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Sample records for clonal hiv sequence

  1. An Evaluation of Phylogenetic Methods for Reconstructing Transmitted HIV Variants using Longitudinal Clonal HIV Sequence Data

    Science.gov (United States)

    McCloskey, Rosemary M.; Liang, Richard H.; Harrigan, P. Richard; Brumme, Zabrina L.

    2014-01-01

    ABSTRACT A population of human immunodeficiency virus (HIV) within a host often descends from a single transmitted/founder virus. The high mutation rate of HIV, coupled with long delays between infection and diagnosis, make isolating and characterizing this strain a challenge. In theory, ancestral reconstruction could be used to recover this strain from sequences sampled in chronic infection; however, the accuracy of phylogenetic techniques in this context is unknown. To evaluate the accuracy of these methods, we applied ancestral reconstruction to a large panel of published longitudinal clonal and/or single-genome-amplification HIV sequence data sets with at least one intrapatient sequence set sampled within 6 months of infection or seroconversion (n = 19,486 sequences, median [interquartile range] = 49 [20 to 86] sequences/set). The consensus of the earliest sequences was used as the best possible estimate of the transmitted/founder. These sequences were compared to ancestral reconstructions from sequences sampled at later time points using both phylogenetic and phylogeny-naive methods. Overall, phylogenetic methods conferred a 16% improvement in reproducing the consensus of early sequences, compared to phylogeny-naive methods. This relative advantage increased with intrapatient sequence diversity (P reconstructing ancestral indel variation, especially within indel-rich regions of the HIV genome. Although further improvements are needed, our results indicate that phylogenetic methods for ancestral reconstruction significantly outperform phylogeny-naive alternatives, and we identify experimental conditions and study designs that can enhance accuracy of transmitted/founder virus reconstruction. IMPORTANCE When HIV is transmitted into a new host, most of the viruses fail to infect host cells. Consequently, an HIV infection tends to be descended from a single “founder” virus. A priority target for the vaccine research, these transmitted/founder viruses are

  2. Evaluating Clonal Expansion of HIV-Infected Cells: Optimization of PCR Strategies to Predict Clonality

    Science.gov (United States)

    Laskey, Sarah B.; Pohlmeyer, Christopher W.; Bruner, Katherine M.; Siliciano, Robert F.

    2016-01-01

    In HIV-infected individuals receiving suppressive antiretroviral therapy, the virus persists indefinitely in a reservoir of latently infected cells. The proliferation of these cells may contribute to the stability of the reservoir and thus to the lifelong persistence of HIV-1 in infected individuals. Because the HIV-1 replication process is highly error-prone, the detection of identical viral genomes in distinct host cells provides evidence for the clonal expansion of infected cells. We evaluated alignments of unique, near-full-length HIV-1 sequences to determine the relationship between clonality in a short region and clonality in the full genome. Although it is common to amplify and sequence short, subgenomic regions of the viral genome for phylogenetic analysis, we show that sequence identity of these amplicons does not guarantee clonality across the full viral genome. We show that although longer amplicons capture more diversity, no subgenomic region can recapitulate the diversity of full viral genomes. Consequently, some identical subgenomic amplicons should be expected even from the analysis of completely unique viral genomes, and the presence of identical amplicons alone is not proof of clonally expanded HIV-1. We present a method for evaluating evidence of clonal expansion in the context of these findings. PMID:27494508

  3. Evaluating Clonal Expansion of HIV-Infected Cells: Optimization of PCR Strategies to Predict Clonality.

    Directory of Open Access Journals (Sweden)

    Sarah B Laskey

    2016-08-01

    Full Text Available In HIV-infected individuals receiving suppressive antiretroviral therapy, the virus persists indefinitely in a reservoir of latently infected cells. The proliferation of these cells may contribute to the stability of the reservoir and thus to the lifelong persistence of HIV-1 in infected individuals. Because the HIV-1 replication process is highly error-prone, the detection of identical viral genomes in distinct host cells provides evidence for the clonal expansion of infected cells. We evaluated alignments of unique, near-full-length HIV-1 sequences to determine the relationship between clonality in a short region and clonality in the full genome. Although it is common to amplify and sequence short, subgenomic regions of the viral genome for phylogenetic analysis, we show that sequence identity of these amplicons does not guarantee clonality across the full viral genome. We show that although longer amplicons capture more diversity, no subgenomic region can recapitulate the diversity of full viral genomes. Consequently, some identical subgenomic amplicons should be expected even from the analysis of completely unique viral genomes, and the presence of identical amplicons alone is not proof of clonally expanded HIV-1. We present a method for evaluating evidence of clonal expansion in the context of these findings.

  4. HIV genetic information and clonal growth

    Science.gov (United States)

    Based on an analysis of blood cells from five HIV-infected individuals, NCI researchers have identified more than 2,400 HIV DNA insertion sites. Analysis of these sites showed that there is extensive clonal expansion (growth) of HIV infected cells.

  5. ClonEvol: clonal ordering and visualization in cancer sequencing.

    Science.gov (United States)

    Dang, H X; White, B S; Foltz, S M; Miller, C A; Luo, J; Fields, R C; Maher, C A

    2017-12-01

    Reconstruction of clonal evolution is critical for understanding tumor progression and implementing personalized therapies. This is often done by clustering somatic variants based on their cellular prevalence estimated via bulk tumor sequencing of multiple samples. The clusters, consisting of the clonal marker variants, are then ordered based on their estimated cellular prevalence to reconstruct clonal evolution trees, a process referred to as 'clonal ordering'. However, cellular prevalence estimate is confounded by statistical variability and errors in sequencing/data analysis, and therefore inhibits accurate reconstruction of the clonal evolution. This problem is further complicated by intra- and inter-tumor heterogeneity. Furthermore, the field lacks a comprehensive visualization tool to facilitate the interpretation of complex clonal relationships. To address these challenges we developed ClonEvol, a unified software tool for clonal ordering, visualization, and interpretation. ClonEvol uses a bootstrap resampling technique to estimate the cellular fraction of the clones and probabilistically models the clonal ordering constraints to account for statistical variability. The bootstrapping allows identification of the sample founding- and sub-clones, thus enabling interpretation of clonal seeding. ClonEvol automates the generation of multiple widely used visualizations for reconstructing and interpreting clonal evolution. ClonEvol outperformed three of the state of the art tools (LICHeE, Canopy and PhyloWGS) for clonal evolution inference, showing more robust error tolerance and producing more accurate trees in a simulation. Building upon multiple recent publications that utilized ClonEvol to study metastasis and drug resistance in solid cancers, here we show that ClonEvol rediscovered relapsed subclones in two published acute myeloid leukemia patients. Furthermore, we demonstrated that through noninvasive monitoring ClonEvol recapitulated the emerging subclones

  6. HIV Sequence Compendium 2015

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    Foley, Brian Thomas [Los Alamos National Lab. (LANL), Los Alamos, NM (United States); Leitner, Thomas Kenneth [Los Alamos National Lab. (LANL), Los Alamos, NM (United States); Apetrei, Cristian [Univ. of Pittsburgh, PA (United States); Hahn, Beatrice [Univ. of Pennsylvania, Philadelphia, PA (United States); Mizrachi, Ilene [National Center for Biotechnology Information, Bethesda, MD (United States); Mullins, James [Univ. of Washington, Seattle, WA (United States); Rambaut, Andrew [Univ. of Edinburgh, Scotland (United Kingdom); Wolinsky, Steven [Northwestern Univ., Evanston, IL (United States); Korber, Bette Tina Marie [Los Alamos National Lab. (LANL), Los Alamos, NM (United States)

    2015-10-05

    This compendium is an annual printed summary of the data contained in the HIV sequence database. We try to present a judicious selection of the data in such a way that it is of maximum utility to HIV researchers. Each of the alignments attempts to display the genetic variability within the different species, groups and subtypes of the virus. This compendium contains sequences published before January 1, 2015. Hence, though it is published in 2015 and called the 2015 Compendium, its contents correspond to the 2014 curated alignments on our website. The number of sequences in the HIV database is still increasing. In total, at the end of 2014, there were 624,121 sequences in the HIV Sequence Database, an increase of 7% since the previous year. This is the first year that the number of new sequences added to the database has decreased compared to the previous year. The number of near complete genomes (>7000 nucleotides) increased to 5834 by end of 2014. However, as in previous years, the compendium alignments contain only a fraction of these. A more complete version of all alignments is available on our website, http://www.hiv.lanl.gov/ content/sequence/NEWALIGN/align.html As always, we are open to complaints and suggestions for improvement. Inquiries and comments regarding the compendium should be addressed to seq-info@lanl.gov.

  7. Clonal expansion of genome-intact HIV-1 in functionally polarized Th1 CD4+ T cells.

    Science.gov (United States)

    Lee, Guinevere Q; Orlova-Fink, Nina; Einkauf, Kevin; Chowdhury, Fatema Z; Sun, Xiaoming; Harrington, Sean; Kuo, Hsiao-Hsuan; Hua, Stephane; Chen, Hsiao-Rong; Ouyang, Zhengyu; Reddy, Kavidha; Dong, Krista; Ndung'u, Thumbi; Walker, Bruce D; Rosenberg, Eric S; Yu, Xu G; Lichterfeld, Mathias

    2017-06-30

    HIV-1 causes a chronic, incurable disease due to its persistence in CD4+ T cells that contain replication-competent provirus, but exhibit little or no active viral gene expression and effectively resist combination antiretroviral therapy (cART). These latently infected T cells represent an extremely small proportion of all circulating CD4+ T cells but possess a remarkable long-term stability and typically persist throughout life, for reasons that are not fully understood. Here we performed massive single-genome, near-full-length next-generation sequencing of HIV-1 DNA derived from unfractionated peripheral blood mononuclear cells, ex vivo-isolated CD4+ T cells, and subsets of functionally polarized memory CD4+ T cells. This approach identified multiple sets of independent, near-full-length proviral sequences from cART-treated individuals that were completely identical, consistent with clonal expansion of CD4+ T cells harboring intact HIV-1. Intact, near-full-genome HIV-1 DNA sequences that were derived from such clonally expanded CD4+ T cells constituted 62% of all analyzed genome-intact sequences in memory CD4 T cells, were preferentially observed in Th1-polarized cells, were longitudinally detected over a duration of up to 5 years, and were fully replication- and infection-competent. Together, these data suggest that clonal proliferation of Th1-polarized CD4+ T cells encoding for intact HIV-1 represents a driving force for stabilizing the pool of latently infected CD4+ T cells.

  8. HIV Sequence Compendium 2010

    Energy Technology Data Exchange (ETDEWEB)

    Kuiken, Carla [Los Alamos National Lab. (LANL), Los Alamos, NM (United States); Foley, Brian [Los Alamos National Lab. (LANL), Los Alamos, NM (United States); Leitner, Thomas [Los Alamos National Lab. (LANL), Los Alamos, NM (United States); Apetrei, Christian [Univ. of Pittsburgh, PA (United States); Hahn, Beatrice [Univ. of Alabama, Tuscaloosa, AL (United States); Mizrachi, Ilene [National Center for Biotechnology Information, Bethesda, MD (United States); Mullins, James [Univ. of Washington, Seattle, WA (United States); Rambaut, Andrew [Univ. of Edinburgh, Scotland (United Kingdom); Wolinsky, Steven [Northwestern Univ., Evanston, IL (United States); Korber, Bette [Los Alamos National Lab. (LANL), Los Alamos, NM (United States)

    2010-12-31

    This compendium is an annual printed summary of the data contained in the HIV sequence database. In these compendia we try to present a judicious selection of the data in such a way that it is of maximum utility to HIV researchers. Each of the alignments attempts to display the genetic variability within the different species, groups and subtypes of the virus. This compendium contains sequences published before January 1, 2010. Hence, though it is called the 2010 Compendium, its contents correspond to the 2009 curated alignments on our website. The number of sequences in the HIV database is still increasing exponentially. In total, at the time of printing, there were 339,306 sequences in the HIV Sequence Database, an increase of 45% since last year. The number of near complete genomes (>7000 nucleotides) increased to 2576 by end of 2009, reflecting a smaller increase than in previous years. However, as in previous years, the compendium alignments contain only a small fraction of these. Included in the alignments are a small number of sequences representing each of the subtypes and the more prevalent circulating recombinant forms (CRFs) such as 01 and 02, as well as a few outgroup sequences (group O and N and SIV-CPZ). Of the rarer CRFs we included one representative each. A more complete version of all alignments is available on our website, http://www.hiv.lanl.gov/content/sequence/NEWALIGN/align.html. Reprints are available from our website in the form of both HTML and PDF files. As always, we are open to complaints and suggestions for improvement. Inquiries and comments regarding the compendium should be addressed to seq-info@lanl.gov.

  9. A comparison of 454 sequencing and clonal sequencing for the characterization of hepatitis C virus NS3 variants

    NARCIS (Netherlands)

    Ho, Cynthia K. Y.; Welkers, Matthijs R. A.; Thomas, Xiomara V.; Sullivan, James C.; Kieffer, Tara L.; Reesink, Henk W.; Rebers, Sjoerd P. H.; de Jong, Menno D.; Schinkel, Janke; Molenkamp, Richard

    2015-01-01

    We compared 454 amplicon sequencing with clonal sequencing for the characterization of intra-host hepatitis C virus (HCV) NS3 variants. Clonal and 454 sequences were obtained from 12 patients enrolled in a clinical phase I study for telaprevir, an NS3-4a protease inhibitor. Thirty-nine datasets were

  10. Reliable reconstruction of HIV-1 whole genome haplotypes reveals clonal interference and genetic hitchhiking among immune escape variants

    Science.gov (United States)

    2014-01-01

    Background Following transmission, HIV-1 evolves into a diverse population, and next generation sequencing enables us to detect variants occurring at low frequencies. Studying viral evolution at the level of whole genomes was hitherto not possible because next generation sequencing delivers relatively short reads. Results We here provide a proof of principle that whole HIV-1 genomes can be reliably reconstructed from short reads, and use this to study the selection of immune escape mutations at the level of whole genome haplotypes. Using realistically simulated HIV-1 populations, we demonstrate that reconstruction of complete genome haplotypes is feasible with high fidelity. We do not reconstruct all genetically distinct genomes, but each reconstructed haplotype represents one or more of the quasispecies in the HIV-1 population. We then reconstruct 30 whole genome haplotypes from published short sequence reads sampled longitudinally from a single HIV-1 infected patient. We confirm the reliability of the reconstruction by validating our predicted haplotype genes with single genome amplification sequences, and by comparing haplotype frequencies with observed epitope escape frequencies. Conclusions Phylogenetic analysis shows that the HIV-1 population undergoes selection driven evolution, with successive replacement of the viral population by novel dominant strains. We demonstrate that immune escape mutants evolve in a dependent manner with various mutations hitchhiking along with others. As a consequence of this clonal interference, selection coefficients have to be estimated for complete haplotypes and not for individual immune escapes. PMID:24996694

  11. Core Genome Multilocus Sequence Typing for Identification of Globally Distributed Clonal Groups and Differentiation of Outbreak Strains of Listeria monocytogenes

    OpenAIRE

    Chen, Yi; Gonzalez-Escalona, Narjol; Hammack, Thomas S.; Allard, Marc W.; Strain, Errol A.; Brown, Eric W.

    2016-01-01

    ABSTRACT Many listeriosis outbreaks are caused by a few globally distributed clonal groups, designated clonal complexes or epidemic clones, of Listeria monocytogenes, several of which have been defined by classic multilocus sequence typing (MLST) schemes targeting 6 to 8 housekeeping or virulence genes. We have developed and evaluated core genome MLST (cgMLST) schemes and applied them to isolates from multiple clonal groups, including those associated with 39 listeriosis outbreaks. The cgMLST...

  12. Ribosomal DNA sequence analysis of different geographically distributed Aloe Vera plants: Comparison with clonally regenerated plants

    International Nuclear Information System (INIS)

    Yagi, A.; Sato, Y.; Miwa, Y.; Kabbash, A.; Moustafa, S.; Shimomura, K.; El-Bassuony, A.

    2006-01-01

    A comparison of the sequences in an internally transcribed spacer (ITS) 1 region of rDNA between clonally regenerated A.vera and same species in Japan, USA and Egypt revealed the presence of two types of nucleotide sequences, 252 and 254 bps. Based on the findings in the ITS 1 region, A.vera having 252 and 254 bps clearly showed a stable sequence similarity, suggesting high conversation of the base peak sequence in the ITS 1 region. However, frequent base substitutions in the 252 bps samples leaves that came from callus tissue and micropropagated plants were observed around the regions of nucleotide positions 66, 99 and 199-201. The minor deviation in clonally regenerated A.vera may be due to the stage of regeneration and cell specification in cases of the callus tissue. In the present study, the base peak sequence of the Its 1 region of rDNA was adopted as a molecular marker for differentiating A.vera plants from geographically distributed and clonally regenerated A.vera plants and it was suggested that the base peak substitutions in the ITS 1 region may arise from the different nutritional and environmental factors in cultivation and plant growth stages. (author)

  13. The Latent Reservoir for HIV-1: How Immunologic Memory and Clonal Expansion Contribute to HIV-1 Persistence

    Science.gov (United States)

    Murray, Alexandra J.; Kwon, Kyungyoon J.; Farber, Donna L.; Siliciano, Robert F.

    2016-01-01

    Combination antiretroviral therapy (ART) for HIV-1 infection reduces plasma virus levels to below the limit of detection of clinical assays. However, even with prolonged suppression of viral replication with ART, viremia rebounds rapidly after treatment interruption. Thus ART is not curative. The principal barrier to cure is a remarkably stable reservoir of latent HIV-1 in resting memory CD4+ T cells. Here we consider explanations for the remarkable stability of the latent reservoir. Stability does not appear to reflect replenishment from new infection events but rather normal physiologic processes that provide for immunologic memory. Of particular importance are proliferative processes that drive clonal expansion of infected cells. Recent evidence suggests that in some infected cells, proliferation is a consequence of proviral integration into host genes associated with cell growth. Efforts to cure HIV-1 infection by targeting the latent reservoir may need to consider the potential of latently infected cells to proliferate. PMID:27382129

  14. Core Genome Multilocus Sequence Typing for Identification of Globally Distributed Clonal Groups and Differentiation of Outbreak Strains of Listeria monocytogenes.

    Science.gov (United States)

    Chen, Yi; Gonzalez-Escalona, Narjol; Hammack, Thomas S; Allard, Marc W; Strain, Errol A; Brown, Eric W

    2016-10-15

    Many listeriosis outbreaks are caused by a few globally distributed clonal groups, designated clonal complexes or epidemic clones, of Listeria monocytogenes, several of which have been defined by classic multilocus sequence typing (MLST) schemes targeting 6 to 8 housekeeping or virulence genes. We have developed and evaluated core genome MLST (cgMLST) schemes and applied them to isolates from multiple clonal groups, including those associated with 39 listeriosis outbreaks. The cgMLST clusters were congruent with MLST-defined clonal groups, which had various degrees of diversity at the whole-genome level. Notably, cgMLST could distinguish among outbreak strains and epidemiologically unrelated strains of the same clonal group, which could not be achieved using classic MLST schemes. The precise selection of cgMLST gene targets may not be critical for the general identification of clonal groups and outbreak strains. cgMLST analyses further identified outbreak strains, including those associated with recent outbreaks linked to contaminated French-style cheese, Hispanic-style cheese, stone fruit, caramel apple, ice cream, and packaged leafy green salad, as belonging to major clonal groups. We further developed lineage-specific cgMLST schemes, which can include accessory genes when core genomes do not possess sufficient diversity, and this provided additional resolution over species-specific cgMLST. Analyses of isolates from different common-source listeriosis outbreaks revealed various degrees of diversity, indicating that the numbers of allelic differences should always be combined with cgMLST clustering and epidemiological evidence to define a listeriosis outbreak. Classic multilocus sequence typing (MLST) schemes targeting internal fragments of 6 to 8 genes that define clonal complexes or epidemic clones have been widely employed to study L. monocytogenes biodiversity and its relation to pathogenicity potential and epidemiology. We demonstrated that core genome MLST

  15. Genetic variation within clonal lineages of Phytophthora infestans revealed through genotyping-by-sequencing, and implications for late blight epidemiology

    Science.gov (United States)

    Genotyping-by-sequencing (GBS) was performed on 257 Phytophthora infestans isolates belonging to four clonal lineages to study within-lineage diversity. The four lineages used in the study included US-8 (n=28), US-11 (n=27), US-23 (n=166), and US-24 (n=36), with isolates originating from 23 of the U...

  16. Multi locus sequence typing of Chlamydiales: clonal groupings within the obligate intracellular bacteria Chlamydia trachomatis.

    Science.gov (United States)

    Pannekoek, Yvonne; Morelli, Giovanna; Kusecek, Barica; Morré, Servaas A; Ossewaarde, Jacobus M; Langerak, Ankie A; van der Ende, Arie

    2008-02-28

    The obligate intracellular growing bacterium Chlamydia trachomatis causes diseases like trachoma, urogenital infection and lymphogranuloma venereum with severe morbidity. Several serovars and genotypes have been identified, but these could not be linked to clinical disease or outcome. The related Chlamydophila pneumoniae, of which no subtypes are recognized, causes respiratory infections worldwide. We developed a multi locus sequence typing (MLST) scheme to understand the population genetic structure and diversity of these species and to evaluate the association between genotype and disease. A collection of 26 strains of C. trachomatis of different serovars and clinical presentation and 18 strains of C. pneumoniae were included in the study. For comparison, sequences of C. abortus, C. psittaci, C. caviae, C. felis, C. pecorum (Chlamydophila), C. muridarum (Chlamydia) and of Candidatus protochlamydia and Simkania negevensis were also included. Sequences of fragments (400 - 500 base pairs) from seven housekeeping genes (enoA, fumC, gatA, gidA, hemN, hlfX, oppA) were analysed. Analysis of allelic profiles by eBurst revealed three non-overlapping clonal complexes among the C. trachomatis strains, while the C. pneumoniae strains formed a single group. An UPGMA tree produced from the allelic profiles resulted in three groups of sequence types. The LGV strains grouped in a single cluster, while the urogenital strains were distributed over two separated groups, one consisted solely of strains with frequent occurring serovars (E, D and F). The distribution of the different serovars over the three groups was not consistent, suggesting exchange of serovar encoding ompA sequences. In one instance, exchange of fumC sequences between strains of different groups was observed. Cluster analyses of concatenated sequences of the Chlamydophila and Chlamydia species together with those of Candidatus Protochlamydia amoebophila and Simkania negevensis resulted in a tree identical to that

  17. Multi locus sequence typing of Chlamydiales: clonal groupings within the obligate intracellular bacteria Chlamydia trachomatis

    Directory of Open Access Journals (Sweden)

    Langerak Ankie A

    2008-02-01

    Full Text Available Abstract Background The obligate intracellular growing bacterium Chlamydia trachomatis causes diseases like trachoma, urogenital infection and lymphogranuloma venereum with severe morbidity. Several serovars and genotypes have been identified, but these could not be linked to clinical disease or outcome. The related Chlamydophila pneumoniae, of which no subtypes are recognized, causes respiratory infections worldwide. We developed a multi locus sequence typing (MLST scheme to understand the population genetic structure and diversity of these species and to evaluate the association between genotype and disease. Results A collection of 26 strains of C. trachomatis of different serovars and clinical presentation and 18 strains of C. pneumoniae were included in the study. For comparison, sequences of C. abortus, C. psittaci, C. caviae, C. felis, C. pecorum (Chlamydophila, C. muridarum (Chlamydia and of Candidatus protochlamydia and Simkania negevensis were also included. Sequences of fragments (400 – 500 base pairs from seven housekeeping genes (enoA, fumC, gatA, gidA, hemN, hlfX, oppA were analysed. Analysis of allelic profiles by eBurst revealed three non-overlapping clonal complexes among the C. trachomatis strains, while the C. pneumoniae strains formed a single group. An UPGMA tree produced from the allelic profiles resulted in three groups of sequence types. The LGV strains grouped in a single cluster, while the urogenital strains were distributed over two separated groups, one consisted solely of strains with frequent occurring serovars (E, D and F. The distribution of the different serovars over the three groups was not consistent, suggesting exchange of serovar encoding ompA sequences. In one instance, exchange of fumC sequences between strains of different groups was observed. Cluster analyses of concatenated sequences of the Chlamydophila and Chlamydia species together with those of Candidatus Protochlamydia amoebophila and Simkania

  18. Genomic sequence and virulence of clonal isolates of vaccinia virus Tiantan, the Chinese smallpox vaccine strain.

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    Qicheng Zhang

    Full Text Available Despite the worldwide eradication of smallpox in 1979, the potential bioterrorism threat from variola virus and the ongoing use of vaccinia virus (VACV as a vector for vaccine development argue for continued research on VACV. In China, the VACV Tiantan strain (TT was used in the smallpox eradication campaign. Its progeny strain is currently being used to develop a human immunodeficiency virus (HIV vaccine. Here we sequenced the full genomes of five TT clones isolated by plaque purification from the TT (752-1 viral stock. Phylogenetic analysis with other commonly used VACV strains showed that TT (752-1 and its clones clustered and exhibited higher sequence diversity than that found in Dryvax clones. The ∼190 kbp genomes of TT appeared to encode 273 open reading frames (ORFs. ORFs located in the middle of the genome were more conserved than those located at the two termini, where many virulence and immunomodulation associated genes reside. Several patterns of nucleotide changes including point mutations, insertions and deletions were identified. The polymorphisms in seven virulence-associated proteins and six immunomodulation-related proteins were analyzed. We also investigated the neuro- and skin- virulence of TT clones in mice and rabbits, respectively. The TT clones exhibited significantly less virulence than the New York City Board of Health (NYCBH strain, as evidenced by less extensive weight loss and morbidity in mice as well as produced smaller skin lesions and lower incidence of putrescence in rabbits. The complete genome sequences, ORF annotations, and phenotypic diversity yielded from this study aid our understanding of the Chinese historic TT strain and are useful for HIV vaccine projects employing TT as a vector.

  19. Genomic sequence and virulence of clonal isolates of vaccinia virus Tiantan, the Chinese smallpox vaccine strain.

    Science.gov (United States)

    Zhang, Qicheng; Tian, Meijuan; Feng, Yi; Zhao, Kai; Xu, Jing; Liu, Ying; Shao, Yiming

    2013-01-01

    Despite the worldwide eradication of smallpox in 1979, the potential bioterrorism threat from variola virus and the ongoing use of vaccinia virus (VACV) as a vector for vaccine development argue for continued research on VACV. In China, the VACV Tiantan strain (TT) was used in the smallpox eradication campaign. Its progeny strain is currently being used to develop a human immunodeficiency virus (HIV) vaccine. Here we sequenced the full genomes of five TT clones isolated by plaque purification from the TT (752-1) viral stock. Phylogenetic analysis with other commonly used VACV strains showed that TT (752-1) and its clones clustered and exhibited higher sequence diversity than that found in Dryvax clones. The ∼190 kbp genomes of TT appeared to encode 273 open reading frames (ORFs). ORFs located in the middle of the genome were more conserved than those located at the two termini, where many virulence and immunomodulation associated genes reside. Several patterns of nucleotide changes including point mutations, insertions and deletions were identified. The polymorphisms in seven virulence-associated proteins and six immunomodulation-related proteins were analyzed. We also investigated the neuro- and skin- virulence of TT clones in mice and rabbits, respectively. The TT clones exhibited significantly less virulence than the New York City Board of Health (NYCBH) strain, as evidenced by less extensive weight loss and morbidity in mice as well as produced smaller skin lesions and lower incidence of putrescence in rabbits. The complete genome sequences, ORF annotations, and phenotypic diversity yielded from this study aid our understanding of the Chinese historic TT strain and are useful for HIV vaccine projects employing TT as a vector.

  20. T cell receptor sequencing of early-stage breast cancer tumors identifies altered clonal structure of the T cell repertoire.

    Science.gov (United States)

    Beausang, John F; Wheeler, Amanda J; Chan, Natalie H; Hanft, Violet R; Dirbas, Frederick M; Jeffrey, Stefanie S; Quake, Stephen R

    2017-11-28

    Tumor-infiltrating T cells play an important role in many cancers, and can improve prognosis and yield therapeutic targets. We characterized T cells infiltrating both breast cancer tumors and the surrounding normal breast tissue to identify T cells specific to each, as well as their abundance in peripheral blood. Using immune profiling of the T cell beta-chain repertoire in 16 patients with early-stage breast cancer, we show that the clonal structure of the tumor is significantly different from adjacent breast tissue, with the tumor containing ∼2.5-fold greater density of T cells and higher clonality compared with normal breast. The clonal structure of T cells in blood and normal breast is more similar than between blood and tumor, and could be used to distinguish tumor from normal breast tissue in 14 of 16 patients. Many T cell sequences overlap between tissue and blood from the same patient, including ∼50% of T cells between tumor and normal breast. Both tumor and normal breast contain high-abundance "enriched" sequences that are absent or of low abundance in the other tissue. Many of these T cells are either not detected or detected with very low frequency in the blood, suggesting the existence of separate compartments of T cells in both tumor and normal breast. Enriched T cell sequences are typically unique to each patient, but a subset is shared between many different patients. We show that many of these are commonly generated sequences, and thus unlikely to play an important role in the tumor microenvironment. Copyright © 2017 the Author(s). Published by PNAS.

  1. Analyzing clonal fidelity of micropropagated Psidium guajava L. plants using simple sequence repeat markers

    Science.gov (United States)

    Micropropagation of Psidium guajava L. (guava) is a viable alternative to currently adopted techniques for large-scale plant propagation of commercial cultivars. Assessment of clonal fidelity in micropropagated plants is the first step towards ensuring genetic uniformity in mass production of planti...

  2. Massively parallel sequencing and genome-wide copy number analysis revealed a clonal relationship in benign metastasizing leiomyoma.

    Science.gov (United States)

    Wu, Ren-Chin; Chao, An-Shine; Lee, Li-Yu; Lin, Gigin; Chen, Shu-Jen; Lu, Yen-Jung; Huang, Huei-Jean; Yen, Chi-Feng; Han, Chien Min; Lee, Yun-Shien; Wang, Tzu-Hao; Chao, Angel

    2017-07-18

    Benign metastasizing leiomyoma (BML) is a rare disease entity typically presenting as multiple extrauterine leiomyomas associated with a uterine leiomyoma. It has been hypothesized that the extrauterine leiomyomata represent distant metastasis of the uterine leiomyoma. To date, the only molecular evidence supporting this hypothesis was derived from clonality analyses based on X-chromosome inactivation assays. Here, we sought to address this issue by examining paired specimens of synchronous pulmonary and uterine leiomyomata from three patients using targeted massively parallel sequencing and molecular inversion probe array analysis for detecting somatic mutations and copy number aberrations. We detected identical non-hot-spot somatic mutations and similar patterns of copy number aberrations (CNAs) in paired pulmonary and uterine leiomyomata from two patients, indicating the clonal relationship between pulmonary and uterine leiomyomata. In addition to loss of chromosome 22q found in the literature, we identified additional recurrent CNAs including losses of chromosome 3q and 11q. In conclusion, our findings of the clonal relationship between synchronous pulmonary and uterine leiomyomas support the hypothesis that BML represents a condition wherein a uterine leiomyoma disseminates to distant extrauterine locations.

  3. Massively parallel sequencing and genome-wide copy number analysis revealed a clonal relationship in benign metastasizing leiomyoma

    Science.gov (United States)

    Lee, Li-Yu; Lin, Gigin; Chen, Shu-Jen; Lu, Yen-Jung; Huang, Huei-Jean; Yen, Chi-Feng; Han, Chien Min; Lee, Yun-Shien; Wang, Tzu-Hao; Chao, Angel

    2017-01-01

    Benign metastasizing leiomyoma (BML) is a rare disease entity typically presenting as multiple extrauterine leiomyomas associated with a uterine leiomyoma. It has been hypothesized that the extrauterine leiomyomata represent distant metastasis of the uterine leiomyoma. To date, the only molecular evidence supporting this hypothesis was derived from clonality analyses based on X-chromosome inactivation assays. Here, we sought to address this issue by examining paired specimens of synchronous pulmonary and uterine leiomyomata from three patients using targeted massively parallel sequencing and molecular inversion probe array analysis for detecting somatic mutations and copy number aberrations. We detected identical non-hot-spot somatic mutations and similar patterns of copy number aberrations (CNAs) in paired pulmonary and uterine leiomyomata from two patients, indicating the clonal relationship between pulmonary and uterine leiomyomata. In addition to loss of chromosome 22q found in the literature, we identified additional recurrent CNAs including losses of chromosome 3q and 11q. In conclusion, our findings of the clonal relationship between synchronous pulmonary and uterine leiomyomas support the hypothesis that BML represents a condition wherein a uterine leiomyoma disseminates to distant extrauterine locations. PMID:28533481

  4. Deep Sequencing of T-cell Receptor DNA as a Biomarker of Clonally Expanded TILs in Breast Cancer after Immunotherapy.

    Science.gov (United States)

    Page, David B; Yuan, Jianda; Redmond, David; Wen, Y Hanna; Durack, Jeremy C; Emerson, Ryan; Solomon, Stephen; Dong, Zhiwan; Wong, Phillip; Comstock, Christopher; Diab, Adi; Sung, Janice; Maybody, Majid; Morris, Elizabeth; Brogi, Edi; Morrow, Monica; Sacchini, Virgilio; Elemento, Olivier; Robins, Harlan; Patil, Sujata; Allison, James P; Wolchok, Jedd D; Hudis, Clifford; Norton, Larry; McArthur, Heather L

    2016-10-01

    In early-stage breast cancer, the degree of tumor-infiltrating lymphocytes (TIL) predicts response to chemotherapy and overall survival. Combination immunotherapy with immune checkpoint antibody plus tumor cryoablation can induce lymphocytic infiltrates and improve survival in mice. We used T-cell receptor (TCR) DNA sequencing to evaluate both the effect of cryoimmunotherapy in humans and the feasibility of TCR sequencing in early-stage breast cancer. In a pilot clinical trial, 18 women with early-stage breast cancer were treated preoperatively with cryoablation, single-dose anti-CTLA-4 (ipilimumab), or cryoablation + ipilimumab. TCRs within serially collected peripheral blood and tumor tissue were sequenced. In baseline tumor tissues, T-cell density as measured by TCR sequencing correlated with TIL scores obtained by hematoxylin and eosin (H&E) staining. However, tumors with little or no lymphocytes by H&E contained up to 3.6 × 10 6 TCR DNA sequences, highlighting the sensitivity of the ImmunoSEQ platform. In this dataset, ipilimumab increased intratumoral T-cell density over time, whereas cryoablation ± ipilimumab diversified and remodeled the intratumoral T-cell clonal repertoire. Compared with monotherapy, cryoablation plus ipilimumab was associated with numerically greater numbers of peripheral blood and intratumoral T-cell clones expanding robustly following therapy. In conclusion, TCR sequencing correlates with H&E lymphocyte scoring and provides additional information on clonal diversity. These findings support further study of the use of TCR sequencing as a biomarker for T-cell responses to therapy and for the study of cryoimmunotherapy in early-stage breast cancer. Cancer Immunol Res; 4(10); 835-44. ©2016 AACR. ©2016 American Association for Cancer Research.

  5. HIV protein sequence hotspots for crosstalk with host hub proteins.

    Directory of Open Access Journals (Sweden)

    Mahdi Sarmady

    Full Text Available HIV proteins target host hub proteins for transient binding interactions. The presence of viral proteins in the infected cell results in out-competition of host proteins in their interaction with hub proteins, drastically affecting cell physiology. Functional genomics and interactome datasets can be used to quantify the sequence hotspots on the HIV proteome mediating interactions with host hub proteins. In this study, we used the HIV and human interactome databases to identify HIV targeted host hub proteins and their host binding partners (H2. We developed a high throughput computational procedure utilizing motif discovery algorithms on sets of protein sequences, including sequences of HIV and H2 proteins. We identified as HIV sequence hotspots those linear motifs that are highly conserved on HIV sequences and at the same time have a statistically enriched presence on the sequences of H2 proteins. The HIV protein motifs discovered in this study are expressed by subsets of H2 host proteins potentially outcompeted by HIV proteins. A large subset of these motifs is involved in cleavage, nuclear localization, phosphorylation, and transcription factor binding events. Many such motifs are clustered on an HIV sequence in the form of hotspots. The sequential positions of these hotspots are consistent with the curated literature on phenotype altering residue mutations, as well as with existing binding site data. The hotspot map produced in this study is the first global portrayal of HIV motifs involved in altering the host protein network at highly connected hub nodes.

  6. Genome-wide-analyses of Listeria monocytogenes from food-processing plants reveals clonal diversity and dates the emergence of persisting sequence types

    DEFF Research Database (Denmark)

    Knudsen, Gitte Maegaard; Nielsen, Jesper Boye; Marvig, Rasmus Lykke

    2017-01-01

    for three persisting sequence types (ST) based on Multi Locus Sequence Typing (MLST) being ST7, ST8 and ST121, long-term persistence of clonal groups was limited, and new clones were introduced continuously, potentially from raw materials. No particular gene could be linked to the persistence phenotype...

  7. B cell clonal lineage alterations upon recombinant HIV-1 envelope immunization of Rhesus macaques

    Science.gov (United States)

    Broadly neutralizing HIV-1 antibodies (bNAbs) isolated from infected subjects display protective potential in animal models. Their elicitation by immunization is thus highly desirable. The HIV-1 envelope glycoprotein (Env) is the sole viral target of bnAbs, but is also targeted by binding, non-neutr...

  8. Whole-exome sequencing reveals the spectrum of gene mutations and the clonal evolution patterns in paediatric acute myeloid leukaemia.

    Science.gov (United States)

    Shiba, Norio; Yoshida, Kenichi; Shiraishi, Yuichi; Okuno, Yusuke; Yamato, Genki; Hara, Yusuke; Nagata, Yasunobu; Chiba, Kenichi; Tanaka, Hiroko; Terui, Kiminori; Kato, Motohiro; Park, Myoung-Ja; Ohki, Kentaro; Shimada, Akira; Takita, Junko; Tomizawa, Daisuke; Kudo, Kazuko; Arakawa, Hirokazu; Adachi, Souichi; Taga, Takashi; Tawa, Akio; Ito, Etsuro; Horibe, Keizo; Sanada, Masashi; Miyano, Satoru; Ogawa, Seishi; Hayashi, Yasuhide

    2016-11-01

    Acute myeloid leukaemia (AML) is a molecularly and clinically heterogeneous disease. Targeted sequencing efforts have identified several mutations with diagnostic and prognostic values in KIT, NPM1, CEBPA and FLT3 in both adult and paediatric AML. In addition, massively parallel sequencing enabled the discovery of recurrent mutations (i.e. IDH1/2 and DNMT3A) in adult AML. In this study, whole-exome sequencing (WES) of 22 paediatric AML patients revealed mutations in components of the cohesin complex (RAD21 and SMC3), BCORL1 and ASXL2 in addition to previously known gene mutations. We also revealed intratumoural heterogeneities in many patients, implicating multiple clonal evolution events in the development of AML. Furthermore, targeted deep sequencing in 182 paediatric AML patients identified three major categories of recurrently mutated genes: cohesion complex genes [STAG2, RAD21 and SMC3 in 17 patients (8·3%)], epigenetic regulators [ASXL1/ASXL2 in 17 patients (8·3%), BCOR/BCORL1 in 7 patients (3·4%)] and signalling molecules. We also performed WES in four patients with relapsed AML. Relapsed AML evolved from one of the subclones at the initial phase and was accompanied by many additional mutations, including common driver mutations that were absent or existed only with lower allele frequency in the diagnostic samples, indicating a multistep process causing leukaemia recurrence. © 2016 John Wiley & Sons Ltd.

  9. Modeling viral coevolution: HIV multi-clonal persistence and competition dynamics

    Science.gov (United States)

    Bagnoli, F.; Liò, P.; Sguanci, L.

    2006-07-01

    The coexistence of different viral strains (quasispecies) within the same host are nowadays observed for a growing number of viruses, most notably HIV, Marburg and Ebola, but the conditions for the formation and survival of new strains have not yet been understood. We present a model of HIV quasispecies competition, which describes the conditions of viral quasispecies coexistence under different immune system conditions. Our model incorporates both T and B cells responses, and we show that the role of B cells is important and additive to that of T cells. Simulations of coinfection (simultaneous infection) and superinfection (delayed secondary infection) scenarios in the early stages (days) and in the late stages of the infection (years) are in agreement with emerging molecular biology findings. The immune response induces a competition among similar phenotypes, leading to differentiation (quasispeciation), escape dynamics and complex oscillations of viral strain abundance. We found that the quasispecies dynamics after superinfection or coinfection has time scales of several months and becomes even slower when the immune system response is weak. Our model represents a general framework to study the speed and distribution of HIV quasispecies during disease progression, vaccination and therapy.

  10. Sequence alignment reveals possible MAPK docking motifs on HIV proteins.

    Directory of Open Access Journals (Sweden)

    Perry Evans

    Full Text Available Over the course of HIV infection, virus replication is facilitated by the phosphorylation of HIV proteins by human ERK1 and ERK2 mitogen-activated protein kinases (MAPKs. MAPKs are known to phosphorylate their substrates by first binding with them at a docking site. Docking site interactions could be viable drug targets because the sequences guiding them are more specific than phosphorylation consensus sites. In this study we use multiple bioinformatics tools to discover candidate MAPK docking site motifs on HIV proteins known to be phosphorylated by MAPKs, and we discuss the possibility of targeting docking sites with drugs. Using sequence alignments of HIV proteins of different subtypes, we show that MAPK docking patterns previously described for human proteins appear on the HIV matrix, Tat, and Vif proteins in a strain dependent manner, but are absent from HIV Rev and appear on all HIV Nef strains. We revise the regular expressions of previously annotated MAPK docking patterns in order to provide a subtype independent motif that annotates all HIV proteins. One revision is based on a documented human variant of one of the substrate docking motifs, and the other reduces the number of required basic amino acids in the standard docking motifs from two to one. The proposed patterns are shown to be consistent with in silico docking between ERK1 and the HIV matrix protein. The motif usage on HIV proteins is sufficiently different from human proteins in amino acid sequence similarity to allow for HIV specific targeting using small-molecule drugs.

  11. Inferring the Clonal Structure of Viral Populations from Time Series Sequencing.

    Directory of Open Access Journals (Sweden)

    Donatien F Chedom

    2015-11-01

    Full Text Available RNA virus populations will undergo processes of mutation and selection resulting in a mixed population of viral particles. High throughput sequencing of a viral population subsequently contains a mixed signal of the underlying clones. We would like to identify the underlying evolutionary structures. We utilize two sources of information to attempt this; within segment linkage information, and mutation prevalence. We demonstrate that clone haplotypes, their prevalence, and maximum parsimony reticulate evolutionary structures can be identified, although the solutions may not be unique, even for complete sets of information. This is applied to a chain of influenza infection, where we infer evolutionary structures, including reassortment, and demonstrate some of the difficulties of interpretation that arise from deep sequencing due to artifacts such as template switching during PCR amplification.

  12. Clonal spread of blaOXA-72-carrying Acinetobacter baumannii sequence type 512 in Taiwan.

    Science.gov (United States)

    Kuo, Han-Yueh; Hsu, Po-Jui; Chen, Jiann-Yuan; Liao, Po-Cheng; Lu, Chia-Wei; Chen, Chang-Hua; Liou, Ming-Li

    2016-07-01

    This is the first report to show an insidious outbreak of armA- and blaOXA-72-carrying Acinetobacter baumannii sequence type 512 (ST512) at a study hospital in northern Taiwan. Multilocus sequence typing revealed that this was a ST512 clone. All of the isolates with ST512 carried a novel 12,056-bp repGR2 in combination with a repGR12-type plasmid. This plasmid, designated pAB-ML, had one copy of the blaOXA-72 gene that was flanked by XerC/XerD-like sites and conferred resistance to carbapenems. Copyright © 2016 Elsevier B.V. and International Society of Chemotherapy. All rights reserved.

  13. Evolution of Sequence Type 4821 Clonal Complex Meningococcal Strains in China from Prequinolone to Quinolone Era, 1972–2013

    Science.gov (United States)

    Guo, Qinglan; Mustapha, Mustapha M.; Chen, Mingliang; Qu, Di; Zhang, Xi; Harrison, Lee H.

    2018-01-01

    The expansion of hypervirulent sequence type 4821 clonal complex (CC4821) lineage Neisseria meningitidis bacteria has led to a shift in meningococcal disease epidemiology in China, from serogroup A (MenA) to MenC. Knowledge of the evolution and genetic origin of the emergent MenC strains is limited. In this study, we subjected 76 CC4821 isolates collected across China during 1972–1977 and 2005–2013 to phylogenetic analysis, traditional genotyping, or both. We show that successive recombination events within genes encoding surface antigens and acquisition of quinolone resistance mutations possibly played a role in the emergence of CC4821 as an epidemic clone in China. MenC and MenB CC4821 strains have spread across China and have been detected in several countries in different continents. Capsular switches involving serogroups B and C occurred among epidemic strains, raising concerns regarding possible increases in MenB disease, given that vaccines in use in China do not protect against MenB. PMID:29553310

  14. Targeted capture massively parallel sequencing analysis of LCIS and invasive lobular cancer: Repertoire of somatic genetic alterations and clonal relationships.

    Science.gov (United States)

    Sakr, Rita A; Schizas, Michail; Carniello, Jose V Scarpa; Ng, Charlotte K Y; Piscuoglio, Salvatore; Giri, Dilip; Andrade, Victor P; De Brot, Marina; Lim, Raymond S; Towers, Russell; Weigelt, Britta; Reis-Filho, Jorge S; King, Tari A

    2016-02-01

    Lobular carcinoma in situ (LCIS) has been proposed as a non-obligate precursor of invasive lobular carcinoma (ILC). Here we sought to define the repertoire of somatic genetic alterations in pure LCIS and in synchronous LCIS and ILC using targeted massively parallel sequencing. DNA samples extracted from microdissected LCIS, ILC and matched normal breast tissue or peripheral blood from 30 patients were subjected to massively parallel sequencing targeting all exons of 273 genes, including the genes most frequently mutated in breast cancer and DNA repair-related genes. Single nucleotide variants and insertions and deletions were identified using state-of-the-art bioinformatics approaches. The constellation of somatic mutations found in LCIS (n = 34) and ILC (n = 21) were similar, with the most frequently mutated genes being CDH1 (56% and 66%, respectively), PIK3CA (41% and 52%, respectively) and CBFB (12% and 19%, respectively). Among 19 LCIS and ILC synchronous pairs, 14 (74%) had at least one identical mutation in common, including identical PIK3CA and CDH1 mutations. Paired analysis of independent foci of LCIS from 3 breasts revealed at least one common mutation in each of the 3 pairs (CDH1, PIK3CA, CBFB and PKHD1L1). LCIS and ILC have a similar repertoire of somatic mutations, with PIK3CA and CDH1 being the most frequently mutated genes. The presence of identical mutations between LCIS-LCIS and LCIS-ILC pairs demonstrates that LCIS is a clonal neoplastic lesion, and provides additional evidence that at least some LCIS are non-obligate precursors of ILC. Copyright © 2015 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

  15. Whole Genome Sequence Analysis Using JSpecies Tool Establishes Clonal Relationships between Listeria monocytogenes Strains from Epidemiologically Unrelated Listeriosis Outbreaks.

    Directory of Open Access Journals (Sweden)

    Laurel S Burall

    Full Text Available In an effort to build a comprehensive genomic approach to food safety challenges, the FDA has implemented a whole genome sequencing effort, GenomeTrakr, which involves the sequencing and analysis of genomes of foodborne pathogens. As a part of this effort, we routinely sequence whole genomes of Listeria monocytogenes (Lm isolates associated with human listeriosis outbreaks, as well as those isolated through other sources. To rapidly establish genetic relatedness of these genomes, we evaluated tetranucleotide frequency analysis via the JSpecies program to provide a cursory analysis of strain relatedness. The JSpecies tetranucleotide (tetra analysis plots standardized (z-score tetramer word frequencies of two strains against each other and uses linear regression analysis to determine similarity (r2. This tool was able to validate the close relationships between outbreak related strains from four different outbreaks. Included in this study was the analysis of Lm strains isolated during the recent caramel apple outbreak and stone fruit incident in 2014. We identified that many of the isolates from these two outbreaks shared a common 4b variant (4bV serotype, also designated as IVb-v1, using a qPCR protocol developed in our laboratory. The 4bV serotype is characterized by the presence of a 6.3 Kb DNA segment normally found in serotype 1/2a, 3a, 1/2c and 3c strains but not in serotype 4b or 1/2b strains. We decided to compare these strains at a genomic level using the JSpecies Tetra tool. Specifically, we compared several 4bV and 4b isolates and identified a high level of similarity between the stone fruit and apple 4bV strains, but not the 4b strains co-identified in the caramel apple outbreak or other 4b or 4bV strains in our collection. This finding was further substantiated by a SNP-based analysis. Additionally, we were able to identify close relatedness between isolates from clinical cases from 1993-1994 and a single case from 2011 as well as

  16. Clonality and Resistome analysis of KPC-producing Klebsiella pneumoniae strain isolated in Korea using whole genome sequencing.

    Science.gov (United States)

    Lee, Yangsoon; Kim, Bong-Soo; Chun, Jongsik; Yong, Ji Hyun; Lee, Yeong Seon; Yoo, Jung Sik; Yong, Dongeun; Hong, Seong Geun; D'Souza, Roshan; Thomson, Kenneth S; Lee, Kyungwon; Chong, Yunsop

    2014-01-01

    We analyzed the whole genome sequence and resistome of the outbreak Klebsiella pneumoniae strain MP14 and compared it with those of K. pneumoniae carbapenemase- (KPC-) producing isolates that showed high similarity in the NCBI genome database. A KPC-2-producing multidrug-resistant (MDR) K. pneumoniae clinical isolate was obtained from a patient admitted to a Korean hospital in 2011. The strain MP14 was resistant to all tested β-lactams including monobactam, amikacin, levofloxacin, and cotrimoxazole, but susceptible to tigecycline and colistin. Resistome analysis showed the presence of β-lactamase genes including bla KPC-2, bla SHV-11, bla TEM-169, and bla OXA-9. MP14 also possessed aac(6'-)Ib, aadA2, and aph(3'-)Ia as aminoglycoside resistance-encoding genes, mph(A) for macrolides, oqxA and oqxB for quinolone, catA1 for phenicol, sul1 for sulfonamide, and dfrA12 for trimethoprim. Both SNP tree and cgMLST analysis showed the close relatedness with the KPC producers (KPNIH strains) isolated from an outbreak in the USA and colistin-resistant strains isolated in Italy. The plasmid-scaffold genes in plasmids pKpQil, pKpQil-IT, pKPN3, or pKPN-IT were identified in MP14, KPNIH, and Italian strains. The KPC-2-producing MDR K. pneumoniae ST258 stain isolated in Korea was highly clonally related with MDR K. pneumoniae strains from the USA and Italy. Global spread of KPC-producing K. pneumoniae is a worrying phenomenon.

  17. Clonality and Resistome Analysis of KPC-Producing Klebsiella pneumoniae Strain Isolated in Korea Using Whole Genome Sequencing

    Directory of Open Access Journals (Sweden)

    Yangsoon Lee

    2014-01-01

    Full Text Available We analyzed the whole genome sequence and resistome of the outbreak Klebsiella pneumoniae strain MP14 and compared it with those of K. pneumoniae carbapenemase- (KPC- producing isolates that showed high similarity in the NCBI genome database. A KPC-2-producing multidrug-resistant (MDR K. pneumoniae clinical isolate was obtained from a patient admitted to a Korean hospital in 2011. The strain MP14 was resistant to all tested β-lactams including monobactam, amikacin, levofloxacin, and cotrimoxazole, but susceptible to tigecycline and colistin. Resistome analysis showed the presence of β-lactamase genes including blaKPC-2, blaSHV-11, blaTEM-169, and blaOXA-9. MP14 also possessed aac(6′-Ib, aadA2, and aph(3′-Ia as aminoglycoside resistance-encoding genes, mph(A for macrolides, oqxA and oqxB for quinolone, catA1 for phenicol, sul1 for sulfonamide, and dfrA12 for trimethoprim. Both SNP tree and cgMLST analysis showed the close relatedness with the KPC producers (KPNIH strains isolated from an outbreak in the USA and colistin-resistant strains isolated in Italy. The plasmid-scaffold genes in plasmids pKpQil, pKpQil-IT, pKPN3, or pKPN-IT were identified in MP14, KPNIH, and Italian strains. The KPC-2-producing MDR K. pneumoniae ST258 stain isolated in Korea was highly clonally related with MDR K. pneumoniae strains from the USA and Italy. Global spread of KPC-producing K. pneumoniae is a worrying phenomenon.

  18. A safe an easy method for building consensus HIV sequences from 454 massively parallel sequencing data.

    Science.gov (United States)

    Fernández-Caballero Rico, Jose Ángel; Chueca Porcuna, Natalia; Álvarez Estévez, Marta; Mosquera Gutiérrez, María Del Mar; Marcos Maeso, María Ángeles; García, Federico

    2018-02-01

    To show how to generate a consensus sequence from the information of massive parallel sequences data obtained from routine HIV anti-retroviral resistance studies, and that may be suitable for molecular epidemiology studies. Paired Sanger (Trugene-Siemens) and next-generation sequencing (NGS) (454 GSJunior-Roche) HIV RT and protease sequences from 62 patients were studied. NGS consensus sequences were generated using Mesquite, using 10%, 15%, and 20% thresholds. Molecular evolutionary genetics analysis (MEGA) was used for phylogenetic studies. At a 10% threshold, NGS-Sanger sequences from 17/62 patients were phylogenetically related, with a median bootstrap-value of 88% (IQR83.5-95.5). Association increased to 36/62 sequences, median bootstrap 94% (IQR85.5-98)], using a 15% threshold. Maximum association was at the 20% threshold, with 61/62 sequences associated, and a median bootstrap value of 99% (IQR98-100). A safe method is presented to generate consensus sequences from HIV-NGS data at 20% threshold, which will prove useful for molecular epidemiological studies. Copyright © 2016 Elsevier España, S.L.U. and Sociedad Española de Enfermedades Infecciosas y Microbiología Clínica. All rights reserved.

  19. Genome-wide-analyses of Listeria monocytogenes from food-processing plants reveal clonal diversity and date the emergence of persisting sequence types.

    Science.gov (United States)

    Knudsen, Gitte M; Nielsen, Jesper Boye; Marvig, Rasmus L; Ng, Yin; Worning, Peder; Westh, Henrik; Gram, Lone

    2017-08-01

    Whole genome sequencing is increasing used in epidemiology, e.g. for tracing outbreaks of food-borne diseases. This requires in-depth understanding of pathogen emergence, persistence and genomic diversity along the food production chain including in food processing plants. We sequenced the genomes of 80 isolates of Listeria monocytogenes sampled from Danish food processing plants over a time-period of 20 years, and analysed the sequences together with 10 public available reference genomes to advance our understanding of interplant and intraplant genomic diversity of L. monocytogenes. Except for three persisting sequence types (ST) based on Multi Locus Sequence Typing being ST7, ST8 and ST121, long-term persistence of clonal groups was limited, and new clones were introduced continuously, potentially from raw materials. No particular gene could be linked to the persistence phenotype. Using time-based phylogenetic analyses of the persistent STs, we estimate the L. monocytogenes evolutionary rate to be 0.18-0.35 single nucleotide polymorphisms/year, suggesting that the persistent STs emerged approximately 100 years ago, which correlates with the onset of industrialization and globalization of the food market. © 2017 Society for Applied Microbiology and John Wiley & Sons Ltd.

  20. Trajectories of Childbearing among HIV Infected Indian Women : A Sequence Analysis Approach

    NARCIS (Netherlands)

    Darak, Shrinivas; Mills, Melinda; Kulkarni, Vinay; Kulkarni, Sanjeevani; Hutter, Inge; Janssen, Fanny

    2015-01-01

    Background HIV infection closely relates to and deeply affects the reproductive career of those infected. However, little is known about the reproductive career trajectories, specifically the interaction of the timing of HIV diagnosis with the timing and sequencing of reproductive events among HIV

  1. Trajectories of childbearing among HIV infected Indian women: A sequence analysis approach

    NARCIS (Netherlands)

    Darak, S.; Mills, M.; Kulkarni, V.; Kulkarni, S.; Hutter, I.; Janssen, F.

    2015-01-01

    Background HIV infection closely relates to and deeply affects the reproductive career of those infected. However, little is known about the reproductive career trajectories, specifically the interaction of the timing of HIV diagnosis with the timing and sequencing of reproductive events among HIV

  2. Trajectories of childbearing among HIV infected Indian women : A sequence analysis approach

    NARCIS (Netherlands)

    S. Darak (Shrinivas); M. Mills (Melinda); V. Kulkarni (Vinay); S. Kulkarni (Sanjeevani); I. Hutter (Inge); F. Janssen (Fanny)

    2015-01-01

    textabstractHIV infection closely relates to and deeply affects the reproductive career of those infected. However, little is known about the reproductive career trajectories, specifically the interaction of the timing of HIV diagnosis with the timing and sequencing of reproductive events among HIV

  3. Clonal hematopoiesis in acquired aplastic anemia

    OpenAIRE

    Ogawa, Seishi

    2016-01-01

    Clonal hematopoiesis (CH) in aplastic anemia (AA) has been closely linked to the evolution of late clonal disorders, including paroxysmal nocturnal hemoglobinuria and myelodysplastic syndromes (MDS)/acute myeloid leukemia (AML), which are common complications after successful immunosuppressive therapy (IST). With the advent of high-throughput sequencing of recent years, the molecular aspect of CH in AA has been clarified by comprehensive detection of somatic mutations that drive clonal evolut...

  4. Multi-virulence-locus sequence typing of Staphylococcus lugdunensis generates results consistent with a clonal population structure and is reliable for epidemiological typing.

    Science.gov (United States)

    Didi, Jennifer; Lemée, Ludovic; Gibert, Laure; Pons, Jean-Louis; Pestel-Caron, Martine

    2014-10-01

    Staphylococcus lugdunensis is an emergent virulent coagulase-negative staphylococcus responsible for severe infections similar to those caused by Staphylococcus aureus. To understand its potentially pathogenic capacity and have further detailed knowledge of the molecular traits of this organism, 93 isolates from various geographic origins were analyzed by multi-virulence-locus sequence typing (MVLST), targeting seven known or putative virulence-associated loci (atlLR2, atlLR3, hlb, isdJ, SLUG_09050, SLUG_16930, and vwbl). The polymorphisms of the putative virulence-associated loci were moderate and comparable to those of the housekeeping genes analyzed by multilocus sequence typing (MLST). However, the MVLST scheme generated 43 virulence types (VTs) compared to 20 sequence types (STs) based on MLST, indicating that MVLST was significantly more discriminating (Simpson's index [D], 0.943). No hypervirulent lineage or cluster specific to carriage strains was defined. The results of multilocus sequence analysis of known and putative virulence-associated loci are consistent with a clonal population structure for S. lugdunensis, suggesting a coevolution of these genes with housekeeping genes. Indeed, the nonsynonymous to synonymous evolutionary substitutions (dN/dS) ratio, the Tajima's D test, and Single-likelihood ancestor counting (SLAC) analysis suggest that all virulence-associated loci were under negative selection, even atlLR2 (AtlL protein) and SLUG_16930 (FbpA homologue), for which the dN/dS ratios were higher. In addition, this analysis of virulence-associated loci allowed us to propose a trilocus sequence typing scheme based on the intragenic regions of atlLR3, isdJ, and SLUG_16930, which is more discriminant than MLST for studying short-term epidemiology and further characterizing the lineages of the rare but highly pathogenic S. lugdunensis. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

  5. ddClone: joint statistical inference of clonal populations from single cell and bulk tumour sequencing data.

    Science.gov (United States)

    Salehi, Sohrab; Steif, Adi; Roth, Andrew; Aparicio, Samuel; Bouchard-Côté, Alexandre; Shah, Sohrab P

    2017-03-01

    Next-generation sequencing (NGS) of bulk tumour tissue can identify constituent cell populations in cancers and measure their abundance. This requires computational deconvolution of allelic counts from somatic mutations, which may be incapable of fully resolving the underlying population structure. Single cell sequencing (SCS) is a more direct method, although its replacement of NGS is impeded by technical noise and sampling limitations. We propose ddClone, which analytically integrates NGS and SCS data, leveraging their complementary attributes through joint statistical inference. We show on real and simulated datasets that ddClone produces more accurate results than can be achieved by either method alone.

  6. HIV sequence diversity during the early phase of infection is associated with HIV DNA reductions during antiretroviral therapy.

    Science.gov (United States)

    Wang, Nidan; Li, Yijia; Han, Yang; Xie, Jing; Li, Taisheng

    2017-06-01

    The association between baseline human immunodeficiency virus (HIV) sequence diversity and HIV DNA decay after the initiation of antiretroviral therapy (ART) remains uncharacterized during the early stages of HIV infection. Samples were obtained from a cohort of 17 patients with early HIV infection (HIV-1 envelope (env) gene was amplified via single genome amplification (SGA) to determine the peripheral plasma HIV quasispecies. We categorized HIV quasispecies into two groups according to baseline viral sequence genetic distance, which was determined by the Poisson-Fitter tool. Total HIV DNA in peripheral blood mononuclear cells (PBMCs), viral load, and T cell subsets were measured prior to and after the initiation of ART. The median SGA sequence number was 17 (range 6-28). At baseline, we identified 7 patients with homogeneous viral populations (designated the Homogeneous group) and 10 patients with heterogeneous viral populations (designated the Heterogeneous group) based on SGA sequences. Both groups exhibited similar HIV DNA decay rates during the first 6 months of ART (P > 0.99), but the Homogenous group experienced more prominent decay than the Heterogeneous group after 6 months (P = 0.037). The Heterogeneous group had higher CD4 cell counts after ART initiation; however, both groups had comparable recovery in terms of CD4/CD8 ratios and CD8 T cell activation levels. Viral population homogeneity upon the initiation of ART is associated with a decrease in HIV DNA levels during ART. J. Med. Virol. 89:982-988, 2017. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

  7. Global epidemiology of capsular group W meningococcal disease (1970-2015): Multifocal emergence and persistence of hypervirulent sequence type (ST)-11 clonal complex.

    Science.gov (United States)

    Mustapha, Mustapha M; Marsh, Jane W; Harrison, Lee H

    2016-03-18

    Following an outbreak in Mecca Saudi Arabia in 2000, meningococcal strains expressing capsular group W (W) emerged as a major cause of invasive meningococcal disease (IMD) worldwide. The Saudi Arabian outbreak strain (Hajj clone) belonging to the ST-11 clonal complex (cc11) is similar to W cc11 causing occasional sporadic disease before 2000. Since 2000, W cc11 has caused large meningococcal disease epidemics in the African meningitis belt and endemic disease in South America, Europe and China. Traditional molecular epidemiologic typing suggested that a majority of current W cc11 burden represented global spread of the Hajj clone. However, recent whole genome sequencing (WGS) analyses revealed significant genetic heterogeneity among global W cc11 strains. While continued spread of the Hajj clone occurs in the Middle East, the meningitis belt and South Africa have co-circulation of the Hajj clone and other unrelated W cc11 strains. Notably, South America, the UK, and France share a genetically distinct W cc11 strain. Other W lineages persist in low numbers in Europe, North America and the meningitis belt. In summary, WGS is helping to unravel the complex genomic epidemiology of group W meningococcal strains. Wider application of WGS and strengthening of global IMD surveillance is necessary to monitor the continued evolution of group W lineages. Copyright © 2016 Elsevier Ltd. All rights reserved.

  8. Importance of Viral Sequence Length and Number of Variable and Informative Sites in Analysis of HIV Clustering.

    Science.gov (United States)

    Novitsky, Vlad; Moyo, Sikhulile; Lei, Quanhong; DeGruttola, Victor; Essex, M

    2015-05-01

    To improve the methodology of HIV cluster analysis, we addressed how analysis of HIV clustering is associated with parameters that can affect the outcome of viral clustering. The extent of HIV clustering and tree certainty was compared between 401 HIV-1C near full-length genome sequences and subgenomic regions retrieved from the LANL HIV Database. Sliding window analysis was based on 99 windows of 1,000 bp and 45 windows of 2,000 bp. Potential associations between the extent of HIV clustering and sequence length and the number of variable and informative sites were evaluated. The near full-length genome HIV sequences showed the highest extent of HIV clustering and the highest tree certainty. At the bootstrap threshold of 0.80 in maximum likelihood (ML) analysis, 58.9% of near full-length HIV-1C sequences but only 15.5% of partial pol sequences (ViroSeq) were found in clusters. Among HIV-1 structural genes, pol showed the highest extent of clustering (38.9% at a bootstrap threshold of 0.80), although it was significantly lower than in the near full-length genome sequences. The extent of HIV clustering was significantly higher for sliding windows of 2,000 bp than 1,000 bp. We found a strong association between the sequence length and proportion of HIV sequences in clusters, and a moderate association between the number of variable and informative sites and the proportion of HIV sequences in clusters. In HIV cluster analysis, the extent of detectable HIV clustering is directly associated with the length of viral sequences used, as well as the number of variable and informative sites. Near full-length genome sequences could provide the most informative HIV cluster analysis. Selected subgenomic regions with a high extent of HIV clustering and high tree certainty could also be considered as a second choice.

  9. Whole genome typing of the recently emerged Canadian serogroup W Neisseria meningitidis sequence type 11 clonal complex isolates associated with invasive meningococcal disease.

    Science.gov (United States)

    Tsang, Raymond S W; Ahmad, Tauqeer; Tyler, Shaun; Lefebvre, Brigitte; Deeks, Shelley L; Gilca, Rodica; Hoang, Linda; Tyrrell, Gregory; Van Caeseele, Paul; Van Domselaar, Gary; Jamieson, Frances B

    2018-04-01

    This study was performed to analyze the Canadian invasive serogroup W Neisseria meningitidis (MenW) sequence type 11 (ST-11) clonal complex (CC) isolates by whole genome typing and to compare Canadian isolates with similar isolates from elsewhere. Whole genome typing of 30 MenW ST-11 CC, 20 meningococcal group C (MenC) ST-11 CC, and 31 MenW ST-22 CC isolates was performed on the Bacterial Isolate Genome Sequence database platform. Canadian MenW ST-11 CC isolates were compared with the 2000 MenW Hajj outbreak strain, as well as with MenW ST-11 CC from other countries. Whole genome typing showed that the Canadian MenW ST-11 CC isolates were distinct from the traditional MenW ST-22 CC; they were not capsule-switched contemporary MenC strains that incorporated MenW capsules. While some recent MenW disease cases in Canada were caused by MenW ST-11 CC isolates showing relatedness to the 2000 MenW Hajj strain, many were non-Hajj isolates similar to current MenW ST-11 isolates found globally. Geographical and temporal variations in genotypes and surface protein antigen genes were found among the MenW ST-11 CC isolates. The current MenW ST-11 isolates did not arise by capsule switching from contemporary MenC ST-11 isolates. Both the Hajj-related and non-Hajj MenW ST-11 CC strains were associated with invasive meningococcal disease in Canada. Copyright © 2018 The Authors. Published by Elsevier Ltd.. All rights reserved.

  10. Whole genome typing of the recently emerged Canadian serogroup W Neisseria meningitidis sequence type 11 clonal complex isolates associated with invasive meningococcal disease

    Directory of Open Access Journals (Sweden)

    Raymond S.W. Tsang

    2018-04-01

    Full Text Available Objectives: This study was performed to analyze the Canadian invasive serogroup W Neisseria meningitidis (MenW sequence type 11 (ST-11 clonal complex (CC isolates by whole genome typing and to compare Canadian isolates with similar isolates from elsewhere. Methods: Whole genome typing of 30 MenW ST-11 CC, 20 meningococcal group C (MenC ST-11 CC, and 31 MenW ST-22 CC isolates was performed on the Bacterial Isolate Genome Sequence database platform. Canadian MenW ST-11 CC isolates were compared with the 2000 MenW Hajj outbreak strain, as well as with MenW ST-11 CC from other countries. Results: Whole genome typing showed that the Canadian MenW ST-11 CC isolates were distinct from the traditional MenW ST-22 CC; they were not capsule-switched contemporary MenC strains that incorporated MenW capsules. While some recent MenW disease cases in Canada were caused by MenW ST-11 CC isolates showing relatedness to the 2000 MenW Hajj strain, many were non-Hajj isolates similar to current MenW ST-11 isolates found globally. Geographical and temporal variations in genotypes and surface protein antigen genes were found among the MenW ST-11 CC isolates. Conclusions: The current MenW ST-11 isolates did not arise by capsule switching from contemporary MenC ST-11 isolates. Both the Hajj-related and non-Hajj MenW ST-11 CC strains were associated with invasive meningococcal disease in Canada. Keywords: Neisseria meningitidis, Invasive meningococcal disease, Whole genome typing

  11. Comparative genomic assessment of Multi-Locus Sequence Typing: rapid accumulation of genomic heterogeneity among clonal isolates of Campylobacter jejuni

    Directory of Open Access Journals (Sweden)

    Nash John HE

    2008-08-01

    Full Text Available Abstract Background Multi-Locus Sequence Typing (MLST has emerged as a leading molecular typing method owing to its high ability to discriminate among bacterial isolates, the relative ease with which data acquisition and analysis can be standardized, and the high portability of the resulting sequence data. While MLST has been successfully applied to the study of the population structure for a number of different bacterial species, it has also provided compelling evidence for high rates of recombination in some species. We have analyzed a set of Campylobacter jejuni strains using MLST and Comparative Genomic Hybridization (CGH on a full-genome microarray in order to determine whether recombination and high levels of genomic mosaicism adversely affect the inference of strain relationships based on the analysis of a restricted number of genetic loci. Results Our results indicate that, in general, there is significant concordance between strain relationships established by MLST and those based on shared gene content as established by CGH. While MLST has significant predictive power with respect to overall genome similarity of isolates, we also found evidence for significant differences in genomic content among strains that would otherwise appear to be highly related based on their MLST profiles. Conclusion The extensive genomic mosaicism between closely related strains has important implications in the context of establishing strain to strain relationships because it suggests that the exact gene content of strains, and by extension their phenotype, is less likely to be "predicted" based on a small number of typing loci. This in turn suggests that a greater emphasis should be placed on analyzing genes of clinical interest as we forge ahead with the next generation of molecular typing methods.

  12. Sequence Quality Analysis Tool for HIV Type 1 Protease and Reverse Transcriptase

    OpenAIRE

    DeLong, Allison K.; Wu, Mingham; Bennett, Diane; Parkin, Neil; Wu, Zhijin; Hogan, Joseph W.; Kantor, Rami

    2012-01-01

    Access to antiretroviral therapy is increasing globally and drug resistance evolution is anticipated. Currently, protease (PR) and reverse transcriptase (RT) sequence generation is increasing, including the use of in-house sequencing assays, and quality assessment prior to sequence analysis is essential. We created a computational HIV PR/RT Sequence Quality Analysis Tool (SQUAT) that runs in the R statistical environment. Sequence quality thresholds are calculated from a large dataset (46,802...

  13. Increased sequence diversity coverage improves detection of HIV-Specific T cell responses

    DEFF Research Database (Denmark)

    Frahm, N.; Kaufmann, D.E.; Yusim, K.

    2007-01-01

    The accurate identification of HIV-specific T cell responses is important for determining the relationship between immune response, viral control, and disease progression. HIV-specific immune responses are usually measured using peptide sets based on consensus sequences, which frequently miss res...

  14. Phylogeny and resistance profiles of HIV-1 POL sequences from rectal biopsies and blood

    DEFF Research Database (Denmark)

    Katzenstein, Terese Lea; Petersen, A B; Storgaard, M

    2010-01-01

    The phylogeny and resistance profiles of human immunodeficiency virus type 1 (HIV-1) protease (PR) and reverse transcriptase (RT) sequences were compared among six patients with HIV-1 who had received numerous treatments. RNA and DNA fractions were obtained from concurrent blood and rectal biopsy...

  15. Use of four next-generation sequencing platforms to determine HIV-1 coreceptor tropism.

    Science.gov (United States)

    Archer, John; Weber, Jan; Henry, Kenneth; Winner, Dane; Gibson, Richard; Lee, Lawrence; Paxinos, Ellen; Arts, Eric J; Robertson, David L; Mimms, Larry; Quiñones-Mateu, Miguel E

    2012-01-01

    HIV-1 coreceptor tropism assays are required to rule out the presence of CXCR4-tropic (non-R5) viruses prior treatment with CCR5 antagonists. Phenotypic (e.g., Trofile™, Monogram Biosciences) and genotypic (e.g., population sequencing linked to bioinformatic algorithms) assays are the most widely used. Although several next-generation sequencing (NGS) platforms are available, to date all published deep sequencing HIV-1 tropism studies have used the 454™ Life Sciences/Roche platform. In this study, HIV-1 co-receptor usage was predicted for twelve patients scheduled to start a maraviroc-based antiretroviral regimen. The V3 region of the HIV-1 env gene was sequenced using four NGS platforms: 454™, PacBio® RS (Pacific Biosciences), Illumina®, and Ion Torrent™ (Life Technologies). Cross-platform variation was evaluated, including number of reads, read length and error rates. HIV-1 tropism was inferred using Geno2Pheno, Web PSSM, and the 11/24/25 rule and compared with Trofile™ and virologic response to antiretroviral therapy. Error rates related to insertions/deletions (indels) and nucleotide substitutions introduced by the four NGS platforms were low compared to the actual HIV-1 sequence variation. Each platform detected all major virus variants within the HIV-1 population with similar frequencies. Identification of non-R5 viruses was comparable among the four platforms, with minor differences attributable to the algorithms used to infer HIV-1 tropism. All NGS platforms showed similar concordance with virologic response to the maraviroc-based regimen (75% to 80% range depending on the algorithm used), compared to Trofile (80%) and population sequencing (70%). In conclusion, all four NGS platforms were able to detect minority non-R5 variants at comparable levels suggesting that any NGS-based method can be used to predict HIV-1 coreceptor usage.

  16. Use of four next-generation sequencing platforms to determine HIV-1 coreceptor tropism.

    Directory of Open Access Journals (Sweden)

    John Archer

    Full Text Available HIV-1 coreceptor tropism assays are required to rule out the presence of CXCR4-tropic (non-R5 viruses prior treatment with CCR5 antagonists. Phenotypic (e.g., Trofile™, Monogram Biosciences and genotypic (e.g., population sequencing linked to bioinformatic algorithms assays are the most widely used. Although several next-generation sequencing (NGS platforms are available, to date all published deep sequencing HIV-1 tropism studies have used the 454™ Life Sciences/Roche platform. In this study, HIV-1 co-receptor usage was predicted for twelve patients scheduled to start a maraviroc-based antiretroviral regimen. The V3 region of the HIV-1 env gene was sequenced using four NGS platforms: 454™, PacBio® RS (Pacific Biosciences, Illumina®, and Ion Torrent™ (Life Technologies. Cross-platform variation was evaluated, including number of reads, read length and error rates. HIV-1 tropism was inferred using Geno2Pheno, Web PSSM, and the 11/24/25 rule and compared with Trofile™ and virologic response to antiretroviral therapy. Error rates related to insertions/deletions (indels and nucleotide substitutions introduced by the four NGS platforms were low compared to the actual HIV-1 sequence variation. Each platform detected all major virus variants within the HIV-1 population with similar frequencies. Identification of non-R5 viruses was comparable among the four platforms, with minor differences attributable to the algorithms used to infer HIV-1 tropism. All NGS platforms showed similar concordance with virologic response to the maraviroc-based regimen (75% to 80% range depending on the algorithm used, compared to Trofile (80% and population sequencing (70%. In conclusion, all four NGS platforms were able to detect minority non-R5 variants at comparable levels suggesting that any NGS-based method can be used to predict HIV-1 coreceptor usage.

  17. Deep sequencing of the vaginal microbiota of women with HIV

    NARCIS (Netherlands)

    R.B.S. Hummelen (Ruben); A.D. Fernandes (Andrew); J.M. Macklaim (Jean); R.J. Dickson (Russell); J. Changalucha (John); G.B. Gloor (Gregory); G.K. Reid (Gregor)

    2010-01-01

    textabstractBackground:Women living with HIV and co-infected with bacterial vaginosis (BV) are at higher risk for transmitting HIV to a partner or newborn. It is poorly understood which bacterial communities constitute BV or the normal vaginal microbiota among this population and how the microbiota

  18. HIV-1 envelope sequence-based diversity measures for identifying recent infections.

    Directory of Open Access Journals (Sweden)

    Alexis Kafando

    Full Text Available Identifying recent HIV-1 infections is crucial for monitoring HIV-1 incidence and optimizing public health prevention efforts. To identify recent HIV-1 infections, we evaluated and compared the performance of 4 sequence-based diversity measures including percent diversity, percent complexity, Shannon entropy and number of haplotypes targeting 13 genetic segments within the env gene of HIV-1. A total of 597 diagnostic samples obtained in 2013 and 2015 from recently and chronically HIV-1 infected individuals were selected. From the selected samples, 249 (134 from recent versus 115 from chronic infections env coding regions, including V1-C5 of gp120 and the gp41 ectodomain of HIV-1, were successfully amplified and sequenced by next generation sequencing (NGS using the Illumina MiSeq platform. The ability of the four sequence-based diversity measures to correctly identify recent HIV infections was evaluated using the frequency distribution curves, median and interquartile range and area under the curve (AUC of the receiver operating characteristic (ROC. Comparing the median and interquartile range and evaluating the frequency distribution curves associated with the 4 sequence-based diversity measures, we observed that the percent diversity, number of haplotypes and Shannon entropy demonstrated significant potential to discriminate recent from chronic infections (p<0.0001. Using the AUC of ROC analysis, only the Shannon entropy measure within three HIV-1 env segments could accurately identify recent infections at a satisfactory level. The env segments were gp120 C2_1 (AUC = 0.806, gp120 C2_3 (AUC = 0.805 and gp120 V3 (AUC = 0.812. Our results clearly indicate that the Shannon entropy measure represents a useful tool for predicting HIV-1 infection recency.

  19. Risk of Breast Cancer with CXCR4-using HIV Defined by V3-Loop Sequencing

    Science.gov (United States)

    Goedert, James J.; Swenson, Luke C.; Napolitano, Laura A.; Haddad, Mojgan; Anastos, Kathryn; Minkoff, Howard; Young, Mary; Levine, Alexandra; Adeyemi, Oluwatoyin; Seaberg, Eric C.; Aouizerat, Bradley; Rabkin, Charles S.; Harrigan, P. Richard; Hessol, Nancy A.

    2014-01-01

    Objective Evaluate the risk of female breast cancer associated with HIV-CXCR4 (X4) tropism as determined by various genotypic measures. Methods A breast cancer case-control study, with pairwise comparisons of tropism determination methods, was conducted. From the Women's Interagency HIV Study repository, one stored plasma specimen was selected from 25 HIV-infected cases near the breast cancer diagnosis date and 75 HIV-infected control women matched for age and calendar date. HIVgp120-V3 sequences were derived by Sanger population sequencing (PS) and 454-pyro deep sequencing (DS). Sequencing-based HIV-X4 tropism was defined using the geno2pheno algorithm, with both high-stringency DS [False-Positive-Rate (FPR 3.5) and 2% X4 cutoff], and lower stringency DS (FPR 5.75, 15% X4 cut-off). Concordance of tropism results by PS, DS, and previously performed phenotyping was assessed with kappa (κ) statistics. Case-control comparisons used exact P-values and conditional logistic regression. Results In 74 women (19 cases, 55 controls) with complete results, prevalence of HIV-X4 by PS was 5% in cases vs 29% in controls (P=0.06, odds ratio 0.14, confidence interval 0.003-1.03). Smaller case-control prevalence differences were found with high-stringency DS (21% vs 36%, P=0.32), lower-stringency DS (16% vs 35%, P=0.18), and phenotyping (11% vs 31%, P=0.10). HIV-X4-tropism concordance was best between PS and lower-stringency DS (93%, κ=0.83). Other pairwise concordances were 82%-92% (κ=0.56-0.81). Concordance was similar among cases and controls. Conclusions HIV-X4 defined by population sequencing (PS) had good agreement with lower stringency deep sequencing and was significantly associated with lower odds of breast cancer. PMID:25321183

  20. New findings on the d(TGGGAG) sequence: Surprising anti-HIV-1 activity.

    Science.gov (United States)

    Romanucci, Valeria; Zarrelli, Armando; Liekens, Sandra; Noppen, Sam; Pannecouque, Christophe; Di Fabio, Giovanni

    2018-02-10

    The biological relevance of tetramolecular G-quadruplexes especially as anti-HIV agents has been extensively reported in the literature over the last years. In the light of our recent results regarding the slow G-quadruplex folding kinetics of ODNs based on d(TGGGAG) sequence, here we report a systematic anti-HIV screening to investigate the impact of the G-quadruplex folding on their anti-HIV activity. In particular, varying the single stranded concentrations of ODNs, it has been tested a pool of ODN sample solutions with different G-quadruplex concentrations. The anti-HIV assays have been designed favouring the limited kinetics involved in the tetramolecular G4-association based on the d(TGGGAG) sequence. Aiming to determine the stoichiometry of G-quadruplex structures in the same experimental conditions of the anti-HIV assays, a native gel electrophoresis was performed. The gel confirmed the G-quadruplex formation for almost all sample solutions while showing the formation of high order G4 structures for the more concentrated ODNs solutions. The most significant result is the discovery of a potent anti-HIV activity of the G-quadruplex formed by the natural d(TGGGAG) sequence (IC 50  = 14 nM) that, until now, has been reported to be completely inactive against HIV infection. Copyright © 2018 Elsevier Masson SAS. All rights reserved.

  1. Cellular specificity of HIV-1 replication can be controlled by LTR sequences

    International Nuclear Information System (INIS)

    Reed-Inderbitzin, Edward; Maury, Wendy

    2003-01-01

    Two well-established determinants of retroviral tropism are envelope sequences that regulate entry and LTR sequences that can regulate viral expression in a cell-specific manner. Studies with human immunodeficiency virus-1 (HIV-1) have demonstrated that tropism of this virus maps primarily to variable envelope sequences. Studies have demonstrated that T cell and macrophage-specific transcription factor binding motifs exist in the upstream region of the LTR U3; however, the ability of the core enhancer/promoter proximal elements (two NF-κB and three Sp1 sites) to function well in macrophages and T cells have led many to conclude that HIV LTR sequences are not primary determinants of HIV tropism. To determine if cellular specificity could be imparted to HIV by the core enhancer elements, the enhancer/promoter proximal region of the HIV LTR was substituted with motifs that control gene expression in a myeloid-specific manner. The enhancer region from equine infectious anemia virus (EIAV) when substituted for the HIV enhancer/promoter proximal region was found to drive expression in a macrophage-specific manner and was responsive to HIV Tat. The addition of a 5' methylation-dependent binding site (MDBP) and a promoter proximal Sp1 motif increased expression without altering cellular specificity. Spacing between the promoter proximal region and the TATA box was also found to influence LTR activity. Infectivity studies using chimeric LTRs within the context of a dual-tropic infectious molecular clone established that these LTRs directed HIV replication and production of infectious virions in macrophages but not primary T cells or T cell lines. This investigation demonstrates that cellular specificity can be imparted onto HIV-1 replication at the level of viral transcription and not entry

  2. Intragenic HIV-1 env sequences that enhance gag expression

    International Nuclear Information System (INIS)

    Suptawiwat, Ornpreya; Sutthent, Ruengpung; Lee, T.-H.; Auewarakul, Prasert

    2003-01-01

    Expression of HIV-1 genes is regulated at multiple levels including the complex RNA splicing and transport mechanisms. Multiple cis-acting elements involved in these regulations have been previously identified in various regions of HIV-1 genome. Here we show that another cis-acting element was present in HIV-1 env region. This element enhanced the expression of Gag when inserted together with Rev response element (RRE) into a truncated HIV-1 genome in the presence of Rev. The enhancing activity was mapped to a 263-bp fragment in the gp41 region downstream to RRE. RNA analysis showed that it might function by promoting RNA stability and Rev-dependent RNA export. The enhancement was specific to Rev-dependent expression, since it did not enhance Gag expression driven by Sam68, a cellular protein that has been shown to be able to substitute for Rev in RNA export function

  3. Sequence quality analysis tool for HIV type 1 protease and reverse transcriptase.

    Science.gov (United States)

    Delong, Allison K; Wu, Mingham; Bennett, Diane; Parkin, Neil; Wu, Zhijin; Hogan, Joseph W; Kantor, Rami

    2012-08-01

    Access to antiretroviral therapy is increasing globally and drug resistance evolution is anticipated. Currently, protease (PR) and reverse transcriptase (RT) sequence generation is increasing, including the use of in-house sequencing assays, and quality assessment prior to sequence analysis is essential. We created a computational HIV PR/RT Sequence Quality Analysis Tool (SQUAT) that runs in the R statistical environment. Sequence quality thresholds are calculated from a large dataset (46,802 PR and 44,432 RT sequences) from the published literature ( http://hivdb.Stanford.edu ). Nucleic acid sequences are read into SQUAT, identified, aligned, and translated. Nucleic acid sequences are flagged if with >five 1-2-base insertions; >one 3-base insertion; >one deletion; >six PR or >18 RT ambiguous bases; >three consecutive PR or >four RT nucleic acid mutations; >zero stop codons; >three PR or >six RT ambiguous amino acids; >three consecutive PR or >four RT amino acid mutations; >zero unique amino acids; or 15% genetic distance from another submitted sequence. Thresholds are user modifiable. SQUAT output includes a summary report with detailed comments for troubleshooting of flagged sequences, histograms of pairwise genetic distances, neighbor joining phylogenetic trees, and aligned nucleic and amino acid sequences. SQUAT is a stand-alone, free, web-independent tool to ensure use of high-quality HIV PR/RT sequences in interpretation and reporting of drug resistance, while increasing awareness and expertise and facilitating troubleshooting of potentially problematic sequences.

  4. Detection of viral sequence fragments of HIV-1 subfamilies yet unknown

    Directory of Open Access Journals (Sweden)

    Stanke Mario

    2011-04-01

    Full Text Available Abstract Background Methods of determining whether or not any particular HIV-1 sequence stems - completely or in part - from some unknown HIV-1 subtype are important for the design of vaccines and molecular detection systems, as well as for epidemiological monitoring. Nevertheless, a single algorithm only, the Branching Index (BI, has been developed for this task so far. Moving along the genome of a query sequence in a sliding window, the BI computes a ratio quantifying how closely the query sequence clusters with a subtype clade. In its current version, however, the BI does not provide predicted boundaries of unknown fragments. Results We have developed Unknown Subtype Finder (USF, an algorithm based on a probabilistic model, which automatically determines which parts of an input sequence originate from a subtype yet unknown. The underlying model is based on a simple profile hidden Markov model (pHMM for each known subtype and an additional pHMM for an unknown subtype. The emission probabilities of the latter are estimated using the emission frequencies of the known subtypes by means of a (position-wise probabilistic model for the emergence of new subtypes. We have applied USF to SIV and HIV-1 sequences formerly classified as having emerged from an unknown subtype. Moreover, we have evaluated its performance on artificial HIV-1 recombinants and non-recombinant HIV-1 sequences. The results have been compared with the corresponding results of the BI. Conclusions Our results demonstrate that USF is suitable for detecting segments in HIV-1 sequences stemming from yet unknown subtypes. Comparing USF with the BI shows that our algorithm performs as good as the BI or better.

  5. nef gene sequence variation among HIV-1-infected African children

    Czech Academy of Sciences Publication Activity Database

    Chakraborty, R.; Reiniš, Milan; Rostron, T.; Philpott, S.; Dong, T.; D'Agostino, A.; Musoke, R.; de Silva, E.; Stumpf, M.; Weiser, B.; Burger, H.; Rowland-Jones, S.L.

    2006-01-01

    Roč. 7, č. 2 (2006), s. 75-84 ISSN 1464-2662 Grant - others:Fogarty International Center, NIH(US) 3D43TW00915; NIH(US) RO1 AI 42555 Institutional research plan: CEZ:AV0Z50520514 Keywords : HIV-1 nef gene * non-clade B * Kenya Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 2.674, year: 2006

  6. Phylogeny and resistance profiles of HIV-1 POL sequences from rectal biopsies and blood

    DEFF Research Database (Denmark)

    Katzenstein, T L; Petersen, A B; Storgaard, M

    2010-01-01

    The phylogeny and resistance profiles of human immunodeficiency virus type 1 (HIV-1) protease (PR) and reverse transcriptase (RT) sequences were compared among six patients with HIV-1 who had received numerous treatments. RNA and DNA fractions were obtained from concurrent blood and rectal biopsy...... samples. Phylogenetic trees and resistance profiles showed that the rectal mucosa and the peripheral blood mononuclear cells (PBMCs) harbored different HIV-1 strains. The resistance-associated mutations found in each strain corresponded to the treatment history of the patients. The resistance mutations...... acquired during earlier treatment regimens were detected in the sequences obtained from the rectal samples and in the PBMCs in several of the patients. Also, differences in the resistance profiles were observed between anatomical sites and between RNA and DNA fractions. Thus, a single sample probably...

  7. Workup of Human Blood Samples for Deep Sequencing of HIV-1 Genomes

    NARCIS (Netherlands)

    Cornelissen, Marion; Gall, Astrid; van der Kuyl, Antoinette; Wymant, Chris; Blanquart, François; Fraser, Christophe; Berkhout, Ben

    2018-01-01

    We describe a detailed protocol for the manual workup of blood (plasma/serum) samples from individuals infected with the human immunodeficiency virus type 1 (HIV-1) for deep sequence analysis of the viral genome. The study optimizing the assay was performed in the context of the BEEHIVE (Bridging

  8. Evaluation of sequence ambiguities of the HIV-1 pol gene as a method to identify recent HIV-1 infection in transmitted drug resistance surveys.

    Science.gov (United States)

    Andersson, Emmi; Shao, Wei; Bontell, Irene; Cham, Fatim; Cuong, Do Duy; Wondwossen, Amogne; Morris, Lynn; Hunt, Gillian; Sönnerborg, Anders; Bertagnolio, Silvia; Maldarelli, Frank; Jordan, Michael R

    2013-08-01

    Identification of recent HIV infection within populations is a public health priority for accurate estimation of HIV incidence rates and transmitted drug resistance at population level. Determining HIV incidence rates by prospective follow-up of HIV-uninfected individuals is challenging and serological assays have important limitations. HIV diversity within an infected host increases with duration of infection. We explore a simple bioinformatics approach to assess viral diversity by determining the percentage of ambiguous base calls in sequences derived from standard genotyping of HIV-1 protease and reverse transcriptase. Sequences from 691 recently infected (≤1 year) and chronically infected (>1 year) individuals from Sweden, Vietnam and Ethiopia were analyzed for ambiguity. A significant difference (p<0.0001) in the proportion of ambiguous bases was observed between sequences from individuals with recent and chronic infection in both HIV-1 subtype B and non-B infection, consistent with previous studies. In our analysis, a cutoff of <0.47% ambiguous base calls identified recent infection with a sensitivity and specificity of 88.8% and 74.6% respectively. 1,728 protease and reverse transcriptase sequences from 36 surveys of transmitted HIV drug resistance performed following World Health Organization guidance were analyzed for ambiguity. The 0.47% ambiguity cutoff was applied and survey sequences were classified as likely derived from recently or chronically infected individuals. 71% of patients were classified as likely to have been infected within one year of genotyping but results varied considerably amongst surveys. This bioinformatics approach may provide supporting population-level information to identify recent infection but its application is limited by infection with more than one viral variant, decreasing viral diversity in advanced disease and technical aspects of population based sequencing. Standardization of sequencing techniques and base calling

  9. HIVBrainSeqDB: a database of annotated HIV envelope sequences from brain and other anatomical sites

    Directory of Open Access Journals (Sweden)

    O'Connor Niall

    2010-12-01

    Full Text Available Abstract Background The population of HIV replicating within a host consists of independently evolving and interacting sub-populations that can be genetically distinct within anatomical compartments. HIV replicating within the brain causes neurocognitive disorders in up to 20-30% of infected individuals and is a viral sanctuary site for the development of drug resistance. The primary determinant of HIV neurotropism is macrophage tropism, which is primarily determined by the viral envelope (env gene. However, studies of genetic aspects of HIV replicating in the brain are hindered because existing repositories of HIV sequences are not focused on neurotropic virus nor annotated with neurocognitive and neuropathological status. To address this need, we constructed the HIV Brain Sequence Database. Results The HIV Brain Sequence Database is a public database of HIV envelope sequences, directly sequenced from brain and other tissues from the same patients. Sequences are annotated with clinical data including viral load, CD4 count, antiretroviral status, neurocognitive impairment, and neuropathological diagnosis, all curated from the original publication. Tissue source is coded using an anatomical ontology, the Foundational Model of Anatomy, to capture the maximum level of detail available, while maintaining ontological relationships between tissues and their subparts. 44 tissue types are represented within the database, grouped into 4 categories: (i brain, brainstem, and spinal cord; (ii meninges, choroid plexus, and CSF; (iii blood and lymphoid; and (iv other (bone marrow, colon, lung, liver, etc. Patient coding is correlated across studies, allowing sequences from the same patient to be grouped to increase statistical power. Using Cytoscape, we visualized relationships between studies, patients and sequences, illustrating interconnections between studies and the varying depth of sequencing, patient number, and tissue representation across studies

  10. Molecular epidemiological analysis of paired pol/env sequences from Portuguese HIV type 1 patients.

    Science.gov (United States)

    Abecasis, Ana B; Martins, Andreia; Costa, Inês; Carvalho, Ana P; Diogo, Isabel; Gomes, Perpétua; Camacho, Ricardo J

    2011-07-01

    The advent of new therapeutic approaches targeting env and the search for efficient anti-HIV-1 vaccines make it necessary to identify the number of recombinant forms using genomic regions that were previously not frequently sequenced. In this study, we have subtyped paired pol and env sequences from HIV-1 strains infecting 152 patients being clinically followed in Portugal. The percentage of strains in which we found discordant subtypes in pol and env was 25.7%. When the subtype in pol and env was concordant (65.1%), the most prevalent subtypes were subtype B (40.8%), followed by subtype C (17.8%) and subtype G (5.3%). The most prevalent recombinant form was CRF14_BGpol/Genv (7.2%).

  11. Use of Four Next-Generation Sequencing Platforms to Determine HIV-1 Coreceptor Tropism

    Czech Academy of Sciences Publication Activity Database

    Archer, J.; Weber, Jan; Henry, K.; Winner, D.; Gibson, R.; Lee, L.; Paxinos, E.; Arts, E. J.; Robertson, D. L.; Mimms, L.; Quinones-Mateu, M. E.

    2012-01-01

    Roč. 7, č. 11 (2012), e49602/1-e49602/17 E-ISSN 1932-6203 R&D Projects: GA MŠk(CZ) LK11207 Institutional research plan: CEZ:AV0Z40550506 Keywords : HIV-1 tropism * V3 region * deep sequencing Subject RIV: EE - Microbiology, Virology Impact factor: 3.730, year: 2012 http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0049602

  12. Genotypic Resistance Tests Sequences Reveal the Role of Marginalized Populations in HIV-1 Transmission in Switzerland.

    Science.gov (United States)

    Shilaih, Mohaned; Marzel, Alex; Yang, Wan Lin; Scherrer, Alexandra U; Schüpbach, Jörg; Böni, Jürg; Yerly, Sabine; Hirsch, Hans H; Aubert, Vincent; Cavassini, Matthias; Klimkait, Thomas; Vernazza, Pietro L; Bernasconi, Enos; Furrer, Hansjakob; Günthard, Huldrych F; Kouyos, Roger

    2016-06-14

    Targeting hard-to-reach/marginalized populations is essential for preventing HIV-transmission. A unique opportunity to identify such populations in Switzerland is provided by a database of all genotypic-resistance-tests from Switzerland, including both sequences from the Swiss HIV Cohort Study (SHCS) and non-cohort sequences. A phylogenetic tree was built using 11,127 SHCS and 2,875 Swiss non-SHCS sequences. Demographics were imputed for non-SHCS patients using a phylogenetic proximity approach. Factors associated with non-cohort outbreaks were determined using logistic regression. Non-B subtype (univariable odds-ratio (OR): 1.9; 95% confidence interval (CI): 1.8-2.1), female gender (OR: 1.6; 95% CI: 1.4-1.7), black ethnicity (OR: 1.9; 95% CI: 1.7-2.1) and heterosexual transmission group (OR:1.8; 95% CI: 1.6-2.0), were all associated with underrepresentation in the SHCS. We found 344 purely non-SHCS transmission clusters, however, these outbreaks were small (median 2, maximum 7 patients) with a strong overlap with the SHCS'. 65% of non-SHCS sequences were part of clusters composed of >= 50% SHCS sequences. Our data suggests that marginalized-populations are underrepresented in the SHCS. However, the limited size of outbreaks among non-SHCS patients in-care implies that no major HIV outbreak in Switzerland was missed by the SHCS surveillance. This study demonstrates the potential of sequence data to assess and extend the scope of infectious-disease surveillance.

  13. Prediction of HIV-1 coreceptor usage (tropism) by sequence analysis using a genotypic approach.

    Science.gov (United States)

    Sierra, Saleta; Kaiser, Rolf; Lübke, Nadine; Thielen, Alexander; Schuelter, Eugen; Heger, Eva; Däumer, Martin; Reuter, Stefan; Esser, Stefan; Fätkenheuer, Gerd; Pfister, Herbert; Oette, Mark; Lengauer, Thomas

    2011-12-01

    Maraviroc (MVC) is the first licensed antiretroviral drug from the class of coreceptor antagonists. It binds to the host coreceptor CCR5, which is used by the majority of HIV strains in order to infect the human immune cells (Fig. 1). Other HIV isolates use a different coreceptor, the CXCR4. Which receptor is used, is determined in the virus by the Env protein (Fig. 2). Depending on the coreceptor used, the viruses are classified as R5 or X4, respectively. MVC binds to the CCR5 receptor inhibiting the entry of R5 viruses into the target cell. During the course of disease, X4 viruses may emerge and outgrow the R5 viruses. Determination of coreceptor usage (also called tropism) is therefore mandatory prior to administration of MVC, as demanded by EMA and FDA. The studies for MVC efficiency MOTIVATE, MERIT and 1029 have been performed with the Trofile assay from Monogram, San Francisco, U.S.A. This is a high quality assay based on sophisticated recombinant tests. The acceptance for this test for daily routine is rather low outside of the U.S.A., since the European physicians rather tend to work with decentralized expert laboratories, which also provide concomitant resistance testing. These laboratories have undergone several quality assurance evaluations, the last one being presented in 2011. For several years now, we have performed tropism determinations based on sequence analysis from the HIV env-V3 gene region (V3). This region carries enough information to perform a reliable prediction. The genotypic determination of coreceptor usage presents advantages such as: shorter turnover time (equivalent to resistance testing), lower costs, possibility to adapt the results to the patients' needs and possibility of analysing clinical samples with very low or even undetectable viral load (VL), particularly since the number of samples analysed with VL < 1000 copies/μl roughly increased in the last years (Fig. 3). The main steps for tropism testing (Fig. 4) demonstrated in

  14. Populations in clonal plants

    Directory of Open Access Journals (Sweden)

    Jussi Tammisola

    1986-12-01

    Full Text Available Population phenomena in higher plants are reviewed critically, particularly in relation to clonality. An array of concepts used in the field are discussed. In contrast to animals, higher plants are modular in structure. Plant populations show hierarchy at two levels: ramets and genets. In addition, their demography is far more complicated, since even the direction of development of a ramet may change by rejuvenation. Therefore, formulae concerning animal populations often require modification for plants. Furthermore, at the zygotic stage, higher plants are generally less mobile than animals. Accordingly, their population processes tend to be more local. Most populations of plants have a genetic structure: alleles and genotypes are spatially aggregated. Due to the short-ranged foraging behaviour of pollinators, genetically non-random pollination prevails. A generalized formula for parent-offspring dispersal variance is derived. It is used to analyze the effect of clonality on genetic patchiness in populations. In self-compatible species, an increase in clonality will tend to increase the degree of patchiness, while in self-incompatible species a decrease may result. Examples of population structure studies in different species are presented. A considerable degree of genetic variation appears to be found also in the populations of species with a strong allocation of resources to clonal growth or apomictic seed production. Some consequences of clonality are considered from the point of view of genetic conservation and plant breeding.

  15. Repair of oxidative DNA base damage in the host genome influences the HIV integration site sequence preference.

    Directory of Open Access Journals (Sweden)

    Geoffrey R Bennett

    Full Text Available Host base excision repair (BER proteins that repair oxidative damage enhance HIV infection. These proteins include the oxidative DNA damage glycosylases 8-oxo-guanine DNA glycosylase (OGG1 and mutY homolog (MYH as well as DNA polymerase beta (Polβ. While deletion of oxidative BER genes leads to decreased HIV infection and integration efficiency, the mechanism remains unknown. One hypothesis is that BER proteins repair the DNA gapped integration intermediate. An alternative hypothesis considers that the most common oxidative DNA base damages occur on guanines. The subtle consensus sequence preference at HIV integration sites includes multiple G:C base pairs surrounding the points of joining. These observations suggest a role for oxidative BER during integration targeting at the nucleotide level. We examined the hypothesis that BER repairs a gapped integration intermediate by measuring HIV infection efficiency in Polβ null cell lines complemented with active site point mutants of Polβ. A DNA synthesis defective mutant, but not a 5'dRP lyase mutant, rescued HIV infection efficiency to wild type levels; this suggested Polβ DNA synthesis activity is not necessary while 5'dRP lyase activity is required for efficient HIV infection. An alternate hypothesis that BER events in the host genome influence HIV integration site selection was examined by sequencing integration sites in OGG1 and MYH null cells. In the absence of these 8-oxo-guanine specific glycosylases the chromatin elements of HIV integration site selection remain the same as in wild type cells. However, the HIV integration site sequence preference at G:C base pairs is altered at several positions in OGG1 and MYH null cells. Inefficient HIV infection in the absence of oxidative BER proteins does not appear related to repair of the gapped integration intermediate; instead oxidative damage repair may participate in HIV integration site preference at the sequence level.

  16. MLST-Based Population Genetic Analysis in a Global Context Reveals Clonality amongst Cryptococcus neoformans var. grubii VNI Isolates from HIV Patients in Southeastern Brazil

    NARCIS (Netherlands)

    Ferreira-Paim, Kennio; Andrade-Silva, Leonardo; Fonseca, Fernanda; Ferreira, Thatiana; Mora, Delio; Andrade-Silva, Juliana; Khan, Aziza; Dao, Aiken; C. Reis, Eduardo; Almeida, Margarete; Maltos, André; Rodrigues Jr, Virmondes; Trilles, Luciana; Rickerts, Volker; Chindamporn, Ariya; Sykes, Jane; Cogliati, Massimo; Nielsen, Kirsten; Boekhout, Teun; L. Silva-Vergara, Mario

    2017-01-01

    Cryptococcosis is an important fungal infection in immunocompromised individuals, especially those infected with HIV. In Brazil, despite the free availability of antiretroviral therapy (ART) in the public health system, the mortality rate due to Cryptococcus neoformans meningitis is still high. To

  17. Identification of HIV Mutation as Diagnostic Biomarker through Next Generation Sequencing.

    Science.gov (United States)

    Shaw, Wen Hui; Lin, Qianqian; Muhammad, Zikry Zhiwei Bin Roslee; Lee, Jia Jun; Khong, Wei Xin; Ng, Oon Tek; Tan, Eng Lee; Li, Peng

    2016-07-01

    Current clinical detection of Human immunodeficiency virus 1 (HIV-1) is used to target viral genes and proteins. However, the immunoassay, such as viral culture or Polymerase Chain Reaction (PCR), lacks accuracy in the diagnosis, as these conventional assays rely on the stable genome and HIV-1 is a highly-mutated virus. Next generation sequencing (NGS) promises to be transformative for the practice of infectious disease, and the rapidly reducing cost and processing time mean that this will become a feasible technology in diagnostic and research laboratories in the near future. The technology offers the superior sensitivity to detect the pathogenic viruses, including unknown and unexpected strains. To leverage the NGS technology in order to improve current HIV-1 diagnosis and genotyping methods. Ten blood samples were collected from HIV-1 infected patients which were diagnosed by RT PCR at Singapore Communicable Disease Centre, Tan Tock Seng Hospital from October 2014 to March 2015. Viral RNAs were extracted from blood plasma and reversed into cDNA. The HIV-1 cDNA samples were cleaned up using a PCR purification kit and the sequencing library was prepared and identified through MiSeq. Two common mutations were observed in all ten samples. The common mutations were identified at genome locations 1908 and 2104 as missense and silent mutations respectively, conferring S37N and S3S found on aspartic protease and reverse transcriptase subunits. The common mutations identified in this study were not previously reported, therefore suggesting the potential for them to be used for identification of viral infection, disease transmission and drug resistance. This was especially the case for, missense mutation S37N which could cause an amino acid change in viral proteases thus reducing the binding affinity of some protease inhibitors. Thus, the unique common mutations identified in this study could be used as diagnostic biomarkers to indicate the origin of infection as being

  18. Eliminating HIV-1 Packaging Sequences from Lentiviral Vector Proviruses Enhances Safety and Expedites Gene Transfer for Gene Therapy.

    Science.gov (United States)

    Vink, Conrad A; Counsell, John R; Perocheau, Dany P; Karda, Rajvinder; Buckley, Suzanne M K; Brugman, Martijn H; Galla, Melanie; Schambach, Axel; McKay, Tristan R; Waddington, Simon N; Howe, Steven J

    2017-08-02

    Lentiviral vector genomic RNA requires sequences that partially overlap wild-type HIV-1 gag and env genes for packaging into vector particles. These HIV-1 packaging sequences constitute 19.6% of the wild-type HIV-1 genome and contain functional cis elements that potentially compromise clinical safety. Here, we describe the development of a novel lentiviral vector (LTR1) with a unique genomic structure designed to prevent transfer of HIV-1 packaging sequences to patient cells, thus reducing the total HIV-1 content to just 4.8% of the wild-type genome. This has been achieved by reconfiguring the vector to mediate reverse-transcription with a single strand transfer, instead of the usual two, and in which HIV-1 packaging sequences are not copied. We show that LTR1 vectors offer improved safety in their resistance to remobilization in HIV-1 particles and reduced frequency of splicing into human genes. Following intravenous luciferase vector administration to neonatal mice, LTR1 sustained a higher level of liver transgene expression than an equivalent dose of a standard lentivirus. LTR1 vectors produce reverse-transcription products earlier and start to express transgenes significantly quicker than standard lentiviruses after transduction. Finally, we show that LTR1 is an effective lentiviral gene therapy vector as demonstrated by correction of a mouse hemophilia B model. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

  19. Analysis of HIV-1 intersubtype recombination breakpoints suggests region with high pairing probability may be a more fundamental factor than sequence similarity affecting HIV-1 recombination.

    Science.gov (United States)

    Jia, Lei; Li, Lin; Gui, Tao; Liu, Siyang; Li, Hanping; Han, Jingwan; Guo, Wei; Liu, Yongjian; Li, Jingyun

    2016-09-21

    With increasing data on HIV-1, a more relevant molecular model describing mechanism details of HIV-1 genetic recombination usually requires upgrades. Currently an incomplete structural understanding of the copy choice mechanism along with several other issues in the field that lack elucidation led us to perform an analysis of the correlation between breakpoint distributions and (1) the probability of base pairing, and (2) intersubtype genetic similarity to further explore structural mechanisms. Near full length sequences of URFs from Asia, Europe, and Africa (one sequence/patient), and representative sequences of worldwide CRFs were retrieved from the Los Alamos HIV database. Their recombination patterns were analyzed by jpHMM in detail. Then the relationships between breakpoint distributions and (1) the probability of base pairing, and (2) intersubtype genetic similarities were investigated. Pearson correlation test showed that all URF groups and the CRF group exhibit the same breakpoint distribution pattern. Additionally, the Wilcoxon two-sample test indicated a significant and inexplicable limitation of recombination in regions with high pairing probability. These regions have been found to be strongly conserved across distinct biological states (i.e., strong intersubtype similarity), and genetic similarity has been determined to be a very important factor promoting recombination. Thus, the results revealed an unexpected disagreement between intersubtype similarity and breakpoint distribution, which were further confirmed by genetic similarity analysis. Our analysis reveals a critical conflict between results from natural HIV-1 isolates and those from HIV-1-based assay vectors in which genetic similarity has been shown to be a very critical factor promoting recombination. These results indicate the region with high-pairing probabilities may be a more fundamental factor affecting HIV-1 recombination than sequence similarity in natural HIV-1 infections. Our

  20. Exploration of the effect of sequence variations located inside the binding pocket of HIV-1 and HIV-2 proteases.

    Science.gov (United States)

    Triki, Dhoha; Billot, Telli; Visseaux, Benoit; Descamps, Diane; Flatters, Delphine; Camproux, Anne-Claude; Regad, Leslie

    2018-04-10

    HIV-2 protease (PR2) is naturally resistant to most FDA (Food and Drug Administration)-approved HIV-1 protease inhibitors (PIs), a major antiretroviral class. In this study, we compared the PR1 and PR2 binding pockets extracted from structures complexed with 12 ligands. The comparison of PR1 and PR2 pocket properties showed that bound PR2 pockets were more hydrophobic with more oxygen atoms and fewer nitrogen atoms than PR1 pockets. The structural comparison of PR1 and PR2 pockets highlighted structural changes induced by their sequence variations and that were consistent with these property changes. Specifically, substitutions at residues 31, 46, and 82 induced structural changes in their main-chain atoms that could affect PI binding in PR2. In addition, the modelling of PR1 mutant structures containing V32I and L76M substitutions revealed a cooperative mechanism leading to structural deformation of flap-residue 45 that could modify PR2 flexibility. Our results suggest that substitutions in the PR1 and PR2 pockets can modify PI binding and flap flexibility, which could underlie PR2 resistance against PIs. These results provide new insights concerning the structural changes induced by PR1 and PR2 pocket variation changes, improving the understanding of the atomic mechanism of PR2 resistance to PIs.

  1. Improved therapy-success prediction with GSS estimated from clinical HIV-1 sequences.

    Science.gov (United States)

    Pironti, Alejandro; Pfeifer, Nico; Kaiser, Rolf; Walter, Hauke; Lengauer, Thomas

    2014-01-01

    Rules-based HIV-1 drug-resistance interpretation (DRI) systems disregard many amino-acid positions of the drug's target protein. The aims of this study are (1) the development of a drug-resistance interpretation system that is based on HIV-1 sequences from clinical practice rather than hard-to-get phenotypes, and (2) the assessment of the benefit of taking all available amino-acid positions into account for DRI. A dataset containing 34,934 therapy-naïve and 30,520 drug-exposed HIV-1 pol sequences with treatment history was extracted from the EuResist database and the Los Alamos National Laboratory database. 2,550 therapy-change-episode baseline sequences (TCEB) were assigned to test set A. Test set B contains 1,084 TCEB from the HIVdb TCE repository. Sequences from patients absent in the test sets were used to train three linear support vector machines to produce scores that predict drug exposure pertaining to each of 20 antiretrovirals: the first one uses the full amino-acid sequences (DEfull), the second one only considers IAS drug-resistance positions (DEonlyIAS), and the third one disregards IAS drug-resistance positions (DEnoIAS). For performance comparison, test sets A and B were evaluated with DEfull, DEnoIAS, DEonlyIAS, geno2pheno[resistance], HIVdb, ANRS, HIV-GRADE, and REGA. Clinically-validated cut-offs were used to convert the continuous output of the first four methods into susceptible-intermediate-resistant (SIR) predictions. With each method, a genetic susceptibility score (GSS) was calculated for each therapy episode in each test set by converting the SIR prediction for its compounds to integer: S=2, I=1, and R=0. The GSS were used to predict therapy success as defined by the EuResist standard datum definition. Statistical significance was assessed using a Wilcoxon signed-rank test. A comparison of the therapy-success prediction performances among the different interpretation systems for test set A can be found in Table 1, while those for test set

  2. High level of APOBEC3F/3G editing in HIV-2 DNA vif and pol sequences from antiretroviral-naive patients.

    Science.gov (United States)

    Bertine, Mélanie; Charpentier, Charlotte; Visseaux, Benoit; Storto, Alexandre; Collin, Gilles; Larrouy, Lucile; Damond, Florence; Matheron, Sophie; Brun-Vézinet, Françoise; Descamps, Diane

    2015-04-24

    In HIV-1, hypermutation introduced by APOBEC3F/3G cytidine deaminase activity leads to defective viruses. In-vivo impact of APOBEC3F/3G editing on HIV-2 sequences remains unknown. The objective of this study was to assess the level of APOBEC3F/3G editing in HIV-2-infected antiretroviral-naive patients. Direct sequencing of vif and pol regions was performed on HIV-2 proviral DNA from antiretroviral-naive patients included in the French Agence Nationale de Recherches sur le SIDA et les hépatites virales CO5 HIV-2 cohort. Hypermutated sequences were identified using Hypermut2.0 program. HIV-1 proviral sequences from Genbank were also assessed. Among 82 antiretroviral-naive HIV-2-infected patients assessed, 15 (28.8%) and five (16.7%) displayed Vif proviral defective sequences in HIV-2 groups A and B, respectively. A lower proportion of defective sequences was observed in protease-reverse transcriptase region. A higher median number of G-to-A mutations was observed in HIV-2 group B than in group A, both in Vif and protease-reverse transcriptase regions (P = 0.02 and P = 0.006, respectively). Compared with HIV-1 Vif sequences, a higher number of Vif defective sequences was observed in HIV-2 group A (P = 0.00001) and group B sequences (P = 0.013). We showed for the first time a high level of APOBEC3F/3G editing in HIV-2 sequences from antiretroviral-naive patients. Our study reported a group effect with a significantly higher level of APOBEC3F/3G editing in HIV-2 group B than in group A sequences.

  3. Clonal study of avian Escherichia coli strains by fliC conserved-DNA-sequence regions analysis Estudo clonal de Escherichia coli aviário por análise de seqüências de DNA conservadas do gene fliC

    Directory of Open Access Journals (Sweden)

    Tatiana Amabile de Campos

    2008-10-01

    Full Text Available The clonal relationship among avian Escherichia coli strains and their genetic proximity with human pathogenic E. coli, Salmonela enterica, Yersinia enterocolitica and Proteus mirabilis, was determined by the DNA sequencing of the conserved 5' and 3'regions fliC gene (flagellin encoded gene. Among 30 commensal avian E. coli strains and 49 pathogenic avian E. coli strains (APEC, 24 commensal and 39 APEC strains harbored fliC gene with fragments size varying from 670bp to 1,900bp. The comparative analysis of these regions allowed the construction of a dendrogram of similarity possessing two main clusters: one compounded mainly by APEC strains and by H-antigens from human E. coli, and another one compounded by commensal avian E. coli strains, S. enterica, and by other H-antigens from human E. coli. Overall, this work demonstrated that fliC conserved regions may be associated with pathogenic clones of APEC strains, and also shows a great similarity among APEC and H-antigens of E. coli strains isolated from humans. These data, can add evidence that APEC strains can exhibit a zoonotic risk.A relação clonal entre linhagens de Escherichia coli de origem aviária e sua proximidade genética com E. coli patogênica para humanos, Salmonella enterica, Yersinia enterocolitica e Proteus mirabilis foi determinada através da utilização das seqüências conservadas 5' e 3' do gene fliC (responsável pela codificação da flagelina. Entre as 30 linhagens comensais de E. coli aviária e as 49 linhagens patogênicas de E. coli para aves (APEC, 24 linhagens comensais e 39 APEC apresentaram o gene fliC, que foi encontrado em tamanhos que variam de 670pb a 1900pb. Um dendrograma representando similaridade genética foi obtido a partir do seqüenciamento das regiões 5' e 3' conservadas do gene fliC das linhagens de E. coli de origem aviária, das seqüências dos antígenos H de E. coli de origem humana, de S. enterica, Y. enterocolitica e de P. mirabilis. A an

  4. Short Communication Phylogenetic Characterization of HIV Type 1 CRF01_AE V3 Envelope Sequences in Pregnant Women in Northern Vietnam

    Science.gov (United States)

    Caridha, Rozina; Ha, Tran Thi Thanh; Gaseitsiwe, Simani; Hung, Pham Viet; Anh, Nguyen Mai; Bao, Nguyen Huy; Khang, Dinh Duy; Hien, Nguyen Tran; Cam, Phung Dac; Chiodi, Francesca

    2012-01-01

    Abstract Characterization of HIV-1 strains is important for surveillance of the HIV-1 epidemic. In Vietnam HIV-1-infected pregnant women often fail to receive the care they are entitled to. Here, we analyzed phylogenetically HIV-1 env sequences from 37 HIV-1-infected pregnant women from Ha Noi (n=22) and Hai Phong (n=15), where they delivered in 2005–2007. All carried CRF01_AE in the gp120 V3 region. In 21 women CRF01_AE was also found in the reverse transcriptase gene. We compared their env gp120 V3 sequences phylogenetically in a maximum likelihood tree to those of 198 other CRF01_AE sequences in Vietnam and 229 from neighboring countries, predominantly Thailand, from the HIV-1 database. Altogether 464 sequences were analyzed. All but one of the maternal sequences colocalized with sequences from northern Vietnam. The maternal sequences had evolved the least when compared to sequences collected in Ha Noi in 2002, as shown by analysis of synonymous and nonsynonymous changes, than to other Vietnamese sequences collected earlier and/or elsewhere. Since the HIV-1 epidemic in women in Vietnam may still be underestimated, characterization of HIV-1 in pregnant women is important to observe how HIV-1 has evolved and follow its molecular epidemiology. PMID:21936713

  5. Molecular epidemiological analysis of env and pol sequences in newly diagnosed HIV type 1-infected, untreated patients in Hungary.

    Science.gov (United States)

    Mezei, Mária; Ay, Eva; Koroknai, Anita; Tóth, Renáta; Balázs, Andrea; Bakos, Agnes; Gyori, Zoltán; Bánáti, Ferenc; Marschalkó, Márta; Kárpáti, Sarolta; Minárovits, János

    2011-11-01

    The aim of our study was to monitor the diversity of HIV-1 strains circulating in Hungary and investigate the prevalence of resistance-associated mutations to reverse transcriptase (RT) and protease (PR) inhibitors in newly diagnosed, drug-naive patients. A total of 30 HIV-1-infected patients without prior antiretroviral treatment diagnosed during the period 2008-2010 were included into this study. Viral subtypes and the presence of RT, PR resistance-associated mutations were established by sequencing. Classification of HIV-1 strains showed that 29 (96.6%) patients were infected with subtype B viruses and one patient (3.3%) with subtype A virus. The prevalence of HIV-1 strains with transmitted drug resistance mutations in newly diagnosed individuals was 16.6% (5/30). This study showed that HIV-1 subtype B is still highly predominant in Hungary and documented a relatively high transmission rate of drug resistance in our country.

  6. SubClonal Hierarchy Inference from Somatic Mutations: Automatic Reconstruction of Cancer Evolutionary Trees from Multi-region Next Generation Sequencing.

    Directory of Open Access Journals (Sweden)

    Noushin Niknafs

    2015-10-01

    Full Text Available Recent improvements in next-generation sequencing of tumor samples and the ability to identify somatic mutations at low allelic fractions have opened the way for new approaches to model the evolution of individual cancers. The power and utility of these models is increased when tumor samples from multiple sites are sequenced. Temporal ordering of the samples may provide insight into the etiology of both primary and metastatic lesions and rationalizations for tumor recurrence and therapeutic failures. Additional insights may be provided by temporal ordering of evolving subclones--cellular subpopulations with unique mutational profiles. Current methods for subclone hierarchy inference tightly couple the problem of temporal ordering with that of estimating the fraction of cancer cells harboring each mutation. We present a new framework that includes a rigorous statistical hypothesis test and a collection of tools that make it possible to decouple these problems, which we believe will enable substantial progress in the field of subclone hierarchy inference. The methods presented here can be flexibly combined with methods developed by others addressing either of these problems. We provide tools to interpret hypothesis test results, which inform phylogenetic tree construction, and we introduce the first genetic algorithm designed for this purpose. The utility of our framework is systematically demonstrated in simulations. For most tested combinations of tumor purity, sequencing coverage, and tree complexity, good power (≥ 0.8 can be achieved and Type 1 error is well controlled when at least three tumor samples are available from a patient. Using data from three published multi-region tumor sequencing studies of (murine small cell lung cancer, acute myeloid leukemia, and chronic lymphocytic leukemia, in which the authors reconstructed subclonal phylogenetic trees by manual expert curation, we show how different configurations of our tools can

  7. SubClonal Hierarchy Inference from Somatic Mutations: Automatic Reconstruction of Cancer Evolutionary Trees from Multi-region Next Generation Sequencing.

    Science.gov (United States)

    Niknafs, Noushin; Beleva-Guthrie, Violeta; Naiman, Daniel Q; Karchin, Rachel

    2015-10-01

    Recent improvements in next-generation sequencing of tumor samples and the ability to identify somatic mutations at low allelic fractions have opened the way for new approaches to model the evolution of individual cancers. The power and utility of these models is increased when tumor samples from multiple sites are sequenced. Temporal ordering of the samples may provide insight into the etiology of both primary and metastatic lesions and rationalizations for tumor recurrence and therapeutic failures. Additional insights may be provided by temporal ordering of evolving subclones--cellular subpopulations with unique mutational profiles. Current methods for subclone hierarchy inference tightly couple the problem of temporal ordering with that of estimating the fraction of cancer cells harboring each mutation. We present a new framework that includes a rigorous statistical hypothesis test and a collection of tools that make it possible to decouple these problems, which we believe will enable substantial progress in the field of subclone hierarchy inference. The methods presented here can be flexibly combined with methods developed by others addressing either of these problems. We provide tools to interpret hypothesis test results, which inform phylogenetic tree construction, and we introduce the first genetic algorithm designed for this purpose. The utility of our framework is systematically demonstrated in simulations. For most tested combinations of tumor purity, sequencing coverage, and tree complexity, good power (≥ 0.8) can be achieved and Type 1 error is well controlled when at least three tumor samples are available from a patient. Using data from three published multi-region tumor sequencing studies of (murine) small cell lung cancer, acute myeloid leukemia, and chronic lymphocytic leukemia, in which the authors reconstructed subclonal phylogenetic trees by manual expert curation, we show how different configurations of our tools can identify either a single

  8. Easy and accurate reconstruction of whole HIV genomes from short-read sequence data with shiver

    Science.gov (United States)

    Blanquart, François; Golubchik, Tanya; Gall, Astrid; Bakker, Margreet; Bezemer, Daniela; Croucher, Nicholas J; Hall, Matthew; Hillebregt, Mariska; Ratmann, Oliver; Albert, Jan; Bannert, Norbert; Fellay, Jacques; Fransen, Katrien; Gourlay, Annabelle; Grabowski, M Kate; Gunsenheimer-Bartmeyer, Barbara; Günthard, Huldrych F; Kivelä, Pia; Kouyos, Roger; Laeyendecker, Oliver; Liitsola, Kirsi; Meyer, Laurence; Porter, Kholoud; Ristola, Matti; van Sighem, Ard; Cornelissen, Marion; Kellam, Paul; Reiss, Peter

    2018-01-01

    Abstract Studying the evolution of viruses and their molecular epidemiology relies on accurate viral sequence data, so that small differences between similar viruses can be meaningfully interpreted. Despite its higher throughput and more detailed minority variant data, next-generation sequencing has yet to be widely adopted for HIV. The difficulty of accurately reconstructing the consensus sequence of a quasispecies from reads (short fragments of DNA) in the presence of large between- and within-host diversity, including frequent indels, may have presented a barrier. In particular, mapping (aligning) reads to a reference sequence leads to biased loss of information; this bias can distort epidemiological and evolutionary conclusions. De novo assembly avoids this bias by aligning the reads to themselves, producing a set of sequences called contigs. However contigs provide only a partial summary of the reads, misassembly may result in their having an incorrect structure, and no information is available at parts of the genome where contigs could not be assembled. To address these problems we developed the tool shiver to pre-process reads for quality and contamination, then map them to a reference tailored to the sample using corrected contigs supplemented with the user’s choice of existing reference sequences. Run with two commands per sample, it can easily be used for large heterogeneous data sets. We used shiver to reconstruct the consensus sequence and minority variant information from paired-end short-read whole-genome data produced with the Illumina platform, for sixty-five existing publicly available samples and fifty new samples. We show the systematic superiority of mapping to shiver’s constructed reference compared with mapping the same reads to the closest of 3,249 real references: median values of 13 bases called differently and more accurately, 0 bases called differently and less accurately, and 205 bases of missing sequence recovered. We also

  9. A novel high-resolution multilocus sequence typing of Giardia intestinalis Assemblage A isolates reveals zoonotic transmission, clonal outbreaks and recombination.

    Science.gov (United States)

    Ankarklev, Johan; Lebbad, Marianne; Einarsson, Elin; Franzén, Oscar; Ahola, Harri; Troell, Karin; Svärd, Staffan G

    2018-06-01

    Molecular epidemiology and genotyping studies of the parasitic protozoan Giardia intestinalis have proven difficult due to multiple factors, such as low discriminatory power in the commonly used genotyping loci, which has hampered molecular analyses of outbreak sources, zoonotic transmission and virulence types. Here we have focused on assemblage A Giardia and developed a high-resolution assemblage-specific multilocus sequence typing (MLST) method. Analyses of sequenced G. intestinalis assemblage A genomes from different sub-assemblages identified a set of six genetic loci with high genetic variability. DNA samples from both humans (n = 44) and animals (n = 18) that harbored Giardia assemblage A infections, were PCR amplified (557-700 bp products) and sequenced at the six novel genetic loci. Bioinformatic analyses showed five to ten-fold higher levels of polymorphic sites than what was previously found among assemblage A samples using the classic genotyping loci. Phylogenetically, a division of two major clusters in assemblage A became apparent, separating samples of human and animal origin. A subset of human samples (n = 9) from a documented Giardia outbreak in a Swedish day-care center, showed full complementarity at nine genetic loci (the six new and the standard BG, TPI and GDH loci), strongly suggesting one source of infection. Furthermore, three samples of human origin displayed MLST profiles that were phylogenetically more closely related to MLST profiles from animal derived samples, suggesting zoonotic transmission. These new genotyping loci enabled us to detect events of recombination between different assemblage A isolates but also between assemblage A and E isolates. In summary, we present a novel and expanded MLST strategy with significantly improved sensitivity for molecular analyses of virulence types, zoonotic potential and source tracking for assemblage A Giardia. Copyright © 2018. Published by Elsevier B.V.

  10. High-resolution deep sequencing reveals biodiversity, population structure, and persistence of HIV-1 quasispecies within host ecosystems

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    Yin Li

    2012-12-01

    Full Text Available Abstract Background Deep sequencing provides the basis for analysis of biodiversity of taxonomically similar organisms in an environment. While extensively applied to microbiome studies, population genetics studies of viruses are limited. To define the scope of HIV-1 population biodiversity within infected individuals, a suite of phylogenetic and population genetic algorithms was applied to HIV-1 envelope hypervariable domain 3 (Env V3 within peripheral blood mononuclear cells from a group of perinatally HIV-1 subtype B infected, therapy-naïve children. Results Biodiversity of HIV-1 Env V3 quasispecies ranged from about 70 to 270 unique sequence clusters across individuals. Viral population structure was organized into a limited number of clusters that included the dominant variants combined with multiple clusters of low frequency variants. Next generation viral quasispecies evolved from low frequency variants at earlier time points through multiple non-synonymous changes in lineages within the evolutionary landscape. Minor V3 variants detected as long as four years after infection co-localized in phylogenetic reconstructions with early transmitting viruses or with subsequent plasma virus circulating two years later. Conclusions Deep sequencing defines HIV-1 population complexity and structure, reveals the ebb and flow of dominant and rare viral variants in the host ecosystem, and identifies an evolutionary record of low-frequency cell-associated viral V3 variants that persist for years. Bioinformatics pipeline developed for HIV-1 can be applied for biodiversity studies of virome populations in human, animal, or plant ecosystems.

  11. Whole-genome sequencing of multiple myeloma from diagnosis to plasma cell leukemia reveals genomic initiating events, evolution, and clonal tides.

    Science.gov (United States)

    Egan, Jan B; Shi, Chang-Xin; Tembe, Waibhav; Christoforides, Alexis; Kurdoglu, Ahmet; Sinari, Shripad; Middha, Sumit; Asmann, Yan; Schmidt, Jessica; Braggio, Esteban; Keats, Jonathan J; Fonseca, Rafael; Bergsagel, P Leif; Craig, David W; Carpten, John D; Stewart, A Keith

    2012-08-02

    The longitudinal evolution of a myeloma genome from diagnosis to plasma cell leukemia has not previously been reported. We used whole-genome sequencing (WGS) on 4 purified tumor samples and patient germline DNA drawn over a 5-year period in a t(4;14) multiple myeloma patient. Tumor samples were acquired at diagnosis, first relapse, second relapse, and end-stage secondary plasma cell leukemia (sPCL). In addition to the t(4;14), all tumor time points also shared 10 common single-nucleotide variants (SNVs) on WGS comprising shared initiating events. Interestingly, we observed genomic sequence variants that waxed and waned with time in progressive tumors, suggesting the presence of multiple independent, yet related, clones at diagnosis that rose and fell in dominance. Five newly acquired SNVs, including truncating mutations of RB1 and ZKSCAN3, were observed only in the final sPCL sample suggesting leukemic transformation events. This longitudinal WGS characterization of the natural history of a high-risk myeloma patient demonstrated tumor heterogeneity at diagnosis with shifting dominance of tumor clones over time and has also identified potential mutations contributing to myelomagenesis as well as transformation from myeloma to overt extramedullary disease such as sPCL.

  12. HIV drug resistance testing among patients failing second line antiretroviral therapy. Comparison of in-house and commercial sequencing.

    Science.gov (United States)

    Chimukangara, Benjamin; Varyani, Bhavini; Shamu, Tinei; Mutsvangwa, Junior; Manasa, Justen; White, Elizabeth; Chimbetete, Cleophas; Luethy, Ruedi; Katzenstein, David

    2017-05-01

    HIV genotyping is often unavailable in low and middle-income countries due to infrastructure requirements and cost. We compared genotype resistance testing in patients with virologic failure, by amplification of HIV pol gene, followed by "in-house" sequencing and commercial sequencing. Remnant plasma samples from adults and children failing second-line ART were amplified and sequenced using in-house and commercial di-deoxysequencing, and analyzed in Harare, Zimbabwe and at Stanford, U.S.A, respectively. HIV drug resistance mutations were determined using the Stanford HIV drug resistance database. Twenty-six of 28 samples were amplified and 25 were successfully genotyped. Comparison of average percent nucleotide and amino acid identities between 23 pairs sequenced in both laboratories were 99.51 (±0.56) and 99.11 (±0.95), respectively. All pairs clustered together in phylogenetic analysis. Sequencing analysis identified 6/23 pairs with mutation discordances resulting in differences in phenotype, but these did not impact future regimens. The results demonstrate our ability to produce good quality drug resistance data in-house. Despite discordant mutations in some sequence pairs, the phenotypic predictions were not clinically significant. Copyright © 2016 Elsevier B.V. All rights reserved.

  13. Generation and Characterization of HIV-1 Transmitted and Founder Virus Consensus Sequence from Intravenous Drug Users in Xinjiang, China.

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    Li, Fan; Ma, Liying; Feng, Yi; Hu, Jing; Ni, Na; Ruan, Yuhua; Shao, Yiming

    2017-06-01

    HIV-1 transmission in intravenous drug users (IDUs) has been characterized by high genetic multiplicity and suggests a greater challenge for HIV-1 infection blocking. We investigated a total of 749 sequences of full-length gp160 gene obtained by single genome sequencing (SGS) from 22 HIV-1 early infected IDUs in Xinjiang province, northwest China, and generated a transmitted and founder virus (T/F virus) consensus sequence (IDU.CON). The T/F virus was classified as subtype CRF07_BC and predicted to be CCR5-tropic virus. The variable region (V1, V2, and V4 loop) of IDU.CON showed length variation compared with the heterosexual T/F virus consensus sequence (HSX.CON) and homosexual T/F virus consensus sequence (MSM.CON). A total of 26 N-linked glycosylation sites were discovered in the IDU.CON sequence, which is less than that of MSM.CON and HSX.CON. Characterization of T/F virus from IDUs highlights the genetic make-up and complexity of virus near the moment of transmission or in early infection preceding systemic dissemination and is important toward the development of an effective HIV-1 preventive methods, including vaccines.

  14. Molecular characterization and clonal diversity of meticillin-resistant Staphylococcus aureus isolated from the community in Spain: emergence of clone sequence type 72.

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    Potel, C; Rey, S; Otero, S; Rubio, J; Álvarez, M

    2016-08-01

    Sequence type 72 meticillin-resistant Staphylococcus aureus (ST72 MRSA) was recently detected in our hospital. Although in Europe this clone is rarely isolated, it is the leading cause of community-associated MRSA infections in Korea, spreading also into hospitals, where it has also emerged as the main MRSA clone recovered from raw meat. We studied MRSA isolated from outpatients in Spain during a nine-year period. More than 70% of the isolates belonged to predominant clones found in hospitals. There was a significant increase in the ST72 prevalence. It appears that boundaries of dominance among MRSA clones have become blurred, demanding continuous surveillance. Copyright © 2016 The Healthcare Infection Society. Published by Elsevier Ltd. All rights reserved.

  15. Clonal hematopoiesis in acquired aplastic anemia.

    Science.gov (United States)

    Ogawa, Seishi

    2016-07-21

    Clonal hematopoiesis (CH) in aplastic anemia (AA) has been closely linked to the evolution of late clonal disorders, including paroxysmal nocturnal hemoglobinuria and myelodysplastic syndromes (MDS)/acute myeloid leukemia (AML), which are common complications after successful immunosuppressive therapy (IST). With the advent of high-throughput sequencing of recent years, the molecular aspect of CH in AA has been clarified by comprehensive detection of somatic mutations that drive clonal evolution. Genetic abnormalities are found in ∼50% of patients with AA and, except for PIGA mutations and copy-neutral loss-of-heterozygosity, or uniparental disomy (UPD) in 6p (6pUPD), are most frequently represented by mutations involving genes commonly mutated in myeloid malignancies, including DNMT3A, ASXL1, and BCOR/BCORL1 Mutations exhibit distinct chronological profiles and clinical impacts. BCOR/BCORL1 and PIGA mutations tend to disappear or show stable clone size and predict a better response to IST and a significantly better clinical outcome compared with mutations in DNMT3A, ASXL1, and other genes, which are likely to increase their clone size, are associated with a faster progression to MDS/AML, and predict an unfavorable survival. High frequency of 6pUPD and overrepresentation of PIGA and BCOR/BCORL1 mutations are unique to AA, suggesting the role of autoimmunity in clonal selection. By contrast, DNMT3A and ASXL1 mutations, also commonly seen in CH in the general population, indicate a close link to CH in the aged bone marrow, in terms of the mechanism for selection. Detection and close monitoring of somatic mutations/evolution may help with prediction and diagnosis of clonal evolution of MDS/AML and better management of patients with AA. © 2016 by The American Society of Hematology.

  16. Combination of multilocus sequence typing and pulsed-field gel electrophoresis reveals an association of molecular clonality with the emergence of extensive-drug resistance (XDR) in Salmonella.

    Science.gov (United States)

    Cao, Yongzhong; Shen, Yongxiu; Cheng, Lingling; Zhang, Xiaorong; Wang, Chao; Wang, Yan; Zhou, Xiaohui; Chao, Guoxiang; Wu, Yantao

    2018-03-01

    Salmonellae is one of the most important foodborne pathogens and becomes resistant to multiple antibiotics, which represents a significant challenge to food industry and public health. However, a molecular signature that can be used to distinguish antimicrobial resistance profile, particularly multi-drug resistance or extensive-drug resistance (XDR). In the current study, 168 isolates from the chicken and pork production chains and ill chickens were characterized by serotyping, antimicrobial susceptibility test, multilocus sequence typing (MLST) and pulsed-field gel electrophoresis (PFGE). The results showed that these isolates belonged to 13 serotypes, 14 multilocus sequence types (STs), 94 PFGE genotypes, and 70 antimicrobial resistant profiles. S. Enteritidis, S. Indiana, and S. Derby were the predominant serotypes, corresponding to the ST11, ST17, and ST40 clones, respectively and the PFGE Cluster A, Cluster E, and Cluster D, respectively. Among the ST11-S. Enteritidis (Cluster A) and the ST40-S. Derby (Cluster D) clones, the majority of isolates were resistant to 4-8 antimicrobial agents, whereas in the ST17S. Indiana (Cluster E) clone, isolates showed extensive-drug resistance (XDR) to 9-16 antimicrobial agents. The bla TEM-1-like gene was prevalent in the ST11 and ST17 clones corresponding to high ampicillin resistance. The bla TEM-1-like , bla CTX-M , bla OXA-1-like , sul1, aaC4, aac(6')-1b, dfrA17, and floR gene complex was highly prevalent among isolates of ST17, corresponding to an XDR phenotype. These results demonstrated the association of the resistant phenotypes and genotypes with ST clone and PFGE cluster. Our results also indicated that the newly identified gene complex comprising bla TEM-1-like , bla CTX-M , bla OXA-1-like , sul1, aaC4, aac(6')-1b, dfrA17, and floR, was responsible for the emergence of the ST17S. Indiana XDR clone. ST17 could be potentially used as a molecular signature to distinguish S. Indiana XDR clone. Copyright © 2017

  17. HIV-1 transmission patterns in antiretroviral therapy-naive, HIV-infected North Americans based on phylogenetic analysis by population level and ultra-deep DNA sequencing.

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    Lisa L Ross

    Full Text Available Factors that contribute to the transmission of human immunodeficiency virus type 1 (HIV-1, especially drug-resistant HIV-1 variants remain a significant public health concern. In-depth phylogenetic analyses of viral sequences obtained in the screening phase from antiretroviral-naïve HIV-infected patients seeking enrollment in EPZ108859, a large open-label study in the USA, Canada and Puerto Rico (ClinicalTrials.gov NCT00440947 were examined for insights into the roles of drug resistance and epidemiological factors that could impact disease dissemination. Viral transmission clusters (VTCs were initially predicted from a phylogenetic analysis of population level HIV-1 pol sequences obtained from 690 antiretroviral-naïve subjects in 2007. Subsequently, the predicted VTCs were tested for robustness by ultra deep sequencing (UDS using pyrosequencing technology and further phylogenetic analyses. The demographic characteristics of clustered and non-clustered subjects were then compared. From 690 subjects, 69 were assigned to 1 of 30 VTCs, each containing 2 to 5 subjects. Race composition of VTCs were significantly more likely to be white (72% vs. 60%; p = 0.04. VTCs had fewer reverse transcriptase and major PI resistance mutations (9% vs. 24%; p = 0.002 than non-clustered sequences. Both men-who-have-sex-with-men (MSM (68% vs. 48%; p = 0.001 and Canadians (29% vs. 14%; p = 0.03 were significantly more frequent in VTCs than non-clustered sequences. Of the 515 subjects who initiated antiretroviral therapy, 33 experienced confirmed virologic failure through 144 weeks while only 3/33 were from VTCs. Fewer VTCs subjects (as compared to those with non-clustering virus had HIV-1 with resistance-associated mutations or experienced virologic failure during the course of the study. Our analysis shows specific geographical and drug resistance trends that correlate well with transmission clusters defined by HIV sequences of similarity

  18. Functional, non-clonal IgMa-restricted B cell receptor interactions with the HIV-1 envelope gp41 membrane proximal external region.

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    Laurent Verkoczy

    Full Text Available The membrane proximal external region (MPER of HIV-1 gp41 has several features that make it an attractive antibody-based vaccine target, but eliciting an effective gp41 MPER-specific protective antibody response remains elusive. One fundamental issue is whether the failure to make gp41 MPER-specific broadly neutralizing antibodies like 2F5 and 4E10 is due to structural constraints with the gp41 MPER, or alternatively, if gp41 MPER epitope-specific B cells are lost to immunological tolerance. An equally important question is how B cells interact with, and respond to, the gp41 MPER epitope, including whether they engage this epitope in a non-canonical manner i.e., by non-paratopic recognition via B cell receptors (BCR. To begin understanding how B cells engage the gp41 MPER, we characterized B cell-gp41 MPER interactions in BALB/c and C57BL/6 mice. Surprisingly, we found that a significant (approximately 7% fraction of splenic B cells from BALB/c, but not C57BL/6 mice, bound the gp41 MPER via their BCRs. This strain-specific binding was concentrated in IgM(hi subsets, including marginal zone and peritoneal B1 B cells, and correlated with enriched fractions (approximately 15% of gp41 MPER-specific IgM secreted by in vitro-activated splenic B cells. Analysis of Igh(a (BALB/c and Igh(b (C57BL/6 congenic mice demonstrated that gp41 MPER binding was controlled by determinants of the Igh(a locus. Mapping of MPER gp41 interactions with IgM(a identified MPER residues distinct from those to which mAb 2F5 binds and demonstrated the requirement of Fc C(H regions. Importantly, gp41 MPER ligation produced detectable BCR-proximal signaling events, suggesting that interactions between gp41 MPER and IgM(a determinants may elicit partial B cell activation. These data suggest that low avidity, non-paratopic interactions between the gp41 MPER and membrane Ig on naïve B cells may interfere with or divert bnAb responses.

  19. Big Bang Tumor Growth and Clonal Evolution.

    Science.gov (United States)

    Sun, Ruping; Hu, Zheng; Curtis, Christina

    2018-05-01

    The advent and application of next-generation sequencing (NGS) technologies to tumor genomes has reinvigorated efforts to understand clonal evolution. Although tumor progression has traditionally been viewed as a gradual stepwise process, recent studies suggest that evolutionary rates in tumors can be variable with periods of punctuated mutational bursts and relative stasis. For example, Big Bang dynamics have been reported, wherein after transformation, growth occurs in the absence of stringent selection, consistent with effectively neutral evolution. Although first noted in colorectal tumors, effective neutrality may be relatively common. Additionally, punctuated evolution resulting from mutational bursts and cataclysmic genomic alterations have been described. In this review, we contrast these findings with the conventional gradualist view of clonal evolution and describe potential clinical and therapeutic implications of different evolutionary modes and tempos. Copyright © 2018 Cold Spring Harbor Laboratory Press; all rights reserved.

  20. Study of HIV-1 subtypes in serodiscordant couples attending an integrated counselling and testing centre in Mumbai using heteroduplex mobility analysis and DNA sequencing

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    Mehta P

    2010-01-01

    Full Text Available Aims: To determine the prevalent subtypes of HIV-1 in serodiscordant couples. Setting: Integrated Counselling and Testing Centre (ICTC, Department of Microbiology. Study Design: Prospective pilot study. Participants: Thirty HIV-1 serodiscordant couples. Inclusion Criteria: a Documentation of HIV-1 infection in one partner and seronegative status in the other, current history of continued unprotected sexual activity within the partnership, demonstration that they have been in a partnership for at least 1 year and are not currently on highly active antiretroviral therapy HAART; b willingness of both partners to provide written informed consent including consent to continued couple counselling for 3 months. Materials and Methods: HIV-1 subtyping was carried out by heteroduplex mobility analysis (HMA by amplifying env region; and DNA sequencing by amplifying gag region. Results: HIV-1 env gene was amplified successfully in 10/30 samples; gag gene, in 25/30 samples; and both env and gag gene were amplified successfully in 5/30 samples. HIV-1 subtype C was detected from 21 samples; subtype B, from 7; and subtype A, from 2. Sample from 1 positive partner was detected as subtype C by env HMA and subtype B by gag sequencing. Conclusion: HIV-1 subtype C was found to be the predominant subtype of HIV-1 in serodiscordant couples attending our ICTC, followed by HIV-1 subtype B and HIV-1 subtype A, respectively. DNA sequencing was found to be the most reliable method for determining the subtypes of HIV-1.

  1. Production of Mucosally Transmissible SHIV Challenge Stocks from HIV-1 Circulating Recombinant Form 01_AE env Sequences.

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    Lawrence J Tartaglia

    2016-02-01

    Full Text Available Simian-human immunodeficiency virus (SHIV challenge stocks are critical for preclinical testing of vaccines, antibodies, and other interventions aimed to prevent HIV-1. A major unmet need for the field has been the lack of a SHIV challenge stock expressing circulating recombinant form 01_AE (CRF01_AE env sequences. We therefore sought to develop mucosally transmissible SHIV challenge stocks containing HIV-1 CRF01_AE env derived from acutely HIV-1 infected individuals from Thailand. SHIV-AE6, SHIV-AE6RM, and SHIV-AE16 contained env sequences that were >99% identical to the original HIV-1 isolate and did not require in vivo passaging. These viruses exhibited CCR5 tropism and displayed a tier 2 neutralization phenotype. These challenge stocks efficiently infected rhesus monkeys by the intrarectal route, replicated to high levels during acute infection, and established chronic viremia in a subset of animals. SHIV-AE16 was titrated for use in single, high dose as well as repetitive, low dose intrarectal challenge studies. These SHIV challenge stocks should facilitate the preclinical evaluation of vaccines, monoclonal antibodies, and other interventions targeted at preventing HIV-1 CRF01_AE infection.

  2. Nucleic acid amplification of HIV-1 integrase sequence subtypes CRF01_AE and B for development of HIV anti-integrase drug resistance genotyping assay

    Science.gov (United States)

    Adlar, F. R.; Bela, B.

    2017-08-01

    To anticipate the potential use of anti-integrase drugs in Indonesia for treatment of HIV-1 infection, the development of a drug resistance genotyping assay for anti-integrase is crucial in identifying the genetic drug resistance profile of Indonesian HIV-1 strains. This experiment aimed to amplify a target region in the integrase gene of Indonesian HIV-1 subtypes CRF01_AE and B that contain genetic mutations known to confer resistance to anti-integrase drug. Eleven archived plasma samples from individuals living with HIV-1 were obtained from the Virology and Cancer Pathobiology Research Center for Health Service (VCPRC FKUI-RSCM) laboratory. One of the plasma samples contained HIV-1 subtype B, and the remaining plasma samples contained subtype CRF01_AE. The target regions for all samples were amplified through RT-PCR, with an annealing temperature of 55 °C, using the primer pair AE_POL 4086F and AE_POL 5232R that were designed by VCPRC FKUI-RSCM. The results of this experiment show that 18.2% (2/11) of the samples were successfully amplified using the one-step RT-PCR. While the primer pair was effective in amplifying the target region in the integrase gene sequence for subtype B (100%; 1/1), it had a low efficacy (10%, 1/10) for subtype CRF01_AE. In conclusion, the primer pair can be used to amplify the target region in Indonesian HIV-1 strain subtypes CRF01_AE and B. However, optimization of the PCR condition and an increased number of samples would help to determine an accurate representation of the efficacy of the primer pair.

  3. Clonality evaluation in human tissues

    Directory of Open Access Journals (Sweden)

    Villamizar-Rivera, Nicolás

    2015-07-01

    Full Text Available Malignant proliferations are usually clonal. While most times the biological potential can be established through routine pathologic and clinical examinations, some cases are difficult to classify. Moreover, in some situations there are dominant clones whose analysis is important, such as in autoimmune diseases and immunodeficiency. This paper presents in an understandable way the main techniques for the study of clonality, namely: evaluation of gene rearrangements of antigen receptor, and evaluation of human antigen receptor gene.

  4. gCUP: rapid GPU-based HIV-1 co-receptor usage prediction for next-generation sequencing.

    Science.gov (United States)

    Olejnik, Michael; Steuwer, Michel; Gorlatch, Sergei; Heider, Dominik

    2014-11-15

    Next-generation sequencing (NGS) has a large potential in HIV diagnostics, and genotypic prediction models have been developed and successfully tested in the recent years. However, albeit being highly accurate, these computational models lack computational efficiency to reach their full potential. In this study, we demonstrate the use of graphics processing units (GPUs) in combination with a computational prediction model for HIV tropism. Our new model named gCUP, parallelized and optimized for GPU, is highly accurate and can classify >175 000 sequences per second on an NVIDIA GeForce GTX 460. The computational efficiency of our new model is the next step to enable NGS technologies to reach clinical significance in HIV diagnostics. Moreover, our approach is not limited to HIV tropism prediction, but can also be easily adapted to other settings, e.g. drug resistance prediction. The source code can be downloaded at http://www.heiderlab.de d.heider@wz-straubing.de. © The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

  5. The highly conserved codon following the slippery sequence supports -1 frameshift efficiency at the HIV-1 frameshift site.

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    Suneeth F Mathew

    Full Text Available HIV-1 utilises -1 programmed ribosomal frameshifting to translate structural and enzymatic domains in a defined proportion required for replication. A slippery sequence, U UUU UUA, and a stem-loop are well-defined RNA features modulating -1 frameshifting in HIV-1. The GGG glycine codon immediately following the slippery sequence (the 'intercodon' contributes structurally to the start of the stem-loop but has no defined role in current models of the frameshift mechanism, as slippage is inferred to occur before the intercodon has reached the ribosomal decoding site. This GGG codon is highly conserved in natural isolates of HIV. When the natural intercodon was replaced with a stop codon two different decoding molecules-eRF1 protein or a cognate suppressor tRNA-were able to access and decode the intercodon prior to -1 frameshifting. This implies significant slippage occurs when the intercodon is in the (perhaps distorted ribosomal A site. We accommodate the influence of the intercodon in a model of frame maintenance versus frameshifting in HIV-1.

  6. Improved detection of CXCR4-using HIV by V3 genotyping: application of population-based and "deep" sequencing to plasma RNA and proviral DNA.

    Science.gov (United States)

    Swenson, Luke C; Moores, Andrew; Low, Andrew J; Thielen, Alexander; Dong, Winnie; Woods, Conan; Jensen, Mark A; Wynhoven, Brian; Chan, Dennison; Glascock, Christopher; Harrigan, P Richard

    2010-08-01

    Tropism testing should rule out CXCR4-using HIV before treatment with CCR5 antagonists. Currently, the recombinant phenotypic Trofile assay (Monogram) is most widely utilized; however, genotypic tests may represent alternative methods. Independent triplicate amplifications of the HIV gp120 V3 region were made from either plasma HIV RNA or proviral DNA. These underwent standard, population-based sequencing with an ABI3730 (RNA n = 63; DNA n = 40), or "deep" sequencing with a Roche/454 Genome Sequencer-FLX (RNA n = 12; DNA n = 12). Position-specific scoring matrices (PSSMX4/R5) (-6.96 cutoff) and geno2pheno[coreceptor] (5% false-positive rate) inferred tropism from V3 sequence. These methods were then independently validated with a separate, blinded dataset (n = 278) of screening samples from the maraviroc MOTIVATE trials. Standard sequencing of HIV RNA with PSSM yielded 69% sensitivity and 91% specificity, relative to Trofile. The validation dataset gave 75% sensitivity and 83% specificity. Proviral DNA plus PSSM gave 77% sensitivity and 71% specificity. "Deep" sequencing of HIV RNA detected >2% inferred-CXCR4-using virus in 8/8 samples called non-R5 by Trofile, and <2% in 4/4 samples called R5. Triplicate analyses of V3 standard sequence data detect greater proportions of CXCR4-using samples than previously achieved. Sequencing proviral DNA and "deep" V3 sequencing may also be useful tools for assessing tropism.

  7. Impact of alemtuzumab on HIV persistence in an HIV-infected individual on antiretroviral therapy with Sezary syndrome.

    Science.gov (United States)

    Rasmussen, Thomas A; McMahon, James; Chang, J Judy; Symons, Jori; Roche, Michael; Dantanarayana, Ashanti; Okoye, Afam; Hiener, Bonnie; Palmer, Sarah; Lee, Wen Shi; Kent, Stephen J; Van Der Weyden, Carrie; Prince, H Miles; Cameron, Paul U; Lewin, Sharon R

    2017-08-24

    To study the effects of alemtuzumab on HIV persistence in an HIV-infected individual on antiretroviral therapy (ART) with Sezary syndrome, a rare malignancy of CD4 T cells. Case report. Blood was collected 30 and 18 months prior to presentation with Sezary syndrome, at the time of presentation and during alemtuzumab. T-cell subsets in malignant (CD7-CD26-TCR-VBeta2+) and nonmalignant cells were quantified by flow cytometry. HIV-DNA in total CD4 T cells, in sorted malignant and nonmalignant CD4 T cells, was quantified by PCR and clonal expansion of HIV-DNA assessed by full-length next-generation sequencing. HIV-hepatitis B virus coinfection was diagnosed and antiretroviral therapy initiated 4 years prior to presentation with Sezary syndrome and primary cutaneous anaplastic large cell lymphoma. The patient received alemtuzumab 10 mg three times per week for 4 weeks but died 6 weeks post alemtuzumab. HIV-DNA was detected in nonmalignant but not in malignant CD4 T cells, consistent with expansion of a noninfected CD4 T-cell clone. Full-length HIV-DNA sequencing demonstrated multiple defective viruses but no identical or expanded sequences. Alemtuzumab extensively depleted T cells, including more than 1 log reduction in total T cells and more than 3 log reduction in CD4 T cells. Finally, alemtuzumab decreased HIV-DNA in CD4 T cells by 57% but HIV-DNA remained detectable at low levels even after depletion of nearly all CD4 T cells. Alemtuzumab extensively depleted multiple T-cell subsets and decreased the frequency of but did not eliminate HIV-infected CD4 T cells. Studying the effects on HIV persistence following immune recovery in HIV-infected individuals who require alemtuzumab for malignancy or in animal studies may provide further insights into novel cure strategies.

  8. HIV drug resistance patterns in pregnant women using next generation sequence in Mozambique.

    Science.gov (United States)

    Rupérez, María; Noguera-Julian, Marc; González, Raquel; Maculuve, Sonia; Bellido, Rocío; Vala, Anifa; Rodríguez, Cristina; Sevene, Esperança; Paredes, Roger; Menéndez, Clara

    2018-01-01

    Few data on HIV resistance in pregnancy are available from Mozambique, one of the countries with the highest HIV toll worldwide. Understanding the patterns of HIV drug resistance in pregnant women might help in tailoring optimal regimens for prevention of mother to child transmission of HIV (pMTCT) and antenatal care. To describe the frequency and characteristics of HIV drug resistance mutations (HIVDRM) in pregnant women with virological failure at delivery, despite pMTCT or antiretroviral therapy (ART). Samples from HIV-infected pregnant women from a rural area in southern Mozambique were analysed. Only women with HIV-1 RNA >400c/mL at delivery were included in the analysis. HIVDRM were determined using MiSeq® (detection threshold 1%) at the first antenatal care (ANC) visit and at the time of delivery. Ninety and 60 samples were available at the first ANC visit and delivery, respectively. At first ANC, 97% of the women had HIV-1 RNA>400c/mL, 39% had CD4+ counts HIV-1 genotyping, less than 20% of women with detectable viremia at delivery had HIVDRM before initiating pMTCT or ART. This suggests that factors other than pre-existing resistance, such as lack of adherence or interruptions of the ANC chain, are also relevant to explain lack of virological suppression at the time of delivery in women receiving antiretrovirals drugs during pregnancy.

  9. Sequence- and interactome-based prediction of viral protein hotspots targeting host proteins: a case study for HIV Nef.

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    Mahdi Sarmady

    Full Text Available Virus proteins alter protein pathways of the host toward the synthesis of viral particles by breaking and making edges via binding to host proteins. In this study, we developed a computational approach to predict viral sequence hotspots for binding to host proteins based on sequences of viral and host proteins and literature-curated virus-host protein interactome data. We use a motif discovery algorithm repeatedly on collections of sequences of viral proteins and immediate binding partners of their host targets and choose only those motifs that are conserved on viral sequences and highly statistically enriched among binding partners of virus protein targeted host proteins. Our results match experimental data on binding sites of Nef to host proteins such as MAPK1, VAV1, LCK, HCK, HLA-A, CD4, FYN, and GNB2L1 with high statistical significance but is a poor predictor of Nef binding sites on highly flexible, hoop-like regions. Predicted hotspots recapture CD8 cell epitopes of HIV Nef highlighting their importance in modulating virus-host interactions. Host proteins potentially targeted or outcompeted by Nef appear crowding the T cell receptor, natural killer cell mediated cytotoxicity, and neurotrophin signaling pathways. Scanning of HIV Nef motifs on multiple alignments of hepatitis C protein NS5A produces results consistent with literature, indicating the potential value of the hotspot discovery in advancing our understanding of virus-host crosstalk.

  10. Comparison of cluster-based and source-attribution methods for estimating transmission risk using large HIV sequence databases.

    Science.gov (United States)

    Le Vu, Stéphane; Ratmann, Oliver; Delpech, Valerie; Brown, Alison E; Gill, O Noel; Tostevin, Anna; Fraser, Christophe; Volz, Erik M

    2018-06-01

    Phylogenetic clustering of HIV sequences from a random sample of patients can reveal epidemiological transmission patterns, but interpretation is hampered by limited theoretical support and statistical properties of clustering analysis remain poorly understood. Alternatively, source attribution methods allow fitting of HIV transmission models and thereby quantify aspects of disease transmission. A simulation study was conducted to assess error rates of clustering methods for detecting transmission risk factors. We modeled HIV epidemics among men having sex with men and generated phylogenies comparable to those that can be obtained from HIV surveillance data in the UK. Clustering and source attribution approaches were applied to evaluate their ability to identify patient attributes as transmission risk factors. We find that commonly used methods show a misleading association between cluster size or odds of clustering and covariates that are correlated with time since infection, regardless of their influence on transmission. Clustering methods usually have higher error rates and lower sensitivity than source attribution method for identifying transmission risk factors. But neither methods provide robust estimates of transmission risk ratios. Source attribution method can alleviate drawbacks from phylogenetic clustering but formal population genetic modeling may be required to estimate quantitative transmission risk factors. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

  11. The C-terminal sequence of IFITM1 regulates its anti-HIV-1 activity.

    Directory of Open Access Journals (Sweden)

    Rui Jia

    Full Text Available The interferon-inducible transmembrane (IFITM proteins inhibit a wide range of viruses. We previously reported the inhibition of human immunodeficiency virus type 1 (HIV-1 strain BH10 by human IFITM1, 2 and 3. It is unknown whether other HIV-1 strains are similarly inhibited by IFITMs and whether there exists viral countermeasure to overcome IFITM inhibition. We report here that the HIV-1 NL4-3 strain (HIV-1NL4-3 is not restricted by IFITM1 and its viral envelope glycoprotein is partly responsible for this insensitivity. However, HIV-1NL4-3 is profoundly inhibited by an IFITM1 mutant, known as Δ(117-125, which is deleted of 9 amino acids at the C-terminus. In contrast to the wild type IFITM1, which does not affect HIV-1 entry, the Δ(117-125 mutant diminishes HIV-1NL4-3 entry by 3-fold. This inhibition correlates with the predominant localization of Δ(117-125 to the plasma membrane where HIV-1 entry occurs. In spite of strong conservation of IFITM1 among most species, mouse IFITM1 is 19 amino acids shorter at its C-terminus as compared to human IFITM1 and, like the human IFITM1 mutant Δ(117-125, mouse IFITM1 also inhibits HIV-1 entry. This is the first report illustrating the role of viral envelope protein in overcoming IFITM1 restriction. The results also demonstrate the importance of the C-terminal region of IFITM1 in modulating the antiviral function through controlling protein subcellular localization.

  12. A comparison of parallel pyrosequencing and sanger clone-based sequencing and its impact on the characterization of the genetic diversity of HIV-1.

    Directory of Open Access Journals (Sweden)

    Binhua Liang

    Full Text Available BACKGROUND: Pyrosequencing technology has the potential to rapidly sequence HIV-1 viral quasispecies without requiring the traditional approach of cloning. In this study, we investigated the utility of ultra-deep pyrosequencing to characterize genetic diversity of the HIV-1 gag quasispecies and assessed the possible contribution of pyrosequencing technology in studying HIV-1 biology and evolution. METHODOLOGY/PRINCIPAL FINDINGS: HIV-1 gag gene was amplified from 96 patients using nested PCR. The PCR products were cloned and sequenced using capillary based Sanger fluorescent dideoxy termination sequencing. The same PCR products were also directly sequenced using the 454 pyrosequencing technology. The two sequencing methods were evaluated for their ability to characterize quasispecies variation, and to reveal sites under host immune pressure for their putative functional significance. A total of 14,034 variations were identified by 454 pyrosequencing versus 3,632 variations by Sanger clone-based (SCB sequencing. 11,050 of these variations were detected only by pyrosequencing. These undetected variations were located in the HIV-1 Gag region which is known to contain putative cytotoxic T lymphocyte (CTL and neutralizing antibody epitopes, and sites related to virus assembly and packaging. Analysis of the positively selected sites derived by the two sequencing methods identified several differences. All of them were located within the CTL epitope regions. CONCLUSIONS/SIGNIFICANCE: Ultra-deep pyrosequencing has proven to be a powerful tool for characterization of HIV-1 genetic diversity with enhanced sensitivity, efficiency, and accuracy. It also improved reliability of downstream evolutionary and functional analysis of HIV-1 quasispecies.

  13. Likelihood-Based Inference of B Cell Clonal Families.

    Directory of Open Access Journals (Sweden)

    Duncan K Ralph

    2016-10-01

    Full Text Available The human immune system depends on a highly diverse collection of antibody-making B cells. B cell receptor sequence diversity is generated by a random recombination process called "rearrangement" forming progenitor B cells, then a Darwinian process of lineage diversification and selection called "affinity maturation." The resulting receptors can be sequenced in high throughput for research and diagnostics. Such a collection of sequences contains a mixture of various lineages, each of which may be quite numerous, or may consist of only a single member. As a step to understanding the process and result of this diversification, one may wish to reconstruct lineage membership, i.e. to cluster sampled sequences according to which came from the same rearrangement events. We call this clustering problem "clonal family inference." In this paper we describe and validate a likelihood-based framework for clonal family inference based on a multi-hidden Markov Model (multi-HMM framework for B cell receptor sequences. We describe an agglomerative algorithm to find a maximum likelihood clustering, two approximate algorithms with various trade-offs of speed versus accuracy, and a third, fast algorithm for finding specific lineages. We show that under simulation these algorithms greatly improve upon existing clonal family inference methods, and that they also give significantly different clusters than previous methods when applied to two real data sets.

  14. Phylogenetic analysis of HIV-1 reverse transcriptase sequences from 382 patients recruited in JJ Hospital of Mumbai, India, between 2002 and 2008.

    Science.gov (United States)

    Deshpande, Alaka; Jauvin, Valerie; Pinson, Patricia; Jeannot, Anne Cecile; Fleury, Herve J

    2009-06-01

    Analysis of reverse transcriptase (RT) sequences of 382 HIV-1 isolates from untreated and treated patients recruited in JJ Hospital (Mumbai, India) between 2002 and 2008 shows that subtype C is largely predominant (98%) and that non-C sequences cluster with A1, B, CRF01_AE, and CRF06_cpx.

  15. Structure-Related Roles for the Conservation of the HIV-1 Fusion Peptide Sequence Revealed by Nuclear Magnetic Resonance.

    Science.gov (United States)

    Serrano, Soraya; Huarte, Nerea; Rujas, Edurne; Andreu, David; Nieva, José L; Jiménez, María Angeles

    2017-10-17

    Despite extensive characterization of the human immunodeficiency virus type 1 (HIV-1) hydrophobic fusion peptide (FP), the structure-function relationships underlying its extraordinary degree of conservation remain poorly understood. Specifically, the fact that the tandem repeat of the FLGFLG tripeptide is absolutely conserved suggests that high hydrophobicity may not suffice to unleash FP function. Here, we have compared the nuclear magnetic resonance (NMR) structures adopted in nonpolar media by two FP surrogates, wtFP-tag and scrFP-tag, which had equal hydrophobicity but contained wild-type and scrambled core sequences LFLGFLG and FGLLGFL, respectively. In addition, these peptides were tagged at their C-termini with an epitope sequence that folded independently, thereby allowing Western blot detection without interfering with FP structure. We observed similar α-helical FP conformations for both specimens dissolved in the low-polarity medium 25% (v/v) 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP), but important differences in contact with micelles of the membrane mimetic dodecylphosphocholine (DPC). Thus, whereas wtFP-tag preserved a helix displaying a Gly-rich ridge, the scrambled sequence lost in great part the helical structure upon being solubilized in DPC. Western blot analyses further revealed the capacity of wtFP-tag to assemble trimers in membranes, whereas membrane oligomers were not observed in the case of the scrFP-tag sequence. We conclude that, beyond hydrophobicity, preserving sequence order is an important feature for defining the secondary structures and oligomeric states adopted by the HIV FP in membranes.

  16. Divergent clonal selection dominates medulloblastoma at recurrence

    Science.gov (United States)

    Morrissy, A. Sorana; Garzia, Livia; Shih, David J. H.; Zuyderduyn, Scott; Huang, Xi; Skowron, Patryk; Remke, Marc; Cavalli, Florence M. G.; Ramaswamy, Vijay; Lindsay, Patricia E.; Jelveh, Salomeh; Donovan, Laura K.; Wang, Xin; Luu, Betty; Zayne, Kory; Li, Yisu; Mayoh, Chelsea; Thiessen, Nina; Mercier, Eloi; Mungall, Karen L.; Ma, Yusanne; Tse, Kane; Zeng, Thomas; Shumansky, Karey; Roth, Andrew J. L.; Shah, Sohrab; Farooq, Hamza; Kijima, Noriyuki; Holgado, Borja L.; Lee, John J. Y.; Matan-Lithwick, Stuart; Liu, Jessica; Mack, Stephen C.; Manno, Alex; Michealraj, K. A.; Nor, Carolina; Peacock, John; Qin, Lei; Reimand, Juri; Rolider, Adi; Thompson, Yuan Y.; Wu, Xiaochong; Pugh, Trevor; Ally, Adrian; Bilenky, Mikhail; Butterfield, Yaron S. N.; Carlsen, Rebecca; Cheng, Young; Chuah, Eric; Corbett, Richard D.; Dhalla, Noreen; He, An; Lee, Darlene; Li, Haiyan I.; Long, William; Mayo, Michael; Plettner, Patrick; Qian, Jenny Q.; Schein, Jacqueline E.; Tam, Angela; Wong, Tina; Birol, Inanc; Zhao, Yongjun; Faria, Claudia C.; Pimentel, José; Nunes, Sofia; Shalaby, Tarek; Grotzer, Michael; Pollack, Ian F.; Hamilton, Ronald L.; Li, Xiao-Nan; Bendel, Anne E.; Fults, Daniel W.; Walter, Andrew W.; Kumabe, Toshihiro; Tominaga, Teiji; Collins, V. Peter; Cho, Yoon-Jae; Hoffman, Caitlin; Lyden, David; Wisoff, Jeffrey H.; Garvin, James H.; Stearns, Duncan S.; Massimi, Luca; Schüller, Ulrich; Sterba, Jaroslav; Zitterbart, Karel; Puget, Stephanie; Ayrault, Olivier; Dunn, Sandra E.; Tirapelli, Daniela P. C.; Carlotti, Carlos G.; Wheeler, Helen; Hallahan, Andrew R.; Ingram, Wendy; MacDonald, Tobey J.; Olson, Jeffrey J.; Van Meir, Erwin G.; Lee, Ji-Yeoun; Wang, Kyu-Chang; Kim, Seung-Ki; Cho, Byung-Kyu; Pietsch, Torsten; Fleischhack, Gudrun; Tippelt, Stephan; Ra, Young Shin; Bailey, Simon; Lindsey, Janet C.; Clifford, Steven C.; Eberhart, Charles G.; Cooper, Michael K.; Packer, Roger J.; Massimino, Maura; Garre, Maria Luisa; Bartels, Ute; Tabori, Uri; Hawkins, Cynthia E.; Dirks, Peter; Bouffet, Eric; Rutka, James T.; Wechsler-Reya, Robert J.; Weiss, William A.; Collier, Lara S.; Dupuy, Adam J.; Korshunov, Andrey; Jones, David T. W.; Kool, Marcel; Northcott, Paul A.; Pfister, Stefan M.; Largaespada, David A.; Mungall, Andrew J.; Moore, Richard A.; Jabado, Nada; Bader, Gary D.; Jones, Steven J. M.; Malkin, David; Marra, Marco A.; Taylor, Michael D.

    2016-01-01

    The development of targeted anti-cancer therapies through the study of cancer genomes is intended to increase survival rates and decrease treatment-related toxicity. We treated a transposon–driven, functional genomic mouse model of medulloblastoma with ‘humanized’ in vivo therapy (microneurosurgical tumour resection followed by multi-fractionated, image-guided radiotherapy). Genetic events in recurrent murine medulloblastoma exhibit a very poor overlap with those in matched murine diagnostic samples (sequencing of 33 pairs of human diagnostic and post-therapy medulloblastomas demonstrated substantial genetic divergence of the dominant clone after therapy (recurrence). In both mice and humans, the dominant clone at recurrence arose through clonal selection of a pre-existing minor clone present at diagnosis. Targeted therapy is unlikely to be effective in the absence of the target, therefore our results offer a simple, proximal, and remediable explanation for the failure of prior clinical trials of targeted therapy. PMID:26760213

  17. Subtype-independent near full-length HIV-1 genome sequencing and assembly to be used in large molecular epidemiological studies and clinical management.

    Science.gov (United States)

    Grossmann, Sebastian; Nowak, Piotr; Neogi, Ujjwal

    2015-01-01

    HIV-1 near full-length genome (HIV-NFLG) sequencing from plasma is an attractive multidimensional tool to apply in large-scale population-based molecular epidemiological studies. It also enables genotypic resistance testing (GRT) for all drug target sites allowing effective intervention strategies for control and prevention in high-risk population groups. Thus, the main objective of this study was to develop a simplified subtype-independent, cost- and labour-efficient HIV-NFLG protocol that can be used in clinical management as well as in molecular epidemiological studies. Plasma samples (n=30) were obtained from HIV-1B (n=10), HIV-1C (n=10), CRF01_AE (n=5) and CRF01_AG (n=5) infected individuals with minimum viral load >1120 copies/ml. The amplification was performed with two large amplicons of 5.5 kb and 3.7 kb, sequenced with 17 primers to obtain HIV-NFLG. GRT was validated against ViroSeq™ HIV-1 Genotyping System. After excluding four plasma samples with low-quality RNA, a total of 26 samples were attempted. Among them, NFLG was obtained from 24 (92%) samples with the lowest viral load being 3000 copies/ml. High (>99%) concordance was observed between HIV-NFLG and ViroSeq™ when determining the drug resistance mutations (DRMs). The N384I connection mutation was additionally detected by NFLG in two samples. Our high efficiency subtype-independent HIV-NFLG is a simple and promising approach to be used in large-scale molecular epidemiological studies. It will facilitate the understanding of the HIV-1 pandemic population dynamics and outline effective intervention strategies. Furthermore, it can potentially be applicable in clinical management of drug resistance by evaluating DRMs against all available antiretrovirals in a single assay.

  18. Transgenerational plasticity in clonal plants

    Czech Academy of Sciences Publication Activity Database

    Latzel, Vít; Klimešová, Jitka

    2010-01-01

    Roč. 24, č. 6 (2010), s. 1537-1543 ISSN 0269-7653 R&D Projects: GA ČR GD206/08/H044; GA ČR GA526/09/0963; GA ČR GPP505/10/P173 Institutional research plan: CEZ:AV0Z60050516 Keywords : maternal effect * evolution * clonal plants Subject RIV: EF - Botanics Impact factor: 2.398, year: 2010

  19. Sieve analysis of breakthrough HIV-1 sequences in HVTN 505 identifies vaccine pressure targeting the CD4 binding site of Env-gp120.

    Science.gov (United States)

    deCamp, Allan C; Rolland, Morgane; Edlefsen, Paul T; Sanders-Buell, Eric; Hall, Breana; Magaret, Craig A; Fiore-Gartland, Andrew J; Juraska, Michal; Carpp, Lindsay N; Karuna, Shelly T; Bose, Meera; LePore, Steven; Miller, Shana; O'Sullivan, Annemarie; Poltavee, Kultida; Bai, Hongjun; Dommaraju, Kalpana; Zhao, Hong; Wong, Kim; Chen, Lennie; Ahmed, Hasan; Goodman, Derrick; Tay, Matthew Z; Gottardo, Raphael; Koup, Richard A; Bailer, Robert; Mascola, John R; Graham, Barney S; Roederer, Mario; O'Connell, Robert J; Michael, Nelson L; Robb, Merlin L; Adams, Elizabeth; D'Souza, Patricia; Kublin, James; Corey, Lawrence; Geraghty, Daniel E; Frahm, Nicole; Tomaras, Georgia D; McElrath, M Juliana; Frenkel, Lisa; Styrchak, Sheila; Tovanabutra, Sodsai; Sobieszczyk, Magdalena E; Hammer, Scott M; Kim, Jerome H; Mullins, James I; Gilbert, Peter B

    2017-01-01

    Although the HVTN 505 DNA/recombinant adenovirus type 5 vector HIV-1 vaccine trial showed no overall efficacy, analysis of breakthrough HIV-1 sequences in participants can help determine whether vaccine-induced immune responses impacted viruses that caused infection. We analyzed 480 HIV-1 genomes sampled from 27 vaccine and 20 placebo recipients and found that intra-host HIV-1 diversity was significantly lower in vaccine recipients (P ≤ 0.04, Q-values ≤ 0.09) in Gag, Pol, Vif and envelope glycoprotein gp120 (Env-gp120). Furthermore, Env-gp120 sequences from vaccine recipients were significantly more distant from the subtype B vaccine insert than sequences from placebo recipients (P = 0.01, Q-value = 0.12). These vaccine effects were associated with signatures mapping to CD4 binding site and CD4-induced monoclonal antibody footprints. These results suggest either (i) no vaccine efficacy to block acquisition of any viral genotype but vaccine-accelerated Env evolution post-acquisition; or (ii) vaccine efficacy against HIV-1s with Env sequences closest to the vaccine insert combined with increased acquisition due to other factors, potentially including the vaccine vector.

  20. Investigating signs of recent evolution in the pool of proviral HIV type 1 DNA during years of successful HAART

    DEFF Research Database (Denmark)

    Mens, Helene; Pedersen, Anders G; Jørgensen, Louise B

    2007-01-01

    In order to shed light on the nature of the persistent reservoir of human immunodeficiency virus type 1 (HIV-1), we investigated signs of recent evolution in the pool of proviral DNA in patients on successful HAART. Pro-viral DNA, corresponding to the C2-V3-C3 region of the HIV-1 env gene...... there were temporal trends indicating ongoing replication and evolution. In summary, it was not possible to detect definitive signs of ongoing evolution in either the bulk-sequenced or the clonal data with the methods employed here, but our results could be consistent with localized expression of archival...

  1. Comprehensive sieve analysis of breakthrough HIV-1 sequences in the RV144 vaccine efficacy trial.

    Science.gov (United States)

    Edlefsen, Paul T; Rolland, Morgane; Hertz, Tomer; Tovanabutra, Sodsai; Gartland, Andrew J; deCamp, Allan C; Magaret, Craig A; Ahmed, Hasan; Gottardo, Raphael; Juraska, Michal; McCoy, Connor; Larsen, Brendan B; Sanders-Buell, Eric; Carrico, Chris; Menis, Sergey; Kijak, Gustavo H; Bose, Meera; Arroyo, Miguel A; O'Connell, Robert J; Nitayaphan, Sorachai; Pitisuttithum, Punnee; Kaewkungwal, Jaranit; Rerks-Ngarm, Supachai; Robb, Merlin L; Kirys, Tatsiana; Georgiev, Ivelin S; Kwong, Peter D; Scheffler, Konrad; Pond, Sergei L Kosakovsky; Carlson, Jonathan M; Michael, Nelson L; Schief, William R; Mullins, James I; Kim, Jerome H; Gilbert, Peter B

    2015-02-01

    The RV144 clinical trial showed the partial efficacy of a vaccine regimen with an estimated vaccine efficacy (VE) of 31% for protecting low-risk Thai volunteers against acquisition of HIV-1. The impact of vaccine-induced immune responses can be investigated through sieve analysis of HIV-1 breakthrough infections (infected vaccine and placebo recipients). A V1/V2-targeted comparison of the genomes of HIV-1 breakthrough viruses identified two V2 amino acid sites that differed between the vaccine and placebo groups. Here we extended the V1/V2 analysis to the entire HIV-1 genome using an array of methods based on individual sites, k-mers and genes/proteins. We identified 56 amino acid sites or "signatures" and 119 k-mers that differed between the vaccine and placebo groups. Of those, 19 sites and 38 k-mers were located in the regions comprising the RV144 vaccine (Env-gp120, Gag, and Pro). The nine signature sites in Env-gp120 were significantly enriched for known antibody-associated sites (p = 0.0021). In particular, site 317 in the third variable loop (V3) overlapped with a hotspot of antibody recognition, and sites 369 and 424 were linked to CD4 binding site neutralization. The identified signature sites significantly covaried with other sites across the genome (mean = 32.1) more than did non-signature sites (mean = 0.9) (p analysis of the breakthrough infections in the RV144 trial, this work describes a set of statistical methods and tools applicable to analysis of breakthrough infection genomes in general vaccine efficacy trials for diverse pathogens.

  2. Clonal sets of a binary relation

    Science.gov (United States)

    Zedam, Lemnaouar; Pérez-Fernández, Raúl; Bouremel, Hassane; De Baets, Bernard

    2018-05-01

    In a recent paper, we have introduced the notion of clone relation of a given binary relation. Intuitively, two elements are said to be "clones" if they are related in the same way w.r.t. every other element. In this paper, we generalize this notion from pairs of elements to sets of elements of any cardinality, resulting in the introduction of clonal sets. We investigate the most important properties of clonal sets, paying particular attention to the introduction of the clonal closure operator, to the analysis of the (lattice) structure of the set of clonal sets and to a distance metric expressing how close two elements are to being clones.

  3. Draft genome sequence of Mycobacterium tuberculosis strain B9741 of Beijing B0/W lineage from HIV positive patient from Siberia

    Directory of Open Access Journals (Sweden)

    K.V. Shur

    2016-12-01

    Full Text Available We report a draft genome sequence of Mycobacterium tuberculosis strain B9741 belonging to Beijing B0/W lineage isolated from a HIV patient from Siberia, Russia. This clinical isolate showed MDR phenotype and resistance to isoniazid, rifampin, streptomycin and pyrazinamide. We analyzed SNPs associated with virulence and resistance. The draft genome sequence and annotation have been deposited at GenBank under the accession NZ_LVJJ00000000.

  4. Bioinformatic analysis of neurotropic HIV envelope sequences identifies polymorphisms in the gp120 bridging sheet that increase macrophage-tropism through enhanced interactions with CCR5

    International Nuclear Information System (INIS)

    Mefford, Megan E.; Kunstman, Kevin; Wolinsky, Steven M.; Gabuzda, Dana

    2015-01-01

    Macrophages express low levels of the CD4 receptor compared to T-cells. Macrophage-tropic HIV strains replicating in brain of untreated patients with HIV-associated dementia (HAD) express Envs that are adapted to overcome this restriction through mechanisms that are poorly understood. Here, bioinformatic analysis of env sequence datasets together with functional studies identified polymorphisms in the β3 strand of the HIV gp120 bridging sheet that increase M-tropism. D197, which results in loss of an N-glycan located near the HIV Env trimer apex, was detected in brain in some HAD patients, while position 200 was estimated to be under positive selection. D197 and T/V200 increased fusion and infection of cells expressing low CD4 by enhancing gp120 binding to CCR5. These results identify polymorphisms in the HIV gp120 bridging sheet that overcome the restriction to macrophage infection imposed by low CD4 through enhanced gp120–CCR5 interactions, thereby promoting infection of brain and other macrophage-rich tissues. - Highlights: • We analyze HIV Env sequences and identify amino acids in beta 3 of the gp120 bridging sheet that enhance macrophage tropism. • These amino acids at positions 197 and 200 are present in brain of some patients with HIV-associated dementia. • D197 results in loss of a glycan near the HIV Env trimer apex, which may increase exposure of V3. • These variants may promote infection of macrophages in the brain by enhancing gp120–CCR5 interactions

  5. Bioinformatic analysis of neurotropic HIV envelope sequences identifies polymorphisms in the gp120 bridging sheet that increase macrophage-tropism through enhanced interactions with CCR5

    Energy Technology Data Exchange (ETDEWEB)

    Mefford, Megan E., E-mail: megan_mefford@hms.harvard.edu [Department of Cancer Immunology and AIDS, Dana-Farber Cancer Institute, Boston, MA (United States); Kunstman, Kevin, E-mail: kunstman@northwestern.edu [Northwestern University Medical School, Chicago, IL (United States); Wolinsky, Steven M., E-mail: s-wolinsky@northwestern.edu [Northwestern University Medical School, Chicago, IL (United States); Gabuzda, Dana, E-mail: dana_gabuzda@dfci.harvard.edu [Department of Cancer Immunology and AIDS, Dana-Farber Cancer Institute, Boston, MA (United States); Department of Neurology (Microbiology and Immunobiology), Harvard Medical School, Boston, MA (United States)

    2015-07-15

    Macrophages express low levels of the CD4 receptor compared to T-cells. Macrophage-tropic HIV strains replicating in brain of untreated patients with HIV-associated dementia (HAD) express Envs that are adapted to overcome this restriction through mechanisms that are poorly understood. Here, bioinformatic analysis of env sequence datasets together with functional studies identified polymorphisms in the β3 strand of the HIV gp120 bridging sheet that increase M-tropism. D197, which results in loss of an N-glycan located near the HIV Env trimer apex, was detected in brain in some HAD patients, while position 200 was estimated to be under positive selection. D197 and T/V200 increased fusion and infection of cells expressing low CD4 by enhancing gp120 binding to CCR5. These results identify polymorphisms in the HIV gp120 bridging sheet that overcome the restriction to macrophage infection imposed by low CD4 through enhanced gp120–CCR5 interactions, thereby promoting infection of brain and other macrophage-rich tissues. - Highlights: • We analyze HIV Env sequences and identify amino acids in beta 3 of the gp120 bridging sheet that enhance macrophage tropism. • These amino acids at positions 197 and 200 are present in brain of some patients with HIV-associated dementia. • D197 results in loss of a glycan near the HIV Env trimer apex, which may increase exposure of V3. • These variants may promote infection of macrophages in the brain by enhancing gp120–CCR5 interactions.

  6. Sequence requirements of the HIV-1 protease flap region determined by saturation mutagenesis and kinetic analysis of flap mutants

    Science.gov (United States)

    Shao, Wei; Everitt, Lorraine; Manchester, Marianne; Loeb, Daniel D.; Hutchison, Clyde A.; Swanstrom, Ronald

    1997-01-01

    The retroviral proteases (PRs) have a structural feature called the flap, which consists of a short antiparallel β-sheet with a turn. The flap extends over the substrate binding cleft and must be flexible to allow entry and exit of the polypeptide substrates and products. We analyzed the sequence requirements of the amino acids within the flap region (positions 46–56) of the HIV-1 PR. The phenotypes of 131 substitution mutants were determined using a bacterial expression system. Four of the mutant PRs with mutations in different regions of the flap were selected for kinetic analysis. Our phenotypic analysis, considered in the context of published structures of the HIV-1 PR with a bound substrate analogs, shows that: (i) Met-46 and Phe-53 participate in hydrophobic interactions on the solvent-exposed face of the flap; (ii) Ile-47, Ile-54, and Val-56 participate in hydrophobic interactions on the inner face of the flap; (iii) Ile-50 has hydrophobic interactions at the distance of both the δ and γ carbons; (iv) the three glycine residues in the β-turn of the flap are virtually intolerant of substitutions. Among these mutant PRs, we have identified changes in both kcat and Km. These results establish the nature of the side chain requirements at each position in the flap and document a role for the flap in both substrate binding and catalysis. PMID:9122179

  7. An integrated genetic data environment (GDE)-based LINUX interface for analysis of HIV-1 and other microbial sequences.

    Science.gov (United States)

    De Oliveira, T; Miller, R; Tarin, M; Cassol, S

    2003-01-01

    Sequence databases encode a wealth of information needed to develop improved vaccination and treatment strategies for the control of HIV and other important pathogens. To facilitate effective utilization of these datasets, we developed a user-friendly GDE-based LINUX interface that reduces input/output file formatting. GDE was adapted to the Linux operating system, bioinformatics tools were integrated with microbe-specific databases, and up-to-date GDE menus were developed for several clinically important viral, bacterial and parasitic genomes. Each microbial interface was designed for local access and contains Genbank, BLAST-formatted and phylogenetic databases. GDE-Linux is available for research purposes by direct application to the corresponding author. Application-specific menus and support files can be downloaded from (http://www.bioafrica.net).

  8. High-throughput monitoring of integration site clonality in preclinical and clinical gene therapy studies

    Directory of Open Access Journals (Sweden)

    Frank A Giordano

    Full Text Available Gene transfer to hematopoietic stem cells with integrating vectors not only allows sustained correction of monogenic diseases but also tracking of individual clones in vivo. Quantitative real-time PCR (qPCR has been shown to be an accurate method to quantify individual stem cell clones, yet due to frequently limited amounts of target material (especially in clinical studies, it is not useful for large-scale analyses. To explore whether vector integration site (IS recovery techniques may be suitable to describe clonal contributions if combined with next-generation sequencing techniques, we designed artificial ISs of different sizes which were mixed to simulate defined clonal situations in clinical settings. We subjected all mixes to either linear amplification–mediated PCR (LAM-PCR or nonrestrictive LAM-PCR (nrLAM-PCR, both combined with 454 sequencing. We showed that nrLAM-PCR/454-detected clonality allows estimating qPCR-detected clonality in vitro. We then followed the kinetics of two clones detected in a patient enrolled in a clinical gene therapy trial using both, nrLAM-PCR/454 and qPCR and also saw nrLAM-PCR/454 to correlate to qPCR-measured clonal contributions. The method presented here displays a feasible high-throughput strategy to monitor clonality in clinical gene therapy trials is at hand.

  9. Comprehensive sieve analysis of breakthrough HIV-1 sequences in the RV144 vaccine efficacy trial.

    Directory of Open Access Journals (Sweden)

    Paul T Edlefsen

    2015-02-01

    Full Text Available The RV144 clinical trial showed the partial efficacy of a vaccine regimen with an estimated vaccine efficacy (VE of 31% for protecting low-risk Thai volunteers against acquisition of HIV-1. The impact of vaccine-induced immune responses can be investigated through sieve analysis of HIV-1 breakthrough infections (infected vaccine and placebo recipients. A V1/V2-targeted comparison of the genomes of HIV-1 breakthrough viruses identified two V2 amino acid sites that differed between the vaccine and placebo groups. Here we extended the V1/V2 analysis to the entire HIV-1 genome using an array of methods based on individual sites, k-mers and genes/proteins. We identified 56 amino acid sites or "signatures" and 119 k-mers that differed between the vaccine and placebo groups. Of those, 19 sites and 38 k-mers were located in the regions comprising the RV144 vaccine (Env-gp120, Gag, and Pro. The nine signature sites in Env-gp120 were significantly enriched for known antibody-associated sites (p = 0.0021. In particular, site 317 in the third variable loop (V3 overlapped with a hotspot of antibody recognition, and sites 369 and 424 were linked to CD4 binding site neutralization. The identified signature sites significantly covaried with other sites across the genome (mean = 32.1 more than did non-signature sites (mean = 0.9 (p < 0.0001, suggesting functional and/or structural relevance of the signature sites. Since signature sites were not preferentially restricted to the vaccine immunogens and because most of the associations were insignificant following correction for multiple testing, we predict that few of the genetic differences are strongly linked to the RV144 vaccine-induced immune pressure. In addition to presenting results of the first complete-genome analysis of the breakthrough infections in the RV144 trial, this work describes a set of statistical methods and tools applicable to analysis of breakthrough infection genomes in general vaccine

  10. Recoding method that removes inhibitory sequences and improves HIV gene expression

    Energy Technology Data Exchange (ETDEWEB)

    Rabadan, Raul; Krasnitz, Michael; Robins, Harlan; Witten, Daniela; Levine, Arnold

    2016-08-23

    The invention relates to inhibitory nucleotide signal sequences or "INS" sequences in the genomes of lentiviruses. In particular the invention relates to the AGG motif present in all viral genomes. The AGG motif may have an inhibitory effect on a virus, for example by reducing the levels of, or maintaining low steady-state levels of, viral RNAs in host cells, and inducing and/or maintaining in viral latency. In one aspect, the invention provides vaccines that contain, or are produced from, viral nucleic acids in which the AGG sequences have been mutated. In another aspect, the invention provides methods and compositions for affecting the function of the AGG motif, and methods for identifying other INS sequences in viral genomes.

  11. Clonal architectures and driver mutations in metastatic melanomas.

    Directory of Open Access Journals (Sweden)

    Li Ding

    Full Text Available To reveal the clonal architecture of melanoma and associated driver mutations, whole genome sequencing (WGS and targeted extension sequencing were used to characterize 124 melanoma cases. Significantly mutated gene analysis using 13 WGS cases and 15 additional paired extension cases identified known melanoma genes such as BRAF, NRAS, and CDKN2A, as well as a novel gene EPHA3, previously implicated in other cancer types. Extension studies using tumors from another 96 patients discovered a large number of truncation mutations in tumor suppressors (TP53 and RB1, protein phosphatases (e.g., PTEN, PTPRB, PTPRD, and PTPRT, as well as chromatin remodeling genes (e.g., ASXL3, MLL2, and ARID2. Deep sequencing of mutations revealed subclones in the majority of metastatic tumors from 13 WGS cases. Validated mutations from 12 out of 13 WGS patients exhibited a predominant UV signature characterized by a high frequency of C->T transitions occurring at the 3' base of dipyrimidine sequences while one patient (MEL9 with a hypermutator phenotype lacked this signature. Strikingly, a subclonal mutation signature analysis revealed that the founding clone in MEL9 exhibited UV signature but the secondary clone did not, suggesting different mutational mechanisms for two clonal populations from the same tumor. Further analysis of four metastases from different geographic locations in 2 melanoma cases revealed phylogenetic relationships and highlighted the genetic alterations responsible for differential drug resistance among metastatic tumors. Our study suggests that clonal evaluation is crucial for understanding tumor etiology and drug resistance in melanoma.

  12. Evaluation of the Roche prototype 454 HIV-1 ultradeep sequencing drug resistance assay in a routine diagnostic laboratory.

    Science.gov (United States)

    Garcia-Diaz, A; Guerrero-Ramos, A; McCormick, A L; Macartney, M; Conibear, T; Johnson, M A; Haque, T; Webster, D P

    2013-10-01

    Studies have shown that low-frequency resistance mutations can influence treatment outcome. However, the lack of a standardized high-throughput assay has precluded their detection in clinical settings. To evaluate the performance of the Roche prototype 454 UDS HIV-1 drug resistance assay (UDS assay) in a routine diagnostic laboratory. 50 plasma samples, previously characterized by population sequencing and that had shown ≥1 resistance associated mutation (RAM), were retrospectively tested by the UDS assay, including 18 B and 32 non-B subtypes; viral loads between 114-1,806,407 cp/ml; drug-naive (n=27) and drug-experienced (n=23) individuals. The UDS assay was successful for 37/50 (74%) samples. It detected all RAMs found by population sequencing at frequencies above 20%. In addition, 39 low-frequency RAMs were exclusively detected by the UDS assay at frequencies below 20% in both drug-naïve (19/26, 73%) and drug-experienced (9/18, 50%) individuals. UDS results would lead to changes from susceptible to resistant to efavirenz (EFV) in one drug-naive individual with suboptimal response to an EFV-containing regimen and from susceptible to resistance to lamivudine (3TC) in one drug naïve subject who subsequently failed a 3TC-containing regimen and in a treatment experienced subject who had failed a 3TC-containing regimen. The UDS assay performed well across a wide range of subtypes and viral loads; it showed perfect agreement with population sequencing for all RAMs analyzed. In addition, the UDS assay detected additional mutations at frequencies below 20% which correlate with patients' treatment history and had in some cases important prognostic implications. Copyright © 2013 Elsevier B.V. All rights reserved.

  13. Investigation of Sequence Clipping and Structural Heterogeneity of an HIV Broadly Neutralizing Antibody by a Comprehensive LC-MS Analysis

    Science.gov (United States)

    Ivleva, Vera B.; Schneck, Nicole A.; Gollapudi, Deepika; Arnold, Frank; Cooper, Jonathan W.; Lei, Q. Paula

    2018-05-01

    CAP256 is one of the highly potent, broadly neutralizing monoclonal antibodies (bNAb) designed for HIV-1 therapy. During the process development of one of the constructs, an unexpected product-related impurity was observed via microfluidics gel electrophoresis. A panel of complementary LC-MS analyses was applied for the comprehensive characterization of CAP256 which included the analysis of the intact and reduced protein, the middle-up approach, and a set of complementary peptide mapping techniques and verification of the disulfide bonds. The designed workflow allowed to identify a clip within a protruding acidic loop in the CDR-H3 region of the heavy chain, which can lead to the decrease of bNAb potency. This characterization explained the origin of the additional species reflected by the reducing gel profile. An intra-loop disulfide bond linking the two fragments was identified, which explained why the non-reducing capillary electrophoresis (CE) profile was not affected. The extensive characterization of CAP256 post-translational modifications was performed to investigate a possible cause of CE profile complexity and to illustrate other structural details related to this molecule's biological function. Two sites of the engineered Tyr sulfation were verified in the antigen-binding loop, and pyroglutamate formation was used as a tool for monitoring the extent of antibody clipping. Overall, the comprehensive LC-MS study was crucial to (1) identify the impurity as sequence clipping, (2) pinpoint the clipping location and justify its susceptibility relative to the molecular structure, (3) lead to an upstream process optimization to mitigate product quality risk, and (4) ultimately re-engineer the sequence to be clip-resistant. [Figure not available: see fulltext.

  14. Stress- and sequence-dependent release into the culture medium of HIV-1 Nef produced in Saccharomyces cerevisiae.

    Science.gov (United States)

    Macreadie, I G; Castelli, L A; Lucantoni, A; Azad, A A

    1995-09-11

    We have produced human immunodeficiency virus type 1 (HIV-1) Nef (a myristylated 206-amino-acid protein) in Saccharomyces cerevisaie and shown that, while Nef is normally found as a predominantly intracellular protein, amounts up to 40 micrograms/ml of Nef are also released into the extracellular medium during stress. By electrophoretic (SDS-PAGE) analysis the extracellular Nef is indistinguishable from intracellular Nef. Conditions of stress that lead to the release of Nef include elevated levels of copper or magnesium ions or growth at elevated temperatures. This release appears to be dependent upon the N-terminal sequences of Nef, including the presence of a myristylation site. Our observations concerning Nef release in yeast suggest new ways in which the behaviour of Nef should be examined in order to gain further insights into the development of AIDS. If the release of Nef is important in the development of AIDS, our work reveals that Nef-associated symptoms may be reduced or delayed by reducing stresses, such as fevers.

  15. Evidence for Within-Host Genetic Recombination among the Human Pegiviral Strains in HIV Infected Subjects.

    Science.gov (United States)

    Wu, Haoming; Padhi, Abinash; Xu, Junqiang; Gong, Xiaoyan; Tien, Po

    2016-01-01

    The non-pathogenic Human Pegivirus (HPgV, formerly GBV-C/HGV), the most prevalent RNA virus worldwide, is known to be associated with reduced morbidity and mortality in HIV-infected individuals. Although previous studies documented its ubiquity and important role in HIV-infected individuals, little is known about the underlying genetic mechanisms that maintain high genetic diversity of HPgV within the HIV-infected individuals. To assess the within-host genetic diversity of HPgV and forces that maintain such diversity within the co-infected hosts, we performed phylogenetic analyses taking into account 229 HPgV partial E1-E2 clonal sequences representing 15 male and 8 female co-infected HIV patients from Hubei province of central China. Our results revealed the presence of eleven strongly supported clades. While nine clades belonged to genotype 3, two clades belonged to genotype 2. Additionally, four clades that belonged to genotype 3 exhibited inter-clade recombination events. The presence of clonal sequences representing multiple clades within the HIV-infected individual provided the evidence of co-circulation of HPgV strains across the region. Of the 23 patients, six patients (i.e., five males and one female) were detected to have HPgV recombinant sequences. Our results also revealed that while male patients shared the viral strains with other patients, viral strains from the female patients had restricted dispersal. Taken together, the present study revealed that multiple infections with divergent HPgV viral strains may have caused within-host genetic recombination, predominantly in male patients, and therefore, could be the major driver in shaping genetic diversity of HPgV.

  16. HIV/HTLV-1 co-infection

    African Journals Online (AJOL)

    result of a lymphoproliferative disorder. In the context of HIV co-infection, lympho- cytosis has been described during early sero- conversion associated with CMV, as well as in HIV/HTLV-1 co-infection where CD4+ lymphocytosis can be caused by both a reactive or clonal expansion. Consequently, patients with untreated ...

  17. Fatal septicemia linked to transmission of MRSA clonal complex 398 in hospital and nursing home, Denmark

    DEFF Research Database (Denmark)

    Nielsen, Rikke Thoft; Kemp, Michael; Holm, Anette

    2016-01-01

    We describe 2 fatal cases of methicillin-resistant Staphylococcus aureus (MRSA) clonal complex 398 septicemia in persons who had no contact with livestock. Whole-genome sequencing of the isolated MRSA strains strongly suggest that both were of animal origin and that the patients had been infected...

  18. Targeted resequencing for analysis of clonal composition of recurrent gene mutations in chronic lymphocytic leukaemia

    NARCIS (Netherlands)

    Jethwa, Alexander; Hüllein, Jennifer; Stolz, Tatjana; Blume, Carolin; Sellner, Leopold; Jauch, Anna; Sill, Martin; Kater, Arnon P.; te Raa, G. Doreen; Geisler, Christian; van Oers, Marinus; Dietrich, Sascha; Dreger, Peter; Ho, Anthony D.; Paruzynski, Anna; Schmidt, Manfred; von Kalle, Christof; Glimm, Hanno; Zenz, Thorsten

    2013-01-01

    Recurrent gene mutations contribute to the pathogenesis of chronic lymphocytic leukaemia (CLL). We developed a next-generation sequencing (NGS) platform to determine the genetic profile, intratumoural heterogeneity, and clonal structure of two independent CLL cohorts. TP53, SF3B1, and NOTCH1 were

  19. Clonal diversity of Staphylococcus aureus originating from the small ruminants goats and sheep

    DEFF Research Database (Denmark)

    Concepción Porrero, M.; Hasman, Henrik; Vela, Ana I.

    2012-01-01

    Staphylococcus aureus is an important pathogen in humans and many animal species. The prevalence of different clonal types in animal species remains largely unknown. We analyzed 267 S. aureus from intramammary infections in goats (47) and sheep (220) by spa typing, multi-locus sequence typing (ML...

  20. Targeted resequencing for analysis of clonal composition of recurrent gene mutations in chronic lymphocytic leukaemia

    DEFF Research Database (Denmark)

    Jethwa, Alexander; Hüllein, Jennifer; Stolz, Tatjana

    2013-01-01

    Recurrent gene mutations contribute to the pathogenesis of chronic lymphocytic leukaemia (CLL). We developed a next-generation sequencing (NGS) platform to determine the genetic profile, intratumoural heterogeneity, and clonal structure of two independent CLL cohorts. TP53, SF3B1, and NOTCH1 were...

  1. Clonal growth and plant species abundance.

    Science.gov (United States)

    Herben, Tomáš; Nováková, Zuzana; Klimešová, Jitka

    2014-08-01

    Both regional and local plant abundances are driven by species' dispersal capacities and their abilities to exploit new habitats and persist there. These processes are affected by clonal growth, which is difficult to evaluate and compare across large numbers of species. This study assessed the influence of clonal reproduction on local and regional abundances of a large set of species and compared the predictive power of morphologically defined traits of clonal growth with data on actual clonal growth from a botanical garden. The role of clonal growth was compared with the effects of seed reproduction, habitat requirements and growth, proxied both by LHS (leaf-height-seed) traits and by actual performance in the botanical garden. Morphological parameters of clonal growth, actual clonal reproduction in the garden and LHS traits (leaf-specific area - height - seed mass) were used as predictors of species abundance, both regional (number of species records in the Czech Republic) and local (mean species cover in vegetation records) for 836 perennial herbaceous species. Species differences in habitat requirements were accounted for by classifying the dataset by habitat type and also by using Ellenberg indicator values as covariates. After habitat differences were accounted for, clonal growth parameters explained an important part of variation in species abundance, both at regional and at local levels. At both levels, both greater vegetative growth in cultivation and greater lateral expansion trait values were correlated with higher abundance. Seed reproduction had weaker effects, being positive at the regional level and negative at the local level. Morphologically defined traits are predictive of species abundance, and it is concluded that simultaneous investigation of several such traits can help develop hypotheses on specific processes (e.g. avoidance of self-competition, support of offspring) potentially underlying clonal growth effects on abundance. Garden

  2. High prevalence of HIV-1 transmitted drug-resistance mutations from proviral DNA massively parallel sequencing data of therapy-naïve chronically infected Brazilian blood donors.

    Directory of Open Access Journals (Sweden)

    Rodrigo Pessôa

    Full Text Available An improved understanding of the prevalence of low-abundance transmitted drug-resistance mutations (TDRM in therapy-naïve HIV-1-infected patients may help determine which patients are the best candidates for therapy. In this study, we aimed to obtain a comprehensive picture of the evolving HIV-1 TDRM across the massive parallel sequences (MPS of the viral entire proviral genome in a well-characterized Brazilian blood donor naïve to antiretroviral drugs.The MPS data from 128 samples used in the analysis were sourced from Brazilian blood donors and were previously classified by less-sensitive (LS or "detuned" enzyme immunoassay as non-recent or longstanding HIV-1 infections. The Stanford HIV Resistance Database (HIVDBv 6.2 and IAS-USA mutation lists were used to interpret the pattern of drug resistance. The minority variants with TDRM were identified using a threshold of ≥ 1.0% and ≤ 20% of the reads sequenced. The rate of TDRM in the MPS data of the proviral genome were compared with the corresponding published consensus sequences of their plasma viruses.No TDRM were detected in the integrase or envelope regions. The overall prevalence of TDRM in the protease (PR and reverse transcriptase (RT regions of the HIV-1 pol gene was 44.5% (57/128, including any mutations to the nucleoside analogue reverse transcriptase inhibitors (NRTI and non-nucleoside analogue reverse transcriptase inhibitors (NNRTI. Of the 57 subjects, 43 (75.4% harbored a minority variant containing at least one clinically relevant TDRM. Among the 43 subjects, 33 (76.7% had detectable minority resistant variants to NRTIs, 6 (13.9% to NNRTIs, and 16 (37.2% to PR inhibitors. The comparison of viral sequences in both sources, plasma and cells, would have detected 48 DNA provirus disclosed TDRM by MPS previously missed by plasma bulk analysis.Our findings revealed a high prevalence of TDRM found in this group, as the use of MPS drastically increased the detection of these

  3. Emergence of clonal hematopoiesis in the majority of patients with acquired aplastic anemia.

    Science.gov (United States)

    Babushok, Daria V; Perdigones, Nieves; Perin, Juan C; Olson, Timothy S; Ye, Wenda; Roth, Jacquelyn J; Lind, Curt; Cattier, Carine; Li, Yimei; Hartung, Helge; Paessler, Michele E; Frank, Dale M; Xie, Hongbo M; Cross, Shanna; Cockroft, Joshua D; Podsakoff, Gregory M; Monos, Dimitrios; Biegel, Jaclyn A; Mason, Philip J; Bessler, Monica

    2015-04-01

    Acquired aplastic anemia (aAA) is a nonmalignant disease caused by autoimmune destruction of early hematopoietic cells. Clonal hematopoiesis is a late complication, seen in 20-25% of older patients. We hypothesized that clonal hematopoiesis in aAA is a more general phenomenon, which can arise early in disease, even in younger patients. To evaluate clonal hematopoiesis in aAA, we used comparative whole exome sequencing of paired bone marrow and skin samples in 22 patients. We found somatic mutations in 16 patients (72.7%) with a median disease duration of 1 year; of these, 12 (66.7%) were patients with pediatric-onset aAA. Fifty-eight mutations in 51 unique genes were found primarily in pathways of immunity and transcriptional regulation. Most frequently mutated was PIGA, with seven mutations. Only two mutations were in genes recurrently mutated in myelodysplastic syndrome. Two patients had oligoclonal loss of the HLA alleles, linking immune escape to clone emergence. Two patients had activating mutations in key signaling pathways (STAT5B (p.N642H) and CAMK2G (p.T306M)). Our results suggest that clonal hematopoiesis in aAA is common, with two mechanisms emerging-immune escape and increased proliferation. Our findings expand conceptual understanding of this nonneoplastic blood disorder. Future prospective studies of clonal hematopoiesis in aAA will be critical for understanding outcomes and for designing personalized treatment strategies. Copyright © 2015 Elsevier Inc. All rights reserved.

  4. Somatic Mutations and Clonal Hematopoiesis in Aplastic Anemia.

    Science.gov (United States)

    Yoshizato, Tetsuichi; Dumitriu, Bogdan; Hosokawa, Kohei; Makishima, Hideki; Yoshida, Kenichi; Townsley, Danielle; Sato-Otsubo, Aiko; Sato, Yusuke; Liu, Delong; Suzuki, Hiromichi; Wu, Colin O; Shiraishi, Yuichi; Clemente, Michael J; Kataoka, Keisuke; Shiozawa, Yusuke; Okuno, Yusuke; Chiba, Kenichi; Tanaka, Hiroko; Nagata, Yasunobu; Katagiri, Takamasa; Kon, Ayana; Sanada, Masashi; Scheinberg, Phillip; Miyano, Satoru; Maciejewski, Jaroslaw P; Nakao, Shinji; Young, Neal S; Ogawa, Seishi

    2015-07-02

    In patients with acquired aplastic anemia, destruction of hematopoietic cells by the immune system leads to pancytopenia. Patients have a response to immunosuppressive therapy, but myelodysplastic syndromes and acute myeloid leukemia develop in about 15% of the patients, usually many months to years after the diagnosis of aplastic anemia. We performed next-generation sequencing and array-based karyotyping using 668 blood samples obtained from 439 patients with aplastic anemia. We analyzed serial samples obtained from 82 patients. Somatic mutations in myeloid cancer candidate genes were present in one third of the patients, in a limited number of genes and at low initial variant allele frequency. Clonal hematopoiesis was detected in 47% of the patients, most frequently as acquired mutations. The prevalence of the mutations increased with age, and mutations had an age-related signature. DNMT3A-mutated and ASXL1-mutated clones tended to increase in size over time; the size of BCOR- and BCORL1-mutated and PIGA-mutated clones decreased or remained stable. Mutations in PIGA and BCOR and BCORL1 correlated with a better response to immunosuppressive therapy and longer and a higher rate of overall and progression-free survival; mutations in a subgroup of genes that included DNMT3A and ASXL1 were associated with worse outcomes. However, clonal dynamics were highly variable and might not necessarily have predicted the response to therapy and long-term survival among individual patients. Clonal hematopoiesis was prevalent in aplastic anemia. Some mutations were related to clinical outcomes. A highly biased set of mutations is evidence of Darwinian selection in the failed bone marrow environment. The pattern of somatic clones in individual patients over time was variable and frequently unpredictable. (Funded by Grant-in-Aid for Scientific Research and others.).

  5. How to use sequence analysis for life course epidemiology? An example on HIV-positive Sub-Saharan migrants in France.

    Science.gov (United States)

    Gosselin, Anne; Desgrées du Loû, Annabel; Lelièvre, Eva

    2018-06-01

    Life course epidemiology is now an established field in social epidemiology; in sociodemography, the quantitative analysis of biographies recently experienced significant trend from event history analysis to sequence analysis. The purpose of this article is to introduce and adapt this methodology to a social epidemiology question, taking the example of the impact of HIV diagnosis on Sub-Saharan migrants' residential trajectories in the Paris region. The sample consists of 640 migrants born in Sub-Saharan Africa receiving HIV care. They were interviewed in healthcare facilities in the Paris region within the PARCOURS project, conducted from 2012 to 2013, using life event history calendars, which recorded year by year their health, family and residential histories. We introduce a two-step methodological approach consisting of (1) sequence analysis by optimal matching to build a typology of migrants' residential pathways before and after diagnosis, and (2) a Cox model of the probability to experience changes in the residential situation. The seven-clusters typology shows that for a majority, the HIV diagnosis did not entail changes in residential situation. However 30% of the migrants experienced a change in their residential situation at time of diagnosis. The Cox model analysis reveals that this residential change was in fact moving in with one's partner (HR 2.99, P<0.000) rather than network rejection. This original combination of sequence analysis and Cox models is a powerful process that could be applied to other themes and constitutes a new approach in the life course epidemiology toolbox. NCT02566148. © Article author(s) (or their employer(s) unless otherwise stated in the text of the article) 2018. All rights reserved. No commercial use is permitted unless otherwise expressly granted.

  6. Age-related cancer mutations associated with clonal hematopoietic expansion

    Science.gov (United States)

    Xie, Mingchao; Lu, Charles; Wang, Jiayin; McLellan, Michael D.; Johnson, Kimberly J.; Wendl, Michael C.; McMichael, Joshua F.; Schmidt, Heather K.; Yellapantula, Venkata; Miller, Christopher A.; Ozenberger, Bradley A.; Welch, John S.; Link, Daniel C.; Walter, Matthew J.; Mardis, Elaine R.; Dipersio, John F.; Chen, Feng; Wilson, Richard K.; Ley, Timothy J.; Ding, Li

    2015-01-01

    Several genetic alterations characteristic of leukemia and lymphoma have been detected in the blood of individuals without apparent hematological malignancies. We analyzed blood-derived sequence data from 2,728 individuals within The Cancer Genome Atlas, and discovered 77 blood-specific mutations in cancer-associated genes, the majority being associated with advanced age. Remarkably, 83% of these mutations were from 19 leukemia/lymphoma-associated genes, and nine were recurrently mutated (DNMT3A, TET2, JAK2, ASXL1, TP53, GNAS, PPM1D, BCORL1 and SF3B1). We identified 14 additional mutations in a very small fraction of blood cells, possibly representing the earliest stages of clonal expansion in hematopoietic stem cells. Comparison of these findings to mutations in hematological malignancies identified several recurrently mutated genes that may be disease initiators. Our analyses show that the blood cells of more than 2% of individuals (5–6% of people older than 70 years) contain mutations that may represent premalignant, initiating events that cause clonal hematopoietic expansion. PMID:25326804

  7. Escherichia coli ST131, an Intriguing Clonal Group

    Science.gov (United States)

    Bertrand, Xavier; Madec, Jean-Yves

    2014-01-01

    SUMMARY In 2008, a previously unknown Escherichia coli clonal group, sequence type 131 (ST131), was identified on three continents. Today, ST131 is the predominant E. coli lineage among extraintestinal pathogenic E. coli (ExPEC) isolates worldwide. Retrospective studies have suggested that it may originally have risen to prominence as early as 2003. Unlike other classical group B2 ExPEC isolates, ST131 isolates are commonly reported to produce extended-spectrum β-lactamases, such as CTX-M-15, and almost all are resistant to fluoroquinolones. Moreover, ST131 E. coli isolates are considered to be truly pathogenic, due to the spectrum of infections they cause in both community and hospital settings and the large number of virulence-associated genes they contain. ST131 isolates therefore seem to contradict the widely held view that high levels of antimicrobial resistance are necessarily associated with a fitness cost leading to a decrease in pathogenesis. Six years after the first description of E. coli ST131, this review outlines the principal traits of ST131 clonal group isolates, based on the growing body of published data, and highlights what is currently known and what we need to find out to provide public health authorities with better information to help combat ST131. PMID:24982321

  8. Rhizome elongation and seagrass clonal growth

    NARCIS (Netherlands)

    Marbà, N.; Duarte, C.M.

    1998-01-01

    A compilation of published and original data on rhizome morphometry, horizontal and vertical elongation rates and branching patterns for 27 seagrass species developing in 192 seagrass stands allowed an examination of the variability of seagrass rhizome and clonal growth programmes across and within

  9. Distribution of clonal growth forms in wetlands

    Czech Academy of Sciences Publication Activity Database

    Sosnová, Monika; van Diggelen, R.; Klimešová, Jitka

    2010-01-01

    Roč. 92, č. 1 (2010), s. 33-39 ISSN 0304-3770 R&D Projects: GA ČR GD206/08/H044 Institutional research plan: CEZ:AV0Z60050516 Keywords : clonal growth organ * the Netherlands * wetland plant community Subject RIV: EF - Botanics Impact factor: 2.087, year: 2010

  10. Quality improvement in Vignoles through clonal selection

    Science.gov (United States)

    Our goal is to select an improved, loose-clustered clone of Vignoles that will contribute to an integrated approach to disease control. Clonal selection has historically proven useful in reducing cluster compactness through a variety of mechanisms, including decreased berry size, lengthening of the ...

  11. Clonal growth and plant species abundance

    Czech Academy of Sciences Publication Activity Database

    Herben, Tomáš; Nováková, Z.; Klimešová, Jitka

    2014-01-01

    Roč. 114, č. 2 (2014), s. 377-388 ISSN 0305-7364 R&D Projects: GA ČR GA526/09/0963 Institutional support: RVO:67985939 Keywords : clonal plants * frequency * plant communities of Central Europe Subject RIV: EF - Botanics Impact factor: 3.654, year: 2014

  12. Clonality analysis of lymphoid proliferations using the BIOMED-2 clonality assays: a single institution experience

    Science.gov (United States)

    Kokovic, Ira; Novakovic, Barbara Jezersek; Cerkovnik, Petra; Novakovic, Srdjan

    2014-01-01

    Background Clonality determination in patients with lymphoproliferative disorders can improve the final diagnosis. The aim of our study was to evaluate the applicative value of standardized BIOMED-2 gene clonality assay protocols for the analysis of clonality of lymphocytes in a group of different lymphoid proliferations. Materials and methods. With this purpose, 121 specimens from 91 patients with suspected lymphoproliferations submitted for routine diagnostics from January to December 2011 were retrospectively analyzed. According to the final diagnosis, our series comprised 32 cases of B-cell lymphomas, 38 cases of non-Hodgkin’s T-cell lymphomas and 51 cases of reactive lymphoid proliferations. Clonality testing was performed using the BIOMED-2 clonality assays. Results The determined sensitivity of the TCR assay was 91.9%, while the sensitivity of the IGH assay was 74.2%. The determined specificity of the IGH assay was 73.3% in the group of lymphomas and 87.2% in the group of reactive lesions. The determined specificity of the TCR assay was 62.5% in the group of lymphomas and 54.3% in the group of reactive lesions. Conclusions In the present study, we confirmed the utility of standardized BIOMED-2 clonality assays for the detection of clonality in a routine diagnostical setting of non-Hodgkin’s lymphomas. Reactions for the detection of the complete IGH rearrangements and reactions for the detection of the TCR rearrangements are a good choice for clonality testing of a wide range of lymphoid proliferations and specimen types while the reactions for the detection of incomplete IGH rearrangements have not shown any additional diagnostic value. PMID:24991205

  13. Sequence-specific activation of the DNA sensor cGAS by Y-form DNA structures as found in primary HIV-1 cDNA.

    Science.gov (United States)

    Herzner, Anna-Maria; Hagmann, Cristina Amparo; Goldeck, Marion; Wolter, Steven; Kübler, Kirsten; Wittmann, Sabine; Gramberg, Thomas; Andreeva, Liudmila; Hopfner, Karl-Peter; Mertens, Christina; Zillinger, Thomas; Jin, Tengchuan; Xiao, Tsan Sam; Bartok, Eva; Coch, Christoph; Ackermann, Damian; Hornung, Veit; Ludwig, Janos; Barchet, Winfried; Hartmann, Gunther; Schlee, Martin

    2015-10-01

    Cytosolic DNA that emerges during infection with a retrovirus or DNA virus triggers antiviral type I interferon responses. So far, only double-stranded DNA (dsDNA) over 40 base pairs (bp) in length has been considered immunostimulatory. Here we found that unpaired DNA nucleotides flanking short base-paired DNA stretches, as in stem-loop structures of single-stranded DNA (ssDNA) derived from human immunodeficiency virus type 1 (HIV-1), activated the type I interferon-inducing DNA sensor cGAS in a sequence-dependent manner. DNA structures containing unpaired guanosines flanking short (12- to 20-bp) dsDNA (Y-form DNA) were highly stimulatory and specifically enhanced the enzymatic activity of cGAS. Furthermore, we found that primary HIV-1 reverse transcripts represented the predominant viral cytosolic DNA species during early infection of macrophages and that these ssDNAs were highly immunostimulatory. Collectively, our study identifies unpaired guanosines in Y-form DNA as a highly active, minimal cGAS recognition motif that enables detection of HIV-1 ssDNA.

  14. Simple PCR assays improve the sensitivity of HIV-1 subtype B drug resistance testing and allow linking of resistance mutations.

    Directory of Open Access Journals (Sweden)

    Jeffrey A Johnson

    Full Text Available BACKGROUND: The success of antiretroviral therapy is known to be compromised by drug-resistant HIV-1 at frequencies detectable by conventional bulk sequencing. Currently, there is a need to assess the clinical consequences of low-frequency drug resistant variants occurring below the detection limit of conventional genotyping. Sensitive detection of drug-resistant subpopulations, however, requires simple and practical methods for routine testing. METHODOLOGY: We developed highly-sensitive and simple real-time PCR assays for nine key drug resistance mutations and show that these tests overcome substantial sequence heterogeneity in HIV-1 clinical specimens. We specifically used early wildtype virus samples from the pre-antiretroviral drug era to measure background reactivity and were able to define highly-specific screening cut-offs that are up to 67-fold more sensitive than conventional genotyping. We also demonstrate that sequencing the mutation-specific PCR products provided a direct and novel strategy to further detect and link associated resistance mutations, allowing easy identification of multi-drug-resistant variants. Resistance mutation associations revealed in mutation-specific amplicon sequences were verified by clonal sequencing. SIGNIFICANCE: Combined, sensitive real-time PCR testing and mutation-specific amplicon sequencing provides a powerful and simple approach that allows for improved detection and evaluation of HIV-1 drug resistance mutations.

  15. CD4-dependent characteristics of coreceptor use and HIV type 1 V3 sequence in a large population of therapy-naive individuals.

    Science.gov (United States)

    Low, Andrew J; Marchant, David; Brumme, Chanson J; Brumme, Zabrina L; Dong, Winnie; Sing, Tobias; Hogg, Robert S; Montaner, Julio S G; Gill, Vikram; Cheung, Peter K; Harrigan, P Richard

    2008-02-01

    We investigated the associations between coreceptor use, V3 loop sequence, and CD4 count in a cross-sectional analysis of a large cohort of chronically HIV-infected, treatment-naive patients. HIV coreceptor usage was determined in the last pretherapy plasma sample for 977 individuals initiating HAART in British Columbia, Canada using the Monogram Trofile Tropism assay. Relative light unit (RLU) readouts from the Trofile assay, as well as HIV V3 loop sequence data, were examined as a function of baseline CD4 cell count for 953 (97%) samples with both phenotype and genotype data available. Median CCR5 RLUs were high for both R5 and X4-capable samples, while CXCR4 RLUs were orders of magnitude lower for X4 samples (p < 0.001). CCR5 RLUs in R5 samples (N = 799) increased with decreasing CD4 count (p < 0.001), but did not vary with plasma viral load (pVL) (p = 0.74). In X4 samples (N = 178), CCR5 RLUs decreased with decreasing CD4 count (p = 0.046) and decreasing pVL (p = 0.097), while CXCR4 RLUs increased with decreasing pVL (p = 0.0008) but did not vary with CD4 (p = 0.96). RLUs varied with the presence of substitutions at V3 loop positions 11, 25, and 6-8. The prevalence and impact of substitutions at codons 25 and 6-8 were CD4 dependent as was the presence of amino acid mixtures in the V3; substitutions at position 11 were CD4 independent. Assay RLU measures predictably vary with both immunological and virological parameters. The ability to predict X4 virus using genotypic determinants at positions 25 and 6-8 of the V3 loop is CD4 dependent, while position 11 appears to be CD4 independent.

  16. Whole genome sequencing reveals mycobacterial microevolution among concurrent isolates from sputum and blood in HIV infected TB patients

    NARCIS (Netherlands)

    Ssengooba, Willy; de Jong, Bouke C.; Joloba, Moses L.; Cobelens, Frank G.; Meehan, Conor J.

    2016-01-01

    In the context of advanced immunosuppression, M. tuberculosis is known to cause detectable mycobacteremia. However, little is known about the intra-patient mycobacterial microevolution and the direction of seeding between the sputum and blood compartments. From a diagnostic study of HIV-infected TB

  17. Sequence similarity between the erythrocyte binding domain 1 of the Plasmodium vivax Duffy binding protein and the V3 loop of HIV-1 strain MN reveals binding residues for the Duffy Antigen Receptor for Chemokines

    OpenAIRE

    Bolton, Michael J; Garry, Robert F

    2011-01-01

    Abstract Background The surface glycoprotein (SU, gp120) of the human immunodeficiency virus (HIV) must bind to a chemokine receptor, CCR5 or CXCR4, to invade CD4+ cells. Plasmodium vivax uses the Duffy Binding Protein (DBP) to bind the Duffy Antigen Receptor for Chemokines (DARC) and invade reticulocytes. Results Variable loop 3 (V3) of HIV-1 SU and domain 1 of the Plasmodium vivax DBP share a sequence similarity. The site of amino acid sequence similarity was necessary, but not sufficient, ...

  18. Drug hypersensitivity in clonal mast cell disorders

    DEFF Research Database (Denmark)

    Bonadonna, P; Pagani, M; Aberer, W

    2015-01-01

    and severity of immediate hypersensitivity reactions. Mastocytosis in adults is associated with a history of anaphylaxis in 22-49%. Fatal anaphylaxis has been described particularly following hymenoptera stings, but also occasionally after the intake of drugs such as nonsteroidal anti-inflammatory drugs......, opioids and drugs in the perioperative setting. However, data on the frequency of drug hypersensitivity in mastocytosis and vice versa are scarce and evidence for an association appears to be limited. Nevertheless, clonal MC disorders should be ruled out in cases of severe anaphylaxis: basal serum...... tryptase determination, physical examination for cutaneous mastocytosis lesions, and clinical characteristics of anaphylactic reaction might be useful for differential diagnosis. In this position paper, the ENDA group performed a literature search on immediate drug hypersensitivity reactions in clonal MC...

  19. Metabolic heterogeneity in clonal microbial populations.

    Science.gov (United States)

    Takhaveev, Vakil; Heinemann, Matthias

    2018-02-21

    In the past decades, numerous instances of phenotypic diversity were observed in clonal microbial populations, particularly, on the gene expression level. Much less is, however, known about phenotypic differences that occur on the level of metabolism. This is likely explained by the fact that experimental tools probing metabolism of single cells are still at an early stage of development. Here, we review recent exciting discoveries that point out different causes for metabolic heterogeneity within clonal microbial populations. These causes range from ecological factors and cell-inherent dynamics in constant environments to molecular noise in gene expression that propagates into metabolism. Furthermore, we provide an overview of current methods to quantify the levels of metabolites and biomass components in single cells. Copyright © 2018 The Authors. Published by Elsevier Ltd.. All rights reserved.

  20. [Chronologic analysis of clonal evolution in acquired aplastic anemia and sMDS].

    Science.gov (United States)

    Yoshizato, Tetsuichi

    2016-04-01

    Acquired aplastic anemia (AA) is a prototype of idiopathic bone marrow failure, which is caused by immune-mediated destruction of hematopoietic progenitors but is also characterized by frequent evolution to clonal myeloid disorders, such as myelodysplastic syndromes or acute myeloid leukemia. However, the chronological behavior of the clonality and its link to myelodysplastic syndrome or acute myeloid leukemia has not been fully explored. To define the clonality and its chronological behavior in AA, we performed targeted sequencing (N=439) in cases with AA. Somatic mutations were detected in 1/3 of our cases. Mutations were most frequently found in DNMT3A, followed by BCOR, PIGA and ASXL1. The prevalence of mutations increased with age. The clone sizes in DNMT3A and ASXL1 were prone to increase, whereas those of BCOR and PIGA were more likely to decrease or remain stable. Mutations in PIGA, BCOR and BCORL1 correlated with a better response to immunosuppressive therapy and more favorable survival. On the other hand, other mutations were associated with worse outcomes. The chronological dynamics of clonality showed marked variability and were not necessarily associated with prognosis.

  1. Analysis of the clonal repertoire of gene-corrected cells in gene therapy.

    Science.gov (United States)

    Paruzynski, Anna; Glimm, Hanno; Schmidt, Manfred; Kalle, Christof von

    2012-01-01

    Gene therapy-based clinical phase I/II studies using integrating retroviral vectors could successfully treat different monogenetic inherited diseases. However, with increased efficiency of this therapy, severe side effects occurred in various gene therapy trials. In all cases, integration of the vector close to or within a proto-oncogene contributed substantially to the development of the malignancies. Thus, the in-depth analysis of integration site patterns is of high importance to uncover potential clonal outgrowth and to assess the safety of gene transfer vectors and gene therapy protocols. The standard and nonrestrictive linear amplification-mediated PCR (nrLAM-PCR) in combination with high-throughput sequencing exhibits technologies that allow to comprehensively analyze the clonal repertoire of gene-corrected cells and to assess the safety of the used vector system at an early stage on the molecular level. It enables clarifying the biological consequences of the vector system on the fate of the transduced cell. Furthermore, the downstream performance of real-time PCR allows a quantitative estimation of the clonality of individual cells and their clonal progeny. Here, we present a guideline that should allow researchers to perform comprehensive integration site analysis in preclinical and clinical studies. Copyright © 2012 Elsevier Inc. All rights reserved.

  2. Clonality and Micro-Diversity of a Nationwide Spreading Genotype of Mycobacterium tuberculosis in Japan

    Science.gov (United States)

    Wada, Takayuki; Iwamoto, Tomotada; Tamaru, Aki; Seto, Junji; Ahiko, Tadayuki; Yamamoto, Kaori; Hase, Atushi; Maeda, Shinji; Yamamoto, Taro

    2015-01-01

    Mycobacterium tuberculosis transmission routes can be estimated from genotypic analysis of clinical isolates from patients. In Japan, still a middle-incidence country of TB, a unique genotype strain designated as ‘M-strain’ has been isolated nationwide recently. To ascertain the history of the wide spread of the strain, 10 clinical isolates from different areas were subjected to genome-wide analysis based on deep sequencers. Results show that all isolates possessed common mutations to those of referential strains. The greatest number of accumulated single nucleotide variants (SNVs) from the oldest coalescence was 13 nucleotides, indicating high clonality of these isolates. When an SNV common to the isolates was used as a surrogate marker of the clone, authentic clonal isolates with variation in a reliable subset of variable number of tandem repeat (VNTR) genotyping method can be selected successfully from clinical isolates populations of M. tuberculosis. When the authentic clones can also be assigned to sub-clonal groups by SNVs derived from the genomic comparison, they are classifiable into three sub-clonal groups with a bias of geographical origins. Feedback from genomic analysis of clinical isolates of M. tuberculosis to genotypic markers will be an efficient strategy for the big data in various settings for public health actions against TB. PMID:25734518

  3. A Clonal Lineage of Fusarium oxysporum Circulates in the Tap Water of Different French Hospitals.

    Science.gov (United States)

    Edel-Hermann, Véronique; Sautour, Marc; Gautheron, Nadine; Laurent, Julie; Aho, Serge; Bonnin, Alain; Sixt, Nathalie; Hartemann, Philippe; Dalle, Frédéric; Steinberg, Christian

    2016-11-01

    Fusarium oxysporum is typically a soilborne fungus but can also be found in aquatic environments. In hospitals, water distribution systems may be reservoirs for the fungi responsible for nosocomial infections. F. oxysporum was previously detected in the water distribution systems of five French hospitals. Sixty-eight isolates from water representative of all hospital units that were previously sampled and characterized by translation elongation factor 1α sequence typing were subjected to microsatellite analysis and full-length ribosomal intergenic spacer (IGS) sequence typing. All but three isolates shared common microsatellite loci and a common two-locus sequence type (ST). This ST has an international geographical distribution in both the water networks of hospitals and among clinical isolates. The ST dominant in water was not detected among 300 isolates of F. oxysporum that originated from surrounding soils. Further characterization of 15 isolates by vegetative compatibility testing allowed us to conclude that a clonal lineage of F. oxysporum circulates in the tap water of the different hospitals. We demonstrated that a clonal lineage of Fusarium oxysporum inhabits the water distribution systems of several French hospitals. This clonal lineage, which appears to be particularly adapted to water networks, represents a potential risk for human infection and raises questions about its worldwide distribution. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

  4. IDEPI: rapid prediction of HIV-1 antibody epitopes and other phenotypic features from sequence data using a flexible machine learning platform.

    Directory of Open Access Journals (Sweden)

    N Lance Hepler

    2014-09-01

    Full Text Available Since its identification in 1983, HIV-1 has been the focus of a research effort unprecedented in scope and difficulty, whose ultimate goals--a cure and a vaccine--remain elusive. One of the fundamental challenges in accomplishing these goals is the tremendous genetic variability of the virus, with some genes differing at as many as 40% of nucleotide positions among circulating strains. Because of this, the genetic bases of many viral phenotypes, most notably the susceptibility to neutralization by a particular antibody, are difficult to identify computationally. Drawing upon open-source general-purpose machine learning algorithms and libraries, we have developed a software package IDEPI (IDentify EPItopes for learning genotype-to-phenotype predictive models from sequences with known phenotypes. IDEPI can apply learned models to classify sequences of unknown phenotypes, and also identify specific sequence features which contribute to a particular phenotype. We demonstrate that IDEPI achieves performance similar to or better than that of previously published approaches on four well-studied problems: finding the epitopes of broadly neutralizing antibodies (bNab, determining coreceptor tropism of the virus, identifying compartment-specific genetic signatures of the virus, and deducing drug-resistance associated mutations. The cross-platform Python source code (released under the GPL 3.0 license, documentation, issue tracking, and a pre-configured virtual machine for IDEPI can be found at https://github.com/veg/idepi.

  5. Recent advances in understanding clonal haematopoiesis in aplastic anaemia.

    Science.gov (United States)

    Stanley, Natasha; Olson, Timothy S; Babushok, Daria V

    2017-05-01

    Acquired aplastic anaemia (AA) is an immune-mediated bone marrow failure disorder inextricably linked to clonal haematopoiesis. The majority of AA patients have somatic mutations and/or structural chromosomal abnormalities detected as early as at diagnosis. In contrast to other conditions linked to clonal haematopoiesis, the clonal signature of AA reflects its immune pathophysiology. The most common alterations are clonal expansions of cells lacking glycophosphotidylinositol-anchored proteins, loss of human leucocyte antigen alleles, and mutations in BCOR/BCORL1, ASXL1 and DNMT3A. Here, we present the current knowledge of clonal haematopoiesis in AA as it relates to aging, inherited bone marrow failure, and the grey-zone overlap of AA and myelodysplastic syndrome (MDS). We conclude by discussing the significance of clonal haematopoiesis both for improved diagnosis of AA, as well as for a more precise, personalized approach to prognostication of outcomes and therapy choices. © 2017 John Wiley & Sons Ltd.

  6. Clonality, virulence and antimicrobial resistance of enteroaggregative Escherichia coli from Mirzapur, Bangladesh.

    Science.gov (United States)

    Chattaway, Marie Anne; Day, Michaela; Mtwale, Julia; White, Emma; Rogers, James; Day, Martin; Powell, David; Ahmad, Marwa; Harris, Ross; Talukder, Kaisar Ali; Wain, John; Jenkins, Claire; Cravioto, Alejandro

    2017-10-01

    This study investigates the virulence and antimicrobial resistance in association with common clonal complexes (CCs) of enteroaggregative Escherichia coli (EAEC) isolated from Bangladesh. The aim was to determine whether specific CCs were more likely to be associated with putative virulence genes and/or antimicrobial resistance. The presence of 15 virulence genes (by PCR) and susceptibility to 18 antibiotics were determined for 151 EAEC isolated from cases and controls during an intestinal infectious disease study carried out between 2007-2011 in the rural setting of Mirzapur, Bangladesh (Kotloff KL, Blackwelder WC, Nasrin D, Nataro JP, Farag TH et al.Clin Infect Dis 2012;55:S232-S245). These data were then analysed in the context of previously determined serotypes and clonal complexes defined by multi-locus sequence typing. Overall there was no association between the presence of virulence or antimicrobial resistance genes in isolates of EAEC from cases versus controls. However, when stratified by clonal complex (CC) one CC associated with cases harboured more virulence factors (CC40) and one CC harboured more resistance genes (CC38) than the average. There was no direct link between the virulence gene content and antibiotic resistance. Strains within a single CC had variable virulence and resistance gene content indicating independent and multiple gene acquisitions over time. In Bangladesh, there are multiple clonal complexes of EAEC harbouring a variety of virulence and resistance genes. The emergence of two of the most successful clones appeared to be linked to either increased virulence (CC40) or antimicrobial resistance (CC38), but increased resistance and virulence were not found in the same clonal complexes.

  7. Prevalence and evolution of low frequency HIV drug resistance mutations detected by ultra deep sequencing in patients experiencing first line antiretroviral therapy failure.

    Science.gov (United States)

    Vandenhende, Marie-Anne; Bellecave, Pantxika; Recordon-Pinson, Patricia; Reigadas, Sandrine; Bidet, Yannick; Bruyand, Mathias; Bonnet, Fabrice; Lazaro, Estibaliz; Neau, Didier; Fleury, Hervé; Dabis, François; Morlat, Philippe; Masquelier, Bernard

    2014-01-01

    Clinical relevance of low-frequency HIV-1 variants carrying drug resistance associated mutations (DRMs) is still unclear. We aimed to study the prevalence of low-frequency DRMs, detected by Ultra-Deep Sequencing (UDS) before antiretroviral therapy (ART) and at virological failure (VF), in HIV-1 infected patients experiencing VF on first-line ART. Twenty-nine ART-naive patients followed up in the ANRS-CO3 Aquitaine Cohort, having initiated ART between 2000 and 2009 and experiencing VF (2 plasma viral loads (VL) >500 copies/ml or one VL >1000 copies/ml) were included. Reverse transcriptase and protease DRMs were identified using Sanger sequencing (SS) and UDS at baseline (before ART initiation) and VF. Additional low-frequency variants with PI-, NNRTI- and NRTI-DRMs were found by UDS at baseline and VF, significantly increasing the number of detected DRMs by 1.35 fold (plow-frequency DRMs modified ARV susceptibility predictions to the prescribed treatment for 1 patient at baseline, in whom low-frequency DRM was found at high frequency at VF, and 6 patients at VF. DRMs found at VF were rarely detected as low-frequency DRMs prior to treatment. The rare low-frequency NNRTI- and NRTI-DRMs detected at baseline that correlated with the prescribed treatment were most often found at high-frequency at VF. Low frequency DRMs detected before ART initiation and at VF in patients experiencing VF on first-line ART can increase the overall burden of resistance to PI, NRTI and NNRTI.

  8. Deep sequencing analysis of HIV-1 reverse transcriptase at baseline and time of failure in patients receiving rilpivirine in the phase III studies ECHO and THRIVE.

    Science.gov (United States)

    Van Eygen, Veerle; Thys, Kim; Van Hove, Carl; Rimsky, Laurence T; De Meyer, Sandra; Aerssens, Jeroen; Picchio, Gaston; Vingerhoets, Johan

    2016-05-01

    Minority variants (1.0-25.0%) were evaluated by deep sequencing (DS) at baseline and virological failure (VF) in a selection of antiretroviral treatment-naïve, HIV-1-infected patients from the rilpivirine ECHO/THRIVE phase III studies. Linkage between frequently emerging resistance-associated mutations (RAMs) was determined. DS (llIumina®) and population sequencing (PS) results were available at baseline for 47 VFs and time of failure for 48 VFs; and at baseline for 49 responders matched for baseline characteristics. Minority mutations were accurately detected at frequencies down to 1.2% of the HIV-1 quasispecies. No baseline minority rilpivirine RAMs were detected in VFs; one responder carried 1.9% F227C. Baseline minority mutations associated with resistance to other non-nucleoside reverse transcriptase inhibitors (NNRTIs) were detected in 8/47 VFs (17.0%) and 7/49 responders (14.3%). Baseline minority nucleoside/nucleotide reverse transcriptase inhibitor (NRTI) RAMs M184V and L210W were each detected in one VF (none in responders). At failure, two patients without NNRTI RAMs by PS carried minority rilpivirine RAMs K101E and/or E138K; and five additional patients carried other minority NNRTI RAMs V90I, V106I, V179I, V189I, and Y188H. Overall at failure, minority NNRTI RAMs and NRTI RAMs were found in 29/48 (60.4%) and 16/48 VFs (33.3%), respectively. Linkage analysis showed that E138K and K101E were usually not observed on the same viral genome. In conclusion, baseline minority rilpivirine RAMs and other NNRTI/NRTI RAMs were uncommon in the rilpivirine arm of the ECHO and THRIVE studies. DS at failure showed emerging NNRTI resistant minority variants in seven rilpivirine VFs who had no detectable NNRTI RAMs by PS. © 2015 Wiley Periodicals, Inc.

  9. The evolutionary ecology of clonally propagated domesticated plants.

    Science.gov (United States)

    McKey, Doyle; Elias, Marianne; Pujol, Benoît; Duputié, Anne

    2010-04-01

    While seed-propagated crops have contributed many evolutionary insights, evolutionary biologists have often neglected clonally propagated crops. We argue that widespread notions about their evolution under domestication are oversimplified, and that they offer rich material for evolutionary studies. The diversity of their wild ancestors, the diverse ecologies of the crop populations themselves, and the intricate mix of selection pressures, acting not only on the parts harvested but also on the parts used by humans to make clonal propagules, result in complex and diverse evolutionary trajectories under domestication. We examine why farmers propagate some plants clonally, and discuss the evolutionary dynamics of sexual reproduction in clonal crops. We explore how their mixed clonal/sexual reproductive systems function, based on the sole example studied in detail, cassava (Manihot esculenta). Biotechnology is now expanding the number of clonal crops, continuing the 10 000-yr-old trend to increase crop yields by propagating elite genotypes. In an era of rapid global change, it is more important than ever to understand how the adaptive potential of clonal crops can be maintained. A key component of strategies for preserving this adaptive potential is the maintenance of mixed clonal/sexual systems, which can be achieved by encouraging and valuing farmer knowledge about the sexual reproductive biology of their clonal crops.

  10. Effects of sequence changes in the HIV-1 gp41 fusion peptide on CCR5 inhibitor resistance

    International Nuclear Information System (INIS)

    Anastassopoulou, Cleo G.; Ketas, Thomas J.; Sanders, Rogier W.; Johan Klasse, Per; Moore, John P.

    2012-01-01

    A rare pathway of HIV-1 resistance to small molecule CCR5 inhibitors such as Vicriviroc (VCV) involves changes solely in the gp41 fusion peptide (FP). Here, we show that the G516V change is critical to VCV resistance in PBMC and TZM-bl cells, although it must be accompanied by either M518V or F519I to have a substantial impact. Modeling VCV inhibition data from the two cell types indicated that G516V allows both double mutants to use VCV-CCR5 complexes for entry. The model further identified F519I as an independent determinant of preference for the unoccupied, high-VCV affinity form of CCR5. From inhibitor-free reversion cultures, we also identified a substitution in the inner domain of gp120, T244A, which appears to counter the resistance phenotype created by the FP substitutions. Examining the interplay of these changes will enhance our understanding of Env complex interactions that influence both HIV-1 entry and resistance to CCR5 inhibitors.

  11. Extensive clonal spread and extreme longevity in saw palmetto, a foundation clonal plant.

    Science.gov (United States)

    Takahashi, Mizuki K; Horner, Liana M; Kubota, Toshiro; Keller, Nathan A; Abrahamson, Warren G

    2011-09-01

    The lack of effective tools has hampered out ability to assess the size, growth and ages of clonal plants. With Serenoa repens (saw palmetto) as a model, we introduce a novel analytical framework that integrates DNA fingerprinting and mathematical modelling to simulate growth and estimate ages of clonal plants. We also demonstrate the application of such life-history information of clonal plants to provide insight into management plans. Serenoa is an ecologically important foundation species in many Southeastern United States ecosystems; yet, many land managers consider Serenoa a troublesome invasive plant. Accordingly, management plans have been developed to reduce or eliminate Serenoa with little understanding of its life history. Using Amplified Fragment Length Polymorphisms, we genotyped 263 Serenoa and 134 Sabal etonia (a sympatric non-clonal palmetto) samples collected from a 20 × 20 m study plot in Florida scrub. Sabal samples were used to assign small field-unidentifiable palmettos to Serenoa or Sabal and also as a negative control for clone detection. We then mathematically modelled clonal networks to estimate genet ages. Our results suggest that Serenoa predominantly propagate via vegetative sprouts and 10,000-year-old genets may be common, while showing no evidence of clone formation by Sabal. The results of this and our previous studies suggest that: (i) Serenoa has been part of scrub associations for thousands of years, (ii) Serenoa invasion are unlikely and (ii) once Serenoa is eliminated from local communities, its restoration will be difficult. Reevaluation of the current management tools and plans is an urgent task. © 2011 Blackwell Publishing Ltd.

  12. Assessing T cell clonal size distribution: a non-parametric approach.

    Directory of Open Access Journals (Sweden)

    Olesya V Bolkhovskaya

    Full Text Available Clonal structure of the human peripheral T-cell repertoire is shaped by a number of homeostatic mechanisms, including antigen presentation, cytokine and cell regulation. Its accurate tuning leads to a remarkable ability to combat pathogens in all their variety, while systemic failures may lead to severe consequences like autoimmune diseases. Here we develop and make use of a non-parametric statistical approach to assess T cell clonal size distributions from recent next generation sequencing data. For 41 healthy individuals and a patient with ankylosing spondylitis, who undergone treatment, we invariably find power law scaling over several decades and for the first time calculate quantitatively meaningful values of decay exponent. It has proved to be much the same among healthy donors, significantly different for an autoimmune patient before the therapy, and converging towards a typical value afterwards. We discuss implications of the findings for theoretical understanding and mathematical modeling of adaptive immunity.

  13. Assessing T cell clonal size distribution: a non-parametric approach.

    Science.gov (United States)

    Bolkhovskaya, Olesya V; Zorin, Daniil Yu; Ivanchenko, Mikhail V

    2014-01-01

    Clonal structure of the human peripheral T-cell repertoire is shaped by a number of homeostatic mechanisms, including antigen presentation, cytokine and cell regulation. Its accurate tuning leads to a remarkable ability to combat pathogens in all their variety, while systemic failures may lead to severe consequences like autoimmune diseases. Here we develop and make use of a non-parametric statistical approach to assess T cell clonal size distributions from recent next generation sequencing data. For 41 healthy individuals and a patient with ankylosing spondylitis, who undergone treatment, we invariably find power law scaling over several decades and for the first time calculate quantitatively meaningful values of decay exponent. It has proved to be much the same among healthy donors, significantly different for an autoimmune patient before the therapy, and converging towards a typical value afterwards. We discuss implications of the findings for theoretical understanding and mathematical modeling of adaptive immunity.

  14. Electrochemical detection of short HIV sequences on chitosan/Fe3O4 nanoparticle based screen printed electrodes

    International Nuclear Information System (INIS)

    Tran, Lam Dai; Nguyen, Binh Hai; Van Hieu, Nguyen; Tran, Hoang Vinh; Nguyen, Huy Le; Nguyen, Phuc Xuan

    2011-01-01

    In this study, a novel CS/Fe 3 O 4 nanobiocomposite-based platform for electrochemical detection of HIV-1 was developed. The most attractive feature of this system is a suitable microenvironment (Fe 3 O 4 nanoparticles) which could contribute to electron transfer and thus sensitivity enhancement when using methylene blue (MB) as an external mediator and Square Wave Voltammetry (SWV), Electrochemical Impedance Spectroscopy (EIS) techniques. The proposed screen printed electrode (SPE) had a low detection limit (as low as 50 pM), acceptable stability and good reproducibility, which would be valuable for clinical diagnosis. In addition, this sensing interface may be feasibly adapted for multiplexed detection of other species of bacterial pathogens.

  15. Geographic and temporal trends in the molecular epidemiology and genetic mechanisms of transmitted HIV-1 drug resistance: an individual-patient- and sequence-level meta-analysis.

    Science.gov (United States)

    Rhee, Soo-Yon; Blanco, Jose Luis; Jordan, Michael R; Taylor, Jonathan; Lemey, Philippe; Varghese, Vici; Hamers, Raph L; Bertagnolio, Silvia; Rinke de Wit, Tobias F; Aghokeng, Avelin F; Albert, Jan; Avi, Radko; Avila-Rios, Santiago; Bessong, Pascal O; Brooks, James I; Boucher, Charles A B; Brumme, Zabrina L; Busch, Michael P; Bussmann, Hermann; Chaix, Marie-Laure; Chin, Bum Sik; D'Aquin, Toni T; De Gascun, Cillian F; Derache, Anne; Descamps, Diane; Deshpande, Alaka K; Djoko, Cyrille F; Eshleman, Susan H; Fleury, Herve; Frange, Pierre; Fujisaki, Seiichiro; Harrigan, P Richard; Hattori, Junko; Holguin, Africa; Hunt, Gillian M; Ichimura, Hiroshi; Kaleebu, Pontiano; Katzenstein, David; Kiertiburanakul, Sasisopin; Kim, Jerome H; Kim, Sung Soon; Li, Yanpeng; Lutsar, Irja; Morris, Lynn; Ndembi, Nicaise; Ng, Kee Peng; Paranjape, Ramesh S; Peeters, Martine; Poljak, Mario; Price, Matt A; Ragonnet-Cronin, Manon L; Reyes-Terán, Gustavo; Rolland, Morgane; Sirivichayakul, Sunee; Smith, Davey M; Soares, Marcelo A; Soriano, Vincent V; Ssemwanga, Deogratius; Stanojevic, Maja; Stefani, Mariane A; Sugiura, Wataru; Sungkanuparph, Somnuek; Tanuri, Amilcar; Tee, Kok Keng; Truong, Hong-Ha M; van de Vijver, David A M C; Vidal, Nicole; Yang, Chunfu; Yang, Rongge; Yebra, Gonzalo; Ioannidis, John P A; Vandamme, Anne-Mieke; Shafer, Robert W

    2015-04-01

    Regional and subtype-specific mutational patterns of HIV-1 transmitted drug resistance (TDR) are essential for informing first-line antiretroviral (ARV) therapy guidelines and designing diagnostic assays for use in regions where standard genotypic resistance testing is not affordable. We sought to understand the molecular epidemiology of TDR and to identify the HIV-1 drug-resistance mutations responsible for TDR in different regions and virus subtypes. We reviewed all GenBank submissions of HIV-1 reverse transcriptase sequences with or without protease and identified 287 studies published between March 1, 2000, and December 31, 2013, with more than 25 recently or chronically infected ARV-naïve individuals. These studies comprised 50,870 individuals from 111 countries. Each set of study sequences was analyzed for phylogenetic clustering and the presence of 93 surveillance drug-resistance mutations (SDRMs). The median overall TDR prevalence in sub-Saharan Africa (SSA), south/southeast Asia (SSEA), upper-income Asian countries, Latin America/Caribbean, Europe, and North America was 2.8%, 2.9%, 5.6%, 7.6%, 9.4%, and 11.5%, respectively. In SSA, there was a yearly 1.09-fold (95% CI: 1.05-1.14) increase in odds of TDR since national ARV scale-up attributable to an increase in non-nucleoside reverse transcriptase inhibitor (NNRTI) resistance. The odds of NNRTI-associated TDR also increased in Latin America/Caribbean (odds ratio [OR] = 1.16; 95% CI: 1.06-1.25), North America (OR = 1.19; 95% CI: 1.12-1.26), Europe (OR = 1.07; 95% CI: 1.01-1.13), and upper-income Asian countries (OR = 1.33; 95% CI: 1.12-1.55). In SSEA, there was no significant change in the odds of TDR since national ARV scale-up (OR = 0.97; 95% CI: 0.92-1.02). An analysis limited to sequences with mixtures at less than 0.5% of their nucleotide positions—a proxy for recent infection—yielded trends comparable to those obtained using the complete dataset. Four NNRTI SDRMs—K101E, K103N, Y181C, and G190A

  16. Sequence similarity between the erythrocyte binding domain of the Plasmodium vivax Duffy binding protein and the V3 loop of HIV-1 strain MN reveals a functional heparin binding motif involved in binding to the Duffy antigen receptor for chemokines

    OpenAIRE

    Bolton, Michael J; Garry, Robert F

    2011-01-01

    Abstract Background The HIV surface glycoprotein gp120 (SU, gp120) and the Plasmodium vivax Duffy binding protein (PvDBP) bind to chemokine receptors during infection and have a site of amino acid sequence similarity in their binding domains that often includes a heparin binding motif (HBM). Infection by either pathogen has been found to be inhibited by polyanions. Results Specific polyanions that inhibit HIV infection and bind to the V3 loop of X4 strains also inhibited DBP-mediated infectio...

  17. Effect of clonal integration on nitrogen cycling in rhizosphere of rhizomatous clonal plant, Phyllostachys bissetii, under heterogeneous light.

    Science.gov (United States)

    Li, Yang; Chen, Jing-Song; Xue, Ge; Peng, Yuanying; Song, Hui-Xing

    2018-07-01

    Clonal integration plays an important role in clonal plant adapting to heterogeneous habitats. It was postulated that clonal integration could exhibit positive effects on nitrogen cycling in the rhizosphere of clonal plant subjected to heterogeneous light conditions. An in-situ experiment was conducted using clonal fragments of Phyllostachys bissetii with two successive ramets. Shading treatments were applied to offspring or mother ramets, respectively, whereas counterparts were treated to full sunlight. Rhizomes between two successive ramets were either severed or connected. Extracellular enzyme activities and nitrogen turnover were measured, as well as soil properties. Abundance of functional genes (archaeal or bacterial amoA, nifH) in the rhizosphere of shaded, offspring or mother ramets were determined using quantitative polymerase chain reaction. Carbon or nitrogen availabilities were significantly influenced by clonal integration in the rhizosphere of shaded ramets. Clonal integration significantly increased extracellular enzyme activities and abundance of functional genes in the rhizosphere of shaded ramets. When rhizomes were connected, higher nitrogen turnover (nitrogen mineralization or nitrification rates) was exhibited in the rhizosphere of shaded offspring ramets. However, nitrogen turnover was significantly decreased by clonal integration in the rhizosphere of shaded mother ramets. Path analysis indicated that nitrogen turnover in the rhizosphere of shaded, offspring or mother ramets were primarily driven by the response of soil microorganisms to dissolved organic carbon or nitrogen. This unique in-situ experiment provided insights into the mechanism of nutrient recycling mediated by clonal integration. It was suggested that effects of clonal integration on the rhizosphere microbial processes were dependent on direction of photosynthates transport in clonal plant subjected to heterogeneous light conditions. Copyright © 2018 Elsevier B.V. All rights

  18. On the ecological genetics of the clonal perennial Agrostis stolonifera

    NARCIS (Netherlands)

    Kik, Christoffel

    1987-01-01

    There have to date been few studies specifically addressed to the evolution of clonal organisms. The present study attempts to fill this gap and aims to analyse the distribution pattern of a clonal plant species, using the wide-spread grass Agrostis stolonifera L.(Creeping Bent) as a model species.

  19. Introduction to special issue on the ecology of clonal plants

    Czech Academy of Sciences Publication Activity Database

    Gross, K. L.; Herben, T.; Klimešová, Jitka

    2017-01-01

    Roč. 52, 3-4 (2017), s. 265-267 ISSN 1211-9520 Institutional support: RVO:67985939 Keywords : Introduction to special issue * clonal plants * clonal meeting Subject RIV: EH - Ecology, Behaviour OBOR OECD: Ecology Impact factor: 1.017, year: 2016

  20. Stomatal characteristics of Eucalyptus grandis clonal hybrids in ...

    African Journals Online (AJOL)

    This study describes the stomatal response occurring during water stress and subsequent recovery of three Eucalyptus grandis clonal hybrids. The aim was to investigate the degree to which stomatal conductance (gs) and stomatal density differ between the clonal hybrids across seasons and in response to water stress.

  1. A zinc(II)-based two-dimensional MOF for sensitive and selective sensing of HIV-1 ds-DNA sequences

    Energy Technology Data Exchange (ETDEWEB)

    Zhao, Hai-Qing; Qiu, Gui-Hua; Liang, Zhen [Guangdong Provincial Key Laboratory of New Drug Screening, School of Pharmaceutical Sciences, Southern Medical University, Guangzhou 510515 (China); Li, Min-Min [The First Affiliated Hospital of Jinan University, Guangzhou 510515 (China); Sun, Bin; Qin, Liang; Yang, Shui-Ping; Chen, Wen-Hua [Guangdong Provincial Key Laboratory of New Drug Screening, School of Pharmaceutical Sciences, Southern Medical University, Guangzhou 510515 (China); Chen, Jin-Xiang, E-mail: jxchen@smu.edu.cn [Guangdong Provincial Key Laboratory of New Drug Screening, School of Pharmaceutical Sciences, Southern Medical University, Guangzhou 510515 (China)

    2016-05-30

    Coordination reaction of a known three-dimensional (3D) polymer precursor {Na_3[Na_9(Cbdcp)_6(H_2O)_1_8]}{sub n} (A, Cbdcp = N-(4-carboxybenzyl)-(3,5-dicarboxyl)pyridinium) with Zn(NO{sub 3}){sub 2}·6H{sub 2}O in H{sub 2}O or H{sub 2}O/DMF at 100 °C and in the presence of aspirin, 5-fluorouracil (5-FU) as modulators, trans-1,2-bis(4-pyridyl)ethylene (bpe) or 1,2-bis(4-pyridyl)ethane (bpea) as ancillary ligands afforded six novel Zn(II)-based metal-organic frameworks (MOFs), that is, {[Zn(Cbdcp)(H_2O)_3]·H_2O}{sub n} (1, 1D zigzag chain), {[Zn(HCbdcp)_2]·H_2O}{sub n} (2, 2D sheet), {[Zn(Cbdcp)(bpe)_1_/_2]·2H_2O}{sub n} (3, 3D polymer), {[Zn(Cbdcp)(bpe)_1_/_2]·2H_2O}{sub n} (4, 2D network), {[Zn(Cbdcp)(bpea)_1_/_2]·2H_2O}{sub n} (5, 3D polymer) and {[Zn(Cbdcp)(bpea)_1_/_2]·2H_2O}{sub n} (6, 2D network). Among them, compound 2 contains aromatic rings, positively charged pyridinium, Zn{sup 2+} cation centers and carboxylic acid groups lined up on the 2D sheet structure with a certain extended surface exposure. The unique structure of 2 facilitates effective association with carboxyfluorescein (FAM) labeled probe single stranded DNA (probe ss-DNA, delineates as P-DNA) to yield a P-DNA@2 system, and leads to fluorescence quenching of FAM via a photoinduced electron transfer process. The P-DNA@2 system is effective and reliable for the detection of human immunodeficiency virus 1 ds-DNA (HIV ds-DNA) sequences and capable of distinguishing complementary HIV ds-DNA from mismatched target sequences with the detection limit as low as 10 pM (S/N = 3). - Graphical abstract: Six water-stable zinc(II) zwitterionic carboxylate compounds with 1D chain, 2D and 3D networks were synthesized. Compound 2 can interact with the probe DNA through noncovalent bonds to form P-DNA@2 system. This system can be used as an effective, fluorescent sensing platform for the detection of HIV ds-DNA with the detection limit as low as 10 pM. - Highlights: • Six water-stable zinc

  2. Cascade detection for the extraction of localized sequence features; specificity results for HIV-1 protease and structure-function results for the Schellman loop.

    Science.gov (United States)

    Newell, Nicholas E

    2011-12-15

    The extraction of the set of features most relevant to function from classified biological sequence sets is still a challenging problem. A central issue is the determination of expected counts for higher order features so that artifact features may be screened. Cascade detection (CD), a new algorithm for the extraction of localized features from sequence sets, is introduced. CD is a natural extension of the proportional modeling techniques used in contingency table analysis into the domain of feature detection. The algorithm is successfully tested on synthetic data and then applied to feature detection problems from two different domains to demonstrate its broad utility. An analysis of HIV-1 protease specificity reveals patterns of strong first-order features that group hydrophobic residues by side chain geometry and exhibit substantial symmetry about the cleavage site. Higher order results suggest that favorable cooperativity is weak by comparison and broadly distributed, but indicate possible synergies between negative charge and hydrophobicity in the substrate. Structure-function results for the Schellman loop, a helix-capping motif in proteins, contain strong first-order features and also show statistically significant cooperativities that provide new insights into the design of the motif. These include a new 'hydrophobic staple' and multiple amphipathic and electrostatic pair features. CD should prove useful not only for sequence analysis, but also for the detection of multifactor synergies in cross-classified data from clinical studies or other sources. Windows XP/7 application and data files available at: https://sites.google.com/site/cascadedetect/home. nacnewell@comcast.net Supplementary information is available at Bioinformatics online.

  3. Insights in Anaphylaxis and Clonal Mast Cell Disorders

    Directory of Open Access Journals (Sweden)

    David González-de-Olano

    2017-07-01

    Full Text Available The prevalence of anaphylaxis among patients with clonal mast cell disorders (MCD is clearly higher comparing to the general population. Due to a lower frequency of symptoms outside of acute episodes, clonal MCD in the absence of skin lesions might sometimes be difficult to identify which may lead to underdiagnosis, and anaphylaxis is commonly the presenting symptom in these patients. Although the release of mast cell (MC mediators upon MC activation might present with a wide variety of symptoms, particular clinical features typically characterize MC mediator release episodes in patients with clonal MCD without skin involvement. Final diagnosis requires a bone marrow study, and it is recommended that this should be done in reference centers. In this article, we address the main triggers for anaphylaxis, risk factors, clinical presentation, diagnosis, and management of patients with MC activation syndromes (MCASs, with special emphasis on clonal MCAS [systemic mastocytosis and mono(clonal MC activations syndromes].

  4. Progenitor strain introduction of Mycobacterium bovis at the wildlife-livestock interface can lead to clonal expansion of the disease in a single ecosystem

    KAUST Repository

    Dippenaar, Anzaan

    2017-04-13

    Mycobacterium bovis infects multiple wildlife species and domesticated cattle across South Africa, and negatively impacts on livestock trade and movement of wildlife for conservation purposes. M. bovis infection was first reported in the Kruger National Park (KNP) in South Africa during the 1990s, and has since spread to infect numerous animal host species throughout the park and across South Africa. Whole genome sequencing data of 17 M. bovis isolates were analyzed to investigate the genomic diversity among M. bovis isolates causing disease in different animal host species from various locations in South Africa. M. bovis strains analyzed in this study are geographic rather than host species-specific. The clonal expansion of M. bovis in the KNP highlights the effect of an introduction of a transmissible infectious disease leading to a rising epidemic in wildlife, and emphasizes the importance of disease control and movement restriction of species that serve as disease reservoirs. In conclusion, the point source introduction of a single M. bovis strain type in the KNP ecosystem lead to an M. bovis outbreak in this area that affects various host species and poses an infection risk in neighboring rural communities where HIV prevalence is high.

  5. Genomic Analysis of the Emergence and Rapid Global Dissemination of the Clonal Group 258 Klebsiella pneumoniae Pandemic.

    Directory of Open Access Journals (Sweden)

    Jolene R Bowers

    Full Text Available Multidrug-resistant Klebsiella pneumoniae producing the KPC carbapenemase have rapidly spread throughout the world, causing severe healthcare-associated infections with limited antimicrobial treatment options. Dissemination of KPC-producing K. pneumoniae is largely attributed to expansion of a single dominant strain, ST258. In this study, we explore phylogenetic relationships and evolution within ST258 and its clonal group, CG258, using whole genome sequence analysis of 167 isolates from 20 countries collected over 17 years. Our results show a common ST258 ancestor emerged from its diverse parental clonal group around 1995 and likely acquired blaKPC prior to dissemination. Over the past two decades, ST258 has remained highly clonal despite diversity in accessory elements and divergence in the capsule polysaccharide synthesis locus. Apart from the large recombination event that gave rise to ST258, few mutations set it apart from its clonal group. However, one mutation occurs in a global transcription regulator. Characterization of outer membrane protein sequences revealed a profile in ST258 that includes a truncated OmpK35 and modified OmpK37. Our work illuminates potential genomic contributors to the pathogenic success of ST258, helps us better understand the global dissemination of this strain, and identifies genetic markers unique to ST258.

  6. How clonal is clonal? Genome plasticity across multicellular segments of a "Candidatus Marithrix sp." filament from sulfidic, briny seafloor sediments in the Gulf of Mexico

    Directory of Open Access Journals (Sweden)

    Verena Salman-Carvalho

    2016-08-01

    Full Text Available Candidatus Marithrix is a recently described lineage within the group of large sulfur bacteria (Beggiatoaceae, Gammaproteobacteria. This group of bacteria comprises vacuolated, attached-living filaments that inhabit the sediment surface around vent and seep sites in the marine environment. A single filament is ca. 100 µm in diameter, several millimeters long, and consists of hundreds of clonal cells, which are considered highly polyploid. Based on these characteristics, Candidatus Marithrix was used as a model organism for the assessment of genomic plasticity along segments of a single filament using next generation sequencing to possibly identify hotspots of microevolution. Using six consecutive segments of a single filament sampled from a mud volcano in the Gulf of Mexico, we recovered ca. 90% of the Candidatus Marithrix genome in each segment. There was a high level of genome conservation along the filament with average nucleotide identities between 99.98-100%. Different approaches to assemble all reads into a complete consensus genome could not fill the gaps. Each of the six segment datasets encoded merely a few hundred unique nucleotides and 5 or less unique genes - the residual content was redundant in all datasets. Besides the overall high genomic identity, we identified a similar number of single nucleotide polymorphisms (SNPs between the clonal segments, which are comparable to numbers reported for other clonal organisms. An increase of SNPs with greater distance of filament segments was not observed. The polyploidy of the cells was apparent when analyzing the heterogeneity of reads within a segment. Here, a strong increase in single nucleotide variants, or 'intrasegmental sequence heterogeneity' (ISH events, was observed. These sites may represent hotspots for genome plasticity, and possibly microevolution, since two thirds of these variants were not co-localized across the genome copies of the multicellular filament.

  7. Spread of clonally related Escherichia coli harboring an IncA/C1 plasmid encoding IMP-8 and its recruitment into an unrelated MCR-1-containing isolate.

    Science.gov (United States)

    Elena, Alan; Cejas, Daniela; Magariños, Francisco; Jewtuchowicz, Virginia; Facente, Andrea; Gutkind, Gabriel; Di Conza, José; Radice, Marcela

    2018-04-16

    Ten IMP-8-producing Escherichia coli isolates were recovered from the surveillance cultures of a neonatal intensive care unit, of which eight were clonally related. A 168.2-kb- bla IMP-8 plasmid was fully sequenced, and it corresponded to the recently described IncA/C1-ST13. This plasmid was detected in all isolates, even in those no clonally related. One unrelated isolate was also resistant to colistin and positive for mcr-1. This marker was located in a 62.7-kb-IncI2 plasmid, which was also fully sequenced. Copyright © 2018 American Society for Microbiology.

  8. Clonality and distribution of clinical Ureaplasma isolates recovered from male patients and infertile couples in China.

    Directory of Open Access Journals (Sweden)

    Zhi Ruan

    Full Text Available Ureaplasma spp. have gained increasing recognition as pathogens in both adult and neonatal patients with multiple clinical presentations. However, the clonality of this organism in the male population and infertile couples in China is largely unknown. In this study, 96 (53 U. parvum and 43 U. urealyticum of 103 Ureaplasma spp. strains recovered from genital specimens from male patients and 15 pairs of infertile couples were analyzed using multilocus sequence typing (MLST/expanded multilocus sequence typing (eMLST schemes. A total of 39 sequence types (STs and 53 expanded sequence types (eSTs were identified, with three predominant STs (ST1, ST9 and ST22 and eSTs (eST16, eST41 and eST82. Moreover, phylogenetic analysis revealed two distinct clusters that were highly congruent with the taxonomic differences between the two Ureaplasma species. We found significant differences in the distributions of both clusters and sub-groups between the male and female patients (P 0.80. However, this concordance was observed only for the detection of U. urealyticum within the infertile couples. In conclusion, the distributions of the clusters and sub-groups significantly differed between the male and female patients. U. urealyticum is more likely to transmit between infertile couples and be associated with clinical manifestations by the specific epidemic clonal lineages.

  9. Clonality and distribution of clinical Ureaplasma isolates recovered from male patients and infertile couples in China.

    Science.gov (United States)

    Ruan, Zhi; Yang, Ting; Shi, Xinyan; Kong, Yingying; Xie, Xinyou; Zhang, Jun

    2017-01-01

    Ureaplasma spp. have gained increasing recognition as pathogens in both adult and neonatal patients with multiple clinical presentations. However, the clonality of this organism in the male population and infertile couples in China is largely unknown. In this study, 96 (53 U. parvum and 43 U. urealyticum) of 103 Ureaplasma spp. strains recovered from genital specimens from male patients and 15 pairs of infertile couples were analyzed using multilocus sequence typing (MLST)/expanded multilocus sequence typing (eMLST) schemes. A total of 39 sequence types (STs) and 53 expanded sequence types (eSTs) were identified, with three predominant STs (ST1, ST9 and ST22) and eSTs (eST16, eST41 and eST82). Moreover, phylogenetic analysis revealed two distinct clusters that were highly congruent with the taxonomic differences between the two Ureaplasma species. We found significant differences in the distributions of both clusters and sub-groups between the male and female patients (P Ureaplasma spp. The present study also attained excellent agreement of the identification of both Ureaplasma species between paired urine and semen specimens from the male partners (k > 0.80). However, this concordance was observed only for the detection of U. urealyticum within the infertile couples. In conclusion, the distributions of the clusters and sub-groups significantly differed between the male and female patients. U. urealyticum is more likely to transmit between infertile couples and be associated with clinical manifestations by the specific epidemic clonal lineages.

  10. HIV integration sites and implications for maintenance of the reservoir.

    Science.gov (United States)

    Symons, Jori; Cameron, Paul U; Lewin, Sharon R

    2018-03-01

    To provide an overview of recent research of how HIV integration relates to productive and latent infection and implications for cure strategies. How and where HIV integrates provides new insights into how HIV persists on antiretroviral therapy (ART). Clonal expansion of infected cells with the same integration site demonstrates that T-cell proliferation is an important factor in HIV persistence, however, the driver of proliferation remains unclear. Clones with identical integration sites harbouring defective provirus can accumulate in HIV-infected individuals on ART and defective proviruses can express RNA and produce protein. HIV integration sites differ in clonally expanded and nonexpanded cells and in latently and productively infected cells and this influences basal and inducible transcription. There is a growing number of cellular proteins that can alter the pattern of integration to favour latency. Understanding these pathways may identify new interventions to eliminate latently infected cells. Using advances in analysing HIV integration sites, T-cell proliferation of latently infected cells is thought to play a major role in HIV persistence. Clonal expansion has been demonstrated with both defective and intact viruses. Production of viral RNA and protein from defective viruses may play a role in driving chronic immune activation. The site of integration may determine the likelihood of proliferation and the degree of basal and induced transcription. Finally, host factors and gene expression at the time of infection may determine the integration site. Together these new insights may lead to novel approaches to elimination of latently infected cells.

  11. Age-related mutations associated with clonal hematopoietic expansion and malignancies.

    Science.gov (United States)

    Xie, Mingchao; Lu, Charles; Wang, Jiayin; McLellan, Michael D; Johnson, Kimberly J; Wendl, Michael C; McMichael, Joshua F; Schmidt, Heather K; Yellapantula, Venkata; Miller, Christopher A; Ozenberger, Bradley A; Welch, John S; Link, Daniel C; Walter, Matthew J; Mardis, Elaine R; Dipersio, John F; Chen, Feng; Wilson, Richard K; Ley, Timothy J; Ding, Li

    2014-12-01

    Several genetic alterations characteristic of leukemia and lymphoma have been detected in the blood of individuals without apparent hematological malignancies. The Cancer Genome Atlas (TCGA) provides a unique resource for comprehensive discovery of mutations and genes in blood that may contribute to the clonal expansion of hematopoietic stem/progenitor cells. Here, we analyzed blood-derived sequence data from 2,728 individuals from TCGA and discovered 77 blood-specific mutations in cancer-associated genes, the majority being associated with advanced age. Remarkably, 83% of these mutations were from 19 leukemia and/or lymphoma-associated genes, and nine were recurrently mutated (DNMT3A, TET2, JAK2, ASXL1, TP53, GNAS, PPM1D, BCORL1 and SF3B1). We identified 14 additional mutations in a very small fraction of blood cells, possibly representing the earliest stages of clonal expansion in hematopoietic stem cells. Comparison of these findings to mutations in hematological malignancies identified several recurrently mutated genes that may be disease initiators. Our analyses show that the blood cells of more than 2% of individuals (5-6% of people older than 70 years) contain mutations that may represent premalignant events that cause clonal hematopoietic expansion.

  12. Loss of heterozygosity drives clonal diversity of Phytophthora capsici in China.

    Directory of Open Access Journals (Sweden)

    Jian Hu

    Full Text Available Phytophthora capsici causes significant loss to pepper (Capsicum annum in China and our goal was to develop single nucleotide polymorphism (SNP markers for P. capsici and characterize genetic diversity nationwide. Eighteen isolates of P. capsici from locations worldwide were re-sequenced and candidate nuclear and mitochondrial SNPs identified. From 2006 to 2012, 276 isolates of P. capsici were recovered from 136 locations in 27 provinces and genotyped using 45 nuclear and 2 mitochondrial SNPs. There were two main mitochondrial haplotypes and 95 multi-locus genotypes (MLGs identified. Genetic diversity was geographically structured with a high level of genotypic diversity in the north and on Hainan Island in the south, suggesting outcrossing contributes to diversity in these areas. The remaining areas of China are dominated by four clonal lineages that share mitochondrial haplotypes, are almost exclusively the A1 or A2 mating type and appear to exhibit extensive diversity based on loss of heterozygosity (LOH. Analysis of SNPs directly from infected peppers confirmed LOH in field populations. One clonal lineage is dominant throughout much of the country. The overall implications for long-lived genetically diverse clonal lineages amidst a widely dispersed sexual population are discussed.

  13. Secondary immunization generates clonally related antigen-specific plasma cells and memory B cells.

    Science.gov (United States)

    Frölich, Daniela; Giesecke, Claudia; Mei, Henrik E; Reiter, Karin; Daridon, Capucine; Lipsky, Peter E; Dörner, Thomas

    2010-09-01

    Rechallenge with T cell-dependent Ags induces memory B cells to re-enter germinal centers (GCs) and undergo further expansion and differentiation into plasma cells (PCs) and secondary memory B cells. It is currently not known whether the expanded population of memory B cells and PCs generated in secondary GCs are clonally related, nor has the extent of proliferation and somatic hypermutation of their precursors been delineated. In this study, after secondary tetanus toxoid (TT) immunization, TT-specific PCs increased 17- to 80-fold on days 6-7, whereas TT-specific memory B cells peaked (delayed) on day 14 with a 2- to 22-fold increase. Molecular analyses of V(H)DJ(H) rearrangements of individual cells revealed no major differences of gene usage and CDR3 length between TT-specific PCs and memory B cells, and both contained extensive evidence of somatic hypermutation with a pattern consistent with GC reactions. This analysis identified clonally related TT-specific memory B cells and PCs. Within clusters of clonally related cells, sequences shared a number of mutations but also could contain additional base pair changes. The data indicate that although following secondary immunization PCs can derive from memory B cells without further somatic hypermutation, in some circumstances, likely within GC reactions, asymmetric mutation can occur. These results suggest that after the fate decision to differentiate into secondary memory B cells or PCs, some committed precursors continue to proliferate and mutate their V(H) genes.

  14. Differential evolution of a CXCR4-using HIV-1 strain in CCR5wt/wt and CCR5∆32/∆32 hosts revealed by longitudinal deep sequencing and phylogenetic reconstruction.

    Science.gov (United States)

    Le, Anh Q; Taylor, Jeremy; Dong, Winnie; McCloskey, Rosemary; Woods, Conan; Danroth, Ryan; Hayashi, Kanna; Milloy, M-J; Poon, Art F Y; Brumme, Zabrina L

    2015-12-03

    Rare individuals homozygous for a naturally-occurring 32 base pair deletion in the CCR5 gene (CCR5∆32/∆32) are resistant to infection by CCR5-using ("R5") HIV-1 strains but remain susceptible to less common CXCR4-using ("X4") strains. The evolutionary dynamics of X4 infections however, remain incompletely understood. We identified two individuals, one CCR5wt/wt and one CCR5∆32/∆32, within the Vancouver Injection Drug Users Study who were infected with a genetically similar X4 HIV-1 strain. While early-stage plasma viral loads were comparable in the two individuals (~4.5-5 log10 HIV-1 RNA copies/ml), CD4 counts in the CCR5wt/wt individual reached a nadir of 250 cells/mm(3) in the CCR5∆32/∆32 individual. Ancestral phylogenetic reconstructions using longitudinal envelope-V3 deep sequences suggested that both individuals were infected by a single transmitted/founder (T/F) X4 virus that differed at only one V3 site (codon 24). While substantial within-host HIV-1 V3 diversification was observed in plasma and PBMC in both individuals, the CCR5wt/wt individual's HIV-1 population gradually reverted from 100% X4 to ~60% R5 over ~4 years whereas the CCR5∆32/∆32 individual's remained consistently X4. Our observations illuminate early dynamics of X4 HIV-1 infections and underscore the influence of CCR5 genotype on HIV-1 V3 evolution.

  15. Effects of Clonal Reproduction on Evolutionary Lag and Evolutionary Rescue.

    Science.gov (United States)

    Orive, Maria E; Barfield, Michael; Fernandez, Carlos; Holt, Robert D

    2017-10-01

    Evolutionary lag-the difference between mean and optimal phenotype in the current environment-is of keen interest in light of rapid environmental change. Many ecologically important organisms have life histories that include stage structure and both sexual and clonal reproduction, yet how stage structure and clonality interplay to govern a population's rate of evolution and evolutionary lag is unknown. Effects of clonal reproduction on mean phenotype partition into two portions: one that is phenotype dependent, and another that is genotype dependent. This partitioning is governed by the association between the nonadditive genetic plus random environmental component of phenotype of clonal offspring and their parents. While clonality slows phenotypic evolution toward an optimum, it can dramatically increase population survival after a sudden step change in optimal phenotype. Increased adult survival slows phenotypic evolution but facilitates population survival after a step change; this positive effect can, however, be lost given survival-fecundity trade-offs. Simulations indicate that the benefits of increased clonality under environmental change greatly depend on the nature of that change: increasing population persistence under a step change while decreasing population persistence under a continuous linear change requiring de novo variation. The impact of clonality on the probability of persistence for species in a changing world is thus inexorably linked to the temporal texture of the change they experience.

  16. An efficient identification strategy of clonal tea cultivars using long-core motif SSR markers.

    Science.gov (United States)

    Wang, Rang Jian; Gao, Xiang Feng; Kong, Xiang Rui; Yang, Jun

    2016-01-01

    Microsatellites, or simple sequence repeats (SSRs), especially those with long-core motifs (tri-, tetra-, penta-, and hexa-nucleotide) represent an excellent tool for DNA fingerprinting. SSRs with long-core motifs are preferred since neighbor alleles are more easily separated and identified from each other, which render the interpretation of electropherograms and the true alleles more reliable. In the present work, with the purpose of characterizing a set of core SSR markers with long-core motifs for well fingerprinting clonal cultivars of tea (Camellia sinensis), we analyzed 66 elite clonal tea cultivars in China with 33 initially-chosen long-core motif SSR markers covering all the 15 linkage groups of tea plant genome. A set of 6 SSR markers were conclusively selected as core SSR markers after further selection. The polymorphic information content (PIC) of the core SSR markers was >0.5, with ≤5 alleles in each marker containing 10 or fewer genotypes. Phylogenetic analysis revealed that the core SSR markers were not strongly correlated with the trait 'cultivar processing-property'. The combined probability of identity (PID) between two random cultivars for the whole set of 6 SSR markers was estimated to be 2.22 × 10(-5), which was quite low, confirmed the usefulness of the proposed SSR markers for fingerprinting analyses in Camellia sinensis. Moreover, for the sake of quickly discriminating the clonal tea cultivars, a cultivar identification diagram (CID) was subsequently established using these core markers, which fully reflected the identification process and provided the immediate information about which SSR markers were needed to identify a cultivar chosen among the tested ones. The results suggested that long-core motif SSR markers used in the investigation contributed to the accurate and efficient identification of the clonal tea cultivars and enabled the protection of intellectual property.

  17. Clonal expansion of CD4+ Cytotoxic T Lymphocytes in IgG4-related disease

    Science.gov (United States)

    Mattoo, Hamid; Mahajan, Vinay S.; Maehara, Takashi; Deshpande, Vikram; Della-Torre, Emanuel; Wallace, Zachary S.; Kulikova, Maria; Drijvers, Jefte M.; Daccache, Joe; Carruthers, Mollie N.; Castellino, Flavia; Stone, James R.; Stone, John H.; Pillai, Shiv

    2016-01-01

    Background IgG4-related disease (IgG4-RD) is a systemic condition of unknown etiology, characterized by highly fibrotic lesions with dense lymphoplasmacytic infiltrates. CD4+ T cells constitute the major inflammatory cell population in IgG4-RD lesions. Objective We used an unbiased approach to characterize CD4+ T cell subsets in IgG4-RD subjects based on their clonal expansion and their ability to infiltrate affected tissue sites. Methods We used flow cytometry to identify CD4+ effector/memory T cells (TEM) in a cohort of 101 IgG4-related disease (IgG4-RD) patients. These expanded cells were characterized by gene expression analysis and flow cytometry. Next-generation sequencing of the T cell receptor β chain gene was performed on CD4+SLAMF7+ CTLs and CD4+GATA3+ TH2 cells in a subset of patients to identify their clonality. Tissue infiltration by specific T cells was examined using quantitative multi-color imaging. Results CD4+ effector/memory T cells with a cytolytic phenotype were expanded in IgG4-RD patients. Next-generation sequencing revealed prominent clonal expansions of these CD4+CTLs but not CD4+GATA3+ memory TH2 cells in subjects with IgG4-RD. The dominant T cells infiltrating a range of inflamed IgG4-RD tissue sites were clonally-expanded CD4+CTLs that expressed SLAMF7, granzyme A, IL-1β, and TGF-β1. Clinical remission induced by rituximab-mediated B cell depletion was associated with a reduction in disease-associated CD4+ CTLs Conclusions IgG4-RD is prominently linked to clonally-expanded, IL-1β, and TGF- β1 secreting, CD4+ CTLs in peripheral blood as well as in inflammatory tissue lesions. These active, terminally-differentiated, cytokine-secreting effector CD4+ T cells are now linked to a human disease characterized by chronic inflammation and fibrosis. PMID:26971690

  18. Anti-HIV-1 B cell responses are dependent on B cell precursor frequency and antigen-binding affinity.

    Science.gov (United States)

    Dosenovic, Pia; Kara, Ervin E; Pettersson, Anna-Klara; McGuire, Andrew T; Gray, Matthew; Hartweger, Harald; Thientosapol, Eddy S; Stamatatos, Leonidas; Nussenzweig, Michel C

    2018-04-16

    The discovery that humans can produce potent broadly neutralizing antibodies (bNAbs) to several different epitopes on the HIV-1 spike has reinvigorated efforts to develop an antibody-based HIV-1 vaccine. Antibody cloning from single cells revealed that nearly all bNAbs show unusual features that could help explain why it has not been possible to elicit them by traditional vaccination and instead would require a sequence of different immunogens. This idea is supported by experiments with genetically modified immunoglobulin (Ig) knock-in mice. Sequential immunization with a series of specifically designed immunogens was required to shepherd the development of bNAbs. However, knock-in mice contain superphysiologic numbers of bNAb precursor-expressing B cells, and therefore how these results can be translated to a more physiologic setting remains to be determined. Here we make use of adoptive transfer experiments using knock-in B cells that carry a synthetic intermediate in the pathway to anti-HIV-1 bNAb development to examine how the relationship between B cell receptor affinity and precursor frequency affects germinal center (GC) B cell recruitment and clonal expansion. Immunization with soluble HIV-1 antigens can recruit bNAb precursor B cells to the GC when there are as few as 10 such cells per mouse. However, at low precursor frequencies, the extent of clonal expansion is directly proportional to the affinity of the antigen for the B cell receptor, and recruitment to GCs is variable and dependent on recirculation.

  19. What's in a Name? AIDS Dementia Complex, HIV-associated ...

    African Journals Online (AJOL)

    Key words: HIV; AIDS; HIV-associated dementia (HAD); HIV-associated neurocognitive disorder (HAND) .... increased survival a mixed picture is becoming more common. ... alternating sequence and memory recall of the four objects.

  20. Clonal outbreaks of [ Pasteurella] pneumotropica biovar Heyl in two mouse colonies

    DEFF Research Database (Denmark)

    Adhikary, Sadhana; Bisgaard, Magne; Dagnæs-Hansen, Frederik

    2017-01-01

    The aim of this study was to document the pathogenic role of biovar Heyl of [Pasteurella] pneumotropica in mouse colonies. Fifty-three isolates associated with mastitis and orbital, cutaneous and vaginal abscesses as well as isolates from the nose and vagina of healthy mice were investigated...... strains with the same rpoB sequence type, as shown by the PFGE profiles. The investigation documented that members of biovar Heyl of [P.] pneumotropica caused disease outbreaks in mouse colonies since the clonality indicated a primary role of [P.] pneumotropica biovar Heyl in the infections observed....

  1. Molecular epidemiological study of HIV-1 CRF01_AE transmission in Hong Kong.

    Science.gov (United States)

    Chen, J H K; Wong, K H; Li, P; Chan, K C; Lee, M P; Lam, H Y; Cheng, V C C; Yuen, K Y; Yam, W C

    2009-08-15

    The objective of this study was to investigate the transmission history of the HIV-1 CRF01_AE epidemics in Hong Kong between 1994 and 2007. A total of 465 HIV-1 CRF01_AE pol sequences were derived from an in-house or a commercial HIV-1 genotyping system. Phylogenies of CRF01_AE sequences were analyzed by the Bayesian coalescent method. CRF01_AE patient population included 363 males (78.1%) and 102 females (21.9%), whereas 65% (314 of 465) were local Chinese. Major transmission routes were heterosexual contact (63%), followed by intravenous drug use (IDU) (19%) and men having sex with men (MSM) (17%). From phylogenetic analysis, local CRF01_AE strains were from multiple origins with 3 separate transmission clusters identified. Cluster 1 consisted mainly of Chinese male IDUs and heterosexuals. Clusters 2 and 3 included mainly local Chinese MSM and non-Chinese Asian IDUs, respectively. Chinese reference isolates available from China (Fujian, Guangxi, or Liaoning) were clonally related to our transmission clusters, demonstrating the epidemiological linkage of CRF01_AE infections between Hong Kong and China. The 3 individual local transmission clusters were estimated to have initiated since late 1980s and late 1990s, causing subsequent epidemics in the early 2000s. This is the first comprehensive molecular epidemiological study of HIV-1 CRF01_AE in Hong Kong. It revealed that MSM contact is becoming a major route of local CRF01_AE transmission in Hong Kong. Epidemiological linkage of CRF01_AE between Hong Kong and China observed in this study indicates the importance of regular molecular epidemiological surveillance for the HIV-1 epidemic in our region.

  2. Comparison of 454 Ultra-Deep Sequencing and Allele-Specific Real-Time PCR with Regard to the Detection of Emerging Drug-Resistant Minor HIV-1 Variants after Antiretroviral Prophylaxis for Vertical Transmission.

    Directory of Open Access Journals (Sweden)

    Andrea Hauser

    Full Text Available Pregnant HIV-infected women were screened for the development of HIV-1 drug resistance after implementation of a triple-antiretroviral transmission prophylaxis as recommended by the WHO in 2006. The study offered the opportunity to compare amplicon-based 454 ultra-deep sequencing (UDS and allele-specific real-time PCR (ASPCR for the detection of drug-resistant minor variants in the HIV-1 reverse transcriptase (RT.Plasma samples from 34 Tanzanian women were previously analysed by ASPCR for key resistance mutations in the viral RT selected by AZT, 3TC, and NVP (K70R, K103N, Y181C, M184V, T215Y/F. In this study, the RT region of the same samples was investigated by amplicon-based UDS for resistance mutations using the 454 GS FLX System.Drug-resistant HIV-variants were identified in 69% (20/29 of women by UDS and in 45% (13/29 by ASPCR. The absolute number of resistance mutations identified by UDS was twice that identified by ASPCR (45 vs 24. By UDS 14 of 24 ASPCR-detected resistance mutations were identified at the same position. The overall concordance between UDS and ASPCR was 61.0% (25/41. The proportions of variants quantified by UDS were approximately 2-3 times lower than by ASPCR. Amplicon generation from samples with viral loads below 20,000 copies/ml failed more frequently by UDS compared to ASPCR (limit of detection = 650 copies/ml, resulting in missing or insufficient sequence coverage.Both methods can provide useful information about drug-resistant minor HIV-1 variants. ASPCR has a higher sensitivity than UDS, but is restricted to single resistance mutations. In contrast, UDS is limited by its requirement for high viral loads to achieve sufficient sequence coverage, but the sequence information reveals the complete resistance patterns within the genomic region analysed. Improvements to the UDS limit of detection are in progress, and UDS could then facilitate monitoring of drug-resistant minor variants in the HIV-1 quasispecies.

  3. Using BOX-PCR to exclude a clonal outbreak of melioidosis

    Directory of Open Access Journals (Sweden)

    Ward Linda

    2007-06-01

    Full Text Available Abstract Background Although melioidosis in endemic regions is usually caused by a diverse range of Burkholderia pseudomallei strains, clonal outbreaks from contaminated potable water have been described. Furthermore B. pseudomallei is classified as a CDC Group B bioterrorism agent. Ribotyping, pulsed-field gel electrophoresis (PFGE and multilocus sequence typing (MLST have been used to identify genetically related B. pseudomallei isolates, but they are time consuming and technically challenging for many laboratories. Methods We have adapted repetitive sequence typing using a BOX A1R primer for typing B. pseudomallei and compared BOX-PCR fingerprinting results on a wide range of well-characterized B. pseudomallei isolates with MLST and PFGE performed on the same isolates. Results BOX-PCR typing compared favourably with MLST and PFGE performed on the same isolates, both discriminating between the majority of multilocus sequence types and showing relatedness between epidemiologically linked isolates from various outbreak clusters. Conclusion Our results suggest that BOX-PCR can be used to exclude a clonal outbreak of melioidosis within 10 hours of receiving the bacterial strains.

  4. NSAIDs modulate clonal evolution in Barrett's esophagus.

    Directory of Open Access Journals (Sweden)

    Rumen L Kostadinov

    2013-06-01

    Full Text Available Cancer is considered an outcome of decades-long clonal evolution fueled by acquisition of somatic genomic abnormalities (SGAs. Non-steroidal anti-inflammatory drugs (NSAIDs have been shown to reduce cancer risk, including risk of progression from Barrett's esophagus (BE to esophageal adenocarcinoma (EA. However, the cancer chemopreventive mechanisms of NSAIDs are not fully understood. We hypothesized that NSAIDs modulate clonal evolution by reducing SGA acquisition rate. We evaluated thirteen individuals with BE. Eleven had not used NSAIDs for 6.2±3.5 (mean±standard deviation years and then began using NSAIDs for 5.6±2.7 years, whereas two had used NSAIDs for 3.3±1.4 years and then discontinued use for 7.9±0.7 years. 161 BE biopsies, collected at 5-8 time points over 6.4-19 years, were analyzed using 1Million-SNP arrays to detect SGAs. Even in the earliest biopsies there were many SGAs (284±246 in 10/13 and 1442±560 in 3/13 individuals and in most individuals the number of SGAs changed little over time, with both increases and decreases in SGAs detected. The estimated SGA rate was 7.8 per genome per year (95% support interval [SI], 7.1-8.6 off-NSAIDs and 0.6 (95% SI 0.3-1.5 on-NSAIDs. Twelve individuals did not progress to EA. In ten we detected 279±86 SGAs affecting 53±30 Mb of the genome per biopsy per time point and in two we detected 1,463±375 SGAs affecting 180±100 Mb. In one individual who progressed to EA we detected a clone having 2,291±78 SGAs affecting 588±18 Mb of the genome at three time points in the last three of 11.4 years of follow-up. NSAIDs were associated with reduced rate of acquisition of SGAs in eleven of thirteen individuals. Barrett's cells maintained relative equilibrium level of SGAs over time with occasional punctuations by expansion of clones having massive amount of SGAs.

  5. Involvement of histone methyltransferase GLP in HIV-1 latency through catalysis of H3K9 dimethylation

    International Nuclear Information System (INIS)

    Ding, Donglin; Qu, Xiying; Li, Lin; Zhou, Xin; Liu, Sijie; Lin, Shiguan; Wang, Pengfei; Liu, Shaohui; Kong, Chuijin; Wang, Xiaohui; Liu, Lin; Zhu, Huanzhang

    2013-01-01

    Understanding the mechanism of HIV-1 latency is crucial to eradication of the viral reservoir in HIV-1-infected individuals. However, the role of histone methyltransferase (HMT) G9a-like protein (GLP) in HIV-1 latency is still unclear. In the present work, we established four clonal cell lines containing HIV-1 vector. We found that the integration sites of most clonal cell lines favored active gene regions. However, we also observed hypomethylation of CpG of HIV 5′LTR in all four clonal cell lines. Additionally, 5′-deoxy-5′-methylthioadenosine (MTA), a broad-spectrum histone methyltransferase inhibitor, was used to examine the role of histone methylation in HIV-1 latency. MTA was found to decrease the level of H3K9 dimethylation, causing reactivation of latent HIV-1 in C11 cells. GLP knockdown by small interfering RNA clearly induced HIV-1 LTR expression. Results suggest that GLP may play a significant role in the maintenance of HIV-1 latency by catalyzing dimethylation of H3K9. - Highlights: ► We have established an in vitro model of HIV-1 latency. ► The integration sites of most clonal cell lines favor in active gene regions. ► Hypomethylation occurs in CpG islands of HIV 5′LTR in all four clonal cell lines. ► MTA can reactivate latent HIV-1 by decreasing the level of H3K9 me2 in C11 cells. ► HMT GLP may play a significant role in the maintenance of HIV-1 latency

  6. PCR-based clonality assessment in patients with lymphocytic ...

    Indian Academy of Sciences (India)

    study was to assess the clinical benefits of clonality testing with previously evaluated .... with the samples, negative controls containing no DNA were ..... 2003 Standardization and quality con- ... In Molecular cloning, a laboratory manual (ed.

  7. Adjusting to global change through clonal growth and epigenetic variation

    Directory of Open Access Journals (Sweden)

    Richard S Dodd

    2016-07-01

    Full Text Available The earth is experiencing major changes in global and regional climates and changes are predicted to accelerate in the future. Many species will be under considerable pressure to evolve, to migrate, or be faced with extinction. Clonal plants would appear to be at a particular disadvantage due to their limited mobility and limited capacity for adaptation. However, they have outlived previous environmental shifts and clonal species have persisted for millenia. Clonal spread offers unique ecological advantages, such as resource sharing, risk sharing, and economies of scale among ramets within genotypes. We suggest that ecological attributes of clonal plants, in tandem with variation in gene regulation through epigenetic mechanisms that facilitate and optimize phenotype variation in response to environmental change may permit them to be well suited to projected conditions.

  8. Discovery of viruses and virus-like pathogens in pistachio using high-throughput sequencing

    Science.gov (United States)

    Pistachio (Pistacia vera L.) trees from the National Clonal Germplasm Repository (NCGR) and orchards in California were surveyed for viruses and virus-like agents by high-throughput sequencing (HTS). Analyses of 60 trees including clonal UCB-1 hybrid rootstock (P. atlantica × P. integerrima) identif...

  9. Recombination of HIV type 1C (C'/C") in Ethiopia: possible link of EthHIV-1C' to subtype C sequences from the high-prevalence epidemics in India and Southern Africa

    NARCIS (Netherlands)

    Pollakis, Georgios; Abebe, Almaz; Kliphuis, Aletta; Rinke de Wit, Tobias F.; Fisseha, Bitew; Tegbaru, Belete; Tesfaye, Girma; Negassa, Hailu; Mengistu, Yohannes; Fontanet, Arnaud L.; Cornelissen, Marion; Goudsmit, Jaap

    2003-01-01

    The magnitude and complexity of the HIV-1 genetic diversity are major challenges for vaccine development. Investigation of the genotypes circulating in areas of high incidence, as well as their interactions, will be a milestone in the development of an efficacious vaccine. Because HIV-1 subtype C

  10. Molecular characterisation of the clonal emergence of high-level ciprofloxacin-monoresistant Haemophilus influenzae in the Region of Southern Denmark

    DEFF Research Database (Denmark)

    Fuursted, Kurt; Hartmeyer, Gitte Nyvang; Stegger, Marc

    2016-01-01

    and were demonstrated by WGS to be clonal belonging to a single clade with an unknown multilocus sequence type (double-locus variant of ST196). The antibiogram demonstrated that they were all monoresistant to ciprofloxacin with a minimum inhibitory concentration (MIC) >32mg/L. In silico resistome analysis...

  11. Epidemiological tracking and population assignment of the non-clonal bacterium, Burkholderia pseudomallei.

    Science.gov (United States)

    Dale, Julia; Price, Erin P; Hornstra, Heidie; Busch, Joseph D; Mayo, Mark; Godoy, Daniel; Wuthiekanun, Vanaporn; Baker, Anthony; Foster, Jeffrey T; Wagner, David M; Tuanyok, Apichai; Warner, Jeffrey; Spratt, Brian G; Peacock, Sharon J; Currie, Bart J; Keim, Paul; Pearson, Talima

    2011-12-01

    Rapid assignment of bacterial pathogens into predefined populations is an important first step for epidemiological tracking. For clonal species, a single allele can theoretically define a population. For non-clonal species such as Burkholderia pseudomallei, however, shared allelic states between distantly related isolates make it more difficult to identify population defining characteristics. Two distinct B. pseudomallei populations have been previously identified using multilocus sequence typing (MLST). These populations correlate with the major foci of endemicity (Australia and Southeast Asia). Here, we use multiple Bayesian approaches to evaluate the compositional robustness of these populations, and provide assignment results for MLST sequence types (STs). Our goal was to provide a reference for assigning STs to an established population without the need for further computational analyses. We also provide allele frequency results for each population to enable estimation of population assignment even when novel STs are discovered. The ability for humans and potentially contaminated goods to move rapidly across the globe complicates the task of identifying the source of an infection or outbreak. Population genetic dynamics of B. pseudomallei are particularly complicated relative to other bacterial pathogens, but the work here provides the ability for broad scale population assignment. As there is currently no independent empirical measure of successful population assignment, we provide comprehensive analytical details of our comparisons to enable the reader to evaluate the robustness of population designations and assignments as they pertain to individual research questions. Finer scale subdivision and verification of current population compositions will likely be possible with genotyping data that more comprehensively samples the genome. The approach used here may be valuable for other non-clonal pathogens that lack simple group-defining genetic characteristics

  12. Epidemiological tracking and population assignment of the non-clonal bacterium, Burkholderia pseudomallei.

    Directory of Open Access Journals (Sweden)

    Julia Dale

    2011-12-01

    Full Text Available Rapid assignment of bacterial pathogens into predefined populations is an important first step for epidemiological tracking. For clonal species, a single allele can theoretically define a population. For non-clonal species such as Burkholderia pseudomallei, however, shared allelic states between distantly related isolates make it more difficult to identify population defining characteristics. Two distinct B. pseudomallei populations have been previously identified using multilocus sequence typing (MLST. These populations correlate with the major foci of endemicity (Australia and Southeast Asia. Here, we use multiple Bayesian approaches to evaluate the compositional robustness of these populations, and provide assignment results for MLST sequence types (STs. Our goal was to provide a reference for assigning STs to an established population without the need for further computational analyses. We also provide allele frequency results for each population to enable estimation of population assignment even when novel STs are discovered. The ability for humans and potentially contaminated goods to move rapidly across the globe complicates the task of identifying the source of an infection or outbreak. Population genetic dynamics of B. pseudomallei are particularly complicated relative to other bacterial pathogens, but the work here provides the ability for broad scale population assignment. As there is currently no independent empirical measure of successful population assignment, we provide comprehensive analytical details of our comparisons to enable the reader to evaluate the robustness of population designations and assignments as they pertain to individual research questions. Finer scale subdivision and verification of current population compositions will likely be possible with genotyping data that more comprehensively samples the genome. The approach used here may be valuable for other non-clonal pathogens that lack simple group-defining genetic

  13. Sequence similarity between the erythrocyte binding domain 1 of the Plasmodium vivax Duffy binding protein and the V3 loop of HIV-1 strain MN reveals binding residues for the Duffy Antigen Receptor for Chemokines

    Directory of Open Access Journals (Sweden)

    Garry Robert F

    2011-01-01

    Full Text Available Abstract Background The surface glycoprotein (SU, gp120 of the human immunodeficiency virus (HIV must bind to a chemokine receptor, CCR5 or CXCR4, to invade CD4+ cells. Plasmodium vivax uses the Duffy Binding Protein (DBP to bind the Duffy Antigen Receptor for Chemokines (DARC and invade reticulocytes. Results Variable loop 3 (V3 of HIV-1 SU and domain 1 of the Plasmodium vivax DBP share a sequence similarity. The site of amino acid sequence similarity was necessary, but not sufficient, for DARC binding and contained a consensus heparin binding site essential for DARC binding. Both HIV-1 and P. vivax can be blocked from binding to their chemokine receptors by the chemokine, RANTES and its analog AOP-RANTES. Site directed mutagenesis of the heparin binding motif in members of the DBP family, the P. knowlesi alpha, beta and gamma proteins abrogated their binding to erythrocytes. Positively charged residues within domain 1 are required for binding of P. vivax and P. knowlesi erythrocyte binding proteins. Conclusion A heparin binding site motif in members of the DBP family may form part of a conserved erythrocyte receptor binding pocket.

  14. Assignment of Streptococcus agalactiae isolates to clonal complexes using a small set of single nucleotide polymorphisms.

    Science.gov (United States)

    Honsa, Erin; Fricke, Thomas; Stephens, Alex J; Ko, Danny; Kong, Fanrong; Gilbert, Gwendolyn L; Huygens, Flavia; Giffard, Philip M

    2008-08-19

    Streptococcus agalactiae (Group B Streptococcus (GBS)) is an important human pathogen, particularly of newborns. Emerging evidence for a relationship between genotype and virulence has accentuated the need for efficient and well-defined typing methods. The objective of this study was to develop a single nucleotide polymorphism (SNP) based method for assigning GBS isolates to multilocus sequence typing (MLST)-defined clonal complexes. It was found that a SNP set derived from the MLST database on the basis of maximization of Simpsons Index of Diversity provided poor resolution and did not define groups concordant with the population structure as defined by eBURST analysis of the MLST database. This was interpreted as being a consequence of low diversity and high frequency horizontal gene transfer. Accordingly, a different approach to SNP identification was developed. This entailed use of the "Not-N" bioinformatic algorithm that identifies SNPs diagnostic for groups of known sequence variants, together with an empirical process of SNP testing. This yielded a four member SNP set that divides GBS into 10 groups that are concordant with the population structure. A fifth SNP was identified that increased the sensitivity for the clinically significant clonal complex 17 to 100%. Kinetic PCR methods for the interrogation of these SNPs were developed, and used to genotype 116 well characterized isolates. A five SNP method for dividing GBS into biologically valid groups has been developed. These SNPs are ideal for high throughput surveillance activities, and combining with more rapidly evolving loci when additional resolution is required.

  15. An Invasive Clonal Plant Benefits from Clonal Integration More than a Co-Occurring Native Plant in Nutrient-Patchy and Competitive Environments

    Science.gov (United States)

    You, Wenhua; Fan, Shufeng; Yu, Dan; Xie, Dong; Liu, Chunhua

    2014-01-01

    Many notorious invasive plants are clonal, however, little is known about the different roles of clonal integration effects between invasive and native plants. Here, we hypothesize that clonal integration affect growth, photosynthetic performance, biomass allocation and thus competitive ability of invasive and native clonal plants, and invasive clonal plants benefit from clonal integration more than co-occurring native plants in heterogeneous habitats. To test these hypotheses, two stoloniferous clonal plants, Alternanthera philoxeroides (invasive), Jussiaea repens (native) were studied in China. The apical parts of both species were grown either with or without neighboring vegetation and the basal parts without competitors were in nutrient- rich or -poor habitats, with stolon connections were either severed or kept intact. Competition significantly reduced growth and photosynthetic performance of the apical ramets in both species, but not the biomass of neighboring vegetation. Without competition, clonal integration greatly improved the growth and photosynthetic performance of both species, especially when the basal parts were in nutrient-rich habitats. When grown with neighboring vegetation, growth of J. repens and photosynthetic performance of both species were significantly enhanced by clonal integration with the basal parts in both nutrient-rich and -poor habitats, while growth and relative neighbor effect (RNE) of A. philoxeroides were greatly improved by clonal integration only when the basal parts were in nutrient-rich habitats. Moreover, clonal integration increased A. philoxeroides's biomass allocation to roots without competition, but decreased it with competition, especially when the basal ramets were in nutrient-rich sections. Effects of clonal integration on biomass allocation of J. repens was similar to that of A. philoxeroides but with less significance. These results supported our hypothesis that invasive clonal plants A. philoxeroides benefits

  16. The sequence of the CA-SP1 junction accounts for the differential sensitivity of HIV-1 and SIV to the small molecule maturation inhibitor 3-O-{3',3'-dimethylsuccinyl}-betulinic acid

    Directory of Open Access Journals (Sweden)

    Aiken Christopher

    2004-06-01

    Full Text Available Abstract Background Despite the effectiveness of currently available antiretroviral therapies in the treatment of HIV-1 infection, a continuing need exists for novel compounds that can be used in combination with existing drugs to slow the emergence of drug-resistant viruses. We previously reported that the small molecule 3-O-{3',3'-dimethylsuccinyl}-betulinic acid (DSB specifically inhibits HIV-1 replication by delaying the processing of the CA-SP1 junction in Pr55Gag. By contrast, SIVmac239 replicates efficiently in the presence of high concentrations of DSB. To determine whether sequence differences in the CA-SP1 junction can fully account for the differential sensitivity of HIV-1 and SIV to DSB, we engineered mutations in this region of two viruses and tested their sensitivity to DSB in replication assays using activated human primary CD4+ T cells. Results Substitution of the P2 and P1 residues of HIV-1 by the corresponding amino acids of SIV resulted in strong resistance to DSB, but the mutant virus replicated with reduced efficiency. Conversely, replication of an SIV mutant containing three amino acid substitutions in the CA-SP1 cleavage site was highly sensitive to DSB, and the mutations resulted in delayed cleavage of the CA-SP1 junction in the presence of the drug. Conclusions These results demonstrate that the CA-SP1 junction in Pr55Gag represents the primary viral target of DSB. They further suggest that the therapeutic application of DSB will be accompanied by emergence of mutant viruses that are highly resistant to the drug but which exhibit reduced fitness relative to wild type HIV-1.

  17. Propagule pressure, habitat conditions and clonal integration influence the establishment and growth of an invasive clonal plant, Alternanthera philoxeroides

    Directory of Open Access Journals (Sweden)

    Wen-Hua eYou

    2016-05-01

    Full Text Available Many notorious invasive plants are clonal, spreading mainly by vegetative propagules. Propagule pressure (the number of propagules may affect the establishment, growth and thus invasion success of these clonal plants, and such effects may also depend on habitat conditions. To understand how propagule pressure, habitat conditions and clonal integration affect the establishment and growth of the invasive clonal plants, an 8-week greenhouse with an invasive clonal plant, Alternanthera philoxeroides was conducted. High (five fragments or low (one fragment propagule pressure was established either in bare soil (open habitat or dense native vegetation of Jussiaea repens (vegetative habitat, with the stolon connections either severed from or connected to the relatively older ramets. High propagule pressure greatly increased the establishment and growth of A. philoxeroides, especially when it grew in vegetative habitats. Surprisingly, high propagule pressure significantly reduced the growth of individual plants of A. philoxeroides in open habitats, whereas it did not affect the individual growth in vegetative habitats. A shift in the intraspecific interaction on A. philoxeroides from competition in open habitats to facilitation in vegetative habitats may be the main reason. Moreover, clonal integration significantly improved the growth of A. philoxeroides only in open habitats, especially with low propagule pressure, whereas it had no effects on the growth and competitive ability of A. philoxeroides in vegetative habitats, suggesting that clonal integration may be of most important for A. philoxeroides to explore new open space and spread. These findings suggest that propagule pressure may be crucial for the invasion success of A. philoxeroides, and such an effect also depends on habitat conditions.

  18. Use of X-Chromosome Inactivation Pattern to Analyze the Clonality of 14 Female Cases of Kaposi Sarcoma.

    Science.gov (United States)

    Yuan, Ding; XiuJuan, Wu; Yan, Zhang; JunQin, Liang; Fang, Xiang; Shirong, Yu; Xiaojing, Kang; Yanyan, Feng; Weidong, Wu; Dong, Luo; Qingli, Lu; DeZhi, Zhang; XiongMing, Pu

    2015-06-16

    Kaposi sarcoma (KS) has features of both neoplastic growth and hyperplastic proliferation. It is the most common tumor seen in patients with HIV infection. Whether KS is a real tumor or a benign hyperplastic disease is not known. Tissues from KS and cutaneous hemangioma lesion DNA were extracted, and then digested with methylation-sensitive restriction endonuclease HpaII. Human androgen receptor gene (HUMARA) was amplified with PCR method and the product was separated on 10% denaturing polyacrylamide gels and stained with ethylene dibromide (EB) to show the polymorphism of HUMARA. Phosphoglycerate kinase (PGK) was amplified and the product was digested by BStXI, agarose gel and EB stained to show the polymorphism of PGK. Finally, we analyzed the clonality of KS. In the 14 patients with KS, heterozygosity of the HUMARA gene was observed in 12 (85.7%) cases. Loss of heterozygosity of HUMARA gene on X-chromosome (without HpaII digestion there were 2 bands, after HpaII digestion there were just 1 of the bands), representing monoclonal origin, was present in 11 cases of Kaposi sarcoma. Heterozygosity of the PGK gene was observed in 5 (35.7%) cases, which all represent monoclonal origin. There was no significant difference according to country, stage, or HIV and HHV-8 (P>0.05). The current findings suggest that Kaposi sarcoma is a clonal neoplasm, not a reactive proliferation.

  19. A high density of tertiary lymphoid structure B cells in lung tumors is associated with increased CD4+ T cell receptor repertoire clonality.

    Science.gov (United States)

    Zhu, Wei; Germain, Claire; Liu, Zheng; Sebastian, Yinong; Devi, Priyanka; Knockaert, Samantha; Brohawn, Philip; Lehmann, Kim; Damotte, Diane; Validire, Pierre; Yao, Yihong; Valge-Archer, Viia; Hammond, Scott A; Dieu-Nosjean, Marie-Caroline; Higgs, Brandon W

    2015-12-01

    T and B cell receptor (TCR and BCR, respectively) Vβ or immunoglobulin heavy chain complementarity-determining region 3 sequencing allows monitoring of repertoire changes through recognition, clonal expansion, affinity maturation, and T or B cell activation in response to antigen. TCR and BCR repertoire analysis can advance understanding of antitumor immune responses in the tumor microenvironment. TCR and BCR repertoires of sorted CD4 + , CD8 + or CD19 + cells in tumor, non-tumoral distant tissue (NT), and peripheral compartments (blood/draining lymph node [P]) from 47 non-small cell lung cancer (NSCLC) patients (age median = 68 y) were sequenced. The clonotype spectra were assessed among different tissues and correlated with clinical and immunological parameters. In all tissues, CD4 + and CD8 + TCR repertoires had greater clonality relative to CD19 + BCR. CD4 + T cells exhibited greater clonality in NT compared to tumor ( p = 0.002) and P ( p 68). Younger patients exhibited greater CD4 + T cell diversity in P compared to older patients ( p = 0.05), and greater CD4 + T cell clonality in tumor relative to P ( p cell clonality in tumor and P, respectively (both p = 0.05), correlated with high density of tumor-associated tertiary lymphoid structure (TLS) B cells, a biomarker of higher overall survival in NSCLC. Results indicate distinct adaptive immune responses in NSCLC, where peripheral T cell diversity is modulated by age, and tumor T cell clonal expansion is favored by the presence of TLSs in the tumor microenvironment.

  20. Clonal selection versus clonal cooperation: the integrated perception of immune objects [version 1; referees: 2 approved

    Directory of Open Access Journals (Sweden)

    Serge Nataf

    2016-09-01

    Full Text Available Analogies between the immune and nervous systems were first envisioned by the immunologist Niels Jerne who introduced the concepts of antigen "recognition" and immune "memory". However, since then, it appears that only the cognitive immunology paradigm proposed by Irun Cohen, attempted to further theorize the immune system functions through the prism of neurosciences. The present paper is aimed at revisiting this analogy-based reasoning. In particular, a parallel is drawn between the brain pathways of visual perception and the processes allowing the global perception of an "immune object". Thus, in the visual system, distinct features of a visual object (shape, color, motion are perceived separately by distinct neuronal populations during a primary perception task. The output signals generated during this first step instruct then an integrated perception task performed by other neuronal networks. Such a higher order perception step is by essence a cooperative task that is mandatory for the global perception of visual objects. Based on a re-interpretation of recent experimental data, it is suggested that similar general principles drive the integrated perception of immune objects in secondary lymphoid organs (SLOs. In this scheme, the four main categories of signals characterizing an immune object (antigenic, contextual, temporal and localization signals are first perceived separately by distinct networks of immunocompetent cells.  Then, in a multitude of SLO niches, the output signals generated during this primary perception step are integrated by TH-cells at the single cell level. This process eventually generates a multitude of T-cell and B-cell clones that perform, at the scale of SLOs, an integrated perception of immune objects. Overall, this new framework proposes that integrated immune perception and, consequently, integrated immune responses, rely essentially on clonal cooperation rather than clonal selection.

  1. Clonal Clusters and Virulence Factors of Group C and G Streptococcus Causing Severe Infections, Manitoba, Canada, 2012-2014.

    Science.gov (United States)

    Lother, Sylvain A; Demczuk, Walter; Martin, Irene; Mulvey, Michael; Dufault, Brenden; Lagacé-Wiens, Philippe; Keynan, Yoav

    2017-07-01

    The incidence of group C and G Streptococcus (GCGS) bacteremia, which is associated with severe disease and death, is increasing. We characterized clinical features, outcomes, and genetic determinants of GCGS bacteremia for 89 patients in Winnipeg, Manitoba, Canada, who had GCGS bacteremia during 2012-2014. Of the 89 patients, 51% had bacteremia from skin and soft tissue, 70% had severe disease features, and 20% died. Whole-genome sequencing analysis was performed on isolates derived from 89 blood samples and 33 respiratory sample controls: 5 closely related genetic lineages were identified as being more likely to cause invasive disease than non-clade isolates (83% vs. 57%, p = 0.002). Virulence factors cbp, fbp, speG, sicG, gfbA, and bca clustered clonally into these clades. A clonal distribution of virulence factors may account for severe and fatal cases of bacteremia caused by invasive GCGS.

  2. Clonal mutations in primary human glial tumors: evidence in support of the mutator hypothesis

    Directory of Open Access Journals (Sweden)

    Sarkar Chitra

    2007-10-01

    Full Text Available Abstract Background A verifiable consequence of the mutator hypothesis is that even low grade neoplasms would accumulate a large number of mutations that do not influence the tumor phenotype (clonal mutations. In this study, we have attempted to quantify the number of clonal mutations in primary human gliomas of astrocytic cell origin. These alterations were identified in tumor tissue, microscopically confirmed to have over 70% neoplastic cells. Methods Random Amplified Polymorphic DNA (RAPD analysis was performed using a set of fifteen 10-mer primers of arbitrary but definite sequences in 17 WHO grade II astrocytomas (low grade diffuse astrocytoma or DA and 16 WHO grade IV astrocytomas (Glioblastoma Multiforme or GBM. The RAPD profile of the tumor tissue was compared with that of the leucocyte DNA of the same patient and alteration(s scored. A quantitative estimate of the overall genomic changes in these tumors was obtained by 2 different modes of calculation. Results The overall change in the tumors was estimated to be 4.24% in DA and 2.29% in GBM by one method and 11.96% and 6.03% in DA and GBM respectively by the other. The difference between high and lower grade tumors was statistically significant by both methods. Conclusion This study demonstrates the presence of extensive clonal mutations in gliomas, more in lower grade. This is consistent with our earlier work demonstrating that technique like RAPD analysis, unbiased for locus, is able to demonstrate more intra-tumor genetic heterogeneity in lower grade gliomas compared to higher grade. The results support the mutator hypothesis proposed by Loeb.

  3. Clonal mutations in primary human glial tumors: evidence in support of the mutator hypothesis

    International Nuclear Information System (INIS)

    Misra, Anjan; Chattopadhyay, Parthaprasad; Chosdol, Kunzang; Sarkar, Chitra; Mahapatra, Ashok K; Sinha, Subrata

    2007-01-01

    A verifiable consequence of the mutator hypothesis is that even low grade neoplasms would accumulate a large number of mutations that do not influence the tumor phenotype (clonal mutations). In this study, we have attempted to quantify the number of clonal mutations in primary human gliomas of astrocytic cell origin. These alterations were identified in tumor tissue, microscopically confirmed to have over 70% neoplastic cells. Random Amplified Polymorphic DNA (RAPD) analysis was performed using a set of fifteen 10-mer primers of arbitrary but definite sequences in 17 WHO grade II astrocytomas (low grade diffuse astrocytoma or DA) and 16 WHO grade IV astrocytomas (Glioblastoma Multiforme or GBM). The RAPD profile of the tumor tissue was compared with that of the leucocyte DNA of the same patient and alteration(s) scored. A quantitative estimate of the overall genomic changes in these tumors was obtained by 2 different modes of calculation. The overall change in the tumors was estimated to be 4.24% in DA and 2.29% in GBM by one method and 11.96% and 6.03% in DA and GBM respectively by the other. The difference between high and lower grade tumors was statistically significant by both methods. This study demonstrates the presence of extensive clonal mutations in gliomas, more in lower grade. This is consistent with our earlier work demonstrating that technique like RAPD analysis, unbiased for locus, is able to demonstrate more intra-tumor genetic heterogeneity in lower grade gliomas compared to higher grade. The results support the mutator hypothesis proposed by Loeb

  4. Maximum-Entropy Models of Sequenced Immune Repertoires Predict Antigen-Antibody Affinity.

    Directory of Open Access Journals (Sweden)

    Lorenzo Asti

    2016-04-01

    Full Text Available The immune system has developed a number of distinct complex mechanisms to shape and control the antibody repertoire. One of these mechanisms, the affinity maturation process, works in an evolutionary-like fashion: after binding to a foreign molecule, the antibody-producing B-cells exhibit a high-frequency mutation rate in the genome region that codes for the antibody active site. Eventually, cells that produce antibodies with higher affinity for their cognate antigen are selected and clonally expanded. Here, we propose a new statistical approach based on maximum entropy modeling in which a scoring function related to the binding affinity of antibodies against a specific antigen is inferred from a sample of sequences of the immune repertoire of an individual. We use our inference strategy to infer a statistical model on a data set obtained by sequencing a fairly large portion of the immune repertoire of an HIV-1 infected patient. The Pearson correlation coefficient between our scoring function and the IC50 neutralization titer measured on 30 different antibodies of known sequence is as high as 0.77 (p-value 10-6, outperforming other sequence- and structure-based models.

  5. HIV RNA and proviral HIV DNA can be detected in semen after 6 months of antiretroviral therapy although HIV RNA is undetectable in blood.

    Science.gov (United States)

    Du, Peiwei; Liu, An; Jiao, Yanmei; Liu, Cuie; Jiang, Taiyi; Zhu, Weijun; Zhu, Yunxia; Wu, Hao; Sun, Lijun

    2016-03-01

    The risk of sexual transmission of HIV is strongly correlated with amounts of genital HIV RNA. Few studies have reported amounts of HIV RNA and HIV DNA in semen in HIV-infected Chinese patients undergoing antiviral treatment (ART). In this observational study, the amounts of HIV RNA and HIV DNA in semen were assessed after six months of ART in HIV-infected Chinese individuals, when HIV RNA was undetectable in blood . This study included 19 HIV-infected Chinese men undergoing ART for six months. Amounts of HIV in paired semen and blood samples were assessed using real-time PCR. The C2-V5 region of the HIV envelope (env) genes was cloned and sequenced and genotype and co-receptor usage predicted based on the sequence. It was found that HIV RNA was undetectable in the plasma of most patients (17/19), whereas HIV RNA could be detected in the semen of most patients (16/19). HIV DNA could be detected in both semen and blood. Genetic diversity of HIV between the seminal and blood compartments was identified. Thus, amounts of HIV RNA and HIV DNA remain high in semen of HIV-infected Chinese patients after six months of ART treatment, even when HIV RNA was undetectable in blood. © 2016 The Societies and John Wiley & Sons Australia, Ltd.

  6. Clonal analysis of stem cells in differentiation and disease.

    Science.gov (United States)

    Colom, Bartomeu; Jones, Philip H

    2016-12-01

    Tracking the fate of individual cells and their progeny by clonal analysis has redefined the concept of stem cells and their role in health and disease. The maintenance of cell turnover in adult tissues is achieved by the collective action of populations of stem cells with an equal likelihood of self-renewal or differentiation. Following injury stem cells exhibit striking plasticity, switching from homeostatic behavior in order to repair damaged tissues. The effects of disease states on stem cells are also being uncovered, with new insights into how somatic mutations trigger clonal expansion in early neoplasia. Copyright © 2016 The Author(s). Published by Elsevier Ltd.. All rights reserved.

  7. Predictive genomics: A cancer hallmark network framework for predicting tumor clinical phenotypes using genome sequencing data

    OpenAIRE

    Wang, Edwin; Zaman, Naif; Mcgee, Shauna; Milanese, Jean-Sébastien; Masoudi-Nejad, Ali; O'Connor, Maureen

    2014-01-01

    We discuss a cancer hallmark network framework for modelling genome-sequencing data to predict cancer clonal evolution and associated clinical phenotypes. Strategies of using this framework in conjunction with genome sequencing data in an attempt to predict personalized drug targets, drug resistance, and metastasis for a cancer patient, as well as cancer risks for a healthy individual are discussed. Accurate prediction of cancer clonal evolution and clinical phenotypes will have substantial i...

  8. Effect of environmental stress on clonal structure of Eucypris virens (Crustacea, Ostracoda)

    OpenAIRE

    Martins M. J. F.; Vandekerkhove J.; Adolfsson S.; Rossetti G.; Namiotko T.; Jokela J.

    2010-01-01

    Environmental stress imposes strong natural selection on clonal populations, promoting evolutionary change in clonal structure. Environmental stress may also lead to reduction in population size, which together with clonal selection may reduce genotypic diversity of the local populations. We examined how clonal structure in wild-collected samples of two parthenogenetic populations of the freshwater ostracod Eucypris virens responded to hypersalinity and starvation, and the combination of the ...

  9. Unexpected heterogeneity derived from Cas9 ribonucleoprotein-introduced clonal cells at the HPRT1 locus.

    Science.gov (United States)

    Sakuma, Tetsushi; Mochida, Keiji; Nakade, Shota; Ezure, Toru; Minagawa, Sachi; Yamamoto, Takashi

    2018-04-01

    Single-cell cloning is an essential technique for establishing genome-edited cell clones mediated by programmable nucleases such as CRISPR-Cas9. However, residual genome-editing activity after single-cell cloning may cause heterogeneity in the clonal cells. Previous studies showed efficient mutagenesis and rapid degradation of CRISPR-Cas9 components in cultured cells by introducing Cas9 ribonucleoproteins (RNPs). In this study, we investigated how the timing for single-cell cloning of Cas9 RNP-transfected cells affected the heterogeneity of the resultant clones. We carried out transfection of Cas9 RNPs targeting several loci in the HPRT1 gene in HCT116 cells, followed by single-cell cloning at 24, 48, 72 hr and 1 week post-transfection. After approximately 3 weeks of incubation, the clonal cells were collected and genotyped by high-resolution microchip electrophoresis and Sanger sequencing. Unexpectedly, long-term incubation before single-cell cloning resulted in highly heterogeneous clones. We used a lipofection method for transfection, and the media containing transfectable RNPs were not removed before single-cell cloning. Therefore, the active Cas9 RNPs were considered to be continuously incorporated into cells during the precloning incubation. Our findings provide a warning that lipofection of Cas9 RNPs may cause continuous introduction of gene mutations depending on the experimental procedures. © 2018 Molecular Biology Society of Japan and John Wiley & Sons Australia, Ltd.

  10. Clonal selection in xenografted TAM recapitulates the evolutionary process of myeloid leukemia in Down syndrome.

    Science.gov (United States)

    Saida, Satoshi; Watanabe, Ken-ichiro; Sato-Otsubo, Aiko; Terui, Kiminori; Yoshida, Kenichi; Okuno, Yusuke; Toki, Tsutomu; Wang, RuNan; Shiraishi, Yuichi; Miyano, Satoru; Kato, Itaru; Morishima, Tatsuya; Fujino, Hisanori; Umeda, Katsutsugu; Hiramatsu, Hidefumi; Adachi, Souichi; Ito, Etsuro; Ogawa, Seishi; Ito, Mamoru; Nakahata, Tatsutoshi; Heike, Toshio

    2013-05-23

    Transient abnormal myelopoiesis (TAM) is a clonal preleukemic disorder that progresses to myeloid leukemia of Down syndrome (ML-DS) through the accumulation of genetic alterations. To investigate the mechanism of leukemogenesis in this disorder, a xenograft model of TAM was established using NOD/Shi-scid, interleukin (IL)-2Rγ(null) mice. Serial engraftment after transplantation of cells from a TAM patient who developed ML-DS a year later demonstrated their self-renewal capacity. A GATA1 mutation and no copy number alterations (CNAs) were detected in the primary patient sample by conventional genomic sequencing and CNA profiling. However, in serial transplantations, engrafted TAM-derived cells showed the emergence of divergent subclones with another GATA1 mutation and various CNAs, including a 16q deletion and 1q gain, which are clinically associated with ML-DS. Detailed genomic analysis identified minor subclones with a 16q deletion or this distinct GATA1 mutation in the primary patient sample. These results suggest that genetically heterogeneous subclones with varying leukemia-initiating potential already exist in the neonatal TAM phase, and ML-DS may develop from a pool of such minor clones through clonal selection. Our xenograft model of TAM may provide unique insight into the evolutionary process of leukemia.

  11. Unforeseen clonal evolution of tumor cell population in recurrent and metastatic dermatofibrosarcoma protuberans.

    Directory of Open Access Journals (Sweden)

    Ensel Oh

    Full Text Available Dermatofibrosarcoma protuberans (DFSP is a very rare soft tissue sarcoma, generally of low-grade malignancy. DFSP is locally aggressive with a high recurrence rate, but metastasis occurs rarely. To investigate the mechanism of metastasis in DFSP, we analyzed the whole exome sequencing data of serial tumor samples obtained from a patient who had a 10-year history of recurrent and metastatic DFSP. Tracking various genomic alterations, namely somatic mutations, copy number variations, and chromosomal rearrangements, we observed a dramatic change in tumor cell population during the occurrence of metastasis in this DFSP case. The new subclone that emerged in metastatic DFSP harbored a completely different set of somatic mutations and new focal amplifications, which had not been observed in the primary clone before metastasis. The COL1A1-PDGFB fusion, characteristic of DFSP, was found in all of the serial samples. Moreover, the break position on the fusion gene was identical in all samples. Based on these observations, we suggest a clonal evolution model to explain the mechanism underlying metastasis in DFSP and identified several candidate target genes responsible for metastatic DFSP by utilizing The Cancer Genome Atlas database. This is the first study to observe clonal evolution in metastatic DFSP and provide insight for a possible therapeutic strategy for imatinib-resistant or metastatic DFSP.

  12. Molecular analysis of immunoglobulin genes reveals frequent clonal relatedness in double monoclonal gammopathies.

    Science.gov (United States)

    Tschumper, R C; Dispenzieri, A; Abraham, R S; Henderson, K J; Jelinek, D F

    2013-04-19

    Monoclonal gammopathies (MGs) are hematological diseases characterized by high levels of a monoclonal immunoglobulin (Ig) or M-protein. Within this group are patients with more than one M-protein, referred to as double MGs (DMGs). The M-proteins in DMG patients may have different heavy chain (HC) isotypes that are associated with different light chains (LCs), or different HCs that are LC matched. In this study, we examined the clonal relatedness of the M-proteins in the latter type in a cohort of 14 DMG patients. By using PCR, we identified 7/14 DMG patients that expressed two Ig HC isotypes with identical Ig HC variable (IGHV), diversity (IGHD), joining (IGHJ), and complementarity determining region (HCDR3) sequences. Two additional DMG patients had two Ig transcripts using the same IGHV, IGHD and IGHJ genes but with slight differences in variable region or HCDR3 mutations. LC analysis confirmed that a single LC was expressed in 3/7 DMG patients with identical HC transcripts and in the two DMGs with highly similar transcripts. The PCR findings were confirmed by immunofluorescence for HC and LC expression. Clonally related HC-dissimilar/LC-matched DMGs may occur often and defines a new subtype of MG that may serve as a tool for studies of disease pathogenesis.

  13. Molecular analysis of immunoglobulin genes reveals frequent clonal relatedness in double monoclonal gammopathies

    International Nuclear Information System (INIS)

    Tschumper, R C; Dispenzieri, A; Abraham, R S; Henderson, K J; Jelinek, D F

    2013-01-01

    Monoclonal gammopathies (MGs) are hematological diseases characterized by high levels of a monoclonal immunoglobulin (Ig) or M-protein. Within this group are patients with more than one M-protein, referred to as double MGs (DMGs). The M-proteins in DMG patients may have different heavy chain (HC) isotypes that are associated with different light chains (LCs), or different HCs that are LC matched. In this study, we examined the clonal relatedness of the M-proteins in the latter type in a cohort of 14 DMG patients. By using PCR, we identified 7/14 DMG patients that expressed two Ig HC isotypes with identical Ig HC variable (IGHV), diversity (IGHD), joining (IGHJ), and complementarity determining region (HCDR3) sequences. Two additional DMG patients had two Ig transcripts using the same IGHV, IGHD and IGHJ genes but with slight differences in variable region or HCDR3 mutations. LC analysis confirmed that a single LC was expressed in 3/7 DMG patients with identical HC transcripts and in the two DMGs with highly similar transcripts. The PCR findings were confirmed by immunofluorescence for HC and LC expression. Clonally related HC-dissimilar/LC-matched DMGs may occur often and defines a new subtype of MG that may serve as a tool for studies of disease pathogenesis

  14. Aging in a long-lived clonal tree.

    Directory of Open Access Journals (Sweden)

    Dilara Ally

    2010-08-01

    Full Text Available From bacteria to multicellular animals, most organisms exhibit declines in survivorship or reproductive performance with increasing age ("senescence". Evidence for senescence in clonal plants, however, is scant. During asexual growth, we expect that somatic mutations, which negatively impact sexual fitness, should accumulate and contribute to senescence, especially among long-lived clonal plants. We tested whether older clones of Populus tremuloides (trembling aspen from natural stands in British Columbia exhibited significantly reduced reproductive performance. Coupling molecular-based estimates of clone age with male fertility data, we observed a significant decline in the average number of viable pollen grains per catkin per ramet with increasing clone age in trembling aspen. We found that mutations reduced relative male fertility in clonal aspen populations by about 5.8 x 10(-5 to 1.6 x 10(-3 per year, leading to an 8% reduction in the number of viable pollen grains, on average, among the clones studied. The probability that an aspen lineage ultimately goes extinct rises as its male sexual fitness declines, suggesting that even long-lived clonal organisms are vulnerable to senescence.

  15. THE EXTENT OF CLONAL STRUCTURE IN DIFFERENT LYMPHOID ORGANS

    NARCIS (Netherlands)

    HERMANS, MHA; WUBBENA, A; KROESE, FGM; HUNT, SV; COWAN, R; OPSTELTEN, D

    1992-01-01

    To gain insight into the clonal organization of lymphoid organs, we studied the distribution in situ of donor-derived cells in near-physiological chimeras. We introduced RT7b fetal liver cells into nonirradiated congenic RT7a neonatal rats. The chimerism 6-20 wk after injection ranged from 0.3 to

  16. Productivity gains by fertilisation in Eucalyptus urophylla clonal ...

    African Journals Online (AJOL)

    Productivity gains by fertilisation in Eucalyptus urophylla clonal plantations across gradients in site and stand conditions. ... The control plot may typically be a permanent plot of an inventory network, providing representative information for a company's decisionmaking. The paired twin-plot receives intensive management ...

  17. Negative plant soil feedback explaining ring formation in clonal plants

    NARCIS (Netherlands)

    Carteni, F.; Marasco, A.; Bonanomi, G.; Mazzoleni, S.; Rietkerk, M.G.; Giannino, F.

    2012-01-01

    Ring shaped patches of clonal plants have been reported in different environments, but the mechanisms underlying such pattern formation are still poorly explained. Water depletion in the inner tussocks zone has been proposed as a possible cause, although ring patterns have been also observed in

  18. Clonal differences in log end splitting in Eucalyptus grandis in ...

    African Journals Online (AJOL)

    This paper discusses the juvenile–mature correlation of log end splitting among Eucalyptus grandis clones from two trials and how differences in splitting relate to differences in wood density, pith-to-bark gradient and growth rate. Two approximately 20-year-old Eucalyptus grandis clonal trials at Bergvliet plantation were ...

  19. Molecular marker analysis to differentiate a clonal selection of ...

    African Journals Online (AJOL)

    Lalit Kumar

    2013-04-03

    Apr 3, 2013 ... Microsatellite and amplified fragment length polymorphism (AFLP) markers were used to differentiate. Manjari Naveen, a clonal selection of Centennial Seedless variety of grape. Twenty one (21) microsatellite primers could not detect variation between parent variety and its clone. AFLP analysis.

  20. Assessment of various strategies for the preservation of clonal ...

    African Journals Online (AJOL)

    Clonal conformity of ramets resulting from the re-cloning of somaplants depended, on one hand, on the floral status of the mother plant at the time of sampling and, on the other hand, on its origin. Re-cloning of abnormal regenerants led, in all cases, to 100 % abnormal offspring. The age of the ramet used as mother palm at ...

  1. The C-terminal tail of the gp41 transmembrane envelope glycoprotein of HIV-1 clades A, B, C, and D may exist in two conformations: an analysis of sequence, structure, and function

    International Nuclear Information System (INIS)

    Hollier, Mark J.; Dimmock, Nigel J.

    2005-01-01

    In addition to the major ectodomain, the gp41 transmembrane glycoprotein of HIV-1 is now known to have a minor ectodomain that is part of the long C-terminal tail. Both ectodomains are highly antigenic, carry neutralizing and non-neutralizing epitopes, and are involved in virus-mediated fusion activity. However, data have so far been biologically based, and derived solely from T cell line-adapted (TCLA), B clade viruses. Here we have carried out sequence and theoretically based structural analyses of 357 gp41 C-terminal sequences of mainly primary isolates of HIV-1 clades A, B, C, and D. Data show that all these viruses have the potential to form a tail loop structure (the minor ectodomain) supported by three, β-sheet, membrane-spanning domains (MSDs). This means that the first (N-terminal) tyrosine-based sorting signal of the gp41 tail is situated outside the cell membrane and is non-functional, and that gp41 that reaches the cell surface may be recycled back into the cytoplasm through the activity of the second tyrosine-sorting signal. However, we suggest that only a minority of cell-associated gp41 molecules - those destined for incorporation into virions - has 3 MSDs and the minor ectodomain. Most intracellular gp41 has the conventional single MSD, no minor ectodomain, a functional first tyrosine-based sorting signal, and in line with current thinking is degraded intracellularly. The gp41 structural diversity suggested here can be viewed as an evolutionary strategy to minimize HIV-1 envelope glycoprotein expression on the cell surface, and hence possible cytotoxicity and immune attack on the infected cell

  2. HIV Molecular Immunology 2014

    Energy Technology Data Exchange (ETDEWEB)

    Yusim, Karina [Los Alamos National Lab. (LANL), Los Alamos, NM (United States); Korber, Bette Tina Marie [Los Alamos National Lab. (LANL), Los Alamos, NM (United States); Barouch, Dan [Beth Israel Deaconess Medical Center, Boston, MA (United States); Koup, Richard [Vaccine Research Center National Institutes of Health (United States); de Boer, Rob [Utrecht Univ. (Netherlands). Dept. of Biology; Moore, John P. [Cornell Univ., Ithaca, NY (United States). Weill Medical College; Brander, Christian [Institucioi Catalana de Recerca i Estudis Avancats (ICREA), Barcelona (Spain); Haynes, Barton F. [Duke Univ., Durham, NC (United States). Duke Human Vaccine Institute and Departments of Medicine, Surgery and Immunology; Walker, Bruce D. [Ragon Institute of Massachusetts General Hospital, Cambridge, MA (United States); Harvard Univ., Cambridge, MA (United States); Massachusetts Inst. of Technology (MIT), Cambridge, MA (United States)

    2015-02-03

    HIV Molecular Immunology is a companion volume to HIV Sequence Compendium. This publication, the 2014 edition, is the PDF version of the web-based HIV Immunology Database (http://www.hiv.lanl.gov/content/immunology/). The web interface for this relational database has many search options, as well as interactive tools to help immunologists design reagents and interpret their results. In the HIV Immunology Database, HIV-specific B-cell and T-cell responses are summarized and annotated. Immunological responses are divided into three parts, CTL, T helper, and antibody. Within these parts, defined epitopes are organized by protein and binding sites within each protein, moving from left to right through the coding regions spanning the HIV genome. We include human responses to natural HIV infections, as well as vaccine studies in a range of animal models and human trials. Responses that are not specifically defined, such as responses to whole proteins or monoclonal antibody responses to discontinuous epitopes, are summarized at the end of each protein section. Studies describing general HIV responses to the virus, but not to any specific protein, are included at the end of each part. The annotation includes information such as crossreactivity, escape mutations, antibody sequence, TCR usage, functional domains that overlap with an epitope, immune response associations with rates of progression and therapy, and how specific epitopes were experimentally defined. Basic information such as HLA specificities for T-cell epitopes, isotypes of monoclonal antibodies, and epitope sequences are included whenever possible. All studies that we can find that incorporate the use of a specific monoclonal antibody are included in the entry for that antibody. A single T-cell epitope can have multiple entries, generally one entry per study. Finally, maps of all defined linear epitopes relative to the HXB2 reference proteins are provided.

  3. Discovering and differentiating new and emerging clonal populations of Chlamydia trachomatis with a novel shotgun cell culture harvest assay.

    Science.gov (United States)

    Somboonna, Naraporn; Mead, Sally; Liu, Jessica; Dean, Deborah

    2008-03-01

    Chlamydia trachomatis is the leading cause of preventable blindness and bacterial sexually transmitted diseases worldwide. Plaque assays have been used to clonally segregate laboratory-adapted C. trachomatis strains from mixed infections, but no assays have been reported to segregate clones from recent clinical samples. We developed a novel shotgun cell culture harvest assay for this purpose because we found that recent clinical samples do not form plaques. Clones were strain-typed by using outer membrane protein A and 16S rRNA sequences. Surprisingly, ocular trachoma reference strain A/SA-1 contained clones of Chlamydophila abortus. C. abortus primarily infects ruminants and pigs and has never been identified in populations where trachoma is endemic. Three clonal variants of reference strain Ba/Apache-2 were also identified. Our findings reflect the importance of clonal isolation in identifying constituents of mixed infections containing new or emerging strains and of viable clones for research to more fully understand the dynamics of in vivo strain-mixing, evolution, and disease pathogenesis.

  4. Geographic and temporal trends in the molecular epidemiology and genetic mechanisms of transmitted HIV-1 drug resistance: an individual-patient- and sequence-level meta-analysis

    NARCIS (Netherlands)

    Rhee, Soo-Yon; Blanco, Jose Luis; Jordan, Michael R.; Taylor, Jonathan; Lemey, Philippe; Varghese, Vici; Hamers, Raph L.; Bertagnolio, Silvia; Rinke de Wit, Tobias F.; Aghokeng, Avelin F.; Albert, Jan; Avi, Radko; Avila-Rios, Santiago; Bessong, Pascal O.; Brooks, James I.; Boucher, Charles A. B.; Brumme, Zabrina L.; Busch, Michael P.; Bussmann, Hermann; Chaix, Marie-Laure; Chin, Bum Sik; D'Aquin, Toni T.; de Gascun, Cillian F.; Derache, Anne; Descamps, Diane; Deshpande, Alaka K.; Djoko, Cyrille F.; Eshleman, Susan H.; Fleury, Herve; Frange, Pierre; Fujisaki, Seiichiro; Harrigan, P. Richard; Hattori, Junko; Holguin, Africa; Hunt, Gillian M.; Ichimura, Hiroshi; Kaleebu, Pontiano; Katzenstein, David; Kiertiburanakul, Sasisopin; Kim, Jerome H.; Kim, Sung Soon; Li, Yanpeng; Lutsar, Irja; Morris, Lynn; Ndembi, Nicaise; Ng, Kee Peng; Paranjape, Ramesh S.; Peeters, Martine; Poljak, Mario; Price, Matt A.; Ragonnet-Cronin, Manon L.; Reyes-Terán, Gustavo; Rolland, Morgane; Sirivichayakul, Sunee; Smith, Davey M.; Soares, Marcelo A.; Soriano, Vincent V.; Ssemwanga, Deogratius; Stanojevic, Maja; Stefani, Mariane A.; Sugiura, Wataru; Sungkanuparph, Somnuek; Tanuri, Amilcar; tee, Kok Keng; Truong, Hong-Ha M.; van de Vijver, David A. M. C.; Vidal, Nicole; Yang, Chunfu; Yang, Rongge; Yebra, Gonzalo; Ioannidis, John P. A.; Vandamme, Anne-Mieke; Shafer, Robert W.

    2015-01-01

    Regional and subtype-specific mutational patterns of HIV-1 transmitted drug resistance (TDR) are essential for informing first-line antiretroviral (ARV) therapy guidelines and designing diagnostic assays for use in regions where standard genotypic resistance testing is not affordable. We sought to

  5. Geographic and Temporal Trends in the Molecular Epidemiology and Genetic Mechanisms of Transmitted HIV-1 Drug Resistance: An Individual-Patient- and Sequence-Level Meta-Analysis

    NARCIS (Netherlands)

    S.Y. Rhee (Soo Yoon); J.L. Blanco (Jose Luis); M.R. Jordan (Michael); J. Taylor (Jonathan); P. Lemey (Philippe); V. Varghese (Vici); R.L. Hamers (Raph); S. Bertagnolio (Silvia); M. De Wit (Meike); A.F. Aghokeng (Avelin); J. Albert (Jan); R. Avi (Radko); S. Avila-Rios (Santiago); P.O. Bessong (Pascal O.); J.I. Brooks (James I.); C.A.B. Boucher (Charles); Z.L. Brumme (Zabrina L.); M.P. Busch (Michael P.); H. Bussmann (Hermann); M.L. Chaix (Marie Laure); B.S. Chin (Bum Sik); T.T. D’Aquin (Toni T.); C. de Gascun (Cillian); A. Derache (Anne); D. Descamps (Diane); A.K. Deshpande (Alaka K.); C.F. Djoko (Cyrille F.); S.H. Eshleman (Susan H.); H. Fleury (Hervé); P. Frange (Pierre); S. Fujisaki (Seiichiro); P. Harrigan (Pr); J. Hattori (Junko); A. Holguin (Africa); G.M. Hunt (Gillian M.); H. Ichimura (Hiroshi); P. Kaleebu (Pontiano); D. Katzenstein (David); S. Kiertiburanakul (Sasisopin); J.H. Kim (Jerome H.); S.S. Kim (Sung Soon); Y. Li (Yanpeng); I. Lutsar (Irja); L. Morris (L.); N. Ndembi (Nicaise); K.P. NG (Kee Peng); R.S. Paranjape (Ramesh S.); M.C. Peeters (Marian); M. Poljak (Mario); M.A. Price (Matt A.); M.L. Ragonnet-Cronin (Manon L.); G. Reyes-Terán (Gustavo); M. Rolland (Morgane); S. Sirivichayakul (Sunee); D.M. Smith (Davey M.); M.A. Soares (Marcelo A.); V. Soriano (Virtudes); D. Ssemwanga (Deogratius); M. Stanojevic (Maja); M.A. Stefani (Mariane A.); W. Sugiura (Wataru); S. Sungkanuparph (Somnuek); A. Tanuri (Amilcar); K.K. Tee (Kok Keng); H.-H.M. Truong (Hong-Ha M.); D.A.M.C. van de Vijver (David); N. Vidal (Nicole); C. Yang (Chunfu); R. Yang (Rongge); G. Yebra (Gonzalo); J.P.A. Ioannidis (John); A.M. Vandamme (Anne Mieke); R.W. Shafer (Robert)

    2015-01-01

    textabstractRegional and subtype-specific mutational patterns of HIV-1 transmitted drug resistance (TDR) are essential for informing first-line antiretroviral (ARV) therapy guidelines and designing diagnostic assays for use in regions where standard genotypic resistance testing is not affordable. We

  6. EuroClonality/BIOMED-2 guidelines for interpretation and reporting of Ig/TCR clonality testing in suspected lymphoproliferations

    NARCIS (Netherlands)

    Langerak, A. W.; Groenen, P. J. T. A.; Brüggemann, M.; Beldjord, K.; Bellan, C.; Bonello, L.; Boone, E.; Carter, G. I.; Catherwood, M.; Davi, F.; Delfau-Larue, M.-H.; Diss, T.; Evans, P. A. S.; Gameiro, P.; Garcia Sanz, R.; Gonzalez, D.; Grand, D.; Håkansson, A.; Hummel, M.; Liu, H.; Lombardia, L.; Macintyre, E. A.; Milner, B. J.; Montes-Moreno, S.; Schuuring, E.; Spaargaren, M.; Hodges, E.; van Dongen, J. J. M.

    2012-01-01

    PCR-based immunoglobulin (Ig)/T-cell receptor (TCR) clonality testing in suspected lymphoproliferations has largely been standardized and has consequently become technically feasible in a routine diagnostic setting. Standardization of the pre-analytical and post-analytical phases is now essential to

  7. Functional specialization of ramets in a clonal plant network functional specialization of ramets in a clonal plant network

    NARCIS (Netherlands)

    Ikegami, M.

    2004-01-01

    Functional specialization of ramets and co-operation between interconnected ramets in heterogeneous environments are studied in this thesis. Since clonal plants can spread horizontally by vegetative growth, a genet has the potential to grow across a heterogeneous environment. Therefore, ramets can

  8. Analysis of clonal expansions through the normal and premalignant human breast epithelium reveals the presence of luminal stem cells.

    Science.gov (United States)

    Cereser, Biancastella; Jansen, Marnix; Austin, Emily; Elia, George; McFarlane, Taneisha; van Deurzen, Carolien Hm; Sieuwerts, Anieta M; Daidone, Maria G; Tadrous, Paul J; Wright, Nicholas A; Jones, Louise; McDonald, Stuart Ac

    2018-01-01

    It is widely accepted that the cell of origin of breast cancer is the adult mammary epithelial stem cell; however, demonstrating the presence and location of tissue stem cells in the human breast has proved difficult. Furthermore, we do not know the clonal architecture of the normal and premalignant mammary epithelium or its cellular hierarchy. Here, we use deficiency in the mitochondrial enzyme cytochrome c oxidase (CCO), typically caused by somatic mutations in the mitochondrial genome, as a means to perform lineage tracing in the human mammary epithelium. PCR sequencing of laser-capture microdissected cells in combination with immunohistochemistry for markers of lineage differentiation was performed to determine the clonal nature of the mammary epithelium. We have shown that in the normal human breast, clonal expansions (defined here by areas of CCO deficiency) are typically uncommon and of limited size, but can occur at any site within the adult mammary epithelium. The presence of a stem cell population was shown by demonstrating multi-lineage differentiation within CCO-deficient areas. Interestingly, we observed infrequent CCO deficiency that was restricted to luminal cells, suggesting that niche succession, and by inference stem cell location, is located within the luminal layer. CCO-deficient areas appeared large within areas of ductal carcinoma in situ, suggesting that the rate of clonal expansion was altered in the premalignant lesion. © 2017 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland. © 2017 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

  9. Population dynamics of HIV-2 in rural West Africa: comparison with HIV-1 and ongoing transmission at the heart of the epidemic

    NARCIS (Netherlands)

    de Silva, Thushan I.; van Tienen, Carla; Onyango, Clayton; Jabang, Abdoulie; Vincent, Tim; Loeff, Maarten F. Schim van der; Coutinho, Roel A.; Jaye, Assan; Rowland-Jones, Sarah; Whittle, Hilton; Cotten, Matthew; Hué, Stéphane

    2013-01-01

    To compare the population dynamics of HIV-2 and HIV-1, and to characterize ongoing HIV-2 transmission in rural Guinea-Bissau. Phylogenetic and phylodynamic analyses using HIV-2 gag and env, and HIV-1 env sequences, combined with epidemiological data from a community cohort. Samples were obtained

  10. Modification of a loop sequence between α-helices 6 and 7 of virus capsid (CA protein in a human immunodeficiency virus type 1 (HIV-1 derivative that has simian immunodeficiency virus (SIVmac239 vif and CA α-helices 4 and 5 loop improves replication in cynomolgus monkey cells

    Directory of Open Access Journals (Sweden)

    Adachi Akio

    2009-08-01

    Full Text Available Abstract Background Human immunodeficiency virus type 1 (HIV-1 productively infects only humans and chimpanzees but not cynomolgus or rhesus monkeys while simian immunodeficiency virus isolated from macaque (SIVmac readily establishes infection in those monkeys. Several HIV-1 and SIVmac chimeric viruses have been constructed in order to develop an animal model for HIV-1 infection. Construction of an HIV-1 derivative which contains sequences of a SIVmac239 loop between α-helices 4 and 5 (L4/5 of capsid protein (CA and the entire SIVmac239 vif gene was previously reported. Although this chimeric virus could grow in cynomolgus monkey cells, it did so much more slowly than did SIVmac. It was also reported that intrinsic TRIM5α restricts the post-entry step of HIV-1 replication in rhesus and cynomolgus monkey cells, and we previously demonstrated that a single amino acid in a loop between α-helices 6 and 7 (L6/7 of HIV type 2 (HIV-2 CA determines the susceptibility of HIV-2 to cynomolgus monkey TRIM5α. Results In the study presented here, we replaced L6/7 of HIV-1 CA in addition to L4/5 and vif with the corresponding segments of SIVmac. The resultant HIV-1 derivatives showed enhanced replication capability in established T cell lines as well as in CD8+ cell-depleted primary peripheral blood mononuclear cells from cynomolgus monkey. Compared with the wild type HIV-1 particles, the viral particles produced from a chimeric HIV-1 genome with those two SIVmac loops were less able to saturate the intrinsic restriction in rhesus monkey cells. Conclusion We have succeeded in making the replication of simian-tropic HIV-1 in cynomolgus monkey cells more efficient by introducing into HIV-1 the L6/7 CA loop from SIVmac. It would be of interest to determine whether HIV-1 derivatives with SIVmac CA L4/5 and L6/7 can establish infection of cynomolgus monkeys in vivo.

  11. Clonal expansion of CD4(+) cytotoxic T lymphocytes in patients with IgG4-related disease.

    Science.gov (United States)

    Mattoo, Hamid; Mahajan, Vinay S; Maehara, Takashi; Deshpande, Vikram; Della-Torre, Emanuel; Wallace, Zachary S; Kulikova, Maria; Drijvers, Jefte M; Daccache, Joe; Carruthers, Mollie N; Castelino, Flavia V; Stone, James R; Stone, John H; Pillai, Shiv

    2016-09-01

    IgG4-related disease (IgG4-RD) is a systemic condition of unknown cause characterized by highly fibrotic lesions with dense lymphoplasmacytic infiltrates. CD4(+) T cells constitute the major inflammatory cell population in IgG4-RD lesions. We used an unbiased approach to characterize CD4(+) T-cell subsets in patients with IgG4-RD based on their clonal expansion and ability to infiltrate affected tissue sites. We used flow cytometry to identify CD4(+) effector/memory T cells in a cohort of 101 patients with IgG4-RD. These expanded cells were characterized by means of gene expression analysis and flow cytometry. Next-generation sequencing of the T-cell receptor β chain gene was performed on CD4(+)SLAMF7(+) cytotoxic T lymphocytes (CTLs) and CD4(+)GATA3(+) TH2 cells in a subset of patients to identify their clonality. Tissue infiltration by specific T cells was examined by using quantitative multicolor imaging. CD4(+) effector/memory T cells with a cytolytic phenotype were expanded in patients with IgG4-RD. Next-generation sequencing revealed prominent clonal expansions of these CD4(+) CTLs but not CD4(+)GATA3(+) memory TH2 cells in patients with IgG4-RD. The dominant T cells infiltrating a range of inflamed IgG4-RD tissue sites were clonally expanded CD4(+) CTLs that expressed SLAMF7, granzyme A, IL-1β, and TGF-β1. Clinical remission induced by rituximab-mediated B-cell depletion was associated with a reduction in numbers of disease-associated CD4(+) CTLs. IgG4-RD is prominently linked to clonally expanded IL-1β- and TGF-β1-secreting CD4(+) CTLs in both peripheral blood and inflammatory tissue lesions. These active, terminally differentiated, cytokine-secreting effector CD4(+) T cells are now linked to a human disease characterized by chronic inflammation and fibrosis. Copyright © 2016 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.

  12. HIV Testing

    Science.gov (United States)

    ... Abroad Treatment Basic Statistics Get Tested Find an HIV testing site near you. Enter ZIP code or city Follow HIV/AIDS CDC HIV CDC HIV/AIDS See RSS | ... All Collapse All Should I get tested for HIV? CDC recommends that everyone between the ages of ...

  13. Covariance of charged amino acids at positions 322 and 440 of HIV-1 Env contributes to coreceptor specificity of subtype B viruses, and can be used to improve the performance of V3 sequence-based coreceptor usage prediction algorithms.

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    Kieran Cashin

    Full Text Available The ability to determine coreceptor usage of patient-derived human immunodeficiency virus type 1 (HIV-1 strains is clinically important, particularly for the administration of the CCR5 antagonist maraviroc. The envelope glycoprotein (Env determinants of coreceptor specificity lie primarily within the gp120 V3 loop region, although other Env determinants have been shown to influence gp120-coreceptor interactions. Here, we determined whether conserved amino acid alterations outside the V3 loop that contribute to coreceptor usage exist, and whether these alterations improve the performance of V3 sequence-based coreceptor usage prediction algorithms. We demonstrate a significant covariant association between charged amino acids at position 322 in V3 and position 440 in the C4 Env region that contributes to the specificity of HIV-1 subtype B strains for CCR5 or CXCR4. Specifically, positively charged Lys/Arg at position 322 and negatively charged Asp/Glu at position 440 occurred more frequently in CXCR4-using viruses, whereas negatively charged Asp/Glu at position 322 and positively charged Arg at position 440 occurred more frequently in R5 strains. In the context of CD4-bound gp120, structural models suggest that covariation of amino acids at Env positions 322 and 440 has the potential to alter electrostatic interactions that are formed between gp120 and charged amino acids in the CCR5 N-terminus. We further demonstrate that inclusion of a "440 rule" can improve the sensitivity of several V3 sequence-based genotypic algorithms for predicting coreceptor usage of subtype B HIV-1 strains, without compromising specificity, and significantly improves the AUROC of the geno2pheno algorithm when set to its recommended false positive rate of 5.75%. Together, our results provide further mechanistic insights into the intra-molecular interactions within Env that contribute to coreceptor specificity of subtype B HIV-1 strains, and demonstrate that incorporation

  14. Clonal progression during the T cell-dependent B cell antibody response depends on the immunoglobulin DH gene segment repertoire.

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    Ahmad eTrad

    2014-08-01

    Full Text Available The diversity of the third complementarity determining region of the Ig H chain is constrained by natural selection of immunoglobulin diversity (DH sequence. To test the functional significance of this constraint in the context of thymus-dependent (TD immune responses, we immunized BALB/c mice with WT or altered DH sequence with 2-phenyloxazolone-coupled chicken serum albumin (phOx-CSA. We chose this antigen because studies of the humoral immune response to the hapten phOx were instrumental in the development of the current theoretical framework on which our understanding of the forces driving TD responses is based. To allow direct comparison, we used the classic approach of generating monoclonal Ab (mAb from various stages of the immune response to phOx to assess the effect of changing the sequence of the DH on clonal expansion, class switching and affinity maturation, which are hallmarks of TD responses. Compared to WT, TD-induced humoral IgM as well as IgG antibody production in the D-altered D-DFS and D-iD strains were significantly reduced. An increased prevalence of IgM producing hybridomas from late primary, secondary, and tertiary memory responses suggested either impaired class switch recombination (CSR or impaired clonal expansion of class switched B cells with phOx reactivity. Neither of the D-altered strains demonstrated the restriction in the VH/VL repertoire, the elimination of VH1 family-encoded antibodies, the focusing of the distribution of CDR-H3 lengths, or the selection for the normally dominant Ox1 clonotype which all are hallmarks of the anti-phOx response in WT mice. These changes in clonal selection and expansion as well as class switch recombination indicate that the genetic constitution of the DH locus, which has been selected by evolution, can strongly influence the functional outcome of a TD humoral response.

  15. Comparative drug susceptibility study of five clonal strains of Trichomonas vaginalis in vitro

    Institute of Scientific and Technical Information of China (English)

    Hemantkumar Somabhai Chaudhari; Prati Pal Singh

    2011-01-01

    Objective: To produce comparative data on a group of Trichomonas vaginalis clonal strains with varied drug responses using identical methods and materials. Methods: Five clonal strains of Trichomonas vaginalis were isolated from reference strain using agar plate technique. The variability of growth kinetic and susceptibility of clonal strain to metronidazole, tinidazole, satranidazole and nitazoxanide were observed in 96 well microtitre plate. Results: Among these clonal strains there was a good correlation between rates of growth with the relative susceptibility of the strains to drugs in vitro. Regarding metronidazole, tinidazole and satranidazole susceptibility, different degrees of susceptibility were determined. However, no difference in nitazoxanide susceptibility was found between the clonal strain tested and a reference strain.Conclusions: This is the first description of biological variability in clonal stock of Trichomonas vaginalis. Different degrees of drug susceptibility were determined among clonal strains tested. Further studies will be necessary to ascertain the importance of this variability in clinical infection.

  16. Evolution and diversity of clonal bacteria: the paradigm of Mycobacterium tuberculosis.

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    Tiago Dos Vultos

    Full Text Available BACKGROUND: Mycobacterium tuberculosis complex species display relatively static genomes and 99.9% nucleotide sequence identity. Studying the evolutionary history of such monomorphic bacteria is a difficult and challenging task. PRINCIPAL FINDINGS: We found that single-nucleotide polymorphism (SNP analysis of DNA repair, recombination and replication (3R genes in a comprehensive selection of M. tuberculosis complex strains from across the world, yielded surprisingly high levels of polymorphisms as compared to house-keeping genes, making it possible to distinguish between 80% of clinical isolates analyzed in this study. Bioinformatics analysis suggests that a large number of these polymorphisms are potentially deleterious. Site frequency spectrum comparison of synonymous and non-synonymous variants and Ka/Ks ratio analysis suggest a general negative/purifying selection acting on these sets of genes that may lead to suboptimal 3R system activity. In turn, the relaxed fidelity of 3R genes may allow the occurrence of adaptive variants, some of which will survive. Furthermore, 3R-based phylogenetic trees are a new tool for distinguishing between M. tuberculosis complex strains. CONCLUSIONS/SIGNIFICANCE: This situation, and the consequent lack of fidelity in genome maintenance, may serve as a starting point for the evolution of antibiotic resistance, fitness for survival and pathogenicity, possibly conferring a selective advantage in certain stressful situations. These findings suggest that 3R genes may play an important role in the evolution of highly clonal bacteria, such as M. tuberculosis. They also facilitate further epidemiological studies of these bacteria, through the development of high-resolution tools. With many more microbial genomes being sequenced, our results open the door to 3R gene-based studies of adaptation and evolution of other, highly clonal bacteria.

  17. Assignment of Streptococcus agalactiae isolates to clonal complexes using a small set of single nucleotide polymorphisms

    Directory of Open Access Journals (Sweden)

    Gilbert Gwendolyn L

    2008-08-01

    Full Text Available Abstract Background Streptococcus agalactiae (Group B Streptococcus (GBS is an important human pathogen, particularly of newborns. Emerging evidence for a relationship between genotype and virulence has accentuated the need for efficient and well-defined typing methods. The objective of this study was to develop a single nucleotide polymorphism (SNP based method for assigning GBS isolates to multilocus sequence typing (MLST-defined clonal complexes. Results It was found that a SNP set derived from the MLST database on the basis of maximisation of Simpsons Index of Diversity provided poor resolution and did not define groups concordant with the population structure as defined by eBURST analysis of the MLST database. This was interpreted as being a consequence of low diversity and high frequency horizontal gene transfer. Accordingly, a different approach to SNP identification was developed. This entailed use of the "Not-N" bioinformatic algorithm that identifies SNPs diagnostic for groups of known sequence variants, together with an empirical process of SNP testing. This yielded a four member SNP set that divides GBS into 10 groups that are concordant with the population structure. A fifth SNP was identified that increased the sensitivity for the clinically significant clonal complex 17 to 100%. Kinetic PCR methods for the interrogation of these SNPs were developed, and used to genotype 116 well characterized isolates. Conclusion A five SNP method for dividing GBS into biologically valid groups has been developed. These SNPs are ideal for high throughput surveillance activities, and combining with more rapidly evolving loci when additional resolution is required.

  18. Characterization of emergent HIV resistance in treatment-naive subjects enrolled in a vicriviroc phase 2 trial.

    Science.gov (United States)

    McNicholas, Paul; Wei, Yi; Whitcomb, Jeannette; Greaves, Wayne; Black, Todd A; Tremblay, Cecile L; Strizki, Julie M

    2010-05-15

    Vicriviroc is a C-C motif chemokine receptor 5 (CCR5) antagonist that is in clinical development for the treatment of human immunodeficiency virus type 1 (HIV-1) infection. This study explored the molecular basis for the development of phenotypically resistant virus. HIV-1 RNA from treatment-naive subjects who experienced virological failure in a phase 2 dose-finding trial was evaluated for coreceptor usage and susceptibility. For viruses that exhibited reduced susceptibility to vicriviroc, envelope clones were phenotypically and genotypically characterized. Twenty-six vicriviroc-treated subjects experienced virological failure; for 24 the virus remained CCR5-tropic, and 2 had dual/X4 virus. Reduced susceptibility to vicriviroc, manifested as decreases in the maximum percent inhibition value (no increase in median inhibitory concentration), was detected in 4 of the 26 subjects who experienced virological failure. Clonal analysis of envelopes in samples from these 4 subjects revealed multiple sequence changes in gp160, principally within the variable domain 1/variable domain 2, variable domain 3, and variable domain 4 loops. However, no consistent pattern of mutations was observed across subjects. In this study, only a small proportion of treatment failures were associated with tropism changes or reduced susceptibility to vicriviroc. Genotypic analysis of cloned env sequences revealed no specific mutational pattern associated with reduced susceptibility to vicriviroc, although numerous changes were observed in the variable domain 3 loop and in other regions of gp160.

  19. Introducing a frameshift mutation to the POL sequence of HIV-1 provirus and evaluation of the immunogenic characteristics of the mutated virions (RINNL4-3).

    Science.gov (United States)

    Zabihollahi, Rezvan; Sadat, Seyed Mehdi; Vahabpour, Rouhollah; Salehi, Mansoor; Azadmanesh, Kayhan; Siadat, Seyed Davar; Saraji, Ali Reza Azizi; Pouriavali, Mohamamd Hassan; Momen, Seyed Bahman; Aghasadeghi, Mohamad Reza

    2012-01-01

    Inactivation of the reverse transcriptase (RT) and integrase (IN) enzymes can abolish the replication of the human immunodeficiency virus (HIV) and, thus, its infectivity. Here, inactivated HIV particles convenient for designing virus-like particle (VLP) based vaccines have been produced. Inactivated HIV-provirus was created by introducing a frame shift mutation. HIV provirus DNA was cut in the pol region by Age I restriction enzyme, followed by filling of sticky ends using the Klenow fragment before ligation. The resulting plasmid was named as pRINNL4-3. HEK-293T cells were used as producer, after being transfected with the modified plasmid. Viral particle production and biological activity were assayed by virus capsid protein (p24) quantification and syncytium formation in MT2 cells, respectively. The immunogenicity of the RINNL4-3 virions was investigated in a mouse model. The mutation was expected to inactivate the virus RT and IN enzymes. The results showed that the VLPs were assembled, as measured by the p24 load of the culture supernatant, and contained functional envelope proteins (Env) as monitored by the syncytium formation. However, these VLPs had no ability to infect target MT2 cells, as well as their VSVG (vesicular stomatitis virus-glycoprotein) pseudotyped counterparts infected HEK-293T cells. A high level of antibody response was observed in immunized mice. Since RINNL4-3 virions are replication incompetent, they are convenient for production and use in biomedical studies. Also, RINNL4-3 is a candidate for a vaccine development due to it contains envelope and structural virus proteins which are crucial for triggering neutralizing antibodies and the cellular immune response.

  20. Genotypic Diversity of Staphylococcus aureus α-Hemolysin Gene (hla and Its Association with Clonal Background: Implications for Vaccine Development.

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    Meng Xiao

    Full Text Available The α-hemolysin, encoded by the hla gene, is a major virulence factor in S. aureus infections. Changes in key amino acid residues of α-hemolysin can result in reduction, or even loss, of toxicity. The aim of this study was to investigate the diversity of the hla gene sequence and the relationship of hla variants to the clonal background of S. aureus isolates. A total of 47 clinical isolates from China were used in this study, supplemented with in silico analysis of 318 well-characterized whole genome sequences from globally distributed isolates. A total of 28 hla genotypes were found, including three unique to isolates from China, 20 found only in the global genomes and five found in both. The hla genotype generally correlated with the clonal background, particularly the multilocus sequence type, but was not related to geographic origin, host source or methicillin-resistance phenotype. In addition, the hla gene showed greater diversity than the seven loci utilized in the MLST scheme for S. aureus. Our investigation has provided genetic data which may be useful for future studies of toxicity, immunogenicity and vaccine development.

  1. Clonal Distribution of Disease-Associated and Healthy Carrier Isolates of Neisseria meningitidis between 1983 and 2005 in Cuba ▿

    Science.gov (United States)

    Climent, Yanet; Yero, Daniel; Martinez, Isabel; Martín, Alejandro; Jolley, Keith A.; Sotolongo, Franklin; Maiden, Martin C. J.; Urwin, Rachel; Pajón, Rolando

    2010-01-01

    In response to epidemic levels of serogroup B meningococcal disease in Cuba during the 1980s, the VA-MENGOC-BC vaccine was developed and introduced into the National Infant Immunization Program in 1991. Since then the incidence of meningococcal disease in Cuba has returned to the low levels recorded before the epidemic. A total of 420 Neisseria meningitidis strains collected between 1983 and 2005 in Cuba were analyzed by multilocus sequence typing (MLST). The set of strains comprised 167 isolated from disease cases and 253 obtained from healthy carriers. By MLST analysis, 63 sequence types (STs) were identified, and 32 of these were reported to be a new ST. The Cuban isolates were associated with 12 clonal complexes; and the most common were ST-32 (246 isolates), ST-53 (86 isolates), and ST-41/44 (36 isolates). This study also showed that the application of VA-MENGOC-BC, the Cuban serogroup B and C vaccine, reduced the frequency and diversity of hypervirulent clonal complexes ST-32 (vaccine serogroup B type-strain) and ST-41/44 and also affected other lineages. Lineages ST-8 and ST-11 were no longer found during the postvaccination period. The vaccine also affected the genetic composition of the carrier-associated meningococcal isolates. The number of carrier isolates belonging to hypervirulent lineages decreased significantly after vaccination, and ST-53, a sequence type common in carriers, became the predominant ST. PMID:20042619

  2. Clonality and evolutionary history of rhabdomyosarcoma.

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    Li Chen

    2015-03-01

    Full Text Available To infer the subclonality of rhabdomyosarcoma (RMS and predict the temporal order of genetic events for the tumorigenic process, and to identify novel drivers, we applied a systematic method that takes into account germline and somatic alterations in 44 tumor-normal RMS pairs using deep whole-genome sequencing. Intriguingly, we find that loss of heterozygosity of 11p15.5 and mutations in RAS pathway genes occur early in the evolutionary history of the PAX-fusion-negative-RMS (PFN-RMS subtype. We discover several early mutations in non-RAS mutated samples and predict them to be drivers in PFN-RMS including recurrent mutation of PKN1. In contrast, we find that PAX-fusion-positive (PFP subtype tumors have undergone whole-genome duplication in the late stage of cancer evolutionary history and have acquired fewer mutations and subclones than PFN-RMS. Moreover we predict that the PAX3-FOXO1 fusion event occurs earlier than the whole genome duplication. Our findings provide information critical to the understanding of tumorigenesis of RMS.

  3. Ex vivo activation of CD4+ T-cells from donors on suppressive ART can lead to sustained production of infectious HIV-1 from a subset of infected cells.

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    John K Bui

    2017-02-01

    Full Text Available The fate of HIV-infected cells after reversal of proviral latency is not well characterized. Simonetti, et al. recently showed that CD4+ T-cells containing intact proviruses can clonally expand in vivo and produce low-level infectious viremia. We hypothesized that reversal of HIV latency by activation of CD4+ T-cells can lead to the expansion of a subset of virus-producing cells rather than their elimination. We established an ex vivo cell culture system involving stimulation of CD4+ T-cells from donors on suppressive antiretroviral therapy (ART with PMA/ionomycin (day 1-7, followed by rest (day 7-21, and then repeat stimulation (day 21-28, always in the presence of high concentrations of raltegravir and efavirenz to effectively block new cycles of viral replication. HIV DNA and virion RNA in the supernatant were quantified by qPCR. Single genome sequencing (SGS of p6-PR-RT was performed to genetically characterize proviruses and virion-associated genomic RNA. The replication-competence of the virions produced was determined by the viral outgrowth assay (VOA and SGS of co-culture supernatants from multiple time points. Experiments were performed with purified CD4+ T-cells from five consecutively recruited donors who had been on suppressive ART for > 2 years. In all experiments, HIV RNA levels in supernatant increased following initial stimulation, decreased or remained stable during the rest period, and increased again with repeat stimulation. HIV DNA levels did not show a consistent pattern of change. SGS of proviruses revealed diverse outcomes of infected cell populations, ranging from their apparent elimination to persistence and expansion. Importantly, a subset of infected cells expanded and produced infectious virus continuously after stimulation. These findings underscore the complexity of eliminating reservoirs of HIV-infected cells and highlight the need for new strategies to kill HIV-infected cells before they can proliferate.

  4. Clonal forestry, heterosis and advanced-generation breeding

    Energy Technology Data Exchange (ETDEWEB)

    Tuskan, G.A.

    1997-08-01

    This report discusses the clonal planting stock offers many advantages to the forest products industry. Advanced-generation breeding strategies should be designed to maximize within-family variance and at the same time allow the capture of heterosis. Certainly there may be a conflict in the choice of breeding strategy based on the trait of interest. It may be that the majority of the traits express heterosis due to overdominance. Alternatively, disease resistance is expressed as the lack of a specific metabolite or infection court then the homozygous recessive genotype may be the most desirable. Nonetheless, as the forest products industry begins to utilize the economic advantages of clonal forestry, breeding strategies will have to be optimized for these commercial plant materials. Here, molecular markers can be used to characterize the nature of heterosis and therefore define the appropriate breeding strategy.

  5. Clonal multiplication of guava through softwood cuttings under mist conditions

    International Nuclear Information System (INIS)

    Kareem, A.; Jaskkani, M.J.; Fatima, B.; Sadia, B.

    2012-01-01

    Guava (psidium guajava l.) is a luscious and important tropical fruit crop. the objective of the study was to develop vegetative propagation system to avoid clonal degradation in guava fruit plants. softwood cuttings from five year old gola accession were prepared from the shoot tops of current season growth, measuring 12 cm in length and carrying 2 to 4 nodes. iba and naa (0, 2000, 4000, 6000, 8000 ppm) were selected to treat cuttings for root induction. cuttings were planted under mist conditions by maintaining temperature at 25 degree c and 85% relative humidity for 25 days. maximum survival percentage (92.17%) of plants at transplanting was noted with 4000 ppm concentration followed by 2000 ppm (85.50%). In general iba 4000 ppm concentration performed better as compared to naa for all parameters studied. This study revealed the potential of clonal propagation of guava through softwood cuttings treated with auxins. (author)

  6. Dynamics of Tumor Heterogeneity Derived from Clonal Karyotypic Evolution

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    Ashley M. Laughney

    2015-08-01

    Full Text Available Numerical chromosomal instability is a ubiquitous feature of human neoplasms. Due to experimental limitations, fundamental characteristics of karyotypic changes in cancer are poorly understood. Using an experimentally inspired stochastic model, based on the potency and chromosomal distribution of oncogenes and tumor suppressor genes, we show that cancer cells have evolved to exist within a narrow range of chromosome missegregation rates that optimizes phenotypic heterogeneity and clonal survival. Departure from this range reduces clonal fitness and limits subclonal diversity. Mapping of the aneuploid fitness landscape reveals a highly favorable, commonly observed, near-triploid state onto which evolving diploid- and tetraploid-derived populations spontaneously converge, albeit at a much lower fitness cost for the latter. Finally, by analyzing 1,368 chromosomal translocation events in five human cancers, we find that karyotypic evolution also shapes chromosomal translocation patterns by selecting for more oncogenic derivative chromosomes. Thus, chromosomal instability can generate the heterogeneity required for Darwinian tumor evolution.

  7. HIV-1 transmission linkage in an HIV-1 prevention clinical trial

    Energy Technology Data Exchange (ETDEWEB)

    Leitner, Thomas [Los Alamos National Laboratory; Campbell, Mary S [UNIV OF WASHINGTON; Mullins, James I [UNIV OF WASHINGTON; Hughes, James P [UNIV OF WASHINGTON; Wong, Kim G [UNIV OF WASHINGTON; Raugi, Dana N [UNIV OF WASHINGTON; Scrensen, Stefanie [UNIV OF WASHINGTON

    2009-01-01

    HIV-1 sequencing has been used extensively in epidemiologic and forensic studies to investigate patterns of HIV-1 transmission. However, the criteria for establishing genetic linkage between HIV-1 strains in HIV-1 prevention trials have not been formalized. The Partners in Prevention HSV/HIV Transmission Study (ClinicaITrials.gov NCT00194519) enrolled 3408 HIV-1 serodiscordant heterosexual African couples to determine the efficacy of genital herpes suppression with acyclovir in reducing HIV-1 transmission. The trial analysis required laboratory confirmation of HIV-1 linkage between enrolled partners in couples in which seroconversion occurred. Here we describe the process and results from HIV-1 sequencing studies used to perform transmission linkage determination in this clinical trial. Consensus Sanger sequencing of env (C2-V3-C3) and gag (p17-p24) genes was performed on plasma HIV-1 RNA from both partners within 3 months of seroconversion; env single molecule or pyrosequencing was also performed in some cases. For linkage, we required monophyletic clustering between HIV-1 sequences in the transmitting and seroconverting partners, and developed a Bayesian algorithm using genetic distances to evaluate the posterior probability of linkage of participants sequences. Adjudicators classified transmissions as linked, unlinked, or indeterminate. Among 151 seroconversion events, we found 108 (71.5%) linked, 40 (26.5%) unlinked, and 3 (2.0%) to have indeterminate transmissions. Nine (8.3%) were linked by consensus gag sequencing only and 8 (7.4%) required deep sequencing of env. In this first use of HIV-1 sequencing to establish endpoints in a large clinical trial, more than one-fourth of transmissions were unlinked to the enrolled partner, illustrating the relevance of these methods in the design of future HIV-1 prevention trials in serodiscordant couples. A hierarchy of sequencing techniques, analysis methods, and expert adjudication contributed to the linkage

  8. Draft genome sequences of seven isolates of Phytophthora ramorum EU2 from Northern Ireland

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    Lourdes de la Mata Saez

    2015-12-01

    Full Text Available Here we present draft-quality genome sequence assemblies for the oomycete Phytophthora ramorum genetic lineage EU2. We sequenced genomes of seven isolates collected in Northern Ireland between 2010 and 2012. Multiple genome sequences from P. ramorum EU2 will be valuable for identifying genetic variation within the clonal lineage that can be useful for tracking its spread.

  9. Clonal and bud bank traits: patterns across temperate plant communities

    Czech Academy of Sciences Publication Activity Database

    Klimešová, Jitka; Herben, Tomáš

    2015-01-01

    Roč. 26, č. 2 (2015), s. 243-253 ISSN 1100-9233 R&D Projects: GA ČR GB14-36079G; GA ČR GA13-17118S; GA ČR GAP505/12/1007 Institutional support: RVO:67985939 Keywords : clonal and bud bank traits * vegetation * central Europe Subject RIV: EH - Ecology, Behaviour Impact factor: 3.151, year: 2015

  10. Nutrient leaching under zero tension in a subtropical clonal eucalypt ...

    African Journals Online (AJOL)

    Little is known about the effects of residue burning or retention on nutrient leaching during the inter-rotation of clonal Eucalyptus grown on the sandy soils of subtropical Zululand, South Africa. A study compared zero-tension nutrient leaching through the top metre of soil at depths of 0.15, 0.5 and 1.0 m in an undisturbed crop ...

  11. Clonal status and clinicopathological observation of cervical minimal deviation adenocarcinoma

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    Lan Miao

    2010-04-01

    Full Text Available Abstract Background Minimal deviation adenocarcinoma (MDA of the uterine cervix is defined as an extremely well differentiated variant of cervical adenocarcinoma, with well-formed glands that resemble benign glands but show distinct nuclear anaplasia or evidence of stromal invasion. Thus, MDA is difficult to differentiate from other cervical hyperplastic lesions. Monoclonality is a major characteristic of most tumors, whereas normal tissue and reactive hyperplasia are polyclonal. Methods The clinicopathological features and clonality of MDA were investigated using laser microdissection and a clonality assay based on the polymorphism of androgen receptor (AR and X-chromosomal inactivation mosaicism in female somatic tissues. Results The results demonstrated that the glands were positive for CEA, Ki-67, and p53 and negative for estrogen receptor (ER, progesterone receptor (PR, and high-risk human papilloma virus (HPV DNA. The index of proliferation for Ki-67 was more than 50%. However, the stromal cells were positive for ER, PR, vimentin, and SM-actin. The clonal assay showed that MDA was monoclonal. Thus, our findings indicate that MDA is a true neoplasm but is not associated with high-risk HPV. Conclusions Diagnosis of MDA depends mainly on its clinical manifestations, the pathological feature that MDA glands are located deeper than the lower level of normal endocervical glands, and immunostaining.

  12. Multiplexing clonality: combining RGB marking and genetic barcoding

    Science.gov (United States)

    Cornils, Kerstin; Thielecke, Lars; Hüser, Svenja; Forgber, Michael; Thomaschewski, Michael; Kleist, Nadja; Hussein, Kais; Riecken, Kristoffer; Volz, Tassilo; Gerdes, Sebastian; Glauche, Ingmar; Dahl, Andreas; Dandri, Maura; Roeder, Ingo; Fehse, Boris

    2014-01-01

    RGB marking and DNA barcoding are two cutting-edge technologies in the field of clonal cell marking. To combine the virtues of both approaches, we equipped LeGO vectors encoding red, green or blue fluorescent proteins with complex DNA barcodes carrying color-specific signatures. For these vectors, we generated highly complex plasmid libraries that were used for the production of barcoded lentiviral vector particles. In proof-of-principle experiments, we used barcoded vectors for RGB marking of cell lines and primary murine hepatocytes. We applied single-cell polymerase chain reaction to decipher barcode signatures of individual RGB-marked cells expressing defined color hues. This enabled us to prove clonal identity of cells with one and the same RGB color. Also, we made use of barcoded vectors to investigate clonal development of leukemia induced by ectopic oncogene expression in murine hematopoietic cells. In conclusion, by combining RGB marking and DNA barcoding, we have established a novel technique for the unambiguous genetic marking of individual cells in the context of normal regeneration as well as malignant outgrowth. Moreover, the introduction of color-specific signatures in barcodes will facilitate studies on the impact of different variables (e.g. vector type, transgenes, culture conditions) in the context of competitive repopulation studies. PMID:24476916

  13. A computational clonal analysis of the developing mouse limb bud.

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    Luciano Marcon

    Full Text Available A comprehensive spatio-temporal description of the tissue movements underlying organogenesis would be an extremely useful resource to developmental biology. Clonal analysis and fate mappings are popular experiments to study tissue movement during morphogenesis. Such experiments allow cell populations to be labeled at an early stage of development and to follow their spatial evolution over time. However, disentangling the cumulative effects of the multiple events responsible for the expansion of the labeled cell population is not always straightforward. To overcome this problem, we develop a novel computational method that combines accurate quantification of 2D limb bud morphologies and growth modeling to analyze mouse clonal data of early limb development. Firstly, we explore various tissue movements that match experimental limb bud shape changes. Secondly, by comparing computational clones with newly generated mouse clonal data we are able to choose and characterize the tissue movement map that better matches experimental data. Our computational analysis produces for the first time a two dimensional model of limb growth based on experimental data that can be used to better characterize limb tissue movement in space and time. The model shows that the distribution and shapes of clones can be described as a combination of anisotropic growth with isotropic cell mixing, without the need for lineage compartmentalization along the AP and PD axis. Lastly, we show that this comprehensive description can be used to reassess spatio-temporal gene regulations taking tissue movement into account and to investigate PD patterning hypothesis.

  14. Detection of Hepatitis B Virus (HBV) Genomes and HBV Drug Resistant Variants by Deep Sequencing Analysis of HBV Genomes in Immune Cell Subsets of HBV Mono-Infected and/or Human Immunodeficiency Virus Type-1 (HIV-1) and HBV Co-Infected Individuals

    Science.gov (United States)

    Lee, Z.; Nishikawa, S.; Gao, S.; Eksteen, J. B.; Czub, M.; Gill, M. J.; Osiowy, C.; van der Meer, F.; van Marle, G.; Coffin, C. S.

    2015-01-01

    The hepatitis B virus (HBV) and the human immunodeficiency virus type 1 (HIV-1) can infect cells of the lymphatic system. It is unknown whether HIV-1 co-infection impacts infection of peripheral blood mononuclear cell (PBMC) subsets by the HBV. Aims To compare the detection of HBV genomes and HBV sequences in unsorted PBMCs and subsets (i.e., CD4+ T, CD8+ T, CD14+ monocytes, CD19+ B, CD56+ NK cells) in HBV mono-infected vs. HBV/HIV-1 co-infected individuals. Methods Total PBMC and subsets isolated from 14 HBV mono-infected (4/14 before and after anti-HBV therapy) and 6 HBV/HIV-1 co-infected individuals (5/6 consistently on dual active anti-HBV/HIV therapy) were tested for HBV genomes, including replication indicative HBV covalently closed circular (ccc)-DNA, by nested PCR/nucleic hybridization and/or quantitative PCR. In CD4+, and/or CD56+ subsets from two HBV monoinfected cases, the HBV polymerase/overlapping surface region was analyzed by next generation sequencing. Results All analyzed whole PBMC from HBV monoinfected and HBV/HIV coinfected individuals were HBV genome positive. Similarly, HBV DNA was detected in all target PBMC subsets regardless of antiviral therapy, but was absent from the CD4+ T cell subset from all HBV/HIV-1 positive cases (PHBV monoinfected cases on tenofovir therapy, mutations at residues associated with drug resistance and/or immune escape (i.e., G145R) were detected in a minor percentage of the population. Summary HBV genomes and drug resistant variants were detectable in PBMC subsets from HBV mono-infected individuals. The HBV replicates in PBMC subsets of HBV/HIV-1 patients except the CD4+ T cell subpopulation. PMID:26390290

  15. Proliferation of latently infected CD4+ T cells carrying replication-competent HIV-1: Potential role in latent reservoir dynamics

    Science.gov (United States)

    Hosmane, Nina N.; Kwon, Kyungyoon J.; Bruner, Katherine M.; Capoferri, Adam A.; Rosenbloom, Daniel I.S.; Keele, Brandon F.; Ho, Ya-Chi

    2017-01-01

    A latent reservoir for HIV-1 in resting CD4+ T lymphocytes precludes cure. Mechanisms underlying reservoir stability are unclear. Recent studies suggest an unexpected degree of infected cell proliferation in vivo. T cell activation drives proliferation but also reverses latency, resulting in productive infection that generally leads to cell death. In this study, we show that latently infected cells can proliferate in response to mitogens without producing virus, generating progeny cells that can release infectious virus. Thus, assays relying on one round of activation underestimate reservoir size. Sequencing of independent clonal isolates of replication-competent virus revealed that 57% had env sequences identical to other isolates from the same patient. Identity was confirmed by full-genome sequencing and was not attributable to limited viral diversity. Phylogenetic and statistical analysis suggested that identical sequences arose from in vivo proliferation of infected cells, rather than infection of multiple cells by a dominant viral species. The possibility that much of the reservoir arises by cell proliferation presents challenges to cure. PMID:28341641

  16. Proliferation of latently infected CD4+ T cells carrying replication-competent HIV-1: Potential role in latent reservoir dynamics.

    Science.gov (United States)

    Hosmane, Nina N; Kwon, Kyungyoon J; Bruner, Katherine M; Capoferri, Adam A; Beg, Subul; Rosenbloom, Daniel I S; Keele, Brandon F; Ho, Ya-Chi; Siliciano, Janet D; Siliciano, Robert F

    2017-04-03

    A latent reservoir for HIV-1 in resting CD4 + T lymphocytes precludes cure. Mechanisms underlying reservoir stability are unclear. Recent studies suggest an unexpected degree of infected cell proliferation in vivo. T cell activation drives proliferation but also reverses latency, resulting in productive infection that generally leads to cell death. In this study, we show that latently infected cells can proliferate in response to mitogens without producing virus, generating progeny cells that can release infectious virus. Thus, assays relying on one round of activation underestimate reservoir size. Sequencing of independent clonal isolates of replication-competent virus revealed that 57% had env sequences identical to other isolates from the same patient. Identity was confirmed by full-genome sequencing and was not attributable to limited viral diversity. Phylogenetic and statistical analysis suggested that identical sequences arose from in vivo proliferation of infected cells, rather than infection of multiple cells by a dominant viral species. The possibility that much of the reservoir arises by cell proliferation presents challenges to cure. © 2017 Hosmane et al.

  17. Physics of HIV

    Science.gov (United States)

    Tristram-Nagle, Stephanie

    2018-05-01

    This review summarizes over a decade of investigations into how membrane-binding proteins from the HIV-1 virus interact with lipid membrane mimics of various HIV and host T-cell membranes. The goal of the work was to characterize at the molecular level both the elastic and structural changes that occur due to HIV protein/membrane interactions, which could lead to new drugs to thwart the HIV virus. The main technique used to study these interactions is diffuse x-ray scattering, which yields the bending modulus, K C, as well as structural parameters such as membrane thickness, area/lipid and position of HIV peptides (parts of HIV proteins) in the membrane. Our methods also yield information about lipid chain order or disorder caused by the peptides. This review focuses on three stages of the HIV-1 life cycle: (1) infection, (2) Tat membrane transport, and (3) budding. In the infection stage, our lab studied three different parts of HIV-1 gp41 (glycoprotein 41 fusion protein): (1) FP23, the N-terminal 23 amino acids that interact non-specifically with the T-cell host membrane to cause fusion of two membranes, and its trimer version, (2) cholesterol recognition amino acid consensus sequence, on the membrane proximal external region near the membrane-spanning domain, and (3) lentiviral lytic peptide 2 on the cytoplasmic C-terminal tail. For Tat transport, we used membrane mimics of the T-cell nuclear membrane as well as simpler models that varied charge and negative curvature. For membrane budding, we varied the myristoylation of the MA31 peptide as well as the negatively charged lipid. These studies show that HIV peptides with different roles in the HIV life cycle affect differently the relevant membrane mimics. In addition, the membrane lipid composition plays an important role in the peptides’ effects.

  18. Frequency and site mapping of HIV-1/SIVcpz, HIV- 2/SIVsmm and ...

    African Journals Online (AJOL)

    out to analyze the effects of various restriction enzymes on the HIV genome. A computer simulated model using Web cutter Version 2.0, and cytogenetic analysis. 339 restriction enzymes from Promega database, 10 HIV-1/SIVcpz genes, 10 HIV-2/SIVsmm genes and 10 other SIV genes. Gene sequences were fed into Web ...

  19. HIV Molecular Immunology 2015

    Energy Technology Data Exchange (ETDEWEB)

    Yusim, Karina [Los Alamos National Lab. (LANL), Los Alamos, NM (United States). Theoretical Division; Korber, Bette Tina [Los Alamos National Lab. (LANL), Los Alamos, NM (United States). Theoretical Division; Brander, Christian [Institucio Catalana de Recerca i Estudis Avancats (ICREA), Barcelona (Spain); Barouch, Dan [Beth Israel Deaconess Medical Center, Boston, MA (United States). Division of Vaccine Research; de Boer, Rob [Utrecht University, Utrecht (Netherlands). Faculty of Biology; Haynes, Barton F. [Duke Univ., Durham, NC (United States). Duke Human Vaccine Institute and Departments of Medicine, Surgery and Immunology; Koup, Richard [National Inst. of Health (NIH), Bethesda, MD (United States). Vaccine Research Center; Moore, John P. [Cornell Univ., Ithaca, NY (United States). Weill Medical College; Walker, Bruce D. [Ragon Institute, Cambridge, MA (United States); Watkins, David [Wisconsin Regional Primate Research Center, Madison, WI (United States)

    2016-04-05

    The scope and purpose of the HIV molecular immunology database: HIV Molecular Immunology is a companion volume to HIV Sequence Compendium. This publication, the 2015 edition, is the PDF version of the web-based HIV Immunology Database (http://www.hiv.lanl.gov/ content/immunology/). The web interface for this relational database has many search options, as well as interactive tools to help immunologists design reagents and interpret their results. In the HIV Immunology Database, HIV-specific B-cell and T-cell responses are summarized and annotated. Immunological responses are divided into three parts, CTL, T helper, and antibody. Within these parts, defined epitopes are organized by protein and binding sites within each protein, moving from left to right through the coding regions spanning the HIV genome. We include human responses to natural HIV infections, as well as vaccine studies in a range of animal models and human trials. Responses that are not specifically defined, such as responses to whole proteins or monoclonal antibody responses to discontinuous epitopes, are summarized at the end of each protein section. Studies describing general HIV responses to the virus, but not to any specific protein, are included at the end of each part. The annotation includes information such as cross-reactivity, escape mutations, antibody sequence, TCR usage, functional domains that overlap with an epitope, immune response associations with rates of progression and therapy, and how specific epitopes were experimentally defined. Basic information such as HLA specificities for T-cell epitopes, isotypes of monoclonal antibodies, and epitope sequences are included whenever possible. All studies that we can find that incorporate the use of a specific monoclonal antibody are included in the entry for that antibody. A single T-cell epitope can have multiple entries, generally one entry per study. Finally, maps of all defined linear epitopes relative to the HXB2 reference proteins

  20. Sequence similarity between the erythrocyte binding domain of the Plasmodium vivax Duffy binding protein and the V3 loop of HIV-1 strain MN reveals a functional heparin binding motif involved in binding to the Duffy antigen receptor for chemokines

    Directory of Open Access Journals (Sweden)

    Bolton Michael J

    2011-11-01

    Full Text Available Abstract Background The HIV surface glycoprotein gp120 (SU, gp120 and the Plasmodium vivax Duffy binding protein (PvDBP bind to chemokine receptors during infection and have a site of amino acid sequence similarity in their binding domains that often includes a heparin binding motif (HBM. Infection by either pathogen has been found to be inhibited by polyanions. Results Specific polyanions that inhibit HIV infection and bind to the V3 loop of X4 strains also inhibited DBP-mediated infection of erythrocytes and DBP binding to the Duffy Antigen Receptor for Chemokines (DARC. A peptide including the HBM of PvDBP had similar affinity for heparin as RANTES and V3 loop peptides, and could be specifically inhibited from heparin binding by the same polyanions that inhibit DBP binding to DARC. However, some V3 peptides can competitively inhibit RANTES binding to heparin, but not the PvDBP HBM peptide. Three other members of the DBP family have an HBM sequence that is necessary for erythrocyte binding, however only the protein which binds to DARC, the P. knowlesi alpha protein, is inhibited by heparin from binding to erythrocytes. Heparitinase digestion does not affect the binding of DBP to erythrocytes. Conclusion The HBMs of DBPs that bind to DARC have similar heparin binding affinities as some V3 loop peptides and chemokines, are responsible for specific sulfated polysaccharide inhibition of parasite binding and invasion of red blood cells, and are more likely to bind to negative charges on the receptor than cell surface glycosaminoglycans.

  1. Next generation sequencing (NGS)technologies and applications

    Energy Technology Data Exchange (ETDEWEB)

    Vuyisich, Momchilo [Los Alamos National Laboratory

    2012-09-11

    NGS technology overview: (1) NGS library preparation - Nucleic acids extraction, Sample quality control, RNA conversion to cDNA, Addition of sequencing adapters, Quality control of library; (2) Sequencing - Clonal amplification of library fragments, (except PacBio), Sequencing by synthesis, Data output (reads and quality); and (3) Data analysis - Read mapping, Genome assembly, Gene expression, Operon structure, sRNA discovery, and Epigenetic analyses.

  2. MDS-associated somatic mutations and clonal hematopoiesis are common in idiopathic cytopenias of undetermined significance.

    Science.gov (United States)

    Kwok, Brian; Hall, Jeff M; Witte, John S; Xu, Yin; Reddy, Prashanti; Lin, Keming; Flamholz, Rachel; Dabbas, Bashar; Yung, Aine; Al-Hafidh, Jenan; Balmert, Emily; Vaupel, Christine; El Hader, Carlos; McGinniss, Matthew J; Nahas, Shareef A; Kines, Julie; Bejar, Rafael

    2015-11-19

    Establishing a diagnosis in patients suspected of having a myelodysplastic syndrome (MDS) can be challenging and could be informed by the identification of somatic mutations. We performed a prospective study to examine the frequency and types of mutations encountered in 144 patients with unexplained cytopenias. Based on bone marrow findings, 17% were diagnosed with MDS, 15% with idiopathic cytopenias of undetermined significance (ICUS) and some evidence of dysplasia, and 69% with ICUS and no dysplasia. Bone marrow DNA was sequenced for mutations in 22 frequently mutated myeloid malignancy genes. Somatic mutations were identified in 71% of MDS patients, 62% of patients with ICUS and some dysplasia, and 20% of ICUS patients and no dysplasia. In total, 35% of ICUS patients carried a somatic mutation or chromosomal abnormality indicative of clonal hematopoiesis. We validated these results in a cohort of 91 lower-risk MDS and 249 ICUS cases identified over a 6-month interval. Mutations were found in 79% of those with MDS, in 45% of those with ICUS with dysplasia, and in 17% of those with ICUS without dysplasia. The spectrum of mutated genes was similar with the exception of SF3B1 which was rarely mutated in patients without dysplasia. Variant allele fractions were comparable between clonal ICUS (CCUS) and MDS as were mean age and blood counts. We demonstrate that CCUS is a more frequent diagnosis than MDS in cytopenic patients. Clinical and mutational features are similar in these groups and may have diagnostic utility once outcomes in CCUS patients are better understood. © 2015 by The American Society of Hematology.

  3. Antimicrobial Susceptibility and Clonality of Clinical Ureaplasma Isolates in the United States.

    Science.gov (United States)

    Fernández, Javier; Karau, Melissa J; Cunningham, Scott A; Greenwood-Quaintance, Kerryl E; Patel, Robin

    2016-08-01

    Ureaplasma urealyticum and Ureaplasma parvum are pathogens involved in urogenital tract and intrauterine infections and also in systemic diseases in newborns and immunosuppressed patients. There is limited information on the antimicrobial susceptibility and clonality of these species. In this study, we report the susceptibility of 250 contemporary isolates of Ureaplasma (202 U. parvum and 48 U. urealyticum isolates) recovered at Mayo Clinic, Rochester, MN. MICs of doxycycline, azithromycin, ciprofloxacin, tetracycline, erythromycin, and levofloxacin were determined by broth microdilution, with MICS of the last three interpreted according to CLSI guidelines. Levofloxacin resistance was found in 6.4% and 5.2% of U. parvum and U. urealyticum isolates, respectively, while 27.2% and 68.8% of isolates, respectively, showed ciprofloxacin MICs of ≥4 μg/ml. The resistance mechanism of levofloxacin-resistant isolates was due to mutations in parC, with the Ser83Leu substitution being most frequent, followed by Glu87Lys. No macrolide resistance was found among the 250 isolates studied; a single U. parvum isolate was tetracycline resistant. tet(M) was found in 10 U. parvum isolates, including the single tetracycline-resistant isolate, as well as in 9 isolates which had low tetracycline and doxycycline MICs. Multilocus sequence typing (MLST) performed on a selection of 46 isolates showed high diversity within the clinical Ureaplasma isolates studied, regardless of antimicrobial susceptibility. The present work extends previous knowledge regarding susceptibility to antimicrobial agents, resistance mechanisms, and clonality of Ureaplasma species in the United States. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

  4. Comparative genome analysis of Pseudogymnoascus spp. reveals primarily clonal evolution with small genome fragments exchanged between lineages.

    Science.gov (United States)

    Leushkin, Evgeny V; Logacheva, Maria D; Penin, Aleksey A; Sutormin, Roman A; Gerasimov, Evgeny S; Kochkina, Galina A; Ivanushkina, Natalia E; Vasilenko, Oleg V; Kondrashov, Alexey S; Ozerskaya, Svetlana M

    2015-05-21

    Pseudogymnoascus spp. is a wide group of fungi lineages in the family Pseudorotiaceae including an aggressive pathogen of bats P. destructans. Although several lineages of P. spp. were shown to produce ascospores in culture, the vast majority of P. spp. demonstrates no evidence of sexual reproduction. P. spp. can tolerate a wide range of different temperatures and salinities and can survive even in permafrost layer. Adaptability of P. spp. to different environments is accompanied by extremely variable morphology and physiology. We sequenced genotypes of 14 strains of P. spp., 5 of which were extracted from permafrost, 1 from a cryopeg, a layer of unfrozen ground in permafrost, and 8 from temperate surface environments. All sequenced genotypes are haploid. Nucleotide diversity among these genomes is very high, with a typical evolutionary distance at synonymous sites dS ≈ 0.5, suggesting that the last common ancestor of these strains lived >50 Mya. The strains extracted from permafrost do not form a separate clade. Instead, each permafrost strain has close relatives from temperate environments. We observed a strictly clonal population structure with no conflicting topologies for ~99% of genome sequences. However, there is a number of short (~100-10,000 nt) genomic segments with the total length of 67.6 Kb which possess phylogenetic patterns strikingly different from the rest of the genome. The most remarkable case is a MAT-locus, which has 2 distinct alleles interspersed along the whole-genome phylogenetic tree. Predominantly clonal structure of genome sequences is consistent with the observations that sexual reproduction is rare in P. spp. Small number of regions with noncanonical phylogenies seem to arise due to some recombination events between derived lineages of P. spp., with MAT-locus being transferred on multiple occasions. All sequenced strains have heterothallic configuration of MAT-locus.

  5. Clonal expansion of the Pseudogymnoascus destructans genotype in North America is accompanied by significant variation in phenotypic expression.

    Directory of Open Access Journals (Sweden)

    Jordan Khankhet

    Full Text Available Pseudogymnoascus destructans is the causative agent of an emerging infectious disease that threatens populations of several North American bat species. The fungal disease was first observed in 2006 and has since caused the death of nearly six million bats. The disease, commonly known as white-nose syndrome, is characterized by a cutaneous infection with P. destructans causing erosions and ulcers in the skin of nose, ears and/or wings of bats. Previous studies based on sequences from eight loci have found that isolates of P. destructans from bats in the US all belong to one multilocus genotype. Using the same multilocus sequence typing method, we found that isolates from eastern and central Canada also had the same genotype as those from the US, consistent with the clonal expansion of P. destructans into Canada. However, our PCR fingerprinting revealed that among the 112 North American isolates we analyzed, three, all from Canada, showed minor genetic variation. Furthermore, we found significant variations among isolates in mycelial growth rate; the production of mycelial exudates; and pigment production and diffusion into agar media. These phenotypic differences were influenced by culture medium and incubation temperature, indicating significant variation in environmental condition--dependent phenotypic expression among isolates of the clonal P. destructans genotype in North America.

  6. Clonal expansion of the Pseudogymnoascus destructans genotype in North America is accompanied by significant variation in phenotypic expression.

    Science.gov (United States)

    Khankhet, Jordan; Vanderwolf, Karen J; McAlpine, Donald F; McBurney, Scott; Overy, David P; Slavic, Durda; Xu, Jianping

    2014-01-01

    Pseudogymnoascus destructans is the causative agent of an emerging infectious disease that threatens populations of several North American bat species. The fungal disease was first observed in 2006 and has since caused the death of nearly six million bats. The disease, commonly known as white-nose syndrome, is characterized by a cutaneous infection with P. destructans causing erosions and ulcers in the skin of nose, ears and/or wings of bats. Previous studies based on sequences from eight loci have found that isolates of P. destructans from bats in the US all belong to one multilocus genotype. Using the same multilocus sequence typing method, we found that isolates from eastern and central Canada also had the same genotype as those from the US, consistent with the clonal expansion of P. destructans into Canada. However, our PCR fingerprinting revealed that among the 112 North American isolates we analyzed, three, all from Canada, showed minor genetic variation. Furthermore, we found significant variations among isolates in mycelial growth rate; the production of mycelial exudates; and pigment production and diffusion into agar media. These phenotypic differences were influenced by culture medium and incubation temperature, indicating significant variation in environmental condition--dependent phenotypic expression among isolates of the clonal P. destructans genotype in North America.

  7. Spatiotemporal Co-existence of Two Mycobacterium ulcerans Clonal Complexes in the Offin River Valley of Ghana.

    Directory of Open Access Journals (Sweden)

    Araceli Lamelas

    2016-07-01

    Full Text Available In recent years, comparative genome sequence analysis of African Mycobacterium ulcerans strains isolated from Buruli ulcer (BU lesion specimen has revealed a very limited genetic diversity of closely related isolates and a striking association between genotype and geographical origin of the patients. Here, we compared whole genome sequences of five M. ulcerans strains isolated in 2004 or 2013 from BU lesions of four residents of the Offin river valley with 48 strains isolated between 2002 and 2005 from BU lesions of individuals residing in the Densu river valley of Ghana. While all M. ulcerans isolates from the Densu river valley belonged to the same clonal complex, members of two distinct clonal complexes were found in the Offin river valley over space and time. The Offin strains were closely related to genotypes from either the Densu region or from the Asante Akim North district of Ghana. These results point towards an occasional involvement of a mobile reservoir in the transmission of M. ulcerans, enabling the spread of bacteria across different regions.

  8. Invasive clonal plant species have a greater root-foraging plasticity than non-invasive ones.

    Science.gov (United States)

    Keser, Lidewij H; Dawson, Wayne; Song, Yao-Bin; Yu, Fei-Hai; Fischer, Markus; Dong, Ming; van Kleunen, Mark

    2014-03-01

    Clonality is frequently positively correlated with plant invasiveness, but which aspects of clonality make some clonal species more invasive than others is not known. Due to their spreading growth form, clonal plants are likely to experience spatial heterogeneity in nutrient availability. Plasticity in allocation of biomass to clonal growth organs and roots may allow these plants to forage for high-nutrient patches. We investigated whether this foraging response is stronger in species that have become invasive than in species that have not. We used six confamilial pairs of native European clonal plant species differing in invasion success in the USA. We grew all species in large pots under homogeneous or heterogeneous nutrient conditions in a greenhouse, and compared their nutrient-foraging response and performance. Neither invasive nor non-invasive species showed significant foraging responses to heterogeneity in clonal growth organ biomass or in aboveground biomass of clonal offspring. Invasive species had, however, a greater positive foraging response in terms of root and belowground biomass than non-invasive species. Invasive species also produced more total biomass. Our results suggest that the ability for strong root foraging is among the characteristics promoting invasiveness in clonal plants.

  9. Electrochemical detection of short HIV sequences on chitosan/Fe{sub 3}O{sub 4} nanoparticle based screen printed electrodes

    Energy Technology Data Exchange (ETDEWEB)

    Tran, Lam Dai, E-mail: lamtd@ims.vast.ac.vn [Institute of Materials Science, Vietnamese Academy of Science and Technology, 18, Hoang Quoc Viet Road (Viet Nam); Nguyen, Binh Hai [Institute of Materials Science, Vietnamese Academy of Science and Technology, 18, Hoang Quoc Viet Road (Viet Nam); Van Hieu, Nguyen [International Training Institute for Materials Science, Hanoi University of Science and Technology, 1, Dai Co Viet Road, Hanoi (Viet Nam); Tran, Hoang Vinh; Nguyen, Huy Le [Faculty of Chemical Technology, Hanoi University of Science and Technology, 1, Dai Co Viet Road, Hanoi (Viet Nam); Nguyen, Phuc Xuan [Institute of Materials Science, Vietnamese Academy of Science and Technology, 18, Hoang Quoc Viet Road (Viet Nam)

    2011-03-12

    In this study, a novel CS/Fe{sub 3}O{sub 4} nanobiocomposite-based platform for electrochemical detection of HIV-1 was developed. The most attractive feature of this system is a suitable microenvironment (Fe{sub 3}O{sub 4} nanoparticles) which could contribute to electron transfer and thus sensitivity enhancement when using methylene blue (MB) as an external mediator and Square Wave Voltammetry (SWV), Electrochemical Impedance Spectroscopy (EIS) techniques. The proposed screen printed electrode (SPE) had a low detection limit (as low as 50 pM), acceptable stability and good reproducibility, which would be valuable for clinical diagnosis. In addition, this sensing interface may be feasibly adapted for multiplexed detection of other species of bacterial pathogens.

  10. Introduction of HIV type 1 into an isolated population

    DEFF Research Database (Denmark)

    Madsen, Tina V; Leitner, Thomas; Lohse, Nicolai

    2007-01-01

    Introduction of HIV-1 into a population may not always give rise to a subsequent epidemic. Greenland is an isolated and sparsely populated island in The Danish Kingdom. We aimed to estimate the number of introductions of HIV-1 into Greenland, the number of subsequent epidemics, and the countries...... from which the virus was introduced. Phylogenetic analyses were performed on three regions of HIV-1 (gag, pol, and env) in samples from 70 Greenlandic patients. Furthermore, we included gene sequences from contemporary Danish HIV-1-infected patients and sequences from the Los Alamos HIV Sequence...... Database. All Greenlandic sequences were subtype B except one sequence found to be a recombinant (probably CRF13). Sequence clusters in the phylogenetic trees indicated that there had been at least nine introductions of HIV-1 into Greenland. One cluster, supported by bootstrap values of 81, 76, and 96...

  11. Effect of Moringa oleifera Lam. leaf powder on the pharmacokinetics of nevirapine in HIV-infected adults: a one sequence cross-over study.

    Science.gov (United States)

    Monera-Penduka, Tsitsi G; Maponga, Charles C; Wolfe, Alan R; Wiesner, Lubbe; Morse, Gene D; Nhachi, Charles F B

    2017-01-01

    Moringa oleifera Lam., an herb commonly consumed by HIV-infected people on antiretroviral therapy, inhibits cytochrome P450 3A4, 1A2 and 2D6 activity in vitro; and may alter the pharmacokinetics (PK) of antiretroviral drugs metabolized via the same pathways. However, in vitro drug interaction activity may not translate to a clinically significant effect. Therefore, the effect of moringa leaf powder on the PK of nevirapine in HIV-infected people was investigated. Adult patients at steady-state dosing with nevirapine were admitted for 12-h intensive PK sampling following a 21-day herbal medicine washout. Blood sampling was repeated after 14 days of nevirapine and moringa (1.85 g leaf powder/day) co-administration. Nevirapine plasma concentrations were determined by liquid chromatography-tandem mass spectrometry. To assess the effect of moringa on nevirapine PK, the change in nevirapine area under the plasma concentration-time curve (AUC) was determined. The mean difference in pre- and post-moringa nevirapine, maximum concentration (C max ) and concentration at 12 h (C 12h ) were also calculated. The PK parameters were compared by assessing the post/pre geometric mean ratios (GMRs) and associated 90% confidence intervals (CIs). Pharmacokinetics analyses were performed on the results from 11 participants for whom complete data were obtained. The post/pre GMRs and associated 90% CIs for nevirapine were 1.07 (1.00-1.14) for the AUC; 1.06 (0.98-1.16) for C max and 1.03 (0.92-1.16) for C 12h . Co-administration of Moringa oleifera Lam. leaf powder at the traditional dose did not significantly alter the steady-state PK of nevirapine. Trial registration number NCT01410058 (ClinicalTrials.gov).

  12. Clonal origins of ETV6-RUNX1+ acute lymphoblastic leukemia

    DEFF Research Database (Denmark)

    Alpar, D.; Wren, D.; Ermini, Luca

    2015-01-01

    Studies on twins with concordant acute lymphoblastic leukemia (ALL) have revealed that ETV6-RUNX1 gene fusion is a common, prenatal genetic event with other driver aberrations occurring subclonally and probably postnatally. The fetal cell type that is transformed by ETV6-RUNX1 is not identified...... by such studies or by the analysis of early B-cell lineage phenotype of derived progeny. Ongoing, clonal immunoglobulin (IG) and cross-lineage T-cell receptor (TCR) gene rearrangements are features of B-cell precursor leukemia and commence at the pro-B-cell stage of normal B-cell lineage development. We reasoned...

  13. 5′ Rapid Amplification of cDNA Ends and Illumina MiSeq Reveals B Cell Receptor Features in Healthy Adults, Adults With Chronic HIV-1 Infection, Cord Blood, and Humanized Mice

    Directory of Open Access Journals (Sweden)

    Eric Waltari

    2018-03-01

    Full Text Available Using 5′ rapid amplification of cDNA ends, Illumina MiSeq, and basic flow cytometry, we systematically analyzed the expressed B cell receptor (BCR repertoire in 14 healthy adult PBMCs, 5 HIV-1+ adult PBMCs, 5 cord blood samples, and 3 HIS-CD4/B mice, examining the full-length variable region of μ, γ, α, κ, and λ chains for V-gene usage, somatic hypermutation (SHM, and CDR3 length. Adding to the known repertoire of healthy adults, Illumina MiSeq consistently detected small fractions of reads with high mutation frequencies including hypermutated μ reads, and reads with long CDR3s. Additionally, the less studied IgA repertoire displayed similar characteristics to that of IgG. Compared to healthy adults, the five HIV-1 chronically infected adults displayed elevated mutation frequencies for all μ, γ, α, κ, and λ chains examined and slightly longer CDR3 lengths for γ, α, and λ. To evaluate the reconstituted human BCR sequences in a humanized mouse model, we analyzed cord blood and HIS-CD4/B mice, which all lacked the typical SHM seen in the adult reference. Furthermore, MiSeq revealed identical unmutated IgM sequences derived from separate cell aliquots, thus for the first time demonstrating rare clonal members of unmutated IgM B cells by sequencing.

  14. 5' Rapid Amplification of cDNA Ends and Illumina MiSeq Reveals B Cell Receptor Features in Healthy Adults, Adults With Chronic HIV-1 Infection, Cord Blood, and Humanized Mice.

    Science.gov (United States)

    Waltari, Eric; Jia, Manxue; Jiang, Caroline S; Lu, Hong; Huang, Jing; Fernandez, Cristina; Finzi, Andrés; Kaufmann, Daniel E; Markowitz, Martin; Tsuji, Moriya; Wu, Xueling

    2018-01-01

    Using 5' rapid amplification of cDNA ends, Illumina MiSeq, and basic flow cytometry, we systematically analyzed the expressed B cell receptor (BCR) repertoire in 14 healthy adult PBMCs, 5 HIV-1+ adult PBMCs, 5 cord blood samples, and 3 HIS-CD4/B mice, examining the full-length variable region of μ, γ, α, κ, and λ chains for V-gene usage, somatic hypermutation (SHM), and CDR3 length. Adding to the known repertoire of healthy adults, Illumina MiSeq consistently detected small fractions of reads with high mutation frequencies including hypermutated μ reads, and reads with long CDR3s. Additionally, the less studied IgA repertoire displayed similar characteristics to that of IgG. Compared to healthy adults, the five HIV-1 chronically infected adults displayed elevated mutation frequencies for all μ, γ, α, κ, and λ chains examined and slightly longer CDR3 lengths for γ, α, and λ. To evaluate the reconstituted human BCR sequences in a humanized mouse model, we analyzed cord blood and HIS-CD4/B mice, which all lacked the typical SHM seen in the adult reference. Furthermore, MiSeq revealed identical unmutated IgM sequences derived from separate cell aliquots, thus for the first time demonstrating rare clonal members of unmutated IgM B cells by sequencing.

  15. IgV gene intraclonal diversification and clonal evolution in B-cell chronic lymphocytic leukaemia.

    Science.gov (United States)

    Bagnara, Davide; Callea, Vincenzo; Stelitano, Caterina; Morabito, Fortunato; Fabris, Sonia; Neri, Antonino; Zanardi, Sabrina; Ghiotto, Fabio; Ciccone, Ermanno; Grossi, Carlo Enrico; Fais, Franco

    2006-04-01

    Intraclonal diversification of immunoglobulin (Ig) variable (V) genes was evaluated in leukaemic cells from a B-cell chronic lymphocytic leukaemia (B-CLL) case over a 2-year period at four time points. Intraclonal heterogeneity was analysed by sequencing 305 molecular clones derived from polymerase chain reaction amplification of B-CLL cell IgV heavy (H) and light (C) chain gene rearrangements. Sequences were compared with evaluating intraclonal variation and the nature of somatic mutations. Although IgV intraclonal variation was detected at all time points, its level decreased with time and a parallel emergence of two more represented V(H)DJ(H) clones was observed. They differed by nine nucleotide substitutions one of which only caused a conservative replacement aminoacid change. In addition, one V(L)J(L) rearrangement became more represented over time. Analyses of somatic mutations suggest antigen selection and impairment of negative selection of neoplastic cells. In addition, a genealogical tree representing a model of clonal evolution of the neoplastic cells was created. It is of note that, during the period of study, the patient showed clinical progression of disease. We conclude that antigen stimulation and somatic hypermutation may participate in disease progression through the selection and expansion of neoplastic subclone(s).

  16. Observing Clonal Dynamics across Spatiotemporal Axes: A Prelude to Quantitative Fitness Models for Cancer.

    Science.gov (United States)

    McPherson, Andrew W; Chan, Fong Chun; Shah, Sohrab P

    2018-02-01

    The ability to accurately model evolutionary dynamics in cancer would allow for prediction of progression and response to therapy. As a prelude to quantitative understanding of evolutionary dynamics, researchers must gather observations of in vivo tumor evolution. High-throughput genome sequencing now provides the means to profile the mutational content of evolving tumor clones from patient biopsies. Together with the development of models of tumor evolution, reconstructing evolutionary histories of individual tumors generates hypotheses about the dynamics of evolution that produced the observed clones. In this review, we provide a brief overview of the concepts involved in predicting evolutionary histories, and provide a workflow based on bulk and targeted-genome sequencing. We then describe the application of this workflow to time series data obtained for transformed and progressed follicular lymphomas (FL), and contrast the observed evolutionary dynamics between these two subtypes. We next describe results from a spatial sampling study of high-grade serous (HGS) ovarian cancer, propose mechanisms of disease spread based on the observed clonal mixtures, and provide examples of diversification through subclonal acquisition of driver mutations and convergent evolution. Finally, we state implications of the techniques discussed in this review as a necessary but insufficient step on the path to predictive modelling of disease dynamics. Copyright © 2018 Cold Spring Harbor Laboratory Press; all rights reserved.

  17. Major clonal lineages in impetigo Staphylococcus aureus strains isolated in Czech and Slovak maternity hospitals.

    Science.gov (United States)

    Růžičková, Vladislava; Pantůček, Roman; Petráš, Petr; Machová, Ivana; Kostýlková, Karla; Doškař, Jiří

    2012-11-01

    One hundred and twenty-seven exfoliative toxin-producing (ET-positive) strains of Staphylococcus aureus collected in 23 Czech and one Slovak maternity hospitals from 1998 to 2011 were genotypically characterized by multilocus sequence typing (MLST), pulsed-field gel electrophoresis (PFGE) profiling, spa gene polymorphism analysis, and ETA-converting prophage carriage, which resulted in the identification of 21 genotypes grouped into 4 clonal complexes (CC). Ninety-one isolates carried the eta gene alone whilst 12 isolates harboured only the etb gene. Two new, to date not defined, spa types (t6644 and t6645) and 2 novel sequence types (ST2194 and ST2195) were identified in the set of strains under study. The predominant CC121 occurred in 13 Czech hospitals. CC15, CC9, and ST88 (CC88) exclusively included eta gene-positive strains while the strains belonging to ST121 harboured the eta and/or etb genes. This study highlights not only significant genomic diversity among impetigo strains and the distribution of major genotypes disseminated in the Czech and Slovak maternity hospitals, but also reveals their impact in epidermolytic infections. Copyright © 2012 Elsevier GmbH. All rights reserved.

  18. Fatal infection in three Grey Slender Lorises (Loris lydekkerianus nordicus) caused by clonally related Trueperella pyogenes.

    Science.gov (United States)

    Nagib, Samy; Glaeser, Stefanie P; Eisenberg, Tobias; Sammra, Osama; Lämmler, Christoph; Kämpfer, Peter; Schauerte, Nicole; Geiger, Christina; Kaim, Ute; Prenger-Berninghoff, Ellen; Becker, André; Abdulmawjood, Amir

    2017-08-29

    Trueperella pyogenes is a worldwide known bacterium causing mastitis, abortion and various other pyogenic infections in domestic animals like ruminants and pigs. In this study we represent the first case report of three unusual fatal infections of Grey Slender Lorises caused by Trueperella pyogenes. Meanwhile, this study represents the first in-depth description of the multilocus sequence analysis (MLSA) on T. pyogenes species. Three Trueperella pyogenes were isolated from three different Grey Slender Lorises, which died within a period of two years at Frankfurt Zoo (Frankfurt am Main - Germany). The three Grey Slender Loris cases were suffering from severe sepsis and died from its complication. During the bacteriological investigation of the three cases, the T. pyogenes were isolated from different organisms in each case. The epidemiological relationship between the three isolates could be shown by four genomic DNA fingerprint methods (ERIC-PCR, BOX-PCR, (GTG) 5 -PCR, and RAPD-PCR) and by multilocus sequence analysis (MLSA) investigating four different housekeeping genes (fusA-tuf-metG-gyrA). In this study, we clearly showed by means of using three different rep-PCRs, by RAPD-PCR and by MLSA that the genomic fingerprinting of the investigated three T. pyogenes have the same clonal origin and are genetically identical. These results suggest that the same isolate contaminated the animal's facility and subsequently caused cross infection between the three different Grey Slender Lorises. To the best of our knowledge, this is the first epidemiological approach concentrating on T. pyogenes using MLSA.

  19. Outbreak of Vibrio parahaemolyticus Sequence Type 120, Peru, 2009.

    Science.gov (United States)

    Gonzalez-Escalona, Narjol; Gavilan, Ronnie G; Toro, Magaly; Zamudio, Maria L; Martinez-Urtaza, Jaime

    2016-07-01

    In 2009, an outbreak of Vibrio parahaemolyticus occurred in Piura, Cajamarca, Lambayeque, and Lima, Peru. Whole-genome sequencing of clinical and environmental samples from the outbreak revealed a new V. parahaemolyticus clone. All the isolates identified belonged to a single clonal complex described exclusively in Asia before its emergence in Peru.

  20. Molecular analysis of critical sequences within the EBNA-2 type 1 gene from Epstein-Barr virus isolates from patients with infectious mononucleosis, tonsillar hyperplasia, and HIV infection

    NARCIS (Netherlands)

    Al-Homsi, A. S.; Berger, C.; van Baarle, D.; Kersten, M. J.; Klein, M. R.; McQuain, C.; van Oers, R.; Knecht, H.

    1998-01-01

    EBNA-2 is the first protein to be detected after infection of primary B lymphocytes by Epstein-Barr virus (EBV) and plays an essential role as transcriptional activator in EBV-induced lymphocyte transformation. We analysed by PCR and sequencing regions of the EBNA-2 type 1 gene from isolates from 13

  1. Women and HIV

    Science.gov (United States)

    ... Consumer Information by Audience For Women Women and HIV: Get the Facts on HIV Testing, Prevention, and Treatment Share Tweet Linkedin Pin ... How can you lower your chance of HIV? HIV Quick Facts What is HIV? HIV is the ...

  2. Limited overlap between phylogenetic HIV and hepatitis C virus clusters illustrates the dynamic sexual network structure of Dutch HIV-infected MSM.

    Science.gov (United States)

    Vanhommerig, Joost W; Bezemer, Daniela; Molenkamp, Richard; Van Sighem, Ard I; Smit, Colette; Arends, Joop E; Lauw, Fanny N; Brinkman, Kees; Rijnders, Bart J; Newsum, Astrid M; Bruisten, Sylvia M; Prins, Maria; Van Der Meer, Jan T; Van De Laar, Thijs J; Schinkel, Janke

    2017-09-24

    MSM are at increased risk for infection with HIV-1 and hepatitis C virus (HCV). Is HIV/HCV coinfection confined to specific HIV transmission networks? A HIV phylogenetic tree was constructed for 5038 HIV-1 subtype B polymerase (pol) sequences obtained from MSM in the AIDS therapy evaluation in the Netherlands cohort. We investigated the existence of HIV clusters with increased HCV prevalence, the HIV phylogenetic density (i.e. the number of potential HIV transmission partners) of HIV/HCV-coinfected MSM compared with HIV-infected MSM without HCV, and the overlap in HIV and HCV phylogenies using HCV nonstructural protein 5B sequences from 183 HIV-infected MSM with acute HCV infection. Five hundred and sixty-three of 5038 (11.2%) HIV-infected MSM tested HCV positive. Phylogenetic analysis revealed 93 large HIV clusters (≥10 MSM), 370 small HIV clusters (2-9 MSM), and 867 singletons with a median HCV prevalence of 11.5, 11.6, and 9.3%, respectively. We identified six large HIV clusters with elevated HCV prevalence (range 23.5-46.2%). Median HIV phylogenetic densities for MSM with HCV (3, interquartile range 1-7) and without HCV (3, interquartile range 1-8) were similar. HCV phylogeny showed 12 MSM-specific HCV clusters (clustersize: 2-39 HCV sequences); 12.7% of HCV infections were part of the same HIV and HCV cluster. We observed few HIV clusters with elevated HCV prevalence, no increase in the HIV phylogenetic density of HIV/HCV-coinfected MSM compared to HIV-infected MSM without HCV, and limited overlap between HIV and HCV phylogenies among HIV/HCV-coinfected MSM. Our data do not support the existence of MSM-specific sexual networks that fuel both the HIV and HCV epidemic.

  3. Analysis of the genetic phylogeny of multifocal prostate cancer identifies multiple independent clonal expansions in neoplastic and morphologically normal prostate tissue.

    Science.gov (United States)

    Cooper, Colin S; Eeles, Rosalind; Wedge, David C; Van Loo, Peter; Gundem, Gunes; Alexandrov, Ludmil B; Kremeyer, Barbara; Butler, Adam; Lynch, Andrew G; Camacho, Niedzica; Massie, Charlie E; Kay, Jonathan; Luxton, Hayley J; Edwards, Sandra; Kote-Jarai, ZSofia; Dennis, Nening; Merson, Sue; Leongamornlert, Daniel; Zamora, Jorge; Corbishley, Cathy; Thomas, Sarah; Nik-Zainal, Serena; O'Meara, Sarah; Matthews, Lucy; Clark, Jeremy; Hurst, Rachel; Mithen, Richard; Bristow, Robert G; Boutros, Paul C; Fraser, Michael; Cooke, Susanna; Raine, Keiran; Jones, David; Menzies, Andrew; Stebbings, Lucy; Hinton, Jon; Teague, Jon; McLaren, Stuart; Mudie, Laura; Hardy, Claire; Anderson, Elizabeth; Joseph, Olivia; Goody, Victoria; Robinson, Ben; Maddison, Mark; Gamble, Stephen; Greenman, Christopher; Berney, Dan; Hazell, Steven; Livni, Naomi; Fisher, Cyril; Ogden, Christopher; Kumar, Pardeep; Thompson, Alan; Woodhouse, Christopher; Nicol, David; Mayer, Erik; Dudderidge, Tim; Shah, Nimish C; Gnanapragasam, Vincent; Voet, Thierry; Campbell, Peter; Futreal, Andrew; Easton, Douglas; Warren, Anne Y; Foster, Christopher S; Stratton, Michael R; Whitaker, Hayley C; McDermott, Ultan; Brewer, Daniel S; Neal, David E

    2015-04-01

    Genome-wide DNA sequencing was used to decrypt the phylogeny of multiple samples from distinct areas of cancer and morphologically normal tissue taken from the prostates of three men. Mutations were present at high levels in morphologically normal tissue distant from the cancer, reflecting clonal expansions, and the underlying mutational processes at work in morphologically normal tissue were also at work in cancer. Our observations demonstrate the existence of ongoing abnormal mutational processes, consistent with field effects, underlying carcinogenesis. This mechanism gives rise to extensive branching evolution and cancer clone mixing, as exemplified by the coexistence of multiple cancer lineages harboring distinct ERG fusions within a single cancer nodule. Subsets of mutations were shared either by morphologically normal and malignant tissues or between different ERG lineages, indicating earlier or separate clonal cell expansions. Our observations inform on the origin of multifocal disease and have implications for prostate cancer therapy in individual cases.

  4. Human and Swine Hosts Share Vancomycin-Resistant Enterococcus faecium CC17 and CC5 and Enterococcus faecalis CC2 Clonal Clusters Harboring Tn1546 on Indistinguishable Plasmids

    DEFF Research Database (Denmark)

    Freitas, Ana R.; Coque, Teresa M.; Novais, Carla

    2011-01-01

    clonally related Enterococcus faecium clonal complex 5 (CC5) isolates (17 sequence type 6 [ST6], 6 ST5, 5 ST185, 1 ST147, and 1 ST493) were obtained from feces of swine and healthy humans. This collection included isolates widespread among pigs of European Union (EU) countries since the mid-1990s. Each ST...... comprised isolates showing similar pulsed-field gel electrophoresis (PFGE) patterns (≤6 bands difference; >82% similarity). Some CC5 PFGE subtype strains from swine were indistinguishable from hospital vancomycin-resistant enterococci (VRE) causing infections. A truncated variant of Tn1546 (encoding...... resistance to vancomycin) and tcrB (coding for resistance to copper) were consistently located on 150- to 190-kb plasmids (rep(pLG1)). E. faecium CC17 (ST132) isolates from pig manure and two clinical samples showed identical PFGE profiles and contained a 60-kb mosaic plasmid (rep(Inc18) plus rep...

  5. Presence and mechanisms of acquired antimicrobial resistance in Belgian Brachyspira hyodysenteriae isolates belonging to different clonal complexes.

    Science.gov (United States)

    Mahu, M; Pasmans, F; Vranckx, K; De Pauw, N; Vande Maele, L; Vyt, Philip; Vandersmissen, Tamara; Martel, A; Haesebrouck, F; Boyen, F

    2017-08-01

    Swine dysentery (SD) is an economically important disease for which antimicrobial treatment still occupies an important place to control outbreaks. However, acquired antimicrobial resistance is increasingly observed in Brachyspira hyodysenteriae. In this study, the Minimal Inhibitory Concentrations (MIC) of six antimicrobial compounds for 30 recent Belgian B. hyodysenteriae isolates were determined using a broth microdilution method. In addition, relevant regions of the 16S rRNA, 23S rRNA and the L3 protein encoding genes were sequenced to reveal mutations associated with acquired resistance. Finally, a phylogeny was reconstructed using minimal spanning tree analysis of multi locus sequence typing of the isolates. For lincomycin, doxycycline, tylosin and tylvalosin, at least 70% of the isolates did not belong to the wild-type population and were considered to have acquired resistance. For valnemulin and tiamulin, this was over 50%. In all isolates with acquired resistance to doxycycline, the G1058C mutation was present in their 16S rRNA gene. All isolates showing acquired resistance to lincomycin and both macrolides displayed the A2058T mutation in their 23S rRNA gene. Other mutations in this gene and the N148S mutation in the L3 protein were present in both wild-type isolates and isolates considered to have acquired resistance. Multi locus sequence analysis revealed a previously undescribed clonal complex, with 4 novel sequence types in which the majority of isolates showed acquired resistance to all tested antimicrobial products. In conclusion, acquired antimicrobial resistance is widespread among Belgian B. hyodysenteriae isolates. The emergence of multi-resistant clonal complexes can pose a threat to swine industry. Copyright © 2017 Elsevier B.V. All rights reserved.

  6. Clonal composition of human ovarian cancer based on copy number analysis reveals a reciprocal relation with oncogenic mutation status.

    Science.gov (United States)

    Sakai, Kazuko; Ukita, Masayo; Schmidt, Jeanette; Wu, Longyang; De Velasco, Marco A; Roter, Alan; Jevons, Luis; Nishio, Kazuto; Mandai, Masaki

    2017-10-01

    Intratumoral heterogeneity of cancer cells remains largely unexplored. Here we investigated the composition of ovarian cancer and its biological relevance. A whole-genome single nucleotide polymorphism array was applied to detect the clonal composition of 24 formalin-fixed, paraffin-embedded samples of human ovarian cancer. Genome-wide segmentation data consisting of the log2 ratio (log2R) and B allele frequency (BAF) were used to calculate an estimate of the clonal composition number (CC number) for each tumor. Somatic mutation profiles of cancer-related genes were also determined for the same 24 samples by next-generation sequencing. The CC number was estimated successfully for 23 of the 24 cancer samples. The mean ± SD value for the CC number was 1.7 ± 1.1 (range of 0-4). A somatic mutation in at least one gene was identified in 22 of the 24 ovarian cancer samples, with the mutations including those in the oncogenes KRAS (29.2%), PIK3CA (12.5%), BRAF (8.3%), FGFR2 (4.2%), and JAK2 (4.2%) as well as those in the tumor suppressor genes TP53 (54.2%), FBXW7 (8.3%), PTEN (4.2%), and RB1 (4.2%). Tumors with one or more oncogenic mutations had a significantly lower CC number than did those without such a mutation (1.0 ± 0.8 versus 2.3 ± 0.9, P = 0.0027), suggesting that cancers with driver oncogene mutations are less heterogeneous than those with other mutations. Our results thus reveal a reciprocal relation between oncogenic mutation status and clonal composition in ovarian cancer using the established method for the estimation of the CC number. Copyright © 2017 The Author(s). Published by Elsevier B.V. All rights reserved.

  7. Viral linkage in HIV-1 seroconverters and their partners in an HIV-1 prevention clinical trial.

    Directory of Open Access Journals (Sweden)

    Mary S Campbell

    2011-03-01

    Full Text Available Characterization of viruses in HIV-1 transmission pairs will help identify biological determinants of infectiousness and evaluate candidate interventions to reduce transmission. Although HIV-1 sequencing is frequently used to substantiate linkage between newly HIV-1 infected individuals and their sexual partners in epidemiologic and forensic studies, viral sequencing is seldom applied in HIV-1 prevention trials. The Partners in Prevention HSV/HIV Transmission Study (ClinicalTrials.gov #NCT00194519 was a prospective randomized placebo-controlled trial that enrolled serodiscordant heterosexual couples to determine the efficacy of genital herpes suppression in reducing HIV-1 transmission; as part of the study analysis, HIV-1 sequences were examined for genetic linkage between seroconverters and their enrolled partners.We obtained partial consensus HIV-1 env and gag sequences from blood plasma for 151 transmission pairs and performed deep sequencing of env in some cases. We analyzed sequences with phylogenetic techniques and developed a Bayesian algorithm to evaluate the probability of linkage. For linkage, we required monophyletic clustering between enrolled partners' sequences and a Bayesian posterior probability of ≥ 50%. Adjudicators classified each seroconversion, finding 108 (71.5% linked, 40 (26.5% unlinked, and 3 (2.0% indeterminate transmissions, with linkage determined by consensus env sequencing in 91 (84%. Male seroconverters had a higher frequency of unlinked transmissions than female seroconverters. The likelihood of transmission from the enrolled partner was related to time on study, with increasing numbers of unlinked transmissions occurring after longer observation periods. Finally, baseline viral load was found to be significantly higher among linked transmitters.In this first use of HIV-1 sequencing to establish endpoints in a large clinical trial, more than one-fourth of transmissions were unlinked to the enrolled partner

  8. Phylogenetic analysis of HIV-1 pol gene: first subgenomic evidence of CRF29-BF among Iranian HIV-1 patients

    Directory of Open Access Journals (Sweden)

    Kazem Baesi

    2014-09-01

    Full Text Available Objective: To identify the dominant subtype among the HIV-1 strains circulation in Iran. Methods: In this cross sectional study 100 HIV positive patients participated. HIV-1 RNA was extracted from plasma. RT nested-PCR was performed and the final products were sequenced and phylogenetically analyzed; reference sequences were downloaded from Los Alamos, aligned with Iranian pol sequences in the study and analyzed by neighbor-joining method. Results: The results of the phylogenetic analysis showed that HIV-1 subtype CRF-35AD was the dominant subtype among HIV-1 infected patients in Iran; this analysis also suggested a new circulating recombinant form that had not previously been identified in Iran: CRF-29BF. Conclusions: The impact of HIV diversity on pathogenesis, transmission and clinical management have been discussed in different studies; therefore, analyses of HIV genetic diversity is required to design effective antiretroviral strategies for different HIV subtypes.

  9. Clonal variation in proliferation rate of cultures of GPK cells.

    Science.gov (United States)

    Riley, P A; Hola, M

    1981-09-01

    Pedigrees of twenty-six clones of a line of keratocytes derived from guinea-pig ear epidermis (GPK cells) were analysed from time-lapse film. The mean interdivision time (IDT) for the culture was 1143 +/- 215 (SD) min. The mean generation rates (mean reciprocal interdivision times) of clones varied over a range of 3.93--10.2 x 10(-4)/min and the standard deviation of the clonal mean generation rates was 16.8% of the average value. Transient intraclonal variations in IDT due to mitoses in a plane perpendicular to the substratum were observed. The data were also analysed on the basis of cell location in sixteen equal zones (quadrats) of the filmed area. The mean generation rate of quadrats was 8.73 x 10(-4)/min (SD = 4.9%). The spatial distribution showed some clustering of cells. The mean local density of the clones (2.25 +/- 0.62 cells/10(-4) cm2) was significantly higher than the quadrat density (1.76 +/- 0.8 cells/10(-4) cm2). There was no significant correlation between clonal density and mean generation rates, whereas for quadrats a significant negative correlation was found (P = 2.7%). The results support the proposition that cell lineage is the major determinant of the proliferation rate of subconfluent cultures.

  10. Competition, salinity, and clonal growth in native and introduced irises.

    Science.gov (United States)

    Mopper, Susan; Wiens, Karen C; Goranova, Greta A

    2016-09-01

    Iris pseudacorus spread rapidly into North America after introduction from Europe in the 1800s and now co-occurs with native I. hexagona in freshwater Louisiana wetlands. Native irises support and interact with multiple trophic levels, whereas I. pseudacorus is classified an invasive pest because it grows aggressively, reduces biodiversity, and displaces native vegetation. Salinity levels are increasing in coastal wetlands worldwide. We examined how salt-stress affects competitive interactions between these conspecifics. We established a three-way full-factorial common-garden experiment that included species (I. pseudacorus, I. hexagona), competition (no competition, intraspecific competition, and interspecific competition), and salinity (0, 4, 8 parts per thousand NaCl), with six replicates per treatment. After 18 mo, Iris pseudacorus produced much more biomass than the native species did (F1, 92 = 71.5, P Interspecific competition did not affect the introduced iris, but biomass of the native was strongly reduced (competition × species interaction: F2, 95 = 76.7, P = 0.002). Salinity significantly reduced biomass of both species (F2, 92 = 21.8, P competitive advantage over the native, regardless of environmental salinity levels. Based on patterns in clonal reproduction, the introduced iris could potentially threaten native iris populations. We are currently investigating seed production and mortality during competition and stress because both clonal and sexual reproduction must be considered when predicting long-term population dynamics. © 2016 Botanical Society of America.

  11. Clonal expansion of renal cell carcinoma-infiltrating T lymphocytes

    DEFF Research Database (Denmark)

    Sittig, Simone; Køllgaard, Tania; Grønbæk, Kirsten

    2013-01-01

    T lymphocytes can mediate the destruction of cancer cells by virtue of their ability to recognize tumor-derived antigenic peptides that are presented on the cell surface in complex with HLA molecules and expand. Thus, the presence of clonally expanded T cells within neoplastic lesions is an indic......T lymphocytes can mediate the destruction of cancer cells by virtue of their ability to recognize tumor-derived antigenic peptides that are presented on the cell surface in complex with HLA molecules and expand. Thus, the presence of clonally expanded T cells within neoplastic lesions...... is an indication of ongoing HLA-restricted T cell-mediated immune responses. Multiple tumors, including renal cell carcinomas (RCCs), are often infiltrated by significant amounts of T cells, the so-called tumor-infiltrating lymphocytes (TILs). In the present study, we analyzed RCC lesions (n = 13) for the presence...... of expanded T-cell clonotypes using T-cell receptor clonotype mapping. Surprisingly, we found that RCCs comprise relatively low numbers of distinct expanded T-cell clonotypes as compared with melanoma lesions. The numbers of different T-cell clonotypes detected among RCC-infiltrating lymphocytes were...

  12. Clonal Spread in Second Growth Stands of Coast Redwood, Sequoia sempervirens

    Science.gov (United States)

    Vladimir Douhovnikoff; Richard S. Dodd

    2007-01-01

    Coast redwood (Sequoia sempervirens) is one of the rare conifers to reproduce successfully through clonal spread. The importance of this mode of reproduction in stand development is largely unknown. Understanding the importance of clonal spread and the spatial structure of clones is crucial for stand management strategies that would aim to maximize...

  13. Virulence, sporulation, and elicitin production in three clonal lineages of Phytophthora ramorum

    Science.gov (United States)

    Phytophthora ramorum populations are clonal and consist of three lineages. Recent studies have shown that the clonal lineages may have varying degrees of aggressiveness on some host species, such as Quercus rubra. In this study, we examined virulence, sporulation and elicitin production of five P. ...

  14. Clonal relation in a case of CLL, ALCL, and Hodgkin composite lymphoma

    NARCIS (Netherlands)

    van den Berg, Anke; Maggio, Ewerton; Rust, R; Kooistra, K; Diepstra, A; Poppema, S

    2002-01-01

    Large cell lymphomas and Hodgkin disease may develop during the course of chronic lymphocytic leukemia (CLL). In some cases the transformed cells are Epstein-Barr virus (EBV)-positive and not clonally related to the CLL cells. In other cases the transformed cells have the same clonal rearrangements

  15. Growth and stem form quality of clonal Pinus taeda following fertilization in the Virginia Piedmont

    Science.gov (United States)

    Jeremy P. Stovall; Colleen A. Carlson; John R. Seiler; Thomas R. Fox

    2013-01-01

    Clonal forestry offers the opportunity to increase yields, enhance uniformity, and improve wood characteristics. Intensive silvicultural practices, including fertilization, will be required to capture the full growth potential of clonal plantations. However, variation in nutrient use efficiency that exists among clones could affect growth responses. Our research...

  16. Clonal neoantigens elicit T cell immunoreactivity and sensitivity to immune checkpoint blockade

    DEFF Research Database (Denmark)

    McGranahan, Nicholas; Furness, Andrew J S; Rosenthal, Rachel

    2016-01-01

    demonstrate a relationship between clonal neoantigen burden and overall survival in primary lung adenocarcinomas. CD8(+)tumor-infiltrating lymphocytes reactive to clonal neoantigens were identified in early-stage non-small cell lung cancer and expressed high levels of PD-1. Sensitivity to PD-1 and CTLA-4...

  17. Novel R tools for analysis of genome-wide population genetic data with emphasis on clonality

    Science.gov (United States)

    To gain a detailed understanding of how plant microbes evolve and adapt to hosts, pesticides, and other factors, knowledge of the population dynamics and evolutionary history of populations is crucial. Plant pathogen populations are often clonal or partially clonal which requires different analytica...

  18. Clonal Growth Forms in Eastern Ladakh, Western Himalayas: Classification and Habitat Preferences

    Czech Academy of Sciences Publication Activity Database

    Klimešová, Jitka; Doležal, Jiří; Dvorský, Miroslav; de Bello, Francesco; Klimeš, Leoš

    2011-01-01

    Roč. 46, 2-3 (2011), 191-217 ISSN 1211-9520 R&D Projects: GA AV ČR IAA600050802 Institutional research plan: CEZ:AV0Z60050516 Keywords : Clonal growth form * Clonal space occupancy strategies * Habitat types Subject RIV: EF - Botanics Impact factor: 1.500, year: 2011

  19. Circulating mutational portrait of cancer: manifestation of aggressive clonal events in both early and late stages

    Directory of Open Access Journals (Sweden)

    Meng Yang

    2017-05-01

    Full Text Available Abstract Background Solid tumors residing in tissues and organs leave footprints in circulation through circulating tumor cells (CTCs and circulating tumor DNAs (ctDNA. Characterization of the ctDNA portraits and comparison with tumor DNA mutational portraits may reveal clinically actionable information on solid tumors that is traditionally achieved through more invasive approaches. Methods We isolated ctDNAs from plasma of patients of 103 lung cancer and 74 other solid tumors of different tissue origins. Deep sequencing using the Guardant360 test was performed to identify mutations in 73 clinically actionable genes, and the results were associated with clinical characteristics of the patient. The mutation profiles of 37 lung cancer cases with paired ctDNA and tumor genomic DNA sequencing were used to evaluate clonal representation of tumor in circulation. Five lung cancer cases with longitudinal ctDNA sampling were monitored for cancer progression or response to treatments. Results Mutations in TP53, EGFR, and KRAS genes are most prevalent in our cohort. Mutation rates of ctDNA are similar in early (I and II and late stage (III and IV cancers. Mutation in DNA repair genes BRCA1, BRCA2, and ATM are found in 18.1% (32/177 of cases. Patients with higher mutation rates had significantly higher mortality rates. Lung cancer of never smokers exhibited significantly higher ctDNA mutation rates as well as higher EGFR and ERBB2 mutations than ever smokers. Comparative analysis of ctDNA and tumor DNA mutation data from the same patients showed that key driver mutations could be detected in plasma even when they were present at a minor clonal population in the tumor. Mutations of key genes found in the tumor tissue could remain in circulation even after frontline radiotherapy and chemotherapy suggesting these mutations represented resistance mechanisms. Longitudinal sampling of five lung cancer cases showed distinct changes in ctDNA mutation portraits that

  20. Analysis of allelic expression patterns in clonal somatic cells by single-cell RNA-seq.

    Science.gov (United States)

    Reinius, Björn; Mold, Jeff E; Ramsköld, Daniel; Deng, Qiaolin; Johnsson, Per; Michaëlsson, Jakob; Frisén, Jonas; Sandberg, Rickard

    2016-11-01

    Cellular heterogeneity can emerge from the expression of only one parental allele. However, it has remained controversial whether, or to what degree, random monoallelic expression of autosomal genes (aRME) is mitotically inherited (clonal) or stochastic (dynamic) in somatic cells, particularly in vivo. Here we used allele-sensitive single-cell RNA-seq on clonal primary mouse fibroblasts and freshly isolated human CD8 + T cells to dissect clonal and dynamic monoallelic expression patterns. Dynamic aRME affected a considerable portion of the cells' transcriptomes, with levels dependent on the cells' transcriptional activity. Notably, clonal aRME was detected, but it was surprisingly scarce (aRME occurs transiently within individual cells, and patterns of aRME are thus primarily scattered throughout somatic cell populations rather than, as previously hypothesized, confined to patches of clonally related cells.

  1. Uncovering the Number and Clonal Dynamics of Mesp1 Progenitors during Heart Morphogenesis

    Directory of Open Access Journals (Sweden)

    Samira Chabab

    2016-01-01

    Full Text Available The heart arises from distinct sources of cardiac progenitors that independently express Mesp1 during gastrulation. The precise number of Mesp1 progenitors that are specified during the early stage of gastrulation, and their clonal behavior during heart morphogenesis, is currently unknown. Here, we used clonal and mosaic tracing of Mesp1-expressing cells combined with quantitative biophysical analysis of the clonal data to define the number of cardiac progenitors and their mode of growth during heart development. Our data indicate that the myocardial layer of the heart derive from ∼250 Mesp1-expressing cardiac progenitors born during gastrulation. Despite arising at different time points and contributing to different heart regions, the temporally distinct cardiac progenitors present very similar clonal dynamics. These results provide insights into the number of cardiac progenitors and their mode of growth and open up avenues to decipher the clonal dynamics of progenitors in other organs and tissues.

  2. Clonal neoantigens elicit T cell immunoreactivity and sensitivity to immune checkpoint blockade

    Science.gov (United States)

    McGranahan, Nicholas; Furness, Andrew J. S.; Rosenthal, Rachel; Ramskov, Sofie; Lyngaa, Rikke; Saini, Sunil Kumar; Jamal-Hanjani, Mariam; Wilson, Gareth A.; Birkbak, Nicolai J.; Hiley, Crispin T.; Watkins, Thomas B. K.; Shafi, Seema; Murugaesu, Nirupa; Mitter, Richard; Akarca, Ayse U.; Linares, Joseph; Marafioti, Teresa; Henry, Jake Y.; Van Allen, Eliezer M.; Miao, Diana; Schilling, Bastian; Schadendorf, Dirk; Garraway, Levi A.; Makarov, Vladimir; Rizvi, Naiyer A.; Snyder, Alexandra; Hellmann, Matthew D.; Merghoub, Taha; Wolchok, Jedd D.; Shukla, Sachet A.; Wu, Catherine J.; Peggs, Karl S.; Chan, Timothy A.; Hadrup, Sine R.; Quezada, Sergio A.; Swanton, Charles

    2016-01-01

    As tumors grow, they acquire mutations, some of which create neoantigens that influence the response of patients to immune checkpoint inhibitors. We explored the impact of neoantigen intratumor heterogeneity (ITH) on antitumor immunity. Through integrated analysis of ITH and neoantigen burden, we demonstrate a relationship between clonal neoantigen burden and overall survival in primary lung adenocarcinomas. CD8+ tumor-infiltrating lymphocytes reactive to clonal neoantigens were identified in early-stage non–small cell lung cancer and expressed high levels of PD-1. Sensitivity to PD-1 and CTLA-4 blockade in patients with advanced NSCLC and melanoma was enhanced in tumors enriched for clonal neoantigens. T cells recognizing clonal neoantigens were detectable in patients with durable clinical benefit. Cytotoxic chemotherapy–induced subclonal neoantigens, contributing to an increased mutational load, were enriched in certain poor responders. These data suggest that neoantigen heterogeneity may influence immune surveillance and support therapeutic developments targeting clonal neoantigens. PMID:26940869

  3. Demographic consequences of greater clonal than sexual reproduction in Dicentra canadensis.

    Science.gov (United States)

    Lin, Chia-Hua; Miriti, Maria N; Goodell, Karen

    2016-06-01

    Clonality is a widespread life history trait in flowering plants that may be essential for population persistence, especially in environments where sexual reproduction is unpredictable. Frequent clonal reproduction, however, could hinder sexual reproduction by spatially aggregating ramets that compete with seedlings and reduce inter-genet pollination. Nevertheless, the role of clonality in relation to variable sexual reproduction in population dynamics is often overlooked. We combined population matrix models and pollination experiments to compare the demographic contributions of clonal and sexual reproduction in three Dicentra canadensis populations, one in a well-forested landscape and two in isolated forest remnants. We constructed stage-based transition matrices from 3 years of census data to evaluate annual population growth rates, λ. We used loop analysis to evaluate the relative contribution of different reproductive pathways to λ. Despite strong temporal and spatial variation in seed set, populations generally showed stable growth rates. Although we detected some pollen limitation of seed set, manipulative pollination treatments did not affect population growth rates. Clonal reproduction contributed significantly more than sexual reproduction to population growth in the forest remnants. Only at the well-forested site did sexual reproduction contribute as much as clonal reproduction to population growth. Flowering plants were more likely to transition to a smaller size class with reduced reproductive potential in the following year than similarly sized nonflowering plants, suggesting energy trade-offs between sexual and clonal reproduction at the individual level. Seed production had negligible effects on growth and tuber production of individual plants. Our results demonstrate that clonal reproduction is vital for population persistence in a system where sexual reproduction is unpredictable. The bias toward clonality may be driven by low fitness returns

  4. Whole transcriptome analysis for T cell receptor-affinity and IRF4-regulated clonal expansion of T cells

    Directory of Open Access Journals (Sweden)

    Wei Shi

    2014-12-01

    Full Text Available Clonal population expansion of T cells during an immune response is dependent on the affinity of the T cell receptor (TCR for its antigen [1]. However, there is little understanding of how this process is controlled transcriptionally. We found that the transcription factor IRF4 was induced in a manner dependent on TCR-affinity and was critical for the clonal expansion and maintenance of effector function of antigen-specific CD8+ T cells. We performed a genome-wide expression profiling experiment using RNA sequencing technology (RNA-seq to interrogate global expression changes when IRF4 was deleted in CD8+ T cells activated with either a low or high affinity peptide ligand. This allowed us not only to determine IRF4-dependent transcriptional changes but also to identify transcripts dependent on TCR-affinity [2]. Here we describe in detail the analyses of the RNA-seq data, including quality control, read mapping, quantification, normalization and assessment of differential gene expression. The RNA-seq data can be accessed from Gene Expression Omnibus database (accession number GSE49929.

  5. Endothelial progenitor cells display clonal restriction in multiple myeloma

    International Nuclear Information System (INIS)

    Braunstein, Marc; Özçelik, Tayfun; Bağişlar, Sevgi; Vakil, Varsha; Smith, Eric LP; Dai, Kezhi; Akyerli, Cemaliye B; Batuman, Olcay A

    2006-01-01

    In multiple myeloma (MM), increased neoangiogenesis contributes to tumor growth and disease progression. Increased levels of endothelial progenitor cells (EPCs) contribute to neoangiogenesis in MM, and, importantly, covary with disease activity and response to treatment. In order to understand the mechanisms responsible for increased EPC levels and neoangiogenic function in MM, we investigated whether these cells were clonal by determining X-chromosome inactivation (XCI) patterns in female patients by a human androgen receptor assay (HUMARA). In addition, EPCs and bone marrow cells were studied for the presence of clonotypic immunoglobulin heavy-chain (IGH) gene rearrangement, which indicates clonality in B cells; thus, its presence in EPCs would indicate a close genetic link between tumor cells in MM and endothelial cells that provide tumor neovascularization. A total of twenty-three consecutive patients who had not received chemotherapy were studied. Screening in 18 patients found that 11 displayed allelic AR in peripheral blood mononuclear cells, and these patients were further studied for XCI patterns in EPCs and hair root cells by HUMARA. In 2 patients whose EPCs were clonal by HUMARA, and in an additional 5 new patients, EPCs were studied for IGH gene rearrangement using PCR with family-specific primers for IGH variable genes (V H ). In 11 patients, analysis of EPCs by HUMARA revealed significant skewing (≥ 77% expression of a single allele) in 64% (n = 7). In 4 of these patients, XCI skewing was extreme (≥ 90% expression of a single allele). In contrast, XCI in hair root cells was random. Furthermore, PCR amplification with V H primers resulted in amplification of the same product in EPCs and bone marrow cells in 71% (n = 5) of 7 patients, while no IGH rearrangement was found in EPCs from healthy controls. In addition, in patients with XCI skewing in EPCs, advanced age was associated with poorer clinical status, unlike patients whose EPCs had random XCI

  6. Multi locus sequence typing of Chlamydiales: clonal groupings within the obligate intracellular bacteria Chlamydia trachomatis

    NARCIS (Netherlands)

    Pannekoek, Yvonne; Morelli, Giovanna; Kusecek, Barica; Morré, Servaas A.; Ossewaarde, Jacobus M.; Langerak, Ankie A.; van der Ende, Arie

    2008-01-01

    BACKGROUND: The obligate intracellular growing bacterium Chlamydia trachomatis causes diseases like trachoma, urogenital infection and lymphogranuloma venereum with severe morbidity. Several serovars and genotypes have been identified, but these could not be linked to clinical disease or outcome.

  7. Aging, Clonality and Rejuvenation of Hematopoietic Stem Cells

    Science.gov (United States)

    Akunuru, Shailaja; Geiger, Hartmut

    2016-01-01

    Aging is associated with reduced organ function and increased disease incidence. Hematopoietic stem cell (HSC) aging driven by both cell intrinsic and extrinsic factors is linked to impaired HSC self-renewal and regeneration, aging-associated immune remodeling, and increased leukemia incidence. Compromised DNA damage responses and increased production of reactive oxygen species have been previously causatively attributed to HSC aging. However, recent paradigm-shifting concepts such as global epigenetic and cytoskeletal polarity shifts, cellular senescence, as well as clonal selection of HSCs upon aging provide new insights into HSC aging mechanisms. Rejuvenating agents that can reprogram the epigenetic status of aged HSCs or senolytic drugs that selectively deplete senescent cells provide promising translational avenues for attenuating hematopoietic aging and potentially, alleviating aging-associated immune remodeling and myeloid malignancies. PMID:27380967

  8. Atherosclerosis and clonal hematopoyesis: A new risk factor.

    Science.gov (United States)

    Páramo Fernández, José A

    Recent research has revealed that clonal hematopoyesis of indeterminate potential (CHIP) characterized by the acquisition of somatic mutations in hematopoietic stem cells, is not only a common age-related disorder and a premalignant condition, but it is also associated with the development of atherosclerotic vascular diseases. Mutations in DNMT3A, TET2 and ASXL1 were each individually associated with coronary heart disease, stroke and coronary calcification. Therefore, CHIP emerges as a new risk factor for atherosclerotic vascular pathologies and its detection may be relevant as a new therapeutic target in order to modify the natural course of the disease. Copyright © 2018 Sociedad Española de Arteriosclerosis. Publicado por Elsevier España, S.L.U. All rights reserved.

  9. Clonal proliferation of cultured nonmalignant and malignant human breast epithelia

    International Nuclear Information System (INIS)

    Smith, H.S.; Lan, S.; Ceriani, R.; Hackett, A.J.; Stampfer, M.R.

    1981-01-01

    We have developed a method for clonal growth of human mammary epithelial cells of both nonmalignant and malignant origin. Plating efficiencies of 1 to 50% were obtained by seeding second-passage mammary epithelial cells on fibroblast feeder layers in an enriched medium composed of various hormones and growth factors, as well as conditioned media from three specific human cell lines. Single mammary epithelial cells seeded sparsely onto the fibroblasts underwent at least eight population doublings to form large, readily visible colonies. Optimal colony formation required both feeder cells and the enriched medium. Epithelial colonies containing at least 16 cells were visible 5 days postseeding, and these colonies continued to grow progressively. Plating efficiency and colony size were similar on ultraviolet-irradiated or nonirradiated fibroblasts. The number of colonies formed was proportional to the number of epithelial cells plated. The colonies were identified as epithelial by the presence of human mammary epithelial antigens

  10. Comparing nonparametric Bayesian tree priors for clonal reconstruction of tumors.

    Science.gov (United States)

    Deshwar, Amit G; Vembu, Shankar; Morris, Quaid

    2015-01-01

    Statistical machine learning methods, especially nonparametric Bayesian methods, have become increasingly popular to infer clonal population structure of tumors. Here we describe the treeCRP, an extension of the Chinese restaurant process (CRP), a popular construction used in nonparametric mixture models, to infer the phylogeny and genotype of major subclonal lineages represented in the population of cancer cells. We also propose new split-merge updates tailored to the subclonal reconstruction problem that improve the mixing time of Markov chains. In comparisons with the tree-structured stick breaking prior used in PhyloSub, we demonstrate superior mixing and running time using the treeCRP with our new split-merge procedures. We also show that given the same number of samples, TSSB and treeCRP have similar ability to recover the subclonal structure of a tumor…

  11. Transcriptomic variation in a coral reveals pathways of clonal organisation

    DEFF Research Database (Denmark)

    K Bay, Line; Nielsen, Henrik Bjørn; Jarmer, Hanne Østergaard

    2009-01-01

    A microarray study was undertaken to examine the potential for clonal gene expression variation in a branching reef building coral, Acropora millepora. The role of small-scale gradients in light and water flow was examined by comparing gene expression levels between branch elevation (tip and base......) and position (centre and edge) of replicate coral colonies (n=3). Analyses of variance revealed that almost 60% of variation in gene expression was present between colonies and 34 genes were considered differentially expressed between colonies (minimum P=6.5 x 10(-4)). These genes are associated with energy...... of corymbose-like branching coral colonies such as A. millepora. Four genes were differentially expressed between the tip and base of branches (P=3.239 x 10(-4)) and were associated with lysosome lipase activity and fluorescence, suggesting that branch tips may encounter higher pathogen loads or levels...

  12. Proviral HIV-genome-wide and pol-gene specific zinc finger nucleases: usability for targeted HIV gene therapy.

    Science.gov (United States)

    Wayengera, Misaki

    2011-07-22

    Infection with HIV, which culminates in the establishment of a latent proviral reservoir, presents formidable challenges for ultimate cure. Building on the hypothesis that ex-vivo or even in-vivo abolition or disruption of HIV-gene/genome-action by target mutagenesis or excision can irreversibly abrogate HIV's innate fitness to replicate and survive, we previously identified the isoschizomeric bacteria restriction enzymes (REases) AcsI and ApoI as potent cleavers of the HIV-pol gene (11 and 9 times in HIV-1 and 2, respectively). However, both enzymes, along with others found to cleave across the entire HIV-1 genome, slice (SX) at palindromic sequences that are prevalent within the human genome and thereby pose the risk of host genome toxicity. A long-term goal in the field of R-M enzymatic therapeutics has thus been to generate synthetic restriction endonucleases with longer recognition sites limited in specificity to HIV. We aimed (i) to assemble and construct zinc finger arrays and nucleases (ZFN) with either proviral-HIV-pol gene or proviral-HIV-1 whole-genome specificity respectively, and (ii) to advance a model for pre-clinically testing lentiviral vectors (LV) that deliver and transduce either ZFN genotype. First, we computationally generated the consensus sequences of (a) 114 dsDNA-binding zinc finger (Zif) arrays (ZFAs or ZifHIV-pol) and (b) two zinc-finger nucleases (ZFNs) which, unlike the AcsI and ApoI homeodomains, possess specificity to >18 base-pair sequences uniquely present within the HIV-pol gene (ZifHIV-polFN). Another 15 ZFNs targeting >18 bp sequences within the complete HIV-1 proviral genome were constructed (ZifHIV-1FN). Second, a model for constructing lentiviral vectors (LVs) that deliver and transduce a diploid copy of either ZifHIV-polFN or ZifHIV-1FN chimeric genes (termed LV- 2xZifHIV-polFN and LV- 2xZifHIV-1FN, respectively) is proposed. Third, two preclinical models for controlled testing of the safety and efficacy of either of these

  13. Clonal Evolution and Clinical Significance of Copy Number Neutral Loss of Heterozygosity of Chromosome Arm 6p in Acquired Aplastic Anemia

    Science.gov (United States)

    Betensky, Marisol; Babushok, Daria; Roth, Jacquelyn J.; Mason, Philip J; Biegel, Jaclyn A.; Busse, Tracy M; Li, Yimei; Lind, Curt; Papazoglou, Anna; Monos, Dimitri; Podsakoff, Gregory; Bessler, Monica; Olson, Timothy S.

    2015-01-01

    Acquired aplastic anemia (aAA) results from the T cell-mediated autoimmune destruction of hematopoietic stem cells. Factors predicting response to immune suppression therapy (IST) or development of myelodysplastic syndrome (MDS) are beginning to be elucidated. Our recent data suggest most patients with aAA treated with IST develop clonal somatic genetic alterations in hematopoietic cells. One frequent acquired abnormality is copy-number neutral loss of heterozygosity on chromosome 6p (6p CN-LOH) involving the human leukocyte antigen (HLA) locus. We hypothesized that because 6p CN-LOH clones may arise from selective pressure to escape immune surveillance through deletion of HLA alleles, the development of 6p CN-LOH may affect response to IST. We used single nucleotide polymorphism array genotyping and targeted next-generation sequencing of HLA alleles to assess frequency of 6p CN-LOH, identity of HLA alleles lost through 6p CN-LOH, and impact of 6p CN-LOH on response to IST. 6p CN-LOH clones were present in 11.3% of patients, remained stable over time, and were not associated with development of MDS-defining cytogenetic abnormalities. Notably, no patient with 6p CN-LOH treated with IST achieved a complete response. In summary, clonal 6p CN-LOH in aAA defines a unique subgroup of patients that may provide insights into hematopoietic clonal evolution. PMID:26702937

  14. Clonal evolution and clinical significance of copy number neutral loss of heterozygosity of chromosome arm 6p in acquired aplastic anemia.

    Science.gov (United States)

    Betensky, Marisol; Babushok, Daria; Roth, Jacquelyn J; Mason, Philip J; Biegel, Jaclyn A; Busse, Tracy M; Li, Yimei; Lind, Curt; Papazoglou, Anna; Monos, Dimitri; Podsakoff, Gregory; Bessler, Monica; Olson, Timothy S

    2016-01-01

    Acquired aplastic anemia (aAA) results from the T cell-mediated autoimmune destruction of hematopoietic stem cells. Factors predicting response to immune suppression therapy (IST) or development of myelodysplastic syndrome (MDS) are beginning to be elucidated. Our recent data suggest most patients with aAA treated with IST develop clonal somatic genetic alterations in hematopoietic cells. One frequent acquired abnormality is copy-number neutral loss of heterozygosity on chromosome 6p (6p CN-LOH) involving the human leukocyte antigen (HLA) locus. We hypothesized that because 6p CN-LOH clones may arise from selective pressure to escape immune surveillance through deletion of HLA alleles, the development of 6p CN-LOH may affect response to IST. We used single nucleotide polymorphism array genotyping and targeted next-generation sequencing of HLA alleles to assess frequency of 6p CN-LOH, identity of HLA alleles lost through 6p CN-LOH, and impact of 6p CN-LOH on response to IST. 6p CN-LOH clones were present in 11.3% of patients, remained stable over time, and were not associated with development of MDS-defining cytogenetic abnormalities. Notably, no patient with 6p CN-LOH treated with IST achieved a complete response. In summary, clonal 6p CN-LOH in aAA defines a unique subgroup of patients that may provide insights into hematopoietic clonal evolution. Copyright © 2016 Elsevier Inc. All rights reserved.

  15. Genetic signatures in the envelope glycoproteins of HIV-1 that associate with broadly neutralizing antibodies.

    Directory of Open Access Journals (Sweden)

    S Gnanakaran

    Full Text Available A steady increase in knowledge of the molecular and antigenic structure of the gp120 and gp41 HIV-1 envelope glycoproteins (Env is yielding important new insights for vaccine design, but it has been difficult to translate this information to an immunogen that elicits broadly neutralizing antibodies. To help bridge this gap, we used phylogenetically corrected statistical methods to identify amino acid signature patterns in Envs derived from people who have made potently neutralizing antibodies, with the hypothesis that these Envs may share common features that would be useful for incorporation in a vaccine immunogen. Before attempting this, essentially as a control, we explored the utility of our computational methods for defining signatures of complex neutralization phenotypes by analyzing Env sequences from 251 clonal viruses that were differentially sensitive to neutralization by the well-characterized gp120-specific monoclonal antibody, b12. We identified ten b12-neutralization signatures, including seven either in the b12-binding surface of gp120 or in the V2 region of gp120 that have been previously shown to impact b12 sensitivity. A simple algorithm based on the b12 signature pattern was predictive of b12 sensitivity/resistance in an additional blinded panel of 57 viruses. Upon obtaining these reassuring outcomes, we went on to apply these same computational methods to define signature patterns in Env from HIV-1 infected individuals who had potent, broadly neutralizing responses. We analyzed a checkerboard-style neutralization dataset with sera from 69 HIV-1-infected individuals tested against a panel of 25 different Envs. Distinct clusters of sera with high and low neutralization potencies were identified. Six signature positions in Env sequences obtained from the 69 samples were found to be strongly associated with either the high or low potency responses. Five sites were in the CD4-induced coreceptor binding site of gp120, suggesting an

  16. Cellular aging (the Hayflick limit) and species longevity: a unification model based on clonal succession.

    Science.gov (United States)

    Juckett, D A

    1987-03-01

    A model is presented which proposes a specific cause-and-effect relationship between a limited cell division potential and the maximum lifespan of humans and other mammals. It is based on the clonal succession hypothesis of Kay which states that continually replicating cell beds (e.g. bone marrow, intestinal crypts, epidermis) could be composed of cells with short, well-defined division potentials. In this model, the cells of these beds are proposed to exist in an ordered hierarchy which establishes a specific sequence for cell divisions throughout the organism's lifespan. The depletion of division potential at all hierarchical levels leads to a loss of bed function and sets an intrinsic limit to species longevity. A specific hierarchy for cell proliferation is defined which allows the calculation of time to bed depletion and, ultimately, to organism mortality. The model allows the existence of a small number (n) of critical cell beds within the organism and defines organism death as the inability of any one of these beds to produce cells. The model is consistent with all major observations related to cellular and organismic aging. In particular, it links the PDLs (population doubling limit) observed for various species to their mean lifespan; it explains the slow decline in PDL as a function of age of the donor; it establishes a thermodynamically stable maximum lifespan for a disease-free population; and it can explain why tissue transplants outlive donors or hosts.

  17. Clonality of bacterial consortia in root canals and subjacent gingival crevices.

    Science.gov (United States)

    Parahitiyawa, Nipuna B; Chu, Frederick C S; Leung, Wai K; Yam, Wing C; Jin, Li Jian; Samaranayake, Lakshman P

    2015-02-01

    No oral niche can be considered to be segregated from the subjacent milieu because of the complex community behavior and nature of the oral biofilms. The aim of this study was to address the paucity of information on how these species are clonally related to the subjacent gingival crevice bacteria. We utilized a metagenomic approach of amplifying 16S rDNA from genomic DNA, cloning, sequencing and analysis using LIBSHUFF software to assess the genetic homogeneity of the bacterial species from two infected root canals and subjacent gingival crevices. The four niches studied yielded 186 clones representing 54 phylotypes. Clone library comparisons using LIBSHUFF software indicated that each niche was inhabited by a unique flora. Further, 42% of the clones were of hitherto unknown phylotypes indicating the extent of bacterial diversity, especially in infected root canals and subjacent gingival crevices. We believe data generated through this novel analytical tool shed new light on understanding oral microbial ecosystems. © 2014 Wiley Publishing Asia Pty Ltd.

  18. HIV-1 vaccines

    Science.gov (United States)

    Excler, Jean-Louis; Robb, Merlin L; Kim, Jerome H

    2014-01-01

    The development of a safe and effective preventive HIV-1 vaccine remains a public health priority. Despite scientific difficulties and disappointing results, HIV-1 vaccine clinical development has, for the first time, established proof-of-concept efficacy against HIV-1 acquisition and identified vaccine-associated immune correlates of risk. The correlate of risk analysis showed that IgG antibodies against the gp120 V2 loop correlated with decreased risk of HIV infection, while Env-specific IgA directly correlated with increased risk. The development of vaccine strategies such as improved envelope proteins formulated with potent adjuvants and DNA and vectors expressing mosaics, or conserved sequences, capable of eliciting greater breadth and depth of potentially relevant immune responses including neutralizing and non-neutralizing antibodies, CD4+ and CD8+ cell-mediated immune responses, mucosal immune responses, and immunological memory, is now proceeding quickly. Additional human efficacy trials combined with other prevention modalities along with sustained funding and international collaboration remain key to bring an HIV-1 vaccine to licensure. PMID:24637946

  19. Clonal diversity and estimation of relative clone age: application to agrobiodiversity of yam (Dioscorea rotundata).

    Science.gov (United States)

    Scarcelli, Nora; Couderc, Marie; Baco, Mohamed N; Egah, Janvier; Vigouroux, Yves

    2013-11-13

    Clonal propagation is a particular reproductive system found in both the plant and animal kingdoms, from human parasites to clonally propagated crops. Clonal diversity provides information about plant and animal evolutionary history, i.e. how clones spread, or the age of a particular clone. In plants, this could provide valuable information about agrobiodiversity dynamics and more broadly about the evolutionary history of a particular crop. We studied the evolutionary history of yam, Dioscorea rotundata. In Africa, Yam is cultivated by tuber clonal propagation. We used 12 microsatellite markers to identify intra-clonal diversity in yam varieties. We then used this diversity to assess the relative ages of clones. Using simulations, we assessed how Approximate Bayesian Computation could use clonal diversity to estimate the age of a clone depending on the size of the sample, the number of independent samples and the number of markers. We then applied this approach to our particular dataset and showed that the relative ages of varieties could be estimated, and that each variety could be ranked by age. We give a first estimation of clone age in an approximate Bayesian framework. However the precise estimation of clone age depends on the precision of the mutation rate. We provide useful information on agrobiodiversity dynamics and suggest recurrent creation of varietal diversity in a clonally propagated crop.

  20. Clonal integration facilitates spread of Paspalum paspaloides from terrestrial to cadmium-contaminated aquatic habitats.

    Science.gov (United States)

    Luo, F-L; Xing, Y-P; Wei, G-W; Li, C-Y; Yu, F-H

    2017-11-01

    Cadmium (Cd) is a hazardous environmental pollutant with high toxicity to plants, which has been detected in many wetlands. Clonal integration (resource translocation) between connected ramets of clonal plants can increase their tolerance to stress. We hypothesised that clonal integration facilitates spread of amphibious clonal plants from terrestrial to Cd-contaminated aquatic habitats. The spread of an amphibious grass Paspalum paspaloides was simulated by growing basal older ramets in uncontaminated soil connected (allowing integration) or not connected (preventing integration) to apical younger ramets of the same fragments in Cd-contaminated water. Cd contamination of apical ramets of P. paspaloides markedly decreased growth and photosynthetic capacity of the apical ramets without connection to the basal ramets, but did not decrease these properties with connection. Cd contamination did not affect growth of the basal ramets without connection to the apical ramets, but Cd contamination of 4 and 12 mg·l -1 significantly increased growth with connection. Consequently, clonal integration increased growth of the apical ramets, basal ramets and whole clones when the apical ramets were grown in Cd-contaminated water of 4 and 12 mg·l -1 . Cd was detected in the basal ramets with connection to the apical ramets, suggesting Cd could be translocated due to clonal integration. Clonal integration, most likely through translocation of photosynthates, can support P. paspaloides to spread from terrestrial to Cd-contaminated aquatic habitats. Amphibious clonal plants with a high ability for clonal integration are particularly useful for re-vegetation of degraded aquatic habitats caused by Cd contamination. © 2017 German Society for Plant Sciences and The Royal Botanical Society of the Netherlands.

  1. Clonal growth strategy, diversity and structure: A spatiotemporal response to sedimentation in tropical Cyperus papyrus swamps.

    Science.gov (United States)

    Geremew, Addisie; Stiers, Iris; Sierens, Tim; Kefalew, Alemayehu; Triest, Ludwig

    2018-01-01

    Land degradation and soil erosion in the upper catchments of tropical lakes fringed by papyrus vegetation can result in a sediment load gradient from land to lakeward. Understanding the dynamics of clonal modules (ramets and genets) and growth strategies of plants on such a gradient in both space and time is critical for exploring a species adaptation and processes regulating population structure and differentiation. We assessed the spatial and temporal dynamics in clonal growth, diversity, and structure of an emergent macrophyte, Cyperus papyrus (papyrus), in response to two contrasting sedimentation regimes by combining morphological traits and genotype data using 20 microsatellite markers. A total of 636 ramets from six permanent plots (18 x 30 m) in three Ethiopian papyrus swamps, each with discrete sedimentation regimes (high vs. low) were sampled for two years. We found that ramets under the high sedimentation regime (HSR) were significantly clumped and denser than the sparse and spreading ramets under the low sedimentation regime (LSR). The HSR resulted in significantly different ramets with short culm height and girth diameter as compared to the LSR. These results indicated that C. papyrus ameliorates the effect of sedimentation by shifting clonal growth strategy from guerrilla (in LSR) to phalanx (in HSR). Clonal richness, size, dominance, and clonal subrange differed significantly between sediment regimes and studied time periods. Each swamp under HSR revealed a significantly high clonal richness (R = 0.80) as compared to the LSR (R = 0.48). Such discrepancy in clonal richness reflected the occurrence of initial and repeated seedling recruitment strategies as a response to different sedimentation regimes. Overall, our spatial and short-term temporal observations highlighted that HSR enhances clonal richness and decreases clonal subrange owing to repeated seedling recruitment and genets turnover.

  2. Prediction for the occurrence of clonal chromosome aberrations in human blood lymphocytes

    International Nuclear Information System (INIS)

    Nakano, M.; Kadama, Y.; Ohtaki, K.; Itoh, M.; Awa, A.; Cologne, J.; Nakamura, N.

    2003-01-01

    Full text: Identical chromosome aberrations among multiple blood lymphocytes in a blood sample (clonal aberrations) are encountered occasionally during cytogenetic examination of radiation-exposed people. Clonal aberrations are found primarily among high-dose exposed people but no systematic surveys were ever conducted. Therefore, the underlying mechanism is unknown. Here we conducted a large-scale screening for detecting clonal aberrations using FISH followed by Q-banding. Examinations of 500 cells from each of 513 A-bomb survivors led us to detect 96 clones. The clonal cell fraction (Cf) varied from 0.6% to 20% among the 500 cells. As the number of clonal event was inversely proportional to Cf, we hypothesized that the progenitor cells vary extensively in the number of offspring that they can produce and relative number of progenitor cells decreases as the increase of treatment, while other genes such as DNA repair proteinsnumber of progenitor cells capable to form clones (Cf >=0.6%) to be 2 (1 to 3) in non-exposed individuals. The number increased to up to 7 among the high-dose exposed survivors. Further, our preliminary results for the origins of 10 clones indicated that both hematopoietic stem cells (HSCs) and mature T cells contributed to the clone formation roughly equally. Thus, the estimated number of 2 in non-exposed individuals is shared as one HSC and one mature T cells. The model could neatly explain the frequency of clones in two reports. Our model predicts that clonal aberrations are rarely found but clonal expansion of T lymphocytes occurs commonly. In fact, clonal expansions of non-aberrant cells are reported using TCR gene rearrangement patterns as a marker. We now understand the rough structure of lymphocyte pool in humans and can predict the probability of detecting a clone if the individual frequency of non-clonal translocations and the number of cells scored are given

  3. An efficient and reproducible method for in vitro clonal multiplication of Rauvolfia tetraphylla L. and evaluation of genetic stability using DNA-based markers.

    Science.gov (United States)

    Faisal, Mohammad; Alatar, Abdulrahman A; Ahmad, Naseem; Anis, Mohammad; Hegazy, Ahmad K

    2012-12-01

    An efficient protocol is described for the rapid in vitro clonal propagation of an endangered medicinal plant, Rauvolfia tetraphylla L., through high frequency shoot induction from nodal explants collected from young shoots of a field grown plant. Effects of growth regulators [6-benzyladenine (BA), kinetin (Kin) 2iP, or α-naphthalene acetic acid (NAA)], carbohydrates, different medium [Murashige and Skoog (MS), Woody Plant Medium (WPM), Gamborg medium (B5), Linsmier and Skoog medium (LS)], and various pH levels on in vitro morphogenesis were investigated. The highest frequency of shoot regeneration (90 %) and maximum number of shoot (35.4 ± 2.3) per explant were observed on WPM medium supplemented with 7.5 μM BA, 2.5 μM NAA, and 30 g/l sucrose at pH 5.8. Well-developed shoots, 4-5 cm in length, were successfully rooted ex vitro at 90 % by a 30-min pulse treatment with 150 μM IBA prior to their transfer in planting substrates. The survival rate of transplantation reached 90 % when transferred to field condition. Genetic stability of micropropagated plantlets was assessed and compared with mother plant using Random Amplified Polymorphic DNA and Inter Simple Sequence Repeats markers. No variation was observed in DNA fingerprinting patterns among the micropropagated plants, which were similar to that of the donor plant illustrating their genetic uniformity and clonal fidelity. This confirms that clonal propagation of this plant using axillary shoot buds can be used for commercial exploitation of the selected genotype where a high degree of fidelity is an essential prerequisite. The work contributed to a better in vitro regeneration and clonal mass multiplication of R. tetraphylla and to develop a strategy for the germplasm conservation of this endangered medicinal plant.

  4. Strongyloidiasis and infective dermatitis alter human T lymphotropic virus-1 clonality in vivo.

    Directory of Open Access Journals (Sweden)

    Nicolas A Gillet

    Full Text Available Human T-lymphotropic Virus-1 (HTLV-1 is a retrovirus that persists lifelong by driving clonal proliferation of infected T-cells. HTLV-1 causes a neuroinflammatory disease and adult T-cell leukemia/lymphoma. Strongyloidiasis, a gastrointestinal infection by the helminth Strongyloides stercoralis, and Infective Dermatitis associated with HTLV-1 (IDH, appear to be risk factors for the development of HTLV-1 related diseases. We used high-throughput sequencing to map and quantify the insertion sites of the provirus in order to monitor the clonality of the HTLV-1-infected T-cell population (i.e. the number of distinct clones and abundance of each clone. A newly developed biodiversity estimator called "DivE" was used to estimate the total number of clones in the blood. We found that the major determinant of proviral load in all subjects without leukemia/lymphoma was the total number of HTLV-1-infected clones. Nevertheless, the significantly higher proviral load in patients with strongyloidiasis or IDH was due to an increase in the mean clone abundance, not to an increase in the number of infected clones. These patients appear to be less capable of restricting clone abundance than those with HTLV-1 alone. In patients co-infected with Strongyloides there was an increased degree of oligoclonal expansion and a higher rate of turnover (i.e. appearance and disappearance of HTLV-1-infected clones. In Strongyloides co-infected patients and those with IDH, proliferation of the most abundant HTLV-1⁺ T-cell clones is independent of the genomic environment of the provirus, in sharp contrast to patients with HTLV-1 infection alone. This implies that new selection forces are driving oligoclonal proliferation in Strongyloides co-infection and IDH. We conclude that strongyloidiasis and IDH increase the risk of development of HTLV-1-associated diseases by increasing the rate of infection of new clones and the abundance of existing HTLV-1⁺ clones.

  5. CCR5 Disruption in Induced Pluripotent Stem Cells Using CRISPR/Cas9 Provides Selective Resistance of Immune Cells to CCR5-tropic HIV-1 Virus.

    Science.gov (United States)

    Kang, HyunJun; Minder, Petra; Park, Mi Ae; Mesquitta, Walatta-Tseyon; Torbett, Bruce E; Slukvin, Igor I

    2015-12-15

    The chemokine (C-C motif) receptor 5 (CCR5) serves as an HIV-1 co-receptor and is essential for cell infection with CCR5-tropic viruses. Loss of functional receptor protects against HIV infection. Here, we report the successful targeting of CCR5 in GFP-marked human induced pluripotent stem cells (iPSCs) using CRISPR/Cas9 with single and dual guide RNAs (gRNAs). Following CRISPER/Cas9-mediated gene editing using a single gRNA, 12.5% of cell colonies demonstrated CCR5 editing, of which 22.2% showed biallelic editing as determined by a Surveyor nuclease assay and direct sequencing. The use of dual gRNAs significantly increased the efficacy of CCR5 editing to 27% with a biallelic gene alteration frequency of 41%. To ensure the homogeneity of gene editing within cells, we used single cell sorting to establish clonal iPSC lines. Single cell-derived iPSC lines with homozygous CCR5 mutations displayed the typical characteristics of pluripotent stem cells and differentiated efficiently into hematopoietic cells, including macrophages. Although macrophages from both wild-type and CCR5-edited iPSCs supported CXCR4-tropic virus replication, macrophages from CCR5-edited iPSCs were uniquely resistant to CCR5-tropic virus challenge. This study demonstrates the feasibility of applying iPSC technology for the study of the role of CCR5 in HIV infection in vitro, and generation of HIV-resistant cells for potential therapeutic applications.

  6. Clonal evolution of AML on novel FMS-like tyrosine kinase-3 (FLT3 inhibitor therapy with evolving actionable targets

    Directory of Open Access Journals (Sweden)

    Pashtoon M. Kasi

    2016-01-01

    Full Text Available For acute myeloid leukemia (AML, identification of activating mutations in the FMS-like tyrosine kinase-3 (FLT3 has led to the development of several FLT3-inhibitors. Here we present clinical and next generation sequencing data at the time of progression of a patient on a novel FLT3-inhibitor clinical trial (ASP2215 to show that employing therapeutic interventions with these novel targeted therapies can lead to consequences secondary to selective pressure and clonal evolution of cancer. We describe novel findings alongside data on treatment directed towards actionable aberrations acquired during the process. (Clinical Trial: NCT02014558; registered at: 〈https://clinicaltrials.gov/ct2/show/NCT02014558〉

  7. Diverse cellular architecture of atherosclerotic plaque derives from clonal expansion of a few medial SMCs

    DEFF Research Database (Denmark)

    Jacobsen, Kevin; Lund, Marie Bek; Shim, Jeong

    2017-01-01

    Fibrous cap smooth muscle cells (SMCs) protect atherosclerotic lesions from rupturing and causing thrombosis, while other plaque SMCs may have detrimental roles in plaque development. To gain insight into recruitment of different plaque SMCs, we mapped their clonal architecture in aggregation...... in the cap and heterogeneous ACTA2– SMCs in the plaque interior, including chondrocyte-like cells and cells with intracellular lipid and crystalline material. Fibrous cap SMCs were invariably arranged in endothelium-aligned clonal sheets, confirming results in the aggregation chimeras. Analysis of the clonal...

  8. Comparison of bacterial culture and 16S rRNA community profiling by clonal analysis and and pyrosequencing for the characterisation of the caries-associated microbiome

    Directory of Open Access Journals (Sweden)

    Kathrin eSchulze-Schweifing

    2014-11-01

    Full Text Available Culture-independent analyses have greatly expanded knowledge regarding the composition of complex bacterial communities including those associated with oral diseases. A consistent finding from such studies, however, has been the under-reporting of members of the phylum Actinobacteria. In this study, five pairs of broad range primers targeting 16S rRNA genes were used in clonal analysis of 6 samples collected from tooth lesions involving dentine in subjects with active caries. Samples were also subjected to cultural analysis and pyrosequencing by means of the 454 platform. A diverse bacterial community of 229 species-level taxa was revealed by culture and clonal analysis, dominated by representatives of the genera Prevotella, Lactobacillus, Selenomonas and Streptococcus. The five most abundant species were: Lactobacillus gasseri, Prevotella denticola, Alloprevotella tannerae, S. mutans and Streptococcus sp. HOT 070, which together made up 31.6 % of the sequences. Two samples were dominated by lactobacilli, while the remaining samples had low numbers of lactobacilli but significantly higher numbers of Prevotella species. The different primer pairs produced broadly similar data but proportions of the phylum Bacteroidetes were significantly higher when primer 1387R was used. All of the primer sets underestimated the proportion of Actinobacteria compared to culture. Pyrosequencing analysis of the samples was performed to a depth of sequencing of 4293 sequences per sample which were identified to 264 species-level taxa, and resulted in significantly higher coverage estimates than the clonal analysis. Pyrosequencing, however, also underestimated the relative abundance of Actinobacteria compared to culture.

  9. Linezolid-resistant enterococci in Polish hospitals: species, clonality and determinants of linezolid resistance.

    Science.gov (United States)

    Gawryszewska, I; Żabicka, D; Hryniewicz, W; Sadowy, E

    2017-07-01

    The significant increase of the linezolid-resistant enterococci (LRE) has been observed in Polish hospitals since 2012 and our study aimed at elucidating the possible reasons for this phenomenon. Polish LRE isolates were analysed by multilocus-sequence typing (MLST) and multiple locus variable-number tandem repeat (VNTR) analysis (MLVA), polymerase chain reaction (PCR) and PCR-restriction fragment length polymorphism (PCR-RFLP) to establish clonal relatedness and mechanism of linezolid resistance, respectively. Fifty analysed LRE (2008-2015) included mostly Enterococcus faecium (82%) and Enterococcus faecalis (16%). Enterococcus faecium belonged to the hospital-adapted lineages 17/18 and 78, while E. faecalis isolates represented ST6, a hospital-associated type, and ST116, found in both humans and food-production animals. The G2576T 23S rRNA mutation was the most frequent (94%) mechanism of linezolid/tedizolid resistance of LRE. None of the isolates carried the plasmid-associated gene of Cfr methyltransferase, whereas optrA, encoding the ABC-type drug transporter, was identified in two E. faecalis isolates. In these isolates, optrA was located on a plasmid, transferable to both E. faecium and E. faecalis, whose partial (36.3 kb) sequence was 100% identical to the pE394 plasmid, identified previously in China in both clinical and farm animal isolates. The optrA-E. faecium transconjugant displayed a significant growth deficiency, in contrast to the optrA-E. faecalis. Our study indicates the role of mutation acquisition by hospital-adapted clones of enterococci as a major driver of increasing resistance to linezolid and tedizolid. Transferability and apparent lack of a biological cost of resistance suggest that E. faecalis may be a natural reservoir of optrA, an emerging mechanism of oxazolidinone resistance.

  10. Clonal structure of Streptococcus sanguinis strains isolated from endocarditis cases and the oral cavity.

    Science.gov (United States)

    Do, T; Gilbert, S C; Klein, J; Warren, S; Wade, W G; Beighton, D

    2011-10-01

    A collection of Streptococcus sanguinis strains from patients with endocarditis (n = 21) and from the oral cavity (n = 34) was subjected to a multi-locus sequence typing analysis using seven housekeeping genes, carbamoyl-phosphate synthetase (carB), Co/Zn/Cd efflux system component (czcD), d-alanyl-d-alanine ligase (ddl), DNA polymerase III (dnaX), glucose-6-phosphate dehydrogenase (gdh), DNA-directed RNA polymerase, beta subunit (rpoB) and superoxide dismutase (sodA). The scheme was expanded by the inclusion of two the putative virulence genes, bacitracin-resistance protein (bacA) and saliva-binding protein (ssaB), to increase strain discrimination. Extensive intra-species recombination was apparent in all genes but inter-species recombination was also apparent with strains apparently harbouring gdh and ddl from unidentified sources and one isolate harboured a sodA allele apparently derived from Streptococcus oralis. The recombination/mutation ratio for the concatenated housekeeping gene sequences was 1.67 (95% confidence limits 1.25-2.72) and for the two virulence genes the r/m ratio was 3.99 (95% confidence limits 1.61-8.72); recombination was the major driver for genetic variation. All isolates were distinct and the endocarditis strains did not form distinct sub-clusters when the data were analysed using ClonalFrame. These data support the widely held opinion that infecting S. sanguinis strains are opportunistic human pathogens. © 2011 John Wiley & Sons A/S.

  11. Somatic HLA mutations expose the role of class I–mediated autoimmunity in aplastic anemia and its clonal complications

    Science.gov (United States)

    Duke, Jamie L.; Xie, Hongbo M.; Stanley, Natasha; Atienza, Jamie; Perdigones, Nieves; Nicholas, Peter; Ferriola, Deborah; Li, Yimei; Huang, Hugh; Ye, Wenda; Morrissette, Jennifer J. D.; Kearns, Jane; Porter, David L.; Podsakoff, Gregory M.; Eisenlohr, Laurence C.; Biegel, Jaclyn A.; Chou, Stella T.; Monos, Dimitrios S.; Bessler, Monica; Olson, Timothy S.

    2017-01-01

    Acquired aplastic anemia (aAA) is an acquired deficiency of early hematopoietic cells, characterized by inadequate blood production, and a predisposition to myelodysplastic syndrome (MDS) and leukemia. Although its exact pathogenesis is unknown, aAA is thought to be driven by human leukocyte antigen (HLA)–restricted T cell immunity, with earlier studies favoring HLA class II-mediated pathways. Using whole-exome sequencing (WES), we recently identified 2 patients with aAA with somatic mutations in HLA class I genes. We hypothesized that HLA class I mutations are pathognomonic for autoimmunity in aAA, but were previously underappreciated because the major histocompatibility complex (MHC) region is notoriously difficult to analyze by WES. Using a combination of targeted deep sequencing of HLA class I genes and single nucleotide polymorphism array (SNP-A) genotyping, we screened 66 patients with aAA for somatic HLA class I loss. We found somatic HLA loss in 11 patients (17%), with 13 loss-of-function mutations in HLA-A*33:03, HLA-A*68:01, HLA-B*14:02, and HLA-B*40:02 alleles. Three patients had more than 1 mutation targeting the same HLA allele. Interestingly, HLA-B*14:02 and HLA-B*40:02 were significantly overrepresented in patients with aAA compared with ethnicity-matched controls. Patients who inherited the targeted HLA alleles, regardless of HLA mutation status, had a more severe disease course with more frequent clonal complications as assessed by WES, SNP-A, and metaphase cytogenetics, and more frequent secondary MDS. The finding of recurrent HLA class I mutations provides compelling evidence for a predominant HLA class I-driven autoimmunity in aAA and establishes a novel link between immunogenetics and clonal evolution of patients with aAA. PMID:28971166

  12. Genomic Structural Variations Affecting Virulence During Clonal Expansion of Pseudomonas syringae pv. actinidiae Biovar 3 in Europe.

    Science.gov (United States)

    Firrao, Giuseppe; Torelli, Emanuela; Polano, Cesare; Ferrante, Patrizia; Ferrini, Francesca; Martini, Marta; Marcelletti, Simone; Scortichini, Marco; Ermacora, Paolo

    2018-01-01

    Pseudomonas syringae pv. actinidiae (Psa) biovar 3 caused pandemic bacterial canker of Actinidia chinensis and Actinidia deliciosa since 2008. In Europe, the disease spread rapidly in the kiwifruit cultivation areas from a single introduction. In this study, we investigated the genomic diversity of Psa biovar 3 strains during the primary clonal expansion in Europe using single molecule real-time (SMRT), Illumina and Sanger sequencing technologies. We recorded evidences of frequent mobilization and loss of transposon Tn6212, large chromosome inversions, and ectopic integration of IS sequences (remarkably ISPsy31, ISPsy36, and ISPsy37). While no phenotype change associated with Tn6212 mobilization could be detected, strains CRAFRU 12.29 and CRAFRU 12.50 did not elicit the hypersensitivity response (HR) on tobacco and eggplant leaves and were limited in their growth in kiwifruit leaves due to insertion of ISPsy31 and ISPsy36 in the hrpS and hrpR genes, respectively, interrupting the hrp cluster. Both strains had been isolated from symptomatic plants, suggesting coexistence of variant strains with reduced virulence together with virulent strains in mixed populations. The structural differences caused by rearrangements of self-genetic elements within European and New Zealand strains were comparable in number and type to those occurring among the European strains, in contrast with the significant difference in terms of nucleotide polymorphisms. We hypothesize a relaxation, during clonal expansion, of the selection limiting the accumulation of deleterious mutations associated with genome structural variation due to transposition of mobile elements. This consideration may be relevant when evaluating strategies to be adopted for epidemics management.

  13. Somatic HLA Mutations Expose the Role of Class I-Mediated Autoimmunity in Aplastic Anemia and its Clonal Complications.

    Science.gov (United States)

    Babushok, Daria V; Duke, Jamie L; Xie, Hongbo M; Stanley, Natasha; Atienza, Jamie; Perdigones, Nieves; Nicholas, Peter; Ferriola, Deborah; Li, Yimei; Huang, Hugh; Ye, Wenda; Morrissette, Jennifer J D; Kearns, Jane; Porter, David L; Podsakoff, Gregory M; Eisenlohr, Laurence C; Biegel, Jaclyn A; Chou, Stella T; Monos, Dimitrios S; Bessler, Monica; Olson, Timothy S

    2017-10-10

    Acquired aplastic anemia (aAA) is an acquired deficiency of early hematopoietic cells, characterized by inadequate blood production, and a predisposition to myelodysplastic syndrome (MDS) and leukemia. Although its exact pathogenesis is unknown, aAA is thought to be driven by Human Leukocyte Antigen (HLA)-restricted T cell immunity, with earlier studies favoring HLA class II-mediated pathways. Using whole exome sequencing (WES), we recently identified two aAA patients with somatic mutations in HLA class I genes. We hypothesized that HLA class I mutations are pathognomonic for autoimmunity in aAA, but were previously underappreciated because the Major Histocompatibility Complex (MHC) region is notoriously difficult to analyze by WES. Using a combination of targeted deep sequencing of HLA class I genes and single nucleotide polymorphism array (SNP-A) genotyping we screened 66 aAA patients for somatic HLA class I loss. We found somatic HLA loss in eleven patients (17%), with thirteen loss-of-function mutations in HLA-A *33:03, HLA-A *68:01, HLA-B *14:02 and HLA-B *40:02 alleles. Three patients had more than one mutation targeting the same HLA allele. Interestingly, HLA-B *14:02 and HLA-B *40:02 were significantly overrepresented in aAA patients, compared to ethnicity-matched controls. Patients who inherited the targeted HLA alleles, regardless of HLA mutation status, had a more severe disease course with more frequent clonal complications as assessed by WES, SNP-A, and metaphase cytogenetics, and more frequent secondary MDS. The finding of recurrent HLA class I mutations provides compelling evidence for a predominant HLA class I-driven autoimmunity in aAA, and establishes a novel link between aAA patients' immunogenetics and clonal evolution.

  14. Mutations Related to Antiretroviral Resistance Identified by Ultra-Deep Sequencing in HIV-1 Infected Children under Structured Interruptions of HAART.

    Directory of Open Access Journals (Sweden)

    Jose Manuel Vazquez-Guillen

    Full Text Available Although Structured Treatment Interruptions (STI are currently not considered an alternative strategy for antiretroviral treatment, their true benefits and limitations have not been fully established. Some studies suggest the possibility of improving the quality of life of patients with this strategy; however, the information that has been obtained corresponds mostly to studies conducted in adults, with a lack of knowledge about its impact on children. Furthermore, mutations associated with antiretroviral resistance could be selected due to sub-therapeutic levels of HAART at each interruption period. Genotyping methods to determine the resistance profiles of the infecting viruses have become increasingly important for the management of patients under STI, thus low-abundance antiretroviral drug-resistant mutations (DRM's at levels under limit of detection of conventional genotyping (<20% of quasispecies could increase the risk of virologic failure. In this work, we analyzed the protease and reverse transcriptase regions of the pol gene by ultra-deep sequencing in pediatric patients under STI with the aim of determining the presence of high- and low-abundance DRM's in the viral rebounds generated by the STI. High-abundance mutations in protease and high- and low-abundance mutations in reverse transcriptase were detected but no one of these are directly associated with resistance to antiretroviral drugs. The results could suggest that the evaluated STI program is virologically safe, but strict and carefully planned studies, with greater numbers of patients and interruption/restart cycles, are still needed to evaluate the selection of DRM's during STI.

  15. Glioblastoma adaptation traced through decline of an IDH1 clonal driver and macro-evolution of a double-minute chromosome

    DEFF Research Database (Denmark)

    Favero, Francesco; McGranahan, Nicholas; Salm, Maximilian P.

    2015-01-01

    approaches. Methods: We describe a 35 year-old female patient with recurrent GBM following surgical removal of the primary tumor, adjuvant treatment with temozolomide, and a 3-year disease-free period. Rapid whole genome sequencing (WGS) of three separate tumour regions at recurrence was performed...... and interpreted relative to WGS of two regions of the primary tumour. Results We found extensive mutational and copy number heterogeneity within the primary tumour. We identified a TP53 mutation and two focal amplifications involving PDGFRA, KIT and CDK4, on chromosomes 4 and 12. A clonal IDH1 R132H mutation...

  16. Population genetics of Phytophthora infestans in Denmark reveals dominantly clonal populations and specific alleles linked to metalaxyl-M resistance

    DEFF Research Database (Denmark)

    Montes, M. S.; Nielsen, Bent Jørgen; Schmidt, S. G.

    2016-01-01

    population of P. infestans was characterized over the course of the 2013 growing season, as was the population genetic structure, using simple sequence repeat (SSR) genotypes and single nucleotide polymorphism (SNP)-based mitochondrial haplotyping of over 80 isolates. Both mating types A1 and A2 were present...... in most fields, but tests for recombination showed that clonal reproduction dominates in Danish populations. Genotype was not linked to haplotype and no differentiation was observed at the haplotype level, but rather between fields. Resistance phenotypes were linked to specific SSR alleles, demonstrating...

  17. Molecular epidemiology of multidrug-resistant Acinetobacter baumannii isolates in a university hospital in Nepal reveals the emergence of a novel epidemic clonal lineage.

    Science.gov (United States)

    Shrestha, Shovita; Tada, Tatsuya; Miyoshi-Akiyama, Tohru; Ohara, Hiroshi; Shimada, Kayo; Satou, Kazuhito; Teruya, Kuniko; Nakano, Kazuma; Shiroma, Akino; Sherchand, Jeevan Bdr; Rijal, Basista Psd; Hirano, Takashi; Kirikae, Teruo; Pokhrel, Bharat Mani

    2015-11-01

    The emergence of multidrug-resistant (MDR) Acinetobacter baumannii has become a serious medical problem worldwide. To clarify the genetic and epidemiological properties of MDR A. baumannii strains isolated from a medical setting in Nepal, 246 Acinetobacter spp. isolates obtained from different patients were screened for MDR A. baumannii by antimicrobial disk susceptibility testing. Whole genomes of the MDR A. baumannii isolates were sequenced by MiSeq™ (Illumina), and the complete genome of one isolate (IOMTU433) was sequenced by PacBio RS II. Phylogenetic trees were constructed from single nucleotide polymorphism concatemers. Multilocus sequence types were deduced and drug resistance genes were identified. Of the 246 Acinetobacter spp. isolates, 122 (49.6%) were MDR A. baumannii, with the majority being resistant to aminoglycosides, carbapenems and fluoroquinolones but not to colistin and tigecycline. These isolates harboured the 16S rRNA methylase gene armA as well as bla(NDM-1), bla(OXA-23) or bla(OXA-58). MDR A. baumannii isolates belonging to clonal complex 1 (CC1) and CC2 as well as a novel clonal complex (CC149) have spread throughout a medical setting in Nepal. The MDR isolates harboured genes encoding carbapenemases (OXA and NDM-1) and a 16S rRNA methylase (ArmA). Copyright © 2015 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved.

  18. HIV Prevention

    Centers for Disease Control (CDC) Podcasts

    2012-02-01

    Dr. Kevin Fenton, Director of CDC’s National Center for HIV/AIDS, Viral Hepatitis, STD, and TB Prevention, talks about steps people can take to protect their health from HIV.  Created: 2/1/2012 by National Center for HIV/AIDS, Viral Hepatitis, STD, and TB Prevention (NCHHSTP).   Date Released: 2/1/2012.

  19. Directional growth of a clonal bromeliad species in response to spatial habitat heterogeneity

    NARCIS (Netherlands)

    Sampaio, M.C.; Araujo, T.F.; Scarano, F.R.; Stuefer, J.F.

    2004-01-01

    Habitat selection by directional growth of plants has previously been investigated but field evidence for this phenomenon is extremely scarce. In this study we demonstrate directional clonal growth in Aechmea nudicaulis, a monocarpic, perennial bromeliad native to spatially heterogeneous sandy

  20. Screening of clonal chromosome aberrations present in A-bomb survivors by FISH method

    International Nuclear Information System (INIS)

    Nakano, Mimako; Kodama, Yoshiaki; Ito, Masahiro; Otaki, Kazuo; Nakamura, Nori

    1997-01-01

    Significance of FISH method for detection of clonal chromosome aberration was reviewed. A clonal chromosome aberration is derived from one abnormal cell clone and gives the influence on the frequency of the aberration. As well, the size and frequency of the aberration give an important information concerning lymphocyte kinetics. FISH method is meaningful for detection of the clonal aberration. Fifteen kinds of clonal aberrations were detected in A-bomb survivors, of which 10 were specifically detected by the method, indicating that its detection rate was 2-3 time as high as the ordinary method. The results were those on the DNA probe on no.1, no.2 and no.3 chromosomes, which consisting of about 23% of the genome. (K.H.)

  1. Implementation of Microfluidic Chip Electrophoresis for the Detection of B-cell Clonality

    Directory of Open Access Journals (Sweden)

    Vazan M

    2016-04-01

    Full Text Available Introduction: A clonal population of B-cells is defined as those cells arising from the mitotic division of a single somatic cell with the same rearrangement of immunoglobulin genes. This gives rise to DNA markers for each individual lymphoid cell and its progenies and enables us to study clonality in different B-cell malignancies using multiplex polymerase chain reaction - PCR. The BIOMED-2 protocol has been implemented for clonality detection in lymphoproliferative diseases and exploits multiplex PCR reaction, subsequently analyzed by heteroduplex analysis (HDA using polyacrylamide gel electrophoresis (PAGE. With the advent of miniaturization and automation of molecular biology methods, lab-on-chip technologies were developed and replace partially the conventional approaches. We tested device for microfluidic chip, which is used for B-cells clonality analysis, using a PCR reaction for three subregions called frameworks (FR of the immunoglobulin heavy locus (IGH gene.

  2. Clonal diversity analysis using SNP microarray: a new prognostic tool for chronic lymphocytic leukemia.

    Science.gov (United States)

    Zhang, Linsheng; Znoyko, Iya; Costa, Luciano J; Conlin, Laura K; Daber, Robert D; Self, Sally E; Wolff, Daynna J

    2011-12-01

    Chronic lymphocytic leukemia (CLL) is a clinically heterogeneous disease. The methods currently used for monitoring CLL and determining conditions for treatment are limited in their ability to predict disease progression, patient survival, and response to therapy. Although clonal diversity and the acquisition of new chromosomal abnormalities during the disease course (clonal evolution) have been associated with disease progression, their prognostic potential has been underappreciated because cytogenetic and fluorescence in situ hybridization (FISH) studies have a restricted ability to detect genomic abnormalities and clonal evolution. We hypothesized that whole genome analysis using high resolution single nucleotide polymorphism (SNP) microarrays would be useful to detect diversity and infer clonal evolution to offer prognostic information. In this study, we used the Infinium Omni1 BeadChip (Illumina, San Diego, CA) array for the analysis of genetic variation and percent mosaicism in 25 non-selected CLL patients to explore the prognostic value of the assessment of clonal diversity in patients with CLL. We calculated the percentage of mosaicism for each abnormality by applying a mathematical algorithm to the genotype frequency data and by manual determination using the Simulated DNA Copy Number (SiDCoN) tool, which was developed from a computer model of mosaicism. At least one genetic abnormality was identified in each case, and the SNP data was 98% concordant with FISH results. Clonal diversity, defined as the presence of two or more genetic abnormalities with differing percentages of mosaicism, was observed in 12 patients (48%), and the diversity correlated with the disease stage. Clonal diversity was present in most cases of advanced disease (Rai stages III and IV) or those with previous treatment, whereas 9 of 13 patients without detected clonal diversity were asymptomatic or clinically stable. In conclusion, SNP microarray studies with simultaneous evaluation

  3. Clonal diversity and fine-scale genetic structure in a high andean treeline population

    Czech Academy of Sciences Publication Activity Database

    Peng, Y.; Macek, P.; Macková, Jana; Romoleroux, K.; Hensen, I.

    2015-01-01

    Roč. 47, č. 1 (2015), s. 59-65 ISSN 0006-3606 Grant - others:GA AV ČR(CZ) IAA601110702; GA MŠk(CZ) LM2010009 Program:IA Institutional support: RVO:60077344 Keywords : AFLP * clonal diversity * clonal propagation * fine-scale genetic structure * Polylepis reticulata * treeline Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 1.944, year: 2015

  4. Clonal Evaluation of Prostate Cancer by ERG/SPINK1 Status to Improve Prognosis Prediction

    Science.gov (United States)

    2017-12-01

    19 NIH Exploiting drivers of androgen receptor signaling negative prostate cancer for precision medicine Goal(s): Identify novel potential drivers...AWARD NUMBER: W81XWH-14-1-0466 TITLE: Clonal evaluation of prostate cancer by ERG/SPINK1 status to improve prognosis prediction PRINCIPAL...Sept 2017 4. TITLE AND SUBTITLE 5a. CONTRACT NUMBER Clonal Evaluation of Prostate Cancer by ERG/SPINK1 Status to Improve Prognosis Prediction 5b

  5. Mutational Profiling Can Establish Clonal or Independent Origin in Synchronous Bilateral Breast and Other Tumors.

    Directory of Open Access Journals (Sweden)

    Lei Bao

    Full Text Available Synchronous tumors can be independent primary tumors or a primary-metastatic (clonal pair, which may have clinical implications. Mutational profiling of tumor DNA is increasingly common in the clinic. We investigated whether mutational profiling can distinguish independent from clonal tumors in breast and other cancers, using a carefully defined test based on the Clonal Likelihood Score (CLS = 100 x # shared high confidence (HC mutations/ # total HC mutations.Statistical properties of a formal test using the CLS were investigated. A high CLS is evidence in favor of clonality; the test is implemented as a one-sided binomial test of proportions. Test parameters were empirically determined using 16,422 independent breast tumor pairs and 15 primary-metastatic tumor pairs from 10 cancer types using The Cancer Genome Atlas.We validated performance of the test with its established parameters, using five published data sets comprising 15,758 known independent tumor pairs (maximum CLS = 4.1%, minimum p-value = 0.48 and 283 known tumor clonal pairs (minimum CLS 13%, maximum p-value 0.99, supporting independence. A plausible molecular mechanism for the shift from hormone receptor positive to triple negative was identified in the clonal pair.We have developed the statistical properties of a carefully defined Clonal Likelihood Score test from mutational profiling of tumor DNA. Under identified conditions, the test appears to reliably distinguish between synchronous tumors of clonal and of independent origin in several cancer types. This approach may have scientific and clinical utility.

  6. Evolutionary genomics and HIV restriction factors.

    Science.gov (United States)

    Pyndiah, Nitisha; Telenti, Amalio; Rausell, Antonio

    2015-03-01

    To provide updated insights into innate antiviral immunity and highlight prototypical evolutionary features of well characterized HIV restriction factors. Recently, a new HIV restriction factor, Myxovirus resistance 2, has been discovered and the region/residue responsible for its activity identified using an evolutionary approach. Furthermore, IFI16, an innate immunity protein known to sense several viruses, has been shown to contribute to the defense to HIV-1 by causing cell death upon sensing HIV-1 DNA. Restriction factors against HIV show characteristic signatures of positive selection. Different patterns of accelerated sequence evolution can distinguish antiviral strategies--offense or defence--as well as the level of specificity of the antiviral properties. Sequence analysis of primate orthologs of restriction factors serves to localize functional domains and sites responsible for antiviral action. We use recent discoveries to illustrate how evolutionary genomic analyses help identify new antiviral genes and their mechanisms of action.

  7. Rational development of radiopharmaceuticals for HIV-1

    International Nuclear Information System (INIS)

    Lau, Chuen-Yen; Maldarelli, Frank; Eckelman, William C.; Neumann, Ronald D.

    2014-01-01

    The global battle against HIV-1 would benefit from a sensitive and specific radiopharmaceutical to localize HIV-infected cells. Ideally, this probe would be able to identify latently infected host cells containing replication competent HIV sequences. Clinical and research applications would include assessment of reservoirs, informing clinical management by facilitating assessment of burden of infection in different compartments, monitoring disease progression and monitoring response to therapy. A “rational” development approach could facilitate efficient identification of an appropriate targeted radiopharmaceutical. Rational development starts with understanding characteristics of the disease that can be effectively targeted and then engineering radiopharmaceuticals to hone in on an appropriate target, which in the case of HIV-1 (HIV) might be an HIV-specific product on or in the host cell, a differentially expressed gene product, an integrated DNA sequence specific enzymatic activity, part of the inflammatory response, or a combination of these. This is different from the current approach that starts with a radiopharmaceutical for a target associated with a disease, mostly from autopsy studies, without a strong rationale for the potential to impact patient care. At present, no targeted therapies are available for HIV latency, although a number of approaches are under study. Here we discuss requirements for a radiopharmaceutical useful in strategies targeting persistently infected cells. The radiopharmaceutical for HIV should be developed based on HIV biology, studied in an animal model and then in humans, and ultimately used in clinical and research settings

  8. Phylogenetic Inference of HIV Transmission Clusters

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    Vlad Novitsky

    2017-10-01

    Full Text Available Better understanding the structure and dynamics of HIV transmission networks is essential for designing the most efficient interventions to prevent new HIV transmissions, and ultimately for gaining control of the HIV epidemic. The inference of phylogenetic relationships and the interpretation of results rely on the definition of the HIV transmission cluster. The definition of the HIV cluster is complex and dependent on multiple factors, including the design of sampling, accuracy of sequencing, precision of sequence alignment, evolutionary models, the phylogenetic method of inference, and specified thresholds for cluster support. While the majority of studies focus on clusters, non-clustered cases could also be highly informative. A new dimension in the analysis of the global and local HIV epidemics is the concept of phylogenetically distinct HIV sub-epidemics. The identification of active HIV sub-epidemics reveals spreading viral lineages and may help in the design of targeted interventions.HIVclustering can also be affected by sampling density. Obtaining a proper sampling density may increase statistical power and reduce sampling bias, so sampling density should be taken into account in study design and in interpretation of phylogenetic results. Finally, recent advances in long-range genotyping may enable more accurate inference of HIV transmission networks. If performed in real time, it could both inform public-health strategies and be clinically relevant (e.g., drug-resistance testing.

  9. Detectable clonal mosaicism and its relationship to aging and cancer

    Science.gov (United States)

    Jacobs, Kevin B; Yeager, Meredith; Zhou, Weiyin; Wacholder, Sholom; Wang, Zhaoming; Rodriguez-Santiago, Benjamin; Hutchinson, Amy; Deng, Xiang; Liu, Chenwei; Horner, Marie-Josephe; Cullen, Michael; Epstein, Caroline G; Burdett, Laurie; Dean, Michael C; Chatterjee, Nilanjan; Sampson, Joshua; Chung, Charles C; Kovaks, Joseph; Gapstur, Susan M; Stevens, Victoria L; Teras, Lauren T; Gaudet, Mia M; Albanes, Demetrius; Weinstein, Stephanie J; Virtamo, Jarmo; Taylor, Philip R; Freedman, Neal D; Abnet, Christian C; Goldstein, Alisa M; Hu, Nan; Yu, Kai; Yuan, Jian-Min; Liao, Linda; Ding, Ti; Qiao, You-Lin; Gao, Yu-Tang; Koh, Woon-Puay; Xiang, Yong-Bing; Tang, Ze-Zhong; Fan, Jin-Hu; Aldrich, Melinda C; Amos, Christopher; Blot, William J; Bock, Cathryn H; Gillanders, Elizabeth M; Harris, Curtis C; Haiman, Christopher A; Henderson, Brian E; Kolonel, Laurence N; Le Marchand, Loic; McNeill, Lorna H; Rybicki, Benjamin A; Schwartz, Ann G; Signorello, Lisa B; Spitz, Margaret R; Wiencke, John K; Wrensch, Margaret; Wu, Xifeng; Zanetti, Krista A; Ziegler, Regina G; Figueroa, Jonine D; Garcia-Closas, Montserrat; Malats, Nuria; Marenne, Gaelle; Prokunina-Olsson, Ludmila; Baris, Dalsu; Schwenn, Molly; Johnson, Alison; Landi, Maria Teresa; Goldin, Lynn; Consonni, Dario; Bertazzi, Pier Alberto; Rotunno, Melissa; Rajaraman, Preetha; Andersson, Ulrika; Freeman, Laura E Beane; Berg, Christine D; Buring, Julie E; Butler, Mary A; Carreon, Tania; Feychting, Maria; Ahlbom, Anders; Gaziano, J Michael; Giles, Graham G; Hallmans, Goran; Hankinson, Susan E; Hartge, Patricia; Henriksson, Roger; Inskip, Peter D; Johansen, Christoffer; Landgren, Annelie; McKean-Cowdin, Roberta; Michaud, Dominique S; Melin, Beatrice S; Peters, Ulrike; Ruder, Avima M; Sesso, Howard D; Severi, Gianluca; Shu, Xiao-Ou; Visvanathan, Kala; White, Emily; Wolk, Alicja; Zeleniuch-Jacquotte, Anne; Zheng, Wei; Silverman, Debra T; Kogevinas, Manolis; Gonzalez, Juan R; Villa, Olaya; Li, Donghui; Duell, Eric J; Risch, Harvey A; Olson, Sara H; Kooperberg, Charles; Wolpin, Brian M; Jiao, Li; Hassan, Manal; Wheeler, William; Arslan, Alan A; Bas Bueno-de-Mesquita, H; Fuchs, Charles S; Gallinger, Steven; Gross, Myron D; Holly, Elizabeth A; Klein, Alison P; LaCroix, Andrea; Mandelson, Margaret T; Petersen, Gloria; Boutron-Ruault, Marie-Christine; Bracci, Paige M; Canzian, Federico; Chang, Kenneth; Cotterchio, Michelle; Giovannucci, Edward L; Goggins, Michael; Bolton, Judith A Hoffman; Jenab, Mazda; Khaw, Kay-Tee; Krogh, Vittorio; Kurtz, Robert C; McWilliams, Robert R; Mendelsohn, Julie B; Rabe, Kari G; Riboli, Elio; Tjønneland, Anne; Tobias, Geoffrey S; Trichopoulos, Dimitrios; Elena, Joanne W; Yu, Herbert; Amundadottir, Laufey; Stolzenberg-Solomon, Rachael Z; Kraft, Peter; Schumacher, Fredrick; Stram, Daniel; Savage, Sharon A; Mirabello, Lisa; Andrulis, Irene L; Wunder, Jay S; García, Ana Patiño; Sierrasesúmaga, Luis; Barkauskas, Donald A; Gorlick, Richard G; Purdue, Mark; Chow, Wong-Ho; Moore, Lee E; Schwartz, Kendra L; Davis, Faith G; Hsing, Ann W; Berndt, Sonja I; Black, Amanda; Wentzensen, Nicolas; Brinton, Louise A; Lissowska, Jolanta; Peplonska, Beata; McGlynn, Katherine A; Cook, Michael B; Graubard, Barry I; Kratz, Christian P; Greene, Mark H; Erickson, Ralph L; Hunter, David J; Thomas, Gilles; Hoover, Robert N; Real, Francisco X; Fraumeni, Joseph F; Caporaso, Neil E; Tucker, Margaret; Rothman, Nathaniel; Pérez-Jurado, Luis A; Chanock, Stephen J

    2012-01-01

    In an analysis of 31,717 cancer cases and 26,136 cancer-free controls drawn from 13 genome-wide association studies (GWAS), we observed large chromosomal abnormalities in a subset of clones from DNA obtained from blood or buccal samples. Mosaic chromosomal abnormalities, either aneuploidy or copy-neutral loss of heterozygosity, of size >2 Mb were observed in autosomes of 517 individuals (0.89%) with abnormal cell proportions between 7% and 95%. In cancer-free individuals, the frequency increased with age; 0.23% under 50 and 1.91% between 75 and 79 (p=4.8×10−8). Mosaic abnormalities were more frequent in individuals with solid-tumors (0.97% versus 0.74% in cancer-free individuals, OR=1.25, p=0.016), with a stronger association for cases who had DNA collected prior to diagnosis or treatment (OR=1.45, p=0.0005). Detectable clonal mosaicism was common in individuals for whom DNA was collected at least one year prior to diagnosis of leukemia compared to cancer-free individuals (OR=35.4, p=3.8×10−11). These findings underscore the importance of the role and time-dependent nature of somatic events in the etiology of cancer and other late-onset diseases. PMID:22561519

  10. Clonal cell populations unresponsive to radiosensitization induced by telomerase inhibition

    International Nuclear Information System (INIS)

    Ju, Yeun-Jin; Shin, Hyun-Jin; Park, Jeong-Eun; Juhn, Kyoung-Mi; Woo, Seon Rang; Kim, Hee-Young; Han, Young-Hoon; Hwang, Sang-Gu; Hong, Sung-Hee; Kang, Chang-Mo; Yoo, Young-Do; Park, Won-Bong; Cho, Myung-Haing; Park, Gil Hong; Lee, Kee-Ho

    2010-01-01

    Research highlights: → In our present manuscript, we have clearly showed an interesting but problematic obstacle of a radiosensitization strategy based on telomerase inhibition by showing that: Clonal population unresponsive to this radiosensitization occasionally arise. → The telomere length of unsensitized clones was reduced, as was that of most sensitized clones. → The unsensitized clones did not show chromosome end fusion which was noted in all sensitized clones. → P53 status is not associated with the occurrence of unsensitized clone. → Telomere end capping in unsensitized clone is operative even under telomerase deficiency. -- Abstract: A combination of a radiotherapeutic regimen with telomerase inhibition is valuable when tumor cells are to be sensitized to radiation. Here, we describe cell clones unresponsive to radiosensitization after telomere shortening. After extensive division of individual transformed clones of mTERC -/- cells, about 22% of clones were unresponsive to radiosensitization even though telomerase action was inhibited. The telomere lengths of unsensitized mTERC -/- clones were reduced, as were those of most sensitized clones. However, the unsensitized clones did not exhibit chromosomal end-to-end fusion to the extent noted in all sensitized clones. Thus, a defense mechanism preventing telomere erosion is operative even when telomeres become shorter under conditions of telomerase deficiency, and results in unresponsiveness to the radiosensitization generally mediated by telomere shortening.

  11. Clonal dominance among T-lymphocyte infiltrates in arthritis

    International Nuclear Information System (INIS)

    Stamenkovic, I.; Stegagno, M.; Wright, K.A.; Krane, S.M.; Amento, E.P.; Colvin, R.B.; Duquesnoy, R.J.; Kurnick, J.T.

    1988-01-01

    Synovial membranes in patients with rheumatoid arthritis as well as other types of chronic destructive inflammatory arthritis contain infiltrates of activated T lymphocytes that probably contribute to the pathogenesis of the disease. In an effort to elucidate the nature of these infiltrates, interleukin 2 (IL-2)-responsive T lymphocytes were grown out of synovial fragments from 14 patients undergoing surgery for advanced destructive inflammatory joint disease. Eleven of the samples examined were from patients with classical rheumatoid arthritis, while three others were obtained from individuals with clinical osteoarthritis. Southern blot analysis of T-cell receptor (TCR) β-chain genes in 13 of 14 cultures showed distinct rearrangements, indicating that each culture was characterized by the predominance of a limited number of clones. T-cell populations from peripheral blood stimulated with a variety of activators and expanded with IL-2 did not demonstrate evidence of similar clonality in long-term culture. These results suggest that a limited number of activated T-cell clones predominate at the site of tissue injury in rheumatoid synovial membranes as well as in other types of destructive inflammatory joint disease. Further characterization of these T-cell clones may aid our understanding of the pathogenesis of these rheumatic disorders

  12. Clonal dominance and transplantation dynamics in hematopoietic stem cell compartments.

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    Peter Ashcroft

    2017-10-01

    Full Text Available Hematopoietic stem cells in mammals are known to reside mostly in the bone marrow, but also transitively passage in small numbers in the blood. Experimental findings have suggested that they exist in a dynamic equilibrium, continuously migrating between these two compartments. Here we construct an individual-based mathematical model of this process, which is parametrised using existing empirical findings from mice. This approach allows us to quantify the amount of migration between the bone marrow niches and the peripheral blood. We use this model to investigate clonal hematopoiesis, which is a significant risk factor for hematologic cancers. We also analyse the engraftment of donor stem cells into non-conditioned and conditioned hosts, quantifying the impact of different treatment scenarios. The simplicity of the model permits a thorough mathematical analysis, providing deeper insights into the dynamics of both the model and of the real-world system. We predict the time taken for mutant clones to expand within a host, as well as chimerism levels that can be expected following transplantation therapy, and the probability that a preconditioned host is reconstituted by donor cells.

  13. Plant Clonal Integration Mediates the Horizontal Redistribution of Soil Resources, Benefiting Neighboring Plants.

    Science.gov (United States)

    Ye, Xue-Hua; Zhang, Ya-Lin; Liu, Zhi-Lan; Gao, Shu-Qin; Song, Yao-Bin; Liu, Feng-Hong; Dong, Ming

    2016-01-01

    Resources such as water taken up by plants can be released into soils through hydraulic redistribution and can also be translocated by clonal integration within a plant clonal network. We hypothesized that the resources from one (donor) microsite could be translocated within a clonal network, released into different (recipient) microsites and subsequently used by neighbor plants in the recipient microsite. To test these hypotheses, we conducted two experiments in which connected and disconnected ramet pairs of Potentilla anserina were grown under both homogeneous and heterogeneous water regimes, with seedlings of Artemisia ordosica as neighbors. The isotopes [(15)N] and deuterium were used to trace the translocation of nitrogen and water, respectively, within the clonal network. The water and nitrogen taken up by P. anserina ramets in the donor microsite were translocated into the connected ramets in the recipient microsites. Most notably, portions of the translocated water and nitrogen were released into the recipient microsite and were used by the neighboring A. ordosica, which increased growth of the neighboring A. ordosica significantly. Therefore, our hypotheses were supported, and plant clonal integration mediated the horizontal hydraulic redistribution of resources, thus benefiting neighboring plants. Such a plant clonal integration-mediated resource redistribution in horizontal space may have substantial effects on the interspecific relations and composition of the community and consequently on ecosystem processes.

  14. Plant clonal integration mediates the horizontal redistribution of soil resources, benefiting neighbouring plants

    Directory of Open Access Journals (Sweden)

    Xuehua eYe

    2016-02-01

    Full Text Available Resources such as water taken up by plants can be released into soils through hydraulic redistribution and can also be translocated by clonal integration within a plant clonal network. We hypothesized that the resources from one (donor microsite could be translocated within a clonal network, released into different (recipient microsites and subsequently used by neighbour plants in the recipient microsite. To test these hypotheses, we conducted two experiments in which connected and disconnected ramet pairs of Potentilla anserina were grown under both homogeneous and heterogeneous water regimes, with seedlings of Artemisia ordosica as neighbours. The isotopes [15N] and deuterium were used to trace the translocation of nitrogen and water, respectively, within the clonal network. The water and nitrogen taken up by P. anserina ramets in the donor microsite were translocated into the connected ramets in the recipient microsites. Most notably, portions of the translocated water and nitrogen were released into the recipient microsite and were used by the neighbouring A. ordosica, which increased growth of the neighbouring A. ordosica significantly. Therefore, our hypotheses were supported, and plant clonal integration mediated the horizontal hydraulic redistribution of resources, thus benefiting neighbouring plants. Such a plant clonal integration-mediated resource redistribution in horizontal space may have substantial effects on the interspecific relations and composition of the community and consequently on ecosystem processes.

  15. A comparison of two measures of HIV diversity in multi-assay algorithms for HIV incidence estimation.

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    Matthew M Cousins

    Full Text Available Multi-assay algorithms (MAAs can be used to estimate HIV incidence in cross-sectional surveys. We compared the performance of two MAAs that use HIV diversity as one of four biomarkers for analysis of HIV incidence.Both MAAs included two serologic assays (LAg-Avidity assay and BioRad-Avidity assay, HIV viral load, and an HIV diversity assay. HIV diversity was quantified using either a high resolution melting (HRM diversity assay that does not require HIV sequencing (HRM score for a 239 base pair env region or sequence ambiguity (the percentage of ambiguous bases in a 1,302 base pair pol region. Samples were classified as MAA positive (likely from individuals with recent HIV infection if they met the criteria for all of the assays in the MAA. The following performance characteristics were assessed: (1 the proportion of samples classified as MAA positive as a function of duration of infection, (2 the mean window period, (3 the shadow (the time period before sample collection that is being assessed by the MAA, and (4 the accuracy of cross-sectional incidence estimates for three cohort studies.The proportion of samples classified as MAA positive as a function of duration of infection was nearly identical for the two MAAs. The mean window period was 141 days for the HRM-based MAA and 131 days for the sequence ambiguity-based MAA. The shadows for both MAAs were <1 year. Both MAAs provided cross-sectional HIV incidence estimates that were very similar to longitudinal incidence estimates based on HIV seroconversion.MAAs that include the LAg-Avidity assay, the BioRad-Avidity assay, HIV viral load, and HIV diversity can provide accurate HIV incidence estimates. Sequence ambiguity measures obtained using a commercially-available HIV genotyping system can be used as an alternative to HRM scores in MAAs for cross-sectional HIV incidence estimation.

  16. A Comparison of Two Measures of HIV Diversity in Multi-Assay Algorithms for HIV Incidence Estimation

    Science.gov (United States)

    Cousins, Matthew M.; Konikoff, Jacob; Sabin, Devin; Khaki, Leila; Longosz, Andrew F.; Laeyendecker, Oliver; Celum, Connie; Buchbinder, Susan P.; Seage, George R.; Kirk, Gregory D.; Moore, Richard D.; Mehta, Shruti H.; Margolick, Joseph B.; Brown, Joelle; Mayer, Kenneth H.; Kobin, Beryl A.; Wheeler, Darrell; Justman, Jessica E.; Hodder, Sally L.; Quinn, Thomas C.; Brookmeyer, Ron; Eshleman, Susan H.

    2014-01-01

    Background Multi-assay algorithms (MAAs) can be used to estimate HIV incidence in cross-sectional surveys. We compared the performance of two MAAs that use HIV diversity as one of four biomarkers for analysis of HIV incidence. Methods Both MAAs included two serologic assays (LAg-Avidity assay and BioRad-Avidity assay), HIV viral load, and an HIV diversity assay. HIV diversity was quantified using either a high resolution melting (HRM) diversity assay that does not require HIV sequencing (HRM score for a 239 base pair env region) or sequence ambiguity (the percentage of ambiguous bases in a 1,302 base pair pol region). Samples were classified as MAA positive (likely from individuals with recent HIV infection) if they met the criteria for all of the assays in the MAA. The following performance characteristics were assessed: (1) the proportion of samples classified as MAA positive as a function of duration of infection, (2) the mean window period, (3) the shadow (the time period before sample collection that is being assessed by the MAA), and (4) the accuracy of cross-sectional incidence estimates for three cohort studies. Results The proportion of samples classified as MAA positive as a function of duration of infection was nearly identical for the two MAAs. The mean window period was 141 days for the HRM-based MAA and 131 days for the sequence ambiguity-based MAA. The shadows for both MAAs were cross-sectional HIV incidence estimates that were very similar to longitudinal incidence estimates based on HIV seroconversion. Conclusions MAAs that include the LAg-Avidity assay, the BioRad-Avidity assay, HIV viral load, and HIV diversity can provide accurate HIV incidence estimates. Sequence ambiguity measures obtained using a commercially-available HIV genotyping system can be used as an alternative to HRM scores in MAAs for cross-sectional HIV incidence estimation. PMID:24968135

  17. The global transmission network of HIV-1.

    Science.gov (United States)

    Wertheim, Joel O; Leigh Brown, Andrew J; Hepler, N Lance; Mehta, Sanjay R; Richman, Douglas D; Smith, Davey M; Kosakovsky Pond, Sergei L

    2014-01-15

    Human immunodeficiency virus type 1 (HIV-1) is pandemic, but its contemporary global transmission network has not been characterized. A better understanding of the properties and dynamics of this network is essential for surveillance, prevention, and eventual eradication of HIV. Here, we apply a simple and computationally efficient network-based approach to all publicly available HIV polymerase sequences in the global database, revealing a contemporary picture of the spread of HIV-1 within and between countries. This approach automatically recovered well-characterized transmission clusters and extended other clusters thought to be contained within a single country across international borders. In addition, previously undescribed transmission clusters were discovered. Together, these clusters represent all known modes of HIV transmission. The extent of international linkage revealed by our comprehensive approach demonstrates the need to consider the global diversity of HIV, even when describing local epidemics. Finally, the speed of this method allows for near-real-time surveillance of the pandemic's progression.

  18. IN-MACA-MCC: Integrated Multiple Attractor Cellular Automata with Modified Clonal Classifier for Human Protein Coding and Promoter Prediction

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    Kiran Sree Pokkuluri

    2014-01-01

    Full Text Available Protein coding and promoter region predictions are very important challenges of bioinformatics (Attwood and Teresa, 2000. The identification of these regions plays a crucial role in understanding the genes. Many novel computational and mathematical methods are introduced as well as existing methods that are getting refined for predicting both of the regions separately; still there is a scope for improvement. We propose a classifier that is built with MACA (multiple attractor cellular automata and MCC (modified clonal classifier to predict both regions with a single classifier. The proposed classifier is trained and tested with Fickett and Tung (1992 datasets for protein coding region prediction for DNA sequences of lengths 54, 108, and 162. This classifier is trained and tested with MMCRI datasets for protein coding region prediction for DNA sequences of lengths 252 and 354. The proposed classifier is trained and tested with promoter sequences from DBTSS (Yamashita et al., 2006 dataset and nonpromoters from EID (Saxonov et al., 2000 and UTRdb (Pesole et al., 2002 datasets. The proposed model can predict both regions with an average accuracy of 90.5% for promoter and 89.6% for protein coding region predictions. The specificity and sensitivity values of promoter and protein coding region predictions are 0.89 and 0.92, respectively.

  19. Population Genomic Analysis of 1,777 Extended-Spectrum Beta-Lactamase-Producing Klebsiella pneumoniae Isolates, Houston, Texas: Unexpected Abundance of Clonal Group 307.

    Science.gov (United States)

    Long, S Wesley; Olsen, Randall J; Eagar, Todd N; Beres, Stephen B; Zhao, Picheng; Davis, James J; Brettin, Thomas; Xia, Fangfang; Musser, James M

    2017-05-16

    Klebsiella pneumoniae is a major human pathogen responsible for high morbidity and mortality rates. The emergence and spread of strains resistant to multiple antimicrobial agents and documented large nosocomial outbreaks are especially concerning. To develop new therapeutic strategies for K. pneumoniae , it is imperative to understand the population genomic structure of strains causing human infections. To address this knowledge gap, we sequenced the genomes of 1,777 extended-spectrum beta-lactamase-producing K. pneumoniae strains cultured from patients in the 2,000-bed Houston Methodist Hospital system between September 2011 and May 2015, representing a comprehensive, population-based strain sample. Strains of largely uncharacterized clonal group 307 (CG307) caused more infections than those of well-studied epidemic CG258. Strains varied markedly in gene content and had an extensive array of small and very large plasmids, often containing antimicrobial resistance genes. Some patients with multiple strains cultured over time were infected with genetically distinct clones. We identified 15 strains expressing the New Delhi metallo-beta-lactamase 1 (NDM-1) enzyme that confers broad resistance to nearly all beta-lactam antibiotics. Transcriptome sequencing analysis of 10 phylogenetically diverse strains showed that the global transcriptome of each strain was unique and highly variable. Experimental mouse infection provided new information about immunological parameters of host-pathogen interaction. We exploited the large data set to develop whole-genome sequence-based classifiers that accurately predict clinical antimicrobial resistance for 12 of the 16 antibiotics tested. We conclude that analysis of large, comprehensive, population-based strain samples can assist understanding of the molecular diversity of these organisms and contribute to enhanced translational research. IMPORTANCE Klebsiella pneumoniae causes human infections that are increasingly difficult to

  20. Acinetobacter baumannii in intensive care unit: A novel system to study clonal relationship among the isolates

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    Leonardis Francesca

    2008-06-01

    Full Text Available Abstract Background The nosocomial infections surveillance system must be strongly effective especially in highly critic areas, such as Intensive Care Units (ICU. These areas are frequently an epidemiological epicentre for transmission of multi-resistant pathogens, like Acinetobacter baumannii. As an epidemic outbreak occurs it is very important to confirm or exclude the genetic relationship among the isolates in a short time. There are several molecular typing systems used with this aim. The Repetitive sequence-based PCR (REP-PCR has been recognized as an effective method and it was recently adapted to an automated format known as the DiversiLab system. Methods In the present study we have evaluated the combination of a newly introduced software package for the control of hospital infection (VIGI@ct with the DiversiLab system. In order to evaluate the reliability of the DiversiLab its results were also compared with those obtained using f-AFLP. Results The combination of VIGI@ct and DiversiLab enabled an earlier identification of an A. baumannii epidemic cluster, through the confirmation of the genetic relationship among the isolates. This cluster regards 56 multi-drug-resistant A. baumannii isolates from several specimens collected from 13 different patients admitted to the ICU in a ten month period. The A. baumannii isolates were clonally related being their similarity included between 97 and 100%. The results of the DiversiLab were confirmed by f-AFLP analysis. Conclusion The early identification of the outbreak has led to the prompt application of operative procedures and precautions to avoid the spread of pathogen. To date, 6 months after the last A. baumannii isolate, no other related case has been identified.

  1. Characterization and clonality of prelymphoma cells of B10 mice treated with fractionated X-irradiation (FX)

    International Nuclear Information System (INIS)

    Muto, M.; Kubo, E.; Sado, T.; Shimizu, T.; Yamagishi, H.

    1992-01-01

    With a combined use of cell separation by cell sorter and intrathymic injection assay, it was shown that prelymphoma cells existed in the subpopulation of thymocytes expressing TL-2 antigen which is not expressed on normal thymocytes of B10. Thy 1.2 or B10. Thy 1.1 mice. We then addressed a question whether all TL-2 + cells undergo neoplastic initiation or pre-neoplastic cells develop infrequently from TL-2 + cells. To investigate this problem and to examine the clonality of prelymphoma cells, thymocytes from individual B10. Thy 1.1 mice at various times after FX were stained with anti TL-2 mAb and the content of TL-2 + cells was evaluated. A graded amount of TL-2 + thymocytes from individual mice was injected into the thymuses of B10. Thy 1.2 mice. Although various numbers of TL-2 + cells appeared in the thymus of individual mice 14 - 28 days after FX, the donor type T cell lymphomas developed when 10 2 - 10 5 of TL-2 + cells from 7 individuals out of 20 mice were injected into the recipient mice. On the other hand, injection of TL-2 + cells from other mice (13 out of 20) did not develop donor type T cell lymphoma in spite of TL-2 + cells appearing in the thymus. These results indicate that all TL-2 + cells did not always undergo neoplastic initiation, and prelymphoma cells might develop infrequently from TL-2 + cells. To evaluate the clonality of prelymphoma cells, high molecular weight DNAs were isolated from the donor-derived T cell lymphomas and the rearrangement of T cell receptors examined by Southern blot analysis. The nucleotide sequences of V-J junctions were also determined by polymerase chain reaction techniques. The results indicated that after irradiation neoplastic initiation might occur oligoclonally in some of the TL-2 + cells. (author)

  2. Clonality and α-a recombination in the Australian Cryptococcus gattii VGII population--an emerging outbreak in Australia.

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    Fabian Carriconde

    Full Text Available BACKGROUND: Cryptococcus gattii is a basidiomycetous yeast that causes life-threatening disease in humans and animals. Within C. gattii, four molecular types are recognized (VGI to VGIV. The Australian VGII population has been in the spotlight since 2005, when it was suggested as the possible origin for the ongoing outbreak at Vancouver Island (British Columbia, Canada, with same-sex mating being suggested as the driving force behind the emergence of this outbreak, and is nowadays hypothesized as a widespread phenomenon in C. gattii. However, an in-depth characterization of the Australian VGII population is still lacking. The present work aimed to define the genetic variability within the Australian VGII population and determine processes shaping its population structure. METHODOLOGY/PRINCIPAL FINDINGS: A total of 54 clinical, veterinary and environmental VGII isolates from different parts of the Australian continent were studied. To place the Australian population in a global context, 17 isolates from North America, Europe, Asia and South America were included. Genetic variability was assessed using the newly adopted international consensus multi-locus sequence typing (MLST scheme, including seven genetic loci: CAP59, GPD1, LAC1, PLB1, SOD1, URA5 and IGS1. Despite the overall clonality observed, the presence of MATa VGII isolates in Australia was demonstrated for the first time in association with recombination in MATα-MATa populations. Our results also support the hypothesis of a "smouldering" outbreak throughout the Australian continent, involving a limited number of VGII genotypes, which is possibly caused by a founder effect followed by a clonal expansion. CONCLUSIONS/SIGNIFICANCE: The detection of sexual recombination in MATα-MATa population in Australia is in accordance with the natural life cycle of C. gattii involving opposite mating types and presents an alternative to the same-sex mating strategy suggested elsewhere. The potential

  3. Adult T-cell leukemia: molecular basis for clonal expansion and transformation of HTLV-1-infected T cells.

    Science.gov (United States)

    Watanabe, Toshiki

    2017-03-02

    Adult T-cell leukemia (ATL) is an aggressive T-cell malignancy caused by human T-cell leukemia virus type 1 (HTLV-1) that develops through a multistep carcinogenesis process involving 5 or more genetic events. We provide a comprehensive overview of recently uncovered information on the molecular basis of leukemogenesis in ATL. Broadly, the landscape of genetic abnormalities in ATL that include alterations highly enriched in genes for T-cell receptor-NF-κB signaling such as PLCG1 , PRKCB , and CARD11 and gain-of function mutations in CCR4 and CCR7 Conversely, the epigenetic landscape of ATL can be summarized as polycomb repressive complex 2 hyperactivation with genome-wide H3K27 me3 accumulation as the basis of the unique transcriptome of ATL cells. Expression of H3K27 methyltransferase enhancer of zeste 2 was shown to be induced by HTLV-1 Tax and NF-κB. Furthermore, provirus integration site analysis with high-throughput sequencing enabled the analysis of clonal composition and cell number of each clone in vivo, whereas multicolor flow cytometric analysis with CD7 and cell adhesion molecule 1 enabled the identification of HTLV-1-infected CD4 + T cells in vivo. Sorted immortalized but untransformed cells displayed epigenetic changes closely overlapping those observed in terminally transformed ATL cells, suggesting that epigenetic abnormalities are likely earlier events in leukemogenesis. These new findings broaden the scope of conceptualization of the molecular mechanisms of leukemogenesis, dissecting them into immortalization and clonal progression. These recent findings also open a new direction of drug development for ATL prevention and treatment because epigenetic marks can be reprogrammed. Mechanisms underlying initial immortalization and progressive accumulation of these abnormalities remain to be elucidated. © 2017 by The American Society of Hematology.

  4. Assessment of clonal fidelity of Tylophora indica (Burm. f.) Merrill "in vitro" plantlets by ISSR molecular markers.

    Science.gov (United States)

    Sharma, Madan Mohan; Verma, Roop Narayan; Singh, Abhijeet; Batra, Amla

    2014-01-01

    Tylophora indica Burm F. Merrill. is widely used against various diseases owing to the presence an array of medicinally important secondary metabolites. Its stem is bitter, stomachic, stimulates bile secretion, enriches the blood and cures diseases like diabetes, fever, flatulence, hypertension, jaundice, leucorrhoea, urinary disease and upper respiratory tract infection. It is neglected for tissue culture work because of deciduous nature of climbing shrub, facing problems for micropropagation. Hence, in vitro regeneration of complete plantlets was done through indirect organogenesis in Tylophora indica. Calli were produced from in vivo leaves of T. indica on MS medium supplemented with 6-Benzylaminopurine (BAP: 2.0 mg l(-1)) and Indole-3-butyric acid (IBA: 0.5 mg l(-1)). The multiple shoots (12.00 ± 1.50) emerged and elongated on MS medium fortified with Thidiazuron (TDZ: 0.1 mg l(-1)). They were rooted on half strength MS medium having IBA (0.5 mg l(-1)) (7.75 ± 0.25) after 20 days of sub-culturing followed by hardening and acclimatization. During indirect regeneration of plants, chances of somaclonal variations may arise. These variations should be identified to produce true to type plants. Plantlets raised through tissue culture were used to validate the clonal fidelity through Inter simple sequence repeat markers (ISSR). Clonal fidelity is a major consideration in commercial micropropagation using in vitro tissue culture methods. During the study, total 71 clear and distinct bands were produced using 6 primers. The banding pattern of each primer was uniform and comparable to mother plant and showed about 93% homology using un-weighted pair group method with arithmetic averaging (UPGMA). ISSR analysis confirmed the genetic stability of in vitro raised plants.

  5. HIV/AIDS Coinfection

    Science.gov (United States)

    ... Coinfection Hepatitis C Coinfection HIV/AIDS Coinfection HIV/AIDS Coinfection Approximately 10% of the HIV-infected population ... Control and Prevention website to learn about HIV/AIDS and Viral Hepatitis guidelines and resources. Home About ...

  6. HIV/AIDS

    Science.gov (United States)

    HIV stands for human immunodeficiency virus. It harms your immune system by destroying the white blood cells ... It is the final stage of infection with HIV. Not everyone with HIV develops AIDS. HIV most ...

  7. HIV and Immunizations

    Science.gov (United States)

    ... AIDS Drugs Clinical Trials Apps skip to content HIV Treatment Home Understanding HIV/AIDS Fact Sheets HIV ... 4 p.m. ET) Send us an email HIV and Immunizations Last Reviewed: February 6, 2018 Key ...

  8. HIV Medication Adherence

    Science.gov (United States)

    ... AIDS Drugs Clinical Trials Apps skip to content HIV Treatment Home Understanding HIV/AIDS Fact Sheets HIV ... 4 p.m. ET) Send us an email HIV Medication Adherence Last Reviewed: January 17, 2018 Key ...

  9. HIV and AIDS

    Science.gov (United States)

    ... Staying Safe Videos for Educators Search English Español HIV and AIDS KidsHealth / For Kids / HIV and AIDS ... actually the virus that causes the disease AIDS. HIV Hurts the Immune System People who are HIV ...

  10. HIV Treatment: The Basics

    Science.gov (United States)

    ... AIDS Drugs Clinical Trials Apps skip to content HIV Treatment Home Understanding HIV/AIDS Fact Sheets HIV ... 4 p.m. ET) Send us an email HIV Treatment: The Basics Last Reviewed: March 22, 2018 ...

  11. HIV and Pregnancy

    Science.gov (United States)

    ... Management Education & Events Advocacy For Patients About ACOG HIV and Pregnancy Home For Patients Search FAQs HIV ... HIV and Pregnancy FAQ113, July 2017 PDF Format HIV and Pregnancy Pregnancy What is human immunodeficiency virus ( ...

  12. HIV and Cardiovascular Disease

    Science.gov (United States)

    ... Select a Language: Fact Sheet 652 HIV and Cardiovascular Disease HIV AND CARDIOVASCULAR DISEASE WHY SHOULD PEOPLE WITH HIV CARE ABOUT CVD? ... OF CVD? WHAT ABOUT CHANGING MEDICATIONS? HIV AND CARDIOVASCULAR DISEASE Cardiovascular disease (CVD) includes a group of problems ...

  13. HIV-1 molecular epidemiology among newly diagnosed HIV-1 individuals in Hebei, a low HIV prevalence province in China.

    Directory of Open Access Journals (Sweden)

    Xinli Lu

    Full Text Available New human immunodeficiency virus type 1 (HIV-1 diagnoses are increasing rapidly in Hebei. The aim of this study presents the most extensive HIV-1 molecular epidemiology investigation in Hebei province in China thus far. We have carried out the most extensive systematic cross-sectional study based on newly diagnosed HIV-1 positive individuals in 2013, and characterized the molecular epidemiology of HIV-1 based on full length gag-partial pol gene sequences in the whole of Hebei. Nine HIV-1 genotypes based on full length gag-partial pol gene sequence were identified among 610 newly diagnosed naïve individuals. The four main genotypes were circulating recombinant form (CRF01_AE (53.4%, CRF07_BC (23.4%, subtype B (15.9%, and unique recombinant forms URFs (4.9%. Within 1 year, three new genotypes (subtype A1, CRF55_01B, CRF65_cpx, unknown before in Hebei, were first found among men who have sex with men (MSM. All nine genotypes were identified in the sexually contracted HIV-1 population. Among 30 URFs, six recombinant patterns were revealed, including CRF01_AE/BC (40.0%, CRF01_AE/B (23.3%, B/C (16.7%, CRF01_AE/C (13.3%, CRF01_AE/B/A2 (3.3% and CRF01_AE/BC/A2 (3.3%, plus two potential CRFs. This study elucidated the complicated characteristics of HIV-1 molecular epidemiology in a low HIV-1 prevalence northern province of China and revealed the high level of HIV-1 genetic diversity. All nine HIV-1 genotypes circulating in Hebei have spread out of their initial risk groups into the general population through sexual contact, especially through MSM. This highlights the urgency of HIV prevention and control in China.

  14. HIV-1 molecular epidemiology among newly diagnosed HIV-1 individuals in Hebei, a low HIV prevalence province in China.

    Science.gov (United States)

    Lu, Xinli; Kang, Xianjiang; Liu, Yongjian; Cui, Ze; Guo, Wei; Zhao, Cuiying; Li, Yan; Chen, Suliang; Li, Jingyun; Zhang, Yuqi; Zhao, Hongru

    2017-01-01

    New human immunodeficiency virus type 1 (HIV-1) diagnoses are increasing rapidly in Hebei. The aim of this study presents the most extensive HIV-1 molecular epidemiology investigation in Hebei province in China thus far. We have carried out the most extensive systematic cross-sectional study based on newly diagnosed HIV-1 positive individuals in 2013, and characterized the molecular epidemiology of HIV-1 based on full length gag-partial pol gene sequences in the whole of Hebei. Nine HIV-1 genotypes based on full length gag-partial pol gene sequence were identified among 610 newly diagnosed naïve individuals. The four main genotypes were circulating recombinant form (CRF)01_AE (53.4%), CRF07_BC (23.4%), subtype B (15.9%), and unique recombinant forms URFs (4.9%). Within 1 year, three new genotypes (subtype A1, CRF55_01B, CRF65_cpx), unknown before in Hebei, were first found among men who have sex with men (MSM). All nine genotypes were identified in the sexually contracted HIV-1 population. Among 30 URFs, six recombinant patterns were revealed, including CRF01_AE/BC (40.0%), CRF01_AE/B (23.3%), B/C (16.7%), CRF01_AE/C (13.3%), CRF01_AE/B/A2 (3.3%) and CRF01_AE/BC/A2 (3.3%), plus two potential CRFs. This study elucidated the complicated characteristics of HIV-1 molecular epidemiology in a low HIV-1 prevalence northern province of China and revealed the high level of HIV-1 genetic diversity. All nine HIV-1 genotypes circulating in Hebei have spread out of their initial risk groups into the general population through sexual contact, especially through MSM. This highlights the urgency of HIV prevention and control in China.

  15. Virtually full-length subtype F and F/D recombinant HIV-1 from Africa and South America

    NARCIS (Netherlands)

    Laukkanen, T.; Carr, J. K.; Janssens, W.; Liitsola, K.; Gotte, D.; McCutchan, F. E.; Op de Coul, E.; Cornelissen, M.; Heyndrickx, L.; van der Groen, G.; Salminen, M. O.

    2000-01-01

    For reliable classification of HIV-1 strains appropriate reference sequences are needed. The HIV-1 genetic subtype F has a wide geographic spread, causing significant epidemics in South America, Africa, and some regions of Europe. Previously only two full-length sequences of each of the HIV-1

  16. Next-Generation Sequencing of Antibody Display Repertoires

    Directory of Open Access Journals (Sweden)

    Romain Rouet

    2018-02-01

    Full Text Available In vitro selection technology has transformed the development of therapeutic monoclonal antibodies. Using methods such as phage, ribosome, and yeast display, high affinity binders can be selected from diverse repertoires. Here, we review strategies for the next-generation sequencing (NGS of phage- and other antibody-display libraries, as well as NGS platforms and analysis tools. Moreover, we discuss recent examples relating to the use of NGS to assess library diversity, clonal enrichment, and affinity maturation.

  17. HIV/AIDS - Multiple Languages

    Science.gov (United States)

    ... HIV - Newly diagnosed with HIV, part 5 - English MP3 Children and HIV - Newly diagnosed with HIV, part 5 - 简体中文 (Chinese, Simplified (Mandarin dialect)) MP3 Children and HIV - Newly diagnosed with HIV, part ...

  18. Basic HIV/AIDS Statistics

    Science.gov (United States)

    ... HIV Syndicated Content Website Feedback HIV/AIDS Basic Statistics Recommend on Facebook Tweet Share Compartir HIV and ... HIV. Interested in learning more about CDC's HIV statistics? Terms, Definitions, and Calculations Used in CDC HIV ...

  19. Characterization of HIV Transmission in South-East Austria.

    Science.gov (United States)

    Hoenigl, Martin; Chaillon, Antoine; Kessler, Harald H; Haas, Bernhard; Stelzl, Evelyn; Weninger, Karin; Little, Susan J; Mehta, Sanjay R

    2016-01-01

    To gain deeper insight into the epidemiology of HIV-1 transmission in South-East Austria we performed a retrospective analysis of 259 HIV-1 partial pol sequences obtained from unique individuals newly diagnosed with HIV infection in South-East Austria from 2008 through 2014. After quality filtering, putative transmission linkages were inferred when two sequences were ≤1.5% genetically different. Multiple linkages were resolved into putative transmission clusters. Further phylogenetic analyses were performed using BEAST v1.8.1. Finally, we investigated putative links between the 259 sequences from South-East Austria and all publicly available HIV polymerase sequences in the Los Alamos National Laboratory HIV sequence database. We found that 45.6% (118/259) of the sampled sequences were genetically linked with at least one other sequence from South-East Austria forming putative transmission clusters. Clustering individuals were more likely to be men who have sex with men (MSM; pAustria had at least one putative inferred linkage with sequences from a total of 69 other countries. In conclusion, analysis of HIV-1 sequences from newly diagnosed individuals residing in South-East Austria revealed a high degree of national and international clustering mainly within MSM. Interestingly, we found that a high number of heterosexual males clustered within MSM networks, suggesting either linkage between risk groups or misrepresentation of sexual risk behaviors by subjects.

  20. Living with HIV

    Science.gov (United States)

    ... Abroad Treatment Basic Statistics Get Tested Find an HIV testing site near you. Enter ZIP code or city Follow HIV/AIDS CDC HIV CDC HIV/AIDS See RSS | ... Syndicated Content Website Feedback HIV/AIDS Living With HIV Language: English (US) Español (Spanish) Recommend on Facebook ...

  1. HIV Risk and Prevention

    Science.gov (United States)

    ... Prevention VIH En Español Get Tested Find an HIV testing site near you. Enter ZIP code or city Follow HIV/AIDS CDC HIV CDC HIV/AIDS See RSS | ... Email Updates on HIV Syndicated Content Website Feedback HIV Risk and Prevention Recommend on Facebook Tweet Share ...

  2. [Lymphocytic Clonal Expansion in Adult Patients with Epstein-Barr Virus-Associated Lymphoproliferative Disease].

    Science.gov (United States)

    Zhong, Feng-Luan; Zhang, Hong-Yu; Zhang, Qian; Feng, Jia; Zhang, Wen-Li; Xu, Lei; Xu, Hai-Chan; Wen, Juan-Juan; Meng, Qing-Xiang

    2017-12-01

    To explore the lymphocytic clonal expansion in adult patients with Epstein-Barr virus-associated lymphoproliferative diseases (EBV+LPD), and to investigate the experimental methods for EBV+LPD cells so as to provide a more objective measure for the diagnosis, classification and prognosis in the early stage of this disease. Peripheral blood samples from 5 patients with EBV+LPD, 4 patients with adult infectious mononucleosis(IM) as negative control and 3 patients with acute NK-cell leukemia(ANKL) as positive control were collected. Prior to immunochemotherapy, viral loads and clonality were analysed by flow cytometry (FCM), T cell receptor gene rearrangement (TCR) was detected by real-time polymerase chain reaction (RT-PCR), and diversity of EB virus terminal repeat (EBV-TR) was detected by Southern blot. FCM showed only 1 case with clonal TCRVβ in 5 patients with EBV+LPD, TCR clonal expansion could be detected both in patients with IM(4 of 4) and 4 patients with EBV+LPD(4 of 5), Out of patients with EBV+LPD, 1 patient displayed a monoclonal band and 2 patients showed oligoclonal bands when detecting EBV-TR by southen blot. Detecting the diversity of EBV-TR by Southern blot may be the most objective way to reflex clonal transformation of EBV+LPD, which is of great benefit to the diagnosis, classification and prognosis in the early stage of this disease.

  3. HIV-1-Specific IgA Monoclonal Antibodies from an HIV-1 Vaccinee Mediate Galactosylceramide Blocking and Phagocytosis

    Science.gov (United States)

    2018-01-01

    ABSTRACT Vaccine-elicited humoral immune responses comprise an array of antibody forms and specificities, with only a fraction contributing to protective host immunity. Elucidation of antibody effector functions responsible for protective immunity against human immunodeficiency virus type 1 (HIV-1) acquisition is a major goal for the HIV-1 vaccine field. Immunoglobulin A (IgA) is an important part of the host defense against pathogens; however, little is known about the role of vaccine-elicited IgA and its capacity to mediate antiviral functions. To identify the antiviral functions of HIV-1-specific IgA elicited by vaccination, we cloned HIV-1 envelope-specific IgA monoclonal antibodies (MAbs) by memory B cell cultures from peripheral blood mononuclear cells from an RV144 vaccinee and produced two IgA clonal cell lines (HG129 and HG130) producing native, nonrecombinant IgA MAbs. The HG129 and HG130 MAbs mediated phagocytosis by monocytes, and HG129 blocked HIV-1 Env glycoprotein binding to galactosylceramide, an alternative HIV-1 receptor. These findings elucidate potential antiviral functions of vaccine-elicited HIV-1 envelope-specific IgA that may act to block HIV-1 acquisition at the portal of entry by preventing HIV-1 binding to galactosylceramide and mediating antibody Fc receptor-mediated virion phagocytosis. Furthermore, these findings highlight the complex and diverse interactions of vaccine-elicited IgA with pathogens that depend on IgA fine specificity and form (e.g., multimeric or monomeric) in the systemic circulation and mucosal compartments. IMPORTANCE Host-pathogen interactions in vivo involve numerous immune mechanisms that can lead to pathogen clearance. Understanding the nature of antiviral immune mechanisms can inform the design of efficacious HIV-1 vaccine strategies. Evidence suggests that both neutralizing and nonneutralizing antibodies can mediate some protection against HIV in animal models. Although numerous studies have characterized the

  4. Novel Method To Identify Source-Associated Phylogenetic Clustering Shows that Listeria monocytogenes Includes Niche-Adapted Clonal Groups with Distinct Ecological Preferences

    DEFF Research Database (Denmark)

    Nightingale, K. K.; Lyles, K.; Ayodele, M.

    2006-01-01

    population are identified (TreeStats test). Analysis of sequence data for 120 L. monocytogenes isolates revealed evidence of clustering between isolates from the same source, based on the phylogenies inferred from actA and inlA (P = 0.02 and P = 0.07, respectively; SourceCluster test). Overall, the Tree...... are biologically valid. Overall, our data show that (i) the SourceCluster and TreeStats tests can identify biologically meaningful source-associated phylogenetic clusters and (ii) L. monocytogenes includes clonal groups that have adapted to infect specific host species or colonize nonhost environments......., including humans, animals, and food. If the null hypothesis that the genetic distances for isolates within and between source populations are identical can be rejected (SourceCluster test), then particular clades in the phylogenetic tree with significant overrepresentation of sequences from a given source...

  5. Clustering patterns of cytotoxic T-lymphocyte epitopes in human immunodeficiency virus type 1 (HIV-1) proteins reveal imprints of immune evasion on HIV-1 global variation

    DEFF Research Database (Denmark)

    Yusim, K.; Kesmir, Can; Gaschen, B.

    2002-01-01

    The human cytotoxic T-lymphocyte (CTL) response to human immunodeficiency virus type 1 (HIV-1) has been intensely studied, and hundreds of CTL epitopes have been experimentally defined, published, and compiled in the HIV Molecular Immunology Database. Maps of CTL epitopes on HIV-1 protein sequenc...

  6. Clonal Dissemination of Extended-Spectrum β-Lactamase (ESBL)-Producing Klebsiella pneumoniae Isolates in a Korean Hospital

    Science.gov (United States)

    Ko, Kwan Soo; Yeom, Joon-Sup; Lee, Mi Young; Peck, Kyong Ran

    2008-01-01

    In this study, we investigated the molecular characteristics of extended-spectrum β-lactamase (ESBL)-producing Klebsiella pneumoniae isolates that were recovered from an outbreak in a Korean hospital. A new multilocus sequence typing (MLST) scheme for K. pneumoniae based on five housekeeping genes was developed and was evaluated for 43 ESBL-producing isolates from an outbreak as well as 38 surveillance isolates from Korea and also a reference strain. Overall, a total of 37 sequence types (STs) and six clonal complexes (CCs) were identified among the 82 K. pneumoniae isolates. The result of MLST analysis was concordant with that of pulsedfield gel electrophoresis. Most of the outbreak isolates belonged to a certain clone (ST2), and they produced SHV-1 and CTX-M14 enzymes, which was a different feature from that of the K. pneumoniae isolates from other Korean hospitals (ST20 and SHV-12). We also found a different distribution of CCs between ESBL-producing and -nonproducing K. pneumoniae isolates. The MLST method we developed in this study could provide unambiguous and well-resolved data for the epidemiologic study of K. pneumoniae. The outbreak isolates showed different molecular characteristics from the other K. pneumoniae isolates from other Korean hospitals. PMID:18303199

  7. Natural controlled HIV infection: Preserved HIV-specific immunity despite undetectable replication competent virus

    International Nuclear Information System (INIS)

    Kloosterboer, Nico; Groeneveld, Paul H.P.; Jansen, Christine A.; Vorst, Teun J.K. van der; Koning, Fransje; Winkel, Carel N.; Duits, Ashley J.; Miedema, Frank; Baarle, Debbie van; Rij, Ronald P. van; Brinkman, Kees; Schuitemaker, Hanneke

    2005-01-01

    Long-term non-progressive HIV infection, characterized by low but detectable viral load and stable CD4 counts in the absence of antiviral therapy, is observed in about 5% of HIV-infected patients. Here we identified four therapy naive individuals who are strongly seropositive for HIV-1 but who lack evidence of detectable HIV p24 antigen, plasma RNA, and proviral DNA in routine diagnostic testing. With an ultrasensitive PCR, we established that frequencies of pol proviral DNA sequences were as low as 0.2-0.5 copies/10 6 PBMC. HIV could not be isolated using up to 30 x 10 6 patient PBMC. One individual was heterozygous for CCR5 Δ32, but CCR5 expression on CD4 + T cells was normal to high in all four individuals. In vitro R5 and X4 HIV-1 susceptibility of CD8-depleted PBMC of all study subjects was significantly lower than the susceptibility of CD8-depleted PBMC of healthy blood donors. All individuals expressed protective HLA-B*58s alleles and showed evidence of HIV-specific cellular immunity either by staining with HLA-B*57 tetramers folded with an HIV RT or gag peptide or after stimulation with HIV-1 p24 gag, RT, or nef peptides in ELIspot analysis. HIV-specific CD4 + T helper cells were demonstrated by proliferation of CD4 + T cells and intracellular staining for IL-2 and IFNγ after stimulation with an HIV-gag peptide pool. Sera of all individuals showed antibody-mediated neutralization of both R5 and X4 HIV-1 variants. These data implicate that very low-level antigen exposure is sufficient for sustained HIV-specific immunity and suggest the possibility of a multi-factorial control of HIV infection

  8. Dissemination of clonal groups of Brachyspira hyodysenteriae amongst pig farms in Spain, and their relationships to isolates from other countries.

    Directory of Open Access Journals (Sweden)

    Jesús Osorio

    Full Text Available BACKGROUND: Swine dysentery (SD is a widespread diarrhoeal disease of pigs caused by infection of the large intestine with the anaerobic intestinal spirochaete Brachyspira hyodysenteriae. Understanding the dynamics of SD, and hence being able to develop more effective measures to counter its spread, depends on the ability to characterise B. hyodysenteriae variants and trace relationships of epidemic strains. METHODOLOGY/PRINCIPAL FINDINGS: A collection of 51 Spanish and 1 Portuguese B. hyodysenteriae isolates was examined using a multilocus sequence typing (MLST scheme based on the sequences of seven conserved genomic loci. The isolates were allocated to 10 sequence types (STs in three major groups of descent. Isolates in four of the STs were widely distributed in farms around Spain. One farm was infected with isolates from more than one ST. Sequence data obtained from PubMLST for 111 other B. hyodysenteriae strains from other countries then were included in the analysis. Two of the predominant STs that were found in Spain also were present in other European countries. The 73 STs were arranged in eleven clonal complexes (Cc containing between 2 and 26 isolates. A population snapshot based on amino acid types (AATs placed 75% of the isolates from 32 of the 48 AATs into one major cluster. The founder type AAT9 included 22 isolates from 10 STs that were recovered in Spain, Australia, Sweden, Germany, Belgium, the UK, Canada, and the USA. CONCLUSIONS/SIGNIFICANCE: This MLST scheme provided sufficient resolution power to unambiguously characterise B. hyodysenteriae isolates, and can be recommended as a routine typing tool that rapidly enables comparisons of isolates. Using this method it was shown that some of the main genetic lineages of B. hyodysenteriae in Spain also occurred in other countries, providing further evidence for international transmission. Finally, analysis of AATs appeared useful for deducing putative ancestral relationships between

  9. Clonal replacement and expansion among invasive meningococcal isolates of serogroup W in France.

    Science.gov (United States)

    Hong, Eva; Barret, Anne-Sophie; Terrade, Aude; Denizon, Mélanie; Antona, Denise; Aouiti-Trabelsi, Myriam; Deghmane, Ala-Eddine; Parent du Châtelet, Isabelle; Levy-Bruhl, Daniel; Taha, Muhamed-Kheir

    2018-02-01

    Neisseria meningitidis group W (NmW) belonging to the clonal complex ST-11 (NmW/cc11) spread in Europe and in France in 2000 and declined thereafter. In France, invasive meningococcal disease (IMD) due to NmW increased again in 2012 and thereafter since 2015. Several sub-lineages of NmW/cc11 are circulating worldwide with successive epidemic waves. We aimed to describe recent epidemiological trends of NmW in France and to explore the microbiological and epidemiological characteristics associated with different NmW/cc11 sub-lineages. The epidemiology of NmW was described based on data collected through mandatory notification of IMD and strain typing data for culture-confirmed and PCR-confirmed cases for the period 2000-2016. All culture-confirmed cases due to NmW from the period 2010-2016 were characterised by whole genome sequencing (WGS). A detailed epidemiological analysis was performed for culture-confirmed cases on the basis of WGS data. During the period 2010-2016, genotyping was obtained for 148 cases including all the 132 culture-confirmed cases, among which 127 were matched with epidemiological data, and 16 PCR-confirmed cases (out of a total of 47 PCR-confirmed cases). An increase in IMD was observed in 2012 and was linked to isolates belonging to the "Anglo-French-Hajj" sub-lineage. These isolates have decreased significantly since 2013 and have been replaced by NmW/cc11 isolates related to the "South American - UK" sub-lineage which caused a marked increase in the number of cases of NmW in 2016. In this sub-lineage, the "original UK strain" was first detected in 2012 and increased thereafter, followed by the recently described "UK 2013-strain". Isolates related to the "South American-UK" sub-lineage represented 45% of all NmW cultured isolates from the whole period 2010-2016 but were the most frequent isolates in 2016, representing 76% of the total NmW typed isolates and 94% of the typed NmW/cc11 isolates. A changing pattern in the epidemiology of Nm

  10. Variation of functional clonal traits along elevation in two fern species

    International Nuclear Information System (INIS)

    Song, Y.B.; Chen, L.Y.; Xiong, W.

    2015-01-01

    Phenotypical plasticity is generally considered among adaptive strategies by which plants can cope with environmental variation in space and time. Although much is known about plasticity in seed plants in terms of functional clonal traits while little is known about ferns. Variation of functional clonal traits of two ferns Dicranopteris dichotoma and Diplopterygium glaucum among plots differing in elevation in a subtropical evergreen broad-leaved forest in southern China was investigated. Along with elevation increasing the two fern species showed similar variation pattern of functional clonal traits: stable spacer length, increasing specific spacer length and decreasing spacer weight per ramet and specific spacer weight. The two ferns species had similar variation pattern of ramet performance traits but different variation pattern of ramet population properties. These results suggest an evolutionary trade-off between functions of foraging for and storing of resources in the two ferns, with a functional preference for the foraging in response to environmental change. (author)

  11. ANALYSIS OF HIV SUBTYPES AND CLINICAL STAGING OF HIV DISEASE/AIDS IN EAST JAVA

    Directory of Open Access Journals (Sweden)

    Yulia Ismail

    2012-04-01

    Full Text Available Human Immunodeficiency Virus type 1 (HIV-1 known to cause Acquired Immune Deficiency Syndrome (AIDS disease are divided into several subtypes (A, B, C, D, F, G, H, J, K and Circulating Recombinant Form (CRF. Different characteristics of subtype of the virus and its interaction with the host can affect the severity of the disease. This study was to analyze HIV-1 subtypes circulating in HIV/AIDS patients from the East Java region descriptively and to analyze its relationship with clinical stadiums of HIV/AIDS. Information from this research was expected to complement the data of mocular epidemiology of HIV in Indonesia. This study utilited blood plasma from patients who had been tested to be HIV positive who sected treatment to or were reffered to the Intermediate Care Unit of Infectious Disease (UPIPI Dr. Soetomo Hospital Surabaya from various area representing the East Java regions. Plasma was separated from blood samples by centrifugation for use in the the molecular biology examination including RNA extraction, nested PCR using specific primer for HIV gp120 env gene region, DNA purifying, DNA sequencing, and homology and phylogenetic analysis. Based on the nucleotide sequence of the HIV gp120 env gene, it was found that the most dominant subtypes in East Java were in one group of Circulating Recombinant Form (CRF that is CRF01_AE, CRF33_01B and CRF34_01B which was also found in Southeast Asia. In the phylogenetic tree, most of HIV samples (30 samples are in the same branch with CRF01_AE, CRF33_01B and CRF34_01B, except for one sample (HIV40 which is in the same branch with subtype B. HIV subtypes are associated with clinical stadiums (disease severity since samples from different stages of HIV disease have the same subtype.

  12. The current MLVA typing scheme for Enterococcus faecium is less discriminatory than MLST and PFGE for epidemic-virulent, hospital-adapted clonal types

    Directory of Open Access Journals (Sweden)

    Klare Ingo

    2007-04-01

    Full Text Available Abstract Background MLVA (multiple-locus variable-number tandem repeat analysis is a reliable typing technique introduced recently to differentiate also isolates of Enterococcus faecium. We used the established VNTR (variable number of tandem repeats scheme to test its suitability to differentiate 58 E. faecium isolates representing mainly outbreaks and clusters of infections and colonizations among patients from 31 German hospitals. All isolates were vancomycin-resistant (vanA type. Typing results for MLVA are compared with results of macrorestriction analysis in PFGE (pulsed-field gel electrophoresis and MLST (multi-locus sequence typing. Results All 51 but one hospital isolates from 1996–2006 were assigned to the clonal complex (CC of epidemic-virulent, hospital-adapted lineages (MLST CC-17; MLVA CC-1 and differed from isolates of sporadic infections and colonizations (n = 7; 1991–1995 and other non-hospital origins (n = 27. Typing of all 58 hospital VRE revealed MLVA as the least discriminatory method (Simpson's diversity index 0.847 when compared to MLST (0.911 and PFGE (0.976. The two most common MLVA types MT-1 (n = 16 and MT-159 (n = 14 combined isolates of several MLST types including also major epidemic, hospital-adapted, clonal types (MT-1: ST-17, ST-18, ST-280, ST-282; MT-159: ST-78, ST-192, ST-203. These data clearly indicate that non-related E. faecium could possess an identical MLVA type being especially critical when MLVA is used to elucidate supposed outbreaks with E. faecium within a single or among different hospitals. Stability of a given MLVA profile MT-12 (ST-117 during an outbreak over a period of five years was also shown. Conclusion MLVA is a suitable method to assign isolates of E. faecium into distinct clonal complexes. To investigate outbreaks the current MLVA typing scheme for E. faecium does not discriminate enough and cannot be recommended as a standard superior to PFGE.

  13. Correlation between HIV-1 genotype and clinical progression in HIV/AIDS patients in Surabaya, Indonesia

    Science.gov (United States)

    Rachman, B. E.; Khairunisa, S. Q.; Witaningrum, A. M.; Yunifiar, M. Q.; Nasronudin

    2018-03-01

    Several factors such as host and viral factors can affect the progression of HIV/AIDS. This study aims to identify the correlation viral factors, especially the HIV-1 subtype with HIV/AIDS progression. Inpatient HIV/AIDS during the period March to September 2017 and willing to participate are included in the study. Historical data of disease and treatment was taken by medical record. Blood samples were amplified, sequenced and undergone phylogenetic analysis. Linear regression analysis was used to estimate beta coefficient (β) and 95%CI of HIV/AIDS progression (measured by the CD4 change rate, ΔCD4 cell count/time span in months).This study has 17 samples. The HIV-1 subtype was dominated by CRF01_AE (81.8%) followed by subtype B (18.2%). There was significant correlation between subtype HIV-1 (p = 0.04) and body mass index (p = 0.038) with HIV/AIDS clinical stage. Many factors were assumed to be correlated with increased rate of CD4, but we only subtype HIV-1 had a significant correlation (p = 0.024) with it. From multivariate analysis, we also found that subtype HIV-1 had a significant correlation (β = 0.788, 95%CI: 17.5-38.6, p = 0.004).

  14. Usefullness of IGH/TCR PCR studies in lymphoproliferative disorders with inconclusive clonality by flow cytometry.

    Science.gov (United States)

    Ribera, Jordi; Zamora, Lurdes; Juncà, Jordi; Rodríguez, Inés; Marcé, Silvia; Cabezón, Marta; Millá, Fuensanta

    2013-07-25

    In up to 5-15% of studies of lymphoproliferative disorders (LPD) flow cytometry (FCM) or immunomorphologic methods cannot discriminate malignant from reactive processes. The aim of this work was to determine the usefulness of PCR for solving these diagnostic uncertainties. We analyzed IGH and TCRγ genes by PCR in 106 samples with inconclusive FCM results. A clonal result was registered in 36/106 studies, with a LPD being confirmed in 27 (75%) of these cases. Specifically, 9/9 IGH clonal and 16/25 TCRγ clonal results were finally diagnosed with LPD. Additionally, 2 clonal TCRγ samples with suspicion of undefined LPD were finally diagnosed with T LPD. Although polyclonal results were obtained in 47 of the cases studied (38 IGH and 9 TCRγ), hematologic neoplasms were diagnosed in 4/38 IGH polyclonal and in 1/9 TCRγ polyclonal studies. There were also 14 PCR polyclonal results (4 IGH, 10 TCRγ), albeit non-conclusive. Of these, 2/4 were eventually diagnosed with B-cell lymphoma and 3/10 with T-cell LPD. In 8 IGH samples the results of PCR techniques were non-informative but in 3/8 cases a B lymphoma was finally confirmed. We concluded that PCR is a useful technique to identify LPD when FCM is inconclusive. A PCR clonal B result is indicative of malignancy but IGH polyclonal and non-conclusive results do not exclude lymphoid neoplasms. Interpretation of T-cell clonality should be based on all the available clinical and analytical data. © 2013 Clinical Cytometry Society. Copyright © 2013 Clinical Cytometry Society.

  15. Clonal relatedness between lobular carcinoma in situ and synchronous malignant lesions

    Science.gov (United States)

    2012-01-01

    Introduction Lobular carcinoma in situ (LCIS) has been accepted as a marker of risk for the development of invasive breast cancer, yet modern models of breast carcinogenesis include LCIS as a precursor of low-grade carcinomas. We provide evidence favoring a clonal origin for LCIS and synchronous estrogen receptor-positive malignant lesions of the ductal and lobular phenotype. Methods Patients with prior LCIS undergoing mastectomy were identified preoperatively from 2003 to 2008. Specimens were widely sampled, and frozen blocks were screened for LCIS and co-existing malignant lesions, and were subject to microdissection. Samples from 65 patients were hybridized to the Affymetrix SNP 6.0 array platform. Cases with both an LCIS sample and an associated ductal carcinoma in situ (DCIS) or invasive tumor sample were evaluated for patterns of somatic copy number changes to assess evidence of clonal relatedness. Results LCIS was identified in 44 of the cases, and among these a DCIS and/or invasive lesion was also identified in 21 cases. A total of 17 tumor pairs had adequate DNA/array data for analysis, including nine pairs of LCIS/invasive lobular cancer, four pairs of LCIS/DCIS, and four pairs of LCIS/invasive ductal cancer. Overall, seven pairs (41%) were judged to be clonally related; in five (29%) evidence suggested clonality but was equivocal, and five (29%) were considered independent. Clonal pairs were observed with all matched lesion types and low and high histological grades. We also show anecdotal evidence of clonality between a patient-matched triplet of LCIS, DCIS, and invasive ductal cancer. Conclusion Our results support the role of LCIS as a precursor in the development of both high-grade and low-grade ductal and lobular cancers. PMID:22776144

  16. PPARγ ligand production is tightly linked to clonal expansion during initiation of adipocyte differentiation

    DEFF Research Database (Denmark)

    Hallenborg, Philip; Petersen, Rasmus Koefoed; Feddersen, Søren

    2014-01-01

    of differentiation. Concomitant with agonist production, murine fibroblasts undergo two rounds of mitosis referred to as mitotic clonal expansion. Here we show that mouse embryonic fibroblasts deficient in either of two cell cycle inhibitors, the transcription factor p53 or its target gene encoding the cyclin...... cycle inhibitory compounds decreased PPAR ligand production in differentiating 3T3-L1 preadipocytes. Furthermore, these inhibitors abolished the release of arachidonic acid induced by the hormonal cocktail initiating adipogenesis. Collectively, our results suggest that murine fibroblasts require clonal...... expansion for PPAR ligand production at the onset of adipocyte differentiation....

  17. Diverse cellular architecture of atherosclerotic plaque derives from clonal expansion of a few medial SMCs

    DEFF Research Database (Denmark)

    Jacobsen, Kevin; Lund, Marie Bek; Shim, Jeong

    2017-01-01

    chimeras of eGFP+Apoe-/- and Apoe-/- mouse embryos and in mice with a mosaic expression of fluorescent proteins in medial SMCs that were rendered atherosclerotic by PCSK9-induced hypercholesterolemia. Fibrous caps in aggregation chimeras were found constructed from large, endothelial-aligned layers...... in the cap and heterogeneous ACTA2- SMCs in the plaque interior, including chondrocyte-like cells and cells with intracellular lipid and crystalline material. Fibrous cap SMCs were invariably arranged in endothelium-aligned clonal sheets, confirming results in the aggregation chimeras. Analysis of the clonal...

  18. Identifying HIV-1 dual infections

    Directory of Open Access Journals (Sweden)

    Cornelissen Marion

    2007-09-01

    Full Text Available Abstract Transmission of human immunodeficiency virus (HIV is no exception to the phenomenon that a second, productive infection with another strain of the same virus is feasible. Experiments with RNA viruses have suggested that both coinfections (simultaneous infection with two strains of a virus and superinfections (second infection after a specific immune response to the first infecting strain has developed can result in increased fitness of the viral population. Concerns about dual infections with HIV are increasing. First, the frequent detection of superinfections seems to indicate that it will be difficult to develop a prophylactic vaccine. Second, HIV-1 superinfections have been associated with accelerated disease progression, although this is not true for all persons. In fact, superinfections have even been detected in persons controlling their HIV infections without antiretroviral therapy. Third, dual infections can give rise to recombinant viruses, which are increasingly found in the HIV-1 epidemic. Recombinants could have increased fitness over the parental strains, as in vitro models suggest, and could exhibit increased pathogenicity. Multiple drug resistant (MDR strains could recombine to produce a pan-resistant, transmittable virus. We will describe in this review what is presently known about super- and re-infection among ambient viral infections, as well as the first cases of HIV-1 superinfection, including HIV-1 triple infections. The clinical implications, the impact of the immune system, and the effect of anti-retroviral therapy will be covered, as will as the timing of HIV superinfection. The methods used to detect HIV-1 dual infections will be discussed in detail. To increase the likelihood of detecting a dual HIV-1 infection, pre-selection of patients can be done by serotyping, heteroduplex mobility assays (HMA, counting the degenerate base codes in the HIV-1 genotyping sequence, or surveying unexpected increases in the

  19. Clinical significance of T-cell clonality in mycosis fungoides and other cutaneous T-cell lymphomas

    NARCIS (Netherlands)

    Muche, Joachim Marcus

    2010-01-01

    The purpose of this thesis was to obtain more insight into T-cell clonality in blood of mycosis fungoides (MF) patients. Investigation of the frequency of blood T-cell clonality clearly indicated early dissemination of neoplastic T-cells into skin and blood as a sign of physiological recirculation.

  20. Synergy against drug-resistant HIV-1 with the microbicide antiretrovirals, dapivirine and tenofovir, in combination.

    Science.gov (United States)

    Schader, Susan M; Colby-Germinario, Susan P; Schachter, Jordana R; Xu, Hongtao; Wainberg, Mark A

    2011-08-24

    To evaluate the candidate antiretroviral microbicide compounds, dapivirine (DAP) and tenofovir (TFV), alone and in combination against the transmission of wild-type and nonnucleoside reverse transcriptase inhibitor (NNRTI)-resistant HIV-1 from different subtypes. We determined single-drug efficacy of the RTIs, DAP and TFV, against subtype B and non-B wild-type and NNRTI-resistant HIV-1 in vitro. To assess breadth of activity, compounds were tested alone and in combination against wild-type and NNRTI-resistant subtype C primary HIV-1 isolates and complimentary clonal HIV-1 from subtypes B, C and CRF02_AG to control for viral variation. Early infection was quantified by counting light units emitted from TZM-bl cells less than 48-h postinfection. Combination ratios were based on drug inhibitory concentrations (IC(50)s) and combined effects were determined by calculating combination indices. Both candidate microbicide antiretrovirals demonstrated potent anti-NNRTI-resistant HIV-1 activity in vitro, albeit the combination protected better than the single-drug treatments. Of particular interest, the DAP with TFV combination exhibited synergy (50% combination index, CI(50) = 0.567) against subtype C NNRTI-resistant HIV-1, whereas additivity (CI(50) = 0.987) was observed against the wild-type counterpart from the same patient. The effect was not compounded by the presence of subdominant viral fractions, as experiments using complimentary clonal subtype C wild-type (CI(50) = 0.968) and NNRTI-resistant (CI(50) = 0.672) HIV-1, in lieu of the patient quasispecies, gave similar results. This study supports the notion that antiretroviral drug combinations may retain antiviral activity against some drug-resistant HIV-1 despite subtype classification and quasispecies diversity.

  1. HIV classification using coalescent theory

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Ming [Los Alamos National Laboratory; Letiner, Thomas K [Los Alamos National Laboratory; Korber, Bette T [Los Alamos National Laboratory

    2008-01-01

    Algorithms for subtype classification and breakpoint detection of HIV-I sequences are based on a classification system of HIV-l. Hence, their quality highly depend on this system. Due to the history of creation of the current HIV-I nomenclature, the current one contains inconsistencies like: The phylogenetic distance between the subtype B and D is remarkably small compared with other pairs of subtypes. In fact, it is more like the distance of a pair of subsubtypes Robertson et al. (2000); Subtypes E and I do not exist any more since they were discovered to be composed of recombinants Robertson et al. (2000); It is currently discussed whether -- instead of CRF02 being a recombinant of subtype A and G -- subtype G should be designated as a circulating recombination form (CRF) nd CRF02 as a subtype Abecasis et al. (2007); There are 8 complete and over 400 partial HIV genomes in the LANL-database which belong neither to a subtype nor to a CRF (denoted by U). Moreover, the current classification system is somehow arbitrary like all complex classification systems that were created manually. To this end, it is desirable to deduce the classification system of HIV systematically by an algorithm. Of course, this problem is not restricted to HIV, but applies to all fast mutating and recombining viruses. Our work addresses the simpler subproblem to score classifications of given input sequences of some virus species (classification denotes a partition of the input sequences in several subtypes and CRFs). To this end, we reconstruct ancestral recombination graphs (ARG) of the input sequences under restrictions determined by the given classification. These restritions are imposed in order to ensure that the reconstructed ARGs do not contradict the classification under consideration. Then, we find the ARG with maximal probability by means of Markov Chain Monte Carlo methods. The probability of the most probable ARG is interpreted as a score for the classification. To our

  2. Horizontal gene transfer of a ColV plasmid has resulted in a dominant avian clonal type of Salmonella enterica serovar Kentucky.

    Directory of Open Access Journals (Sweden)

    Timothy J Johnson

    Full Text Available Salmonella enterica continues to be a significant cause of foodborne gastrointestinal illness in humans. A wide variety of Salmonella serovars have been isolated from production birds and from retail poultry meat. Recently, though, S. enterica subsp. enterica serovar Kentucky has emerged as one of the prominent Salmonella serovars isolated from broiler chickens. Recent work suggests that its emergence apparently coincides with its acquisition of a ColV virulence plasmid. In the present study, we examined 902 Salmonella isolates belonging to 59 different serovars for the presence of this plasmid. Of the serovars examined, the ColV plasmid was found only among isolates belonging to the serovars Kentucky (72.9%, Typhimurium (15.0% and Heidelberg (1.7%. We demonstrated that a single PFGE clonal type of S. Kentucky harbors this plasmid, and acquisition of this plasmid by S. Kentucky significantly increased its ability to colonize the chicken cecum and cause extraintestinal disease. Comparison of the completed sequences of three ColV plasmids from S. Kentucky isolated from different geographical locales, timepoints and sources revealed a nearly identical genetic structure with few single nucleotide changes or insertions/deletions. Overall, it appears that the ColV plasmid was recently acquired by a single clonal type S. Kentucky and confers to its host enhanced colonization and fitness capabilities. Thus, the potential for horizontal gene transfer of virulence and fitness factors to Salmonella from other enteric bacteria exists in poultry, representing a potential human health hazard.

  3. Reconstructing a B-cell clonal lineage. I. Statistical inference of unobserved ancestors [v1; ref status: indexed, http://f1000r.es/z6

    Directory of Open Access Journals (Sweden)

    Thomas B Kepler

    2013-04-01

    Full Text Available One of the key phenomena in the adaptive immune response to infection and immunization is affinity maturation, during which antibody genes are mutated and selected, typically resulting in a substantial increase in binding affinity to the eliciting antigen. Advances in technology on several fronts have made it possible to clone large numbers of heavy-chain light-chain pairs from individual B cells and thereby identify whole sets of clonally related antibodies. These collections could provide the information necessary to reconstruct their own history - the sequence of changes introduced into the lineage during the development of the clone - and to study affinity maturation in detail. But the success of such a program depends entirely on accurately inferring the founding ancestor and the other unobserved intermediates. Given a set of clonally related immunoglobulin V-region genes, the method described here allows one to compute the posterior distribution over their possible ancestors, thereby giving a thorough accounting of the uncertainty inherent in the reconstruction. I demonstrate the application of this method on heavy-chain and light-chain clones, assess the reliability of the inference, and discuss the sources of uncertainty.

  4. High prevalence of non-clonal imipenem-nonsusceptible Enterobacter spp. isolates in Korea and their association with porin down-regulation.

    Science.gov (United States)

    Lee, Ji-Young; Hong, Yoon-Kyoung; Lee, Haejeong; Ko, Kwan Soo

    2017-01-01

    We investigated the prevalence and clonal distribution of imipenem-nonsusceptible Enterobacter clinical isolates from hospitals in Korea and the contributions of various mechanisms to imipenem nonsusceptibility. The in vitro antimicrobial susceptibility to imipenem of 357 non-duplicated Enterobacter isolates obtained from eight geographically distant tertiary care hospitals in Korea was evaluated. Imipenem-nonsusceptible Enterobacter isolates were genotyped. Additionally, β-lactamase genes were screened using PCR, and the expression of efflux pump and porin genes was investigated using quantitative RT-PCR. A total of 31 isolates (8.7%) were not susceptible to imipenem. Clonal diversity of 17 imipenem-nonsusceptible E. cloacae isolates was demonstrated by multilocus sequence typing. Fourteen imipenem-nonsusceptible E. aerogenes isolates were found to be distantly genetically related by an ERIC-PCR analysis. Expression levels of porin ompD and ompK35 genes were decreased in all imipenem-nonsusceptible E. cloacae and E. aerogenes isolates. However, only two isolates were found positive for bla IMP and bla VIM genes, and expression of the efflux pump gene, acrB, was not associated with reduced imipenem susceptibility. Imipenem resistance seems to have occurred independently in most of the imipenem-nonsusceptible isolates in this study, and decreased porin expression was found to be the main mechanism underlying this reduced susceptibility to imipenem. Copyright © 2016 Elsevier Inc. All rights reserved.

  5. Sequence assembly

    DEFF Research Database (Denmark)

    Scheibye-Alsing, Karsten; Hoffmann, S.; Frankel, Annett Maria

    2009-01-01

    Despite the rapidly increasing number of sequenced and re-sequenced genomes, many issues regarding the computational assembly of large-scale sequencing data have remain unresolved. Computational assembly is crucial in large genome projects as well for the evolving high-throughput technologies and...... in genomic DNA, highly expressed genes and alternative transcripts in EST sequences. We summarize existing comparisons of different assemblers and provide a detailed descriptions and directions for download of assembly programs at: http://genome.ku.dk/resources/assembly/methods.html....

  6. Genome Sequencing

    DEFF Research Database (Denmark)

    Sato, Shusei; Andersen, Stig Uggerhøj

    2014-01-01

    The current Lotus japonicus reference genome sequence is based on a hybrid assembly of Sanger TAC/BAC, Sanger shotgun and Illumina shotgun sequencing data generated from the Miyakojima-MG20 accession. It covers nearly all expressed L. japonicus genes and has been annotated mainly based on transcr......The current Lotus japonicus reference genome sequence is based on a hybrid assembly of Sanger TAC/BAC, Sanger shotgun and Illumina shotgun sequencing data generated from the Miyakojima-MG20 accession. It covers nearly all expressed L. japonicus genes and has been annotated mainly based...

  7. Clonal deleted latent membrane protein 1 variants of Epstein-Barr virus are predominant in European extranodal NK/T lymphomas and disappear during successful treatment.

    Science.gov (United States)

    Halabi, Mohamad Adnan; Jaccard, Arnaud; Moulinas, Rémi; Bahri, Racha; Al Mouhammad, Hazar; Mammari, Nour; Feuillard, Jean; Ranger-Rogez, Sylvie

    2016-08-15

    Extranodal natural killer/T-cell lymphomas (NK/TL), rare in Europe, are Epstein-Barr virus (EBV) associated lymphomas with poor outcomes. Here, we determined the virus type and analyzed the EBV latent membrane protein-1 (LMP1) gene sequence in NK/TL from French patients. Six clones of viral LMP1 were sequenced by Sanger technology in blood from 13 patients before treatment with an l-asparaginase based regimen and, for 8 of them, throughout the treatment. Blood LMP1 sequences from 21 patients without any known malignancy were tested as controls. EBV Type A was identified for 11/13 patients and for all controls. Before treatment, a clonal LMP1 gene containing a 30 bp deletion (del30) was found in 46.1% of NK/TL and only in 4.8% of controls. Treatment was less effective in these patients who died more rapidly than the others. Patients with a deleted strain evolving toward a wild-type strain during treatment reached complete remission. The LMP1 gene was sequenced by highly sensitive next-generation sequencing technology in five NK/TL nasopharyngeal biopsies, two of them originating from the previous patients. Del30 was present in 100% of the biopsies; two viruses at least coexisted in three biopsies. These results suggest that del30 may be associated with poor prognosis NK/TL and that strain evolution could be used as a potential marker to monitor treatment. © 2016 UICC.

  8. A novel strategy for efficient production of anti-V3 human scFvs against HIV-1 clade C

    Directory of Open Access Journals (Sweden)

    Kumar Rajesh

    2012-11-01

    Full Text Available Abstract Background Production of human monoclonal antibodies that exhibit broadly neutralizing activity is needed for preventing HIV-1 infection, however only a few such antibodies have been generated till date. Isolation of antibodies by the hybridoma technology is a cumbersome process with fewer yields. Further, the loss of unstable or slowly growing clones which may have unique binding specificities often occurs during cloning and propagation and the strongly positive clones are often lost. This has been avoided by the process described in this paper, wherein, by combining the strategy of EBV transformation and recombinant DNA technology, we constructed human single chain variable fragments (scFvs against the third variable region (V3 of the clade C HIV-1 envelope. Results An antigen specific phage library of 7000 clones was constructed from the enriched V3- positive antibody secreting EBV transformed cells. By ligation of the digested scFv DNA into phagemid vector and bio panning against the HIV-1 consensus C and B V3 peptides followed by random selection of 40 clones, we identified 15 clones that showed V3 reactivity in phage ELISA. DNA fingerprinting analysis and sequencing showed that 13 out of the 15 clones were distinct. Expression of the positive clones was tested by SDS-PAGE and Western blot. All the 13 anti-V3 scFvs showed cross-reactivity against both the clade C and B V3 peptides and did not show any reactivity against other unrelated peptides in ELISA. Preliminary neutralization assays indicated varying degrees of neutralization of clade C and B viruses. EBV transformation, followed by antigen selection of lines to identify specific binders, enabled the selection of phage from un-cloned lines for scFv generation, thus avoiding the problems of hybridoma technology. Moreover, as the clones were pretested for antigen binding, a comparatively small library sufficed for the selection of a considerable number of unique antigen binding

  9. Novel avian oropharyngeal trichomonads isolated from European turtle doves (Streptopelia turtur) and racing pigeons (Columba livia): genetic and morphometric characterisation of clonal cultures.

    Science.gov (United States)

    Martínez-Herrero, M C; Garijo-Toledo, M M; Liebhart, D; Ganas, P; Martínez-Díaz, R A; Ponce-Gordo, F; Carrero-Ruiz, A; Hess, M; Gómez-Muñoz, M T

    2017-11-01

    Extensive diversity has been described within the avian oropharyngeal trichomonad complex in recent years. In this study we developed clonal cultures from four isolates selected by their different ITS1/5.8S/ITS2 (ITS) genotype and their association with gross lesions of avian trichomonosis. Isolates were obtained from an adult racing pigeon and a nestling of Eurasian eagle owl with macroscopic lesions, and from a juvenile wood pigeon and an European turtle dove without clinical signs. Multi-locus sequence typing analysis of the ITS, small subunit of ribosomal rRNA (SSUrRNA) and Fe-hydrogenase (Fe-hyd) genes together with a morphological study by optical and scanning electron microscopy was performed. No significant differences in the structures were observed with scanning electron microscopy. However, the genetic characterisation revealed novel sequence types for the SSUrRNA region and Fe-hyd gene. Two clones were identified as Trichomonas gallinae in the MLST analysis, but the clones from the racing pigeon and European turtle dove showed higher similarity with Trichomonas tenax and Trichomonas canistomae than with T. gallinae at their ITS region, respectively. SSUrRNA sequences grouped all the clones in a clade that includes T. gallinae, T. tenax and T. canistomae. Further diversity was detected within the Fe-hyd locus, with a clear separation from T. gallinae of the clones obtained from the racing pigeon and the European turtle dove. In addition, morphometric comparison by optical microscopy with clonal cultures of T. gallinae revealed significant statistical differences on axostyle projection length in the clone from the European turtle dove. Morphometric and genetic data indicate that possible new species within the Trichomonas genus were detected. Taking in consideration the diversity in Trichomonas species present in the oral cavity of birds, a proper genetic analysis is highly recommended when outbreaks occur. Copyright © 2017 Elsevier B.V. All rights

  10. Cyclophilin B enhances HIV-1 infection

    Energy Technology Data Exchange (ETDEWEB)

    DeBoer, Jason; Madson, Christian J. [Department of Medical Microbiology and Immunology, Creighton University, Omaha, NE (United States); Belshan, Michael, E-mail: michaelbelshan@creighton.edu [Department of Medical Microbiology and Immunology, Creighton University, Omaha, NE (United States); The Nebraska Center for Virology, University of Nebraska, Lincoln, NE (United States)

    2016-02-15

    Cyclophilin B (CypB) is a member of the immunophilin family and intracellular chaperone. It predominantly localizes to the ER, but also contains a nuclear localization signal and is secreted from cells. CypB has been shown to interact with the Gag protein of human immunodeficiency type 1 (HIV-1). Several proteomic and genetic studies identified it as a potential factor involved in HIV replication. Herein, we show that over-expression of CypB enhances HIV infection by increasing nuclear import of viral DNA. This enhancement was unaffected by cyclosporine treatment and requires the N-terminus of the protein. The N-terminus contains an ER leader sequence, putative nuclear localization signal, and is required for secretion. Deletion of the N-terminus resulted in mislocalization from the ER and suppression of HIV infection. Passive transfer experiments showed that secreted CypB did not impact HIV infection. Combined, these experiments show that intracellular CypB modulates a pathway of HIV nuclear import. - Highlights: • CypB has been identified in several proteomic studies of HIV-1 infection. • CypB expression is upregulated in activated and infected T-cells. • Over-expression of CypB enhances HIV nuclear import and infection. • The N-terminus of CypB is necessary for these effects.

  11. Cyclophilin B enhances HIV-1 infection

    International Nuclear Information System (INIS)

    DeBoer, Jason; Madson, Christian J.; Belshan, Michael

    2016-01-01

    Cyclophilin B (CypB) is a member of the immunophilin family and intracellular chaperone. It predominantly localizes to the ER, but also contains a nuclear localization signal and is secreted from cells. CypB has been shown to interact with the Gag protein of human immunodeficiency type 1 (HIV-1). Several proteomic and genetic studies identified it as a potential factor involved in HIV replication. Herein, we show that over-expression of CypB enhances HIV infection by increasing nuclear import of viral DNA. This enhancement was unaffected by cyclosporine treatment and requires the N-terminus of the protein. The N-terminus contains an ER leader sequence, putative nuclear localization signal, and is required for secretion. Deletion of the N-terminus resulted in mislocalization from the ER and suppression of HIV infection. Passive transfer experiments showed that secreted CypB did not impact HIV infection. Combined, these experiments show that intracellular CypB modulates a pathway of HIV nuclear import. - Highlights: • CypB has been identified in several proteomic studies of HIV-1 infection. • CypB expression is upregulated in activated and infected T-cells. • Over-expression of CypB enhances HIV nuclear import and infection. • The N-terminus of CypB is necessary for these effects.

  12. Clonally Expanding Thymocytes Having Lineage Capability in Gamma-Ray-Induced Mouse Atrophic Thymus

    International Nuclear Information System (INIS)

    Yamamoto, Takashi; Morita, Shin-ichi; Go, Rieka; Obata, Miki; Katsuragi, Yoshinori; Fujita, Yukari; Maeda, Yoshitaka; Yokoyama, Minesuke; Aoyagi, Yutaka; Ichikawa, Hitoshi; Mishima, Yukio; Kominami, Ryo

    2010-01-01

    Purpose: To characterize, in the setting of γ-ray-induced atrophic thymus, probable prelymphoma cells showing clonal growth and changes in signaling, including DNA damage checkpoint. Methods and Materials: A total of 111 and 45 mouse atrophic thymuses at 40 and 80 days, respectively, after γ-irradiation were analyzed with polymerase chain reaction for D-J rearrangements at the TCRβ locus, flow cytometry for cell cycle, and Western blotting for the activation of DNA damage checkpoints. Results: Limited D-J rearrangement patterns distinct from normal thymus were detected at high frequencies (43 of 111 for 40-day thymus and 21 of 45 for 80-day thymus). Those clonally expanded thymocytes mostly consisted of CD4 + CD8 + double-positive cells, indicating the retention of lineage capability. They exhibited pausing at a late G1 phase of cell cycle progression but did not show the activation of DNA damage checkpoints such as γH2AX, Chk1/2, or p53. Of interest is that 17 of the 52 thymuses showing normal D-J rearrangement patterns at 40 days after irradiation showed allelic loss at the Bcl11b tumor suppressor locus, also indicating clonal expansion. Conclusion: The thymocytes of clonal growth detected resemble human chronic myeloid leukemia in possessing self-renewal and lineage capability, and therefore they can be a candidate of the lymphoma-initiating cells.

  13. In vitro clonal propagation of the neem tree ( Azadirachta indica A ...

    African Journals Online (AJOL)

    In vitro clonal propagation of the neem tree (Azadirachta indica A. Juss.) M Shahin-uz-zaman, M Ashrafuzzaman, MS Haque, LN Luna. Abstract. A study was conducted with root and shoot tip explants of neem to develop an efficient protocol of regeneration. Shoot tips and root tips from 10 - 20 days old seedlings of neem ...

  14. Epigenetic memory as a basis for intelligent behavior in clonal plants

    Czech Academy of Sciences Publication Activity Database

    Latzel, Vít; González, Alejandra Pilar Rendina; Rosenthal, J.

    2016-01-01

    Roč. 7, AUG 31 (2016), s. 1-7, č. článku 1354. ISSN 1664-462X R&D Projects: GA ČR(CZ) GA14-06802S Institutional support: RVO:67985939 Keywords : epigenetic variability * memory * clonal plant s Subject RIV: EF - Botanics Impact factor: 4.298, year: 2016

  15. Descriptions of new varieties recently distributed from the Citrus Clonal Protection Program

    Science.gov (United States)

    The Citrus Clonal Protection Program (CCPP) is operated through the Department of Plant Pathology and Microbiology at University of California (UC) Riverside and is funded in large part by The California Citrus Research Board (CRB). The CCPP processes citrus propagative material in two phases. First...

  16. Clonal genotype of Geomyces destructans among bats with White Nose Syndrome, New York, USA.

    Science.gov (United States)

    Rajkumar, Sunanda S; Li, Xiaojiang; Rudd, Robert J; Okoniewski, Joseph C; Xu, Jianping; Chaturvedi, Sudha; Chaturvedi, Vishnu

    2011-07-01

    The dispersal mechanism of Geomyces destructans, which causes geomycosis (white nose syndrome) in hibernating bats, remains unknown. Multiple gene genealogic analyses were conducted on 16 fungal isolates from diverse sites in New York State during 2008-2010. The results are consistent with the clonal dispersal of a single G. destructans genotype.

  17. Clonal Genotype of Geomyces destructans among Bats with White Nose Syndrome, New York, USA

    OpenAIRE

    Rajkumar, Sunanda S.; Li, Xiaojiang; Rudd, Robert J.; Okoniewski, Joseph C.; Xu, Jianping; Chaturvedi, Sudha; Chaturvedi, Vishnu

    2011-01-01

    The dispersal mechanism of Geomyces destructans, which causes geomycosis (white nose syndrome) in hibernating bats, remains unknown. Multiple gene genealogic analyses were conducted on 16 fungal isolates from diverse sites in New York State during 2008–2010. The results are consistent with the clonal dispersal of a single G. destructans genotype.

  18. Diverse cellular architecture of atherosclerotic plaque derives from clonal expansion of a few medial SMCs.

    Science.gov (United States)

    Jacobsen, Kevin; Lund, Marie Bek; Shim, Jeong; Gunnersen, Stine; Füchtbauer, Ernst-Martin; Kjolby, Mads; Carramolino, Laura; Bentzon, Jacob Fog

    2017-10-05

    Fibrous cap smooth muscle cells (SMCs) protect atherosclerotic lesions from rupturing and causing thrombosis, while other plaque SMCs may have detrimental roles in plaque development. To gain insight into recruitment of different plaque SMCs, we mapped their clonal architecture in aggregation chimeras of eGFP+Apoe-/- and Apoe-/- mouse embryos and in mice with a mosaic expression of fluorescent proteins in medial SMCs that were rendered atherosclerotic by PCSK9-induced hypercholesterolemia. Fibrous caps in aggregation chimeras were found constructed from large, endothelial-aligned layers of either eGFP+ or nonfluorescent SMCs, indicating substantial clonal expansion of a few cells. Similarly, plaques in mice with SMC-restricted Confetti expression showed oligoclonal SMC populations with little intermixing between the progeny of different medial SMCs. Phenotypes comprised both ACTA2+ SMCs in the cap and heterogeneous ACTA2- SMCs in the plaque interior, including chondrocyte-like cells and cells with intracellular lipid and crystalline material. Fibrous cap SMCs were invariably arranged in endothelium-aligned clonal sheets, confirming results in the aggregation chimeras. Analysis of the clonal structure showed that a low number of local medial SMCs partake in atherosclerosis and that single medial SMCs can produce several different SMC phenotypes in plaque. The combined results show that few medial SMCs proliferate to form the entire phenotypically heterogeneous plaque SMC population in murine atherosclerosis.

  19. CLO-PLA: the database of clonal and bud bank traits of Central European flora

    Czech Academy of Sciences Publication Activity Database

    Klimešová, Jitka; de Bello, F.

    2009-01-01

    Roč. 20, č. 3 (2009), s. 511-516 ISSN 1100-9233 R&D Projects: GA ČR(CZ) GA526/06/0723 Institutional research plan: CEZ:AV0Z60050516 Keywords : bud bank * clonal traits * database Subject RIV: EF - Botanics Impact factor: 2.376, year: 2009

  20. Clonal splitters and integrators in harsh environments of the Trans-Himalaya

    Czech Academy of Sciences Publication Activity Database

    Klimeš, Leoš

    2008-01-01

    Roč. 22, č. 3 (2008), s. 351-367 ISSN 0269-7653 R&D Projects: GA ČR GA206/03/1219 Institutional research plan: CEZ:AV0Z60050516 Keywords : Ladakh * altitude * clonal growth Subject RIV: EF - Botanics Impact factor: 3.448, year: 2008

  1. Production and carbon allocation in a clonal Eucalyptus plantation with water and nutrient manipulations

    Science.gov (United States)

    Jose Luiz Stape; Dan Binkley; Michael G. Ryan

    2008-01-01

    We examined resource limitations on growth and carbon allocation in a fast-growing, clonal plantation of Eucalyptus grandis urophylla in Brazil by characterizing responses to annual rainfall, and response to irrigation and fertililization for 2 years. Productivity measures included gross primary production (GPP), total belowground carbon allocation (...

  2. Integration Site and Clonal Expansion in Human Chronic Retroviral Infection and Gene Therapy

    Science.gov (United States)

    Niederer, Heather A.; Bangham, Charles R. M.

    2014-01-01

    Retroviral vectors have been successfully used therapeutically to restore expression of genes in a range of single-gene diseases, including several primary immunodeficiency disorders. Although clinical trials have shown remarkable results, there have also been a number of severe adverse events involving malignant outgrowth of a transformed clonal population. This clonal expansion is influenced by the integration site profile of the viral integrase, the transgene expressed, and the effect of the viral promoters on the neighbouring host genome. Infection with the pathogenic human retrovirus HTLV-1 also causes clonal expansion of cells containing an integrated HTLV-1 provirus. Although the majority of HTLV-1-infected people remain asymptomatic, up to 5% develop an aggressive T cell malignancy. In this review we discuss recent findings on the role of the genomic integration site in determining the clonality and the potential for malignant transformation of cells carrying integrated HTLV-1 or gene therapy vectors, and how these results have contributed to the understanding of HTLV-1 pathogenesis and to improvements in gene therapy vector safety. PMID:25365582

  3. Searching for the relevance of clonal and bud bank traits across floras and communities

    Czech Academy of Sciences Publication Activity Database

    Klimešová, Jitka; de Bello, Francesco; Herben, Tomáš

    2011-01-01

    Roč. 46, 2-3 (2011), 109-115 ISSN 1211-9520 R&D Projects: GA ČR GA526/09/0963; GA ČR GA526/07/0808 Institutional research plan: CEZ:AV0Z60050516 Keywords : clonal growth * bud bank * database Subject RIV: EF - Botanics Impact factor: 1.500, year: 2011

  4. Age and distribution of an evergreen clonal shrub in the Coweeta basin: Rhododendron maximum L

    Science.gov (United States)

    Katherine J. Elliott; James M. Vose

    2012-01-01

    Rhododendron maximum L. is an evergreen, clonal shrub that forms a dominant sub-canopy layer and is a key species in southern Appalachian forests. We investigated the age and distribution of R. maximum across the Coweeta Basin, a 1626 ha watershed in western North Carolina. We selected 16 perennial, second-order streams and used a Global Positioning System to establish...

  5. Clonal vs leaf-height-seed (LHS) traits: which are filtered more strongly across habitats?

    Czech Academy of Sciences Publication Activity Database

    E.-Vojtkó, A.; Freitag, M.; Bricca, A.; Martello, F.; Compañ, J. M.; Küttim, M.; Kun, R.; de Bello, Francesco; Klimešová, Jitka; Götzenberger, Lars

    2017-01-01

    Roč. 52, 3-4 (2017), s. 269-281 ISSN 1211-9520 R&D Projects: GA ČR(CZ) GA16-15012S Institutional support: RVO:67985939 Keywords : environmental filtering * functional diversity * clonal traits Subject RIV: EH - Ecology, Behaviour OBOR OECD: Ecology Impact factor: 1.017, year: 2016

  6. Reverse Transcriptase Mechanism of Somatic Hypermutation: 60 Years of Clonal Selection Theory

    Directory of Open Access Journals (Sweden)

    Edward J. Steele

    2017-11-01

    Full Text Available The evidence for the reverse transcriptase mechanism of somatic hypermutation is substantial and multifactorial. In this 60th anniversary year of the publication of Sir MacFarlane Burnet’s Clonal Selection Theory, the evidence is briefly reviewed and updated.

  7. Rapid clonal analysis of recurrent tuberculosis by direct MIRU-VNTR typing on stored isolates

    Directory of Open Access Journals (Sweden)

    de Viedma Darío

    2007-07-01

    Full Text Available Abstract Background The application of molecular tools to the analysis of tuberculosis has revealed examples of clonal complexity, such as exogenous reinfection, coinfection, microevolution or compartmentalization. The detection of clonal heterogeneity by standard genotyping approaches is laborious and often requires expertise. This restricts the rapid availability of Mycobacterium tuberculosis (MTB genotypes for clinical or therapeutic decision-making. A new PCR-based technique, MIRU-VNTR, has made it possible to genotype MTB in a time frame close to real-time fingerprinting. Our purpose was to evaluate the capacity of this technique to provide clinicians with a rapid discrimination between reactivation and exogenous reinfection and whether MIRU-VNTR makes it possible to obtain data directly from stored MTB isolates from recurrent episodes. Results We detected differences, between the MIRUtypes of recurrent isolates in 38.5% (5/13 of the cases studied. These included cases of i exogenous reinfection, often with more resistant strains, ii likely examples of microevolution, leading to the appearance of new clonal variants and iii a combination of microevolution, coinfection and competition. Conclusion MIRU-VNTR rapidly obtained clinically useful genotyping data in a challenging situation, directly from stored MTB isolates without subculturing them or purifying their DNA. Our results also mean that MIRU-VNTR could be applied for easy, rapid and affordable massive screening of collections of stored MTB isolates, which could establish the real dimension of clonal heterogeneity in MTB infection.

  8. Implications of self/non-self discrimination for spatial patterning of clonal plants

    Czech Academy of Sciences Publication Activity Database

    Herben, Tomáš; Novoplansky, A.

    2008-01-01

    Roč. 22, č. 3 (2008), s. 337-350 ISSN 0269-7653 R&D Projects: GA ČR GA206/06/0098 Institutional research plan: CEZ:AV0Z60050516 Keywords : clonal plants * clustering * competition * facilitation Subject RIV: EF - Botanics Impact factor: 3.448, year: 2008

  9. Physiological integration affects growth form and competitive ability in clonal plants

    Czech Academy of Sciences Publication Activity Database

    Herben, Tomáš

    2004-01-01

    Roč. 18, - (2004), s. 493-520 ISSN 0269-7653 R&D Projects: GA ČR(CZ) GA206/02/0953 Institutional research plan: CEZ:AV0Z6005908 Keywords : competitive ability * Physiological integration * clonal plants Subject RIV: EF - Botanics Impact factor: 3.215, year: 2004

  10. Clonal diversity and genetic differentiation of Maianthemum bifolium among forest fragments of different age

    NARCIS (Netherlands)

    Arens, P.F.P.; Grashof-Bokdam, C.J.; Sluis, van der T.; Smulders, M.J.M.

    2005-01-01

    Maianthemum bifolium Schmidt (May Lily) is a woodland species with low colonisation ability and high demands for seedling establishment conditions. To study the colonisation process, we analysed the clonal organisation and population structure of Maianthemum bifolium populations in a number of

  11. OCCURRENCE OF ANTIBIOTIC-RESISTANT UROPATHOGENIC ESCHERICHIA COLI CLONAL GROUP A IN WASTEWATER EFFLUENTS

    Science.gov (United States)

    Isolates of Escherichia coli belonging to clonal group A (CGA), a recently described disseminated cause of drug-resistant urinary tract infections in humans, were present in four of seven sewage effluents collected from geographically dispersed areas of the United States. ...

  12. Reproduction by seed and clonality in plants: correlated syndromes or independent strategies?

    Czech Academy of Sciences Publication Activity Database

    Herben, Tomáš; Tackenberg, O.; Klimešová, Jitka

    2016-01-01

    Roč. 104, č. 6 (2016), s. 1696-1706 ISSN 0022-0477 R&D Projects: GA ČR(CZ) GA16-19245S; GA ČR GB14-36079G Institutional support: RVO:67985939 Keywords : bud bank traits * clonal traits * CLOPLA database * dispersal * phylogenetic regressions Subject RIV: EF - Botanics Impact factor: 5.813, year: 2016

  13. Clonal growth forms in Arctic plants and their habitat preferences: a study from Petuniabukta, Spitsbergen

    Czech Academy of Sciences Publication Activity Database

    Klimešová, Jitka; Doležal, Jiří; Prach, Karel; Košnar, J.

    2012-01-01

    Roč. 33, č. 4 (2012), 421-442 ISSN 0138-0338 R&D Projects: GA MŠk LA341; GA AV ČR IAA600050802 Institutional research plan: CEZ:AV0Z60050516 Institutional support: RVO:67985939 Keywords : clonality * plants * Arctic Subject RIV: EH - Ecology, Behaviour Impact factor: 0.745, year: 2012

  14. Prevalence, serotype, virulence characteristics, clonality and antibiotic susceptibility of pathogenic Yersinia enterocolitica from swine feces

    Science.gov (United States)

    Introduction: Swine are the only known animal reservoir of Yersinia enterocolitica (YE), a human pathogen. Since YE is a fecal organism of swine, the primary goal of this study was to evaluate the prevalence, serotype, virulence plasmid (pYV)-associated characteristics, clonality, and antibiotic su...

  15. Get Tested for HIV

    Science.gov (United States)

    ... AIDS: What is HIV/AIDS? Women and HIV/AIDS Next section ... Tested? Why do I need to get tested for HIV? The only way to know if you have HIV is to get tested. Many people with HIV don’t have any symptoms. In the United States, about 1 in 7 ...

  16. Transmission of clonal chromosomal abnormalities in human hematopoietic stem and progenitor cells surviving radiation exposure

    Energy Technology Data Exchange (ETDEWEB)

    Kraft, Daniela, E-mail: d.kraft@gsi.de [GSI Helmholtz Center for Heavy Ion Research, Department of Biophysics, Planckstr. 1, 64291 Darmstadt (Germany); Institute for Transfusion Medicine und Immunohematology, DRK-Blutspendedienst Baden-Wuerttemberg—Hessen, Johann Wolfgang Goethe-University Hospital, Sandhofstrasse 1, 60528 Frankfurt (Germany); Ritter, Sylvia, E-mail: s.ritter@gsi.de [GSI Helmholtz Center for Heavy Ion Research, Department of Biophysics, Planckstr. 1, 64291 Darmstadt (Germany); Durante, Marco, E-mail: m.durante@gsi.de [GSI Helmholtz Center for Heavy Ion Research, Department of Biophysics, Planckstr. 1, 64291 Darmstadt (Germany); Institute for Condensed Matter Physics, Physics Department, Technical University Darmstadt, Hochschulstraße 6-8, 64289 Darmstadt (Germany); Seifried, Erhard, E-mail: e.seifried@blutspende.de [Institute for Transfusion Medicine und Immunohematology, DRK-Blutspendedienst Baden-Wuerttemberg—Hessen, Johann Wolfgang Goethe-University Hospital, Sandhofstrasse 1, 60528 Frankfurt (Germany); Fournier, Claudia, E-mail: c.fournier@gsi.de [GSI Helmholtz Center for Heavy Ion Research, Department of Biophysics, Planckstr. 1, 64291 Darmstadt (Germany); Tonn, Torsten, E-mail: t.tonn@blutspende.de [Institute for Transfusion Medicine und Immunohematology, DRK-Blutspendedienst Baden-Wuerttemberg—Hessen, Johann Wolfgang Goethe-University Hospital, Sandhofstrasse 1, 60528 Frankfurt (Germany); Technische Universität Dresden, Med. Fakultät Carl Gustav Carus, Institute for Transfusion Medicine Dresden, German Red Cross Blood Donation Service North-East, Blasewitzer Straße 68/70, 01307 Dresden (Germany)

    2015-07-15

    Highlights: • Radiation induced formation and transmission of chromosomal aberrations were assessed. • Cytogenetic analysis was performed in human CD34+ HSPC by mFISH. • We report transmission of stable aberrations in irradiated, clonally expanded HSPC. • Unstable aberrations in clonally expanded HSPC occur independently of irradiation. • Carbon ions and X-rays bear a similar risk for propagation of cytogenetic changes. - Abstract: In radiation-induced acute myeloid leukemia (rAML), clonal chromosomal abnormalities are often observed in bone marrow cells of patients, suggesting that their formation is crucial in the development of the disease. Since rAML is considered to originate from hematopoietic stem and progenitor cells (HSPC), we investigated the frequency and spectrum of radiation-induced chromosomal abnormalities in human CD34{sup +} cells. We then measured stable chromosomal abnormalities, a possible biomarker of leukemia risk, in clonally expanded cell populations which were grown for 14 days in a 3D-matrix (CFU-assay). We compared two radiation qualities used in radiotherapy, sparsely ionizing X-rays and densely ionizing carbon ions (29 and 60–85 keV/μm, doses between 0.5 and 4 Gy). Only a negligible number of de novo arising, unstable aberrations (≤0.05 aberrations/cell, 97% breaks) were measured in the descendants of irradiated HSPC. However, stable aberrations were detected in colonies formed by irradiated HSPC. All cells of the affected colonies exhibited one or more identical aberrations, indicating their clonal origin. The majority of the clonal rearrangements (92%) were simple exchanges such as translocations (77%) and pericentric inversions (15%), which are known to contribute to the development of rAML. Carbon ions were more efficient in inducing cell killing (maximum of ∼30–35% apoptotic cells for 2 Gy carbon ions compared to ∼25% for X-rays) and chromosomal aberrations in the first cell-cycle after exposure (∼70% and

  17. HIV-1 genetic diversity and its distribution characteristics among newly diagnosed HIV-1 individuals in Hebei province, China.

    Science.gov (United States)

    Lu, Xinli; Zhao, Cuiying; Wang, Wei; Nie, Chenxi; Zhang, Yuqi; Zhao, Hongru; Chen, Suliang; Cui, Ze

    2016-01-01

    Since the first HIV-1 case in 1989, Hebei province has presented a clearly rising trend of HIV-1 prevalence, and HIV-1 genetic diversity has become the vital barrier to HIV prevention and control in this area. To obtain detailed information of HIV-1 spread in different populations and in different areas of Hebei, a cross-sectional HIV-1 molecular epidemiological investigation was performed across the province. Blood samples of 154 newly diagnosed HIV-1 individuals were collected from ten prefectures in Hebei using stratified sampling. Partial gag and env genes were amplified and sequenced. HIV-1 genotypes were identified by phylogenetic tree analyses. Among the 139 subjects genotyped, six HIV-1 subtypes were identified successfully, including subtype B (41.0 %), CRF01_AE (40.3 %), CRF07_BC (11.5 %), CRF08_BC (4.3 %), unique recombinant forms (URFs) (1.4 %) and subtype C (1.4 %). Subtype B was identified as the most frequent subtype. Two URF recombination patterns were the same as CRF01_AE/B. HIV-1 genotype distribution showed a significant statistical difference in different demographic characteristics, such as source (P  0.05). The differences in HIV-1 genotype distribution were closely associated with transmission routes. Particularly, all six subtype strains were found in heterosexuals, showing that HIV-1 has spread from the high-risk populations to the general populations in Hebei, China. In addition, CRF01_AE instead of subtype B has become the major strain of HIV-1 infection among homosexuals. Our study revealed HIV-1 evolution and genotype distribution by investigating newly diagnosed HIV-1 individuals in Hebei, China. This study provides important information to enhance the strategic plan for HIV prevention and control in China.

  18. CXCR4-using HIV variants in a cohort of Black men who have sex with men: HIV Prevention Trials Network 061.

    Science.gov (United States)

    Chen, Iris; Huang, Wei; Connor, Matthew B; Frantzell, Arne; Cummings, Vanessa; Beauchamp, Geetha G; Griffith, Sam; Fields, Sheldon D; Scott, Hyman M; Shoptaw, Steven; Del Rio, Carlos; Magnus, Manya; Mannheimer, Sharon; Tieu, Hong-Van; Wheeler, Darrell P; Mayer, Kenneth H; Koblin, Beryl A; Eshleman, Susan H

    2016-07-01

    To evaluate factors associated with HIV tropism among Black men who have sex with men (MSM) in the United States enrolled in a clinical study (HIV Prevention Trials Network 061). HIV tropism was analyzed using a phenotypic assay (Trofile assay, Monogram Biosciences). Samples were analyzed from 43 men who were HIV infected at enrollment and reported either exclusive insertive intercourse or exclusive receptive intercourse; samples were also analyzed from 20 men who were HIV uninfected at enrollment and seroconverted during the study. Clonal analysis of individual viral variants was performed for seroconverters who had dual/mixed (DM) viruses. DM viruses were detected in samples from 11 (26%) of the 43 HIV-infected men analyzed at the enrollment visit; HIV tropism did not differ between those reporting exclusive insertive vs receptive intercourse. DM viruses were also detected in five (25%) of the 20 seroconverters. DM viruses were associated with lower CD4 cell counts. Seroconverters with DM viruses had dual-tropic viruses only or mixed populations of CCR5- and dual-tropic viruses. DM viruses were frequently detected among Black MSM in this study, including seroconverters. Further studies are needed to understand factors driving transmission and selection of CXCR4- and dual-tropic viruses among Black MSM.

  19. Drugs + HIV, Learn the Link

    Medline Plus

    Full Text Available ... of HIV in the United States, please visit: https://www.aids.gov/hiv-aids-basics/hiv-aids- ... HIV, STD, and TB Prevention. About HIV/AIDS. ( https://www.cdc.gov/actagainstaids/basics/whatishiv.html ). Atlanta, ...

  20. High resolution melting analysis (HRM) for the assessment of clonality in feline B-cell lymphomas.

    Science.gov (United States)

    Henrich, Manfred; Scheffold, Svenja; Hecht, Werner; Reinacher, Manfred

    2018-06-01

    Analysis of clonality is gaining importance in diagnosing lymphomas in veterinary medicine. Usually, PCR for the analysis of antigen receptor rearrangement (PARR) is followed by electrophoretic separation of the PCR products. Aim of this study was to test the feasibility of HRM for the assessment of clonality in B-cell lymphomas of cats. High resolution melting analysis differentiates PCR products by their different melting point using the decrease in fluorescence of an intercalating dye during melting of the PCR product. Additionally, the method is easy to use with no post-PCR manipulation of the samples. Forty-seven feline B-cell lymphomas and 31 reactive lymphatic proliferations of cats were investigated by PARR followed either by capillary electrophoresis or an HRM assay. To objectify the interpretation of the HRM results a recently published mathematical approach was applied to the melting curve. To overcome discrepancies between the visual interpretation and the mathematical approach, the latter was modified to include testing of reproducibility and recognition of pseudoclonality. In 11 of 47 lymphoma cases clonal populations were detectable by HRM assay compared to 14 of 47 lymphomas in which clonal populations were detected by capillary electrophoresis assay. Neither of the methods showed a clonal pattern in any of the reactive samples. However, the HRM assay showed a unique pattern in cases of follicular lymphatic hyperplasia that had no corresponding pattern in capillary electrophoresis. The capillary electrophoresis assay could identify 3 lymphomas that were not detected by the HRM assay and is therefore regarded superior to the HRM assay. The comparison however, was hampered by the overall bad performance of the PARR, that might be the consequence of insufficient primer binding due to somatic hypermutation of the binding sites during antigen stimulated proliferation of the B lymphocytes. Copyright © 2018 Elsevier B.V. All rights reserved.

  1. Bacterial clonal diagnostics as a tool for evidence-based empiric antibiotic selection.

    Directory of Open Access Journals (Sweden)

    Veronika Tchesnokova

    Full Text Available Despite the known clonal distribution of antibiotic resistance in many bacteria, empiric (pre-culture antibiotic selection still relies heavily on species-level cumulative antibiograms, resulting in overuse of broad-spectrum agents and excessive antibiotic/pathogen mismatch. Urinary tract infections (UTIs, which account for a large share of antibiotic use, are caused predominantly by Escherichia coli, a highly clonal pathogen. In an observational clinical cohort study of urgent care patients with suspected UTI, we assessed the potential for E. coli clonal-level antibiograms to improve empiric antibiotic selection. A novel PCR-based clonotyping assay was applied to fresh urine samples to rapidly detect E. coli and the urine strain's clonotype. Based on a database of clonotype-specific antibiograms, the acceptability of various antibiotics for empiric therapy was inferred using a 20%, 10%, and 30% allowed resistance threshold. The test's performance characteristics and possible effects on prescribing were assessed. The rapid test identified E. coli clonotypes directly in patients' urine within 25-35 minutes, with high specificity and sensitivity compared to culture. Antibiotic selection based on a clonotype-specific antibiogram could reduce the relative likelihood of antibiotic/pathogen mismatch by ≥ 60%. Compared to observed prescribing patterns, clonal diagnostics-guided antibiotic selection could safely double the use of trimethoprim/sulfamethoxazole and minimize fluoroquinolone use. In summary, a rapid clonotyping test showed promise for improving empiric antibiotic prescribing for E. coli UTI, including reversing preferential use of fluoroquinolones over trimethoprim/sulfamethoxazole. The clonal diagnostics approach merges epidemiologic surveillance, antimicrobial stewardship, and molecular diagnostics to bring evidence-based medicine directly to the point of care.

  2. Bacterial clonal diagnostics as a tool for evidence-based empiric antibiotic selection.

    Science.gov (United States)

    Tchesnokova, Veronika; Avagyan, Hovhannes; Rechkina, Elena; Chan, Diana; Muradova, Mariya; Haile, Helen Ghirmai; Radey, Matthew; Weissman, Scott; Riddell, Kim; Scholes, Delia; Johnson, James R; Sokurenko, Evgeni V

    2017-01-01

    Despite the known clonal distribution of antibiotic resistance in many bacteria, empiric (pre-culture) antibiotic selection still relies heavily on species-level cumulative antibiograms, resulting in overuse of broad-spectrum agents and excessive antibiotic/pathogen mismatch. Urinary tract infections (UTIs), which account for a large share of antibiotic use, are caused predominantly by Escherichia coli, a highly clonal pathogen. In an observational clinical cohort study of urgent care patients with suspected UTI, we assessed the potential for E. coli clonal-level antibiograms to improve empiric antibiotic selection. A novel PCR-based clonotyping assay was applied to fresh urine samples to rapidly detect E. coli and the urine strain's clonotype. Based on a database of clonotype-specific antibiograms, the acceptability of various antibiotics for empiric therapy was inferred using a 20%, 10%, and 30% allowed resistance threshold. The test's performance characteristics and possible effects on prescribing were assessed. The rapid test identified E. coli clonotypes directly in patients' urine within 25-35 minutes, with high specificity and sensitivity compared to culture. Antibiotic selection based on a clonotype-specific antibiogram could reduce the relative likelihood of antibiotic/pathogen mismatch by ≥ 60%. Compared to observed prescribing patterns, clonal diagnostics-guided antibiotic selection could safely double the use of trimethoprim/sulfamethoxazole and minimize fluoroquinolone use. In summary, a rapid clonotyping test showed promise for improving empiric antibiotic prescribing for E. coli UTI, including reversing preferential use of fluoroquinolones over trimethoprim/sulfamethoxazole. The clonal diagnostics approach merges epidemiologic surveillance, antimicrobial stewardship, and molecular diagnostics to bring evidence-based medicine directly to the point of care.

  3. Contrasting HIV phylogenetic relationships and V3 loop protein similarities

    Energy Technology Data Exchange (ETDEWEB)

    Korber, B. (Los Alamos National Lab., NM (United States) Santa Fe Inst., NM (United States)); Myers, G. (Los Alamos National Lab., NM (United States))

    1992-01-01

    At least five distinct sequence subtypes of HIV-I can be identified from the major centers of the AMS pandemic. While it is too early to tell whether these subtypes are serologically or phenotypically similar or distinct in terms of properties such as pathogenicity and transmissibility, we can begin to investigate their potential for phenotypic divergence at the protein sequence level. Phylogenetic analysis of HIV DNA sequences is being widely used to examine lineages of different viral strains as they evolve and spread throughout the globe. We have identified five distinct HIV-1 subtypes (designated A-E), or clades, based on phylogenetic clustering patterns generated from genetic information from both the gag and envelope (env) genes from a spectrum of international isolates. Our initial observations concerning both HIV-1 and HIV-2 sequences indicate that conserved patterns in protein chemistry may indeed exist across distant lineages. Such patterns in V3 loop amino acid chemistry may be indicative of stable lineages or convergence within this highly variable, though functionally and immunologically critical, region. We think that there may be parallels between the apparently stable HIV-2 V3 lineage and the previously mentioned HIV-1 V3 loops which are very similar at the protein level despite being distant by cladistic analysis, and which do not possess the distinctive positively charged residues. Highly conserved V3 loop protein sequences are also encountered in SIVAGMs and CIVs (chimpanzee viral strains), which do not appear to be pathogenic in their wild-caught natural hosts.

  4. Contrasting HIV phylogenetic relationships and V3 loop protein similarities

    Energy Technology Data Exchange (ETDEWEB)

    Korber, B. [Los Alamos National Lab., NM (United States)]|[Santa Fe Inst., NM (United States); Myers, G. [Los Alamos National Lab., NM (United States)

    1992-12-31

    At least five distinct sequence subtypes of HIV-I can be identified from the major centers of the AMS pandemic. While it is too early to tell whether these subtypes are serologically or phenotypically similar or distinct in terms of properties such as pathogenicity and transmissibility, we can begin to investigate their potential for phenotypic divergence at the protein sequence level. Phylogenetic analysis of HIV DNA sequences is being widely used to examine lineages of different viral strains as they evolve and spread throughout the globe. We have identified five distinct HIV-1 subtypes (designated A-E), or clades, based on phylogenetic clustering patterns generated from genetic information from both the gag and envelope (env) genes from a spectrum of international isolates. Our initial observations concerning both HIV-1 and HIV-2 sequences indicate that conserved patterns in protein chemistry may indeed exist across distant lineages. Such patterns in V3 loop amino acid chemistry may be indicative of stable lineages or convergence within this highly variable, though functionally and immunologically critical, region. We think that there may be parallels between the apparently stable HIV-2 V3 lineage and the previously mentioned HIV-1 V3 loops which are very similar at the protein level despite being distant by cladistic analysis, and which do not possess the distinctive positively charged residues. Highly conserved V3 loop protein sequences are also encountered in SIVAGMs and CIVs (chimpanzee viral strains), which do not appear to be pathogenic in their wild-caught natural hosts.

  5. Inference with viral quasispecies diversity indices: clonal and NGS approaches.

    Science.gov (United States)

    Gregori, Josep; Salicrú, Miquel; Domingo, Esteban; Sanchez, Alex; Esteban, Juan I; Rodríguez-Frías, Francisco; Quer, Josep

    2014-04-15

    Given the inherent dynamics of a viral quasispecies, we are often interested in the comparison of diversity indices of sequential samples of a patient, or in the comparison of diversity indices of virus in groups of patients in a treated versus control design. It is then important to make sure that the diversity measures from each sample may be compared with no bias and within a consistent statistical framework. In the present report, we review some indices often used as measures for viral quasispecies complexity and provide means for statistical inference, applying procedures taken from the ecology field. In particular, we examine the Shannon entropy and the mutation frequency, and we discuss the appropriateness of different normalization methods of the Shannon entropy found in the literature. By taking amplicons ultra-deep pyrosequencing (UDPS) raw data as a surrogate of a real hepatitis C virus viral population, we study through in-silico sampling the statistical properties of these indices under two methods of viral quasispecies sampling, classical cloning followed by Sanger sequencing (CCSS) and next-generation sequencing (NGS) such as UDPS. We propose solutions specific to each of the two sampling methods-CCSS and NGS-to guarantee statistically conforming conclusions as free of bias as possible. josep.gregori@gmail.com Supplementary information: Supplementary data are available at Bioinformatics online. © The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

  6. Asymptomatic HIV infection

    Science.gov (United States)

    ... of HIV/AIDS during which there are no symptoms of HIV infection. During this phase, the immune system in someone with HIV slowly weakens, but the person has no symptoms. How long this phase lasts depends on how ...

  7. HIV and Pulmonary Hypertension

    Science.gov (United States)

    ... What do I need to know about pulmonary hypertension in connection with HIV? Although pulmonary hypertension and ... Should an HIV patient be tested for pulmonary hypertension? HIV patients know that medical supervision is critical ...

  8. HIV epidemiology. The early spread and epidemic ignition of HIV-1 in human populations.

    Science.gov (United States)

    Faria, Nuno R; Rambaut, Andrew; Suchard, Marc A; Baele, Guy; Bedford, Trevor; Ward, Melissa J; Tatem, Andrew J; Sousa, João D; Arinaminpathy, Nimalan; Pépin, Jacques; Posada, David; Peeters, Martine; Pybus, Oliver G; Lemey, Philippe

    2014-10-03

    Thirty years after the discovery of HIV-1, the early transmission, dissemination, and establishment of the virus in human populations remain unclear. Using statistical approaches applied to HIV-1 sequence data from central Africa, we show that from the 1920s Kinshasa (in what is now the Democratic Republic of Congo) was the focus of early transmission and the source of pre-1960 pandemic viruses elsewhere. Location and dating estimates were validated using the earliest HIV-1 archival sample, also from Kinshasa. The epidemic histories of HIV-1 group M and nonpandemic group O were similar until ~1960, after which group M underwent an epidemiological transition and outpaced regional population growth. Our results reconstruct the early dynamics of HIV-1 and emphasize the role of social changes and transport networks in the establishment of this virus in human populations. Copyright © 2014, American Association for the Advancement of Science.

  9. Conservation of HIV-1 T cell epitopes across time and clades

    DEFF Research Database (Denmark)

    Levitz, Lauren; Koita, Ousmane A; Sangare, Kotou

    2012-01-01

    HIV genomic sequence variability has complicated efforts to generate an effective globally relevant vaccine. Regions of the viral genome conserved in sequence and across time may represent the "Achilles' heel" of HIV. In this study, highly conserved T-cell epitopes were selected using immunoinfor...

  10. Recognition of HIV-1 peptides by host CTL is related to HIV-1 similarity to human proteins.

    Directory of Open Access Journals (Sweden)

    Morgane Rolland

    Full Text Available BACKGROUND: While human immunodeficiency virus type 1 (HIV-1-specific cytotoxic T lymphocytes preferentially target specific regions of the viral proteome, HIV-1 features that contribute to immune recognition are not well understood. One hypothesis is that similarities between HIV and human proteins influence the host immune response, i.e., resemblance between viral and host peptides could preclude reactivity against certain HIV epitopes. METHODOLOGY/PRINCIPAL FINDINGS: We analyzed the extent of similarity between HIV-1 and the human proteome. Proteins from the HIV-1 B consensus sequence from 2001 were dissected into overlapping k-mers, which were then probed against a non-redundant database of the human proteome in order to identify segments of high similarity. We tested the relationship between HIV-1 similarity to host encoded peptides and immune recognition in HIV-infected individuals, and found that HIV immunogenicity could be partially modulated by the sequence similarity to the host proteome. ELISpot responses to peptides spanning the entire viral proteome evaluated in 314 individuals showed a trend indicating an inverse relationship between the similarity to the host proteome and the frequency of recognition. In addition, analysis of responses by a group of 30 HIV-infected individuals against 944 overlapping peptides representing a broad range of individual HIV-1B Nef variants, affirmed that the degree of similarity to the host was significantly lower for peptides with reactive epitopes than for those that were not recognized. CONCLUSIONS/SIGNIFICANCE: Our results suggest that antigenic motifs that are scarcely represented in human proteins might represent more immunogenic CTL targets not selected against in the host. This observation could provide guidance in the design of more effective HIV immunogens, as sequences devoid of host-like features might afford superior immune reactivity.

  11. Occurrence, species distribution, antimicrobial resistance and clonality of methicillin- and erythromycin-resistant staphylococci in the nasal cavity of domestic animals

    DEFF Research Database (Denmark)

    Bagcigil, Funda A.; Moodley, Arshnee; Baptiste, Keith E.

    2007-01-01

    beta-Lactams and macrolides are important antibiotics for treatment of staphylococcal infections in both humans and animals. The aim of the study was to investigate the occurrence, species distribution and clonality of methicillin and erythromycin-resistant staphylococci in the nasal cavity of dogs......, horses, pigs, and cattle in Denmark. Nasal swabs were collected from a total of 400 animals, including 100 individuals of each species. Methicillin and erythromycin-resistant staphylococci were isolated on selective media, identified by 16S rDNA sequencing, and typed by pulsed field gel electrophoresis...... (PFGE). Methicillin-resistant coagulase-negative staphylococci (MRCoNS) harbouring mecA were isolated from horses (50%) and dogs (13%), but not from food animals. The species identified were S. haemolyticus (n = 21), S. vitulinus (n = 19), S. sciuri (n = 13), S. epidermidis (n = 8), and S. warneri (n...

  12. Clonal variation of DNA repair in a human glioma cell line This study was supported by the Cancer Research Campaign and the Bob Champion Cancer Trust

    International Nuclear Information System (INIS)

    Powell, Simon; McMillan, T.J.

    1991-01-01

    Clonal heterogeneity in response to ionizing radiation was found for a human glioma cell line, IN859. The authors have investigated the most sensitive clone, the most resistant clone and the parent line for differences in DNA repair fidelity using the method of plasmid reconstitution. Significant differences in repair fidelity were found between the two clones, and between the sensitive clone and the parent line. The resistant clone and the parent lines showed the greater repair fidelity. A comparison of two different restriction enzymes, which cleave the plasmid with blunt or cohesive-ended double-strand breaks, did not reveal differences in repair fidelity. Equal numbers of plasmids were integrated in each cell line, but the sensitive clone showed a higher frequency of misrepair of cleaved plasmid. Misrepair was characterized by partial or complete loss of sequence at the site of plasmid cleavage. It is concluded that the radiosensitive clone exhibits increased misrepair. (author). 15 refs.; 5 figs.; 2 tabs

  13. A Livestock-Associated, Multidrug-Resistant, Methicillin-Resistant Staphylococcus aureus Clonal Complex 97 Lineage Spreading in Dairy Cattle and Pigs in Italy

    DEFF Research Database (Denmark)

    Feltrin, Fabiola; Alba, Patricia; Kraushaar, Britta

    2016-01-01

    by macrorestriction pulsed-field gel electrophoresis (PFGE) analysis, multilocus sequence typing (MLST), spa typing, staphylococcal cassette chromosome mec (SCCmec) typing, and antimicrobial resistance pattern analysis. Virulence and resistance genes were investigated by PCR and microarray analysis. Most...... resistance, fluoroquinolone resistance (n = 33), tet(K) in 32/37 tet(M)-positive isolates, and blaZ in almost all MRSA isolates. Few host-associated differences were detected among CC97 MRSA isolates: their extensive MDR nature in both pigs and dairy cattle may be a consequence of a spillback from pigs......Pandemic methicillin-resistant Staphylococcus aureus (MRSA) clonal complex 97 (CC97) lineages originated from livestock-to-human host jumps. In recent years, CC97 has become one of the major MRSA lineages detected in Italian farmed animals. The aim of this study was to characterize and analyze...

  14. Transmission of clonal hepatitis C virus genomes reveals the dominant but transitory role of CD8¿ T cells in early viral evolution

    DEFF Research Database (Denmark)

    Callendret, Benoît; Bukh, Jens; Eccleston, Heather B

    2011-01-01

    occurred slowly over several years of chronic infection. Together these observations indicate that during acute hepatitis C, virus evolution was driven primarily by positive selection pressure exerted by CD8(+) T cells. This influence of immune pressure on viral evolution appears to subside as chronic......The RNA genome of the hepatitis C virus (HCV) diversifies rapidly during the acute phase of infection, but the selective forces that drive this process remain poorly defined. Here we examined whether Darwinian selection pressure imposed by CD8(+) T cells is a dominant force driving early amino acid...... replacement in HCV viral populations. This question was addressed in two chimpanzees followed for 8 to 10 years after infection with a well-defined inoculum composed of a clonal genotype 1a (isolate H77C) HCV genome. Detailed characterization of CD8(+) T cell responses combined with sequencing of recovered...

  15. HIV coreceptor phenotyping in the clinical setting.

    Science.gov (United States)

    Low, Andrew J; Swenson, Luke C; Harrigan, P Richard

    2008-01-01

    The introduction of CCR5 antagonists increases the options available for constructing antiretroviral regimens. However, this option is coupled with the caveat that patients should be tested for HIV coreceptor tropism prior to initiating CCR5 antagonist-based therapy. Failure to screen for CXCR4 usage increases the risk of using an ineffective drug, thus reducing the likelihood of viral suppression and increasing their risk for developing antiretroviral resistance. This review discusses current and future methods of determining HIV tropism, with a focus on their utility in the clinical setting for screening purposes. Some of these methods include recombinant phenotypic tests, such as the Monogram Trofile assay, as well as genotype-based predictors, heteroduplex tracking assays, and flow cytometry based methods. Currently, the best evidence supports the use of phenotypic methods, although other methods of screening for HIV coreceptor usage prior to the administration of CCR5 antagonists may reduce costs and increase turnaround time over phenotypic methods. The presence of low levels of X4 virus is a challenge to all assay methods, resulting in reduced sensitivity in clinical, patient-derived samples when compared to clonally derived samples. Gaining a better understanding of the output of these assays and correlating them with clinical progression and therapy response will provide some indication on how both genotype-based, and phenotypic assays for determining HIV coreceptor usage can be improved. In addition, leveraging new technologies capable of detecting low-level minority species may provide the most significant advances in ensuring that individuals with low levels of dual/mixed tropic virus are not inadvertently prescribed CCR5 antagonists.

  16. Significant spread of extensively drug-resistant Acinetobacter baumannii genotypes of clonal complex 92 among intensive care unit patients in a university hospital in southern Iran.

    Science.gov (United States)

    Saffari, Fereshteh; Monsen, Tor; Karmostaji, Afsaneh; Azimabad, Fahimeh Bahadori; Widerström, Micael

    2017-11-01

    Infections associated with Acinetobacter baumannii represent an increasing threat in healthcare settings. Therefore, we investigated the epidemiological relationship between clinical isolates of A. baumannii obtained from patients in a university hospital in Bandar Abbas in southern Iran. Sixty-four consecutive non-duplicate clinical isolates collected during 2014-2015 were subjected to susceptibility testing, clonal relationship analysis using PFGE, multilocus variable-number tandem-repeat analysis (MLVA) and multilocus sequence typing (MLST), and examined for the presence of carbapenemases and integrons. Almost all A. baumannii isolates were extensively drug-resistant (XDR; 98 %) and carried an OXA carbapenemase gene (blaOXA-23-like; 98 %) and class 1 integrons (48 %). PFGE and MLST analysis identified three major genotypes, all belonging to clonal complex 92 (CC92): sequence type 848 (ST848) (n=23), ST451 (n=16) and ST195 (n=8). CC92 has previously been documented in the hospital setting in northern Iran, and ST195 has been reported in Arab States of the Persian Gulf. These data suggest national and global transmission of A. baumannii CC92. This report demonstrates the occurrence and potential spread of closely related XDR genotypes of A. baumannii CC92 within a university hospital in southern Iran. These genotypes were found in the majority of the investigated isolates, showed high prevalence of blaOXA-23 and integron class 1, and were associated with stay in the intensive care unit. Very few treatment options remain for healthcare-adapted XDR A. baumannii, and hence effective measures are desperately needed to reduce the spread of these strains and resultant infections in the healthcare setting.

  17. Enhanced discrimination of highly clonal ST22-methicillin-resistant Staphylococcus aureus IV isolates achieved by combining spa, dru, and pulsed-fi