isolation, expansion, differentiating induction and preservation of human umbilical cord mesenchymal stem cells

    Institute of Scientific and Technical Information of China (English)

    LI Dong-rui; CAI Jian-hui

    2012-01-01

    Objective This literature review aims to summarize the methods of isolation,expansion,differentiation and preservation of human umbilical cord mesenchymal stem cells (hUCMSCs),for comprehensive understanding and practical use in preclinical research and clinical trials.Data sources All the literature reviewed was published over the last 10 years and is listed in PubMed and Chinese National Knowledge Infrastructure (CNKI).Studies were retrieved using the key word "human umbilical cord mesenchymal stem cells".Results Explants culture and enzymatic digestion are two methods to isolate hUCMSCs from WJ and there are modifications to improve these methods.Culture conditions may affect the expansion and differentiating orientations of hUCMSCs.In addition,hUCMSCs can maintain their multi-potential effects after being properly frozen and thawed.Conclusion Considering their multi-potential,convenient and non-invasive accessibility,low immunogenicity and the reported therapeutic effects in several different preclinical animal models,hUCMSCs have immense scope in regeneration medicine as a substitute for MSCs derived from bone marrow or umbilical cord blood.

  20. Identification of high immunoreactive proteins from Streptococcus agalactiae isolates recognized by human serum antibodies.

    Science.gov (United States)

    Brzychczy-Wloch, Monika; Gorska, Sabina; Brzozowska, Ewa; Gamian, Andrzej; Heczko, Piotr B; Bulanda, Malgorzata

    2013-12-01

    The aim of the studies was to identify immunogenic proteins of Streptococcus agalactiae (group B streptococcus; GBS) isolates. Investigation of the immunoreactivity with human sera allowed us to determine major immunogenic proteins which might be potential candidates for the development of vaccine. For the study, we have selected 60 genetically different, well-characterized GBS clinical isolates. The proteins immunoreactivity with 24 human sera from patients with GBS infections, carriers, and control group without GBS was detected by SDS-PAGE and Western blotting. As a result, some major immunogenic proteins were identified, of which four proteins with molecular masses of about 45 to 50 kDa, which exhibited the highest immunoreactivity features, were analyzed by LC-MS/MS. The proteins were identified by comparative analysis of peptides masses using MASCOT and statistical analysis. The results showed known molecules such as enolase (47.4 kDa), aldehyde dehydrogenase (50.6 kDa), and ones not previously described such as trigger factor (47 kDa) and elongation factor Tu (44 kDa). The preliminary results indicated that some GBS proteins that elicit protective immunity hold promise not only as components in a vaccine as antigens but also as carriers or adjuvants in polysaccharide conjugate vaccines, but more studies are needed.

  1. ISOLATION AND EXPANSION OF HUMAN EMBRYONIC NEURAL STEM/PROGENITOR CELLS IN VITRO

    Institute of Scientific and Technical Information of China (English)

    2002-01-01

    Objective: To isolate, culture and identify human embryonic neural stem cells and to establish a practical passaging method. Method:The cerebral cortex cells were isolated from aborted embryos (11~13 weeks)by mechanical dissociation, and cultured in DMEM/F12 culture medium supplemented with N2 and growth factors for proliferation. Upon passaging, the neurospheres were pipetted gentlely to separate them into several cell masses and then grown in growth medium. The cells were grown in DMEM/F12 medium with serum (without growth factors) to induce differentiation. The stem cell, neuron, astrocyte and oligodendrocyte were identified by immunocytochemistry with antibodies to vimentin, MAP2, GFAP and GalC, respectively. Results:The primary cells grew together and formed neurospheres at 5th~7th day. They were all vimentin positive and could be passaged for at least 8passages. After passaging, the cell masses grew up and formed new neurospheres rapidly. These cells could differentiated into MAP2 ( + ), GFAP( + ) or GalC( + ) cells. Conclusion: The neural stem cells from human embryonic cerebral cortex have the capacity of proliferation and multi - differentiation in vitro. The passaging methods we used in this experiment were practical and convenient.

  2. Isolated corneal papilloma-like lesion associated with human papilloma virus type 6.

    Science.gov (United States)

    Park, Choul Yong; Kim, Eo-Jin; Choi, Jong Sun; Chuck, Roy S

    2011-05-01

    To report a case of a corneal papilloma-like lesion associated with human papilloma virus type 6. A 48-year-old woman presented with a 2-year history of ocular discomfort and gradual visual deterioration in her right eye. Ophthalmic examination revealed an elevated, semitranslucent, well-defined vascularized mass approximately 4 × 2.5 mm in size localized to the right cornea. The surface of the mass appeared smooth and many small, shallow, and irregular elevations were noted. An excisional biopsy was performed. The underlying cornea was markedly thinned, and fine ramifying vasculature was also noted on the exposed corneal stroma. Typical koilocytic change was observed on the histopathologic examination. Polymerase chain reaction revealed the existence of human papilloma virus type 6 DNA. Here we describe a case of an isolated corneal papilloma-like lesion. Although the corneal extension of the limbal or the conjunctival papillomas has been commonly observed, an isolated corneal papilloma-like lesion with underlying stromal destruction has only rarely been reported.

  3. Efficient Methods To Isolate Human Monoclonal Antibodies from Memory B Cells and Plasma Cells.

    Science.gov (United States)

    Corti, Davide; Lanzavecchia, Antonio

    2014-10-01

    In this article, we highlight the advantages of isolating human monoclonal antibodies from the human memory B cells and plasma cell repertoires by using high-throughput cellular screens. Memory B cells are immortalized with high efficiency using Epstein-Barr virus (EBV) in the presence of a toll-like receptor (TLR) agonist, while plasma cells are maintained in single-cell cultures by using interleukin 6 (IL-6) or stromal cells. In both cases, multiple parallel assays, including functional assays, can be used to identify rare cells that produce antibodies with unique properties. Using these methods, we have isolated potent and broadly neutralizing antibodies against a variety of viruses, in particular, a pan-influenza-A-neutralizing antibody and an antibody that neutralizes four different paramyxoviruses. Given the high throughput and the possibility of directly screening for function (rather than just binding), these methods are instrumental to implement a target-agnostic approach to identify the most effective antibodies and, consequently, the most promising targets for vaccine design. This approach is exemplified by the identification of unusually potent cytomegalovirus-neutralizing antibodies that led to the identification of the target, a pentameric complex that we are developing as a candidate vaccine.

  4. Chromosome protein framework from proteome analysis of isolated human metaphase chromosomes.

    Science.gov (United States)

    Fukui, Kiichi; Uchiyama, Susumu

    2007-01-01

    We have presented a structural model of the chromosome based on its constituent proteins. Development of a method of mass isolation for intact human metaphase chromosomes and proteome analysis by mass spectrometry of the isolated chromosomal proteins enabled us to develop a four-layer structural model of human metaphase chromosomes. The model consists of four layers, each with different chromosomal protein sets, i.e., chromosome coating proteins (CCPs), chromosome peripheral proteins (CPPs), chromosome structural proteins (CSPs), and chromosome fibrous proteins (CFPs). More than 200 identified proteins have been classified and assigned to the four layers with each layer occupying a distinct region of the chromosome. CCPs are localized at the most outer regions of the chromosomes and they attach to the regions tentatively and occasionally. CCPs include mostly mitochondrial and cytoplasmic proteins, e.g., 70 kDa heat shock protein 9B and Hsp60. CPPs are also localized at the peripheral regions of the chromosomes, but as the essential part of the chromosomes. CPPs include nucleolin, lamin A/C, fibrillarin, etc. CSPs are the primary chromosomal structure proteins, and include topoisomerase IIalpha, condensin subunits, histones, etc. CFPs have a fibrous nature, e.g., beta-actin, vimentin, myosin II, tublin, etc. A data set of these proteins, which we developed, contains essential chromosome proteins with classified information based on this four-layer model and presents useful leads for further studies on chromosomal structure and function.

  5. Effects of isolation and confinement on humans-implications for manned space explorations.

    Science.gov (United States)

    Pagel, J I; Choukèr, A

    2016-06-15

    Human psychology and physiology are significantly altered by isolation and confinement. In light of planned exploration class interplanetary missions, the related adverse effects on the human body need to be explored and defined as they have a large impact on a mission's success. Terrestrial space analogs offer an excellent controlled environment to study some of these stressors during a space mission in isolation without the complex environment of the International Space Station. Participants subjected to these space analog conditions can encounter typical symptoms ranging from neurocognitive changes, fatigue, misaligned circadian rhythm, sleep disorders, altered stress hormone levels, and immune modulatory changes. This review focuses on both the psychological and the physiological responses observed in participants of long-duration spaceflight analog studies, such as Mars500 or Antarctic winter-over. They provide important insight into similarities and differences encountered in each simulated setting. The identification of adverse effects from confinement allows not only the crew to better prepare for but also to design feasible countermeasures that will help support space travelers during exploration class missions in the future. Copyright © 2016 the American Physiological Society.

  6. Genetic heterogeneity and subtyping of human Hepatitis E virus isolates from Uruguay.

    Science.gov (United States)

    Mirazo, Santiago; Ramos, Natalia; Russi, José Carlos; Arbiza, Juan

    2013-05-01

    Hepatitis E virus (HEV) infection is an important public health concern in many developing countries causing waterborne outbreaks, as well as sporadic autochthonous hepatitis. It is transmitted primarily by the fecal-oral route. However, zoonotic transmission from animal reservoirs to human has also been suggested. Genotype 3 is the most frequent genotype found in South America and the HEV epidemiology in this region seems to be very complex. However, data about the molecular characterization of HEV isolates of the region is still lacking and further investigation is needed. Our study characterized human HEV strains detected in a 1-year period in Uruguay, by extensive sequence analysis of three regions of the HEV genome. Uruguayan strains were closely related to a set of European strains and in turn, were dissimilar to Brazilian, Argentinean and Bolivian isolates. Additionally, the co-circulation of viral subtypes 3i and 3h was observed. Circulation of subtype 3i had been reported in Argentina and Bolivia whereas sequences of subtype 3h are rare and had never been reported in Latin America. In order to contribute to shedding light over the molecular epidemiology of this emergent infection in the region, we thoroughly analyzed the genetic variability of HEV strains detected in Uruguay, providing the largest dataset of sequences of HEV ever reported in a country in South America.

  7. Inhibition of isolated human myometrium contractility by minoxidil and reversal by glibenclamide.

    Science.gov (United States)

    Prabhakaran, S S; Dhanasekar, K R; Thomas, E; Jose, R; Peedicayil, J; Samuel, P

    2010-03-01

    This study investigated the ability of the antihypertensive drug minoxidil to inhibit potassium chloride (KCl)-induced contractility of the isolated human myometrium. Twelve strips of myometrium obtained from 12 patients who underwent hysterectomy were triggered to contract with 55 mM KCl before and after incubation with 3 concentrations (1, 3 and 10 microM) of minoxidil. The percent inhibition by minoxidil on the extent of contraction, and the area under the contractile curve of KCl-induced contraction of the myometrial strips was determined. Furthermore, the effect of 10 microM glibenclamide on the inhibition generated by 3 microM minoxidil on KCl-induced contractility was studied. It was found that minoxidil produced a concentration-dependent inhibition of KCl-induced contractility of the myometrium and that glibenclamide reversed this inhibitory effect. These results suggest that the inhibitory effect of minoxidil on isolated human myometrium contractility may prove useful in clinical conditions requiring relaxation of the myometrium. 2010 Prous Science, S.A.U. or its licensors. All rights reserved.

  8. Potentially hypervirulent Clostridium difficile PCR ribotype 078 lineage isolates in pigs and possible implications for humans in Taiwan.

    Science.gov (United States)

    Wu, Ying-Chen; Lee, Jen-Jie; Tsai, Bo-Yang; Liu, Yi-Fen; Chen, Chih-Ming; Tien, Ni; Tsai, Pei-Jane; Chen, Ter-Hsin

    2016-02-01

    Clostridium difficile is a human and animal pathogen. Recently, the incidence of community-acquired C. difficile infection has increased, and many studies have indicated that C. difficile might be food-borne. The correlation between C. difficile infection in humans and in animals has been a topic of debate. The objective of this study was to determine the genetic relatedness of C. difficile from human and pigs in Taiwan. We investigated the molecular epidemiology of C. difficile in healthy humans and pigs from 2011 to 2015. The isolation rate of C. difficile from pigs in 13 commercial farms was 49% (100/204), and a high proportion of hypervirulent (C. difficile carrying tcdA, tcdB, and cdtA/B genes and a 39-bp deletion in the tcdC gene) ribotype 078 lineage isolates (90%, 90/100; including 078, 126, 127, and 066-like isolates) were identified. In addition, the C. difficile ribotype 127 isolates from pigs typically exhibited moxifloxacin resistance (37/43; 86%). In healthy humans, the isolation rate was 4.3% (3/69), and all healthy human isolates were non-toxigenic. In particular, we compared the porcine isolates with two patient strains (ribotype 127) obtained from two hospitals in central Taiwan. The multilocus variable number tandem repeat analysis revealed a high genetic relatedness between ribotype 127 from patients and pigs. This study indicated that isolates of the ribotype 078 lineage, and especially ribotype 127, were widely distributed in pig farms and showed a high frequency of moxifloxacin resistance. The closely related ribotype 127 from patients and pigs may have had a common origin or low diversity. In conclusion, C. difficile ribotype 127 is a noteworthy pathogen in pigs and poses a potential public health threat.

  9. Characterization of functional properties of Enterococcus faecium strains isolated from human gut.

    Science.gov (United States)

    İspirli, Hümeyra; Demirbaş, Fatmanur; Dertli, Enes

    2015-11-01

    The aim of this work was to characterize the functional properties of Enterococcus faecium strains identified after isolation from human faeces. Of these isolates, strain R13 showed the best resistance to low pH, bile salts, and survival in the simulated in vitro digestion assay, and demonstrated an important level of adhesion to hexadecane as a potential probiotic candidate. Analysis of the antibiotic resistance of E. faecium strains indicated that in general these isolates were sensitive to the tested antibiotics and no strain appeared to be resistant to vancomycin. Examination of the virulence determinants for E. faecium strains demonstrated that all strains contained the virulence genes common in gut- and food-originated enterococci, and strain R13 harboured the lowest number of virulence genes. Additionally, no strain contained the genes related to cytolysin metabolism and showed hemolytic activity. The antimicrobial role of E. faecium strains was tested against several pathogens, in which different levels of inhibitory effects were observed, and strain R13 was inhibitory to all tested pathogens. PCR screening of genes encoding enterocin A and B indicated the presence of these genes in E. faecium strains. Preliminary characterization of bacteriocins revealed that their activity was lost after proteolytic enzyme treatments, but no alteration in antimicrobial activity was observed at different pHs (3.5 to 9.5) and after heat treatments. In conclusion, this study revealed the functional characteristics of E. faecium R13 as a gut isolate, and this strain could be developed as a new probiotic after further tests.

  10. Antimicrobial susceptibilities of Listeria monocytogenes human strains isolated from 1970 to 2008 in Brazil

    Directory of Open Access Journals (Sweden)

    Cristhiane Moura Falavina dos Reis

    2011-04-01

    Full Text Available INTRODUCTION: Listeria monocytogenes is the causative agent of listeriosis, a foodborne illness that affects mainly pregnant women, the elderly and immunocompromised patients. The primary treatment is a combination of ampicillin with an aminoglycoside, in addition to a second-choice drug represented by chloramphenicol, erythromycin, tetracycline and rifampicin. The aim of this study was to analyze the antimicrobial susceptibility profile of strains isolated from human sources in the last four decades. METHODS: Sixty-eight strains were selected from the culture collection of the Laboratory of Bacterial Zoonoses/LABZOO/FIOCRUZ isolated in different regions of Brazil from 1970 to 2008 and primarily isolated from cerebrospinal fluid and blood culture. Susceptibility tests to antimicrobials drugs were evaluated using the criteria established by Soussy using the Kirby-Bauer method and E-Test strips were used to determine the minimum inhibitory concentration (MIC. RESULTS: Among the strains tested, serovar L4b (60.3% was the most prevalent, followed by serovar 1/2a (20.6%, 1/2b (13.2% and the more uncommon serovars 1/2c, 3b and 4ab (5.9%. All strains were susceptible to ampicillin, cephalothin, erythromycin, gentamicin, teicoplanin and vancomycin. Only one strain (1.5% showed resistance to rifampin, and two (3% were resistant to trimethoprim-sulfamethoxazole. MICs with values up to 2μg/ml reinforce the need for microbiological surveillance. CONCLUSIONS: The study demonstrated low prevalence of strains resistant to the antimicrobial drugs indicated in the treatment of human listeriosis. Monitoring antimicrobial resistance profile is still very important to determine adequate treatment, especially in immunocompromised patients.

  11. Two novel EHEC/EAEC hybrid strains isolated from human infections.

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    Rita Prager

    Full Text Available The so far highest number of life-threatening hemolytic uremic syndrome was associated with a food-borne outbreak in 2011 in Germany which was caused by an enterohemorrhagic Escherichia coli (EHEC of the rare serotype O104:H4. Most importantly, the outbreak strain harbored genes characteristic of both EHEC and enteroaggregative E. coli (EAEC. Such strains have been described seldom but due to the combination of virulence genes show a high pathogenicity potential. To evaluate the importance of EHEC/EAEC hybrid strains in human disease, we analyzed the EHEC strain collection of the German National Reference Centre for Salmonella and other Bacterial Enteric Pathogens (NRC. After exclusion of O104:H4 EHEC/EAEC strains, out of about 2400 EHEC strains sent to NRC between 2008 and 2012, two strains exhibited both EHEC and EAEC marker genes, specifically were stx2 and aatA positive. Like the 2011 outbreak strain, one of the novel EHEC/EAEC harbored the Shiga toxin gene type stx2a. The strain was isolated from a patient with bloody diarrhea in 2010, was serotyped as O59:H-, belonged to MLST ST1136, and exhibited genes for type IV aggregative adherence fimbriae (AAF. The second strain was isolated from a patient with diarrhea in 2012, harbored stx2b, was typed as Orough:H-, and belonged to MLST ST26. Although the strain conferred the aggregative adherence phenotype, no known AAF genes corresponding to fimbrial types I to V were detected. In summary, EHEC/EAEC hybrid strains are currently rarely isolated from human disease cases in Germany and two novel EHEC/EAEC of rare serovars/MLST sequence types were characterized.

  12. 76 FR 51374 - Direct Discovery of HLA Associated Influenza Epitopes Isolated From Human Cells for Vaccine and...

    Science.gov (United States)

    2011-08-18

    ... HUMAN SERVICES Food and Drug Administration Direct Discovery of HLA Associated Influenza Epitopes Isolated From Human Cells for Vaccine and Therapeutic Evaluation and Development (U01) AGENCY: Food and... will provide the regulatory science to facilitate development and evaluation of direct discovery of...

  13. Isolation and characterization of the human collagen α1(I)-like gene from a cosmid library.

    NARCIS (Netherlands)

    E.H. Weiss (Elisabeth); K.S.E. Cheah (Kathryn); F.G. Grosveld (Frank); H.H.M. Dahl; E. Solomon; R.A. Flavell (Richard)

    1994-01-01

    textabstractWe have isolated a human collagen alpha 1(I)-like gene from a cosmid library. The clone which contains 37kb of human DNA has been shown to contain this gene by DNA sequencing, hybrid arrest and hybrid selection assays and Northern blot hybridizations. The collagen gene sequence extends

  14. Isolation and characterization of the human collagen α1(I)-like gene from a cosmid library.

    NARCIS (Netherlands)

    E.H. Weiss (Elisabeth); K.S.E. Cheah (Kathryn); F.G. Grosveld (Frank); H.H.M. Dahl; E. Solomon; R.A. Flavell (Richard)

    1994-01-01

    textabstractWe have isolated a human collagen alpha 1(I)-like gene from a cosmid library. The clone which contains 37kb of human DNA has been shown to contain this gene by DNA sequencing, hybrid arrest and hybrid selection assays and Northern blot hybridizations. The collagen gene sequence extends t

  15. Isolation of human anti-serum albumin Fab antibodies with an extended serum-half life.

    Science.gov (United States)

    Kang, Hyeon-Ju; Kim, Hye-Jin; Cha, Sang-Hoon

    2016-01-01

    The serum albumin (SA) has been exploited to generate long-acting biotherapeutics by taking advantage of the FcRn-mediated recycling mechanism in a direct or an indirect way. Since Fab fragments have been proven to be clinically safe for human usage, we assumed that human anti-SA Fab antibodies could have a great potential as a carrier molecule to extend the serum half-life of therapeutic proteins. We, herein, had attempted to isolate anti-SA Fab antibodies from HuDVFab-8L antibody library via a phage display technology, and identified eight discrete human Fab antibodies. One of the Fab antibodies, SL335, showed the strongest binding reactivity to human SA with nM range of affinity at both pH 6 and pH 7.4, and cross-reacted to SAs from various species including rat, mouse, canine and monkey. The in vivo pharmacokinetic assay using a rat model indicated that SL335 has approximately 10 fold longer serum half-life and 26 to 44-fold increase in AUC0 → ∞ compared to the negative control Fab molecule in both intravenous and subcutaneous administrations. Knowing that Fabs have proven to be safe in clinics for a long time, SL335 seems to have a great potential in generating long-acting protein drugs by tagging effector molecules with either chemical conjugation or genetic fusion.

  16. Genotyping of cystic echinococcosis isolates from clinical samples of human and domestic animals

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    S.A. Fadhil

    2016-12-01

    Full Text Available Cystic hydatid disease is a cosmopolitan important disease in both human and animals. Many strains were investigated in this parasite. The aim of study was to characterize genotype variations of Echinococcus granulosus isolates collected from human and domestic animals in Al-Qadisiyah province/ Iraq based on sequencing of nad1 mitochondrial gene. Eighty hydatid cysts of human (12, sheep (15, cattle (36, and camels (17 were collected from hospital and slaughter house of the province, during October 2014 to June 2015; microscopic examination was made for cysts fluid to determine the fertility. DNAs extraction was done for each sample in addition to purify and concentrate of extracted DNA samples was performed to determine nad1 (400bp gene used conventional PCR method. Phylogenetic analysis was performed using NCBI-Blast Alignment identification and Unweighted Pair Group Method with Arithmetic Mean. Twenty five (10 from human and 5 from each studied animals samples were chosen due to their fertility and high DNA purity, in which three strains (genotypes were investigated including sheep strain (G1 40%, buffalo strain (G3 48% and camel strain (G6 12%, where human samples related to G1(20% and G3(80%; sheep samples related to G1(80% and G3(20%; cattle samples related to G1(60%, G3 (20% and G6 (20%; camels samples related to G1(20%, G3(40% and G6(40%. The dominant strain is a buffalo strain (G3; both of buffalo strain (G3 and sheep strain (G1 represented the actual source of human infection. There is no host specificity of detected genotypes.

  17. Profile of Shiga toxin-producing Escherichia coli strains isolated from dogs and cats and genetic relationships with isolates from cattle, meat and humans.

    Science.gov (United States)

    Bentancor, A; Rumi, M V; Carbonari, C; Gerhardt, E; Larzábal, M; Vilte, D A; Pistone-Creydt, V; Chinen, I; Ibarra, C; Cataldi, A; Mercado, E C

    2012-05-04

    Pets can be reservoirs of Shiga toxin-producing Escherichia coli (STEC) strains. The aim of this study was to examine nine strains belonging to several serotypes (O91:H21, O91:H16, O178:H19, O8:H19, O22:H8, O22:HNT, ONT:H8), previously recovered from cats or dogs. To this end, we assessed a set of additional virulence genes (stx(2) subtype, subAB, ehxA, eae and saa), cytotoxic activity, and genetic relationships with strains isolated from cattle, meat and humans using pulsed-field gel electrophoresis (PFGE). Most of the isolates carried the stx(2) and/or stx(2vh-b) sequences, while only the O91:H21 isolate presented the mucus-activatable stx(2d) variant, as confirmed by sequencing the genes of subunits A and B. All the strains showed cytotoxic activity in cultured cells. One of the two O178:H19, selected for its high level of cytotoxicity in Vero cells, showed the ability to cause functional alterations in the human colon mucosa in vitro. None of the strains possessed the subAB, eae or saa genes and only the strains belonging to serotype O8:H19 carried the ehxA gene. The isolates shared 90-100% similarity by PFGE to epidemiologically unrelated strains of the corresponding serotypes recovered from cattle, meat or humans. Our results demonstrate that dogs and cats may have a role in the infection of humans by STEC, probably serving as a vehicle for bovine strains in the cycle of human infection, and thus emphasize the health risks for owners and their families. Copyright © 2011 Elsevier B.V. All rights reserved.

  18. Diversity of Vibrio navarrensis Revealed by Genomic Comparison: Veterinary Isolates Are Related to Strains Associated with Human Illness and Sewage Isolates While Seawater Strains Are More Distant

    Directory of Open Access Journals (Sweden)

    Keike Schwartz

    2017-09-01

    Full Text Available Strains of Vibrio navarrensis are present in aquatic environments like seawater, rivers, and sewage. Recently, strains of this species were identified in human clinical specimens. In this study, V. navarrensis strains isolated from livestock in Germany were characterized that were found in aborted fetuses and/or placentas after miscarriages. The veterinary strains were analyzed using phenotypical and genotypical methods and compared to isolates from marine environments of the Baltic Sea and North Sea. The investigated phenotypical traits were similar in all German strains. Whole genome sequencing (WGS was used to evaluate a phylogenetic relationship by performing a single nucleotide polymorphism (SNP analysis. For the SNP analysis, WGS data of two American human pathogenic strains and two Spanish environmental isolates from sewage were included. A phylogenetic analysis of concatenated sequences of five protein-coding housekeeping genes (gyrB, pyrH, recA, atpA, and rpoB, was additionally performed. Both phylogenetic analyses reveal a greater distance of the environmental seawater strains to the other strains. The phylogenetic tree constructed from concatenated sequences of housekeeping genes places veterinary, human pathogenic and Spanish sewage strains into one cluster. Presence and absence of virulence-associated genes were investigated based on WGS data and confirmed by PCR. However, this analysis showed no clear pattern for the potentially pathogenic strains. The detection of V. navarrensis in human clinical specimens strongly suggests that this species should be regarded as a potential human pathogen. The identification of V. navarrensis strains in domestic animals implicates a zoonotic potential of this species. This could indicate a potential threat for humans, as according to the “One Health” concept, human, animal, and environmental health are linked. Future studies are necessary to search for reservoirs of these bacteria in the

  19. Isolation and Culture of Human Endothelial Cells from Micro- and Macro-vessels.

    Science.gov (United States)

    Hewett, Peter W

    2016-01-01

    The endothelium from different vascular beds exhibits a high degree of phenotypic heterogeneity. Endothelial cells (EC) can be harvested easily from large vessels by mechanical removal or collagenase digestion. In particular, the human umbilical vein has been used due to its wide availability, and the study of ECs derived from it has undoubtedly greatly advanced our knowledge of vascular biology. However, the majority of the body's endothelium (>95 %) forms the microvasculature, and it is these cells providing the interface between the blood and tissues that play a critical role in the development of new blood vessels. This has led to the establishment of techniques for the isolation of microvascular ECs (MEC) from different tissues to provide more physiologically relevant in vitro models of angiogenesis and EC function.In this chapter the use of superparamagnetic beads (Dynabeads) coated with anti-PECAM-1 (CD31) antibodies (PECA-beads) to culture MECs from human adipose tissue is described along with the standard methods used to characterize them. Adipose tissue is an ideal source of MECs as it is composed mainly of adipocytes with a very rich microvasculature and is easy to disaggregate. Furthermore, it can be obtained in large quantities during plastic surgery procedures. Adipose obtained at reduction mammoplasty or abdominoplasty is first dissected free of the connective tissue, minced finely, and subjected to collagenase type II digestion. The adipocytes are removed by centrifugation to obtain a microvessel rich pellet, which is further disaggregated with trypsin/EDTA solution. Following filtration to remove fragments of the connective tissue, the pellet is incubated with PECA-beads and microvessel fragments/ECs and washed and harvested using a magnet. In addition, the adaptation of this basic technique for the isolation of the human lung and stomach MECs is also described along with common methods for the preparation of large vessel endothelial cells.

  20. Acetaldehyde production and other ADH-related characteristics of aerobic bacteria isolated from hypochlorhydric human stomach.

    Science.gov (United States)

    Väkeväinen, S; Tillonen, J; Blom, M; Jousimies-Somer, H; Salaspuro, M

    2001-03-01

    Acetaldehyde is a known local carcinogen in the digestive tract in humans. Bacterial overgrowth in the hypochlorhydric stomach enhances production of acetaldehyde from ethanol in vivo after alcohol ingestion. Therefore, microbially produced acetaldehyde may be a potential risk factor for alcohol-related gastric and cardiac cancers. This study was aimed to investigate which bacterial species and/or groups are responsible for acetaldehyde formation in the hypochlorhydric human stomach and to characterize their alcohol dehydrogenase (ADH) enzymes. After 7 days of treatment with 30 mg of lansoprazole twice a day, a gastroscopy was performed on eight volunteers to obtain hypochlorhydric gastric juice. Samples were cultured and bacteria were isolated and identified; thereafter, their acetaldehyde production capacity was measured gas chromatographically by incubating intact bacterial suspensions with ethanol at 37 degrees C. Cytosolic ADH activities, Km values, and protein concentration were determined spectrophotometrically. Acetaldehyde production of the isolated bacterial strains (n = 51) varied from less than 1 to 13,690 nmol of acetaldehyde/10(9) colony-forming units/hr. ADH activity of the strains that produced more than 100 nmol of acetaldehyde/10(9) colony-forming units/hr (n = 23) varied from 3.9 to 1253 nmol of nicotinamide adenine dinucleotide per minute per milligram of protein, and Km values for ethanol ranged from 0.65 to 116 mM and from 0.5 to 3.1 M (high Km). There was a statistically significant correlation (r = 0.64, p Streptococcus salivarius, whereas nearly all Stomatococcus, Staphylococcus, and other Streptococcus species had a very low capacity to produce acetaldehyde. This study demonstrated that certain bacterial species or groups that originate from the oral cavity are responsible for the bulk of acetaldehyde production in the hypochlorhydric stomach. These findings provide new information with the respect to the local production of carcinogenic

  1. Easily-handled method to isolate mesenchymal stem cells from coagulated human bone marrow samples

    Institute of Scientific and Technical Information of China (English)

    Heng-Xiang; Wang; Zhi-Yong; Li; Zhi-Kun; Guo; Zi-Kuan; Guo

    2015-01-01

    AIM:To establish an easily-handled method to isolate mesenchymal stem cells(MSCs) from coagulated human bone marrow samples. METHODS: Thrombin was added to aliquots of seven heparinized human bone marrow samples to mimic marrow coagulation. The clots were untreated,treated with urokinase or mechanically cut into pieces before culture for MSCs. The un-coagulated samples and the clots were also stored at 4 ℃ for 8 or 16 h before the treatment. The numbers of colony-forming unit-fibroblast(CFU-F) in the different samples were determined. The adherent cells from different groups were passaged and their surface profile was analyzed with flow cytometry. Their capacities of in vitro osteogenesis and adipogenesis were observed after the cells were exposed to specific inductive agents.RESULTS: The average CFU-F number of urokinasetreated samples(16.85 ± 11.77/106) was comparable to that of un-coagulated control samples(20.22 ± 10.65/106,P = 0.293),which was significantly higher than those of mechanically-cut clots(6.5 ± 5.32/106,P < 0.01) and untreated clots(1.95 ± 1.86/106,P < 0.01). The CFU-F numbers decreased after samples were stored,but those of control and urokinase-treated clots remained higher than the other two groups. Consistently,the numbers of the attached cells at passage 0 were higher in control and urokinase-treated clots than those of mechanically-cut clots and untreated clots.The attached cells were fibroblast-like in morphology and homogenously positive for CD44,CD73 and CD90,and negative for CD31 and CD45. Also,they could be induced to differentiate into osteoblasts and adipocytes in vitro. CONCLUSION: Urokinase pretreatment is an optimal strategy to isolate MSCs from human bone marrow samples that are poorly aspirated and clotted.

  2. Virulence determinants invA and spvC in salmonellae isolated from poultry products, wastewater, and human sources.

    Science.gov (United States)

    Swamy, S C; Barnhart, H M; Lee, M D; Dreesen, D W

    1996-10-01

    The presence of two virulence foci, invA and spvC, in Salmonella isolates obtained from poultry, wastewater, and human sources was determined. All isolates (n = 245) were positive for the invA gene sequence. Differences in degree of invasiveness were apparent with the Madin Darby canine kidney cell line, as only 79 of 159 randomly selected isolates (49.7%) tested were invasive at > 0.1% of the inoculum. 25% were invasive between 0.1 and 1.0% of the inoculum, and 24.5% were invasive at > 1.0% of the inoculum. There was a significant correlation between degree of invasion and source from which the isolate was recovered but no correlation between geographic origin of poultry isolates and degree of invasion. Only 37 of 245 isolates (15.1%) hybridized with the spvC DNA probe. All isolates that were recovered from a commercial egg production environment and chicken eggs and whose sequences exhibited homology with the spvC gene sequence were determined to be either Salmonella enteritidis PT 23 or PT 13. The sequences of few isolates from ceca and none from wastewater or humans demonstrated homology with the spvC gene.

  3. Isolation and genotyping of viable Toxoplasma gondii from sheep and goats in Ethiopia destined for human consumption.

    Science.gov (United States)

    Gebremedhin, Endrias Zewdu; Abdurahaman, Mukarim; Tessema, Tesfaye Sisay; Tilahun, Getachew; Cox, Eric; Goddeeris, Bruno; Dorny, Pierre; De Craeye, Stephane; Dardé, Marie-Laure; Ajzenberg, Daniel

    2014-09-04

    Toxoplasma gondii is an obligate intracellular protozoan parasite that infects humans and a broad spectrum of warm-blooded vertebrates. The present study was undertaken with the objectives of isolation and determining the genotypes of T. gondii strains from sheep and goats slaughtered in East and West Shewa Zones of Oromia Regional State, Central Ethiopia. Hearts of 47 sheep and 44 goats that were seropositive in the Direct Agglutination Test (DAT) were bioassayed in mice. A multiplex PCR assay with 15 microsatellite markers was employed for genotyping of T. gondii isolates from sheep and goats. Viable T. gondii were isolated from 47 (51.65%) animals, 27 sheep and 20 goats. Most isolates caused sub-clinical infections in mice, however, 2 sheep and 1 goat isolates were mouse-virulent, killing mice between 19-27 days post-inoculation. The success of T. gondii isolation in mice increased significantly (P = 0.0001) with higher DAT antibody titers in sheep and goats. Genotyping revealed that 29 (87.88%) of the 33 isolates were Type II, 3 (9.09%) were Type III and 1 (3.03%) was atypical. Three strains (one type II, one type III, and the atypical genotype) were virulent for mice. T. gondii tissue cysts in sheep and goats slaughtered for human consumption are widespread. This is the first report on isolation and genotyping of T. gondii from sheep and goats of Ethiopia.

  4. Molecular characterisation of Sporothrix schenckii isolates from humans and cats involved in the sporotrichosis epidemic in Rio de Janeiro, Brazil

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    Rosani Santos Reis

    2009-08-01

    Full Text Available An epidemic of sporotrichosis, a subcutaneous mycosis caused by the fungus Sporothrix schenckii, is ongoing in Rio de Janeiro, Brazil, in which cases of human infection are related to exposure to cats. In an attempt to demonstrate the zoonotic character of this epidemic using molecular methodology, we characterised by DNA-based typing methods 19 human and 25 animal S. schenckii isolates from the epidemic, as well as two control strains. To analyse the isolates, the random amplified polymorphic DNA (RAPD technique was performed using three different primers, together with DNA fingerprinting using the minisatellite derived from the wild-type phage M13 core-sequence. The analyses generated amplicons with considerable polymorphism. Although isolates exhibited high levels of genetic relatedness, they could be clustered into 5-10 genotypes. The RAPD profiles of epidemic S. schenckii isolates could be distinguished from that of the United States isolate, displaying 20% similarity to each primer and 60% when amplified with the M13 primer. DNA fingerprinting of S. schenckii isolated from the nails (42.8% and the oral cavities (66% of cats were identical to related human samples, suggesting that there is a common infection source for animals and humans in this epidemic. It is clear that cats act as a vehicle for dissemination of S. schenckii.

  5. Prevalence and molecular characterization of Clostridium difficile isolates from a pig slaughterhouse, pork, and humans in Taiwan.

    Science.gov (United States)

    Wu, Ying-Chen; Chen, Chih-Ming; Kuo, Chih-Jung; Lee, Jen-Jie; Chen, Pin-Chun; Chang, Yi-Chih; Chen, Ter-Hsin

    2017-02-02

    Clostridium difficile causes antibiotic-associated diarrhea in both humans and animals. The ribotype 078, predominant in food animals, is associated with community-acquired C. difficile infection, and C. difficile is suggested to be a foodborne pathogen. Recently, the C. difficile ribotype 078 lineage emerged in patients and pigs in Taiwan. This study aimed to investigate the prevalence and molecular characterization of C. difficile isolated from a pig slaughterhouse, retail meat, ready-to-eat meals, and humans in Taiwan. We collected samples from one slaughterhouse (n=422), 29 retail markets (raw pork, n=62; ready-to-eat pork, n=65), and one hospital (non-diarrheal humans, stool, n=317) in 2015. The isolated C. difficile were subjected to ribotyping and multilocus variable-number tandem-repeat analysis (MLVA). In the slaughterhouse, the isolation rate from carcasses was high (23%, 21/92) and ribotype 126 dominated. Scalding water was found to have C. difficile contamination (44%, 4/9), and two of the seven isolates were ribotype 126. The isolation rates from raw pork and ready-to-eat pork were between 20% and 29%. Ribotypes 126, 127, and 014 were found in raw pork, whereas ribotype 078 was not identified in this study. Eight isolates-seven non-toxigenic isolates and one ribotype 017-were found in non-diarrheal human samples. Notably, MLVA showed that ribotype 126 isolates from the slaughterhouse, pig stool, colons, carcasses, and scalding water were closely genetically related, indicating serious risk for cross-contamination. However, the genetic evidence of foodborne transmission from carcasses to food and humans is still lacking. Copyright © 2016. Published by Elsevier B.V.

  6. Prevalence of virulence and antimicrobial resistance genes in Salmonella spp. isolated from commercial chickens and human clinical isolates from South Africa and Brazil.

    Science.gov (United States)

    Zishiri, Oliver T; Mkhize, Nelisiwe; Mukaratirwa, Samson

    2016-05-26

    Salmonellosis is a significant public health concern around the world. The injudicious use of antimicrobial agents in poultry production for treatment, growth promotion and prophylaxis has resulted in the emergence of drug resistant strains of Salmonella. The current study was conducted to investigate the prevalence of virulence and antimicrobial resistance genes from Salmonella isolated from South African and Brazilian broiler chickens as well as human clinical isolates. Out of a total of 200 chicken samples that were collected from South Africa 102 (51%) tested positive for Salmonella using the InvA gene. Of the overall 146 Salmonella positive samples that were screened for the iroB gene most of them were confirmed to be Salmonella enterica with the following prevalence rates: 85% of human clinical samples, 68.6% of South African chicken isolates and 70.8% of Brazilian chicken samples. All Salmonella isolates obtained were subjected to antimicrobial susceptibility testing with 10 antibiotics. Salmonella isolates from South African chickens exhibited resistance to almost all antimicrobial agents used, such as tetracycline (93%), trimethoprim-sulfamthoxazole (84%), trimethoprim (78.4%), kanamycin (74%), gentamicin (48%), ampicillin (47%), amoxicillin (31%), chloramphenicol (31%), erythromycin (18%) and streptomycin (12%). All samples were further subjected to PCR in order to screen some common antimicrobial and virulence genes of interest namely spiC, pipD, misL, orfL, pse-1, tet A, tet B, ant (3")-la, sul 1 and sul. All Salmonella positive isolates exhibited resistance to at least one antimicrobial agent; however, antimicrobial resistance patterns demonstrated that multiple drug resistance was prevalent. The findings provide evidence that broiler chickens are colonised by pathogenic Salmonella harbouring antimicrobial resistance genes. Therefore, it is evident that there is a need for prudent use of antimicrobial agents in poultry production systems in order to

  7. Ingrown toenail relief drug products for over-the-counter human use. Final rule.

    Science.gov (United States)

    2003-05-07

    The Food and Drug Administration (FDA) is issuing a final rule establishing conditions under which over-the-counter (OTC) ingrown toenail relief drug products containing sodium sulfide 1 percent in a gel vehicle are generally recognized as safe and effective and not misbranded. This rule also amends the regulation that lists nonmonograph active ingredients in OTC drug products for ingrown toenail relief by removing sodium sulfide from that list. This final rule is part of FDA's ongoing review of OTC drug products.

  8. Molecular characterization showed limited genetic diversity among Salmonella Enteritidis isolated from humans and animals in Malaysia.

    Science.gov (United States)

    Ngoi, Soo Tein; Thong, Kwai Lin

    2013-12-01

    Salmonella enterica serovar Enteritidis (S. Enteritidis) is the most common causative agent of non-typhoidal salmonellosis in Malaysia. We aimed to characterize S. Enteritidis isolated from humans and animals by analyzing their antimicrobial resistance profiles and genotypes. A total of 111 strains were characterized using multiple-locus variable-number tandem repeat analysis, pulsed-field gel electrophoresis, and antimicrobial susceptibility testing. Both typing methods revealed that genetically similar S. Enteritidis strains had persisted among human and animal populations within the period of study (2003-2008). Only 39% of the strains were multi-drug resistant (i.e., resistant to 3 or more classes of antimicrobial agents), with a majority (73%) of these in low-risk phase (multiple antibiotic resistant index <0.20). Limited genetic diversity among clinical and zoonotic S. Enteritidis suggested that animals are possible sources of human salmonellosis. The degree of multi-drug resistance among the strains was generally low during the study period.

  9. Neisseria cinerea isolates can adhere to human epithelial cells by type IV pilus-independent mechanisms.

    Science.gov (United States)

    Wörmann, Mirka E; Horien, Corey L; Johnson, Errin; Liu, Guangyu; Aho, Ellen; Tang, Christoph M; Exley, Rachel M

    2016-03-01

    In pathogenic Neisseria species the type IV pili (Tfp) are of primary importance in host-pathogen interactions. Tfp mediate initial bacterial attachment to cell surfaces and formation of microcolonies via pilus-pilus interactions. Based on genome analysis, many non-pathogenic Neisseria species are predicted to express Tfp, but aside from studies on Neisseria elongata, relatively little is known about the formation and function of pili in these organisms. Here, we have analysed pilin expression and the role of Tfp in Neisseria cinerea. This non-pathogenic species shares a close taxonomic relationship to the pathogen Neisseria meningitidis and also colonizes the human oropharyngeal cavity. Through analysis of non-pathogenic Neisseria genomes we identified two genes with homology to pilE, which encodes the major pilin of N. meningitidis. We show which of the two genes is required for Tfp expression in N. cinerea and that Tfp in this species are required for DNA competence, similar to other Neisseria. However, in contrast to the meningococcus, deletion of the pilin gene did not impact the association of N. cinerea to human epithelial cells, demonstrating that N. cinerea isolates can adhere to human epithelial cells by Tfp-independent mechanisms.

  10. Tissue engineering potential of human dermis-isolated adult stem cells from multiple anatomical locations.

    Science.gov (United States)

    Kwon, Heenam; Haudenschild, Anne K; Brown, Wendy E; Vapniarsky, Natalia; Paschos, Nikolaos K; Arzi, Boaz; Hu, Jerry C; Athanasiou, Kyriacos A

    2017-01-01

    Abundance and accessibility render skin-derived stem cells an attractive cell source for tissue engineering applications. Toward assessing their utility, the variability of constructs engineered from human dermis-isolated adult stem (hDIAS) cells was examined with respect to different anatomical locations (foreskin, breast, and abdominal skin), both in vitro and in a subcutaneous, athymic mouse model. All anatomical locations yielded hDIAS cells with multi-lineage differentiation potentials, though adipogenesis was not seen for foreskin-derived hDIAS cells. Using engineered cartilage as a model, tissue engineered constructs from hDIAS cells were compared. Construct morphology differed by location. The mechanical properties of human foreskin- and abdominal skin-derived constructs were similar at implantation, remaining comparable after 4 additional weeks of culture in vivo. Breast skin-derived constructs were not mechanically testable. For all groups, no signs of abnormality were observed in the host. Addition of aggregate redifferentiation culture prior to construct formation improved chondrogenic differentiation of foreskin-derived hDIAS cells, as evident by increases in glycosaminoglycan and collagen contents. More robust Alcian blue staining and homogeneous cell populations were also observed compared to controls. Human DIAS cells elicited no adverse host responses, reacted positively to chondrogenic regimens, and possessed multi-lineage differentiation potential with the caveat that efficacy may differ by anatomical origin of the skin. Taken together, these results suggest that hDIAS cells hold promise as a potential cell source for a number of tissue engineering applications.

  11. A novel tandem repeat sequence located on human chromosome 4p: isolation and characterization.

    Science.gov (United States)

    Kogi, M; Fukushige, S; Lefevre, C; Hadano, S; Ikeda, J E

    1997-06-01

    In an effort to analyze the genomic region of the distal half of human chromosome 4p, to where Huntington disease and other diseases have been mapped, we have isolated the cosmid clone (CRS447) that was likely to contain a region with specific repeat sequences. Clone CRS447 was subjected to detailed analysis, including chromosome mapping, restriction mapping, and DNA sequencing. Chromosome mapping by both a human-CHO hybrid cell panel and FISH revealed that CRS447 was predominantly located in the 4p15.1-15.3 region. CRS447 was shown to consist of tandem repeats of 4.7-kb units present on chromosome 4p. A single EcoRI unit was subcloned (pRS447), and the complete sequence was determined as 4752 nucleotides. When pRS447 was used as a probe, the number of copies of this repeat per haploid genome was estimated to be 50-70. Sequence analysis revealed that it contained two internal CA repeats and one putative ORF. Database search established that this sequence was unreported. However, two homologous STS markers were found in the database. We concluded that CRS447/pRS447 is a novel tandem repeat sequence that is mainly specific to human chromosome 4p.

  12. Antimicrobial Resistance and Molecular Typing of Salmonella Stanley Isolated from Humans, Foods, and Environment.

    Science.gov (United States)

    Yang, Xiaowei; Kuang, Dai; Meng, Jianghong; Pan, Haijian; Shen, Junqing; Zhang, Jing; Shi, Weimin; Chen, Qi; Shi, Xianming; Xu, Xuebin; Zhang, Jianmin

    2015-12-01

    Salmonella enterica serovar Stanley is an important serovar that has been increasingly identified in human salmonellosis. The present study aimed to investigate the antimicrobial resistance and molecular typing of 88 Salmonella Stanley strains isolated from humans (diarrhea patients, n = 64; and healthy carrier, n = 1), foods (aquatic products, n = 16; vegetable, n = 1; and pork, n = 1), and environment (waste water, n = 2; and river water, n = 3) in Shanghai, China from 2006 to 2012. Nearly half of the strains were resistant to sulfafurazole (43/88, 48.9%), and many were resistant to streptomycin (35/88, 39.8%), tetracycline (22/88, 25%), and nalidixic acid (19/88, 21.6%). Approximately a quarter of the strains (24/88, 27.3%) were resistant to more than three antimicrobials, and five had ACSSuT resistance type. Six clusters (A-F) were identified by pulsed-field gel electrophoresis (PFGE) with 80% similarity. Interestingly, strains in the same cluster identified by PFGE possessed similar antibiotic resistance patterns. PFGE typing also indicated that aquatic products might serve as a transmission reservoir for Salmonella Stanley infections in humans.

  13. Selective Inhibition of Bakuchicin Isolated from Psoralea corylifolia on CYP1A in Human Liver Microsomes

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    Sun Joo Kim

    2016-01-01

    Full Text Available Bakuchicin is a furanocoumarin isolated from Psoralea corylifolia and shows several biological activities. Although there have been studies on the biological effects of bakuchicin, its modulation potency of CYP activities has not been previously investigated. Here, we investigated the inhibitory effects of bakuchicin on the activities of CYP isoforms by using a cocktail of probe substrates in pooled human liver microsomes (HLMs and human recombinant cDNA-expressed CYP. Bakuchicin strongly inhibited CYP1A-mediated phenacetin O-deethylation with an IC50 value of 0.43 μM in HLMs. It was confirmed by human recombinant cDNA-expressed CYP1A1 and CYP1A2 with a Ki value of 0.11 μM and 0.32 μM, respectively. A Lineweaver-Burk plot indicated that the inhibition mechanism of bakuchicin was competitive inhibition. Overall, this is the first study to investigate the potential CYP1A1 and CYP1A2 inhibition associated with bakuchicin and to report its competitive inhibitory effects on HLMs.

  14. Isolation and characterization of lactobacilli from human faeces and indigenous fermented foods for their potential application as probiotics.

    Science.gov (United States)

    Mandal, Hemanti; Jariwala, Ruchi; Bagchi, Tamishraha

    2016-04-01

    This study was conducted to select Lactobacillus strains from various sources on the basis of their probiotic attributes, such as acid and bile tolerance, binding to intestinal cells, and antimicrobial activity. Twelve isolates were obtained from human and food sources and were evaluated against standard probiotic Lactobacillus rhamnosus GG (LGG). Isolates were also studied for their antibiotic susceptibility. Isolate Lactobacillus fermentum GPI-6 showed the best survival profile at 0.3% and 1% bile salt, as compared with LGG. Isolates Lactobacillus plantarum GRI-2 and Lactobacillus salivarius GPI-4 showed no reduction in survival rate at pH 2.5. As expected, isolates showed strain-specific differences when comparing various attributes. Isolates GPI-4, GPI-7, and FA-5 showed better adhesion to HT-29, while isolate GPI-4 adhered better to Caco-2 cells than did LGG. However, when studying their ability to compete with Escherichia coli O26:H11, isolates GPI-6 and GPI-7 significantly inhibited E. coli adhesion to both HT-29 and Caco-2 cells compared with LGG. In conclusion, isolates GPI-4, GPI-7, and FA-5 showed excellent binding ability and antagonistic activity and better tolerance to acidic pH (pH 2.5) and to different bile salt concentrations in comparison with LGG, and hence, they could be considered as potential probiotic candidates.

  15. Anti-proliferative effect of a compound isolated from Cassia auriculata against human colon cancer cell line HCT 15.

    Science.gov (United States)

    Esakkirajan, M; Prabhu, N M; Arulvasu, C; Beulaja, M; Manikandan, R; Thiagarajan, R; Govindaraju, K; Prabhu, D; Dinesh, D; Babu, G; Dhanasekaran, G

    2014-01-01

    The compound was isolated from leaves of Cassia auriculata and its structure was characterized using high-performance liquid chromatography (HPLC), liquid chromatography mass spectrometry (LC-MS), UV-vis spectroscopy (UV-vis), fourier transform infrared spectroscopy (FT-IR) and nuclear magnetic resonance spectroscopy (NMR). 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay, cytotoxicity, nuclear morphology and lactate dehydrogenase assay of isolated compound was tested against human colon cancer cell line HCT 15. The isolated compound, 4-(4-chlorobenzyl)-2,3,4,5,6,7-hexahydro-7-(2-ethoxyphenyl)benzo[h][1,4,7]triazecin-8(1H)-one at 25μg/ml concentration and by 48h showed 50% inhibition of human colon cancer cells (HCT 15). The results suggest that isolated compound from C. auriculata has potential to prevent colon cancer cell line.

  16. Final report. Human artificial episomal chromosome (HAEC) for building large genomic libraries

    Energy Technology Data Exchange (ETDEWEB)

    Jean-Michael H. Vos

    1999-12-09

    Collections of human DNA fragments are maintained for research purposes as clones in bacterial host cells. However for unknown reasons, some regions of the human genome appear to be unclonable or unstable in bacteria. Their team has developed a system using episomes (extrachromosomal, autonomously replication DNA) that maintains large DNA fragments in human cells. This human artificial episomal chromosomal (HAEC) system may prove useful for coverage of these especially difficult regions. In the broader biomedical community, the HAEC system also shows promise for use in functional genomics and gene therapy. Recent improvements to the HAEC system and its application to mapping, sequencing, and functionally studying human and mouse DNA are summarized. Mapping and sequencing the human genome and model organisms are only the first steps in determining the function of various genetic units critical for gene regulation, DNA replication, chromatin packaging, chromosomal stability, and chromatid segregation. Such studies will require the ability to transfer and manipulate entire functional units into mammalian cells.

  17. Characterization of influenza A (H7N9 viruses isolated from human cases imported into Taiwan.

    Directory of Open Access Journals (Sweden)

    Ji-Rong Yang

    Full Text Available A novel avian influenza A (H7N9 virus causes severe human infections and was first identified in March 2013 in China. The H7N9 virus has exhibited two epidemiological peaks of infection, occurring in week 15 of 2013 and week 5 of 2014. Taiwan, which is geographically adjacent to China, faces a large risk of being affected by this virus. Through extensive surveillance, launched in April 2013, four laboratory-confirmed H7N9 cases imported from China have been identified in Taiwan. The H7N9 virus isolated from imported case 1 in May 2013 (during the first wave was found to be closest genetically to a virus from wild birds and differed from the prototype virus, A/Anhui/1/2013, in the MP gene. The other three imported cases were detected in December 2013 and April 2014 (during the second wave. The viruses isolated from cases 2 and 4 were similar in the compositions of their 6 internal genes and distinct from A/Anhui/1/2013 in the PB2 and MP genes, whereas the virus isolated from case 3 exhibited a novel reassortment that has not been identified previously and was different from A/Anhui/1/2013 in the PB2, PA and MP genes. The four imported H7N9 viruses share similar antigenicity with A/Anhui/1/2013, and their HA and NA genes grouped together in their respective phylogenies. In contrast with the HA and NA genes, which exhibited a smaller degree of diversity, the internal genes were heterogeneous and provided potential distinctions between transmission sources in terms of both geography and hosts. It is important to strengthen surveillance of influenza and to share viral genetic data in real-time for reducing the threat of rapid and continuing evolution of H7N9 viruses.

  18. Comparative genomics of Tunisian Leishmania major isolates causing human cutaneous leishmaniasis with contrasting clinical severity.

    Science.gov (United States)

    Ghouila, Amel; Guerfali, Fatma Z; Atri, Chiraz; Bali, Aymen; Attia, Hanene; Sghaier, Rabiaa M; Mkannez, Ghada; Dickens, Nicholas J; Laouini, Dhafer

    2017-06-01

    Zoonotic cutaneous leishmaniasis caused by Leishmania (L.) major parasites affects urban and suburban areas in the center and south of Tunisia where the disease is endemo-epidemic. Several cases were reported in human patients for which infection due to L. major induced lesions with a broad range of severity. However, very little is known about the mechanisms underlying this diversity. Our hypothesis is that parasite genomic variability could, in addition to the host immunological background, contribute to the intra-species clinical variability observed in patients and explain the lesion size differences observed in the experimental model. Based on several epidemiological, in vivo and in vitro experiments, we focused on two clinical isolates showing contrasted severity in patients and BALB/c experimental mice model. We used DNA-seq as a high-throughput technology to facilitate the identification of genetic variants with discriminating potential between both isolates. Our results demonstrate that various levels of heterogeneity could be found between both L. major isolates in terms of chromosome or gene copy number variation (CNV), and that the intra-species divergence could surprisingly be related to single nucleotide polymorphisms (SNPs) and Insertion/Deletion (InDels) events. Interestingly, we particularly focused here on genes affected by both types of variants and correlated them with the observed gene CNV. Whether these differences are sufficient to explain the severity in patients is obviously still open to debate, but we do believe that additional layers of -omic information is needed to complement the genomic screen in order to draw a more complete map of severity determinants.

  19. Bordetella pertussis naturally occurring isolates with altered lipooligosaccharide structure fail to fully mature human dendritic cells.

    Science.gov (United States)

    Brummelman, Jolanda; Veerman, Rosanne E; Hamstra, Hendrik Jan; Deuss, Anna J M; Schuijt, Tim J; Sloots, Arjen; Kuipers, Betsy; van Els, Cécile A C M; van der Ley, Peter; Mooi, Frits R; Han, Wanda G H; Pinelli, Elena

    2015-01-01

    Bordetella pertussis is a Gram-negative bacterium and the causative agent of whooping cough. Despite high vaccination coverage, outbreaks are being increasingly reported worldwide. Possible explanations include adaptation of this pathogen, which may interfere with recognition by the innate immune system. Here, we describe innate immune recognition and responses to different B. pertussis clinical isolates. By using HEK-Blue cells transfected with different pattern recognition receptors, we found that 3 out of 19 clinical isolates failed to activate Toll-like receptor 4 (TLR4). These findings were confirmed by using the monocytic MM6 cell line. Although incubation with high concentrations of these 3 strains resulted in significant activation of the MM6 cells, it was found to occur mainly through interaction with TLR2 and not through TLR4. When using live bacteria, these 3 strains also failed to activate TLR4 on HEK-Blue cells, and activation of MM6 cells or human monocyte-derived dendritic cells was significantly lower than activation induced by the other 16 strains. Mass spectrum analysis of the lipid A moieties from these 3 strains indicated an altered structure of this molecule. Gene sequence analysis revealed mutations in genes involved in lipid A synthesis. Findings from this study indicate that B. pertussis isolates that do not activate TLR4 occur naturally and that this phenotype may give this bacterium an advantage in tempering the innate immune response and establishing infection. Knowledge on the strategies used by this pathogen in evading the host immune response is essential for the improvement of current vaccines or for the development of new ones.

  20. Chromosome region-specific libraries for human genome analysis. Final progress report, 1 March 1991--28 February 1994

    Energy Technology Data Exchange (ETDEWEB)

    Kao, F.T.

    1994-04-01

    The objectives of this grant proposal include (1) development of a chromosome microdissection and PCR-mediated microcloning technology, (2) application of this microtechnology to the construction of region-specific libraries for human genome analysis. During this grant period, the authors have successfully developed this microtechnology and have applied it to the construction of microdissection libraries for the following chromosome regions: a whole chromosome 21 (21E), 2 region-specific libraries for the long arm of chromosome 2, 2q35-q37 (2Q1) and 2q33-q35 (2Q2), and 4 region-specific libraries for the entire short arm of chromosome 2, 2p23-p25 (2P1), 2p21-p23 (2P2), 2p14-p16 (wP3) and 2p11-p13 (2P4). In addition, 20--40 unique sequence microclones have been isolated and characterized for genomic studies. These region-specific libraries and the single-copy microclones from the library have been used as valuable resources for (1) isolating microsatellite probes in linkage analysis to further refine the disease locus; (2) isolating corresponding clones with large inserts, e.g. YAC, BAC, P1, cosmid and phage, to facilitate construction of contigs for high resolution physical mapping; and (3) isolating region-specific cDNA clones for use as candidate genes. These libraries are being deposited in the American Type Culture Collection (ATCC) for general distribution.

  1. Procedure for conducting a human-reliability analysis for nuclear power plants. Final report

    Energy Technology Data Exchange (ETDEWEB)

    Bell, B.J.; Swain, A.D.

    1983-05-01

    This document describes in detail a procedure to be followed in conducting a human reliability analysis as part of a probabilistic risk assessment when such an analysis is performed according to the methods described in NUREG/CR-1278, Handbook for Human Reliability Analysis with Emphasis on Nuclear Power Plant Applications. An overview of the procedure describing the major elements of a human reliability analysis is presented along with a detailed description of each element and an example of an actual analysis. An appendix consists of some sample human reliability analysis problems for further study.

  2. Isolation of human salivary extracellular vesicles by iodixanol density gradient ultracentrifugation and their characterizations

    Directory of Open Access Journals (Sweden)

    Kazuya Iwai

    2016-05-01

    Full Text Available Diagnostic methods that focus on the extracellular vesicles (EVs present in saliva have been attracting great attention because of their non-invasiveness. EVs contain biomolecules such as proteins, messenger RNA (mRNA and microRNA (miRNA, which originate from cells that release EVs, making them an ideal source for liquid biopsy. Although there have been many reports on density-based fractionation of EVs from blood and urine, the number of reports on EVs from saliva has been limited, most probably because of the difficulties in separating EVs from viscous saliva using density gradient centrifugation. This article establishes a protocol for the isolation of EVs from human saliva using density gradient centrifugation. The fractionated salivary EVs were characterized by atomic force microscopy, western blot and reverse transcription polymerase chain reaction. The results indicate that salivary EVs have a smaller diameter (47.8±12.3 nm and higher density (1.11 g/ml than EVs isolated from conditioned cell media (74.0±23.5 nm and 1.06 g/ml, respectively. Additionally, to improve the throughput of density-based fractionation of EVs, the original protocol was further modified by using a fixed angle rotor instead of a swinging rotor. It was also confirmed that several miRNAs were expressed strongly in the EV-marker-expressing fractions.

  3. Isolation of human salivary extracellular vesicles by iodixanol density gradient ultracentrifugation and their characterizations

    Science.gov (United States)

    Iwai, Kazuya; Minamisawa, Tamiko; Suga, Kanako; Yajima, Yasutomo; Shiba, Kiyotaka

    2016-01-01

    Diagnostic methods that focus on the extracellular vesicles (EVs) present in saliva have been attracting great attention because of their non-invasiveness. EVs contain biomolecules such as proteins, messenger RNA (mRNA) and microRNA (miRNA), which originate from cells that release EVs, making them an ideal source for liquid biopsy. Although there have been many reports on density-based fractionation of EVs from blood and urine, the number of reports on EVs from saliva has been limited, most probably because of the difficulties in separating EVs from viscous saliva using density gradient centrifugation. This article establishes a protocol for the isolation of EVs from human saliva using density gradient centrifugation. The fractionated salivary EVs were characterized by atomic force microscopy, western blot and reverse transcription polymerase chain reaction. The results indicate that salivary EVs have a smaller diameter (47.8±12.3 nm) and higher density (1.11 g/ml) than EVs isolated from conditioned cell media (74.0±23.5 nm and 1.06 g/ml, respectively). Additionally, to improve the throughput of density-based fractionation of EVs, the original protocol was further modified by using a fixed angle rotor instead of a swinging rotor. It was also confirmed that several miRNAs were expressed strongly in the EV-marker-expressing fractions. PMID:27193612

  4. Isolation of human immunodeficiency virus from genital ulcers in Nairobi prostitutes.

    Science.gov (United States)

    Kreiss, J K; Coombs, R; Plummer, F; Holmes, K K; Nikora, B; Cameron, W; Ngugi, E; Ndinya Achola, J O; Corey, L

    1989-09-01

    Recent epidemiologic studies have implicated genital/anorectal ulcer disease as an important cofactor for acquisition and transmission of human immunodeficiency virus (HIV) during sexual intercourse. To better understand the mechanism for the association between genital ulcers and HIV, exudates from 62 genital ulcers of 56 HIV-seropositive prostitutes in Nairobi (Kenya) were cultured for HIV. Twenty-six ulcer cultures could not be evaluated for the presence of HIV because of bacterial or fungal contamination. HIV was isolated from 4 (11%) of the 36 remaining uncontaminated ulcer cultures (2 introital, 1 vaginal, and 1 cervical) from 4 separate women. HIV was isolated from the cervical os from only 2 of the 4 women. HIV p24 antigen was detected in exudate from 1 of the 4 culture-positive ulcers and 0 of 32 culture-negative ulcers. Genital ulcers in seropositive patients should be regarded as potential sources of HIV, which could be important in transmission of HIV during intercourse. Public health measures aimed at controlling sexually transmitted genital ulcer diseases should be an integral part of acquired immunodeficiency syndrome (AIDS) prevention programs.

  5. In vitro characterization of human dental pulp stem cells isolated by three different methods

    Directory of Open Access Journals (Sweden)

    Ji-Hyun Jang

    2016-11-01

    Full Text Available Objectives In this study, we characterized human dental pulp cells (HDPCs obtained by different culture methods to establish the most suitable methodology for dental tissue engineering and regenerative endodontic applications. Materials and Methods HDPCs were isolated by the outgrowth method (HDPCs-OG, the enzymatic digestion method (collagenase/dispase/trypsin, HDPCs-ED, or the combination of both methods (HDPCs-Combined. The expression of mesenchymal stem cell markers (CD105, CD90, and CD73 was investigated. In vitro differentiation capacities of HDPCs into adipogenic, osteogenic, and chondrogenic lineages were compared. Differentiation markers were analyzed by quantitative reverse-transcription polymerase chain reaction (RT-PCR and western blotting. Results Our data indicated that whole HDPCs-ED, HPDCs-OG, and HDPCs-Combined could be differentiated into adipogenic, chrondrogenic, and osteogenic cell types. However, we found that the methods for isolating and culturing HDPCs influence the differentiation capacities of cells. HDPCs-OG and HDPCs-ED were preferably differentiated into adipogenic and osteogenic cells, respectively. Differentiation markers shown by RT-PCR and western blotting analysis were mostly upregulated in the treated groups compared with the control groups. Conclusions Our findings confirmed that cell populations formed by two different culture methods and the combined culture method exhibited different properties. The results of this study could provide an insight into regenerative endodontic treatment using HDPCs.

  6. Isolation of Anti-Ricin Protective Antibodies Exhibiting High Affinity from Immunized Non-Human Primates

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    Tal Noy-Porat

    2016-03-01

    Full Text Available Ricin, derived from the castor bean plant Ricinus communis, is one of the most potent and lethal toxins known, against which there is no available antidote. To date, the use of neutralizing antibodies is the most promising post-exposure treatment for ricin intoxication. The aim of this study was to isolate high affinity anti-ricin antibodies that possess potent toxin-neutralization capabilities. Two non-human primates were immunized with either a ricin-holotoxin- or subunit-based vaccine, to ensure the elicitation of diverse high affinity antibodies. By using a comprehensive set of primers, immune scFv phage-displayed libraries were constructed and panned. A panel of 10 antibodies (five directed against the A subunit of ricin and five against the B subunit was isolated and reformatted into a full-length chimeric IgG. All of these antibodies were found to neutralize ricin in vitro, and several conferred full protection to ricin-intoxicated mice when given six hours after exposure. Six antibodies were found to possess exceptionally high affinity toward the toxin, with KD values below pM (koff < 1 × 10−7 s−1 that were well correlated with their ability to neutralize ricin. These antibodies, alone or in combination, could be used for the development of a highly-effective therapeutic preparation for post-exposure treatment of ricin intoxication.

  7. Genetic similarity between cysticerci of Taenia solium isolated from human brain and from pigs.

    Science.gov (United States)

    Hinojosa-Juarez, Araceli Consuelo; Sandoval-Balanzario, Miguel; McManus, Donald Peter; Monroy-Ostria, Amalia

    2008-09-01

    Mitochondrial (mt) cox1 and ribosomal ITS1 DNA sequences from Taenia solium cysticercus isolates from pigs and cysticerci (racemose and cellulose types) from patients with neurocysticercosis were amplified by the polymerase chain reaction (PCR). The amplicons were sequenced in order to determine the genetic relationship between these types of cysticerci. Phylogenetic trees were constructed and evolutionary distances were calculated. ITS1 and mt cox1 cysticerci sequence data were compared with previously published Taenia spp. sequences. The variation in the ITS1 and cox1 sequences of samples collected from Mexico was minimal, regardless of geographical origin, size or colour of cysticerci from either pigs or human brain. These results suggest that the racemose and cellulose types represent genetically identical metacestodes of T. solium. Alignment of the mt cox1 sequences of the Mexican samples with sequences of other Taenia taxa showed that most were very similar to T. solium from Mexico and T. solium from Colombia; one T. solium Mexican isolate and Taenia hydatigena were placed in the same group close to Taenia crassiceps. The ITS1 sequences for the Mexican T. solium samples indicated the majority were in the same group as the Latin American T. solium. Two Mexican T. solium samples and T. solium from Philippines were placed together in a different group.

  8. Chromatographic Monoliths for High-Throughput Immunoaffinity Isolation of Transferrin from Human Plasma

    Directory of Open Access Journals (Sweden)

    Irena Trbojević-Akmačić

    2016-06-01

    Full Text Available Changes in protein glycosylation are related to different diseases and have a potential as diagnostic and prognostic disease biomarkers. Transferrin (Tf glycosylation changes are common marker for congenital disorders of glycosylation. However, biological interindividual variability of Tf N-glycosylation and genes involved in glycosylation regulation are not known. Therefore, high-throughput Tf isolation method and large scale glycosylation studies are needed in order to address these questions. Due to their unique chromatographic properties, the use of chromatographic monoliths enables very fast analysis cycle, thus significantly increasing sample preparation throughput. Here, we are describing characterization of novel immunoaffinity-based monolithic columns in a 96-well plate format for specific high-throughput purification of human Tf from blood plasma. We optimized the isolation and glycan preparation procedure for subsequent ultra performance liquid chromatography (UPLC analysis of Tf N-glycosylation and managed to increase the sensitivity for approximately three times compared to initial experimental conditions, with very good reproducibility. This work is licensed under a Creative Commons Attribution 4.0 International License.

  9. Characteristics of Quinolone Resistance in Escherichia coli Isolates from Humans, Animals, and the Environment in the Czech Republic

    Science.gov (United States)

    Röderova, Magdalena; Halova, Dana; Papousek, Ivo; Dolejska, Monika; Masarikova, Martina; Hanulik, Vojtech; Pudova, Vendula; Broz, Petr; Htoutou-Sedlakova, Miroslava; Sauer, Pavel; Bardon, Jan; Cizek, Alois; Kolar, Milan; Literak, Ivan

    2017-01-01

    Escherichia coli is a common commensal bacterial species of humans and animals that may become a troublesome pathogen causing serious diseases. The aim of this study was to characterize the quinolone resistance phenotypes and genotypes in E. coli isolates of different origin from one area of the Czech Republic. E. coli isolates were obtained from hospitalized patients and outpatients, chicken farms, retailed turkeys, rooks wintering in the area, and wastewaters. Susceptibility of the isolates grown on the MacConkey agar with ciprofloxacin (0.05 mg/L) to 23 antimicrobial agents was determined. The presence of plasmid-mediated quinolone resistance (PMQR) and ESBL genes was tested by PCR and sequencing. Specific mutations in gyrA, gyrB, parC, and parE were also examined. Multilocus sequence typing and pulsed-field gel electrophoresis were performed to assess the clonal relationship. In total, 1050 E. coli isolates were obtained, including 303 isolates from humans, 156 from chickens, 105 from turkeys, 114 from the rooks, and 372 from wastewater samples. PMQR genes were detected in 262 (25%) isolates. The highest occurrence was observed in isolates from retailed turkey (49% of the isolates were positive) and inpatients (32%). The qnrS1 gene was the most common PMQR determinant identified in 146 (56%) followed by aac(6′)-Ib-cr in 77 (29%), qnrB19 in 41 (16%), and qnrB1 in 9 (3%) isolates. All isolates with high level of ciprofloxacin resistance (>32 mg/L) carried double or triple mutations in gyrA combined with single or double mutations in parC. The most frequently identified substitutions were Ser(83)Leu; Asp(87)Asn in GyrA, together with Ser(80)Ile, or Glu(84)Val in ParC. Majority of these isolates showed resistance to beta-lactams and multiresistance phenotype was found in 95% isolates. Forty-eight different sequence types among 144 isolates analyzed were found, including five major clones ST131 (26), ST355 (19), ST48 (13), ST95 (10), and ST10 (5). No isolates

  10. Isolation of PCR ready-human DNA using copper nanoparticles from skeletal remains.

    Science.gov (United States)

    Lodha, Anand; Ansari, Niha; Shah, Shahil; Rao, M V; Menon, Shobhana K

    2017-01-01

    Present study represents a novel approach of PCR ready-human DNA extraction method from skeletal remains using copper nanoparticles (CuNPs) for personnel identification. To achieve rapid, cost effective, sensitive and non-hazardous method for DNA extraction we utilized CuNPs synthesized using microwave. The applicability of this approach was first tested in blood samples and afterwards, this system was extended to skeletal remains' samples also. This method yields good quality DNA that are ready for PCR reactions from small quantities of blood and skeletal remains. Consequently, even small quantities of nanoparticles could be potentially utilized for a highly efficient isolation of DNA from skeletal remains as well as from ancient archaeological samples. The present method has the advantages that it is quick with high yield, inexpensive, robust, environment friendly and does not require use of hazardous organic solvents. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

  11. Seasonality of Campylobacter jejuni isolates associated with human campylobacteriosis in the Manawatu region, New Zealand.

    Science.gov (United States)

    Friedrich, A; Marshall, J C; Biggs, P J; Midwinter, A C; French, N P

    2016-03-01

    A 9-year time-series of genotyped human campylobacteriosis cases from the Manawatu region of New Zealand was used to investigate strain-type seasonality. The data were collected from 2005 to 2013 and the samples were multi-locus sequence-typed (MLST). The four most prevalent clonal complexes (CCs), consisting of 1215 isolates, were CC48, CC21, CC45 and CC61. Seasonal decomposition and Poisson regression with autocorrelated errors, were used to display and test for seasonality of the most prevalent CCs. Of the four examined CCs, only CC45 showed a marked seasonal (summer) peak. The association of CC45 with summer peaks has been observed in other temperate countries, but has previously not been identified in New Zealand. This is the first in-depth study over a long time period employing MLST data to examine strain-type-associated seasonal patterns of C. jejuni infection in New Zealand.

  12. Isolation and evaluation of cytogenetic effect of Brahmi saponins on cultured human lymphocytes exposed in vitro.

    Science.gov (United States)

    Kalachaveedu, Mangathayaru; Papacchan, Sunu; Sanyal, Sudip; Koshy, Teena; Telapolu, Srivani

    2015-01-01

    Major saponins of Brahmi (Bacopa monniera, Fam: Scrophulariaceae) - bacosides A and B - were isolated from the total methanol extract and characterised based on melting point, TLC, IR, (1)H NMR and (13)C NMR. They were evaluated for their in vitro cytogenetic effects on human peripheral blood lymphocytes by chromosomal aberration (CA) assay and sister chromatid exchange (SCE) assay. The frequency of chromatid type aberrations and reciprocal interchanges between sister chromatids in the treated cells was scored in comparison to the untreated control. At 30 μg/mL dose, bacoside A showed a statistically significant increase in the frequency of both CA and SCE and bacoside B showed an increase only in SCE. Our report of the genotoxicity of the saponins is significant in view of the reports of anticancer activity of Brahmi extracts.

  13. Isolation of Osteosarcoma-Associated Human Antibodies from a Combinatorial Fab Phage Display Library

    Directory of Open Access Journals (Sweden)

    Carmela Dantas-Barbosa

    2009-01-01

    Full Text Available Osteosarcoma, a highly malignant disease, is the most common primary bone tumor and is frequently found in children and adolescents. In order to isolate antibodies against osteosarcoma antigens, a combinatorial osteosarcoma Fab library displayed on the surface of phages was used. After three rounds of selection on the surface of tumor cells, several osteosarcoma-reactive Fabs were detected. From these Fabs, five were better characterized, and despite having differences in their VH (heavy chain variable domain and Vκ (kappa chain variable domain regions, they all bound to a protein with the same molecular mass. Further analysis by cell ELISA and immunocytochemistry suggested that the Fabs recognize a membrane-associated tumor antigen expressed in higher amounts in neoplasic cells than in normal tissue. These results suggest that the human Fabs selected in this work are a valuable tool for the study of this neoplasia.

  14. Spacial isolation of protein kinase C activation in thrombin stimulated human platelets.

    Science.gov (United States)

    Crouch, M F; Lapetina, E G

    1988-10-14

    Thrombin stimulation of human platelets is associated with turnover of inositol phospholipids, mobilization of intracellular Ca2+ stores, and activation of protein kinase C. However, within 5 minutes, the thrombin receptor desensitizes, but can be re-coupled to its effectors by stimulation of alpha 2-adrenergic receptors (Crouch and Lapetina, J. Biol. Chem. 263, 3363-3371, 1988). This effect of epinephrine was found to be inhibited by preincubation of platelets with phorbol ester, suggesting that protein kinase C was inhibitory. However, since thrombin also activated protein kinase C and epinephrine was active following thrombin stimulation of platelets, this implied that thrombin activation of protein kinase C may have been spacially isolated near the thrombin receptor and could not inactivate alpha 2-receptor activity. In the present paper, we have tested this possibility, and we present evidence which strongly favours the possibility that protein kinase C activation by receptors induces its local translocation to the cell membrane.

  15. Assessment of an ad hoc procedure for isolation and characterization of human albuminome.

    Science.gov (United States)

    Scumaci, Domenica; Gaspari, Marco; Saccomanno, Milena; Argirò, Giuseppe; Quaresima, Barbara; Faniello, Concetta M; Ricci, Pietrantonio; Costanzo, Francesco; Cuda, Giovanni

    2011-11-01

    The dynamic range of plasma protein abundance, ranging from milligrams to picograms per milliliter, makes characterization of this proteome nearly impossible with current analytical methods. Plasma preprocessing by high-abundance protein depletion may concomitantly remove important diagnostic information. This article describes an original chromatographic procedure to isolate proteins bound to human serum albumin (HSA). Using HSA as an "affinity agent", we significantly improved the detection and identification of HSA ligands by two-dimensional liquid chromatography tandem mass spectrometry (2D LC-MS/MS). Some of the characterized species were not previously reported in published blood databases. Albumin-binding proteins may be classified as belonging to several putative functional categories and span a wide variety of predicted physiological functions.

  16. Molecular characterization of human Trypanosoma cruzi isolates from endemic areas in Panama

    Directory of Open Access Journals (Sweden)

    Octavio E Sousa

    2006-06-01

    Full Text Available The present work provides information on Trypanosoma cruzi genotype circulating in endemic areas of Chagas disease in Panama. A total of 26 crude stocks of T. cruzi, isolated from the blood of persons with different clinical profiles of Chagas disease were collected and crio-conserved until used. Most of the stocks had been characterized by means of isoenzyme electrophoresis on cellulose acetate membranes. The clinical profiles of infected persons included 9 (34.6% asymptomatic and 17 acute (65.4% including 5 (19.2% fatal cases, 2 under 5 years old and 3 adults. A multiplex-PCR assay based on the amplification of the non-transcribed spacer of the mini-exon gene was performed. All stocks of T. cruzi included in the study were found to correspond to Tc I group. This result supports the predominance of T. cruzi-I in the transmission cycles affecting the human population in the Republic of Panama.

  17. Isolation and identification of quercetin degrading bacteria from human fecal microbes.

    Directory of Open Access Journals (Sweden)

    Zhichao Zhang

    Full Text Available Quercetin has a wide range of biological properties. The gut microflora can often modulate its biological activity and their potential health effects. There still is a lack of information about gut bacteria involving in this process. The strains of gut microbes from human feces that can transform quercetin were isolated and identified by in vitro fermentation. The results showed that Escherichia coli, Stretococcus lutetiensis, Lactobacillus acidophilus, Weissella confusa, Enterococcus gilvus, Clostridium perfringens and Bacteroides fragilis have the various ability of degrading quercetin. Among them, C. perfringens and B. fragilis were discovered to have the strongest ability of degrading quercetin. Additionally, quercetin can't inhibit the growth of C. perfringens. In conclusion, many species of gut microbiota can degrade quercetin, but their ability are different.

  18. Evaluation of Escherichia coli isolates from healthy chickens to determine their potential risk to poultry and human health.

    Science.gov (United States)

    Stromberg, Zachary R; Johnson, James R; Fairbrother, John M; Kilbourne, Jacquelyn; Van Goor, Angelica; Curtiss, Roy; Mellata, Melha

    2017-01-01

    Extraintestinal pathogenic Escherichia coli (ExPEC) strains are important pathogens that cause diverse diseases in humans and poultry. Some E. coli isolates from chicken feces contain ExPEC-associated virulence genes, so appear potentially pathogenic; they conceivably could be transmitted to humans through handling and/or consumption of contaminated meat. However, the actual extraintestinal virulence potential of chicken-source fecal E. coli is poorly understood. Here, we assessed whether fecal E. coli isolates from healthy production chickens could cause diseases in a chicken model of avian colibacillosis and three rodent models of ExPEC-associated human infections. From 304 E. coli isolates from chicken fecal samples, 175 E. coli isolates were screened by PCR for virulence genes associated with human-source ExPEC or avian pathogenic E. coli (APEC), an ExPEC subset that causes extraintestinal infections in poultry. Selected isolates genetically identified as ExPEC and non-ExPEC isolates were assessed in vitro for virulence-associated phenotypes, and in vivo for disease-causing ability in animal models of colibacillosis, sepsis, meningitis, and urinary tract infection. Among the study isolates, 13% (40/304) were identified as ExPEC; the majority of these were classified as APEC and uropathogenic E. coli, but none as neonatal meningitis E. coli. Multiple chicken-source fecal ExPEC isolates resembled avian and human clinical ExPEC isolates in causing one or more ExPEC-associated illnesses in experimental animal infection models. Additionally, some isolates that were classified as non-ExPEC were able to cause ExPEC-associated illnesses in animal models, and thus future studies are needed to elucidate their mechanisms of virulence. These findings show that E. coli isolates from chicken feces contain ExPEC-associated genes, exhibit ExPEC-associated in vitro phenotypes, and can cause ExPEC-associated infections in animal models, and thus may pose a health threat to

  19. Evaluation of Escherichia coli isolates from healthy chickens to determine their potential risk to poultry and human health

    Science.gov (United States)

    Johnson, James R.; Fairbrother, John M.; Kilbourne, Jacquelyn; Van Goor, Angelica; Curtiss, Roy; Mellata, Melha

    2017-01-01

    Extraintestinal pathogenic Escherichia coli (ExPEC) strains are important pathogens that cause diverse diseases in humans and poultry. Some E. coli isolates from chicken feces contain ExPEC-associated virulence genes, so appear potentially pathogenic; they conceivably could be transmitted to humans through handling and/or consumption of contaminated meat. However, the actual extraintestinal virulence potential of chicken-source fecal E. coli is poorly understood. Here, we assessed whether fecal E. coli isolates from healthy production chickens could cause diseases in a chicken model of avian colibacillosis and three rodent models of ExPEC-associated human infections. From 304 E. coli isolates from chicken fecal samples, 175 E. coli isolates were screened by PCR for virulence genes associated with human-source ExPEC or avian pathogenic E. coli (APEC), an ExPEC subset that causes extraintestinal infections in poultry. Selected isolates genetically identified as ExPEC and non-ExPEC isolates were assessed in vitro for virulence-associated phenotypes, and in vivo for disease-causing ability in animal models of colibacillosis, sepsis, meningitis, and urinary tract infection. Among the study isolates, 13% (40/304) were identified as ExPEC; the majority of these were classified as APEC and uropathogenic E. coli, but none as neonatal meningitis E. coli. Multiple chicken-source fecal ExPEC isolates resembled avian and human clinical ExPEC isolates in causing one or more ExPEC-associated illnesses in experimental animal infection models. Additionally, some isolates that were classified as non-ExPEC were able to cause ExPEC-associated illnesses in animal models, and thus future studies are needed to elucidate their mechanisms of virulence. These findings show that E. coli isolates from chicken feces contain ExPEC-associated genes, exhibit ExPEC-associated in vitro phenotypes, and can cause ExPEC-associated infections in animal models, and thus may pose a health threat to

  20. Assessment and characterization of biofilm formation among human isolates of Streptococcus dysgalactiae subsp. equisimilis.

    Science.gov (United States)

    Genteluci, Gabrielle Limeira; Silva, Ligia Guedes; Souza, Maria Clara; Glatthardt, Thaís; de Mattos, Marcos Corrêa; Ejzemberg, Regina; Alviano, Celuta Sales; Figueiredo, Agnes Marie Sá; Ferreira-Carvalho, Bernadete Teixeira

    2015-12-01

    The capacity to form biofilm is considered a protective mechanism that allows the bacteria to survive and proliferate in hostile environments, facilitating the maintenance of the infectious process. Recently, biofilm has become a topic of interest in the study of the human pathogen group A Streptococcus (GAS). Although GAS has not been associated with infection on medical implants, the presence of microcolonies embedded in an extracellular matrix on infected tissues has been reported. Despite the similarity between GAS and Streptococcus dysgalactiae subspecies equisimilis (SDSE), there are no studies in the literature describing the production of biofilm by SDSE. In this work, we assessed and characterized biofilm development among SDSE human isolates of group C. The in vitro data showed that 59.3% of the 118 isolates tested were able to form acid-induced biofilm on glass, and 28% formed it on polystyrene surfaces. More importantly, biofilm was also formed in a foreign body model in mice. The biofilm structure was analyzed by confocal laser scanning microscopy, transmission electron microscopy, and scanning electron microscopy. Long fibrillar-like structures were observed by scanning electron microscopy. Additionally, the expression of a pilus associated gene of SDSE was increased for in vitro sessile cells compared with planktonics, and when sessile cells were collected from biofilms formed in the animal model compared with that of in vitro model. Results obtained from the immunofluorescence microscopy indicated the biofilm was immunogenic. Our data also suggested a role for proteins, exopolysaccharide and extracellular DNA in the formation and accumulation of biofilm by SDSE.

  1. Using organotypic (raft) epithelial tissue cultures for the biosynthesis and isolation of infectious human papillomaviruses.

    Science.gov (United States)

    Ozbun, Michelle A; Patterson, Nicole A

    2014-08-01

    Papillomaviruses have a strict tropism for epithelial cells, and they are fully reliant on cellular differentiation for completion of their life cycles, resulting in the production of progeny virions. Thus, a permissive environment for full viral replication in vitro-wherein virion morphogenesis occurs under cooperative viral and cellular cues-requires the cultivation of epithelium. Presented in the first section of this unit is a protocol to grow differentiating epithelial tissues that mimic many important morphological and biochemical aspects of normal skin. The technique involves growing epidermal cells atop a dermal equivalent consisting of live fibroblasts and a collagen lattice. Epithelial stratification and differentiation ensues when the keratinocyte-dermal equivalent is placed at the air-liquid interface. The apparent floating nature of the cell-matrix in this method led to the nickname "raft" cultures. The general technique can be applied to normal low passage keratinocytes, to cells stably transfected with papillomavirus genes or genomes, or keratinocytes established from neoplastic lesions. However, infectious papillomavirus particles have only been isolated from organotypic epithelial cultures initiated with cells that maintain oncogenic human papillomavirus genomes in an extrachomosomal replicative form. The second section of this unit is dedicated to a virion isolation method that minimizes aerosol and skin exposure to these human carcinogens. Although the focus of the protocols is on the growth of tissues that yields infectious papillomavirus progeny, this culture system facilitates the investigation of these fastidious viruses during their complex replicative cycles, and raft tissues can be manipulated and harvested at any point during the process. Importantly, a single-step virus growth cycle is achieved in this process, as it is unlikely that progeny virions are released to initiate subsequent rounds of infection.

  2. Human genome libraries. Final progress report, February 1, 1994--August 31, 1997

    Energy Technology Data Exchange (ETDEWEB)

    Kao, Fa-Ten

    1998-01-01

    The goal of this program is to use a novel technology of chromosome microdissection and microcloning to construct chromosome region-specific libraries as resources for various human genome program studies. Region specific libraries have been constructed for the entire human chromosomes 2 and 18.

  3. Forecasting Performance in Organizations: An Application of Current-Value Human Resources Accounting. Final Report.

    Science.gov (United States)

    Pecorella, Patricia A.; And Others

    A methodology to describe current-value human resources accounting (HRA) was developed to aid management in decision making and provide information about the effects of organizational policies and practices on the value of the organizations' human resources. A two-phase activity was designed to investigate the nature of the relationship between…

  4. Detection of vitamin D binding protein on the surface of cytotrophoblasts isolated from human placentae

    Energy Technology Data Exchange (ETDEWEB)

    Nestler, J.E.; McLeod, J.F.; Kowalski, M.A.; Strauss, J.F. 3d.; Haddad, J.G. Jr.

    1987-05-01

    Vitamin D binding protein (DBP), a Mr 56,000-58,000 alpha 2-glycoprotein, is the major serum protein involved in the transport of vitamin D sterols. Recently it has been suggested that DBP may also be involved in immunoglobulin G binding to cells. Because the trophoblast is involved in the transport of molecules such as vitamin D and immunoglobulin G to the fetus, we asked whether DBP could be detected on the surface of human placental trophoblast cells. Cytotrophoblasts purified from human term placentae were fixed and made permeant with Triton X-100 and examined by indirect immunofluorescence after incubation with a monoclonal antibody to DBP. Greater than 90% of these cells stained positively, whereas no staining was observed with nonimmune antiserum. The presence of DBP on/in the surface of cytotrophoblasts could also be demonstrated by fluorescent cytometry. When cell surface-associated proteins of cytotrophoblasts were radioiodinated, a Mr 57,000 radiolabeled protein could be immunoisolated from the cell lysate with a purified monospecific polyclonal antibody to DBP. Immunoisolation of this radiolabeled protein was prevented by the addition of excess unlabeled human DBP to the cell lysate before incubation with antibody. This Mr 57,000 radiolabeled protein could also be isolated by affinity chromatography selecting for proteins that bind to globular actin. When cytotrophoblasts were incubated with (/sup 35/S)methionine for 3 or 18 h, active synthesis of DBP could not be demonstrated by immunoisolation techniques. These studies demonstrate the presence of DBP on the surface of well washed, human cytotrophoblasts. This DBP may be maternally derived, since active synthesis of DBP could not be demonstrated.

  5. Salmonella enterica serotype Infantis in southern Italy from 2000-2005: molecular typing of isolates from human and non human sources

    Directory of Open Access Journals (Sweden)

    Caterina Mammina

    2007-03-01

    Full Text Available Serotype Infantis ranks within the top-five Salmonella serotypes in many European countries. An association between this serotype and the poultry ecosystem is apparent in some geographic areas and could account for its persistent or emergent role as causative agent of human salmonellosis. Recently, in southern Italy isolation from eggs and poultry has become increasingly frequent, and in one case the egg yolk also cultured positive. Molecular typing was performed to assess the possible relationship among isolates from human, food and animal sources. Isolates of serotype Infantis identified in southern Italy between 2000 and 2005 rom various sources were submitted to pulsed field gel electrophoresis (PFGE by a CHEF-Mapper apparatus fter endonuclease digestion of DNA by XbaI. PFGE patterns of isolates from swine and poultry reservoirs showed distinctive PFGE profiles and human solates shared pulsotypes with isolates from both sources. Moreover, egg isolates from different farms ppeared very similar to each other. Epidemiological investigation and risk assessment can obtain reliable and useful pointers from he pplication of molecular epidemiology techniques.

  6. Antimicrobial drug resistance of Salmonella isolates from meat and humans, Denmark

    DEFF Research Database (Denmark)

    Skov, Marianne; Andersen, Jens Strodl; Aabo, Søren

    2007-01-01

    We compared 8,144 Salmonella isolates collected from meat imported to or produced in Denmark, as well as from Danish patients. Isolates from imported meat showed a higher rate of antimicrobial drug resistance, including multidrug resistance, than did isolates from domestic meat. Isolates from hum...

  7. Handbook of human-reliability analysis with emphasis on nuclear power plant applications. Final report

    Energy Technology Data Exchange (ETDEWEB)

    Swain, A D; Guttmann, H E

    1983-08-01

    The primary purpose of the Handbook is to present methods, models, and estimated human error probabilities (HEPs) to enable qualified analysts to make quantitative or qualitative assessments of occurrences of human errors in nuclear power plants (NPPs) that affect the availability or operational reliability of engineered safety features and components. The Handbook is intended to provide much of the modeling and information necessary for the performance of human reliability analysis (HRA) as a part of probabilistic risk assessment (PRA) of NPPs. Although not a design guide, a second purpose of the Handbook is to enable the user to recognize error-likely equipment design, plant policies and practices, written procedures, and other human factors problems so that improvements can be considered. The Handbook provides the methodology to identify and quantify the potential for human error in NPP tasks.

  8. Pheromone-responsive conjugative vancomycin resistance plasmids in Enterococcus faecalis isolates from humans and chicken feces.

    Science.gov (United States)

    Lim, Suk-Kyung; Tanimoto, Koichi; Tomita, Haruyoshi; Ike, Yasuyoshi

    2006-10-01

    The drug resistances and plasmid contents of a total of 85 vancomycin-resistant enterococcus (VRE) strains that had been isolated in Korea were examined. Fifty-four of the strains originated from samples of chicken feces, and 31 were isolated from hospital patients in Korea. Enterococcus faecalis KV1 and KV2, which had been isolated from a patient and a sample of chicken feces, respectively, were found to carry the plasmids pSL1 and pSL2, respectively. The plasmids transferred resistances to vancomycin, gentamicin, kanamycin, streptomycin, and erythromycin to E. faecalis strains at a high frequency of about 10(-3) per donor cell during 4 hours of broth mating. E. faecalis strains containing each of the pSL plasmids formed clumps after 2 hours of incubation in broth containing E. faecalis FA2-2 culture filtrate (i.e., the E. faecalis sex pheromone), and the plasmid subsequently transferred to the recipient strain in a 10-min short mating in broth, indicating that the plasmids are responsive to E. faecalis pheromones. The pSL plasmids did not respond to any of synthetic pheromones for the previously characterized plasmids. The pheromone specific for pSL plasmids has been designated cSL1. Southern hybridization analysis showed that specific FspI fragments from each of the pSL plasmids hybridized with the aggregation substance gene (asa1) of the pheromone-responsive plasmid pAD1, indicating that the plasmids had a gene homologous to asa1. The restriction maps of the plasmids were identical, and the size of the plasmids was estimated to be 128.1 kb. The plasmids carried five drug resistance determinants for vanA, ermB, aph(3'), aph(6'), and aac(6')/aph(2'), which encode resistance to vancomycin, erythromycin, kanamycin, streptomycin, and gentamicin/kanamycin, respectively. Nucleotide sequence analyses of the drug resistance determinants and their flanking regions are described in this report. The results described provide evidence for the exchange of genetic information

  9. MLST genotypes of Campylobacter jejuni isolated from broiler products, dairy cattle and human campylobacteriosis cases in Lithuania.

    Science.gov (United States)

    Ramonaite, Sigita; Tamuleviciene, Egle; Alter, Thomas; Kasnauskyte, Neringa; Malakauskas, Mindaugas

    2017-06-15

    Campylobacter (C.) jejuni is the leading cause of human campylobacteriosis worldwide. We performed a molecular epidemiological study to investigate the genetic relationship among C. jejuni strains isolated from human diarrhoeal patients, broiler products and dairy cattle in Lithuania. The C. jejuni isolates from human clinical cases, dairy cattle and broiler products were genotyped using multilocus sequence typing (MLST). Allele numbers for each housekeeping gene, sequence type (ST), and clonal complex (CC) were assigned by submitting the DNA sequences to the C. jejuni MLST database ( http://pubmlst.org/campylobacter ). Based on the obtained sequence data of the housekeeping genes a phylogenetic analysis of the strains was performed and a minimum spanning tree (MST) was calculated. Among the 262 C. jejuni strains (consisting of 43 strains isolated from dairy cattle, 102 strains isolated from broiler products and 117 clinical human C. jejuni strains), 82 different MLST sequence types and 22 clonal complexes were identified. Clonal complexes CC21 and CC353 predominated among the C. jejuni strains. On ST-level, five sequence types (ST-5, ST-21, ST-50, ST-464 and ST-6410) were dominating and these five STs accounted for 35.9% (n = 94) of our isolates. In addition, 51 (19.5%) C. jejuni strains representing 27 (32.9%) STs were reported for the first time in the PubMLST database ( http://pubmlst.org/campylobacter ). The highest Czekanowski index or proportional similarity index (PSI) was calculated for C. jejuni strains isolated from human campylobacteriosis cases and broiler products (PSI = 0.32) suggesting a strong link between broiler strains and human cases. The PSI of dairy cattle and human samples was lower (PSI = 0.11), suggesting a weaker link between bovine strains and human cases. The calculated Simpson's index of all C. jejuni isolates showed a high genetic diversity (D = 0.96). Our results suggest that broiler products are the most important source of

  10. Detection of sul1, sul2 and sul3 in sulphonamide resistant Escherichia coli isolates obtained from healthy humans, pork and pigs in Denmark.

    Science.gov (United States)

    Hammerum, Anette M; Sandvang, Dorthe; Andersen, Sigrid R; Seyfarth, Anne Mette; Porsbo, Lone Jannok; Frimodt-Møller, Niels; Heuer, Ole E

    2006-02-01

    The occurrence of sulphonamide resistance was investigated in 998 Escherichia coli isolates, obtained from pig faeces collected at slaughter, Danish pork collected at retail outlets and from faeces from healthy persons in Denmark. In total 18% (n=35), 20% (n=38) and 26% (n=161) of the E. coli isolates obtained from humans, pork and pigs, respectively, were resistant to sulphonamide. All sulphonamide resistant E. coli isolates were investigated for the presence of sul1, sul2, sul3 and intI1 genes by PCR. The sul1 gene was detected in 40% (n=14), 29% (n=11) and 55% (n=88) of the sulphonamide resistant isolates from humans, pork and pigs, respectively. The sul2 gene was detected in 80% (n=28), 76% (n=29) and 50% (n=81) of isolates from humans, pork and pigs, respectively. None of the human isolates were PCR-positive for sul3, whereas sul3 was present in 5% of the pork isolates and 11% of the pig isolates. Of the 113 sul1 positive isolates, 97 carried the integron-associated integrase gene intI1. All 20 sul3 positive isolates were positive for intI1, and in 12 of these isolates sul3 was the only sulphonamide resistance gene detected. The origin of sul1 and sul2 found in isolates from healthy humans is speculative, but their spread from pigs to humans via the food chain is possible.

  11. Successful isolation, in vitro expansion and characterization of stem cells from Human Dental Pulp

    Directory of Open Access Journals (Sweden)

    Preethy SP

    2010-01-01

    Full Text Available BACKGROUND: Recent studies have shown that mesenchymal stem cells isolated from post natal human dental pulp, (Dental pulp stem cells-DPSCs which is from permanent teeth and SHED (stem cells from human exfoliated deciduous teeth,the Periodontal ligament stem cells (PDLSC and Stem cells from root Apical papilla(SCAPhave the potential to differentiate into cells of a variety of tissues including heart, muscle, cartilage, bone, nerve, salivary glands, teeth etc(1,2,3,4.This multipotential ability of DPSCs is being researched for clinical application for treating a variety of diseases like myocardial infarction, muscular dystrophy, neuro-degenerative disorders, cartilage replacement, tooth regeneration and for repair of bone defects to mention a few. Moreover, the isolation of stem cells from teeth is minimally invasive, readily accessible and the non immunogenic characteristic of dental stem cells has paved the way for efforts to store the exfoliated deciduous teeth or milk teeth which is usually discarded, for use in the future. In this study we have isolated and expanded in vitro, the cells obtained from human dental pulp. MATERIALS AND METHODS: After obtaining written informed consent, 24 teeth that were extracted for therapeutic or cosmetic reasons from 16 patients were used in this study. The specimens were transported from the clinic to NCRM lab taking 6 to 48 Hrs. For removal of the pulp tissue, the teeth were split obliquely at the Cementoenamel junction and the pulp tissue was isolated using brooches. The extracted pulp tissues were subjected to digestion using Collagenase type-I and type II at 37˚C for 15- 30 minutes. The digested cells were filtered with 70µm filter and centrifuged at 1800 rpm for 10 minutes. The pellet was then suspended in Dulbecco’s modified Eagle’s medium (DMEM/Ham’s F12 supplemented with 15% fetal bovine serum , 100 U/ml penicillin, 100 µg/ml streptomycin,2 m M L -glutamine, and 2 m M nonessential amino

  12. Diversity of Virulence Factors Associated with West Australian Methicillin-Sensitive Staphylococcus aureus Isolates of Human Origin

    Directory of Open Access Journals (Sweden)

    Charlene Babra Waryah

    2016-01-01

    Full Text Available An extensive array of virulence factors associated with S. aureus has contributed significantly to its success as a major nosocomial pathogen in hospitals and community causing variety of infections in affected patients. Virulence factors include immune evading capsular polysaccharides, poly-N-acetyl glucosamine, and teichoic acid in addition to damaging toxins including hemolytic toxins, enterotoxins, cytotoxins, exfoliative toxin, and microbial surface components recognizing adhesive matrix molecules (MSCRAMM. In this investigation, 31 West Australian S. aureus isolates of human origin and 6 controls were analyzed for relative distribution of virulence-associated genes using PCR and/or an immunoassay kit and MSCRAMM by PCR-based typing. Genes encoding MSCRAMM, namely, Spa, ClfA, ClfB, SdrE, SdrD, IsdA, and IsdB, were detected in >90% of isolates. Gene encoding α-toxin was detected in >90% of isolates whereas genes encoding β-toxin and SEG were detectable in 50–60% of isolates. Genes encoding toxin proteins, namely, SEA, SEB, SEC, SED, SEE, SEH, SEI, SEJ, TSST, PVL, ETA, and ETB, were detectable in >50% of isolates. Use of RAPD-PCR for determining the virulence factor-based genetic relatedness among the isolates revealed five cluster groups confirming genetic diversity among the MSSA isolates, with the greatest majority of the clinical S. aureus (84% isolates clustering in group IIIa.

  13. Inter-Individual Variability in Human Response to Low-Dose Ionizing Radiation, Final Report

    Energy Technology Data Exchange (ETDEWEB)

    Rocke, David [Univ. of California, Davis, CA (United States)

    2016-08-01

    In order to investigate inter-individual variability in response to low-dose ionizing radiation, we are working with three models, 1) in-vivo irradiated human skin, for which we have a realistic model, but with few subjects, all from a previous project, 2) ex-vivo irradiated human skin, for which we also have a realistic model, though with the limitations involved in keeping skin pieces alive in media, and 3) MatTek EpiDermFT skin plugs, which provides a more realistic model than cell lines, which is more controllable than human samples.

  14. The Human Genome Project: Information access, management, and regulation. Final report

    Energy Technology Data Exchange (ETDEWEB)

    McInerney, J.D.; Micikas, L.B.

    1996-08-31

    The Human Genome Project is a large, internationally coordinated effort in biological research directed at creating a detailed map of human DNA. This report describes the access of information, management, and regulation of the project. The project led to the development of an instructional module titled The Human Genome Project: Biology, Computers, and Privacy, designed for use in high school biology classes. The module consists of print materials and both Macintosh and Windows versions of related computer software-Appendix A contains a copy of the print materials and discs containing the two versions of the software.

  15. cDNA/STS map of human genome. Final technical report

    Energy Technology Data Exchange (ETDEWEB)

    NONE

    1995-11-01

    The human gene identification and transcript mapping project has generated over 3,000 3`ESTs derived from human brain cDNA libraries and mapped over 300 of these. The data have been submitted to the appropriate gene sequence and mapping databases. Clones are either available from Greg Lennon at Lawrence Livermore or from ATCC. A summary of this work is provided and a News and Views article from the same issue is included which highlights this paper. The strategy developed by this laboratory is now being used by an international consortium to generate the first comprehensive human gene (transcript) map over the next year or two.

  16. Combining cell lines to optimize isolation of human enterovirus from clinical specimens: report of 25 years of experience.

    Science.gov (United States)

    Prim, Núria; Rodríguez, Graciela; Margall, Núria; Del Cuerpo, Margarita; Trallero, Gloria; Rabella, Núria

    2013-01-01

    Cell culture is still the gold standard for the diagnosis of human enteroviruses (HEVs) although molecular techniques are required for detection of some serotypes. Due to the diversity of HEVs, a single cell line is not susceptible to all serotypes, and several lines are required to optimize the isolation of HEVs. In this study, the results of HEV isolation during the last 25 years are reported. A total of 1,192 HEVs were isolated and isolation rates varied depending on the cell line used. MRC5 cells yielded the best results (70.7%), followed by A549 cells (52.6%), RD cells (37.5%), and HEp-2 cells (29.7%). A total of 521 HEVs were characterized, and HEV-B was the most frequent species (81%). Polioviruses (PV) and HEV-A were isolated less frequently (17% and 1%, respectively). None of the cell lines detected all the enteroviruses. MRC5 cells were the most susceptible for isolation of echoviruses (85.7%) and PVs (85.4%), whereas HEp2 was the most susceptible for Coxsackieviruses B (82.6%). Some serotypes were isolated in one cell line only. 40.5% of echoviruses were isolated in MRC5 cells whereas 42.3% and 23.9% of Coxsackieviruses B were isolated in RD cells and HEp2 cells, respectively. Although A549 cells did not achieve the best performance for any enterovirus serotypes, they isolated 52.6% of the total HEVs. In view of these results, MRC5 cells, A549 cells, and RD cells should be combined to optimize isolation of HEVs. Copyright © 2012 Wiley Periodicals, Inc.

  17. Activation of GPER-1 estradiol receptor downregulates production of testosterone in isolated rat Leydig cells and adult human testis.

    Directory of Open Access Journals (Sweden)

    Laurent Vaucher

    Full Text Available PURPOSE: Estradiol (E2 modulates testicular functions including steroidogenesis, but the mechanisms of E2 signaling in human testis are poorly understood. GPER-1 (GPR30, a G protein-coupled membrane receptor, mediates rapid genomic and non-genomic response to estrogens. The aim of this study was to evaluate GPER-1 expression in the testis, and its role in estradiol dependent regulation of steroidogenesis in isolated rat Leydig cells and human testis. MATERIALS AND METHODS: Isolated Leydig cells (LC from adult rats and human testicular tissue were used in this study. Expression and localization studies of GPER-1 were performed with qRT-PCR, immunofluorescence, immunohistochemistry and Western Blot. Luteinizing Hormone (LH -stimulated, isolated LC were incubated with estradiol, G-1 (GPER-1-selective agonist, and estrogen receptor antagonist ICI 182,780. Testosterone production was measured with radioimmunoassay. LC viability after incubation with G-1 was measured using 3-(4,5-dimethylthiazol-2-yl-5-(3-carboxymethoxyphenyl-2-(4-sulfophenyl-2H-tetrazolium, inner salt (MTS assay. RESULTS: GPER-1 mRNA is abundantly expressed in rat LC and human testis. Co-localization experiments showed high expression levels of GPER-1 protein in LC. E2-dependent activation of GPER-1 lowers testosterone production in isolated rats LCs and in human testis, with statistically and clinically significant drops in testosterone production by 20-30% as compared to estradiol-naïve LC. The exposure to G-1 does not affect viability of isolated LCs. CONCLUSIONS: Our results indicate that activation of GPER-1 lowers testosterone levels in the rat and human testis. The expression of GPER-1 in human testis, which lack ERα, makes it an exciting target for developing new agents affecting testosterone production in men.

  18. Antitumour Effects of Isocurcumenol Isolated from Curcuma zedoaria Rhizomes on Human and Murine Cancer Cells

    Science.gov (United States)

    Lakshmi, S.; Padmaja, G.; Remani, P.

    2011-01-01

    Curcuma zedoaria belonging to the family Zingiberaceae has been used in the traditional system of medicine in India and Southwest Asia in treating many human ailments and is found to possess many biological activities. The rationale of the present study was to isolate, identify, and characterize antitumour principles from the rhizomes of Curcuma zedoaria, to assess its cytotoxic effects on human and murine cancer cells, to determine its apoptosis inducing capacity in cancer cells, and to evaluate its tumour reducing properties in in vivo mice models. Isocurcumenol was characterized as the active compound by spectroscopy and was found to inhibit the proliferation of cancer cells without inducing significant toxicity to the normal cells. Fluorescent staining exhibited the morphological features of apoptosis in the compound-treated cancer cells. In vivo tumour reduction studies revealed that a dose of 35.7 mg/kg body weight significantly reduced the ascitic tumour in DLA-challenged mice and increased the lifespan with respect to untreated control mice. PMID:27429805

  19. Isolation and characterization of a human apoptosis-inducing gene with yeast two-hybrid system

    Institute of Scientific and Technical Information of China (English)

    齐兵; 齐义鹏; Masuo; Yutsudo; 刘青珍

    2000-01-01

    asy gene is a novel apoptosis-inducing gene, but its mechanism is unclear. To investigate the mechanism of asy inducing apoptosis, a novel gene encoding ASY interacting protein (asyip) is isolated from human lung cell line (WI-38) cDNA library with yeast two-hybrid system. The asyip gene is constitutively expressed as two mRNA transcripts with the size of 1.8 and 2.7 kb in various human tissues at different levels. Sequence analysis of full-length cDNA reveals that the two alternative transcripts of asyip gene contain common 5’ end and different 3’ end, and share a common open reading frame encoding a polypeptide of 236 amino acids. Two protein kinase C phosphorylation sites and two casein kinase II phosphorylation sites are found in ASYIP amino acid sequence. Two highly hydrophobic regions encoding potentially two transmembrane domains are present. The ASYIP protein contains a C-terminal endoplasmic reticulum retrieval signal (Lys-Lys-Lys-Ala-Glu). Immunoprecipitation assay confirmed the interaction of

  20. Isolation and characterization of a human apoptosis-inducing gene with yeast two-hybrid system

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    asy gene is a novel apoptosis-inducing gene,but its mechanism is unclear.To investigate the mechanism of asy inducing apoptosis,a novel gene encoding ASY interacting protein (asyip) is isolated from human lung cell line (WI-38) cDNA library with yeast two-hybrid system.The asyip gene is constitutively expressed as two mRNA transcripts with the size of 1.8 and 2.7 kb in various human tissues at different levels.Sequence analysis of full-length cDNA reveals that the two alternative transcripts of asyip gene contain common 5' end and different 3' end,and share a common open reading frame encoding a polypeptide of 236 amino acids.Two protein kinase C phosphorylation sites and two casein kinase II phosphorylation sites are found in ASYIP amino acid sequence.Two highly hydrophobic regions encoding potentially two transmembrane domains are present.The ASYIP protein contains a C-terminal endoplasmic reticulum retrieval signal (Lys-Lys-Lys-Ala-Glu).Immunoprecipitation assay confirmed the interaction of ASY and ASYIP in mammalian cells.Compared with asy gene,overexpression of asyip gene can inhibit growth of tumor cell Saos2 and induce cell apoptosis with a low efficiency.

  1. Isolation of osteogenic progenitors from human amniotic fluid using a single step culture protocol

    Directory of Open Access Journals (Sweden)

    Salini Vincenzo

    2009-02-01

    Full Text Available Abstract Background Stem cells isolated from amniotic fluid are known to be able to differentiate into different cells types, being thus considered as a potential tool for cellular therapy of different human diseases. In the present study, we report a novel single step protocol for the osteoblastic differentiation of human amniotic fluid cells. Results The described protocol is able to provide osteoblastic cells producing nodules of calcium mineralization within 18 days from withdrawal of amniotic fluid samples. These cells display a complete expression of osteogenic markers (COL1, ONC, OPN, OCN, OPG, BSP, Runx2 within 30 days from withdrawal. In order to test the ability of these cells to proliferate on surfaces commonly used in oral osteointegrated implantology, we carried out cultures onto different test disks, namely smooth copper, machined titanium and Sandblasted and Acid Etching titanium (SLA titanium. Electron microscopy analysis evidenced the best cell growth on this latter surface. Conclusion The described protocol provides an efficient and time-saving tool for the production of osteogenic cells from amniotic fluid that in the future could be used in oral osteointegrated implantology.

  2. Gliotoxin Isolated from Marine Fungus Aspergillus sp. Induces Apoptosis of Human Cervical Cancer and Chondrosarcoma Cells

    Directory of Open Access Journals (Sweden)

    Van-Tinh Nguyen

    2013-12-01

    Full Text Available Gliotoxin, a secondary metabolite produced by marine fungus Aspergillus sp., possesses various biological activities including anticancer activity. However, the mechanism underlying gliotoxin-induced cytotoxicity on human cervical cancer (Hela and human chondrosarcoma (SW1353 cells remains unclear. In this study, we focused on the effect of gliotoxin induction on apoptosis, the activating expressions of caspase family enzymes in the cells. Apoptotic cell levels were measured through DAPI and Annexin V/Propidium Iodide (PI double staining analysis. The apoptotic protein expression of Bcl-2 and caspase family was detected by Western blot in Hela and SW1353 cells. Our results showed that gliotoxin treatment inhibited cell proliferation and induced significant morphological changes. Gliotoxin induced apoptosis was further confirmed by DNA fragmentation, chromatin condensation and disrupted mitochondrial membrane potential. Gliotoxin-induced activation of caspase-3, caspase-8 and caspase-9, down-regulation of Bcl-2, up-regulation of Bax and cytochromec (cyt c release showed evidence for the gliotoxin activity on apoptosis. These findings suggest that gliotoxin isolated from marine fungus Aspergillus sp. induced apoptosis in Hela and SW1353 cells via the mitochondrial pathway followed by downstream events leading to apoptotic mode of cell death.

  3. Virulence genes and genetic relationship of L. monocytogenes isolated from human and food sources in Brazil

    Directory of Open Access Journals (Sweden)

    Rosana Macedo de Almeida

    Full Text Available ABSTRACT The herein presented assay provided a bacteriological and molecular characterization of 100 samples of L. monocytogenes isolated from human (43 and food (57 sources, from several regions of Brazil, and collected between 1975 and 2013. Antigenic characterization defined 49% of serotype 4b samples, followed by 28% of serotype 1/2b, 14% of serotype 1/2c, 8% of serotype 1/2a, and 1% of serotype 3b. Both type of samples from human and food origin express the same serotype distribution. Multiplex PCR analysis showed 13 strains of type 4b with the amplification profile 4b-VI (Variant I. Virulence genes hly, inlA, inlB, inlC, inlJ, actA, plcA, and prfA were detected in all samples, highlighting a deletion of 105pb on the actA gene in 23% of serotype 4b samples. Macrorestriction profile with ApaI at PFGE showed 55 pulsotypes, with the occurrence of the same pulsotype in hospitalized patients in São Paulo in 1992 and 1997, and two other highly related pulsotypes in patients hospitalized in Rio de Janeiro in 2008. Recognized pulsotypes in listeriosis cases have also been detected in food. Thus, the prevalence of a serotype and the persistence of certain pulsotypes herald future problems.

  4. Isolation and characterization of a new mutant human cell line unresponsive to alpha and beta interferons.

    Science.gov (United States)

    John, J; McKendry, R; Pellegrini, S; Flavell, D; Kerr, I M; Stark, G R

    1991-08-01

    Previously we described human cell line 2fTGH, in which expression of guanine phosphoribosyltransferase is tightly controlled by the upstream region of interferon (IFN)-stimulated human gene 6-16. After mutagenesis of 2fTGH and selection with 6-thioguanine and IFN-alpha, we isolated 11.1, a recessive mutant that does not respond to IFN-alpha. We now describe U2, a second recessive mutant, selected similarly, that complements 11.1. U2 had no response to IFN-alpha or IFN-beta, and its response to IFN-gamma was partially defective. Although many genes did respond to IFN-gamma in U2, the 9-27 gene did not and the antiviral response of U2 cells to IFN-gamma was greatly reduced. Band shift assays showed that none of the transcription factors normally induced in 2fTGH cells by IFN-alpha (E and M) or IFN-gamma (G) were induced in U2. However, extracts of untreated U2 cells gave rise to a novel band that was increased by treatment with IFN-gamma but not IFN-alpha. Band shift complementation assays revealed that untreated and IFN-gamma-treated U2 cells lack the functional E gamma subunit of transcription factor E and that IFN-alpha-treated U2 cells do contain the functional E alpha subunit.

  5. Occurrence, Virulence Factors, Antimicrobial Resistance, and Genotyping of Staphylococcus aureus Strains Isolated from Chicken Products and Humans.

    Science.gov (United States)

    El Bayomi, Rasha M; Ahmed, Heba A; Awadallah, Maysa A I; Mohsen, Rasha A; Abd El-Ghafar, Abeer E; Abdelrahman, Mahmoud A

    2016-03-01

    Staphylococcus aureus in food is a consequence of inadequate hygienic handling and processing, posing a potential risk to public health. The current study aimed to characterize virulence factors, as well as antimicrobial resistance of Staphylococcus aureus and methicillin-resistant S. aureus (MRSA) isolated from retail chicken products and hand swabs from vendors in Egypt. In addition, genetic relatedness of the isolates from chicken and humans was evaluated by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) using protein A as a target. A total of 110 samples were collected from chicken products (n = 80) and vendors (n = 30). Overall, 30 (37.5%) chicken products samples were positive for S. aureus, whereas hand swabs from meat handlers revealed that 18 (60%) were positive. Ten MRSA strains were characterized by the presence of the mecA gene, comprising seven isolates from chicken and three from humans. Virulence-associated factors were evaluated by PCR, revealing that 31.3% of S. aureus isolates harbored the Panton-Valentine leukocidin (PVL) gene, whereas 10.4% were positive for the sea and sed genes each, and only two isolates were positive for γ-hemolysin-associated gene. Genotyping using spa PCR-RFLP showed identical restriction banding patterns of MRSA isolates of human and chicken meat origin, indicating the genetic relatedness of the isolates. To the best of our knowledge, this is the first study to characterize PVL-positive MRSA from chicken products and to utilize spa-RFLP for evaluating the genetic relatedness between MRSA of human and chicken origin in Egypt.

  6. Human Genome Teacher Networking Project, Final Report, April 1, 1992 - March 31, 1998

    Energy Technology Data Exchange (ETDEWEB)

    Collins, Debra

    1999-10-01

    Project to provide education regarding ethical legal and social implications of Human Genome Project to high school science teachers through two consecutive summer workshops, in class activities, and peer teaching workshops.

  7. Core Genome Multilocus Sequence Typing Scheme for Stable, Comparative Analyses of Campylobacter jejuni and C. coli Human Disease Isolates

    Science.gov (United States)

    Bray, James E.; Jolley, Keith A.; McCarthy, Noel D.

    2017-01-01

    ABSTRACT Human campylobacteriosis, caused by Campylobacter jejuni and C. coli, remains a leading cause of bacterial gastroenteritis in many countries, but the epidemiology of campylobacteriosis outbreaks remains poorly defined, largely due to limitations in the resolution and comparability of isolate characterization methods. Whole-genome sequencing (WGS) data enable the improvement of sequence-based typing approaches, such as multilocus sequence typing (MLST), by substantially increasing the number of loci examined. A core genome MLST (cgMLST) scheme defines a comprehensive set of those loci present in most members of a bacterial group, balancing very high resolution with comparability across the diversity of the group. Here we propose a set of 1,343 loci as a human campylobacteriosis cgMLST scheme (v1.0), the allelic profiles of which can be assigned to core genome sequence types. The 1,343 loci chosen were a subset of the 1,643 loci identified in the reannotation of the genome sequence of C. jejuni isolate NCTC 11168, chosen as being present in >95% of draft genomes of 2,472 representative United Kingdom campylobacteriosis isolates, comprising 2,207 (89.3%) C. jejuni isolates and 265 (10.7%) C. coli isolates. Validation of the cgMLST scheme was undertaken with 1,478 further high-quality draft genomes, containing 150 or fewer contiguous sequences, from disease isolate collections: 99.5% of these isolates contained ≥95% of the 1,343 cgMLST loci. In addition to the rapid and effective high-resolution analysis of large numbers of diverse isolates, the cgMLST scheme enabled the efficient identification of very closely related isolates from a well-defined single-source campylobacteriosis outbreak. PMID:28446571

  8. Human-system interface design review guideline -- Process and guidelines: Final report. Revision 1, Volume 1

    Energy Technology Data Exchange (ETDEWEB)

    None

    1996-06-01

    NUREG-0700, Revision 1, provides human factors engineering (HFE) guidance to the US Nuclear Regulatory Commission staff for its: (1) review of the human system interface (HSI) design submittals prepared by licensees or applications for a license or design certification of commercial nuclear power plants, and (2) performance of HSI reviews that could be undertaken as part of an inspection or other type of regulatory review involving HSI design or incidents involving human performance. The guidance consists of a review process and HFE guidelines. The document describes those aspects of the HSI design review process that are important to the identification and resolution of human engineering discrepancies that could adversely affect plant safety. Guidance is provided that could be used by the staff to review an applicant`s HSI design review process or to guide the development of an HSI design review plan, e.g., as part of an inspection activity. The document also provides detailed HFE guidelines for the assessment of HSI design implementations. NUREG-0700, Revision 1, consists of three stand-alone volumes. Volume 1 consists of two major parts. Part 1 describes those aspects of the review process of the HSI design that are important to identifying and resolving human engineering discrepancies. Part 2 contains detailed guidelines for a human factors engineering review which identify criteria for assessing the implementation of an applicant`s or licensee`s HSI design.

  9. Studies on adenosine triphosphate transphosphorylases. Human isoenzymes of adenylate kinase: isolation and physicochemical comparison of the crystalline human ATP-AMP transphosphorylases from muscle and liver.

    Science.gov (United States)

    Kuby, S A; Fleming, G; Frischat, A; Cress, M C; Hamada, M

    1983-02-10

    Procedures are described for the isolation, in crystalline form, of the adenylate kinases from autopsy samples of human muscle and from human liver. Weight average molecular weights were determined by sedimentation equilibrium to be 22,000 (+/- 700) and 25,450 (+/- 160) for the human muscle and liver isoenzymes, respectively. By sodium dodecyl sulfate-polyacrylamide gel electrophoresis, their molecular weights were estimated to be 21,700 and 26,500 for the muscle and liver enzymes, respectively. Both isoenzymes are accordingly monomeric proteins in their native state. Amino acid analyses are reported here for the normal human liver, calf liver, and rabbit liver adenylate kinases and compared with the normal human muscle, calf muscle, and rabbit muscle myokinases. The liver types as a group and the muscle types as a group show a great deal of homology, but some distinct differences are evident between the liver and muscle enzyme groups, especially in the number of residues of His, Pro, half-cystine, and the presence of tryptophan in the liver enzymes. The normal human liver adenylate kinase, as isolated in this report, has proved to be similar in its properties, if not identical, to the adenylate kinase isolated directly from human liver mitochondria (Hamada, M., Sumida, M., Okuda, H., Watanabe, T., Nojima, M., and Kuby, S. A. (1982) J. Biol. Chem. 257, 13120-13128). Therefore, the liver-type adenylate kinase may be considered a mitochondrial type.

  10. Protocol for Isolation of Primary Human Hepatocytes and Corresponding Major Populations of Non-parenchymal Liver Cells.

    Science.gov (United States)

    Kegel, Victoria; Deharde, Daniela; Pfeiffer, Elisa; Zeilinger, Katrin; Seehofer, Daniel; Damm, Georg

    2016-03-30

    Beside parenchymal hepatocytes, the liver consists of non-parenchymal cells (NPC) namely Kupffer cells (KC), liver endothelial cells (LEC) and hepatic Stellate cells (HSC). Two-dimensional (2D) culture of primary human hepatocyte (PHH) is still considered as the "gold standard" for in vitro testing of drug metabolism and hepatotoxicity. It is well-known that the 2D monoculture of PHH suffers from dedifferentiation and loss of function. Recently it was shown that hepatic NPC play a central role in liver (patho-) physiology and the maintenance of PHH functions. Current research focuses on the reconstruction of in vivo tissue architecture by 3D- and co-culture models to overcome the limitations of 2D monocultures. Previously we published a method to isolate human liver cells and investigated the suitability of these cells for their use in cell cultures in Experimental Biology and Medicine(1). Based on the broad interest in this technique the aim of this article was to provide a more detailed protocol for the liver cell isolation process including a video, which will allow an easy reproduction of this technique. Human liver cells were isolated from human liver tissue samples of surgical interventions by a two-step EGTA/collagenase P perfusion technique. PHH were separated from the NPC by an initial centrifugation at 50 x g. Density gradient centrifugation steps were used for removal of dead cells. Individual liver cell populations were isolated from the enriched NPC fraction using specific cell properties and cell sorting procedures. Beside the PHH isolation we were able to separate KC, LEC and HSC for further cultivation. Taken together, the presented protocol allows the isolation of PHH and NPC in high quality and quantity from one donor tissue sample. The access to purified liver cell populations could allow the creation of in vivo like human liver models.

  11. Human adaptation to isolated and confined environments: Preliminary findings of a seven month Antarctic winter-over human factors study

    Science.gov (United States)

    Evans, Gary W.; Stokols, Daniel; Carrere, Sybil

    1988-01-01

    This field study was conducted during the last decade of an austral winter-over at Palmer Station in the Antarctic. The purpose of the study was to understand temporal patterns in physiological arousal and psychological mood over the course of the mission. The investigators were principally interested in how people adapted over time to chronic and acute stressors, and how people use and modify their built environment. Physiological and psychological data were collected several times a week, and information on behavior and the use of physical facilities was collected monthly. Physiological and psychological data were compared with social changes in the setting toward the development of a sequential model of human-environment transactional relationships. Based on the study results, guidelines for design of future isolated and confined environments (ICEs) included: plan space for items which make people feel at home, provide materials to allow people to personalize their environment, allow for flexible environments, provide areas for visual and auditory privacy, equip areas for socializing and remove them from private areas, and provide facilities for exercise and for projects involving physical activity. The study offers guidelines about patterns of adaption that could be expected in an ICE, discusses how these settings can be programmed to facilitate successful adjustment, and provides information about how to design future ICE habitats to maximize a healthy living environment.

  12. Probiotic properties of Lactobacillus rhamnosus and Lactobacillus paracasei isolated from human faeces.

    Science.gov (United States)

    Verdenelli, Maria Cristina; Ghelfi, Francesca; Silvi, Stefania; Orpianesi, Carla; Cecchini, Cinzia; Cresci, Alberto

    2009-09-01

    The possibility of using microbes to maintain health, and to prevent or treat disease is a topic as old as microbiology. The research of novel probiotic strains is important in order to satisfy the increasing request of the market and to obtain functional products in which the probiotic cultures are more active and with better probiotic characteristics than those already present on the market. In this study, the probiotic potential of Lactobacillus strains isolated from Italian elderly human faeces was investigated. The Lactobacillus strains were identified and examined for resistance to gastric acidity and bile toxicity, adhesion to HT-29 cells, antimicrobial activities, antibiotic susceptibility and plasmid profile. Survival of the strains through human intestine was examined in a 3 months human feeding trial. Two strains, Lactobacillus rhamnosus IMC 501 and Lactobacillus paracasei IMC 502, tolerated well low pH and bile acids. In antimicrobial activity assays, both strains showed inhibitory properties towards selected potential harmful microorganisms, particularly against Candida albicans. The two selected strains expressed high in vitro adherence to HT-29 cells increasing this characteristic when they are used in combination and they were resistant to vamcomycin, colistin sulphate, gentamicin, oxolinic acid and kanamycin. Moreover, the two strains could be recovered from stools of volunteers after the feeding trials. Lactobacillus rhamnosus IMC 501 and L. paracasei IMC 502 present favourable strain-specific properties for their utilisation as probiotics in functional foods and the high adhesion ability of the L. rhamnosus IMC 501 and L. paracasei IMC 502 used in combination, confirmed by both in vitro and in vivo study, indicate that the two bacterial strains could be used as health-promoting bacteria.

  13. Isolation of a hemidesmosome-rich fraction from a human squamous cell carcinoma cell line

    Energy Technology Data Exchange (ETDEWEB)

    Hirako, Yoshiaki, E-mail: s47526a@cc.nagoya-u.ac.jp [Division of Biological Science, Graduate School of Science, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8602 (Japan); Yonemoto, Yuki; Yamauchi, Tomoe [Division of Biological Science, Graduate School of Science, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8602 (Japan); Nishizawa, Yuji; Kawamoto, Yoshiyuki [Department of Biomedical Sciences, Chubu University, 1200 Matsumoto-cho, Kasugai 487-8501 (Japan); Owaribe, Katsushi [Division of Biological Science, Graduate School of Science, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8602 (Japan)

    2014-06-10

    Hemidesmosomes are cell-to-matrix adhesion complexes anchoring keratinocytes to basement membranes. For the first time, we present a method to prepare a fraction from human cultured cells that are highly enriched in hemidesmosomal proteins. Using DJM-1 cells derived from human squamous cell carcinoma, accumulation of hemidesmosomes was observed when these cells were cultured for more than 10 days in a commercial serum-free medium without supplemental calcium. Electron microscopy demonstrated that numerous electron-dense adhesion structures were present along the basal cell membranes of DJM-1 cells cultured under the aforementioned conditions. After removing cellular materials using an ammonia solution, hemidesmosomal proteins and deposited extracellular matrix were collected and separated by electrophoresis. There were eight major polypeptides, which were determined to be plectin, BP230, BP180, integrin α6 and β4 subunits, and laminin-332 by immunoblotting and mass spectrometry. Therefore, we designated this preparation as a hemidesmosome-rich fraction. This fraction contained laminin-332 exclusively in its unprocessed form, which may account for the promotion of laminin deposition, and minimal amounts of Lutheran blood group protein, a nonhemidesmosomal transmembrane protein. This hemidesmosome-rich fraction would be useful not only for biological research on hemidesmosomes but also for developing a serum test for patients with blistering skin diseases. - Highlights: • A defined condition promoted accumulation of hemidesmosomes in human cultured cells. • A fraction isolated from the cells contained eight major polypeptides. • The polypeptides were the five major hemidesmosome proteins and laminin-332. • The cultured cells deposited laminin-332 in its unprocessed form under the condition. • We report a method to prepare a fraction highly enriched in hemidesmosome proteins.

  14. Lipid-protein interactions in plasma membranes of fiber cells isolated from the human eye lens.

    Science.gov (United States)

    Raguz, Marija; Mainali, Laxman; O'Brien, William J; Subczynski, Witold K

    2014-03-01

    The protein content in human lens membranes is extremely high, increases with age, and is higher in the nucleus as compared with the cortex, which should strongly affect the organization and properties of the lipid bilayer portion of intact membranes. To assess these effects, the intact cortical and nuclear fiber cell plasma membranes isolated from human lenses from 41- to 60-year-old donors were studied using electron paramagnetic resonance spin-labeling methods. Results were compared with those obtained for lens lipid membranes prepared from total lipid extracts from human eyes of the same age group [Mainali, L., Raguz, M., O'Brien, W. J., and Subczynski, W. K. (2013) Biochim. Biophys. Acta]. Differences were considered to be mainly due to the effect of membrane proteins. The lipid-bilayer portions of intact membranes were significantly less fluid than lipid bilayers of lens lipid membranes, prepared without proteins. The intact membranes were found to contain three distinct lipid environments termed the bulk lipid domain, boundary lipid domain, and trapped lipid domain. However, the cholesterol bilayer domain, which was detected in cortical and nuclear lens lipid membranes, was not detected in intact membranes. The relative amounts of bulk and trapped lipids were evaluated. The amount of lipids in domains uniquely formed due to the presence of membrane proteins was greater in nuclear membranes than in cortical membranes. Thus, it is evident that the rigidity of nuclear membranes is greater than that of cortical membranes. Also the permeability coefficients for oxygen measured in domains of nuclear membranes were significantly lower than appropriate coefficients measured in cortical membranes. Relationships between the organization of lipids into lipid domains in fiber cells plasma membranes and the organization of membrane proteins are discussed.

  15. Genotypic characteristics of hydatid cysts isolated from humans in East Azerbaijan Province (2011-2013

    Directory of Open Access Journals (Sweden)

    Amir Vahedi

    2014-08-01

    Full Text Available Introduction: Cystic echinococcosis (CE is one of the important helminthic diseases of human and animals, which causes by Echinococcus granulosus. Canids are its definite and grazers especially sheep, and cattle, and also wild herbivores are its intermediate hosts. Human can also be accidentally infected by a parasite. This study aimed to investigate genotypes of the hydatid cysts isolated from hydatidosis patients in order to confine the source of the infection, 2013. Methods: In this cross-sectional study 55 paraffin blocks of identified hydatid cysts have been undergone genotyping using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP technique. The ITS1 region of rDNA has been amplified using BD1 forward and 4s reverse primers. PCR products have been digested using HpaII and RsaI restriction endonucleases. RFLP products studied using gel electrophoresis. Data were analyzed using SPSS for Windows using the chi-square test. Results: About 29 (52.72%, 16 (29.1%, 3 (5.45%, 3 (5.45%, 1 (1.81%, 1 (1.81%, 1 (1.81% and 1 (1.81% out of 55 hydatid cysts were located in lung, liver, spleen, kidney, heart, pancreas, brain, and femore, respectively. The frequency of hydatidosis observed higher in patients from rural areas (P = 0.013; odds ratio = 0.599; 95% confidence interval: 0.28, 1.27. Based on RFLP results, the entire studied hydatid cysts identified as sheep strain (G1. Conclusion: According to the results of the present observation, it can be concluded that the majority of cases of human hydatidosis in East Azerbaijan Province are caused by sheep strain (G1 of E. granulosus, which indicates the sheep-doge cycle in the studied area.

  16. [Multiplexing mapping of human cDNAs]. Final report, September 1, 1991--February 28, 1994

    Energy Technology Data Exchange (ETDEWEB)

    1994-04-01

    Using PCR with automated product analysis, 329 human brain cDNA sequences have been assigned to individual human chromosomes. Primers were designed from single-pass cDNA sequences expressed sequence tags (ESTs). Primers were used in PCR reactions with DNA from somatic cell hybrid mapping panels as templates, often with multiplexing. Many ESTs mapped match sequence database records. To evaluate of these matches, the position of the primers relative to the matching region (In), the BLAST scores and the Poisson probability values of the EST/sequence record match were determined. In cases where the gene product was stringently identified by the sequence match had already been mapped, the gene locus determined by EST was consistent with the previous position which strongly supports the validity of assigning unknown genes to human chromosomes based on the EST sequence matches. In the present cases mapping the ESTs to a chromosome can also be considered to have mapped the known gene product: rolipram-sensitive cAMP phosphodiesterase, chromosome 1; protein phosphatase 2A{beta}, chromosome 4; alpha-catenin, chromosome 5; the ELE1 oncogene, chromosome 10q11.2 or q2.1-q23; MXII protein, chromosome l0q24-qter; ribosomal protein L18a homologue, chromosome 14; ribosomal protein L3, chromosome 17; and moesin, Xp11-cen. There were also ESTs mapped that were closely related to non-human sequence records. These matches therefore can be considered to identify human counterparts of known gene products, or members of known gene families. Examples of these include membrane proteins, translation-associated proteins, structural proteins, and enzymes. These data then demonstrate that single pass sequence information is sufficient to design PCR primers useful for assigning cDNA sequences to human chromosomes. When the EST sequence matches previous sequence database records, the chromosome assignments of the EST can be used to make preliminary assignments of the human gene to a chromosome.

  17. Isolation and Evaluation of Marine Actinomycetes from Mangrove Forests in South of Iran against Some Human Bacterial Pathogens

    Directory of Open Access Journals (Sweden)

    Farshid Kafilzadeh

    2013-04-01

    Full Text Available Background & Objectives: Recent studies have shown that aquatic actinomycetes can be a source of new biological products such as antibiotics and i n dustrial products. This study was designed to examine the aquatic actinomycetes isolated from mangrove forests in South of Iran and their antibacterial activities against some human pathogens.   Methods: In this study 115 samples were randomly taken from different places of a mangrove forests in South of Iran. Isolation was based on serial dilution of the samples and plating them on starch casein agar medium. Agar well diffusion and disc diffusion assays were used to examine the antibacterial activity of the isolated purified aquatic actinomycetes.   Results: The aquatic actinomycetes were isolated from 83 samples (70%. Of them, 66 (80 percent showed antibacterial activity and 17 (20% could not inhibit the human pathogenic bacteria. The diameter of the inhibitory zones (ZOI ranged from 4 to 11 mm and the biggest zone belonged to B acillus cereus (p≤0.05.   Conclusion: The findings showed that the various and useful aquatic actinomycetes for production of new antibiotic compounds are isolated easily from the mangrove forests in South of Iran. Considering the vast spreading of mangrove forests in South of Iran and the economic and simplicity of isolation of actinomycetes for industrial usage, these source can be an important and new place for research and industry.

  18. Characterization of the virulence, growth temperature and antibiotic resistance of the Campylobacter jejuni IAL 2383 strain isolated from humans

    Directory of Open Access Journals (Sweden)

    B.B. Fonseca

    2014-01-01

    Full Text Available The objective of this study was to characterize the C. jejuni IAL2383 strain isolated from humans in Brazil. Transcripts for the racR, dnaJ and ciaB genes were found and flaA, plda and cadF genes were present in the genome and bacteria was sensitive to most of the important antimicrobials used to treat humans. C. jejuni IAL2383 is a good experimental model to analyze the interactions with cells.

  19. Effects of growth temperature on the ingestion and killing of clinical isolates of Listeria monocytogenes by human neutrophils.

    OpenAIRE

    Stecha, P F; Heynen, C A; Roll, J T; Brown, J F; Czuprynski, C J

    1989-01-01

    In this study, we compared three human isolates (F5380, Scott A, and Murray B) and one laboratory strain (EGD) of Listeria monocytogenes for their resistance to ingestion and killing by human neutrophils. We observed no substantial difference in killing among these strains when they were grown at 37 degrees C. Because it is likely that listerial growth occurs at lower temperatures during food-borne outbreaks of listeriosis, we also compared these strains after they were grown at 22 and 4 degr...

  20. BEC, a novel enterotoxin of Clostridium perfringens found in human clinical isolates from acute gastroenteritis outbreaks.

    Science.gov (United States)

    Yonogi, Shinya; Matsuda, Shigeaki; Kawai, Takao; Yoda, Tomoko; Harada, Tetsuya; Kumeda, Yuko; Gotoh, Kazuyoshi; Hiyoshi, Hirotaka; Nakamura, Shota; Kodama, Toshio; Iida, Tetsuya

    2014-06-01

    Clostridium perfringens is a causative agent of food-borne gastroenteritis for which C. perfringens enterotoxin (CPE) has been considered an essential factor. Recently, we experienced two outbreaks of food-borne gastroenteritis in which non-CPE producers of C. perfringens were strongly suspected to be the cause. Here, we report a novel enterotoxin produced by C. perfringens isolates, BEC (binary enterotoxin of C. perfringens). Culture supernatants of the C. perfringens strains showed fluid-accumulating activity in rabbit ileal loop and suckling mouse assays. Purification of the enterotoxic substance in the supernatants and high-throughput sequencing of genomic DNA of the strains revealed BEC, composed of BECa and BECb. BECa and BECb displayed limited amino acid sequence similarity to other binary toxin family members, such as the C. perfringens iota toxin. The becAB genes were located on 54.5-kb pCP13-like plasmids. Recombinant BECb (rBECb) alone had fluid-accumulating activity in the suckling mouse assay. Although rBECa alone did not show enterotoxic activity, rBECa enhanced the enterotoxicity of rBECb when simultaneously administered in suckling mice. The entertoxicity of the mutant in which the becB gene was disrupted was dramatically decreased compared to that of the parental strain. rBECa showed an ADP-ribosylating activity on purified actin. Although we have not directly evaluated whether BECb delivers BECa into cells, rounding of Vero cells occurred only when cells were treated with both rBECa and rBECb. These results suggest that BEC is a novel enterotoxin of C. perfringens distinct from CPE, and that BEC-producing C. perfringens strains can be causative agents of acute gastroenteritis in humans. Additionally, the presence of becAB on nearly identical plasmids in distinct lineages of C. perfringens isolates suggests the involvement of horizontal gene transfer in the acquisition of the toxin genes.

  1. Characterization of lipases from Staphylococcus aureus and Staphylococcus epidermidis isolated from human facial sebaceous skin.

    Science.gov (United States)

    Xie, Winny; Khosasih, Vivia; Suwanto, Antonius; Kim, Hyung Kwoun

    2012-01-01

    Two staphylococcal lipases were obtained from Staphylococcus epidermidis S2 and Staphylococcus aureus S11 isolated from sebaceous areas on the skin of the human face. The molecular mass of both enzymes was estimated to be 45 kDa by SDS-PAGE. S2 lipase displayed its highest activity in the hydrolysis of olive oil at 32 degrees C and pH 8, whereas S11 lipase showed optimal activity at 31 degrees C and pH 8.5. The S2 lipase showed the property of cold-adaptation, with activation energy of 6.52 kcal/mol. In contrast, S11 lipase's activation energy, at 21 kcal/mol, was more characteristic of mesophilic lipases. S2 lipase was stable up to 45° C and within the pH range from 5 to 9, whereas S11 lipase was stable up to 50 degrees C and from pH 6 to 10. Both enzymes had high activity against tributyrin, waste soybean oil, and fish oil. Sequence analysis of the S2 lipase gene showed an open reading frame of 2,067 bp encoding a signal peptide (35 aa), a pro-peptide (267 aa), and a mature enzyme (386 aa); the S11 lipase gene, at 2,076 bp, also encoded a signal peptide (37 aa), pro-peptide (255 aa), and mature enzyme (399 aa). The two enzymes maintained amino acid sequence identity of 98-99% with other similar staphylococcal lipases. Their microbial origins and biochemical properties may make these staphylococcal lipases isolated from facial sebaceous skin suitable for use as catalysts in the cosmetic, medicinal, food, or detergent industries.

  2. Comparison of methods for the isolation of human breast epithelial and myoepithelial cells.

    Science.gov (United States)

    Zubeldia-Plazaola, Arantzazu; Ametller, Elisabet; Mancino, Mario; Prats de Puig, Miquel; López-Plana, Anna; Guzman, Flavia; Vinyals, Laia; Pastor-Arroyo, Eva M; Almendro, Vanessa; Fuster, Gemma; Gascón, Pedro

    2015-01-01

    Two lineages, epithelial, and myoepithelial cells are the main cell populations in the normal mammary gland and in breast cancer. Traditionally, cancer research has been performed using commercial cell lines, but primary cell cultures obtained from fresh breast tissue are a powerful tool to study more reliably new aspects of mammary gland biology, including normal and pathological conditions. Nevertheless, the methods described to date have some technical problems in terms of cell viability and yield, which hamper work with primary mammary cells. Therefore, there is a need to optimize technology for the proper isolation of epithelial and myoepithelial cells. For this reason, we compared four methods in an effort to improve the isolation and primary cell culture of different cell populations of human mammary epithelium. The samples were obtained from healthy tissue of patients who had undergone mammoplasty or mastectomy surgery. We based our approaches on previously described methods, and incorporated additional steps to ameliorate technical efficiency and increase cell survival. We determined cell growth and viability by phase-contrast images, growth curve analysis and cell yield, and identified cell-lineage specific markers by flow cytometry and immunofluorescence in 3D cell cultures. These techniques allowed us to better evaluate the functional capabilities of these two main mammary lineages, using CD227/K19 (epithelial cells) and CD10/K14 (myoepithelial cells) antigens. Our results show that slow digestion at low enzymatic concentration combined with the differential centrifugation technique is the method that best fits the main goal of the present study: protocol efficiency and cell survival yield. In summary, we propose some guidelines to establish primary mammary epithelial cell lines more efficiently and to provide us with a strong research instrument to better understand the role of different epithelial cell types in the origin of breast cancer.

  3. Binding of furosemide to albumin isolated from human fetal and adult serum.

    Science.gov (United States)

    Viani, A; Cappiello, M; Silvestri, D; Pacifici, G M

    1991-01-01

    Albumin was isolated from pooled fetal serum from 58 placentas obtained at normal delivery at term and from pooled adult plasma from 8 individuals. Albumin isolation was carried out by means of PEG precipitation followed by ion-exchange chromatography on DEAE-Sephadex A 50 and then on SP-Sephadex C 50. The electrophoresis on SDS-polyacrylamide gels showed only one spot that comigrated with commercial human albumin. Binding to albumin was measured by equilibrium dialysis of an aliquot of albumin solution (0.7 ml) against the same volume of 0.13 M sodium orthophosphate buffer (pH 7.4). At a total concentration of 2 micrograms/ml (therapeutic range), the unbound fraction of furosemide was 2.71% (fetal albumin) and 2.51% (adult albumin). Two classes of binding sites for furosemide were observed in fetal and adult albumin. The number of binding sites (moles of furosemide per mole of albumin) was 1.22 (fetal albumin) and 1.58 (adult albumin) for the high-affinity site and 2.97 (fetal albumin) and 3.25 (adult albumin) for the low-affinity site. The association constants (M-1) were 3.1 X 10(4) (fetal albumin) and 2.6 X 10(4) (adult albumin) for the high-affinity set of sites and 0.83 X 10(4) (fetal albumin) and 1.0 X 10(4) (adult albumin) low-affinity site. The displacement of furosemide from albumin was studied with therapeutic concentrations of several drugs. Valproic acid, salicylic acid, azapropazone and tolbutamide had the highest displacing effects which were significantly higher with fetal than with adult albumin.

  4. Characterization and Differentiation of Stem Cells Isolated from Human Newborn Foreskin Tissue.

    Science.gov (United States)

    Somuncu, Özge Sezin; Taşlı, Pakize Neslihan; Şişli, Hatice Burcu; Somuncu, Salih; Şahin, Fikrettin

    2015-11-01

    Circumcision is described as a cultural, medical, and religious process which states surgical removal of the foreskin either partly or fully. Cells isolated from the circumcised tissues are referred as foreskin cells. They have been thought as feeder cell lines for embryonic stem cells. Their fibroblastic properties were also utilized for several experiments. The waste tissues that remain after the circumcision thought to have stem cell properties. Therefore, there have been very few attempts to expose their stem cell properties without turning them into induced pluripotent stem cells. Although stem cell isolation from prepuce and their mesenchymal multilineage differentiation potential have been presented many times in the literature, the current study explored hematopoietical phenotype of newborn foreskin stem cells for the first time. According to the results, human newborn foreskin stem cells (hnFSSCs) were identified by their capability to turn into all three germ layer cell types under in vitro conditions. In addition, these cells have exhibited a stable phenotype and have remained as a monolayer in vitro. hnFSSCs suggested to carry different treatment potentials for bone damages, cartilage problems, nerve damages, lesion formations, and other diseases that are derive from mesodermal, endodermal, and ectodermal origins. Owing to the location of the tissue in the body and differentiation capabilities of hnFSSCs, these cells can be considered as easily obtainable and utilizable even better than the other stem cell sources. In addition, hnFSSCs offers a great potential for tissue engineering approaches due to exhibiting embryonic stem cell-like characteristics, not having any ethical issues, and teratoma induction as in embryonic stem cell applications.

  5. Extortion subdues human players but is finally punished in the prisoner's dilemma.

    Science.gov (United States)

    Hilbe, Christian; Röhl, Torsten; Milinski, Manfred

    2014-05-29

    Extortion is the practice of obtaining advantages through explicit forces and threats. Recently, it was demonstrated that even the repeated prisoner's dilemma, one of the key models to explain mutual cooperation, allows for implicit forms of extortion. According to the theory, extortioners demand and receive an excessive share of any surplus, which allows them to outperform any adapting co-player. To explore the performance of such strategies against humans, we have designed an economic experiment in which participants were matched either with an extortioner or with a generous co-player. Although extortioners succeeded against each of their human opponents, extortion resulted in lower payoffs than generosity. Human subjects showed a strong concern for fairness: they punished extortion by refusing to fully cooperate, thereby reducing their own, and even more so, the extortioner's gains. Thus, the prospects of extorting others in social relationships seem limited; in the long run, generosity is more profitable.

  6. Hybrids of Shigatoxigenic and Enterotoxigenic Escherichia coli (STEC/ETEC) Among Human and Animal Isolates in Finland.

    Science.gov (United States)

    Nyholm, O; Heinikainen, S; Pelkonen, S; Hallanvuo, S; Haukka, K; Siitonen, A

    2015-11-01

    Diarrhoeagenic Escherichia coli (DEC) cause serious foodborne infections in humans. Total of 450 Shigatoxigenic E. coli (STEC) strains isolated from humans, animals and environment in Finland were examined by multiplex PCR targeting the virulence genes of various DEC pathogroups simultaneously. One per cent (3/291) of the human STEC and 14% (22/159) of the animal and environmental STEC had genes typically present in enterotoxigenic E. coli (ETEC). The strains possessed genes encoding both Shiga toxin 1 and/or 2 (stx1 and/or stx2 ) and ETEC-specific heat-stable (ST) enterotoxin Ia (estIa). The identified stx subtypes were stx1a, stx1c, stx2a, stx2d and stx2g. The three human STEC/ETEC strains were isolated from the patients with haemolytic uraemic syndrome and diarrhoea and from an asymptomatic carrier. The animal STEC/ETEC strains were isolated from cattle and moose. The human and animal STEC/ETEC strains belonged to 11 serotypes, of which O2:H27, O15:H16, O101:H-, O128:H8 and O141:H8 have previously been described to be associated with human disease. Identification of multiple virulence genes offers further information for assessing the virulence potential of STEC and other DEC. The emergence of novel hybrid pathogens should be taken into account in the patient care and epidemiological surveillance.

  7. An experimental evaluation of in vitro immunomodulatory activity of isolated compound of Ricinus communis on human neutrophils

    Directory of Open Access Journals (Sweden)

    Arvind Kumar

    2011-01-01

    Full Text Available In the present study, the in vitro immunomodulatory activity of Ricinus communis Linn (Euphorbiaceae was determined on human neutrophils. The isolated compound (tannin of R. communis leaves was screened for its possible immunomodulatory activity by carrying out nitroblue tetrazolium test, phagocytosis of killed Candida albicans, neutrophil locomotion and chemotaxis. The isolated compound was tested at concentrations, viz. 10 μg/ml, 20 μg/ml, 40 μg/ml, 100 μg/ml and 1000 μg/ml. The isolated compound of R. communis showed predominantly significant activity on human neutrophils in all the parameters tested, which was comparable to the standard and control at different concentrations, indicating the possible immunostimulating effect.

  8. Molecular screening of virulence genes in extraintestinal pathogenic Escherichia coli isolated from human blood culture in Brazil.

    Science.gov (United States)

    Koga, Vanessa L; Tomazetto, Geizecler; Cyoia, Paula S; Neves, Meiriele S; Vidotto, Marilda C; Nakazato, Gerson; Kobayashi, Renata K T

    2014-01-01

    Extraintestinal pathogenic Escherichia coli (ExPEC) is one of the main etiological agents of bloodstream infections caused by Gram-negative bacilli. In the present study, 20 E. coli isolates from human hemocultures were characterized to identify genetic features associated with virulence (pathogenicity islands markers, phylogenetic group, virulence genes, plasmid profiles, and conjugative plasmids) and these results were compared with commensal isolates. The most prevalent pathogenicity island, in strains from hemoculture, were PAI IV536, described by many researchers as a stable island in enterobacteria. Among virulence genes, iutA gene was found more frequently and this gene enconding the aerobactin siderophore receptor. According to the phylogenetic classification, group B2 was the most commonly found. Additionally, through plasmid analysis, 14 isolates showed plasmids and 3 of these were shown to be conjugative. Although in stool samples of healthy people the presence of commensal strains is common, human intestinal tract may serve as a reservoir for ExPEC.

  9. An atypical Clostridium strain related to the Clostridium botulinum group III strain isolated from a human blood culture.

    Science.gov (United States)

    Bouvet, Philippe; Ruimy, Raymond; Bouchier, Christiane; Faucher, Nathalie; Mazuet, Christelle; Popoff, Michel R

    2014-01-01

    A nontoxigenic strain isolated from a fatal human case of bacterial sepsis was identified as a Clostridium strain from Clostridium botulinum group III, based on the phenotypic characters and 16S rRNA gene sequence, and was found to be related to the mosaic C. botulinum D/C strain according to a multilocus sequence analysis of 5 housekeeping genes.

  10. Draft Genome Sequences of Two Propionibacterium acnes Strains Isolated from Progressive Macular Hypomelanosis Lesions of Human Skin

    DEFF Research Database (Denmark)

    Petersen, Rolf; Lomholt, Hans B.; Scholz, Christian F. P.;

    2015-01-01

    Propionibacterium acnes is a Gram-positive bacterium that is prevalent on human skin. It has been associated with skin disorders such as acne vulgaris and progressive macular hypomelanosis (PMH). Here, we report draft genome sequences of two type III P. acnes strains, PMH5 and PMH7, isolated from...

  11. Draft Genome Sequences of Salmonella enterica Isolates Containing Incompatibility Group I1 Plasmids from Swine, Poultry, and Human Sources.

    Science.gov (United States)

    Kaldhone, Pravin R; Khajanchi, Bijay K; Han, Jing; Nayak, Rajesh; Ricke, Steven C; Foley, Steven L

    2017-09-28

    The draft genome sequences of eight Salmonella enterica isolates from various sources were evaluated for the influence of incompatibility group I1 (IncI1) plasmids on virulence. Strains SE142, SE143, SE144, and SE146 originated from swine, SE36N and SE89N from poultry-related sources, and SE991 and SE1148 from human patients.

  12. Genetic Features Differentiating Bovine, Food, and Human Isolates of Shiga Toxin-Producing Escherichia coli O157 in The Netherlands

    NARCIS (Netherlands)

    Franz, E.; Hoek, van A.H.A.M.; Wal, van der F.J.; Boer, de A.G.; Zwartkruis-Nahuis, A.; Zwaluw, van der K.; Aarts, H.J.M.; Heuvelink, A.E.

    2012-01-01

    The frequency of Escherichia coli O157 genotypes among bovine, food, and human clinical isolates from The Netherlands was studied. Genotyping included the lineage-specific polymorphism assay (LSPA6), the Shiga-toxin-encoding bacteriophage insertion site assay (SBI), and PCR detection and/or subtypin

  13. Draft Genome Sequences of Two Propionibacterium acnes Strains Isolated from Progressive Macular Hypomelanosis Lesions of Human Skin

    DEFF Research Database (Denmark)

    Petersen, Rolf; Lomholt, Hans B.; Scholz, Christian F. P.

    2015-01-01

    Propionibacterium acnes is a Gram-positive bacterium that is prevalent on human skin. It has been associated with skin disorders such as acne vulgaris and progressive macular hypomelanosis (PMH). Here, we report draft genome sequences of two type III P. acnes strains, PMH5 and PMH7, isolated from...

  14. Isolation and characterization of human salivary gland cells for stem cell transplantation to reduce radiation-induced hyposalivation

    NARCIS (Netherlands)

    Feng, Jielin; van der Zwaag, Marianne; Stokman, Monique A.; van Os, Ronald; Coppes, Robert P.

    2009-01-01

    Background: Recently, we showed that transplantation of 100-300 c-Kit(+) stem cells isolated from cultured salispheres ameliorates radiation-damage in murine salivary glands. The aim of this study is to optimize and translate these findings from mice to man. Methods: Mouse and human non-malignant pa

  15. Polyphasic taxonomic approach in the description of Alishewanella fetalis gen. nov., sp nov., isolated from a human foetus

    DEFF Research Database (Denmark)

    Vogel, Birte Fonnesbech; Venkateswaran, K.; Christensen, H.

    2000-01-01

    A taxonomically unique bacterium is described on the basis of a physiological and biochemical characterization, fatty acid profiling and sequence analyses of 16S rRNA and gyrase B (gyrB) genes. This non-motile, non-fermentative bacterium was isolated from a human foetus in Uppsala, Sweden, and or...

  16. Complete, closed genome sequences of 10 Salmonella enterica subsp. enterica serovar Typhimurium strains isolated from human and bovine sources

    Science.gov (United States)

    Salmonella enterica are a leading cause of enterocolitis for humans and animals. S. enterica serovar Typhimurium infects a broad range of hosts. To facilitate genomic comparisons among isolates from different sources, we present the complete genome sequences of ten S. Typhimurium strains, five each...

  17. Complete Genome Sequence of Lactobacillus oris J-1, a Potential Probiotic Isolated from the Human Oral Microbiome

    Science.gov (United States)

    2016-01-01

    Lactobacilli can exert health-promoting effects in the human oral microbiome through many mechanisms, including pathogen inhibition, maintenance of microbial balance, immunomodulation, and enhancement of the epithelial barrier function. Here, we present the complete genome sequence of a potential probiotic, Lactobacillus oris J-1, that was isolated from the oral cavity of a health child. PMID:27634996

  18. Comparative analysis of ESBL-positive Escherichia coli isolates from animals and humans from the UK, The Netherlands and Germany

    NARCIS (Netherlands)

    Wu, G.; Day, M.J.; Mafura, T.; Nunez-Garcia, J.; Fenner, J.J.; Sharma, M.; Essen-Zandbergen, van A.; Rodriguez, I.; Dierikx, C.M.; Mevius, D.J.

    2013-01-01

    The putative virulence and antimicrobial resistance gene contents of extended spectrum ß-lactamase (ESBL)-positive E. coli (n=629) isolated between 2005 and 2009 from humans, animals and animal food products in Germany, The Netherlands and the UK were compared using a microarray approach to test the

  19. Effect of cold and warm ischaemia on drug metabolism in isolated hepatocytes and slices from human and monkey liver

    NARCIS (Netherlands)

    Olinga, Peter; Hof, I.H; de Jager, M.H; de Jong, Kurt; Slooff, M.JH; Meijer, D.K F; Groothuis, Geny; Merema, M.T.

    1998-01-01

    1. The influence of short-term cold storage in University of Wisconsin organ preservation solution (UW) on the ability to metabolize lidocaine, testosterone and 7-ethoxycoumarin in isolated human and cynomolgus monkey (Macaca fascicularis) hepatocytes and liver slices has been investigated. 2. The h

  20. Isolation and Partial Characterization of Human Amniotic Epithelial Cells: The Effect of Trypsin

    Science.gov (United States)

    Tabatabaei, Meraj; Mosaffa, Nariman; Nikoo, Shohreh; Bozorgmehr, Mahmood; Ghods, Roya; Kazemnejad, Somaieh; Rezania, Simin; Keshavarzi, Bahareh; Arefi, Soheila; Ramezani-Tehrani, Fahimeh; Mirzadegan, Ebrahim; Zarnani, Amir-Hassan

    2014-01-01

    Background Despite the extensive information available in the literature, cell surface marker signature of human Amniotic Epithelial Cells (hAECs) remains controversial. The aim of the present study was to characterize immunophenotypic features, proliferative capacity and immunogenicity of hAECs. We also tested whether expression of some cell surface markers is influenced by the type of trypsin used for tissue digestion. Methods Single cell suspensions of amniotic membranes from four human placentas were isolated by enzymatic digestion and expression of CD9, CD10, CD29, CD34, CD38, CD44, CD45, CD73, CD105, CD133, HLA-I, HLA-DR, HLA-G, SSEA-4, STRO-1 and OCT-4 was then evaluated by flow cytometry. The differential impact of four trypsin types on the yield and expression of CD105 and HLA-I was also determined. The proliferative capacity of cultured hAECs was assessed and compared in the presence and absence of Epidermal Growth Factor (EGF). To test their immunogenicity, hAECs were injected into Balb/c mice and the reactivity of hyperimmunized sera was examined by immunofluorescence staining. Results Nearly all purified cells expressed mesenchymal markers, CD9, CD10, CD29, and CD73 and the embryonic marker, SSEA-4. A large proportion of the cells also expressed STRO-1 and OCT-4. The purified cells also expressed HLA-G and HLA-I. A very small proportion of hAECs expressed CD34, CD38, CD44, CD133 and HLA-DR. The type of trypsin used for enzymatic digestion affected both the percentage and expression of HLA-I and CD105. hAECs revealed substantial proliferative capacity only when cultured in the medium supplemented with EGF. These cells were shown to be capable of inducing high amounts of anti-donor antibodies. Conclusion Here we provided evidence that hAECs are immunogenic cells with high level of HLA-I expression. Furthermore, this work highlighted the impact of isolation procedure on the immunophenotype of hAEC. PMID:24523953

  1. Target acquisition: Human observer performance studies and TARGAC model validation (Final Report)

    NARCIS (Netherlands)

    Valeton, J.M.; Bijl, P.; Gillespie, P.

    1995-01-01

    Human target acquisition performance was studied using the thermal imagery that was collected during Battlefield Emissives Sources Trials under the European Theater Weather and Obscurants, (BEST TWO), organized by NATO AC243/Panel4/RSG.l5 in 1990. Recognition and identification probabilities were me

  2. Annotated Bibliography of the Air Force Human Resources Laboratory Technical Reports--1979. Final Report.

    Science.gov (United States)

    Barlow, Esther M.

    This annotated bibliography presents summaries of 81 reports on personnel and training research conducted by the Air Force Human Resources Laboratory (AFHRL). Topics addressed include electronics, aeronautics, computers, mathematics, and operational research, as they relate to the selection, motivation, training, retention, education, utilization,…

  3. Target acquisition: Human observer performance studies and TARGAC model validation (Final Report)

    NARCIS (Netherlands)

    Valeton, J.M.; Bijl, P.; Gillespie, P.

    1995-01-01

    Human target acquisition performance was studied using the thermal imagery that was collected during Battlefield Emissives Sources Trials under the European Theater Weather and Obscurants, (BEST TWO), organized by NATO AC243/Panel4/RSG.l5 in 1990. Recognition and identification probabilities were

  4. Human Cytomegalovirus and Human Umbilical Vein Endothelial Cells: Restriction of Primary Isolation to Blood Samples and Susceptibilities of Clinical Isolates from Other Sources to Adaptation

    OpenAIRE

    2002-01-01

    In immunocompromised patients with disseminated infection, human cytomegalovirus (HCMV) is widespread in the microvascular endothelium of multiple organs. Human umbilical vein endothelial cells (HUVEC) were used in parallel to human embryonic lung fibroblasts (HELF) to recover HCMV from blood samples of immunocompromised patients. Using the shell vial technique, comparable median numbers of p72-positive HUVEC and HELF cells were found with the 26 HCMV-positive buffy coat samples out of 150 ex...

  5. Tissue specific characteristics of cells isolated from human and rat tendons and ligaments

    Directory of Open Access Journals (Sweden)

    Scutt A

    2008-07-01

    Full Text Available Abstract Background Tendon and ligament injuries are common and costly in terms of surgery and rehabilitation. This might be improved by using tissue engineered constructs to accelerate the repair process; a method used successfully for skin wound healing and cartilage repair. Progress in this field has however been limited; possibly due to an over-simplistic choice of donor cell. For tissue engineering purposes it is often assumed that all tendon and ligament cells are similar despite their differing roles and biomechanics. To clarify this, we have characterised cells from various tendons and ligaments of human and rat origin in terms of proliferation, response to dexamethasone and cell surface marker expression. Methods Cells isolated from tendons by collagenase digestion were plated out in DMEM containing 10% fetal calf serum, penicillin/streptomycin and ultraglutamine. Cell number and collagen accumulation were by determined methylene blue and Sirius red staining respectively. Expression of cell surface markers was established by flow cytometry. Results In the CFU-f assay, human PT-derived cells produced more and bigger colonies suggesting the presence of more progenitor cells with a higher proliferative capacity. Dexamethasone had no effect on colony number in ACL or PT cells but 10 nM dexamethasone increased colony size in ACL cultures whereas higher concentrations decreased colony size in both ACL and PT cultures. In secondary subcultures, dexamethasone had no significant effect on PT cultures whereas a stimulation was seen at low concentrations in the ACL cultures and an inhibition at higher concentrations. Collagen accumulation was inhibited with increasing doses in both ACL and PT cultures. This differential response was also seen in rat-derived cells with similar differences being seen between Achilles, Patellar and tail tendon cells. Cell surface marker expression was also source dependent; CD90 was expressed at higher levels by PT

  6. Knowledge of the human immunodeficiency virus among final year dental students.

    Science.gov (United States)

    Gilbert, A D; Nuttall, N M

    1994-08-01

    A sound basis of knowledge about HIV infection and AIDS is essential to allow students to develop as dentists who undertake appropriate measures during clinical practice. In addition, it is also likely that possessing appropriate information may instil confidence in their own ability to diagnose and then manage patients infected by HIV. A questionnaire designed to test the knowledge of final year dental students in the UK was completed by 60.5% of students in 15 out of the 16 dental schools in the UK. Generally, the students rated the teaching they had received about cross-infection precautions, virology, sterilization practice and procedures and recognition of blood-borne virus risk groups as adequate or more than adequate. However, there was a lower degree of satisfaction expressed for instruction in the management of blood-borne virus carriers and the performance of barrier dentistry. Most dental students were aware of the association of hairy leukoplakia, oral Kaposi's sarcoma, oral candidiasis as a whole, and thrush as one clinical variant, with HIV infection but there was a much lower level of knowledge of erythematous candidiasis, HIV-associated salivary gland disease, oral melanotic hyperpigmentation and idiopathic thrombocytopenic purpura. This study highlights some important gaps in the knowledge of final year dental students about HIV and AIDS.

  7. Human Clinical Isolates of Corynebacterium diphtheriae and Corynebacterium ulcerans Collected in Canada from 1999 to 2003 but Not Fitting Reporting Criteria for Cases of Diphtheria

    OpenAIRE

    DeWinter, Leanne M.; Bernard, Kathryn A.; Marc G Romney

    2005-01-01

    A 5-year collection of Corynebacterium diphtheriae and Corynebacterium ulcerans human clinical isolates yielded nine isolates from blood cultures of patients with invasive infections, stressing the importance of C. diphtheriae as a serious blood-borne pathogen. Seven percent of C. diphtheriae and 100% of C. ulcerans isolates produced diphtheria toxin, demonstrating that toxigenic corynebacteria continue to circulate.

  8. Human clinical isolates of Corynebacterium diphtheriae and Corynebacterium ulcerans collected in Canada from 1999 to 2003 but not fitting reporting criteria for cases of diphtheria.

    Science.gov (United States)

    Dewinter, Leanne M; Bernard, Kathryn A; Romney, Marc G

    2005-07-01

    A 5-year collection of Corynebacterium diphtheriae and Corynebacterium ulcerans human clinical isolates yielded nine isolates from blood cultures of patients with invasive infections, stressing the importance of C. diphtheriae as a serious blood-borne pathogen. Seven percent of C. diphtheriae and 100% of C. ulcerans isolates produced diphtheria toxin, demonstrating that toxigenic corynebacteria continue to circulate.

  9. [Antimicrobial Susceptibility and Resistance Mutations in Campylobacter jejuni and C. coli Isolates from Human and Meat Sources].

    Science.gov (United States)

    Oishi, Akira; Murakami, Koichi; Etoh, Yoshiki; Sera, Nobuyuki; Horikawa, Kazumi

    2015-03-01

    Recently, there has been a marked increase in the number of reports of fluoroquinolone-resistant Campylobacter jejuni and Campylobacter coli. The aim of this study was to evaluate the prevalence of antimicrobial resistance and its genetic determinants in Campylobacter species isolated from meat and human subjects in Fukuoka Prefecture, Japan. Between 2011 and 2013, 55 and 64 isolates were collected from meat (chicken meat and beef liver) and humans, respectively, in this prefecture. Antimicrobial susceptibility tests were conducted using the agar dilution method in accordance with the Clinical and Laboratory Standards Institute guidelines, using the following 11 antimicrobial agents : cephalexin, cefoxitin, nalidixic acid, ciprofloxacin, levofloxacin, tetracycline, minocycline, ampicillin, streptomycin, kanamycin and erythromycin. The susceptibility rates of the isolates to three quinolones (nalidixic acid, ciprofloxacin, levofloxacin) were 43.7%, 41.2%, 40.3%, respectively. All the isolates were multidrug resistant. Whereas 46.9%-51.6% of the human isolates were resistant to one or more of the quinolones, only 32.7%-34.5% of the meat isolates were resistant to one or more of the drugs. DNA sequencing showed that of the 50 quinolone resistant isolates 44 had position 86 isoleucine (Ile) substituted for threonine (Thr) in the GyrA protein (Thr86Ile). This amino acid substitution resulted from ACA to ATA and ACT to ATT mutations of codon 86 in C. jejuni and C. coli, respectively. Furthermore, two of the four C. jejuni isolates lacking the Thr86Ile mutation had combined Ser22Gly-Asn203Ser substitutions, while the remaining two isolates had combined Ser22Gly-Asn203Ser-Ala 206Val substitutions. These four isolates also had cmeABC sequences that differed from the quinolone sensitive C. jejuni ATCC33560(T) strain. In conclusion, C. jejuni and C. coli have relatively high quinolone resistance, and are resistant to other antibiotics. The new combination of amino acid

  10. Isolating gait-related movement artifacts in electroencephalography during human walking

    Science.gov (United States)

    Kline, Julia E.; Huang, Helen J.; Snyder, Kristine L.; Ferris, Daniel P.

    2015-08-01

    Objective. High-density electroencephelography (EEG) can provide an insight into human brain function during real-world activities with walking. Some recent studies have used EEG to characterize brain activity during walking, but the relative contributions of movement artifact and electrocortical activity have been difficult to quantify. We aimed to characterize movement artifact recorded by EEG electrodes at a range of walking speeds and to test the efficacy of artifact removal methods. We also quantified the similarity between movement artifact recorded by EEG electrodes and a head-mounted accelerometer. Approach. We used a novel experimental method to isolate and record movement artifact with EEG electrodes during walking. We blocked electrophysiological signals using a nonconductive layer (silicone swim cap) and simulated an electrically conductive scalp on top of the swim cap using a wig coated with conductive gel. We recorded motion artifact EEG data from nine young human subjects walking on a treadmill at speeds from 0.4 to 1.6 m s-1. We then tested artifact removal methods including moving average and wavelet-based techniques. Main results. Movement artifact recorded with EEG electrodes varied considerably, across speed, subject, and electrode location. The movement artifact measured with EEG electrodes did not correlate well with head acceleration. All of the tested artifact removal methods attenuated low-frequency noise but did not completely remove movement artifact. The spectral power fluctuations in the movement artifact data resembled data from some previously published studies of EEG during walking. Significance. Our results suggest that EEG data recorded during walking likely contains substantial movement artifact that: cannot be explained by head accelerations; varies across speed, subject, and channel; and cannot be removed using traditional signal processing methods. Future studies should focus on more sophisticated methods for removal of EEG

  11. Final Report: Latent Expression of Genetic Damage in Human Lung Cells

    Energy Technology Data Exchange (ETDEWEB)

    Cornforth, Michael N.

    1999-02-28

    This project was aimed at furthering understanding of the latent effects of ionizing radiation. The underlying premise was that such latent (i.e., delayed) effects stemmed from radiation-induced genetic instability. As model system to investigate certain aspects of genomic instability, they proposed to look at chromosomal instability involving quasi-targeted radiation-induced breakpoints in the vicinity of the HPRT gene in EJ30 human epithelial cells. Using whole chromosome painting of the X chromosome, the authors were able to show that about 15% of randomly selected 6-thioguanine resistant (6TG{prime}) mutants involved translocations in the terminal portion of Xq. Subsequent analysis, using human genomic YAC probes confirmed that all the translocations were either within (or near Xq26.1), the cytogenetic location of HPRT, whereas none were found elsewhere involving the X chromosome.

  12. Human Power Vehicle Program. Final report, June 15, 1993--June 14, 1995

    Energy Technology Data Exchange (ETDEWEB)

    Crowell, J.; Graves, P.

    1995-11-01

    The Human Power Vehicle Program was an intensive, five day a week, four week program designed to give middle school students the opportunity to ``be engineers``. During the month of July, Delta College, the Macro Michigan Multicultural Pre-Technical Education Partnership (M3PEP), and the United States Department of Energy sponsored a four-week learning experience in human-powered vehicles. This unique experience introduced students to the physiology of exercise, the mechanics of the bicycle, and the physics and mathematics of the bicycle. Students also participated in a three day bike tour. The Program used the Bike Lab facility at Delta College`s International Centre in Saginaw, Michigan. Students had the opportunity to explore the development and refinement of the bicycle design and to investigate it`s power machine-the human body. Interactive instruction was conducted in groups to assure that all students experienced the satisfaction of understanding the bicycle. The purpose of the Program was to increase minority students` awareness and appreciation of mathematics and science. The premise behind the Program was that engineers and scientists are made, not born. The Program was open to all minority youth, grades 8 and 9, and was limited to 25 students. Students were selected to participate based upon their interest, desire, maturity, and attitude.

  13. Induction of apoptosis on human hepatocarcinoma cell lines by an alkyl resorcinol isolated from Lithraea molleoides

    Institute of Scientific and Technical Information of China (English)

    Luciana Barbini; Paula Lopez; Julieta Ruffa; Virginia Martino; Graciela Ferraro; Rodolfo Campos; Lucia Cavallaro

    2006-01-01

    AIM: To study the mechanism of cytotoxicity of a new active 5-alkyl resorcinol [1, 3-dihydroxy-5- (tridec-4', 7'-dienyl) benzene] isolated from Lithraea molleoides leaves on liver tumor cells.METHODS: Human hepatocarcinoma cell lines (HepG2and Hep3B) in culture were treated with inhibitory concentrations, 50% of the compound, for 24 h. The induction of apoptosis was detected in treated cells by analysis of DNA fragmentation, DNA content, and acridine orange and propidium iodide staining.RESULTS: After 24 h of 5-alkyl resorcinol treatment,both cell lines showed: (1) the typical morphological alterations of apoptosis; (2) DNA fragmentation, detected by laddering and appearance of a subG0 population by flow cytometry; and (3) condensed and fragmented nuclei by acridine orange-propidium iodide staining.CONCLUSION: Based on the results, this compound exerts its cytotoxic effect in both hepatocellular cell lines through apoptotic cell death. For Hep3B, cells with mutated p53 and Fas, apoptosis would proceed by p53-or Fas-independent pathways.

  14. Pathogenic Vibrio Strains Isolated from Human Stool and Water Samples from Western Kenya

    Directory of Open Access Journals (Sweden)

    Roselida Achieng Owuor

    2016-03-01

    Full Text Available Objective: Investigate the type of pathogenic Vibrio strains from water and stool samples collected from Migori, SonduMiriu, Nyando and Yala regions in Western Kenya. Methods: A total of 811 samples (596 water and 215 stool samples were collected during the study periods of May to December 2013 and August to September 2014. Pathogenic Vibrio strains were identified through culturing in TCBS Agar, followed by oxidation, string and serological (polyvalent tests, respectively. The PCR analysis was done using combined primers targeting Vibrionaceae 16SrRNA and species specific primers for V. vulnificus and V. cholerae. Results: The results showed the presence of V. vulnificus and V. cholerae. However, V. parahaemolyticus was not found in any of the samples. The PCR results for 16SrRNA, Vib 1, and Vib 2 showed polymorphism in the genes, this was an indication of cross combination of genes from more than one strain in one isolate. Conclusion: The study showed the presence of V. cholerae (Ogawa and Inaba in water and human stool samples. Type B V. vulnificus was detected in the water sample collected from River Migori. This information is of essence in controlling and managing cholera in the western part of Kenya. J Microbiol Infect Dis 2016;6(1: 1-7

  15. Sequence and phylogenetic analysis of the VP4 gene of human rotaviruses isolated in Paraguay.

    Science.gov (United States)

    Espínola, E E; Amarilla, A; Arbiza, J; Parra, G I

    2008-01-01

    Nucleotide and amino acid analyzes of the VP4 gene of human rotaviruses isolated both in Paraguay and worldwide were carried out in order to increase our knowledge about the complex pattern of evolution of this virus in nature. Paraguayan strains bearing the P[8] genotype were grouped in the lineages P[8]-1, P[8]-2, and P[8]-3. Regardless of the year of detection, all of the G4 and G9 strains were related to lineage P[8]-3, whereas the G1 strains were related to the three lineages detected in Paraguay; this fact reinforces the notion of the existence of constraints within specific populations of rotavirus strains except for the G1 strains. In addition, we propose a phylogenetic classification for the P[4] strains in five different lineages (i.e. P[4]-1 to P[4]-5). The findings presented in this paper reinforce the importance of a continuous surveillance of rotavirus strains in order to predict the possible variants that will circulate in a country, and ultimately improve current vaccination programs.

  16. Selective isolation and differentiation of a stromal population of human embryonic stem cells with osteogenic potential

    DEFF Research Database (Denmark)

    Harkness, Linda M; Mahmood, Amer; Ditzel, Nicholas

    2011-01-01

    The derivation of osteogenic cells from human embryonic stem cells (hESC) has been hampered by the absence of easy and reproducible protocols. hESC grown in feeder-free conditions, often show a sub population of fibroblast-like, stromal cells growing between the colonies. Thus, we examined...... the possibility that these cells represent a population of stromal (mesenchymal) stem cells (hESC-stromal). Two in house derived hES cell lines (Odense3 and KMEB3) as well as an externally derived cell line (Hues8) were transitioned to feeder-free conditions. A sub population of fibroblast-like cells established...... between the hESC colonies were isolated by selective adherence to hyaluronic acid-coated plates (100μg/ml) and were characterized using a combination of FACS analysis and staining. The cells were CD44(+), CD29(+), CD73(+), CD166(+), CD146(+), and CD105(+); and, Oct4(-), CD34(-), CD45(-) and CXCR4(-). When...

  17. Identification and characterization of toxin-antitoxin systems in strains of Lactobacillus rhamnosus isolated from humans.

    Science.gov (United States)

    Klimina, K M; Kjasova, D K; Poluektova, E U; Krügel, H; Leuschner, Y; Saluz, H-P; Danilenko, V N

    2013-08-01

    The toxin-antitoxin gene systems (TASs) are present in the genomes of the overwhelming majority of bacteria and archaea. These systems are involved in various cellular regulatory processes (including stress response), and have not been previously investigated in Lactobacilli. We identified 6 putative TASs with toxins belonging to the MazE and RelE superfamilies (PemK1-А1Lrh, PemK2-А2Lrh, PemK3-RelB2Lrh, RelE1Lrh, RelB3-RelE3Lrh, and YefM-YoeBLrh) in the genomes of annotated strains of Lactobacillus rhamnosus. PCR analyses revealed that all systems were found in the genomes of 15 strains of L. rhamnosus isolated from humans in central Russia. These strains were highly heterogeneous with respect to the presence of TASs, as well as their nucleotide and amino acid sequences. In three cases, the relE1 genes contained IS3 elements. TAS heterogeneity may be used to reveal inter-genus differences between strains. Cloning of the toxin genes of 3 TASs inhibited Escherichia coli growth, thus confirming their functionality. Cell growth arrest caused by expression of the toxin genes could be reverted by the expression of a cognate antitoxins. Transcription of toxin-antitoxin loci in L. rhamnosus was shown by RT-PCR. Copyright © 2013 Elsevier Ltd. All rights reserved.

  18. Isolation of a rice gene homologous to the human putative tumor suppressor gene QM

    Institute of Scientific and Technical Information of China (English)

    2002-01-01

    QM gene was originally isolated from human by Dowdy et al during a search for a wilms′ tumor suppressor gene. Researches of QM gene focused mainly on animals and yeasts, little was known about plant QM gene. For better understanding of QM gene in rice, a QM homologous fragment was used as a probe to screen rice (Oryza sativa subsp. indica c.v. Guanglu′ ai 4) genomic DNA library,and two clones were obtained. One of them, OSQM2, encoded a highly basic protein of 184 amino acids, the sequence was about 3.1 kb long with a very special promoter region compared with other known QM genes. Seven potential G boxes could be found between -690 and -230. G box, which contains a ACGT core motif, had been reported in many plants to act as a cis acting DNA element in the regulation of genes in a variety of environmental conditions, such as ABA regulated gene expression, red light, UV light, anaerobiosis, and wounding etc. Two closely linked DRE related motifs (dehydration responsive element) could also be found between -182 and 173, which had a CCGAC conserved sequence and had been identified in many cold and drought responsive genes in Arabidopsis. Six MYC recognition sequences with the conserved motif NCANNTGN were also presented, which might be essential for ABA and drought responsive expression of the plant genes.

  19. Induction of apoptosis on human hepatocarcinoma cell lines by an alkyl resorcinol isolated from Lithraea molleoides

    Science.gov (United States)

    Barbini, Luciana; Lopez, Paula; Ruffa, Julieta; Martino, Virginia; Ferraro, Graciela; Campos, Rodolfo; Cavallaro, Lucia

    2006-01-01

    AIM: To study the mechanism of cytotoxicity of a new active 5-alkyl resorcinol [1, 3-dihydroxy-5- (tridec-4’, 7’-dienyl) benzene] isolated from Lithraea molleoides leaves on liver tumor cells. METHODS: Human hepatocarcinoma cell lines (HepG2 and Hep3B) in culture were treated with inhibitory concentrations, 50% of the compound, for 24 h. The induction of apoptosis was detected in treated cells by analysis of DNA fragmentation, DNA content, and acridine orange and propidium iodide staining. RESULTS: After 24 h of 5-alkyl resorcinol treatment, both cell lines showed: (1) the typical morphological alterations of apoptosis; (2) DNA fragmentation, detected by laddering and appearance of a subG0 population by flow cytometry; and (3) condensed and fragmented nuclei by acridine orange-propidium iodide staining. CONCLUSION: Based on the results, this compound exerts its cytotoxic effect in both hepatocellular cell lines through apoptotic cell death. For Hep3B, cells with mutated p53 and Fas, apoptosis would proceed by p53- or Fas-independent pathways. PMID:17009393

  20. [Isolation of isoforms of apolipoprotein CIII from human serum by chromatofocusing].

    Science.gov (United States)

    Fang, D; Gong, R; O, K

    1999-03-01

    This study aimed to isolate isoforms of apolipoprotein (apo) C III from human serum. 24-hour fasting serum from normal and hyperlipidemic subjects was pooled and subjected to ultracentrifugation at plasma density for 20 hours. Very low density lipoprotein (VLDL) was collected at density of d < 1.006 g/ml, and it was delipidated by ethanol and ether. The delipidated apo-VLDL was dissolved in a solution containing 7.2 mol/L urea and 20 mmol/L dithiothreitol. The insoluble apo B was removed by centrifugation. The soluble apo-VLDL was applied to PBE94 column, and eluted with elution buffer containing polybuffer 74 and 8 mol/L urea (1:8, pH4.0). After pooled, the eluted peaks of apolipoproteins were applied to column chromatography of hydroxylapatite to remove the polybuffer. The purified isoforms of apoC III and the purified apo C I, C II and E, were characterized by isoelectrofocusing and west blot. The results showed that the purified apoC III1, C III2, and C II were pure.

  1. Characterization of H5N1 influenza viruses isolated from humans in vitro

    Directory of Open Access Journals (Sweden)

    Kameoka Masanori

    2010-06-01

    Full Text Available Abstract Since December 1997, highly pathogenic avian influenza A H5N1viruses have swept through poultry populations across Asian countries and been transmitted into African and European countries. We characterized 6 avian influenza H5N1 viruses isolated from humans in 2004 in Thailand. A highly pathogenic (HP KAN353 strain showed faster replication and higher virulence in embryonated eggs compared to other strains, especially compared to the low pathogenic (LP SP83 strain. HP KAN353 also showed strong cytopathogenicity compared to SP83 in Madin-Darby canine kidney cells. Interestingly, LP SP83 induced smaller plaques compared to other strains, especially HP KAN353. PB2 amino acid 627E may contribute to low virulence, whereas either PB2 amino acid 627 K or the combination of 627E/701N seems to be associated with high virulence. The in vitro assays used in this study may provide the basis for assessing the pathogenesis of influenza H5N1 viruses in vivo.

  2. Development of a Potential Probiotic Fresh Cheese Using Two Lactobacillus salivarius Strains Isolated from Human Milk

    Directory of Open Access Journals (Sweden)

    Nivia Cárdenas

    2014-01-01

    Full Text Available Cheeses have been proposed as a good alternative to other fermented milk products for the delivery of probiotic bacteria to the consumer. The objective of this study was to assess the survival of two Lactobacillus salivarius strains (CECT5713 and PS2 isolated from human milk during production and storage of fresh cheese for 28 days at 4°C. The effect of such strains on the volatile compounds profile, texture, and other sensorial properties, including an overall consumer acceptance, was also investigated. Both L. salivarius strains remained viable in the cheeses throughout the storage period and a significant reduction in their viable counts was only observed after 21 days. Globally, the addition of the L. salivarius strains did not change significantly neither the chemical composition of the cheese nor texture parameters after the storage period, although cheeses manufactured with L. salivarius CECT5713 presented significantly higher values of hardness. A total of 59 volatile compounds were identified in the headspace of experimental cheeses, and some L. salivarius-associated differences could be identified. All cheeses presented good results of acceptance after the sensory evaluation. Consequently, our results indicated that fresh cheese can be a good vehicle for the two L. salivarius strains analyzed in this study.

  3. Effect of human isolated probiotic bacteria on preventing Campylobacter jejuni colonization of poultry.

    Science.gov (United States)

    Cean, Ada; Stef, Lavinia; Simiz, Eliza; Julean, Calin; Dumitrescu, Gabi; Vasile, Aida; Pet, Elena; Drinceanu, Dan; Corcionivoschi, Nicolae

    2015-02-01

    This study was performed in order to determine whether human isolated probiotic bacteria can be effective in reducing Campylobacter jejuni infection of chicken intestinal cells, in vitro, and in decreasing its colonization abilities within the chicken gut. Our results show that the probiotic strains Lactobacillus paracasei J. R, L. rhamnosus 15b, L. lactis Y, and L. lactis FOa had a significant effect on C. jejuni invasion of chicken primary cells, with the strongest inhibitory effect detected when a combination of four was administered. In regard to the in vivo effect, using all four strains in one combination prevented mucus colonization in the duodenum and cecum. Moreover, the pathogen load in the lumen of these two compartments was significantly reduced. When probiotics were introduced during the early growth period, the presence of the pathogen in feces was increased (p>0.05), but when they were given during the last week of growth, there was no significant effect. In conclusion, our data indicate that these four new probiotic strains are able to cause modifications in the chicken intestinal mucosa and can reduce the ability of C. jejuni to invade, in vitro, and to colonize, in vivo. These probiotics are now proven to be effective even when introduced in broiler's feed 7 days before slaughter, which makes them cost-effective for the producers.

  4. Identification, characterization and description of Arcobacter faecis sp. nov., isolated from a human waste septic tank.

    Science.gov (United States)

    Whiteduck-Léveillée, Kerri; Whiteduck-Léveillée, Jenni; Cloutier, Michel; Tambong, James T; Xu, Renlin; Topp, Edward; Arts, Michael T; Chao, Jerry; Adam, Zaky; Lévesque, C André; Lapen, David R; Villemur, Richard; Khan, Izhar U H

    2016-03-01

    A study on the taxonomic classification of Arcobacter species was performed on the cultures isolated from various fecal sources where an Arcobacter strain AF1078(T) from human waste septic tank near Ottawa, Ontario, Canada was characterized using a polyphasic approach. Genetic investigations including 16S rRNA, atpA, cpn60, gyrA, gyrB and rpoB gene sequences of strain AF1078(T) are unique in comparison with other arcobacters. Phylogenetic analysis based on the 16S rRNA gene sequence revealed that the strain is most closely related to Arcobacter lanthieri and Arcobacter cibarius. Analyses of atpA, cpn60, gyrA, gyrB and rpoB gene sequences suggested that strain AF1078(T) formed a phylogenetic lineage independent of other species in the genus. Whole-genome sequence, DNA-DNA hybridization, fatty acid profile and phenotypic analysis further supported the conclusion that strain AF1078(T) represents a novel Arcobacter species, for which the name Arcobacter faecis sp. nov. is proposed, with type strain AF1078(T) (=LMG 28519(T); CCUG 66484(T)).

  5. Isolation and Characterization of 2'-amino-modified RNA Aptamers for Human TNFα

    Institute of Scientific and Technical Information of China (English)

    Xinrui Yan; Xuwen Gao; Zhiqing Zhang

    2004-01-01

    Human tumor necrosis factor α (hTNFα), a pleiotropic cytokine with activities ranging from host defense mechanisms in infection and injury to severe toxicity in septic shock or other related diseases, is a promising target for drug screening. Using the SELEX (systematic evolution of ligands by exponential enrichment) process, we isolated oligonucleotide ligands (aptamers) with high affinities for hTNFα. Aptamers were selected from a starting pool of 40 randomized sequences composed of about 1015 RNA molecules. Representative aptamers were truncated to the minimal length with high affinity for hTNFα and were further modified by replacement of 2'-OH with 2'-F and 2'-NH2 at all ribopurine positions. These modified RNA aptamers were resistant to nuclease. The specificity of these aptamers for hTNFαwas confirmed, and their activity to inhibit the cytotoxicity of hTNFα on mouse L929 cells was determined. Results demonstrated that four 2'-NH2-modified aptamers bound to hTNFα with high affinity and blocked the binding of hTNFα to its receptor, thus protecting the L929 cells from the cytotoxicity of hTNFα. Oligonucleotide aptamers described here are potential therapeutics and diagnostics for hTNFc-related diseases.

  6. Biochemical and serological characterization of mycoplasma strains isolated from the genital tracts of humans in Nigeria.

    Science.gov (United States)

    Agbakoba, N R; Adetosoye, A I; Adewole, I F

    2006-06-01

    Fifty-five (55) Mycoplasma strains isolated from the genital tracts of humans were biochemically characterized using various biochemical tests and also serologically identified by growth inhibition technique using 5 mycoplasma antisera namely M. hominis PG2 1: M. genitalium G37: M. penetrans GTU54 and 2 strains of M. fermentans PG18 (HRC 6-62-S-170 and MB713-501-069). Biochemically, 43 (78.2%) strains were identified as Mycoplasma hominis, 8 (14.5%) strains as M. fermentans and 4 (7.3%) as M. penetrans. The M. hominis strains hydrolyzed only arginine while the M. fermentans and M. penetrans strains in addition to arginine hydrolysis also broke down glucose fermentatively and oxidatively. The M. fermentans strains showed varying reactions to phosphatase activity and to the reduction of tetrazolium chloride. Serologically, 4 (7.3%) mycoplasma strains were confirmed as M. penetrans GTU54 and of the 8 M. fermentans strains, 4 (7.3%) were identified as M. fermentans PG18 serotype HRC 6-62-S-170 and the other 4 (7.3%) as M. fermentans PG18 serotype MB 713-501-069. Only 13 (30.2%) of the 43 M. hominis strains were identified as M. hominis serotype PG2 1. None was identified as M. genitalium. The heterogeneity of the mycoplasma strains especially M. hominis was observed in this study and the need for the use of multiple antisera in growth inhibition test is hereby supported.

  7. Effect of pulmonary vein isolation on the distribution of complex fractionated electrograms in humans.

    Science.gov (United States)

    Roux, Jean-François; Gojraty, Sattar; Bala, Rupa; Liu, Christopher F; Dixit, Sanjay; Hutchinson, Mathew D; Garcia, Fermin; Lin, David; Callans, David J; Riley, Michael; Marchlinski, Francis; Gerstenfeld, Edward P

    2009-02-01

    Targeting of complex fractionated electrograms (CFEs) has been used as an adjunctive strategy to pulmonary vein isolation (PVI) in patients with persistent atrial fibrillation (AF). However, it is unclear whether CFEs should be targeted before or after PVI. The purpose of this study was to examine the effect of PVI on CFE distribution in humans. We compared left atrial (LA) CFE maps acquired using the NavX system before and after PVI in patients with persistent AF. CFE maps were constructed from bipolar electrograms acquired from a circular mapping catheter. At each point, the mean AF cycle length (CL) was calculated automatically by averaging the intervals between deflections over a 4-second window. Sites with mean CL CFE+. A total of 22 consecutive patients (82% male, age 58 +/- 9 years) were studied. At baseline, 47% of the LA was encompassed by electrograms with CL CFE characteristics, with an increase in mean LA AF CL (144 ms pre-PVI vs. 214 ms post-PVI; P CFE+ LA surface area (47% vs 23%; P CFE burden after PVI in both PV (50% vs. 6%; P CFE. To limit extensive LA ablation, PVI should be performed before targeting CFE when a combined approach is pursued.

  8. Isolation and Multiple Differentiation Potential Assessment of Human Gingival Mesenchymal Stem Cells

    Directory of Open Access Journals (Sweden)

    Yuan Gao

    2014-11-01

    Full Text Available The aim of this study was to isolate human mesenchymal stem cells (MSCs from the gingiva (GMSCs and confirm their multiple differentiation potentials, including the odontogenic lineage. GMSCs, periodontal ligament stem cells (PDLSCs and dermal stem cells (DSCs cultures were analyzed for cell shape, cell cycle, colony-forming unit-fibroblast (CFU-F and stem cell markers. Cells were then induced for osteogenic and adipogenic differentiation and analyzed for differentiation markers (alkaline phosphatase (ALP activity, mineralization nodule formation and Runx2, ALP, osteocalcin (OCN and collagen I expressions for the osteogenic differentiation, and lipid vacuole formation and PPARγ-2 expression for the adipogenic differentiation. Besides, the odontogenic differentiation potential of GMSCs induced with embryonic tooth germ cell-conditioned medium (ETGC-CM was observed. GMSCs, PDLSCs and DSCs were all stromal origin. PDLSCs showed much higher osteogenic differentiation ability but lower adipogenic differentiation potential than DSCs. GMSCs showed the medial osteogenic and adipogenic differentiation potentials between those of PDLSCs and DSCs. GMSCs were capable of expressing the odontogenic genes after ETGC-CM induction. This study provides evidence that GMSCs can be used in tissue engineering/regeneration protocols as an approachable stem cell source.

  9. Isolation of novel human cDNA (hGMF-gamma) homologous to Glia Maturation Factor-beta gene.

    Science.gov (United States)

    Asai, K; Fujita, K; Yamamoto, M; Hotta, T; Morikawa, M; Kokubo, M; Moriyama, A; Kato, T

    1998-03-13

    A novel full-length human cDNA homologous to Glia Maturation Factor-beta (GMF-beta) gene was isolated. Sequence analysis of the entire cDNA revealed an open reading frame of 426 nucleotides with a deduced protein sequence of 142 amino acid residues. The deduced amino acid sequences of its putative product is highly homologous to human GMF-beta (82% identity) and named for GMF-gamma. Northern blot analysis indicated that a message of 0.9 kb long, but not 4.1 kb of GMF-beta, is predominantly expressed in human lung, heart, and placenta.

  10. Isolation of human monocytes by double gradient centrifugation and their differentiation to macrophages in teflon-coated cell culture bags.

    Science.gov (United States)

    Menck, Kerstin; Behme, Daniel; Pantke, Mathias; Reiling, Norbert; Binder, Claudia; Pukrop, Tobias; Klemm, Florian

    2014-09-09

    Human macrophages are involved in a plethora of pathologic processes ranging from infectious diseases to cancer. Thus they pose a valuable tool to understand the underlying mechanisms of these diseases. We therefore present a straightforward protocol for the isolation of human monocytes from buffy coats, followed by a differentiation procedure which results in high macrophage yields. The technique relies mostly on commonly available lab equipment and thus provides a cost and time effective way to obtain large quantities of human macrophages. Briefly, buffy coats from healthy blood donors are subjected to a double density gradient centrifugation to harvest monocytes from the peripheral blood. These monocytes are then cultured in fluorinated ethylene propylene (FEP) Teflon-coated cell culture bags in the presence of macrophage colony-stimulating factor (M-CSF). The differentiated macrophages can be easily harvested and used for subsequent studies and functional assays. Important methods for quality control and validation of the isolation and differentiation steps will be highlighted within the protocol. In summary, the protocol described here enables scientists to routinely and reproducibly isolate human macrophages without the need for cost intensive tools. Furthermore, disease models can be studied in a syngeneic human system circumventing the use of murine macrophages.

  11. Detection and viability of Campylobacter species isolates from different species of poultry and humans in Sokoto State, Nigeria

    Directory of Open Access Journals (Sweden)

    I. O. Nwankwo

    2016-04-01

    Full Text Available Aim: The study was conducted to determine the prevalence and viability of Campylobacter species isolates from different species of poultry and humans in Sokoto State, Nigeria. Materials and Methods: A cross-sectional study was performed in the live birds markets, humans on admission and at outpatient clinics in the randomly selected hospitals in Sokoto State. Isolation and characterization of Campylobacter species were performed using standard culture isolation techniques and biochemical characterization. A total of 798 (506 cloacal and 292 fecal swabs from poultry and humans, respectively, were collected and analyzed. The viability of 307 isolates stored in 15% glycerol and 85% tryptone broth at −20°C was determined after 7-13 months. Results: A total of 312 (39% were positive for Campylobacter species which comprises 119 (30%, 20 (30%, 3 (14%, 9 (56%, 1 (50%, and 160 (55% in chicken, guinea fowls, pigeons, ducks, turkey, and humans, respectively. The total of 38 (24%, 63 (39%, and 59 (37% humans and 29 (19%, 79 (52%, and 44 (29% poultry isolates were positive for Campylobacter jejuni, Campylobacter Coli, and Campylobacter Lari, respectively. A total of 261 (85% of the stored isolates were still viable on re-isolation with the viability rates of 41 (95%, 67 (85%, and 17 (59% at 7, 9, and 13 months of storage, respectively. There was a negative correlation between months of storage and viability rates. However, there was no significant statistical association (p>0.05 between prevalence rate and species of poultry. Conclusion: Campylobacter species have been detected with varying degree of prevalence in both poultry and humans and their ability to survive freezing at −20°C (95% for up to 7 months has been revealed in the study. This is not only a concern to food and livestock industries but also a concern to the public health at large, especially, in view of the study area being considered one of the largest livestock producers in Nigeria

  12. Effects of chronic triclosan exposure upon the antimicrobial susceptibility of 40 ex-situ environmental and human isolates.

    Science.gov (United States)

    Ledder, R G; Gilbert, P; Willis, C; McBain, A J

    2006-05-01

    Triclosan (TCS) exposure of Escherichia coli selects for tolerant clones, mutated in their enoyl-acyl carrier protein reductase (FabI). It has been inferred that this phenomenon is widespread amongst bacterial genera and might be associated with resistance to third party agents. Ex-situ, low passage isolates of enteric, human axilla, human oral origin and bacteria isolated from a domestic drain, together with selected type cultures were exposed to escalating concentrations of TCS over 10 passages using a gradient plate technique. One fresh faecal isolate of E. coli was included as a positive control. TCS susceptibility was determined for all strains before and after exposure, whilst enteric isolates were additionally assessed for susceptibility towards chlorhexidine, tetracycline, chloramphenicol, nalidixic acid and ciprofloxacin, and the oral isolates towards chlorhexidine, tetracycline and metronidazole. Triclosan exposure of E. coli markedly decreased TCS susceptibility. TCS susceptibility also decreased for Klebsiella oxytoca, Aranicola proteolyticus and Stenotrophomonas maltophilia. Susceptibility of the remaining 35 strains to TCS and the other test agents remained unchanged. These data suggest that selection for high level resistance by TCS exposure is not widespread and appears to be confined to certain enteric bacteria, especially E. coli. Change in TCS susceptibility did not affect susceptibility towards chemically unrelated antimicrobials. Acquired high-level TCS resistance is not a widespread phenomenon.

  13. Isolation and maintenance of Balantidium coli (Malmsteim, 1857) cultured from fecal samples of pigs and non-human primates.

    Science.gov (United States)

    Barbosa, Alynne da Silva; Bastos, Otilio Machado Pereira; Uchôa, Claudia M Antunes; Pissinatti, Alcides; Ferreira Filho, Paulo Ricardo; Dib, Lais Verdan; Azevedo, Eduarda Peixoto; de Siqueira, Mayara Perlingeiro; Cardozo, Matheus Lessa; Amendoeira, Maria Regina Reis

    2015-06-15

    Balantidium coli is a protozoa that can determine dysentery in humans, pigs and non-human primates having zoonotic potential. The lack of standardization in isolation and maintenance hinders the development of research on its biology and epidemiology. This study is aimed to standardize the isolation and maintenance of this parasite from animal feces, in culture medium, Pavlova modified. From 2012 to 2014, 1905 fecal samples were collected from captive animals of Rio de Janeiro. Were selected for isolation samples with a minimum of 10 trophozoites and/or 30 cysts of B. coli, totaling 88 pigs, 26 Cynomolgus and 90 rhesus macaques. In the presence of cysts, the sample was homogenized in saline solution, 500 μL was removed and inoculated into culture medium. The material that contained trophozoites the inoculum was made from 240 μL of fecal solution. All inoculate tubes with the subcultures were kept at 36°C, and sterile rice starch was always added to the medium. The parasites isolate from pigs, 34%, and from Cynomolgus 38.4% were maintained in vitro for a period of more than 24 months. These procedures proved to be adequate for isolation and maintenance of B. coli from different animals, they were found to be inexpensive and easy to perform.

  14. Distribution of phylogroups and co-resistance to antimicrobial agents in ampicillin resistant Escherichia coli isolated from healthy humans and from patients with bacteraemia

    DEFF Research Database (Denmark)

    Haugaard, A.; Hammerum, A. M.; Porsbo, Lone Jannok;

    In 2002-03, 31 ampicillin resistant faecal isolates were collected from healthy humans. Moreover, 31 ampicillin resistant blood isolates from patients with bacte-raemia were collected in 2000-02. All isolates were tested positive for the pres-ence of blaTEM. Isolates were characterized by minimum...... inhibitory concentration to antimicrobial agents and examined by PCR to determine their phylogroups. The phylotyping grouped the faecal samples into A (13%), B1 (10%), B2 (42%), D (19%), NT (16%) while the blood isolates grouped into A (16%), B1 (0%), B2 (48%), D (32%) and NT (3%). The frequency...... of resistance in faecal and blood isolates (F/B) was: tetracycline (48%/48%), gentamicin (0%/10%), ciprofloxacin (3%,13%), sulfonamide (68%/77%) and trimethoprim (39%/39%). Conclusion: B2 was the most prevalent phylogroup found both in faecal isolates collected from healthy humans and in blood isolates from...

  15. Characterization of vancomycin-resistant and vancomycin-susceptible Enterococcus faecium isolates from humans, chickens and pigs by RiboPrinting and pulsed-field gel electrophoresis

    DEFF Research Database (Denmark)

    Hammerum, Anette Marie; Fussing, Vivian; Aarestrup, Frank Møller;

    2000-01-01

    Forty-eight vancomycin-resistant and 35 vancomycin-sensitive Danish Enterococcus faecium isolates obtained from pigs, chickens and humans, as well as the human vanA reference isolate BM4147, were characterized by EcoRI RiboPrinting and Smal pulsed-field gel electrophoresis. RiboPrinting of the 84...

  16. Characterization of vancomycin-resistant and vancomycin-susceptible Enterococcus faecium isolates from humans, chickens and pigs by RiboPrinting and pulsed-field gel electrophoresis

    DEFF Research Database (Denmark)

    Hammerum, Anette Marie; Fussing, Vivian; Aarestrup, Frank Møller

    2000-01-01

    Forty-eight vancomycin-resistant and 35 vancomycin-sensitive Danish Enterococcus faecium isolates obtained from pigs, chickens and humans, as well as the human vanA reference isolate BM4147, were characterized by EcoRI RiboPrinting and Smal pulsed-field gel electrophoresis. RiboPrinting of the 84...

  17. Platelet-rich plasma promotes the development of isolated human primordial and primary follicles to the preantral stage.

    Science.gov (United States)

    Hosseini, Laleh; Shirazi, Abolfazl; Naderi, Mohammad Mehdi; Shams-Esfandabadi, Naser; Borjian Boroujeni, Sara; Sarvari, Ali; Sadeghnia, Samaneh; Behzadi, Bahareh; Akhondi, Mohammad Mehdi

    2017-10-01

    This study aimed to assess the effects of platelet-rich plasma (PRP) on growth and survival of isolated early human follicles in a three-dimensional culture system. After fresh and vitrified-warmed ovarian tissue was digested, isolated early preantral follicles and ovarian cells were separately encapsulated in 1% alginate (w/v). The encapsulated follicles and ovarian cells were cultured together in a medium supplemented with foetal bovine serum (FBS), PRP, PRP + FBS, or human serum albumin (HSA) for 10 days. Growth and survival of the follicles were assessed by measurement of diameter and staining with trypan blue. Follicular integrity was assessed by histological analysis. After culturing, all follicles increased in size, but growth rate was greater in follicles isolated from fresh samples than those from vitrified-warmed ones (P media were significantly higher than those of other groups (growth P media supplementation with PRP can better support viability and growth of isolated human early preantral follicles in vitro. Copyright © 2017 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.

  18. Leptospira Serovars for Diagnosis of Leptospirosis in Humans and Animals in Africa: Common Leptospira Isolates and Reservoir Hosts.

    Science.gov (United States)

    Mgode, Georgies F; Machang'u, Robert S; Mhamphi, Ginethon G; Katakweba, Abdul; Mulungu, Loth S; Durnez, Lies; Leirs, Herwig; Hartskeerl, Rudy A; Belmain, Steven R

    2015-12-01

    The burden of leptospirosis in humans and animals in Africa is higher than that reported from other parts of the world. However, the disease is not routinely diagnosed in the continent. One of major factors limiting diagnosis is the poor availability of live isolates of locally circulating Leptospira serovars for inclusion in the antigen panel of the gold standard microscopic agglutination test (MAT) for detecting antibodies against leptospirosis. To gain insight in Leptospira serovars and their natural hosts occurring in Tanzania, concomitantly enabling the improvement of the MAT by inclusion of fresh local isolates, a total of 52 Leptospira isolates were obtained from fresh urine and kidney homogenates, collected between 1996 and 2006 from small mammals, cattle and pigs. Isolates were identified by serogrouping, cross agglutination absorption test (CAAT), and molecular typing. Common Leptospira serovars with their respective animal hosts were: Sokoine (cattle and rodents); Kenya (rodents and shrews); Mwogolo (rodents); Lora (rodents); Qunjian (rodent); serogroup Grippotyphosa (cattle); and an unknown serogroup from pigs. Inclusion of local serovars particularly serovar Sokoine in MAT revealed a 10-fold increase in leptospirosis prevalence in Tanzania from 1.9% to 16.9% in rodents and 0.26% to 10.75% in humans. This indicates that local serovars are useful for diagnosis of human and animal leptospirosis in Tanzania and other African countries.

  19. Leptospira Serovars for Diagnosis of Leptospirosis in Humans and Animals in Africa: Common Leptospira Isolates and Reservoir Hosts.

    Directory of Open Access Journals (Sweden)

    Georgies F Mgode

    2015-12-01

    Full Text Available The burden of leptospirosis in humans and animals in Africa is higher than that reported from other parts of the world. However, the disease is not routinely diagnosed in the continent. One of major factors limiting diagnosis is the poor availability of live isolates of locally circulating Leptospira serovars for inclusion in the antigen panel of the gold standard microscopic agglutination test (MAT for detecting antibodies against leptospirosis. To gain insight in Leptospira serovars and their natural hosts occurring in Tanzania, concomitantly enabling the improvement of the MAT by inclusion of fresh local isolates, a total of 52 Leptospira isolates were obtained from fresh urine and kidney homogenates, collected between 1996 and 2006 from small mammals, cattle and pigs. Isolates were identified by serogrouping, cross agglutination absorption test (CAAT, and molecular typing. Common Leptospira serovars with their respective animal hosts were: Sokoine (cattle and rodents; Kenya (rodents and shrews; Mwogolo (rodents; Lora (rodents; Qunjian (rodent; serogroup Grippotyphosa (cattle; and an unknown serogroup from pigs. Inclusion of local serovars particularly serovar Sokoine in MAT revealed a 10-fold increase in leptospirosis prevalence in Tanzania from 1.9% to 16.9% in rodents and 0.26% to 10.75% in humans. This indicates that local serovars are useful for diagnosis of human and animal leptospirosis in Tanzania and other African countries.

  20. Leptospira Serovars for Diagnosis of Leptospirosis in Humans and Animals in Africa: Common Leptospira Isolates and Reservoir Hosts

    Science.gov (United States)

    Mgode, Georgies F.; Machang’u, Robert S.; Mhamphi, Ginethon G.; Katakweba, Abdul; Mulungu, Loth S.; Durnez, Lies; Leirs, Herwig; Hartskeerl, Rudy A.; Belmain, Steven R.

    2015-01-01

    The burden of leptospirosis in humans and animals in Africa is higher than that reported from other parts of the world. However, the disease is not routinely diagnosed in the continent. One of major factors limiting diagnosis is the poor availability of live isolates of locally circulating Leptospira serovars for inclusion in the antigen panel of the gold standard microscopic agglutination test (MAT) for detecting antibodies against leptospirosis. To gain insight in Leptospira serovars and their natural hosts occurring in Tanzania, concomitantly enabling the improvement of the MAT by inclusion of fresh local isolates, a total of 52 Leptospira isolates were obtained from fresh urine and kidney homogenates, collected between 1996 and 2006 from small mammals, cattle and pigs. Isolates were identified by serogrouping, cross agglutination absorption test (CAAT), and molecular typing. Common Leptospira serovars with their respective animal hosts were: Sokoine (cattle and rodents); Kenya (rodents and shrews); Mwogolo (rodents); Lora (rodents); Qunjian (rodent); serogroup Grippotyphosa (cattle); and an unknown serogroup from pigs. Inclusion of local serovars particularly serovar Sokoine in MAT revealed a 10-fold increase in leptospirosis prevalence in Tanzania from 1.9% to 16.9% in rodents and 0.26% to 10.75% in humans. This indicates that local serovars are useful for diagnosis of human and animal leptospirosis in Tanzania and other African countries. PMID:26624890

  1. Mapping and sequencing the human genome: Science, ethics, and public policy. Final report

    Energy Technology Data Exchange (ETDEWEB)

    McInerney, J.D.

    1993-03-31

    Development of Mapping and Sequencing the Human Genome: Science, Ethics, and Public Policy followed the standard process of curriculum development at the Biological Sciences Curriculum Study (BSCS), the process is described. The production of this module was a collaborative effort between BSCS and the American Medical Association (AMA). Appendix A contains a copy of the module. Copies of reports sent to the Department of Energy (DOE) during the development process are contained in Appendix B; all reports should be on file at DOE. Appendix B also contains copies of status reports submitted to the BSCS Board of Directors.

  2. The Human Genome Project and Mental Retardation: An Educational Program. Final Progress Report

    Energy Technology Data Exchange (ETDEWEB)

    Davis, Sharon

    1999-05-03

    The Arc, a national organization on mental retardation, conducted an educational program for members, many of whom have a family member with a genetic condition causing mental retardation. The project informed members about the Human Genome scientific efforts, conducted training regarding ethical, legal and social implications and involved members in issue discussions. Short reports and fact sheets on genetic and ELSI topics were disseminated to 2,200 of the Arc's leaders across the country and to other interested individuals. Materials produced by the project can e found on the Arc's web site, TheArc.org.

  3. Avoidance of Maternal Cell Contamination and Overgrowth in Isolating Fetal Chorionic Villi Mesenchymal Stem Cells from Human Term Placenta.

    Science.gov (United States)

    Sardesai, Varda S; Shafiee, Abbas; Fisk, Nicholas M; Pelekanos, Rebecca A

    2017-04-01

    Human placenta is rich in mesenchymal stem/stromal cells (MSC), with their origin widely presumed fetal. Cultured placental MSCs are confounded by a high frequency of maternal cell contamination. Our recent systematic review concluded that only a small minority of placental MSC publications report fetal/maternal origin, and failed to discern a specific methodology for isolation of fetal MSC from term villi. We determined isolation conditions to yield fetal and separately maternal MSC during ex vivo expansion from human term placenta. MSCs were isolated via a range of methods in combination; selection from various chorionic regions, different commercial media, mononuclear cell digest and/or explant culture. Fetal and maternal cell identities were quantitated in gender-discordant pregnancies by XY chromosome fluorescence in situ hybridization. We first demonstrated reproducible maternal cell contamination in MSC cultures from all chorionic anatomical locations tested. Cultures in standard media rapidly became composed entirely of maternal cells despite isolation from fetal villi. To isolate pure fetal cells, we validated a novel isolation procedure comprising focal dissection from the cotyledonary core, collagenase/dispase digestion and explant culture in endothelial growth media that selected, and provided a proliferative environment, for fetal MSC. Comparison of MSC populations within the same placenta confirmed fetal to be smaller, more osteogenic and proliferative than maternal MSC. We conclude that in standard media, fetal chorionic villi-derived MSC (CV-MSC) do not grow readily, whereas maternal MSC proliferate to result in maternal overgrowth during culture. Instead, fetal CV-MSCs require isolation under specific conditions, which has implications for clinical trials using placental MSC. Stem Cells Translational Medicine 2017;6:1070-1084.

  4. Evaluation of a single procedure allowing the isolation of enteropathogenic Yersinia along with other bacterial enteropathogens from human stools.

    Science.gov (United States)

    Savin, Cyril; Leclercq, Alexandre; Carniel, Elisabeth

    2012-01-01

    Enteropathogenic Yersinia are among the most frequent agents of human diarrhea in temperate and cold countries. However, the incidence of yersiniosis is largely underestimated because of the peculiar growth characteristics of pathogenic Yersinia, which make their isolation from poly-contaminated samples difficult. The use of specific procedures for Yersinia isolation is required, but is expensive and time consuming, and therefore is not systematically performed in clinical pathology laboratories. A means to circumvent this problem would be to use a single procedure for the isolation of all bacterial enteropathogens. Since the Statens Serum Institut enteric medium (SSI) has been reported to allow the growth at 37°C of most gram-negative bacteria, including Yersinia, our study aimed at evaluating its performances for Yersinia isolation, as compared to the commonly used Yersinia-specific semi-selective Cefsulodin-Irgasan-Novobiocin medium (CIN) incubated at 28°C. Our results show that Yersinia pseudotuberculosis growth was strongly inhibited on SSI at 37°C, and therefore that this medium is not suitable for the isolation of this species. All Yersinia enterocolitica strains tested grew on SSI, while some non-pathogenic Yersinia species were inhibited. The morphology of Y. enterocolitica colonies on SSI allowed their differentiation from various other gram-negative bacteria commonly isolated from stool samples. However, in artificially contaminated human stools, the recovery of Y. enterocolitica colonies on SSI at 37°C was difficult and was 3 logs less sensitive than on CIN at 28°C. Therefore, despite its limitations, the use of a specific procedure (CIN incubated at 28°C) is still required for an efficient isolation of enteropathogenic Yersinia from stools.

  5. Streptococcus agalactiae isolates of serotypes Ia, III and V from human and cow are able to infect tilapia.

    Science.gov (United States)

    Chen, Ming; Wang, Rui; Luo, Fu-Guang; Huang, Yan; Liang, Wan-Wen; Huang, Ting; Lei, Ai-Ying; Gan, Xi; Li, Li-Ping

    2015-10-22

    Recent studies have shown that group B streptococcus (GBS) may be infectious across hosts. The purpose of this study is to investigate the pathogenicity of clinical GBS isolates with serotypes Ia, III and V from human and cow to tilapia and the evolutionary relationship among these GBS strains of different sources. A total of 27 clinical GBS isolates from human (n=10), cow (n=2) and tilapia (n=15) were analyzed using serotyping, multi-locus sequence typing (MLST) and pulsed-field gel electrophoresis (PFGE). Among them, 15 isolates were tested for their pathogenicity to tilapia. The results showed that five human GBS strains (2 serotype III, 2 serotype Ia and 1 serotype V) infected tilapia with mortality rate ranging from 56.67% to 100%, while the other five human GBS strains tested were unable to infect tilapia. In addition, two cow GBS strains C001 and C003 of serotype III infected tilapia. However, they had significantly lower pathogenicity than the five human strains. Furthermore, human GBS strains H005 and H008, which had very strong ability to infect tilapia, had the same PFGE pattern. MLST analysis showed that the five human and the two cow GBS strains that were able to infect tilapia belonged to clonal complexes CC19, CC23 and CC103. The study for the first time confirmed that human or cow GBS clonal complexes CC19, CC23 and CC103 containing strains with serotypes Ia, III and V could infect tilapia and induce clinical signs under experimental conditions.

  6. whole-genome sequence of livestock-associated st398 methicillin-resistant staphylococcus aureus Isolated from Humans in Canada.

    Science.gov (United States)

    Golding, George R; Bryden, Louis; Levett, Paul N; McDonald, Ryan R; Wong, Alice; Graham, Morag R; Tyler, Shaun; Van Domselaar, Gary; Mabon, Philip; Kent, Heather; Butaye, Patrick; Smith, Tara C; Kadlec, Kristina; Schwarz, Stefan; Weese, Scott J; Mulvey, Michael R

    2012-12-01

    Despite reports of high colonization rates of ST398 livestock-associated methicillin-resistant Staphylococcus aureus (LA-MRSA) among pigs and pig farmers, the incidence of LA-MRSA infection in the general population in Canada appears to be rare in comparison to that in some European countries. In this study, the complete genome sequence of a Canadian representative LA-MRSA isolate (08BA02176) from a human postoperative surgical site infection was acquired and compared to the sequenced genome of an LA-MRSA isolate (S0385) from Europe to identify genetic traits that may explain differences in the success of these particular strains in some locales.

  7. Identification of Brucella spp. isolated from human brucellosis in Malaysia using high-resolution melt (HRM) analysis.

    Science.gov (United States)

    Mohamed Zahidi, Jama'ayah; Bee Yong, Tay; Hashim, Rohaidah; Mohd Noor, Azura; Hamzah, Siti Hawa; Ahmad, Norazah

    2015-04-01

    Molecular approaches have been investigated to overcome difficulties in identification and differentiation of Brucella spp. using conventional phenotypic methods. In this study, high-resolution melt (HRM) analysis was used for rapid identification and differentiation of members of Brucella genus. A total of 41 Brucella spp. isolates from human brucellosis were subjected to HRM analysis using 4 sets of primers, which identified 40 isolates as Brucella melitensis and 1 as Brucella canis. The technique utilized low DNA concentration and was highly reproducible. The assay is shown to be a useful diagnostic tool, which can rapidly differentiate Brucella up to species level. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

  8. A human factors engineering evaluation of the Multi-Function Waste Tank Facility. Final report

    Energy Technology Data Exchange (ETDEWEB)

    Donohoo, D.T. [Pacific Northwest Lab., Richland, WA (United States); Sarver, T.L. [ARES Corp., San Francisco, CA (United States)

    1995-06-05

    This report documents the methods and results of a human factors engineering (HFE) review conducted on the Multi-Function Waste Tank Facility (MWTF), Westinghouse Hanford Company (WHC) Project 236A, to be constructed at the U.S. Department of Energy (DOE) facility at Hanford, Washington. This HFE analysis of the MWTF was initiated by WHC to assess how well the current facility and equipment design satisfies the needs of its operations and maintenance staff and other potential occupants, and to identify areas of the design that could benefit from improving the human interfaces at the facility. Safe and effective operations, including maintenance, is a primary goal for the MWTF. Realization of this goal requires that the MWTF facility, equipment, and operations be designed in a manner that is consistent with the abilities and limitations of its operating personnel. As a consequence, HFE principles should be applied to the MWTF design, construction, its operating procedures, and its training. The HFE review was focused on the 200-West Area facility as the design is further along than that of the 200-East Area. The review captured, to the greatest extent feasible at this stage of design, all aspects of the facility activities and included the major topics generally associated with HFE (e.g., communication, working environment). Lessons learned from the review of the 200 West facility will be extrapolated to the 200-East Area, as well as generalized to the Hanford Site.

  9. Further search for supersymmetry at $\\sqrt{s}$ = 7 TeV in final states with jets, missing transverse momentum and isolated leptons with the ATLAS detector

    CERN Document Server

    Aad, Georges; Abbott, Brad; Abdallah, Jalal; Abdel Khalek, Samah; Abdelalim, Ahmed Ali; Abdinov, Ovsat; Aben, Rosemarie; Abi, Babak; Abolins, Maris; AbouZeid, Ossama; Abramowicz, Halina; Abreu, Henso; Acharya, Bobby Samir; Adamczyk, Leszek; Adams, David; Addy, Tetteh; Adelman, Jahred; Adomeit, Stefanie; Adragna, Paolo; Adye, Tim; Aefsky, Scott; Aguilar-Saavedra, Juan Antonio; Agustoni, Marco; Aharrouche, Mohamed; Ahlen, Steven; Ahles, Florian; Ahmad, Ashfaq; Ahsan, Mahsana; Aielli, Giulio; Akdogan, Taylan; Åkesson, Torsten Paul Ake; Akimoto, Ginga; Akimov, Andrei; Alam, Mohammad; Alam, Muhammad Aftab; Albert, Justin; Albrand, Solveig; Aleksa, Martin; Aleksandrov, Igor; Alessandria, Franco; Alexa, Calin; Alexander, Gideon; Alexandre, Gauthier; Alexopoulos, Theodoros; Alhroob, Muhammad; Aliev, Malik; Alimonti, Gianluca; Alison, John; Allbrooke, Benedict; Allport, Phillip; Allwood-Spiers, Sarah; Almond, John; Aloisio, Alberto; Alon, Raz; Alonso, Alejandro; Alonso, Francisco; Altheimer, Andrew David; Alvarez Gonzalez, Barbara; Alviggi, Mariagrazia; Amako, Katsuya; Amelung, Christoph; Ammosov, Vladimir; Amor Dos Santos, Susana Patricia; Amorim, Antonio; Amram, Nir; Anastopoulos, Christos; Ancu, Lucian Stefan; Andari, Nansi; Andeen, Timothy; Anders, Christoph Falk; Anders, Gabriel; Anderson, Kelby; Andreazza, Attilio; Andrei, George Victor; Andrieux, Marie-Laure; Anduaga, Xabier; Anger, Philipp; Angerami, Aaron; Anghinolfi, Francis; Anisenkov, Alexey; Anjos, Nuno; Annovi, Alberto; Antonaki, Ariadni; Antonelli, Mario; Antonov, Alexey; Antos, Jaroslav; Anulli, Fabio; Aoki, Masato; Aoun, Sahar; Aperio Bella, Ludovica; Apolle, Rudi; Arabidze, Giorgi; Aracena, Ignacio; Arai, Yasuo; Arce, Ayana; Arfaoui, Samir; Arguin, Jean-Francois; Arik, Engin; Arik, Metin; Armbruster, Aaron James; Arnaez, Olivier; Arnal, Vanessa; Arnault, Christian; Artamonov, Andrei; Artoni, Giacomo; Arutinov, David; Asai, Shoji; Asfandiyarov, Ruslan; Ask, Stefan; Åsman, Barbro; Asquith, Lily; Assamagan, Ketevi; Astbury, Alan; Atkinson, Markus; Aubert, Bernard; Auge, Etienne; Augsten, Kamil; Aurousseau, Mathieu; Avolio, Giuseppe; Avramidou, Rachel Maria; Axen, David; Azuelos, Georges; Azuma, Yuya; Baak, Max; Baccaglioni, Giuseppe; Bacci, Cesare; Bach, Andre; Bachacou, Henri; Bachas, Konstantinos; Backes, Moritz; Backhaus, Malte; Backus Mayes, John; Badescu, Elisabeta; Bagnaia, Paolo; Bahinipati, Seema; Bai, Yu; Bailey, David; Bain, Travis; Baines, John; Baker, Oliver Keith; Baker, Mark; Baker, Sarah; Banas, Elzbieta; Banerjee, Piyali; Banerjee, Swagato; Banfi, Danilo; Bangert, Andrea Michelle; Bansal, Vikas; Bansil, Hardeep Singh; Barak, Liron; Baranov, Sergei; Barbaro Galtieri, Angela; Barber, Tom; Barberio, Elisabetta Luigia; Barberis, Dario; Barbero, Marlon; Bardin, Dmitri; Barillari, Teresa; Barisonzi, Marcello; Barklow, Timothy; Barlow, Nick; Barnett, Bruce; Barnett, Michael; Baroncelli, Antonio; Barone, Gaetano; Barr, Alan; Barreiro, Fernando; Barreiro Guimarães da Costa, João; Barrillon, Pierre; Bartoldus, Rainer; Barton, Adam Edward; Bartsch, Valeria; Basye, Austin; Bates, Richard; Batkova, Lucia; Batley, Richard; Battaglia, Andreas; Battistin, Michele; Bauer, Florian; Bawa, Harinder Singh; Beale, Steven; Beau, Tristan; Beauchemin, Pierre-Hugues; Beccherle, Roberto; Bechtle, Philip; Beck, Hans Peter; Becker, Anne Kathrin; Becker, Sebastian; Beckingham, Matthew; Becks, Karl-Heinz; Beddall, Andrew; Beddall, Ayda; Bedikian, Sourpouhi; Bednyakov, Vadim; Bee, Christopher; Beemster, Lars; Begel, Michael; Behar Harpaz, Silvia; Behera, Prafulla; Beimforde, Michael; Belanger-Champagne, Camille; Bell, Paul; Bell, William; Bella, Gideon; Bellagamba, Lorenzo; Bellina, Francesco; Bellomo, Massimiliano; Belloni, Alberto; Beloborodova, Olga; Belotskiy, Konstantin; Beltramello, Olga; Benary, Odette; Benchekroun, Driss; Bendtz, Katarina; Benekos, Nektarios; Benhammou, Yan; Benhar Noccioli, Eleonora; Benitez Garcia, Jorge-Armando; Benjamin, Douglas; Benoit, Mathieu; Bensinger, James; Benslama, Kamal; Bentvelsen, Stan; Berge, David; Bergeaas Kuutmann, Elin; Berger, Nicolas; Berghaus, Frank; Berglund, Elina; Beringer, Jürg; Bernat, Pauline; Bernhard, Ralf; Bernius, Catrin; Berry, Tracey; Bertella, Claudia; Bertin, Antonio; Bertolucci, Federico; Besana, Maria Ilaria; Besjes, Geert-Jan; Besson, Nathalie; Bethke, Siegfried; Bhimji, Wahid; Bianchi, Riccardo-Maria; Bianco, Michele; Biebel, Otmar; Bieniek, Stephen Paul; Bierwagen, Katharina; Biesiada, Jed; Biglietti, Michela; Bilokon, Halina; Bindi, Marcello; Binet, Sebastien; Bingul, Ahmet; Bini, Cesare

    2012-01-01

    This work presents a new inclusive search for supersymmetry (SUSY) by the ATLAS experiment at the LHC in proton-proton collisions at a center-of-mass energy $\\sqrt{s}$ = 7 TeV in final states with jets, missing transverse momentum and one or more isolated electrons and/or muons. The search is based on data from the full 2011 data-taking period, corresponding to an integrated luminosity of 4.7 inverse fb. Single- and multi-lepton channels are treated together in one analysis. An increase in sensitivity is obtained by simultaneously fitting the number of events in statistically independent signal regions, and the shapes of distributions within those regions. A dedicated signal region is introduced to be sensitive to decay cascades of SUSY particles with small mass differences ("compressed SUSY"). Background uncertainties are constrained by fitting to the jet multiplicity distribution in background control regions. Observations are consistent with Standard Model expectations, and limits are set or extended on a ...

  10. Isolated limb infusion with melphalan and dactinomycin for regional melanoma and soft-tissue sarcoma of the extremity: final report of a phase II clinical trial.

    Science.gov (United States)

    Brady, Mary S; Brown, Karen; Patel, Ami; Fisher, Charles; Marx, Will

    2009-04-01

    Isolated limb infusion (ILI) is a minimally invasive technique of delivering regional chemotherapy in patients with advanced melanoma or soft-tissue sarcoma of the limb. We report the final results of the first clinical trial of ILI in North America (NCT00004250). Eligible patients had recurrent melanoma or unresectable soft-tissue sarcoma of the limb. Angiographic catheters were positioned just above the knee or elbow of the extremity. General anesthesia was performed, a proximal tourniquet inflated, and a normothermic, low flow, hypoxic infusion of melphalan and dactinomycin circulated through the involved limb for 20 min. Tumor response and morbidity were assessed using standard criteria. Thirty-seven patients were accrued to the trial and 44 ILIs were performed (eight patients had two ILIs); one patient was not treated. Of the 32 evaluable patients, 17 (53%) had a significant response at 3 months: 25% of patients had a complete response and 28% of patients had a partial response. The median duration of complete response was 1 year (5-32 months). Morbidity was acceptable, with peak erythema, edema, and pain experienced at 2 weeks and considered 'moderate' in most patients. No patients developed compartment syndrome or required amputation because of ILI. ILI is well tolerated. More than half of the treated patients experienced a complete or partial response.

  11. Search for top squarks in final states with one isolated lepton in $\\sqrt{s}$ = 13 TeV pp collisions with the ATLAS detector

    CERN Document Server

    Yamazaki, Tomohiro; The ATLAS collaboration

    2017-01-01

    The result of a search for the direct pair production of top squarks, the supersymmetric partner of the top-quark, in final states with one isolated electron or muon, jets, and missing transverse momentum is reported. The search uses data from $pp$ collisions recorded at the LHC with the ATLAS detector at a centre-of-mass energy of $\\sqrt{s}$ = 13 TeV correspoinding to an integrated luminosity of 36 $\\mathrm{fb}^{-1}$. Dedicated analysis for the Higgsino LSP model is performed considering small mass splitting between electroweakinos and the realistic branching ratio of a top squark. No significant excess over the Standard Model predictions is observed. Exclusion limits at 95\\% confidence level in the Higgsino LSP model are set. The top squark mass is excluded up to 810 GeV for 2 GeV mass splitting between the chargino and the lightest neutralino. The results are also reinterpreted to set exclusion limits in a Bino-Higgsino mixed LSP model.

  12. Evaluation of Existing Methods for Human Blood mRNA Isolation and Analysis for Large Studies

    Science.gov (United States)

    Meyer, Anke; Paroni, Federico; Günther, Kathrin; Dharmadhikari, Gitanjali; Ahrens, Wolfgang; Kelm, Sørge; Maedler, Kathrin

    2016-01-01

    Aims Prior to implementing gene expression analyses from blood to a larger cohort study, an evaluation to set up a reliable and reproducible method is mandatory but challenging due to the specific characteristics of the samples as well as their collection methods. In this pilot study we optimized a combination of blood sampling and RNA isolation methods and present reproducible gene expression results from human blood samples. Methods The established PAXgeneTM blood collection method (Qiagen) was compared with the more recent TempusTM collection and storing system. RNA from blood samples collected by both systems was extracted on columns with the corresponding Norgen and PAX RNA extraction Kits. RNA quantity and quality was compared photometrically, with Ribogreen and by Real-Time PCR analyses of various reference genes (PPIA, β-ACTIN and TUBULIN) and exemplary of SIGLEC-7. Results Combining different sampling methods and extraction kits caused strong variations in gene expression. The use of PAXgeneTM and TempusTM collection systems resulted in RNA of good quality and quantity for the respective RNA isolation system. No large inter-donor variations could be detected for both systems. However, it was not possible to extract sufficient RNA of good quality with the PAXgeneTM RNA extraction system from samples collected by TempusTM collection tubes. Comparing only the Norgen RNA extraction methods, RNA from blood collected either by the TempusTM or PAXgeneTM collection system delivered sufficient amount and quality of RNA, but the TempusTM collection delivered higher RNA concentration compared to the PAXTM collection system. The established Pre-analytix PAXgeneTM RNA extraction system together with the PAXgeneTM blood collection system showed lowest CT-values, i.e. highest RNA concentration of good quality. Expression levels of all tested genes were stable and reproducible. Conclusions This study confirms that it is not possible to mix or change sampling or

  13. Final Oocyte Maturation in Assisted Reproduction with Human Chorionic Gonadotropin and Gonadotropin-releasing Hormone agonist (Dual Trigger).

    Science.gov (United States)

    Oliveira, Sofia Andrade de; Calsavara, Vinícius Fernando; Cortés, Gemma Castillón

    2016-12-01

    Final oocyte maturation with Human Chorionic Gonadotropin (hCG) and ovarian stimulation with Follicle Stimulation Hormone (FSH) combined with Gonadotrophin-releasing Hormone (GnRH) antagonist to block Luteinizing hormone (LH) surge is a standard procedure of in vitro Fertilization (IVF) and Intracytoplasmic Sperm Injection (ICSI). However, GnRH agonist has been replacing the use of hCG in certain situations, especially in patients at risk of Ovarian Hyperstimulation Syndrome (OHSS). Some studies have also shown advantages in the combined use of GnRH agonist concurrently with hCG in inducing final oocyte maturation, a treatment known as "Dual Trigger". In theory, this method combines the advantages of both induction regimens, and it has brought promising results. The objective of this study is to compare Dual Trigger with the use of hCG alone or the use of GnRH agonist alone. A systematic review of articles on Dual Trigger and a retrospective cohort study comparing the three methods of induction of final oocyte maturation have been conducted. It has been found that Dual Triggering for poor responder patients had a statistically significant increase in the number of retrieved oocytes, mature oocytes, and fertilized embryos in the positive beta hCG rate, implantation rate, and newborn/transferred embryo (TE) rate.

  14. Genotyping of Giardia duodenalis isolates from human subjects in Zabul, using PCR-RFLP

    Directory of Open Access Journals (Sweden)

    Maryam Abedi

    2016-09-01

    Full Text Available Objective: To uncover the molecular prevalence of Giardia duodenalis by PCR-restriction fragment length polymorphism (RFLP in Zabul city, Iran. Methods: Twenty-four stool samples were collected from 215 patients with suspected giardiasis by microscopic examination. To increase the sensitivity of the PCR, the total genomic DNA from isolates was extracted by applying glass beads and the QIAamp Kit. A one-step PCRRFLP method, targeting the glutamate dehydrogenase gene, was utilized to differentiate the assemblages A and B among isolates. Results: The PCR fragment was determined from 30 isolates, RFLP assay of 24 isolates showed 24 (100 isolates as Genotype B group BIII. Conclusions: The results with the glutamate dehydrogenase gene assay demonstrated that the predominant subtype of Giardia duodenalis in the area is BIII, which showed animals are the main reservoir of the isolates in this area.

  15. Isolation of fungi belonging to the genera Geotrichum and Trichosporum from human dermal lesions.

    Science.gov (United States)

    Restrepo, A; De Uribe, L

    1976-08-30

    Isolates of Geotrichum and Trichosporum spp. obtained from patients with a variety of dermal lesions were studied. Among 2,202 cases examined, microorganisms of these genera were recovered from 100 (4,5%); there were 38 isolated of Geotrichum- and 62 of Trichosporum- spp. Most isolations were obtained from nails: 52 cases. The species most frequently found were T. beigelii (25 cases) and G. candidum (30 cases). In 50 of the patients, these fungi were isolated in pure culture, in an additional 40 Trichosporum spp. were found. Mixed cultures with C. albicans were observed in 28 patients, with other Candida spp. in 16 and with dermatophytes in 6. Among the patients whose isolations occurred in pure cultures, the number of colonies recovered was large in 20 cases, 1 with Geotrichum candidum - 19 with Trichosporum (16 T. beigelii, 3 T. capitatum). The relationship between the isolated yeast-like fungi and the dermal lesion was considered to be direct in these 20 patients.

  16. PSC-RANTES blocks R5 human immunodeficiency virus infection of Langerhans cells isolated from individuals with a variety of CCR5 diplotypes.

    Science.gov (United States)

    Kawamura, Tatsuyoshi; Bruse, Shannon E; Abraha, Awet; Sugaya, Makoto; Hartley, Oliver; Offord, Robin E; Arts, Eric J; Zimmerman, Peter A; Blauvelt, Andrew; Bruce, Shannon E

    2004-07-01

    Topical microbicides that effectively block interactions between CCR5(+) immature Langerhans cells (LC) residing within genital epithelia and R5 human immunodeficiency virus (HIV) may decrease sexual transmission of HIV. Here, we investigated the ability of synthetic RANTES analogues (AOP-, NNY-, and PSC-RANTES) to block R5 HIV infection of human immature LC by using a skin explant model. In initial experiments using activated peripheral blood mononuclear cells, each analogue compound demonstrated marked antiviral activity against two R5 HIV isolates. Next, we found that 20-min preincubation of skin explants with each RANTES analogue blocked R5 HIV infection of LC in a dose-dependent manner (1 to 100 nM) and that PSC-RANTES was the most potent of these compounds. Similarly, preincubation of LC with each analogue was able to block LC-mediated infection of cocultured CD4(+) T cells. Competition experiments between primary R5 and X4 HIV isolates showed blocking of R5 HIV by PSC-RANTES and no evidence of increased propagation of X4 HIV, data that are consistent with the specificity of PSC-RANTES for CCR5 and the CCR5(+) CXCR4(-) phenotype of immature LC. Finally, when CCR5 genetic polymorphism data were integrated with results from the in vitro LC infection studies, PSC-RANTES was found to be equally effective in inhibiting R5 HIV in LC isolated from individuals with CCR5 diplotypes known to be associated with low, intermediate, and high cell surface levels of CCR5. In summary, PSC-RANTES is a potent inhibitor of R5 HIV infection in immature LC, suggesting that it may be useful as a topical microbicide to block sexual transmission of HIV.

  17. Decay of linkage disequilibrium within genes across HGDP-CEPH human samples: most population isolates do not show increased LD

    Directory of Open Access Journals (Sweden)

    Navarro Arcadi

    2009-07-01

    Full Text Available Abstract Background It is well known that the pattern of linkage disequilibrium varies between human populations, with remarkable geographical stratification. Indirect association studies routinely exploit linkage disequilibrium around genes, particularly in isolated populations where it is assumed to be higher. Here, we explore both the amount and the decay of linkage disequilibrium with physical distance along 211 gene regions, most of them related to complex diseases, across 39 HGDP-CEPH population samples, focusing particularly on the populations defined as isolates. Within each gene region and population we use r2 between all possible single nucleotide polymorphism (SNP pairs as a measure of linkage disequilibrium and focus on the proportion of SNP pairs with r2 greater than 0.8. Results Although the average r2 was found to be significantly different both between and within continental regions, a much higher proportion of r2 variance could be attributed to differences between continental regions (2.8% vs. 0.5%, respectively. Similarly, while the proportion of SNP pairs with r2 > 0.8 was significantly different across continents for all distance classes, it was generally much more homogenous within continents, except in the case of Africa and the Americas. The only isolated populations with consistently higher LD in all distance classes with respect to their continent are the Kalash (Central South Asia and the Surui (America. Moreover, isolated populations showed only slightly higher proportions of SNP pairs with r2 > 0.8 per gene region than non-isolated populations in the same continent. Thus, the number of SNPs in isolated populations that need to be genotyped may be only slightly less than in non-isolates. Conclusion The "isolated population" label by itself does not guarantee a greater genotyping efficiency in association studies, and properties other than increased linkage disequilibrium may make these populations interesting in

  18. Decay of linkage disequilibrium within genes across HGDP-CEPH human samples: most population isolates do not show increased LD

    Science.gov (United States)

    Bosch, Elena; Laayouni, Hafid; Morcillo-Suarez, Carlos; Casals, Ferran; Moreno-Estrada, Andrés; Ferrer-Admetlla, Anna; Gardner, Michelle; Rosa, Araceli; Navarro, Arcadi; Comas, David; Graffelman, Jan; Calafell, Francesc; Bertranpetit, Jaume

    2009-01-01

    Background It is well known that the pattern of linkage disequilibrium varies between human populations, with remarkable geographical stratification. Indirect association studies routinely exploit linkage disequilibrium around genes, particularly in isolated populations where it is assumed to be higher. Here, we explore both the amount and the decay of linkage disequilibrium with physical distance along 211 gene regions, most of them related to complex diseases, across 39 HGDP-CEPH population samples, focusing particularly on the populations defined as isolates. Within each gene region and population we use r2 between all possible single nucleotide polymorphism (SNP) pairs as a measure of linkage disequilibrium and focus on the proportion of SNP pairs with r2 greater than 0.8. Results Although the average r2 was found to be significantly different both between and within continental regions, a much higher proportion of r2 variance could be attributed to differences between continental regions (2.8% vs. 0.5%, respectively). Similarly, while the proportion of SNP pairs with r2 > 0.8 was significantly different across continents for all distance classes, it was generally much more homogenous within continents, except in the case of Africa and the Americas. The only isolated populations with consistently higher LD in all distance classes with respect to their continent are the Kalash (Central South Asia) and the Surui (America). Moreover, isolated populations showed only slightly higher proportions of SNP pairs with r2 > 0.8 per gene region than non-isolated populations in the same continent. Thus, the number of SNPs in isolated populations that need to be genotyped may be only slightly less than in non-isolates. Conclusion The "isolated population" label by itself does not guarantee a greater genotyping efficiency in association studies, and properties other than increased linkage disequilibrium may make these populations interesting in genetic epidemiology. PMID

  19. Final report for the endowment of simulator agents with human-like episodic memory LDRD.

    Energy Technology Data Exchange (ETDEWEB)

    Speed, Ann Elizabeth; Lippitt, Carl Edward; Thomas, Edward Victor; Xavier, Patrick Gordon; Forsythe, Christi A.; Schaller, Mark J.; Schoenwald, David Alan

    2003-12-01

    This report documents work undertaken to endow the cognitive framework currently under development at Sandia National Laboratories with a human-like memory for specific life episodes. Capabilities have been demonstrated within the context of three separate problem areas. The first year of the project developed a capability whereby simulated robots were able to utilize a record of shared experience to perform surveillance of a building to detect a source of smoke. The second year focused on simulations of social interactions providing a queriable record of interactions such that a time series of events could be constructed and reconstructed. The third year addressed tools to promote desktop productivity, creating a capability to query episodic logs in real time allowing the model of a user to build on itself based on observations of the user's behavior.

  20. Filling knowledge gaps in radiation protection methodologies for non-human biota. Final summary report

    Energy Technology Data Exchange (ETDEWEB)

    Brown, J.; Gjelsvik, R. (Norwegian Radiation Protection Authority (Norway)); Holm, E. (Univ. of Lund (Sweden)); Roos, P. (Technical Univ. of Denmark, Risoe National Lab. for Sustainable Energy, Roskilde (Denmark)); Saxen, R.; Outola, I. (Radiation and Nuclear Safety Authority (Finland))

    2009-03-15

    The activities of the GAPRAD project are summarised in this report. The background and rationale to GAPRAD are presented and explained. Most notably this relates to a lack of information on naturally occuring radionuclides in terrestrial and aquatic systems that have direct applicability for use in environmental impact assessments. Results from field activities are presented from the Dovrefjell area in Norway (terrestrial study) and selected lake and brackish water systems in Finland. The data mainly concern activity concentrations of Po-210 in environmental media and selected biota allowing concentration ratios to be derived where appropriate. Furthermore, details in relation to Po-210 uptake and biokinetics in humans based on experimental work conducted within the project are presented. (au)

  1. Postmarketing safety reports for human drug and biological products; electronic submission requirements. Final rule.

    Science.gov (United States)

    2014-06-10

    The Food and Drug Administration (FDA or we) is amending its postmarketing safety reporting regulations for human drug and biological products to require that persons subject to mandatory reporting requirements submit safety reports in an electronic format that FDA can process, review, and archive. FDA is taking this action to improve the Agency's systems for collecting and analyzing postmarketing safety reports. The change will help the Agency to more rapidly review postmarketing safety reports, identify emerging safety problems, and disseminate safety information in support of FDA's public health mission. In addition, the amendments will be a key element in harmonizing FDA's postmarketing safety reporting regulations with international standards for the electronic submission of safety information.

  2. Evolutionary Medicine and Future of Humanity: Will Evolution Have the Final Word?

    Directory of Open Access Journals (Sweden)

    Maciej Henneberg

    2013-06-01

    Full Text Available Evolutionary medicine in its classical form assumes that since cultural evolution is faster than biological evolution, ailments of modern people are a result of mismatch between adaptations to the past environments and current situations. A core principle is that we, humans, having evolved for millions of years in a specific natural environment (environment of evolutionary adaptation EEA are biologically adapted to this past environment and the ancient lifestyle. This adaptation to the past produces major mismatch of our bodies with the present, highly anthropic and thus “artificial” living conditions. This article provides two areas of possible future evolution, diet and physical activity levels which have been dramatically altered in industrialised societies. Consequently, micro-evolution is an on-going process.

  3. In Vitro Antimicrobial Susceptibility of Staphylococcus pseudintermedius Isolates of Human and Animal Origin

    Science.gov (United States)

    Wu, Max T.; Westblade, Lars F.; Robertson, Amy E.; Wallace, Meghan A.; Burd, Eileen M.; Hindler, Janet A.

    2016-01-01

    MIC results for 115 Staphylococcus intermedius group isolates are presented. Of these, 33% were methicillin resistant, among which 51.4% were susceptible to doxycycline, 29.7% to clindamycin, and 21.6% to trimethoprim-sulfamethoxazole. All of the isolates were susceptible to ceftaroline, daptomycin, linezolid, nitrofurantoin, quinupristin-dalfopristin, rifampin, tigecycline, and vancomycin. Of all the isolates, 82.6%, 67.8%, and 23.5% were susceptible to ciprofloxacin, erythromycin, and penicillin, respectively. No isolates harbored mupA or qacA/B genes, which suggested a lack of resistance to mupirocin or chlorhexidine. PMID:26962087

  4. Pheno- and genotyping of Staphylococcus epidermidis isolated from bovine milk and human skin

    DEFF Research Database (Denmark)

    Thorberg, B. M.; Kuhn, I.; Aarestrup, Frank Møller

    2006-01-01

    The purpose of this study was to improve our knowledge concerning the epidemiology and strain diversity of Staphylococcus epidermidis isolated from bovine milk in commercial dairy herds. A total of 341 S. epidermidis isolates obtained from cows' milk (317), farmers (17) and patients (7) were...... different S. epidermidis types exist in milk samples. Antibiotic resistance patterns matched the SmaI profiles closely in the two herds, but poorly in the routinely collected milk samples. Isolates from herd I showed one to five patterns, depending on the typing method used. Isolates from the milker's skin...

  5. New Paracoccidioides brasiliensis isolate reveals unexpected genomic variability in this human pathogen.

    Science.gov (United States)

    Carrero, Lilia L; Niño-Vega, Gustavo; Teixeira, Marcus M; Carvalho, Maria Jose A; Soares, Célia M A; Pereira, Maristela; Jesuino, Rosália S A; McEwen, Juan G; Mendoza, Leonel; Taylor, John W; Felipe, Maria Sueli; San-Blas, Gioconda

    2008-05-01

    By means of genealogical concordance phylogenetic species recognition (GCPSR), we have investigated coding and non-coding regions from various genes and the ITS sequences of 7 new and 14 known isolates of Paracoccidioides brasiliensis. Such isolates grouped within the three phylogenetic groups recently reported in the genus Paracoccidioides, with one single exception, i.e., Pb01, a strain that has been the subject of intense molecular studies for many years. This isolate clearly separates from all other Paracoccidioides isolates in phylogenetic analyses and greatly increases the