Multilocus Sequence Typing Analysis of Staphylococcus lugdunensis Implies a Clonal Population Structure

PubMed Central

Chassain, Benoît; Lemée, Ludovic; Didi, Jennifer; Thiberge, Jean-Michel; Brisse, Sylvain; Pons, Jean-Louis

2012-01-01

Staphylococcus lugdunensis is recognized as one of the major pathogenic species within the genus Staphylococcus, even though it belongs to the coagulase-negative group. A multilocus sequence typing (MLST) scheme was developed to study the genetic relationships and population structure of 87 S. lugdunensis isolates from various clinical and geographic sources by DNA sequence analysis of seven housekeeping genes (aroE, dat, ddl, gmk, ldh, recA, and yqiL). The number of alleles ranged from four (gmk and ldh) to nine (yqiL). Allelic profiles allowed the definition of 20 different sequence types (STs) and five clonal complexes. The 20 STs lacked correlation with geographic source. Isolates recovered from hematogenic infections (blood or osteoarticular isolates) or from skin and soft tissue infections did not cluster in separate lineages. Penicillin-resistant isolates clustered mainly in one clonal complex, unlike glycopeptide-tolerant isolates, which did not constitute a distinct subpopulation within S. lugdunensis. Phylogenies from the sequences of the seven individual housekeeping genes were congruent, indicating a predominantly mutational evolution of these genes. Quantitative analysis of the linkages between alleles from the seven loci revealed a significant linkage disequilibrium, thus confirming a clonal population structure for S. lugdunensis. This first MLST scheme for S. lugdunensis provides a new tool for investigating the macroepidemiology and phylogeny of this unusually virulent coagulase-negative Staphylococcus. PMID:22785196

Clonal growth and plant species abundance.

PubMed

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 performance parameters provide a practical approach to assessing the roles of clonal growth morphological traits (and LHS traits) for large sets of species. © The Author 2014. Published by Oxford University Press on behalf of the Annals of Botany Company. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Origins of multicellular evolvability in snowflake yeast

PubMed Central

Ratcliff, William C.; Fankhauser, Johnathon D.; Rogers, David W.; Greig, Duncan; Travisano, Michael

2015-01-01

Complex life has arisen through a series of ‘major transitions’ in which collectives of formerly autonomous individuals evolve into a single, integrated organism. A key step in this process is the origin of higher-level evolvability, but little is known about how higher-level entities originate and gain the capacity to evolve as an individual. Here we report a single mutation that not only creates a new level of biological organization, but also potentiates higher-level evolvability. Disrupting the transcription factor ACE2 in Saccharomyces cerevisiae prevents mother–daughter cell separation, generating multicellular ‘snowflake’ yeast. Snowflake yeast develop through deterministic rules that produce geometrically defined clusters that preclude genetic conflict and display a high broad-sense heritability for multicellular traits; as a result they are preadapted to multicellular adaptation. This work demonstrates that simple microevolutionary changes can have profound macroevolutionary consequences, and suggests that the formation of clonally developing clusters may often be the first step to multicellularity. PMID:25600558

Establishment of high reciprocal connectivity between clonal cortical neurons is regulated by the Dnmt3b DNA methyltransferase and clustered protocadherins.

PubMed

Tarusawa, Etsuko; Sanbo, Makoto; Okayama, Atsushi; Miyashita, Toshio; Kitsukawa, Takashi; Hirayama, Teruyoshi; Hirabayashi, Takahiro; Hasegawa, Sonoko; Kaneko, Ryosuke; Toyoda, Shunsuke; Kobayashi, Toshihiro; Kato-Itoh, Megumi; Nakauchi, Hiromitsu; Hirabayashi, Masumi; Yagi, Takeshi; Yoshimura, Yumiko

2016-12-02

The specificity of synaptic connections is fundamental for proper neural circuit function. Specific neuronal connections that underlie information processing in the sensory cortex are initially established without sensory experiences to a considerable extent, and then the connections are individually refined through sensory experiences. Excitatory neurons arising from the same single progenitor cell are preferentially connected in the postnatal cortex, suggesting that cell lineage contributes to the initial wiring of neurons. However, the postnatal developmental process of lineage-dependent connection specificity is not known, nor how clonal neurons, which are derived from the same neural stem cell, are stamped with the identity of their common neural stem cell and guided to form synaptic connections. We show that cortical excitatory neurons that arise from the same neural stem cell and reside within the same layer preferentially establish reciprocal synaptic connections in the mouse barrel cortex. We observed a transient increase in synaptic connections between clonal but not nonclonal neuron pairs during postnatal development, followed by selective stabilization of the reciprocal connections between clonal neuron pairs. Furthermore, we demonstrate that selective stabilization of the reciprocal connections between clonal neuron pairs is impaired by the deficiency of DNA methyltransferase 3b (Dnmt3b), which determines DNA-methylation patterns of genes in stem cells during early corticogenesis. Dnmt3b regulates the postnatal expression of clustered protocadherin (cPcdh) isoforms, a family of adhesion molecules. We found that cPcdh deficiency in clonal neuron pairs impairs the whole process of the formation and stabilization of connections to establish lineage-specific connection reciprocity. Our results demonstrate that local, reciprocal neural connections are selectively formed and retained between clonal neurons in layer 4 of the barrel cortex during postnatal development, and that Dnmt3b and cPcdhs are required for the establishment of lineage-specific reciprocal connections. These findings indicate that lineage-specific connection reciprocity is predetermined by Dnmt3b during embryonic development, and that the cPcdhs contribute to postnatal cortical neuron identification to guide lineage-dependent synaptic connections in the neocortex.

Aging, clonal hematopoiesis and preleukemia: not just bad luck?

PubMed

Shlush, Liran I; Zandi, Sasan; Itzkovitz, Shalev; Schuh, Andre C

2015-11-01

Chronological human aging is associated with a number of changes in the hematopoietic system, occurring at many levels from stem to mature cells, and the marrow microenvironment as well. This review will focus mainly on the aging of hematopoietic stem and progenitor cells (HSPCs), and on the associated increases in the incidence of hematological malignancies. HSPCs manifest reduced function and acquire molecular changes with chronological aging. Furthermore, while for many years it has been known that the human hematopoietic system becomes increasingly clonal with chronological aging (clonal hematopoiesis), only in the last few years has it become clear that clonal hematopoiesis may result from the accumulation of preleukemic mutations in HSPCs. Such mutations confer a selective advantage that leads to clonal hematopoiesis, and that may occasionally result in the development of leukemia, and define the existence of both preleukemic stem cells, and of 'preleukemia' as a clinical entity. While it is well appreciated that clonal hematopoiesis is very common in the elderly, several questions remain unanswered: why and how does clonal hematopoiesis develop? How is clonal hematopoiesis related to the age-related changes observed in the hematopoietic system? And why do only some individuals with clonal hematopoiesis develop leukemia?

Dispersion of Multidrug-Resistant Enterococcus faecium Isolates Belonging to Major Clonal Complexes in Different Portuguese Settings▿

PubMed Central

Freitas, Ana R.; Novais, Carla; Ruiz-Garbajosa, Patricia; Coque, Teresa M.; Peixe, Luísa

2009-01-01

The population structure of 56 Enterococcus faecium isolates selected from a collection of enterococci from humans, animals, and the environment in Portugal (1997 to 2007) was analyzed by multilocus sequence typing. We identified 41 sequence types clustering into CC17, CC5, CC9, CC22 and CC94, all clonal lineages comprising isolates from different hosts. Our findings highlight the role of community-associated hosts as reservoirs of enterococci able to cause human infections. PMID:19447948

Clonal evolution revealed by whole genome sequencing in a case of primary myelofibrosis transformed to secondary acute myeloid leukemia.

PubMed

Engle, E K; Fisher, D A C; Miller, C A; McLellan, M D; Fulton, R S; Moore, D M; Wilson, R K; Ley, T J; Oh, S T

2015-04-01

Clonal architecture in myeloproliferative neoplasms (MPNs) is poorly understood. Here we report genomic analyses of a patient with primary myelofibrosis (PMF) transformed to secondary acute myeloid leukemia (sAML). Whole genome sequencing (WGS) was performed on PMF and sAML diagnosis samples, with skin included as a germline surrogate. Deep sequencing validation was performed on the WGS samples and an additional sample obtained during sAML remission/relapsed PMF. Clustering analysis of 649 validated somatic single-nucleotide variants revealed four distinct clonal groups, each including putative driver mutations. The first group (including JAK2 and U2AF1), representing the founding clone, included mutations with high frequency at all three disease stages. The second clonal group (including MYB) was present only in PMF, suggesting the presence of a clone that was dispensable for transformation. The third group (including ASXL1) contained mutations with low frequency in PMF and high frequency in subsequent samples, indicating evolution of the dominant clone with disease progression. The fourth clonal group (including IDH1 and RUNX1) was acquired at sAML transformation and was predominantly absent at sAML remission/relapsed PMF. Taken together, these findings illustrate the complex clonal dynamics associated with disease evolution in MPNs and sAML.

Clonal architecture of secondary acute myeloid leukemia defined by single-cell sequencing.

PubMed

Hughes, Andrew E O; Magrini, Vincent; Demeter, Ryan; Miller, Christopher A; Fulton, Robert; Fulton, Lucinda L; Eades, William C; Elliott, Kevin; Heath, Sharon; Westervelt, Peter; Ding, Li; Conrad, Donald F; White, Brian S; Shao, Jin; Link, Daniel C; DiPersio, John F; Mardis, Elaine R; Wilson, Richard K; Ley, Timothy J; Walter, Matthew J; Graubert, Timothy A

2014-07-01

Next-generation sequencing has been used to infer the clonality of heterogeneous tumor samples. These analyses yield specific predictions-the population frequency of individual clones, their genetic composition, and their evolutionary relationships-which we set out to test by sequencing individual cells from three subjects diagnosed with secondary acute myeloid leukemia, each of whom had been previously characterized by whole genome sequencing of unfractionated tumor samples. Single-cell mutation profiling strongly supported the clonal architecture implied by the analysis of bulk material. In addition, it resolved the clonal assignment of single nucleotide variants that had been initially ambiguous and identified areas of previously unappreciated complexity. Accordingly, we find that many of the key assumptions underlying the analysis of tumor clonality by deep sequencing of unfractionated material are valid. Furthermore, we illustrate a single-cell sequencing strategy for interrogating the clonal relationships among known variants that is cost-effective, scalable, and adaptable to the analysis of both hematopoietic and solid tumors, or any heterogeneous population of cells.

Next-generation sequencing for typing and detection of resistance genes: performance of a new commercial method during an outbreak of extended-spectrum-beta-lactamase-producing Escherichia coli.

PubMed

Veenemans, J; Overdevest, I T; Snelders, E; Willemsen, I; Hendriks, Y; Adesokan, A; Doran, G; Bruso, S; Rolfe, A; Pettersson, A; Kluytmans, J A J W

2014-07-01

Next-generation sequencing (NGS) has the potential to provide typing results and detect resistance genes in a single assay, thus guiding timely treatment decisions and allowing rapid tracking of transmission of resistant clones. We evaluated the performance of a new NGS assay (Hospital Acquired Infection BioDetection System; Pathogenica) during an outbreak of sequence type 131 (ST131) Escherichia coli infections in a nursing home in The Netherlands. The assay was performed on 56 extended-spectrum-beta-lactamase (ESBL) E. coli isolates collected during 2 prevalence surveys (March and May 2013). Typing results were compared to those of amplified fragment length polymorphism (AFLP), whereby we visually assessed the agreement of the BioDetection phylogenetic tree with clusters defined by AFLP. A microarray was considered the gold standard for detection of resistance genes. AFLP identified a large cluster of 31 indistinguishable isolates on adjacent departments, indicating clonal spread. The BioDetection phylogenetic tree showed that all isolates of this outbreak cluster were strongly related, while the further arrangement of the tree also largely agreed with other clusters defined by AFLP. The BioDetection assay detected ESBL genes in all but 1 isolate (sensitivity, 98%) but was unable to discriminate between ESBL and non-ESBL TEM and SHV beta-lactamases or to specify CTX-M genes by group. The performance of the hospital-acquired infection (HAI) BioDetection System for typing of E. coli isolates compared well with the results of AFLP. Its performance with larger collections from different locations, and for typing of other species, was not evaluated and needs further study. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

Inferring a Population Structure for Staphylococcus epidermidis from Multilocus Sequence Typing Data▿

PubMed Central

Miragaia, M.; Thomas, J. C.; Couto, I.; Enright, M. C.; de Lencastre, H.

2007-01-01

Despite its importance as a human pathogen, information on population structure and global epidemiology of Staphylococcus epidermidis is scarce and the relative importance of the mechanisms contributing to clonal diversification is unknown. In this study, we addressed these issues by analyzing a representative collection of S. epidermidis isolates from diverse geographic and clinical origins using multilocus sequence typing (MLST). Additionally, we characterized the mobile element (SCCmec) carrying the genetic determinant of methicillin resistance. The 217 S. epidermidis isolates from our collection were split by MLST into 74 types, suggesting a high level of genetic diversity. Analysis of MLST data using the eBURST algorithm revealed the existence of nine epidemic clonal lineages that were disseminated worldwide. One single clonal lineage (clonal complex 2) comprised 74% of the isolates, whereas the remaining isolates were clustered into 8 minor clonal lineages and 13 singletons. According to our evolutionary model, SCCmec was acquired at least 56 times by S. epidermidis. Although geographic dissemination of S. epidermidis strains and the value of the index of association between the alleles, 0.2898 (P < 0.05), support the clonality of S. epidermidis species, examination of the sequence changes at MLST loci during clonal diversification showed that recombination gives rise to new alleles approximately twice as frequently as point mutations. We suggest that S. epidermidis has a population with an epidemic structure, in which nine clones have emerged upon a recombining background and evolved quickly through frequent transfer of genetic mobile elements, including SCCmec. PMID:17220222

Identification of Novel Sequence Types among Staphylococcus haemolyticus Isolated from Variety of Infections in India.

PubMed

Panda, Sasmita; Jena, Smrutiti; Sharma, Savitri; Dhawan, Benu; Nath, Gopal; Singh, Durg Vijai

2016-01-01

The aim of this study was to determine sequence types of 34 S. haemolyticus strains isolated from a variety of infections between 2013 and 2016 in India by MLST. The MEGA5.2 software was used to align and compare the nucleotide sequences. The advanced cluster analysis was performed to define the clonal complexes. MLST analysis showed 24 new sequence types (ST) among S. haemolyticus isolates, irrespective of sources and place of isolation. The finding of this study allowed to set up an MLST database on the PubMLST.org website using BIGSdb software and made available at http://pubmlst.org/shaemolyticus/. The data of this study thus suggest that MLST can be used to study population structure and diversity among S. haemolyticus isolates.

Identification of Novel Sequence Types among Staphylococcus haemolyticus Isolated from Variety of Infections in India

PubMed Central

Panda, Sasmita; Jena, Smrutiti; Sharma, Savitri; Dhawan, Benu; Nath, Gopal

2016-01-01

The aim of this study was to determine sequence types of 34 S. haemolyticus strains isolated from a variety of infections between 2013 and 2016 in India by MLST. The MEGA5.2 software was used to align and compare the nucleotide sequences. The advanced cluster analysis was performed to define the clonal complexes. MLST analysis showed 24 new sequence types (ST) among S. haemolyticus isolates, irrespective of sources and place of isolation. The finding of this study allowed to set up an MLST database on the PubMLST.org website using BIGSdb software and made available at http://pubmlst.org/shaemolyticus/. The data of this study thus suggest that MLST can be used to study population structure and diversity among S. haemolyticus isolates. PMID:27824930

An atlas of B-cell clonal distribution in the human body.

PubMed

Meng, Wenzhao; Zhang, Bochao; Schwartz, Gregory W; Rosenfeld, Aaron M; Ren, Daqiu; Thome, Joseph J C; Carpenter, Dustin J; Matsuoka, Nobuhide; Lerner, Harvey; Friedman, Amy L; Granot, Tomer; Farber, Donna L; Shlomchik, Mark J; Hershberg, Uri; Luning Prak, Eline T

2017-09-01

B-cell responses result in clonal expansion, and can occur in a variety of tissues. To define how B-cell clones are distributed in the body, we sequenced 933,427 B-cell clonal lineages and mapped them to eight different anatomic compartments in six human organ donors. We show that large B-cell clones partition into two broad networks-one spans the blood, bone marrow, spleen and lung, while the other is restricted to tissues within the gastrointestinal (GI) tract (jejunum, ileum and colon). Notably, GI tract clones display extensive sharing of sequence variants among different portions of the tract and have higher frequencies of somatic hypermutation, suggesting extensive and serial rounds of clonal expansion and selection. Our findings provide an anatomic atlas of B-cell clonal lineages, their properties and tissue connections. This resource serves as a foundation for studies of tissue-based immunity, including vaccine responses, infections, autoimmunity and cancer.

Multilocus Sequence Typing Reveals a New Cluster of Closely Related Candida tropicalis Genotypes in Italian Patients With Neurological Disorders.

PubMed

Scordino, Fabio; Giuffrè, Letterio; Barberi, Giuseppina; Marino Merlo, Francesca; Orlando, Maria Grazia; Giosa, Domenico; Romeo, Orazio

2018-01-01

Candida tropicalis is a pathogenic yeast that has emerged as an important cause of candidemia especially in elderly patients with hematological malignancies. Infections caused by this species are mainly reported from Latin America and Asian-Pacific countries although recent epidemiological data revealed that C. tropicalis accounts for 6-16.4% of the Candida bloodstream infections (BSIs) in Italy by representing a relevant issue especially for patients receiving long-term hospital care. The aim of this study was to describe the genetic diversity of C. tropicalis isolates contaminating the hands of healthcare workers (HCWs) and hospital environments and/or associated with BSIs occurring in patients with different neurological disorders and without hematological disease. A total of 28 C. tropicalis isolates were genotyped using multilocus sequence typing analysis of six housekeeping ( ICL1, MDR1, SAPT2, SAPT4, XYR1 , and ZWF1 ) genes and data revealed the presence of only eight diploid sequence types (DSTs) of which 6 (75%) were completely new. Four eBURST clonal complexes (CC2, CC10, CC11, and CC33) contained all DSTs found in this study and the CC33 resulted in an exclusive, well-defined, clonal cluster from Italy. In conclusion, C. tropicalis could represent an important cause of BSIs in long-term hospitalized patients with no underlying hematological disease. The findings of this study also suggest a potential horizontal transmission of a specific C. tropicalis clone through hands of HCWs and expand our understanding of the molecular epidemiology of this pathogen whose population structure is still far from being fully elucidated as its complexity increases as different categories of patients and geographic areas are examined.

Application of biomarkers in cancer risk management: evaluation from stochastic clonal evolutionary and dynamic system optimization points of view.

PubMed

Li, Xiaohong; Blount, Patricia L; Vaughan, Thomas L; Reid, Brian J

2011-02-01

Aside from primary prevention, early detection remains the most effective way to decrease mortality associated with the majority of solid cancers. Previous cancer screening models are largely based on classification of at-risk populations into three conceptually defined groups (normal, cancer without symptoms, and cancer with symptoms). Unfortunately, this approach has achieved limited successes in reducing cancer mortality. With advances in molecular biology and genomic technologies, many candidate somatic genetic and epigenetic "biomarkers" have been identified as potential predictors of cancer risk. However, none have yet been validated as robust predictors of progression to cancer or shown to reduce cancer mortality. In this Perspective, we first define the necessary and sufficient conditions for precise prediction of future cancer development and early cancer detection within a simple physical model framework. We then evaluate cancer risk prediction and early detection from a dynamic clonal evolution point of view, examining the implications of dynamic clonal evolution of biomarkers and the application of clonal evolution for cancer risk management in clinical practice. Finally, we propose a framework to guide future collaborative research between mathematical modelers and biomarker researchers to design studies to investigate and model dynamic clonal evolution. This approach will allow optimization of available resources for cancer control and intervention timing based on molecular biomarkers in predicting cancer among various risk subsets that dynamically evolve over time.

ELKS, a Protein Structurally Related to the Active Zone-associated Protein CAST, Is Expressed in Pancreatic β Cells and Functions in Insulin Exocytosis: Interaction of ELKS with Exocytotic Machinery Analyzed by Total Internal Reflection Fluorescence MicroscopyV⃞

PubMed Central

Ohara-Imaizumi, Mica; Ohtsuka, Toshihisa; Matsushima, Satsuki; Akimoto, Yoshihiro; Nishiwaki, Chiyono; Nakamichi, Yoko; Kikuta, Toshiteru; Nagai, Shintaro; Kawakami, Hayato; Watanabe, Takashi; Nagamatsu, Shinya

2005-01-01

The cytomatrix at the active zone (CAZ) has been implicated in defining the site of Ca2+-dependent exocytosis of neurotransmitters. Here, we demonstrate the expression and function of ELKS, a protein structurally related to the CAZ protein CAST, in insulin exocytosis. The results of confocal and immunoelectron microscopic analysis showed that ELKS is present in pancreatic β cells and is localized close to insulin granules docked on the plasma membrane-facing blood vessels. Total internal reflection fluorescence microscopy imaging in insulin-producing clonal cells revealed that the ELKS clusters are less dense and unevenly distributed than syntaxin 1 clusters, which are enriched in the plasma membrane. Most of the ELKS clusters were on the docking sites of insulin granules that were colocalized with syntaxin 1 clusters. Total internal reflection fluorescence images of single-granule motion showed that the fusion events of insulin granules mostly occurred on the ELKS cluster, where repeated fusion was sometimes observed. When the Bassoon-binding region of ELKS was introduced into the cells, the docking and fusion of insulin granules were markedly reduced. Moreover, attenuation of ELKS expression by small interfering RNA reduced the glucose-evoked insulin release. These data suggest that the CAZ-related protein ELKS functions in insulin exocytosis from pancreatic β cells. PMID:15888548

Subtyping of Clostridium difficile PCR ribotypes 591, 106 and 002, the dominant strain types circulating in Medellin, Colombia.

PubMed

Salazar, Clara Lina; Reyes, Catalina; Cienfuegos-Gallet, Astrid Vanessa; Best, Emma; Atehortua, Santiago; Sierra, Patricia; Correa, Margarita M; Fawley, Warren N; Paredes-Sabja, Daniel; Wilcox, Mark; Gonzalez, Angel

2018-01-01

We aimed to achieve a higher typing resolution within the three dominant Clostridium difficile ribotypes (591,106 and 002) circulating in Colombia. A total of 50 C. difficile isolates we had previously typed by PCR-ribotyping, representing the major three ribotypes circulating in Colombia, were analyzed. Twenty-seven isolates of ribotype 591, 12 of ribotype 106 and 11 of ribotype 002 were subtyped by multiple locus variable-number tandem-repeat analysis (MLVA). The presence of the PaLoc genes (tcdA/tcdB), toxin production in culture and antimicrobial susceptibility were also determined. From the total C. difficile ribotypes analyzed, 20 isolates (74%) of ribotype 591, nine (75%) of ribotype 106 and five (45.5%) of ribotype 002 were recovered from patients with Clostridium difficile infection (CDI). MLVA allowed us to recognize four and two different clonal complexes for ribotypes 591 and 002, respectively, having a summed tandem-repeat difference (STRD) <2, whereas none of the ribotype 106 isolates were grouped in a cluster or clonal complex having a STRD >10. Six ribotype 591 and three ribotype 002 isolates belonging to a defined clonal complex were isolated on the same week in two different hospitals. All ribotypes harbored either tcdA+/tcdB+ or tcdA-/tcdB+ PaLoc genes. Moreover, 94% of the isolates were positive for toxin in culture. All isolates were susceptible to vancomycin and metronidazole, while 75% to 100% of the isolates were resistant to clindamycin, and less than 14.8% of ribotype 591 isolates were resistant to moxifloxacina. No significant differences were found among ribotypes with respect to demographic and clinical patients' data; however, our results demonstrated a high molecular heterogeneity of C. difficile strains circulating in Colombia.

Subtyping of Clostridium difficile PCR ribotypes 591, 106 and 002, the dominant strain types circulating in Medellin, Colombia

PubMed Central

Salazar, Clara Lina; Reyes, Catalina; Cienfuegos-Gallet, Astrid Vanessa; Best, Emma; Atehortua, Santiago; Sierra, Patricia; Correa, Margarita M.; Fawley, Warren N.; Paredes-Sabja, Daniel; Wilcox, Mark

2018-01-01

We aimed to achieve a higher typing resolution within the three dominant Clostridium difficile ribotypes (591,106 and 002) circulating in Colombia. A total of 50 C. difficile isolates we had previously typed by PCR-ribotyping, representing the major three ribotypes circulating in Colombia, were analyzed. Twenty-seven isolates of ribotype 591, 12 of ribotype 106 and 11 of ribotype 002 were subtyped by multiple locus variable-number tandem-repeat analysis (MLVA). The presence of the PaLoc genes (tcdA/tcdB), toxin production in culture and antimicrobial susceptibility were also determined. From the total C. difficile ribotypes analyzed, 20 isolates (74%) of ribotype 591, nine (75%) of ribotype 106 and five (45.5%) of ribotype 002 were recovered from patients with Clostridium difficile infection (CDI). MLVA allowed us to recognize four and two different clonal complexes for ribotypes 591 and 002, respectively, having a summed tandem-repeat difference (STRD) <2, whereas none of the ribotype 106 isolates were grouped in a cluster or clonal complex having a STRD >10. Six ribotype 591 and three ribotype 002 isolates belonging to a defined clonal complex were isolated on the same week in two different hospitals. All ribotypes harbored either tcdA+/tcdB+ or tcdA-/tcdB+ PaLoc genes. Moreover, 94% of the isolates were positive for toxin in culture. All isolates were susceptible to vancomycin and metronidazole, while 75% to 100% of the isolates were resistant to clindamycin, and less than 14.8% of ribotype 591 isolates were resistant to moxifloxacina. No significant differences were found among ribotypes with respect to demographic and clinical patients’ data; however, our results demonstrated a high molecular heterogeneity of C. difficile strains circulating in Colombia. PMID:29649308

Case Series Description and Genomic Characterization of Invasive Group A Streptococcal Infections in Pediatric Patients.

PubMed

Dien Bard, Jennifer; Mongkolrattanothai, Kanokporn; Kachroo, Priyanka; Beres, Stephen; Olsen, Randall J

2017-06-01

We report an unusual cluster of invasive group A Streptococcus infections in 6 pediatric patients and demonstrate that the strains were derived from diverse genetic backgrounds, confirming the occurrence of a community cluster rather than a clonal outbreak. Whole genome sequencing provided a rapid and comprehensive view of group A Streptococcus genotypes and helped guide our institutional response and public health maneuvers.

Clustering of Tuberculosis Cases Based on Variable-Number Tandem-Repeat Typing in Relation to the Population Structure of Mycobacterium tuberculosis in the Netherlands

PubMed Central

Sloot, Rosa; Borgdorff, Martien W.; de Beer, Jessica L.; van Ingen, Jakko; Supply, Philip

2013-01-01

The population structure of 3,776 Mycobacterium tuberculosis isolates was determined using variable-number tandem-repeat (VNTR) typing. The degree of clonality was so high that a more relaxed definition of clustering cannot be applied. Among recent immigrants with non-Euro-American isolates, transmission is overestimated if based on identical VNTR patterns. PMID:23658260

Clonality and serotypes of Streptococcus mutans among children by multilocus sequence typing

PubMed Central

Momeni, Stephanie S.; Whiddon, Jennifer; Cheon, Kyounga; Moser, Stephen A.; Childers, Noel K.

2015-01-01

Studies using multilocus sequence typing (MLST) have demonstrated that Streptococcus mutans isolates are genetically diverse. Our laboratory previously demonstrated clonality of S. mutans using MLST but could not discount the possibility of sampling bias. In this study, the clonality of randomly selected S. mutans plaque isolates from African American children was examined using MLST. Serotype and presence of collagen-binding proteins (CBP) cnm/cbm were also assessed. One hundred S. mutans isolates were randomly selected for MLST analysis. Sequence analysis was performed and phylogenetic trees were generated using START2 and MEGA. Thirty-four sequence types (ST) were identified of which 27 were unique to this population. Seventy-five percent of the isolates clustered into 16 clonal groups. Serotypes observed were c (n=84), e (n=3), and k (n=11). The prevalence of S. mutans isolates serotype k was notably high at 17.5%. All isolates were cnm/cbm negative. The clonality of S. mutans demonstrated in this study illustrates the importance of localized populations studies and are consistent with transmission. The prevalence of serotype k, a recently proposed systemic pathogen, observed in this study is higher than reported in most populations and is the first report of S. mutans serotype k in a US population. PMID:26443288

Deep sequencing is an appropriate tool for the selection of unique Hepatitis C virus (HCV) variants after single genomic amplification.

PubMed

Guinoiseau, Thibault; Moreau, Alain; Hohnadel, Guillaume; Ngo-Giang-Huong, Nicole; Brulard, Celine; Vourc'h, Patrick; Goudeau, Alain; Gaudy-Graffin, Catherine

2017-01-01

Hepatitis C virus (HCV) evolves rapidly in a single host and circulates as a quasispecies wich is a complex mixture of genetically distinct virus's but closely related namely variants. To identify intra-individual diversity and investigate their functional properties in vitro, it is necessary to define their quasispecies composition and isolate the HCV variants. This is possible using single genome amplification (SGA). This technique, based on serially diluted cDNA to amplify a single cDNA molecule (clonal amplicon), has already been used to determine individual HCV diversity. In these studies, positive PCR reactions from SGA were directly sequenced using Sanger technology. The detection of non-clonal amplicons is necessary for excluding them to facilitate further functional analysis. Here, we compared Next Generation Sequencing (NGS) with De Novo assembly and Sanger sequencing for their ability to distinguish clonal and non-clonal amplicons after SGA on one plasma specimen. All amplicons (n = 42) classified as clonal by NGS were also classified as clonal by Sanger sequencing. No double peaks were seen on electropherograms for non-clonal amplicons with position-specific nucleotide variation below 15% by NGS. Altogether, NGS circumvented many of the difficulties encountered when using Sanger sequencing after SGA and is an appropriate tool to reliability select clonal amplicons for further functional studies.

Deep sequencing is an appropriate tool for the selection of unique Hepatitis C virus (HCV) variants after single genomic amplification

PubMed Central

Guinoiseau, Thibault; Moreau, Alain; Hohnadel, Guillaume; Ngo-Giang-Huong, Nicole; Brulard, Celine; Vourc’h, Patrick; Goudeau, Alain; Gaudy-Graffin, Catherine

2017-01-01

Hepatitis C virus (HCV) evolves rapidly in a single host and circulates as a quasispecies wich is a complex mixture of genetically distinct virus’s but closely related namely variants. To identify intra-individual diversity and investigate their functional properties in vitro, it is necessary to define their quasispecies composition and isolate the HCV variants. This is possible using single genome amplification (SGA). This technique, based on serially diluted cDNA to amplify a single cDNA molecule (clonal amplicon), has already been used to determine individual HCV diversity. In these studies, positive PCR reactions from SGA were directly sequenced using Sanger technology. The detection of non-clonal amplicons is necessary for excluding them to facilitate further functional analysis. Here, we compared Next Generation Sequencing (NGS) with De Novo assembly and Sanger sequencing for their ability to distinguish clonal and non-clonal amplicons after SGA on one plasma specimen. All amplicons (n = 42) classified as clonal by NGS were also classified as clonal by Sanger sequencing. No double peaks were seen on electropherograms for non-clonal amplicons with position-specific nucleotide variation below 15% by NGS. Altogether, NGS circumvented many of the difficulties encountered when using Sanger sequencing after SGA and is an appropriate tool to reliability select clonal amplicons for further functional studies. PMID:28362878

Clonal development and organization of the adult Drosophila central brain.

PubMed

Yu, Hung-Hsiang; Awasaki, Takeshi; Schroeder, Mark David; Long, Fuhui; Yang, Jacob S; He, Yisheng; Ding, Peng; Kao, Jui-Chun; Wu, Gloria Yueh-Yi; Peng, Hanchuan; Myers, Gene; Lee, Tzumin

2013-04-22

The insect brain can be divided into neuropils that are formed by neurites of both local and remote origin. The complexity of the interconnections obscures how these neuropils are established and interconnected through development. The Drosophila central brain develops from a fixed number of neuroblasts (NBs) that deposit neurons in regional clusters. By determining individual NB clones and pursuing their projections into specific neuropils, we unravel the regional development of the brain neural network. Exhaustive clonal analysis revealed 95 stereotyped neuronal lineages with characteristic cell-body locations and neurite trajectories. Most clones show complex projection patterns, but despite the complexity, neighboring clones often coinnervate the same local neuropil or neuropils and further target a restricted set of distant neuropils. These observations argue for regional clonal development of both neuropils and neuropil connectivity throughout the Drosophila central brain. Copyright © 2013 Elsevier Ltd. All rights reserved.

Gene flow contributes to diversification of the major fungal pathogen Candida albicans.

PubMed

Ropars, Jeanne; Maufrais, Corinne; Diogo, Dorothée; Marcet-Houben, Marina; Perin, Aurélie; Sertour, Natacha; Mosca, Kevin; Permal, Emmanuelle; Laval, Guillaume; Bouchier, Christiane; Ma, Laurence; Schwartz, Katja; Voelz, Kerstin; May, Robin C; Poulain, Julie; Battail, Christophe; Wincker, Patrick; Borman, Andrew M; Chowdhary, Anuradha; Fan, Shangrong; Kim, Soo Hyun; Le Pape, Patrice; Romeo, Orazio; Shin, Jong Hee; Gabaldon, Toni; Sherlock, Gavin; Bougnoux, Marie-Elisabeth; d'Enfert, Christophe

2018-06-08

Elucidating population structure and levels of genetic diversity and recombination is necessary to understand the evolution and adaptation of species. Candida albicans is the second most frequent agent of human fungal infections worldwide, causing high-mortality rates. Here we present the genomic sequences of 182 C. albicans isolates collected worldwide, including commensal isolates, as well as ones responsible for superficial and invasive infections, constituting the largest dataset to date for this major fungal pathogen. Although, C. albicans shows a predominantly clonal population structure, we find evidence of gene flow between previously known and newly identified genetic clusters, supporting the occurrence of (para)sexuality in nature. A highly clonal lineage, which experimentally shows reduced fitness, has undergone pseudogenization in genes required for virulence and morphogenesis, which may explain its niche restriction. Candida albicans thus takes advantage of both clonality and gene flow to diversify.

Epidemiologic and Genotypic Review of Carbapenemase-Producing Organisms in British Columbia, Canada, between 2008 and 2014

PubMed Central

Sekirov, Inna; Croxen, Matthew A.; Ng, Corrinne; Azana, Robert; Chang, Yin; Mataseje, Laura; Boyd, David; Mangat, Chand; Mack, Benjamin; Tadros, Manal; Brodkin, Elizabeth; Kibsey, Pamela; Stefanovic, Aleksandra; Champagne, Sylvie; Mulvey, Michael R.

2015-01-01

Carbapenemase-producing organisms (CPOs) are a serious emerging problem for health care facilities worldwide. Owing to their resistance to most antimicrobial therapies, CPOs are difficult to treat and pose a challenge for infection prevention and control. Since 2010, lab-based surveillance for CPOs and PCR-based testing were implemented in British Columbia (BC), Canada. A review of CPOs in BC from 2008 to March 2014 was done to characterize the resistance mechanisms and possible clonal strain transmission and to compare pulsed-field gel electrophoresis (PFGE), multilocus sequence typing (MLST), and plasmid restriction fragment length polymorphism (RFLP) as molecular typing tools. During this study period, a total of 177 CPO cases were identified. Patient demographics and travel history were reviewed, and a descriptive analysis was carried out. PFGE profiles, MLST, and plasmid RFLP analysis for a subset of Escherichia coli, Klebsiella pneumoniae, and Enterobacter species isolates were obtained and analyzed. Our findings demonstrate that CPOs have been increasing in number in BC over time, from 1 isolate/year retrospectively identified in 2008 and 2009 to 82 isolates in 2013 and 30 isolates in the first quarter of 2014. Overall, K. pneumoniae isolates lack clonality, although some seemingly related clusters have been found. Plasmid analysis showed evidence of the spread of plasmids carrying carbapenemase-encoding genes between the examined isolates. Analysis of Enterobacter cloacae isolates revealed a more clonal nature of these CPOs in BC. The presence of related clusters provides evidence of interpatient organism transmission both within and between institutions. Although in our study, NDM-harboring E. cloacae isolates appeared to spread clonally, the spread of carbapenem resistance in K. pneumoniae seems to be plasmid mediated. PMID:26607987

Clonal success of piliated penicillin nonsusceptible pneumococci

PubMed Central

Sjöström, K.; Blomberg, C.; Fernebro, J.; Dagerhamn, J.; Morfeldt, E.; Barocchi, M. A.; Browall, S.; Moschioni, M.; Andersson, M.; Henriques, F.; Albiger, B.; Rappuoli, Rino; Normark, S.; Henriques-Normark, B.

2007-01-01

Antibiotic resistance in pneumococci is due to the spread of strains belonging to a limited number of clones. The Spain9V-3 clone of sequence type (ST)156 is one of the most successful clones with reduced susceptibility to penicillin [pneumococci nonsusceptible to penicillin (PNSP)]. In Sweden during 2000–2003, a dramatic increase in the number of PNSP isolates was observed. Molecular characterization of these isolates showed that a single clone of sequence type ST156 increased from 40% to 80% of all serotype 14, thus causing the serotype expansion. Additionally, during the same time period, we examined the clonal composition of two serotypes 9V and 19F: all 9V and 20% of 19F isolates belonged to the clonal cluster of ST156, and overall ≈50% of all PNSP belonged to the ST156 clonal cluster. Moreover, microarray and PCR analysis showed that all ST156 isolates, irrespective of capsular type, carried the rlrA pilus islet. This islet was also found to be present in the penicillin-sensitive ST162 clone, which is believed to be the drug-susceptible ancestor of ST156. Competitive experiments between related ST156 serotype 19F strains confirmed that those containing the rlrA pilus islet were more successful in an animal model of carriage. We conclude that the pilus island is an important biological factor common to ST156 isolates and other successful PNSP clones. In Sweden, a country where the low antibiotic usage does not explain the spread of resistant strains, at least 70% of all PNSP isolates collected during year 2003 carried the pilus islet. PMID:17644611

The genetics of myelodysplastic syndrome: from clonal haematopoiesis to secondary leukaemia.

PubMed

Sperling, Adam S; Gibson, Christopher J; Ebert, Benjamin L

2017-01-01

Myelodysplastic syndrome (MDS) is a clonal disease that arises from the expansion of mutated haematopoietic stem cells. In a spectrum of myeloid disorders ranging from clonal haematopoiesis of indeterminate potential (CHIP) to secondary acute myeloid leukaemia (sAML), MDS is distinguished by the presence of peripheral blood cytopenias, dysplastic haematopoietic differentiation and the absence of features that define acute leukaemia. More than 50 recurrently mutated genes are involved in the pathogenesis of MDS, including genes that encode proteins involved in pre-mRNA splicing, epigenetic regulation and transcription. In this Review we discuss the molecular processes that lead to CHIP and further clonal evolution to MDS and sAML. We also highlight the ways in which these insights are shaping the clinical management of MDS, including classification schemata, prognostic scoring systems and therapeutic approaches.

Population structure of the butternut canker fungus, Ophiognomonia clavigignenti-juglandacearum, in North American forests

PubMed Central

Broders, K D; Boraks, A; Sanchez, A M; Boland, G J

2012-01-01

The occurrence of multiple introduction events, or sudden emergence from a host jump, of forest pathogens may be an important factor in successful establishment in a novel environment or on a new host; however, few studies have focused on the introduction and emergence of fungal pathogens in forest ecosystems. While Ophiognomonia clavigignenti-juglandacearum (Oc-j), the butternut canker fungus, has caused range-wide mortality of butternut trees in North America since its first observation in 1967, the history of its emergence and spread across the United States and Canada remains unresolved. Using 17 single nucleotide polymorphic loci, we investigated the genetic population structure of 101 isolates of Oc-j from across North America. Clustering analysis revealed that the Oc-j population in North America is made up of three differentiated genetic clusters of isolates, and these genetic clusters were found to have a strong clonal structure. These results, in combination with the geographic distribution of the populations, suggest that Oc-j was introduced or has emerged in North America on more than one occasion, and these clonal lineages have since proliferated across much of the range of butternut. No evidence of genetic recombination was observed in the linkage analysis, and conservation of the distinct genetic clusters in regions where isolates from two or more genetic clusters are present, would indicate a very minimal or non-existent role of sexual recombination in populations of Oc-j in North America. PMID:23139872

Proposal of a Consensus Set of Hypervariable Mycobacterial Interspersed Repetitive-Unit–Variable-Number Tandem-Repeat Loci for Subtyping of Mycobacterium tuberculosis Beijing Isolates

PubMed Central

Allix-Béguec, Caroline; Wahl, Céline; Hanekom, Madeleine; Nikolayevskyy, Vladyslav; Drobniewski, Francis; Maeda, Shinji; Campos-Herrero, Isolina; Mokrousov, Igor; Niemann, Stefan; Kontsevaya, Irina; Rastogi, Nalin; Samper, Sofia; Sng, Li-Hwei; Warren, Robin M.

2014-01-01

Mycobacterium tuberculosis Beijing strains represent targets of special importance for molecular surveillance of tuberculosis (TB), especially because they are associated with spread of multidrug resistance in some world regions. Standard 24-locus mycobacterial interspersed repetitive-unit–variable-number tandem-repeat (MIRU-VNTR) typing lacks resolution power for accurately discriminating closely related clones that often compose Beijing strain populations. Therefore, we evaluated a set of 7 additional, hypervariable MIRU-VNTR loci for better resolution and tracing of such strains, using a collection of 535 Beijing isolates from six world regions where these strains are known to be prevalent. The typeability and interlaboratory reproducibility of these hypervariable loci were lower than those of the 24 standard loci. Three loci (2163a, 3155, and 3336) were excluded because of their redundant variability and/or more frequent noninterpretable results compared to the 4 other markers. The use of the remaining 4-locus set (1982, 3232, 3820, and 4120) increased the number of types by 52% (from 223 to 340) and reduced the clustering rate from 58.3 to 36.6%, when combined with the use of the standard 24-locus set. Known major clonal complexes/24-locus-based clusters were all subdivided, although the degree of subdivision varied depending on the complex. Only five single-locus variations were detected among the hypervariable loci of an additional panel of 92 isolates, representing 15 years of clonal spread of a single Beijing strain in a geographically restricted setting. On this calibrated basis, we propose this 4-locus set as a consensus for subtyping Beijing clonal complexes and clusters, after standard typing. PMID:24172154

Proposal of a consensus set of hypervariable mycobacterial interspersed repetitive-unit-variable-number tandem-repeat loci for subtyping of Mycobacterium tuberculosis Beijing isolates.

PubMed

Allix-Béguec, Caroline; Wahl, Céline; Hanekom, Madeleine; Nikolayevskyy, Vladyslav; Drobniewski, Francis; Maeda, Shinji; Campos-Herrero, Isolina; Mokrousov, Igor; Niemann, Stefan; Kontsevaya, Irina; Rastogi, Nalin; Samper, Sofia; Sng, Li-Hwei; Warren, Robin M; Supply, Philip

2014-01-01

Mycobacterium tuberculosis Beijing strains represent targets of special importance for molecular surveillance of tuberculosis (TB), especially because they are associated with spread of multidrug resistance in some world regions. Standard 24-locus mycobacterial interspersed repetitive-unit-variable-number tandem-repeat (MIRU-VNTR) typing lacks resolution power for accurately discriminating closely related clones that often compose Beijing strain populations. Therefore, we evaluated a set of 7 additional, hypervariable MIRU-VNTR loci for better resolution and tracing of such strains, using a collection of 535 Beijing isolates from six world regions where these strains are known to be prevalent. The typeability and interlaboratory reproducibility of these hypervariable loci were lower than those of the 24 standard loci. Three loci (2163a, 3155, and 3336) were excluded because of their redundant variability and/or more frequent noninterpretable results compared to the 4 other markers. The use of the remaining 4-locus set (1982, 3232, 3820, and 4120) increased the number of types by 52% (from 223 to 340) and reduced the clustering rate from 58.3 to 36.6%, when combined with the use of the standard 24-locus set. Known major clonal complexes/24-locus-based clusters were all subdivided, although the degree of subdivision varied depending on the complex. Only five single-locus variations were detected among the hypervariable loci of an additional panel of 92 isolates, representing 15 years of clonal spread of a single Beijing strain in a geographically restricted setting. On this calibrated basis, we propose this 4-locus set as a consensus for subtyping Beijing clonal complexes and clusters, after standard typing.

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

PubMed Central

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

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 < 0.001). Moreover, 66.7% and 40.0% of the male and female partners of the infertile couples tested positive for 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. PMID:28859153

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

PubMed

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 < 0.001). Moreover, 66.7% and 40.0% of the male and female partners of the infertile couples tested positive for 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.

Probing Prokaryotic Social Behaviors with Bacterial “Lobster Traps”

PubMed Central

Connell, Jodi L.; Wessel, Aimee K.; Parsek, Matthew R.; Ellington, Andrew D.; Whiteley, Marvin; Shear, Jason B.

2010-01-01

Bacteria are social organisms that display distinct behaviors/phenotypes when present in groups. These behaviors include the abilities to construct antibiotic-resistant sessile biofilm communities and to communicate with small signaling molecules (quorum sensing [QS]). Our understanding of biofilms and QS arises primarily from in vitro studies of bacterial communities containing large numbers of cells, often greater than 108 bacteria; however, in nature, bacteria often reside in dense clusters (aggregates) consisting of significantly fewer cells. Indeed, bacterial clusters containing 101 to 105 cells are important for transmission of many bacterial pathogens. Here, we describe a versatile strategy for conducting mechanistic studies to interrogate the molecular processes controlling antibiotic resistance and QS-mediated virulence factor production in high-density bacterial clusters. This strategy involves enclosing a single bacterium within three-dimensional picoliter-scale microcavities (referred to as bacterial “lobster traps”) defined by walls that are permeable to nutrients, waste products, and other bioactive small molecules. Within these traps, bacteria divide normally into extremely dense (1012 cells/ml) clonal populations with final population sizes similar to that observed in naturally occurring bacterial clusters. Using these traps, we provide strong evidence that within low-cell-number/high-density bacterial clusters, QS is modulated not only by bacterial density but also by population size and flow rate of the surrounding medium. We also demonstrate that antibiotic resistance develops as cell density increases, with as few as ~150 confined bacteria exhibiting an antibiotic-resistant phenotype similar to biofilm bacteria. Together, these findings provide key insights into clinically relevant phenotypes in low-cell-number/high-density bacterial populations. PMID:21060734

Assessment of clonality and serotypes of Streptococcus mutans among children by multilocus sequence typing.

PubMed

Momeni, Stephanie S; Whiddon, Jennifer; Cheon, Kyounga; Moser, Stephen A; Childers, Noel K

2015-12-01

Studies using multilocus sequence typing (MLST) have demonstrated that Streptococcus mutans isolates are genetically diverse. Our laboratory previously demonstrated clonality of S. mutans using MLST but could not discount the possibility of sampling bias. In this study, the clonality of randomly selected S. mutans plaque isolates from African-American children was examined using MLST. Serotype and the presence of collagen-binding proteins (CBPs) encoded by cnm/cbm were also assessed. One-hundred S. mutans isolates were randomly selected for MLST analysis. Sequence analysis was performed and phylogenetic trees were generated using start2 and mega. Thirty-four sequence types were identified, of which 27 were unique to this population. Seventy-five per cent of the isolates clustered into 16 clonal groups. The serotypes observed were c (n = 84), e (n = 3), and k (n = 11). The prevalence of S. mutans isolates of serotype k was notably high, at 17.5%. All isolates were cnm/cbm negative. The clonality of S. mutans demonstrated in this study illustrates the importance of localized population studies and are consistent with transmission. The prevalence of serotype k, a recently proposed systemic pathogen, observed in this study, is higher than reported in most populations and is the first report of S. mutans serotype k in a United States population. © 2015 Eur J Oral Sci.

Polymerase chain reaction-based clonality testing in tissue samples with reactive lymphoproliferations: usefulness and pitfalls. A report of the BIOMED-2 Concerted Action BMH4-CT98-3936.

PubMed

Langerak, A W; Molina, T J; Lavender, F L; Pearson, D; Flohr, T; Sambade, C; Schuuring, E; Al Saati, T; van Dongen, J J M; van Krieken, J H J M

2007-02-01

Lymphoproliferations are generally diagnosed via histomorphology and immunohistochemistry. Although mostly conclusive, occasionally the differential diagnosis between reactive lesions and malignant lymphomas is difficult. In such cases molecular clonality studies of immunoglobulin (Ig)/T-cell receptor (TCR) rearrangements can be useful. Here we address the issue of clonality assessment in 106 histologically defined reactive lesions, using the standardized BIOMED-2 Ig/TCR multiplex polymerase chain reaction (PCR) heteroduplex and GeneScan assays. Samples were reviewed nationally, except 10% random cases and cases with clonal results selected for additional international panel review. In total 75% (79/106) only showed polyclonal Ig/TCR targets (type I), whereas another 15% (16/106) represent probably polyclonal cases, with weak Ig/TCR (oligo)clonality in an otherwise polyclonal background (type II). Interestingly, in 10% (11/106) clear monoclonal Ig/TCR products were observed (types III/IV), which prompted further pathological review. Clonal cases included two missed lymphomas in national review and nine cases that could be explained as diagnostically difficult cases or probable lymphomas upon additional review. Our data show that the BIOMED-2 Ig/TCR multiplex PCR assays are very helpful in confirming the polyclonal character in the vast majority of reactive lesions. However, clonality detection in a minority should lead to detailed pathological review, including close interaction between pathologist and molecular biologist.

Population Structure in Nontypeable Haemophilus influenzae

PubMed Central

LaCross, Nathan C.; Marrs, Carl F.; Gilsdorf, Janet R.

2013-01-01

Nontypeable Haemophilus influenzae (NTHi) frequently colonize the human pharynx asymptomatically, and are an important cause of otitis media in children. Past studies have identified typeable H. influenzae as being clonal, but the population structure of NTHi has not been extensively characterized. The research presented here investigated the diversity and population structure in a well-characterized collection of NTHi isolated from the middle ears of children with otitis media or the pharynges of healthy children in three disparate geographic regions. Multilocus sequence typing identified 109 unique sequence types among 170 commensal and otitis media-associated NTHi isolates from Finland, Israel, and the US. The largest clonal complex contained only five sequence types, indicating a high level of genetic diversity. The eBURST v3, ClonalFrame 1.1, and structure 2.3.3 programs were used to further characterize diversity and population structure from the sequence typing data. Little clustering was apparent by either disease state (otitis media or commensalism) or geography in the ClonalFrame phylogeny. Population structure was clearly evident, with support for eight populations when all 170 isolates were analyzed. Interestingly, one population contained only commensal isolates, while two others consisted solely of otitis media isolates, suggesting associations between population structure and disease. PMID:23266487

Poor clinical outcome for meningitis caused by Haemophilus influenzae serotype A strains containing the IS1016-bexA deletion.

PubMed

Lima, Josilene B T; Ribeiro, Guilherme S; Cordeiro, Soraia M; Gouveia, Edilane L; Salgado, Kátia; Spratt, Brian G; Godoy, Daniel; Reis, Mitermayer G; Ko, Albert I; Reis, Joice N

2010-11-15

Since the introduction of Haemophilus influenzae type b (Hib) conjugate vaccines, meningitis caused by serotypes other than Hib has gained in importance. We conducted active hospital-based surveillance for meningitis over an 11-year period in Salvador, Brazil. H. influenzae isolates were serotyped and analyzed by polymerase chain reaction, pulsed-field gel electrophoresis, and DNA sequencing to identify strains with a specific deletion (IS1016) in the bexA gene (IS1016-bexA). We identified 43 meningitis cases caused by non-type b H. influenzae: 28 (65%) were caused by type a (Hia), 9 (21%) were caused by noncapsulated strains, and 3 (7%) each were caused by types e and f. Hia isolates clustered in 2 clonal groups; clonal group A strains (n = 9) had the IS1016-bexA deletion. Among children <5 years of age, meningitis caused by Hia from clonal group A had higher case-fatality than meningitis caused by clonal group B. Despite small numbers, these results indicate that the presence of the IS1016-bexA deletion is associated with enhanced virulence in non-type b H. influenzae.

ICUS/CCUS/CHIP: basics & beyond.

PubMed

Jain, Mili; Tripathi, Anil

2017-10-01

Patients presenting with idiopathic cytopenia with non-diagnostic marrow morphology and a normal karyotype pose a diagnostic and therapeutic challenge. Additional diagnostic information from mutation analysis could provide important clinical insights. However, one has to be cautious during such diagnostic interpretations in view of the recent documentation of clonal somatic mutations in healthy elder individuals. Whether to regard clonality synonymous with malignant proliferation or a manifestation of ageing process is to be judged carefully. Areas covered: The review covers defining criteria and diagnostic work up for Idiopathic cytopenia of undetermined significance (ICUS), Clonal cytopenia of undetermined significance (CCUS), Clonal hematopoiesis of indeterminate potential (CHIP). It also presents the results from previous reports on this subject. In addition the evolution and potential impact of these entities is discussed. Expert commentary: Current evidence does not support the use of somatic mutations as presumptive evidence of myelodysplastic syndrome (MDS). Including CCUS under the category of MDS requires further insight on natural disease course. Longitudinal follow up study on ICUS, CCUS, CHIP may eventually identify the pathological significance of the clonal mutations. An absence of mutation however may still be useful as good predictor of not having MDS.

Recent emergence of clonal group O25b:K1:H4-B2-ST131 ibeA strains among Escherichia coli poultry isolates, including CTX-M-9-producing strains, and comparison with clinical human isolates.

PubMed

Mora, Azucena; Herrera, Alexandra; Mamani, Rosalia; López, Cecilia; Alonso, María Pilar; Blanco, Jesús E; Blanco, Miguel; Dahbi, Ghizlane; García-Garrote, Fernando; Pita, Julia María; Coira, Amparo; Bernárdez, María Isabel; Blanco, Jorge

2010-11-01

To discern the possible spread of the Escherichia coli O25b:H4-ST131 clonal group in poultry and the zoonotic potential of avian strains, we made a retrospective search of our strain collection and compared the findings for those strains with the findings for current strains. Thus, we have characterized a collection of 19 avian O25b:H4-ST131 E. coli strains isolated from 1995 to 2010 which, interestingly, harbored the ibeA gene. Using this virulence gene as a criterion for selection, we compared those 19 avian strains with 33 human O25b:H4-ST131 ibeA-positive E. coli strains obtained from patients with extraintestinal infections (1993 to 2009). All 52 O25b:H4-ST131 ibeA-positive E. coli strains shared the fimH, kpsMII, malX, and usp genes but showed statistically significant differences in nine virulence factors, namely, papGIII, cdtB, sat, and kpsMII K5, which were associated with human strains, and iroN, kpsMII K1, cvaC, iss, and tsh, which were associated with strains of avian origin. The XbaI macrorestriction profiles of the 52 E. coli O25b:H4-ST131 ibeA-positive strains revealed 11 clusters (clusters I to XI) of >85% similarity, with four clusters including strains of human and avian origin. Cluster VII (90.9% similarity) grouped 10 strains (7 avian and 3 human strains) that mostly produced CTX-M-9 and that also shared the same virulence profile. Finally, we compared the macrorestriction profiles of the 12 CTX-M-9-producing O25b:H4-ST131 ibeA strains (7 avian and 5 human strains) identified among the 52 strains with those of 15 human O25b:H4-ST131 CTX-M-14-, CTX-M-15-, and CTX-M-32-producing strains that proved to be negative for ibeA and showed that they clearly differed in the level of similarity from the CTX-M-9-producing strains. In conclusion, E. coli clonal group O25b:H4-ST131 ibeA has recently emerged among avian isolates with the new acquisition of the K1 capsule antigen and includes CTX-M-9-producing strains. This clonal group represents a real zoonotic risk that has crossed the barrier between human and avian hosts.

Clonal diversity and detection of carbapenem resistance encoding genes among multidrug-resistant Acinetobacter baumannii isolates recovered from patients and environment in two intensive care units in a Moroccan hospital.

PubMed

Uwingabiye, Jean; Lemnouer, Abdelhay; Roca, Ignasi; Alouane, Tarek; Frikh, Mohammed; Belefquih, Bouchra; Bssaibis, Fatna; Maleb, Adil; Benlahlou, Yassine; Kassouati, Jalal; Doghmi, Nawfal; Bait, Abdelouahed; Haimeur, Charki; Louzi, Lhoussain; Ibrahimi, Azeddine; Vila, Jordi; Elouennass, Mostafa

2017-01-01

Carbapenem-resistant Acinetobacter baumannii has recently been defined by the World Health Organization as a critical pathogen. The aim of this study was to compare clonal diversity and carbapenemase-encoding genes of A. baumannii isolates collected from colonized or infected patients and hospital environment in two intensive care units (ICUs) in Morocco. The patient and environmental sampling was carried out in the medical and surgical ICUs of Mohammed V Military teaching hospital from March to August 2015. All A. baumannii isolates recovered from clinical and environmental samples, were identified using routine microbiological techniques and Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry. Antimicrobial susceptibility testing was performed using disc diffusion method. The carbapenemase-encoding genes were screened for by PCR. Clonal relatedness was analyzed by digestion of the DNA with low frequency restriction enzymes and pulsed field gel electrophoresis (PFGE) and the multi locus sequence typing (MLST) was performed on two selected isolates from two major pulsotypes. A total of 83 multidrug-resistant A. baumannii isolates were collected: 47 clinical isolates and 36 environmental isolates. All isolates were positive for the bla OXA51-like and bla OXA23-like genes. The coexistence of bla NDM-1 /bla OXA-23-like and bla OXA 24-like /bla OXA-23-like were detected in 27 (32.5%) and 2 (2.4%) of A. baumannii isolates, respectively. The environmental samples and the fecally-colonized patients were significantly identified ( p  < 0.05) as the most common sites of isolation of NDM-1-harboring isolates. PFGE grouped all isolates into 9 distinct clusters with two major groups (0007 and 0008) containing up to 59% of the isolates. The pulsotype 0008 corresponds to sequence type (ST) 195 while pulsotype 0007 corresponds to ST 1089.The genetic similarity between the clinical and environmental isolates was observed in 80/83 = 96.4% of all isolates, belonging to 7 pulsotypes. This study shows that the clonal spread of environmental A. baumannii isolates is related to that of clinical isolates recovered from colonized or infected patients, being both associated with a high prevalence of the bla OXA23-like and bla NDM-1 genes. These findings emphasize the need for prioritizing the bio-cleaning of the hospital environment to control and prevent the dissemination of A. baumannii clonal lineages.

Clonal diversity analysis using SNP microarray: a new prognostic tool for chronic lymphocytic leukemia.

PubMed

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 of genomic alterations and mosaic distribution of clones can be used to assess apparent clonal evolution via analysis of clonal diversity. Since clonal evolution in CLL is strongly correlated with disease progression, whole genome SNP microarray analysis provides a new comprehensive and reliable prognostic tool for CLL patients. Copyright © 2011 Elsevier Inc. All rights reserved.

The clonal and mutational evolution spectrum of primary triple-negative breast cancers.

PubMed

Shah, Sohrab P; Roth, Andrew; Goya, Rodrigo; Oloumi, Arusha; Ha, Gavin; Zhao, Yongjun; Turashvili, Gulisa; Ding, Jiarui; Tse, Kane; Haffari, Gholamreza; Bashashati, Ali; Prentice, Leah M; Khattra, Jaswinder; Burleigh, Angela; Yap, Damian; Bernard, Virginie; McPherson, Andrew; Shumansky, Karey; Crisan, Anamaria; Giuliany, Ryan; Heravi-Moussavi, Alireza; Rosner, Jamie; Lai, Daniel; Birol, Inanc; Varhol, Richard; Tam, Angela; Dhalla, Noreen; Zeng, Thomas; Ma, Kevin; Chan, Simon K; Griffith, Malachi; Moradian, Annie; Cheng, S-W Grace; Morin, Gregg B; Watson, Peter; Gelmon, Karen; Chia, Stephen; Chin, Suet-Feung; Curtis, Christina; Rueda, Oscar M; Pharoah, Paul D; Damaraju, Sambasivarao; Mackey, John; Hoon, Kelly; Harkins, Timothy; Tadigotla, Vasisht; Sigaroudinia, Mahvash; Gascard, Philippe; Tlsty, Thea; Costello, Joseph F; Meyer, Irmtraud M; Eaves, Connie J; Wasserman, Wyeth W; Jones, Steven; Huntsman, David; Hirst, Martin; Caldas, Carlos; Marra, Marco A; Aparicio, Samuel

2012-04-04

Primary triple-negative breast cancers (TNBCs), a tumour type defined by lack of oestrogen receptor, progesterone receptor and ERBB2 gene amplification, represent approximately 16% of all breast cancers. Here we show in 104 TNBC cases that at the time of diagnosis these cancers exhibit a wide and continuous spectrum of genomic evolution, with some having only a handful of coding somatic aberrations in a few pathways, whereas others contain hundreds of coding somatic mutations. High-throughput RNA sequencing (RNA-seq) revealed that only approximately 36% of mutations are expressed. Using deep re-sequencing measurements of allelic abundance for 2,414 somatic mutations, we determine for the first time-to our knowledge-in an epithelial tumour subtype, the relative abundance of clonal frequencies among cases representative of the population. We show that TNBCs vary widely in their clonal frequencies at the time of diagnosis, with the basal subtype of TNBC showing more variation than non-basal TNBC. Although p53 (also known as TP53), PIK3CA and PTEN somatic mutations seem to be clonally dominant compared to other genes, in some tumours their clonal frequencies are incompatible with founder status. Mutations in cytoskeletal, cell shape and motility proteins occurred at lower clonal frequencies, suggesting that they occurred later during tumour progression. Taken together, our results show that understanding the biology and therapeutic responses of patients with TNBC will require the determination of individual tumour clonal genotypes.

Clonal Architecture of Secondary Acute Myeloid Leukemia

PubMed Central

Walter, Matthew J.; Shen, Dong; Ding, Li; Shao, Jin; Koboldt, Daniel C.; Chen, Ken; Larson, David E.; McLellan, Michael D.; Dooling, David; Abbott, Rachel; Fulton, Robert; Magrini, Vincent; Schmidt, Heather; Kalicki-Veizer, Joelle; O’Laughlin, Michelle; Fan, Xian; Grillot, Marcus; Witowski, Sarah; Heath, Sharon; Frater, John L.; Eades, William; Tomasson, Michael; Westervelt, Peter; DiPersio, John F.; Link, Daniel C.; Mardis, Elaine R.; Ley, Timothy J.; Wilson, Richard K.; Graubert, Timothy A.

2012-01-01

BACKGROUND The myelodysplastic syndromes are a group of hematologic disorders that often evolve into secondary acute myeloid leukemia (AML). The genetic changes that underlie progression from the myelodysplastic syndromes to secondary AML are not well understood. METHODS We performed whole-genome sequencing of seven paired samples of skin and bone marrow in seven subjects with secondary AML to identify somatic mutations specific to secondary AML. We then genotyped a bone marrow sample obtained during the antecedent myelodysplastic-syndrome stage from each subject to determine the presence or absence of the specific somatic mutations. We identified recurrent mutations in coding genes and defined the clonal architecture of each pair of samples from the myelodysplastic-syndrome stage and the secondary-AML stage, using the allele burden of hundreds of mutations. RESULTS Approximately 85% of bone marrow cells were clonal in the myelodysplastic-syndrome and secondary-AML samples, regardless of the myeloblast count. The secondary-AML samples contained mutations in 11 recurrently mutated genes, including 4 genes that have not been previously implicated in the myelodysplastic syndromes or AML. In every case, progression to acute leukemia was defined by the persistence of an antecedent founding clone containing 182 to 660 somatic mutations and the outgrowth or emergence of at least one subclone, harboring dozens to hundreds of new mutations. All founding clones and subclones contained at least one mutation in a coding gene. CONCLUSIONS Nearly all the bone marrow cells in patients with myelodysplastic syndromes and secondary AML are clonally derived. Genetic evolution of secondary AML is a dynamic process shaped by multiple cycles of mutation acquisition and clonal selection. Recurrent gene mutations are found in both founding clones and daughter subclones. (Funded by the National Institutes of Health and others.) PMID:22417201

Clonally Related Neisseria gonorrhoeae Isolates with Decreased Susceptibility to the Extended-Spectrum Cephalosporin Cefotaxime in Amsterdam, the Netherlands

PubMed Central

Heymans, Raymond; Bruisten, Sylvia M.; Golparian, Daniel; Unemo, Magnus; de Vries, Henry J. C.

2012-01-01

From 2006 to 2008, Neisseria gonorrhoeae isolates were identified with decreased susceptibility to the extended-spectrum cephalosporin (ESC) cefotaxime among visitors of the Amsterdam sexually transmitted infections (STI) clinic, the Netherlands. Spread, clonality, and characteristics of 202 isolates were examined using antibiograms, conventional penA mosaic gene PCR, and N. gonorrhoeae multiple-locus variable-number tandem repeat analysis (NG-MLVA). A strictly defined subset was further characterized by N. gonorrhoeae multiantigen sequence typing (NG-MAST) and sequencing of ESC resistance determinants (penA, mtrR, and porB1b). Seventy-four N. gonorrhoeae isolates with a cefotaxime MIC of >0.125 μg/ml (group A), 54 with a cefotaxime MIC of 0.125 μg/ml (group B), and a control group of 74 with a cefotaxime MIC of <0.125 μg/ml (group C) were included. Fifty-three clonally related penA mosaic-positive isolates (penicillin-binding protein 2 type XXXIV) were identified in group A (n = 47 isolates; 64%) and B (n = 6 isolates; 11%). The 53 penA mosaic-positive isolates were predominantly NG-MAST ST1407 (87%) and contained an mtrR promoter A deletion (98%) and porB1b alterations G101K/A102N. All were assigned to the same NG-MLVA cluster that comprised in total 56 isolates. A correlation was found between decreased cefotaxime susceptibility and ST1407 that was highly prevalent among visitors of the Amsterdam STI clinic. The rapid spread of this strain, which also has been identified in many other countries, might be facilitated by high-risk sexual behavior and should be monitored closely to identify potential treatment failure. Quality-assured surveillance of ESC susceptibility on the national and international levels and exploration of new drugs and/or strategies for treatment of gonorrhea are crucial. PMID:22214779

A reassessment of IgM memory subsets in humans

PubMed Central

Bagnara, Davide; Squillario, Margherita; Kipling, David; Mora, Thierry; Walczak, Aleksandra M.; Da Silva, Lucie; Weller, Sandra; Dunn-Walters, Deborah K.; Weill, Jean-Claude; Reynaud, Claude-Agnès

2015-01-01

From paired blood and spleen samples from three adult donors we performed high-throughput V-h sequencing of human B-cell subsets defined by IgD and CD27 expression: IgD+CD27+ (“MZ”), IgD−CD27+(“memory”, including IgM (“IgM-only”), IgG and IgA) and IgD−CD27− cells (“double-negative”, including IgM, IgG and IgA). 91,294 unique sequences clustered in 42,670 clones, revealing major clonal expansions in each of these subsets. Among these clones, we further analyzed those shared sequences from different subsets or tissues for Vh-gene mutation, H-CDR3-length, and Vh/Jh usage, comparing these different characteristics with all sequences from their subset of origin, for which these parameters constitute a distinct signature. The IgM-only repertoire profile differed notably from that of MZ B cells by a higher mutation frequency, and lower Vh4 and higher Jh6 gene usage. Strikingly, IgM sequences from clones shared between the MZ and the memory IgG/IgA compartments showed a mutation and repertoire profile of IgM-only and not of MZ B cells. Similarly, all IgM clonal relationships (between MZ, IgM-only, and double-negative compartments) involved sequences with the characteristics of IgM-only B cells. Finally, clonal relationships between tissues suggested distinct recirculation characteristics between MZ and switched B cells. The “IgM-only” subset (including cells with its repertoire signature but higher IgD or lower CD27 expression levels) thus appear as the only subset showing precursor-product relationships with CD27+ switched memory B cells, indicating that they represent germinal center-derived IgM memory B cells, and that IgM memory and MZ B cells constitute two distinct entities. PMID:26355154

A Reassessment of IgM Memory Subsets in Humans.

PubMed

Bagnara, Davide; Squillario, Margherita; Kipling, David; Mora, Thierry; Walczak, Aleksandra M; Da Silva, Lucie; Weller, Sandra; Dunn-Walters, Deborah K; Weill, Jean-Claude; Reynaud, Claude-Agnès

2015-10-15

From paired blood and spleen samples from three adult donors, we performed high-throughput VH sequencing of human B cell subsets defined by IgD and CD27 expression: IgD(+)CD27(+) ("marginal zone [MZ]"), IgD(-)CD27(+) ("memory," including IgM ["IgM-only"], IgG and IgA) and IgD(-)CD27(-) cells ("double-negative," including IgM, IgG, and IgA). A total of 91,294 unique sequences clustered in 42,670 clones, revealing major clonal expansions in each of these subsets. Among these clones, we further analyzed those shared sequences from different subsets or tissues for VH gene mutation, H-CDR3-length, and VH/JH usage, comparing these different characteristics with all sequences from their subset of origin for which these parameters constitute a distinct signature. The IgM-only repertoire profile differed notably from that of MZ B cells by a higher mutation frequency and lower VH4 and higher JH6 gene usage. Strikingly, IgM sequences from clones shared between the MZ and the memory IgG/IgA compartments showed a mutation and repertoire profile of IgM-only and not of MZ B cells. Similarly, all IgM clonal relationships (among MZ, IgM-only, and double-negative compartments) involved sequences with the characteristics of IgM-only B cells. Finally, clonal relationships between tissues suggested distinct recirculation characteristics between MZ and switched B cells. The "IgM-only" subset (including cells with its repertoire signature but higher IgD or lower CD27 expression levels) thus appear as the only subset showing precursor-product relationships with CD27(+) switched memory B cells, indicating that they represent germinal center-derived IgM memory B cells and that IgM memory and MZ B cells constitute two distinct entities. Copyright © 2015 by The American Association of Immunologists, Inc.

How Often Do They Have Sex? A Comparative Analysis of the Population Structure of Seven Eukaryotic Microbial Pathogens

PubMed Central

Tomasini, Nicolás; Lauthier, Juan José; Ayala, Francisco José; Tibayrenc, Michel; Diosque, Patricio

2014-01-01

The model of predominant clonal evolution (PCE) proposed for micropathogens does not state that genetic exchange is totally absent, but rather, that it is too rare to break the prevalent PCE pattern. However, the actual impact of this “residual” genetic exchange should be evaluated. Multilocus Sequence Typing (MLST) is an excellent tool to explore the problem. Here, we compared online available MLST datasets for seven eukaryotic microbial pathogens: Trypanosoma cruzi, the Fusarium solani complex, Aspergillus fumigatus, Blastocystis subtype 3, the Leishmania donovani complex, Candida albicans and Candida glabrata. We first analyzed phylogenetic relationships among genotypes within each dataset. Then, we examined different measures of branch support and incongruence among loci as signs of genetic structure and levels of past recombination. The analyses allow us to identify three types of genetic structure. The first was characterized by trees with well-supported branches and low levels of incongruence suggesting well-structured populations and PCE. This was the case for the T. cruzi and F. solani datasets. The second genetic structure, represented by Blastocystis spp., A. fumigatus and the L. donovani complex datasets, showed trees with weakly-supported branches but low levels of incongruence among loci, whereby genetic structuration was not clearly defined by MLST. Finally, trees showing weakly-supported branches and high levels of incongruence among loci were observed for Candida species, suggesting that genetic exchange has a higher evolutionary impact in these mainly clonal yeast species. Furthermore, simulations showed that MLST may fail to show right clustering in population datasets even in the absence of genetic exchange. In conclusion, these results make it possible to infer variable impacts of genetic exchange in populations of predominantly clonal micro-pathogens. Moreover, our results reveal different problems of MLST to determine the genetic structure in these organisms that should be considered. PMID:25054834

Ruminant Rhombencephalitis-Associated Listeria monocytogenes Alleles Linked to a Multilocus Variable-Number Tandem-Repeat Analysis Complex ▿ †

PubMed Central

Balandyté, Lina; Brodard, Isabelle; Frey, Joachim; Oevermann, Anna; Abril, Carlos

2011-01-01

Listeria monocytogenes is among the most important food-borne pathogens and is well adapted to persist in the environment. To gain insight into the genetic relatedness and potential virulence of L. monocytogenes strains causing central nervous system (CNS) infections, we used multilocus variable-number tandem-repeat analysis (MLVA) to subtype 183 L. monocytogenes isolates, most from ruminant rhombencephalitis and some from human patients, food, and the environment. Allelic-profile-based comparisons grouped L. monocytogenes strains mainly into three clonal complexes and linked single-locus variants (SLVs). Clonal complex A essentially consisted of isolates from human and ruminant brain samples. All but one rhombencephalitis isolate from cattle were located in clonal complex A. In contrast, food and environmental isolates mainly clustered into clonal complex C, and none was classified as clonal complex A. Isolates of the two main clonal complexes (A and C) obtained by MLVA were analyzed by PCR for the presence of 11 virulence-associated genes (prfA, actA, inlA, inlB, inlC, inlD, inlE, inlF, inlG, inlJ, and inlC2H). Virulence gene analysis revealed significant differences in the actA, inlF, inlG, and inlJ allelic profiles between clinical isolates (complex A) and nonclinical isolates (complex C). The association of particular alleles of actA, inlF, and newly described alleles of inlJ with isolates from CNS infections (particularly rhombencephalitis) suggests that these virulence genes participate in neurovirulence of L. monocytogenes. The overall absence of inlG in clinical complex A and its presence in complex C isolates suggests that the InlG protein is more relevant for the survival of L. monocytogenes in the environment. PMID:21984240

Vibrio parahaemolyticus isolates from southeastern Chinese coast are genetically diverse with circulation of clonal complex 3 strains since 2002.

PubMed

Yu, Ying; Hu, Weizhao; Wu, Beibei; Zhang, Peipei; Chen, Jianshun; Wang, Shuna; Fang, Weihuan

2011-11-01

Multilocus sequence typing (MLST) was used to examine the clonal relationship and genetic diversity of 71 Vibrio parahaemolyticus isolates from clinical and seafood-related sources in southeastern Chinese coast between 2002 and 2009. The tested isolates fell into 61 sequence types (STs). Of 17 clinical isolates, 7 belonged to ST3 of the pandemic clonal complex 3, with 3 strains isolated in 2002. Although there was no apparent clonal relationship found between clinical strains and those from seafood-related sources positive with pathogenic markers, there were clonal relationships between clinical strains from this study and those from environmental sources in other parts of China. Phylogenetic analysis showed that strains of 112 STs (61 STs from this study and 51 retrieved from PUBMLST database covering different continents) could be divided into four branches. The vast majority of our isolates and those from other countries were genetically diverse and clustered into two major branches of mixed distribution (of geographic origins and sample sources), whereas five STs representing six isolates split as two minor branches because of divergence of their recA genes, which had 80%-82% nucleotide identity to typical V. parahaemolyticus strains and 73.3%-76.9% identity to the CDS24 of a Vibrio sp. plasmid p23023, indicating that the recA gene might have recombined by lateral gene transfer. This was further supported by a high ratio of recombination to mutation (3.038) for recA. In conclusion, MLST with fully extractable database is a powerful system for analysis of clonal relationship for strains of a particular region in a national or global scale as well as between clinical and environmental or food-related strains.

Based Upon Repeat Pattern (BURP): an algorithm to characterize the long-term evolution of Staphylococcus aureus populations based on spa polymorphisms.

PubMed

Mellmann, Alexander; Weniger, Thomas; Berssenbrügge, Christoph; Rothgänger, Jörg; Sammeth, Michael; Stoye, Jens; Harmsen, Dag

2007-10-29

For typing of Staphylococcus aureus, DNA sequencing of the repeat region of the protein A (spa) gene is a well established discriminatory method for outbreak investigations. Recently, it was hypothesized that this region also reflects long-term epidemiology. However, no automated and objective algorithm existed to cluster different repeat regions. In this study, the Based Upon Repeat Pattern (BURP) implementation that is a heuristic variant of the newly described EDSI algorithm was investigated to infer the clonal relatedness of different spa types. For calibration of BURP parameters, 400 representative S. aureus strains with different spa types were characterized by MLST and clustered using eBURST as "gold standard" for their phylogeny. Typing concordance analysis between eBURST and BURP clustering (spa-CC) were performed using all possible BURP parameters to determine their optimal combination. BURP was subsequently evaluated with a strain collection reflecting the breadth of diversity of S. aureus (JCM 2002; 40:4544). In total, the 400 strains exhibited 122 different MLST types. eBURST grouped them into 23 clonal complexes (CC; 354 isolates) and 33 singletons (46 isolates). BURP clustering of spa types using all possible parameter combinations and subsequent comparison with eBURST CCs resulted in concordances ranging from 8.2 to 96.2%. However, 96.2% concordance was reached only if spa types shorter than 8 repeats were excluded, which resulted in 37% excluded spa types. Therefore, the optimal combination of the BURP parameters was "exclude spa types shorter than 5 repeats" and "cluster spa types into spa-CC if cost distances are less than 4" exhibiting 95.3% concordance to eBURST. This algorithm identified 24 spa-CCs, 40 singletons, and excluded only 7.8% spa types. Analyzing the natural population with these parameters, the comparison of whole-genome micro-array groupings (at the level of 0.31 Pearson correlation index) and spa-CCs gave a concordance of 87.1%; BURP spa-CCs vs. manually grouped spa types resulted in 95.7% concordance. BURP is the first automated and objective tool to infer clonal relatedness from spa repeat regions. It is able to extract an evolutionary signal rather congruent to MLST and micro-array data.

Partner-Drug Resistance and Population Substructuring of Artemisinin-Resistant Plasmodium falciparum in Cambodia

PubMed Central

Parobek, Christian M.; Parr, Jonathan B.; Brazeau, Nicholas F.; Lon, Chanthap; Chaorattanakawee, Suwanna; Gosi, Panita; Barnett, Eric J.; Norris, Lauren D.; Meshnick, Steven R.; Spring, Michele D.; Lanteri, Charlotte A.; Bailey, Jeffrey A.; Saunders, David L.; Lin, Jessica T.

2017-01-01

Abstract Plasmodium falciparum in western Cambodia has developed resistance to artemisinin and its partner drugs, causing frequent treatment failure. Understanding this evolution can inform the deployment of new therapies. We investigated the genetic architecture of 78 falciparum isolates using whole-genome sequencing, correlating results to in vivo and ex vivo drug resistance and exploring the relationship between population structure, demographic history, and partner drug resistance. Principle component analysis, network analysis and demographic inference identified a diverse central population with three clusters of clonally expanding parasite populations, each associated with specific K13 artemisinin resistance alleles and partner drug resistance profiles which were consistent with the sequential deployment of artemisinin combination therapies in the region. One cluster displayed ex vivo piperaquine resistance and mefloquine sensitivity with a high rate of in vivo failure of dihydroartemisinin-piperaquine. Another cluster displayed ex vivo mefloquine resistance and piperaquine sensitivity with high in vivo efficacy of dihydroartemisinin-piperaquine. The final cluster was clonal and displayed intermediate sensitivity to both drugs. Variations in recently described piperaquine resistance markers did not explain the difference in mean IC90 or clinical failures between the high and intermediate piperaquine resistance groups, suggesting additional loci may be involved in resistance. The results highlight an important role for partner drug resistance in shaping the P. falciparum genetic landscape in Southeast Asia and suggest that further work is needed to evaluate for other mutations that drive piperaquine resistance. PMID:28854635

Validation of the REMA score for predicting mast cell clonality and systemic mastocytosis in patients with systemic mast cell activation symptoms.

PubMed

Alvarez-Twose, I; González-de-Olano, D; Sánchez-Muñoz, L; Matito, A; Jara-Acevedo, M; Teodosio, C; García-Montero, A; Morgado, J M; Orfao, A; Escribano, L

2012-01-01

A variable percentage of patients with systemic mast cell (MC) activation symptoms meet criteria for systemic mastocytosis (SM). We prospectively evaluated the clinical utility of the REMA score versus serum baseline tryptase (sBt) levels for predicting MC clonality and SM in 158 patients with systemic MC activation symptoms in the absence of mastocytosis in the skin (MIS). World Health Organization criteria for SM were applied in all cases. MC clonality was defined as the presence of KIT-mutated MC or by a clonal HUMARA test. The REMA score consisted of the assignment of positive or negative points as follows: male (+1), female (-1), sBt <15 μg/l (-1) or >25 μg/l (+2), presence (-2) or absence (+1) of pruritus, hives or angioedema and presence (+3) of presyncope or syncope. Efficiency of the REMA score for predicting MC clonality and SM was assessed by receiver operating characteristic (ROC) curve analyses and compared to those obtained by means of sBt levels alone. Molecular studies revealed the presence of clonal MC in 68/80 SM cases and in 11/78 patients who did not meet the criteria for SM. ROC curve analyses confirmed the greater sensitivity and a similar specificity of the REMA score versus sBt levels (84 vs. 59% and 74 vs. 70% for MC clonality and 87 vs. 62% and 73 vs. 71% for SM, respectively). Our results confirm the clinical utility of the REMA score to predict MC clonality and SM in patients suffering from systemic MC activation symptoms without MIS. Copyright © 2011 S. Karger AG, Basel.

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

PubMed

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.

Clonal structure in Ichthyobacterium seriolicida, the causative agent of bacterial haemolytic jaundice in yellowtail, Seriola quinqueradiata, inferred from molecular epidemiological analysis.

PubMed

Matsuyama, T; Fukuda, Y; Sakai, T; Tanimoto, N; Nakanishi, M; Nakamura, Y; Takano, T; Nakayasu, C

2017-08-01

Bacterial haemolytic jaundice caused by Ichthyobacterium seriolicida has been responsible for mortality in farmed yellowtail, Seriola quinqueradiata, in western Japan since the 1980s. In this study, polymorphic analysis of I. seriolicida was performed using three molecular methods: amplified fragment length polymorphism (AFLP) analysis, multilocus sequence typing (MLST) and multiple-locus variable-number tandem repeat analysis (MLVA). Twenty-eight isolates were analysed using AFLP, while 31 isolates were examined by MLST and MLVA. No polymorphisms were identified by AFLP analysis using EcoRI and MseI, or by MLST of internal fragments of eight housekeeping genes. However, MLVA revealed variation in repeat numbers of three elements, allowing separation of the isolates into 16 sequence types. The unweighted pair group method using arithmetic averages cluster analysis of the MLVA data identified four major clusters, and all isolates belonged to clonal complexes. It is likely that I. seriolicida populations share a common ancestor, which may be a recently introduced strain. © 2016 John Wiley & Sons Ltd.

Emergence of carbapenem non-susceptible multidrug resistant Acinetobacter baumannii strains of clonal complexes 103(B) and 92(B) harboring OXA-type carbapenemases and metallo-β-lactamases in Southern India.

PubMed

Saranathan, Rajagopalan; Vasanth, Vaidyanathan; Vasanth, Thamodharan; Shabareesh, Pidathala Raghavendra Venkata; Shashikala, P; Devi, Chandrakesan Sheela; Kalaivani, Ramakrishnan; Asir, Johny; Sudhakar, Pagal; Prashanth, K

2015-05-01

The molecular epidemiology and carbapenem resistance mechanisms of clinical isolates of Acinetobacter baumannii obtained from a south Indian tertiary care hospital were investigated by repetitive extragenic palindromic sequence PCR (REP-PCR) and multi-locus sequence typing (MLST). Analysis of resistant determinants was achieved by PCR screening for the presence of genes encoding OXA-carbapenemases, metallo-β-lactamases (MBLs) and efflux pumps. REP-PCR generated around eight clusters of high heterogeneity; of these, two major clusters (I and V) appeared to be clonal in origin. Analysis of representative isolates from different clusters by MLST revealed that most of the isolates belonged to sequence type 103 of CC103(B) . Second most prevalent ST belonged to clonal complex (CC) 92(B) which is also referred to as international clone II. Most of the isolates were multi-drug resistant, being susceptible only to polymyxin-B and newer quinolones. Class D β-lactamases such as blaOXA-51-like (100%), blaOXA-23-like (56.8%) and blaOXA-24-like (14.8%) were found to be predominant, followed by a class B β-lactamase, namely blaIMP-1 (40.7%); none of the isolates had blaOXA-58 like, blaNDM-1 or blaSIM-1 . Genes of efflux-pump adeABC were predominant, most of isolates being biofilm producers that were PCR-positive for autoinducer synthase gene (>94%). Carbapenem non-susceptible isolates were highly diverse and present throughout the hospital irrespective of type of ward or intensive care unit. Although previous reports have documented diverse resistant mechanisms in A. baumannii, production of MBL and OXA-type of carbapenamases were found to be the predominant mechanism(s) of carbapenem resistance identified in strains isolated from Southern India. © 2015 The Societies and Wiley Publishing Asia Pty Ltd.

Clonal analysis reveals a common origin between nonsomite-derived neck muscles and heart myocardium

PubMed Central

Lescroart, Fabienne; Hamou, Wissam; Francou, Alexandre; Théveniau-Ruissy, Magali; Kelly, Robert G.; Buckingham, Margaret

2015-01-01

Neck muscles constitute a transition zone between somite-derived skeletal muscles of the trunk and limbs, and muscles of the head, which derive from cranial mesoderm. The trapezius and sternocleidomastoid neck muscles are formed from progenitor cells that have expressed markers of cranial pharyngeal mesoderm, whereas other muscles in the neck arise from Pax3-expressing cells in the somites. Mef2c-AHF-Cre genetic tracing experiments and Tbx1 mutant analysis show that nonsomitic neck muscles share a gene regulatory network with cardiac progenitor cells in pharyngeal mesoderm of the second heart field (SHF) and branchial arch-derived head muscles. Retrospective clonal analysis shows that this group of neck muscles includes laryngeal muscles and a component of the splenius muscle, of mixed somitic and nonsomitic origin. We demonstrate that the trapezius muscle group is clonally related to myocardium at the venous pole of the heart, which derives from the posterior SHF. The left clonal sublineage includes myocardium of the pulmonary trunk at the arterial pole of the heart. Although muscles derived from the first and second branchial arches also share a clonal relationship with different SHF-derived parts of the heart, neck muscles are clonally distinct from these muscles and define a third clonal population of common skeletal and cardiac muscle progenitor cells within cardiopharyngeal mesoderm. By linking neck muscle and heart development, our findings highlight the importance of cardiopharyngeal mesoderm in the evolution of the vertebrate heart and neck and in the pathophysiology of human congenital disease. PMID:25605943

Clonal analysis reveals a common origin between nonsomite-derived neck muscles and heart myocardium.

PubMed

Lescroart, Fabienne; Hamou, Wissam; Francou, Alexandre; Théveniau-Ruissy, Magali; Kelly, Robert G; Buckingham, Margaret

2015-02-03

Neck muscles constitute a transition zone between somite-derived skeletal muscles of the trunk and limbs, and muscles of the head, which derive from cranial mesoderm. The trapezius and sternocleidomastoid neck muscles are formed from progenitor cells that have expressed markers of cranial pharyngeal mesoderm, whereas other muscles in the neck arise from Pax3-expressing cells in the somites. Mef2c-AHF-Cre genetic tracing experiments and Tbx1 mutant analysis show that nonsomitic neck muscles share a gene regulatory network with cardiac progenitor cells in pharyngeal mesoderm of the second heart field (SHF) and branchial arch-derived head muscles. Retrospective clonal analysis shows that this group of neck muscles includes laryngeal muscles and a component of the splenius muscle, of mixed somitic and nonsomitic origin. We demonstrate that the trapezius muscle group is clonally related to myocardium at the venous pole of the heart, which derives from the posterior SHF. The left clonal sublineage includes myocardium of the pulmonary trunk at the arterial pole of the heart. Although muscles derived from the first and second branchial arches also share a clonal relationship with different SHF-derived parts of the heart, neck muscles are clonally distinct from these muscles and define a third clonal population of common skeletal and cardiac muscle progenitor cells within cardiopharyngeal mesoderm. By linking neck muscle and heart development, our findings highlight the importance of cardiopharyngeal mesoderm in the evolution of the vertebrate heart and neck and in the pathophysiology of human congenital disease.

Oriented clonal cell dynamics enables accurate growth and shaping of vertebrate cartilage.

PubMed

Kaucka, Marketa; Zikmund, Tomas; Tesarova, Marketa; Gyllborg, Daniel; Hellander, Andreas; Jaros, Josef; Kaiser, Jozef; Petersen, Julian; Szarowska, Bara; Newton, Phillip T; Dyachuk, Vyacheslav; Li, Lei; Qian, Hong; Johansson, Anne-Sofie; Mishina, Yuji; Currie, Joshua D; Tanaka, Elly M; Erickson, Alek; Dudley, Andrew; Brismar, Hjalmar; Southam, Paul; Coen, Enrico; Chen, Min; Weinstein, Lee S; Hampl, Ales; Arenas, Ernest; Chagin, Andrei S; Fried, Kaj; Adameyko, Igor

2017-04-17

Cartilaginous structures are at the core of embryo growth and shaping before the bone forms. Here we report a novel principle of vertebrate cartilage growth that is based on introducing transversally-oriented clones into pre-existing cartilage. This mechanism of growth uncouples the lateral expansion of curved cartilaginous sheets from the control of cartilage thickness, a process which might be the evolutionary mechanism underlying adaptations of facial shape. In rod-shaped cartilage structures (Meckel, ribs and skeletal elements in developing limbs), the transverse integration of clonal columns determines the well-defined diameter and resulting rod-like morphology. We were able to alter cartilage shape by experimentally manipulating clonal geometries. Using in silico modeling, we discovered that anisotropic proliferation might explain cartilage bending and groove formation at the macro-scale.

Clonal evolution and clinical significance of copy number neutral loss of heterozygosity of chromosome arm 6p in acquired aplastic anemia.

PubMed

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.

Clonal Evolution and Clinical Significance of Copy Number Neutral Loss of Heterozygosity of Chromosome Arm 6p in Acquired Aplastic Anemia

PubMed Central

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

CRISPR-cas loci profiling of Cronobacter sakazakii pathovars.

PubMed

Ogrodzki, Pauline; Forsythe, Stephen James

2016-12-01

Cronobacter sakazakii sequence types 1, 4, 8 and 12 are associated with outbreaks of neonatal meningitis and necrotizing enterocolitis infections. However clonality results in strains which are indistinguishable using conventional methods. This study investigated the use of clustered regularly interspaced short palindromic repeats (CRISPR)-cas loci profiling for epidemiological investigations. Seventy whole genomes of C. sakazakii strains from four clonal complexes which were widely distributed temporally, geographically and origin of source were profiled. All strains encoded the same type I-E subtype CRISPR-cas system with a total of 12 different CRISPR spacer arrays. This study demonstrated the greater discriminatory power of CRISPR spacer array profiling compared with multilocus sequence typing, which will be of use in source attribution during Cronobacter outbreak investigations.

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

PubMed

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.

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

PubMed Central

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

2008-01-01

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. PMID:18710585

A molecular surveillance reveals the prevalence of Vibrio cholerae O139 isolates in China from 1993 to 2012.

PubMed

Zhang, Ping; Zhou, Haijian; Diao, Baowei; Li, Fengjuan; Du, Pengcheng; Li, Jie; Kan, Biao; Morris, J Glenn; Wang, Duochun

2014-04-01

Vibrio cholerae serogroup O139 was first identified in 1992 in India and Bangladesh, in association with major epidemics of cholera in both countries; cases were noted shortly thereafter in China. We characterized 211 V. cholerae O139 isolates that were isolated at multiple sites in China between 1993 and 2012 from patients (n = 92) and the environment (n = 119). Among clinical isolates, 88 (95.7%) of 92 were toxigenic, compared with 47 (39.5%) of 119 environmental isolates. Toxigenic isolates carried the El Tor CTX prophage and toxin-coregulated pilus A gene (tcpA), as well as the Vibrio seventh pandemic island I (VSP-I) and VSP-II. Among a subset of 42 toxigenic isolates screened by multilocus sequence typing (MLST), all were in the same sequence type as a clinical isolate (MO45) from the original Indian outbreak. Nontoxigenic isolates, in contrast, generally lacked VSP-I and -II, and fell within 13 additional sequence types in two clonal complexes distinct from the toxigenic isolates. In further pulsed-field gel electrophoresis (PFGE) (with NotI digestion) studies, toxigenic isolates formed 60 pulsotypes clustered in one group, while the nontoxigenic isolates formed 43 pulsotypes which clustered into 3 different groups. Our data suggest that toxigenic O139 isolates from widely divergent geographic locations, while showing some diversity, have maintained a relatively tight clonal structure across a 20-year time span. Nontoxigenic isolates, in contrast, exhibited greater diversity, with multiple clonal lineages, than did their toxigenic counterparts.

Population clustering and clonal structure evidence the relict state of Ulmus minor Mill. in the Balearic Islands

PubMed Central

Fuentes-Utrilla, P; Valbuena-Carabaña, M; Ennos, R; Gil, L

2014-01-01

Field elm (Ulmus minor) is a riparian tree that grows in rare, small populations scattered along temporary watercourses in the Balearic Islands, nowadays mostly covered with Mediterranean vegetation. Agriculture and farming on the fertile land along the periodically flooded plains have reduced the elm populations to sparse tree lines along the creek beds. The presence of field elm in this very anthropic landscape has led some authors to consider it as an introduced species in the Balearics. However, pollen data suggest these elms may be the remains of larger populations experiencing continuous population shrinkage during the Holocene, and hence be native to the isles. In this paper, we apply genetic markers to assess whether field elm is or is not indigenous to the Balearic Islands. We compare the genetic variation in nine nuclear microsatellites of six Balearic populations (three in each of the largest islands, Majorca and Minorca) with that of three natural Iberian populations located in two regions, one geologically (Baetic mountains, SE Iberia) and another historically (Catalonia, NE Iberia) related to the islands. Principal coordinates analysis and Bayesian clustering methods reveal a strong genetic differentiation of the Balearic populations from the Iberian ones, and even among islands, which support their native origin. Genotypic variation in the islands is very low and clonal reproduction is very high compared with the mainland, as it is frequently observed in populations of clonal species where sexual reproduction is limited. We discuss the practical implications of these findings for the conservation of elm genetic resources of these findings. PMID:24619184

Genetic variation among Staphylococcus aureus strains from Norwegian bulk milk.

PubMed

Jørgensen, H J; Mørk, T; Caugant, D A; Kearns, A; Rørvik, L M

2005-12-01

Strains of Staphylococcus aureus obtained from bovine (n = 117) and caprine (n = 114) bulk milk were characterized and compared with S. aureus strains from raw-milk products (n = 27), bovine mastitis specimens (n = 9), and human blood cultures (n = 39). All isolates were typed by pulsed-field gel electrophoresis (PFGE). In addition, subsets of isolates were characterized using multilocus sequence typing (MLST), multiplex PCR (m-PCR) for genes encoding nine of the staphylococcal enterotoxins (SE), and the cloverleaf method for penicillin resistance. A variety of genotypes were observed, and greater genetic diversity was found among bovine than caprine bulk milk isolates. Certain genotypes, with a wide geographic distribution, were common to bovine and caprine bulk milk and may represent ruminant-specialized S. aureus. Isolates with genotypes indistinguishable from those of strains from ruminant mastitis were frequently found in bulk milk, and strains with genotypes indistinguishable from those from bulk milk were observed in raw-milk products. This indicates that S. aureus from infected udders may contaminate bulk milk and, subsequently, raw-milk products. Human blood culture isolates were diverse and differed from isolates from other sources. Genotyping by PFGE, MLST, and m-PCR for SE genes largely corresponded. In general, isolates with indistinguishable PFGE banding patterns had the same SE gene profile and isolates with identical SE gene profiles were placed together in PFGE clusters. Phylogenetic analyses agreed with the division of MLST sequence types into clonal complexes, and isolates within the same clonal complex had the same SE gene profile. Furthermore, isolates within PFGE clusters generally belonged to the same clonal complex.

Genetic Variation among Staphylococcus aureus Strains from Norwegian Bulk Milk

PubMed Central

Jørgensen, H. J.; Mørk, T.; Caugant, D. A.; Kearns, A.; Rørvik, L. M.

2005-01-01

Strains of Staphylococcus aureus obtained from bovine (n = 117) and caprine (n = 114) bulk milk were characterized and compared with S. aureus strains from raw-milk products (n = 27), bovine mastitis specimens (n = 9), and human blood cultures (n = 39). All isolates were typed by pulsed-field gel electrophoresis (PFGE). In addition, subsets of isolates were characterized using multilocus sequence typing (MLST), multiplex PCR (m-PCR) for genes encoding nine of the staphylococcal enterotoxins (SE), and the cloverleaf method for penicillin resistance. A variety of genotypes were observed, and greater genetic diversity was found among bovine than caprine bulk milk isolates. Certain genotypes, with a wide geographic distribution, were common to bovine and caprine bulk milk and may represent ruminant-specialized S. aureus. Isolates with genotypes indistinguishable from those of strains from ruminant mastitis were frequently found in bulk milk, and strains with genotypes indistinguishable from those from bulk milk were observed in raw-milk products. This indicates that S. aureus from infected udders may contaminate bulk milk and, subsequently, raw-milk products. Human blood culture isolates were diverse and differed from isolates from other sources. Genotyping by PFGE, MLST, and m-PCR for SE genes largely corresponded. In general, isolates with indistinguishable PFGE banding patterns had the same SE gene profile and isolates with identical SE gene profiles were placed together in PFGE clusters. Phylogenetic analyses agreed with the division of MLST sequence types into clonal complexes, and isolates within the same clonal complex had the same SE gene profile. Furthermore, isolates within PFGE clusters generally belonged to the same clonal complex. PMID:16332822

Novel squalene-producing thraustochytrids found in mangrove water.

PubMed

Otagiri, Masato; Khalid, Ammara; Moriya, Shigeharu; Osada, Hiroyuki; Takahashi, Shunji

2017-10-01

On extended screening of squalene-producing strains in Okinawa mangrove water, we identified 14 novel squalene-producing thraustochytrids from 172 unialgal clonal isolates. The novel thraustochytrids produced 13.9-7.54 mg squalene/g dry cell weight. Eight isolates were found to belong to potentially novel squalene-producing genera, forming a monophyletic cluster independent from any known thraustochytrids.

A genomic island harboring arsenic resistance genes varies in gene content and is located in different chromosomal loci among Listeria monocytogenes strains

USDA-ARS?s Scientific Manuscript database

In the foodborne pathogen Listeria monocytogenes, arsenic resistance has been often encountered among certain clonal groups of serotype 4b and was earlier found to be strongly associated with an arsenic resistance gene cluster within a 35 kb chromosomal region, designated Listeria genomic island 2 (...

Critical analysis of the stringent complete response in multiple myeloma: contribution of sFLC and bone marrow clonality

PubMed Central

Paiva, Bruno; López-Anglada, Lucía; Mateos, María-Victoria; Cedena, Teresa; Vidríales, María-Belén; Sáez-Gómez, María Auxiliadora; Contreras, Teresa; Oriol, Albert; Rapado, Inmaculada; Teruel, Ana-Isabel; Cordón, Lourdes; Blanchard, María Jesús; Bengoechea, Enrique; Palomera, Luis; de Arriba, Felipe; Cueto-Felgueroso, Cecilia; Orfao, Alberto; Bladé, Joan; San Miguel, Jesús F.; Lahuerta, Juan José

2015-01-01

Stringent complete response (sCR) criteria are used in multiple myeloma as a deeper response category compared with CR, but prospective validation is lacking, it is not always clear how evaluation of clonality is performed, and is it not known what the relative clinical influence is of the serum free light chain ratio (sFLCr) and bone marrow (BM) clonality to define more sCR. To clarify this controversy, we focused on 94 patients that reached CR, of which 69 (73%) also fulfilled the sCR criteria. Patients with sCR displayed slightly longer time to progression (median, 62 vs 53 months, respectively; P = .31). On analyzing this contribution to the prognosis of sFLCr or clonality, it was found that the sFLCr does not identify patients in CR at distinct risk; by contrast, low-sensitive multiparametric flow cytometry (MFC) immunophenotyping (2 colors), which is equivalent to immunohistochemistry, identifies a small number of patients (5 cases) with high residual tumor burden and dismal outcome; nevertheless, using traditional 4-color MFC, persistent clonal BM disease was detectable in 36% of patients, who, compared with minimal residual disease-negative cases, had a significantly inferior outcome. These results show that the current definition of sCR should be revised. PMID:26089396

Application of the two-stage clonal expansion model in characterizing the joint effect of exposure to two carcinogens

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zielinski, J.M.; Krewski, D.

1992-12-31

In this paper, we describe application of the two-stage clonal expansion model to characterize the joint effect of exposure to two carcinogens. This biologically based model of carcinogenesis provides a useful framework for the quantitative description of carcinogenic risks and for defining agents that act as initiators, promoters, and completers. Depending on the mechanism of action, the agent-specific relative risk following exposure to two carcinogens can be additive, multiplicative, or supramultiplicative, with supra-additive relative risk indicating a synergistic effect between the two agents. Maximum-likelihood methods for fitting the two-stage clonal expansion model with intermittent exposure to two carcinogens are describedmore » and illustrated, using data on lung-cancer mortality among Colorado uranium miners exposed to both radon and tobacco smoke.« less

Beating the odds: The poisson distribution of all input cells during limiting dilution grossly underestimates whether a cell line is clonally-derived or not.

PubMed

Zhou, Yizhou; Shaw, David; Lam, Cynthia; Tsukuda, Joni; Yim, Mandy; Tang, Danming; Louie, Salina; Laird, Michael W; Snedecor, Brad; Misaghi, Shahram

2017-09-23

Establishing that a cell line was derived from a single cell progenitor and defined as clonally-derived for the production of clinical and commercial therapeutic protein drugs has been the subject of increased emphasis in cell line development (CLD). Several regulatory agencies have expressed that the prospective probability of clonality for CHO cell lines is assumed to follow the Poisson distribution based on the input cell count. The probability of obtaining monoclonal progenitors based on the Poisson distribution of all cells suggests that one round of limiting dilution may not be sufficient to assure the resulting cell lines are clonally-derived. We experimentally analyzed clonal derivatives originating from single cell cloning (SCC) via one round of limiting dilution, following our standard legacy cell line development practice. Two cell populations with stably integrated DNA spacers were mixed and subjected to SCC via limiting dilution. Cells were cultured in the presence of selection agent, screened, and ranked based on product titer. Post-SCC, the growing cell lines were screened by PCR analysis for the presence of identifying spacers. We observed that the percentage of nonclonal populations was below 9%, which is considerably lower than the determined probability based on the Poisson distribution of all cells. These results were further confirmed using fluorescence imaging of clonal derivatives originating from SCC via limiting dilution of mixed cell populations expressing GFP or RFP. Our results demonstrate that in the presence of selection agent, the Poisson distribution of all cells clearly underestimates the probability of obtaining clonally-derived cell lines. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 2017. © 2017 American Institute of Chemical Engineers.

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

PubMed

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 and provides a rapid reference for epidemiologists wishing to track the origin of infection without the need to compile population data and learn population assignment algorithms.

Generation of a conditional analog-sensitive kinase in human cells using CRISPR/Cas9-mediated genome engineering.

PubMed

Moyer, Tyler C; Holland, Andrew J

2015-01-01

The ability to rapidly and specifically modify the genome of mammalian cells has been a long-term goal of biomedical researchers. Recently, the clustered, regularly interspaced, short palindromic repeats (CRISPR)/Cas9 system from bacteria has been exploited for genome engineering in human cells. The CRISPR system directs the RNA-guided Cas9 nuclease to a specific genomic locus to induce a DNA double-strand break that may be subsequently repaired by homology-directed repair using an exogenous DNA repair template. Here we describe a protocol using CRISPR/Cas9 to achieve bi-allelic insertion of a point mutation in human cells. Using this method, homozygous clonal cell lines can be constructed in 5-6 weeks. This method can also be adapted to insert larger DNA elements, such as fluorescent proteins and degrons, at defined genomic locations. CRISPR/Cas9 genome engineering offers exciting applications in both basic science and translational research. Copyright © 2015 Elsevier Inc. All rights reserved.

Spatial genetic structure reflects extensive clonality, low genotypic diversity and habitat fragmentation in Grevillea renwickiana (Proteaceae), a rare, sterile shrub from south-eastern Australia

PubMed Central

James, Elizabeth A.; McDougall, Keith L.

2014-01-01

Background and Aims The association of clonality, polyploidy and reduced fecundity has been identified as an extinction risk for clonal plants. Compromised sexual reproduction limits both their ability to adapt to new conditions and their capacity to disperse to more favourable environments. Grevillea renwickiana is a prostrate, putatively sterile shrub reliant on asexual reproduction. Dispersal is most likely limited by the rate of clonal expansion via rhizomes. The nine localized populations constituting this species provide an opportunity to examine the extent of clonality and spatial genotypic diversity to evaluate its evolutionary prospects. Methods Ten microsatellite loci were used to compare genetic and genotypic diversity across all sites with more intensive sampling at four locations (n = 185). The spatial distribution of genotypes and chloroplast DNA haplotypes based on the trnQ–rps16 intergenic spacer region were compared. Chromosome counts provided a basis for examining genetic profiles inconsistent with diploidy. Key Results Microsatellite analysis identified 46 multilocus genotypes (MLGs) in eight multilocus clonal lineages (MLLs). MLLs are not shared among sites, with two exceptions. Spatial autocorrelation was significant to 1·6 km. Genotypic richness ranged from 0 to 0·33. Somatic mutation is likely to contribute to minor variation between MLGs within clonal lineages. The eight chloroplast haplotypes identified were correlated with eight MLLs defined by ordination and generally restricted to single populations. Triploidy is the most likely reason for tri-allelic patterns. Conclusions Grevillea renwickiana comprises few genetic individuals. Sterility has most likely been induced by triploidy. Extensive lateral suckering in long-lived sterile clones facilitates the accumulation of somatic mutations, which contribute to the measured genetic diversity. Genetic conservation value may not be a function of population size. Despite facing evolutionary stagnation, sterile clonal species can play a vital role in mitigating ecological instability as floras respond to rapid environmental change. PMID:24737718

Clonal hematopoiesis as determined by the HUMARA assay is a marker for acquired mutations in epigenetic regulators in older women.

PubMed

Wiedmeier, Julia Erin; Kato, Catherine; Zhang, Zhenzhen; Lee, Hyunjung; Dunlap, Jennifer; Nutt, Eric; Rattray, Rogan; McKay, Sarah; Eide, Christopher; Press, Richard; Mori, Motomi; Druker, Brian; Dao, Kim-Hien

2016-09-01

Recent large cohort studies revealed that healthy older individuals harbor somatic mutations that increase their risk for hematologic malignancy and all-cause cardiovascular deaths. The majority of these mutations are in chromatin and epigenetic regulatory genes (CERGs). CERGs play a key role in regulation of DNA methylation (DNMT3A and TET2) and histone function (ASXL1) and in clonal proliferation of hematopoietic stem cells. We hypothesize that older women manifesting clonal hematopoiesis, defined here as a functional phenomenon in which a hematopoietic stem cell has acquired a survival and proliferative advantage, harbor a higher frequency of somatic mutations in CERGs. The human androgen receptor gene (HUMARA) assay was used in our study to detect the presence of nonrandom X inactivation in women, a marker for clonal hematopoiesis. In our pilot study, we tested 127 blood samples from women ≥65 years old without a history of invasive cancer or hematologic malignancies. Applying stringent qualitative criteria, we found that 26% displayed clonal hematopoiesis; 52.8% displayed polyclonal hematopoiesis; and 21.3% had indeterminate patterns (too close to call by qualitative assessment). Using Illumina MiSeq next-generation sequencing, we identified somatic mutations in CERGs in 15.2% of subjects displaying clonal hematopoiesis (three ASXL1 and two DNMT3A mutations with an average variant allele frequency of 15.7%, range: 6.3%-23.3%). In a more limited sequencing analysis, we evaluated the frequency of ASXL1 mutations by Sanger sequencing and found mutations in 9.7% of the clonal samples and 0% of the polyclonal samples. By comparing several recent studies (with some caveats as described), we determined the fold enrichment of detecting CERG mutations by using the HUMARA assay as a functional screen for clonal hematopoiesis. We conclude that a functional assay of clonal hematopoiesis is enriching for older women with somatic mutations in CERGs, particularly for ASXL1 and TET2 mutations and less so for DNMT3A mutations. Copyright © 2016 ISEH - International Society for Experimental Hematology. Published by Elsevier Inc. All rights reserved.

Processing-Dependent and Clonal Contamination Patterns of Listeria monocytogenes in the Cured Ham Food Chain Revealed by Genetic Analysis

PubMed Central

Morganti, Marina; Scaltriti, Erika; Cozzolino, Paolo; Bolzoni, Luca; Casadei, Gabriele; Pierantoni, Marco; Foni, Emanuela

2015-01-01

The quantitative and qualitative patterns of environmental contamination by Listeria monocytogenes were investigated in the production chain of dry-cured Parma ham. Standard arrays of surfaces were sampled in processing facilities during a single visit per plant in the three compartments of the food chain, i.e., ham production (19 plants) and postproduction, which was divided into deboning (43 plants) and slicing (25 plants) steps. The numbers of sampled surfaces were 384 in ham production, with 25 positive for L. monocytogenes, and 1,084 in postproduction, with 83 positives. Statistical analysis of the prevalence of contaminated surfaces showed that in ham production, contamination was higher at the beginning of processing and declined significantly toward the end, while in postproduction, prevalence rose toward the end of processing. Prevalence was higher in the deboning facilities than in slicing facilities and was dependent on the type of surface (floor/drainage > clothing > equipment). The qualitative pattern of contamination was investigated through an analysis of the survey isolates and a set of isolates derived from routine monitoring, including longitudinal isolations. Pulsed-field gel electrophoresis (PFGE) and whole-genome single-nucleotide polymorphism (SNP) analysis revealed a remarkable clonality of L. monocytogenes within plants, with the detection of 16 plant-specific clones out of 17 establishments with multiple isolates. Repeated detections of clonal isolates >6 months apart were also observed. Six was the maximum number of between-isolate differences in core SNPs observed within these clones. Based on the same six-SNP threshold, three clusters of clonal isolates, shared by six establishments, were also identified. The spread of L. monocytogenes within and between plants, as indicated by its clonal behavior, is a matter of concern for the hygienic management of establishments. PMID:26590278

Oriented clonal cell dynamics enables accurate growth and shaping of vertebrate cartilage

PubMed Central

Kaucka, Marketa; Zikmund, Tomas; Tesarova, Marketa; Gyllborg, Daniel; Hellander, Andreas; Jaros, Josef; Kaiser, Jozef; Petersen, Julian; Szarowska, Bara; Newton, Phillip T; Dyachuk, Vyacheslav; Li, Lei; Qian, Hong; Johansson, Anne-Sofie; Mishina, Yuji; Currie, Joshua D; Tanaka, Elly M; Erickson, Alek; Dudley, Andrew; Brismar, Hjalmar; Southam, Paul; Coen, Enrico; Chen, Min; Weinstein, Lee S; Hampl, Ales; Arenas, Ernest; Chagin, Andrei S; Fried, Kaj; Adameyko, Igor

2017-01-01

Cartilaginous structures are at the core of embryo growth and shaping before the bone forms. Here we report a novel principle of vertebrate cartilage growth that is based on introducing transversally-oriented clones into pre-existing cartilage. This mechanism of growth uncouples the lateral expansion of curved cartilaginous sheets from the control of cartilage thickness, a process which might be the evolutionary mechanism underlying adaptations of facial shape. In rod-shaped cartilage structures (Meckel, ribs and skeletal elements in developing limbs), the transverse integration of clonal columns determines the well-defined diameter and resulting rod-like morphology. We were able to alter cartilage shape by experimentally manipulating clonal geometries. Using in silico modeling, we discovered that anisotropic proliferation might explain cartilage bending and groove formation at the macro-scale. DOI: http://dx.doi.org/10.7554/eLife.25902.001 PMID:28414273

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

PubMed

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.

Orthogonal typing methods identify genetic diversity among Belgian Campylobacter jejuni strains isolated over a decade from poultry and cases of sporadic human illness.

PubMed

Elhadidy, Mohamed; Arguello, Hector; Álvarez-Ordóñez, Avelino; Miller, William G; Duarte, Alexandra; Martiny, Delphine; Hallin, Marie; Vandenberg, Olivier; Dierick, Katelijne; Botteldoorn, Nadine

2018-06-20

Campylobacter jejuni is a zoonotic pathogen commonly associated with human gastroenteritis. Retail poultry meat is a major food-related transmission source of C. jejuni to humans. The present study investigated the genetic diversity, clonal relationship, and strain risk-analysis of 403 representative C. jejuni isolates from chicken broilers (n = 204) and sporadic cases of human diarrhea (n = 199) over a decade (2006-2015) in Belgium, using multilocus sequence typing (MLST), PCR binary typing (P-BIT), and identification of lipooligosaccharide (LOS) biosynthesis locus classes. A total of 123 distinct sequence types (STs), clustered in 28 clonal complexes (CCs) were assigned, including ten novel sequence types that were not previously documented in the international database. Sequence types ST-48, ST-21, ST-50, ST-45, ST-464, ST-2274, ST-572, ST-19, ST-257 and ST-42 were the most prevalent. Clonal complex 21 was the main clonal complex in isolates from humans and chickens. Among observed STs, a total of 35 STs that represent 72.2% (291/403) of the isolates were identified in both chicken and human isolates confirming considerable epidemiological relatedness; these 35 STs also clustered together in the most prevalent CCs. A majority of the isolates harbored sialylated LOS loci associated with potential neuropathic outcomes in humans. Although the concordance between MLST and P-BIT, determined by the adjusted Rand and Wallace coefficients, showed low congruence between both typing methods. The discriminatory power of P-BIT and MLST was similar, with Simpson's diversity indexes of 0.978 and 0.975, respectively. Furthermore, P-BIT could provide additional epidemiological information that would provide further insights regarding the potential association to human health from each strain. In addition, certain clones could be linked to specific clinical symptoms. Indeed, LOS class E was associated with less severe infections. Moreover, ST-572 was significantly associated with clinical infections occurring after travelling abroad. Ultimately, the data generated from this study will help to better understand the molecular epidemiology of C. jejuni infection. Copyright © 2018. Published by Elsevier B.V.

Selfish Spermatogonial Selection: Evidence from an Immunohistochemical Screen in Testes of Elderly Men

PubMed Central

Turner, Gareth D. H.; Dudka-Ruszkowska, Wioleta; Taylor, Stephen; Meyts, Ewa Rajpert-De; Goriely, Anne; Wilkie, Andrew O. M.

2012-01-01

The dominant congenital disorders Apert syndrome, achondroplasia and multiple endocrine neoplasia–caused by specific missense mutations in the FGFR2, FGFR3 and RET proteins respectively–represent classical examples of paternal age-effect mutation, a class that arises at particularly high frequencies in the sperm of older men. Previous analyses of DNA from randomly selected cadaveric testes showed that the levels of the corresponding FGFR2, FGFR3 and RET mutations exhibit very uneven spatial distributions, with localised hotspots surrounded by large mutation-negative areas. These studies imply that normal testes are mosaic for clusters of mutant cells: these clusters are predicted to have altered growth and signalling properties leading to their clonal expansion (selfish spermatogonial selection), but DNA extraction eliminates the possibility to study such processes at a tissue level. Using a panel of antibodies optimised for the detection of spermatocytic seminoma, a rare tumour of spermatogonial origin, we demonstrate that putative clonal events are frequent within normal testes of elderly men (mean age: 73.3 yrs) and can be classed into two broad categories. We found numerous small (less than 200 cells) cellular aggregations with distinct immunohistochemical characteristics, localised to a portion of the seminiferous tubule, which are of uncertain significance. However more infrequently we identified additional regions where entire seminiferous tubules had a circumferentially altered immunohistochemical appearance that extended through multiple serial sections that were physically contiguous (up to 1 mm in length), and exhibited enhanced staining for antibodies both to FGFR3 and a marker of downstream signal activation, pAKT. These findings support the concept that populations of spermatogonia in individual seminiferous tubules in the testes of older men are clonal mosaics with regard to their signalling properties and activation, thus fulfilling one of the specific predictions of selfish spermatogonial selection. PMID:22879958

Critical analysis of the stringent complete response in multiple myeloma: contribution of sFLC and bone marrow clonality.

PubMed

Martínez-López, Joaquín; Paiva, Bruno; López-Anglada, Lucía; Mateos, María-Victoria; Cedena, Teresa; Vidríales, María-Belén; Sáez-Gómez, María Auxiliadora; Contreras, Teresa; Oriol, Albert; Rapado, Inmaculada; Teruel, Ana-Isabel; Cordón, Lourdes; Blanchard, María Jesús; Bengoechea, Enrique; Palomera, Luis; de Arriba, Felipe; Cueto-Felgueroso, Cecilia; Orfao, Alberto; Bladé, Joan; San Miguel, Jesús F; Lahuerta, Juan José

2015-08-13

Stringent complete response (sCR) criteria are used in multiple myeloma as a deeper response category compared with CR, but prospective validation is lacking, it is not always clear how evaluation of clonality is performed, and is it not known what the relative clinical influence is of the serum free light chain ratio (sFLCr) and bone marrow (BM) clonality to define more sCR. To clarify this controversy, we focused on 94 patients that reached CR, of which 69 (73%) also fulfilled the sCR criteria. Patients with sCR displayed slightly longer time to progression (median, 62 vs 53 months, respectively; P = .31). On analyzing this contribution to the prognosis of sFLCr or clonality, it was found that the sFLCr does not identify patients in CR at distinct risk; by contrast, low-sensitive multiparametric flow cytometry (MFC) immunophenotyping (2 colors), which is equivalent to immunohistochemistry, identifies a small number of patients (5 cases) with high residual tumor burden and dismal outcome; nevertheless, using traditional 4-color MFC, persistent clonal BM disease was detectable in 36% of patients, who, compared with minimal residual disease-negative cases, had a significantly inferior outcome. These results show that the current definition of sCR should be revised. © 2015 by The American Society of Hematology.

Marked heterogeneity in growth characteristics of myoblast clonal cultures and myoblast mixed cultures obtained from the same individual.

PubMed

Maier, Andrea B; Cohen, Ron; Blom, Joke; van Heemst, Diana; Westendorp, Rudi G J

2012-01-01

Sarcopenia is defined as an age-related decrease in skeletal muscle mass and function while adjacent satellite cells are unable to compensate for this loss. However, myoblast cultures can be established even in the presence of sarcopenia. It is yet unknown whether satellite cells from failing muscle in older age are equally affected, as human satellite cells have been assessed using myoblast mixed cultures and not by using myoblast clonal cultures. We questioned to what extent myoblast mixed cultures reflect the in vivo characteristics of single satellite cells from adult skeletal muscle. We established a myoblast mixed culture and three myoblast clonal cultures out of the same muscle biopsy and cultured these cells for 100 days. Replicative capacity and oxidative stress resistance were compared. We found marked heterogeneity between the myoblast clonal cultures that all had a significantly lower replicative capacity when compared to the mixed culture. Replicative capacity of the clonal cultures was inversely related to the β-galactosidase activity after exposure to oxidative stress. Addition of L-carnosine enhanced the remaining replicative capacity in all cultures with a concomitant marginal decrease in β-galactosidase activity. It is concluded that myoblast mixed cultures in vitro do not reflect the marked heterogeneity between single isolated satellite cells. The consequences of the heterogeneity on muscle performance remain to be established. Copyright © 2011 S. Karger AG, Basel.

Genomic insights from whole genome sequencing of four clonal outbreak Campylobacter jejuni assessed within the global C. jejuni population.

PubMed

Clark, Clifford G; Berry, Chrystal; Walker, Matthew; Petkau, Aaron; Barker, Dillon O R; Guan, Cai; Reimer, Aleisha; Taboada, Eduardo N

2016-12-03

Whole genome sequencing (WGS) is useful for determining clusters of human cases, investigating outbreaks, and defining the population genetics of bacteria. It also provides information about other aspects of bacterial biology, including classical typing results, virulence, and adaptive strategies of the organism. Cell culture invasion and protein expression patterns of four related multilocus sequence type 21 (ST21) C. jejuni isolates from a significant Canadian water-borne outbreak were previously associated with the presence of a CJIE1 prophage. Whole genome sequencing was used to examine the genetic diversity among these isolates and confirm that previous observations could be attributed to differential prophage carriage. Moreover, we sought to determine the presence of genome sequences that could be used as surrogate markers to delineate outbreak-associated isolates. Differential carriage of the CJIE1 prophage was identified as the major genetic difference among the four outbreak isolates. High quality single-nucleotide variant (hqSNV) and core genome multilocus sequence typing (cgMLST) clustered these isolates within expanded datasets consisting of additional C. jejuni strains. The number and location of homopolymeric tract regions was identical in all four outbreak isolates but differed from all other C. jejuni examined. Comparative genomics and PCR amplification enabled the identification of large chromosomal inversions of approximately 93 kb and 388 kb within the outbreak isolates associated with transducer-like proteins containing long nucleotide repeat sequences. The 93-kb inversion was characteristic of the outbreak-associated isolates, and the gene content of this inverted region displayed high synteny with the reference strain. The four outbreak isolates were clonally derived and differed mainly in the presence of the CJIE1 prophage, validating earlier findings linking the prophage to phenotypic differences in virulence assays and protein expression. The identification of large, genetically syntenous chromosomal inversions in the genomes of outbreak-associated isolates provided a unique method for discriminating outbreak isolates from the background population. Transducer-like proteins appear to be associated with the chromosomal inversions. CgMLST and hqSNV analysis also effectively delineated the outbreak isolates within the larger C. jejuni population structure.

Clinical and epidemiological analysis of Campylobacter fetus subsp. fetus infections in humans and comparative genetic analysis with strains isolated from cattle.

PubMed

Escher, Robert; Brunner, Colette; von Steiger, Niklaus; Brodard, Isabelle; Droz, Sara; Abril, Carlos; Kuhnert, Peter

2016-05-14

Campylobacter fetus subspecies fetus (CFF) is an important pathogen for both cattle and humans. We performed a systematic epidemiological and clinical study of patients and evaluated the genetic relatedness of 17 human and 17 bovine CFF isolates by using different genotyping methods. In addition, the serotype, the dissemination of the genomic island containing a type IV secretion system (T4SS) and resistance determinants for tetracycline and streptomycin were also evaluated. The isolates from patients diagnosed with CFF infection as well as those from faecal samples of healthy calves were genotyped using pulsed-field gel electrophoresis (PFGE), multilocus sequence typing (MLST), as well as single locus sequence typing (SLST) targeting cmp1 and cmp2 genes encoding two major outer membrane proteins in CFF. The presence of the genomic island and identification of serotype was determined by PCRs targeting genes of the T4SS and the sap locus, respectively. Tetracycline and streptomycin resistance phenotypes were determined by minimal inhibitory concentration. Clinical data obtained from medical records and laboratory data were supplemented by data obtained via telephone interviews with the patients and treating physicians. PFGE analysis defined two major clusters; cluster A containing 16 bovine (80 %) isolates and cluster B containing 13 human (92 %) isolates, suggesting a host preference. Further genotypic analysis using MLST, SLST as well as sap and T4SS PCR showed the presence of genotypically identical isolates in cattle and humans. The low diversity observed within the cmp alleles of CFF corroborates the clonal nature of this pathogen. The genomic island containing the tetracycline and streptomycin resistance determinants was found in 55 % of the isolates in cluster A and correlated with phenotypic antibiotic resistance. Most human and bovine isolates were separated on two phylogenetic clusters. However, several human and bovine isolates were identical by diverse genotyping methods, indicating a possible link between strains from these two hosts.

First Insight into the Population Structure of Mycobacterium tuberculosis in Saudi Arabia▿ †

PubMed Central

Al-Hajoj, Sahal A. M.; Zozio, Thierry; Al-Rabiah, Fahad; Mohammad, Viquar; Al-Nasser, Maryam; Sola, Christophe; Rastogi, Nalin

2007-01-01

This study constitutes a first attempt to describe the genetic population structure and drug resistance of the tubercle bacilli circulating in Saudi Arabia. A total of 1,505 clinical isolates of M. tuberculosis, isolated between 2002 and 2005 from seven regions of Saudi Arabia, were studied. The sample studied showed a male-to-female sex ratio of 1.27, with half of the cases among foreign-born individuals and 47% within the 21- to 40-year-old age group; a total resistance rate of 19.7%; and multiple drug resistance of 4.5%. Upon spoligotyping, a total of 387 individual patterns were obtained (clustering rate, 86.4%; 182 clusters containing between 2 and 130 isolates per cluster). A total of 94% of the strains matched the spoligotype patterns in an international database. Nearly 81% of the isolates in this study belonged to established phylogeographic clades: Central Asian (CAS), 22.5%; ill-defined T clade, 19.5%; East African-Indian (EAI), 13.5%; Haarlem, 7.5%; Latin American-Mediterranean, 7.2%; Beijing, 4.4%; Manu, 2.7%; X, 0.9%; and Bovis, 0.9%. Two clonal complexes with unique spoligotyping signatures (octal codes 703777707770371 and 467777377413771) specific to Saudi Arabia were identified. These belonged to the CAS and EAI clades, respectively, as confirmed upon secondary typing using mycobacterial interspersed repetitive units (MIRUs). The results obtained underline the predominance of historic clones of principal genetic group 1, which are responsible for roughly 45% of all tuberculosis cases in Saudi Arabia. The high rate of clustering observed might be an indication of rapid ongoing transmission within certain communities and/or subpopulations in Saudi Arabia; nonetheless, spoligotyping is known to overestimate clustering, and only a systematic second-line typing, such as MIRUs, coupled with a better tuberculosis registry and epidemiological investigations would allow us to know the exact rate of ongoing transmission and associated risk factors in Saudi Arabia. PMID:17507515

Stenotrophomonas maltophilia in Mexico: antimicrobial resistance, biofilm formation and clonal diversity.

PubMed

Flores-Treviño, Samantha; Gutiérrez-Ferman, Jessica Lizzeth; Morfín-Otero, Rayo; Rodríguez-Noriega, Eduardo; Estrada-Rivadeneyra, Diego; Rivas-Morales, Catalina; Llaca-Díaz, Jorge M; Camacho-Ortíz, Adrián; Mendoza-Olazarán, Soraya; Garza-González, Elvira

2014-11-01

Stenotrophomonas maltophilia is an important multidrug-resistant nosocomial pathogen associated with high mortality. Our aim was to examine antimicrobial susceptibility, biofilm production and clonal relatedness of clinical isolates of S. maltophilia. S. maltophilia isolates were collected between 2006 and 2013 from two tertiary care hospitals in Mexico. Antimicrobial susceptibility was evaluated by the broth microdilution method. PCR was used to determine the presence of β-lactamase genes L1 and L2. Biofilm formation was assessed with crystal violet staining. Clonal relatedness was determined by PFGE. Among the 119 collected S. maltophilia isolates, 73 (61.3%) were from the respiratory tract. Resistance levels exceeded 75% for imipenem, meropenem, ampicillin, aztreonam, gentamicin and tobramycin. Resistance to trimethoprim-sulfamethoxazole was 32.8%. L1 and L2 genes were detected in 77.1% (91/118) and 66.9% (79/118) of isolates, respectively. All S. maltophilia strains were able to produce biofilms. Strains were classified as weak (47.9%, 57/119), moderate (38.7%, 46/119), or strong (13.4%, 16/119) biofilm producers. A total of 89 distinct PFGE types were identified and 21.6% (22/102) of the isolates were distributed in nine clusters. This is the first study in Mexico to reveal characteristics of clinical isolates of S. maltophilia. Clonal diversity data indicate low cross-transmission of S. maltophilia in a hospital setting. The high antibiotic resistance underscores the need for continuous surveillance of S. maltophilia in hospital settings in Mexico. © 2014 The Authors.

Population Structure of Listeria monocytogenes Serotype 4b Isolates from Sporadic Human Listeriosis Cases in the United States from 2003 to 2008

PubMed Central

Ward, Todd J.; Graves, Lewis M.; Tarr, Cheryl L.; Siletzky, Robin M.; Kathariou, Sophia

2014-01-01

Listeria monocytogenes can cause severe food-borne disease (listeriosis). Numerous outbreaks have involved three serotype 4b epidemic clones (ECs): ECI, ECII, and ECIa. However, little is known about the population structure of L. monocytogenes serotype 4b from sporadic listeriosis in the United States, even though most cases of human listeriosis are in fact sporadic. Here we analyzed 136 serotype 4b isolates from sporadic cases in the United States, 2003 to 2008, utilizing multiple tools including multilocus genotyping, pulsed-field gel electrophoresis, and sequence analysis of the inlAB locus. ECI, ECII, and ECIa were frequently encountered (32, 17, and 7%, respectively). However, annually 30 to 68% of isolates were outside these ECs, and several novel clonal groups were identified. An estimated 33 and 17% of the isolates, mostly among the ECs, were resistant to cadmium and arsenic, respectively, but resistance to benzalkonium chloride was uncommon (3%) among the sporadic isolates. The frequency of clonal groups fluctuated within the 6-year study period, without consistent trends. However, on several occasions, temporal clusters of isolates with indistinguishable genotypes were detected, suggesting the possibility of hidden multistate outbreaks. Our analysis suggests a complex population structure of serotype 4b L. monocytogenes from sporadic disease, with important contributions by ECs and several novel clonal groups. Continuous monitoring will be needed to assess long-term trends in clonality patterns and population structure of L. monocytogenes from sporadic listeriosis. PMID:24705322

Population structure of Listeria monocytogenes serotype 4b isolates from sporadic human listeriosis cases in the United States from 2003 to 2008.

PubMed

Lee, Sangmi; Ward, Todd J; Graves, Lewis M; Tarr, Cheryl L; Siletzky, Robin M; Kathariou, Sophia

2014-06-01

Listeria monocytogenes can cause severe food-borne disease (listeriosis). Numerous outbreaks have involved three serotype 4b epidemic clones (ECs): ECI, ECII, and ECIa. However, little is known about the population structure of L. monocytogenes serotype 4b from sporadic listeriosis in the United States, even though most cases of human listeriosis are in fact sporadic. Here we analyzed 136 serotype 4b isolates from sporadic cases in the United States, 2003 to 2008, utilizing multiple tools including multilocus genotyping, pulsed-field gel electrophoresis, and sequence analysis of the inlAB locus. ECI, ECII, and ECIa were frequently encountered (32, 17, and 7%, respectively). However, annually 30 to 68% of isolates were outside these ECs, and several novel clonal groups were identified. An estimated 33 and 17% of the isolates, mostly among the ECs, were resistant to cadmium and arsenic, respectively, but resistance to benzalkonium chloride was uncommon (3%) among the sporadic isolates. The frequency of clonal groups fluctuated within the 6-year study period, without consistent trends. However, on several occasions, temporal clusters of isolates with indistinguishable genotypes were detected, suggesting the possibility of hidden multistate outbreaks. Our analysis suggests a complex population structure of serotype 4b L. monocytogenes from sporadic disease, with important contributions by ECs and several novel clonal groups. Continuous monitoring will be needed to assess long-term trends in clonality patterns and population structure of L. monocytogenes from sporadic listeriosis.

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

PubMed

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.

Capturing neutral and adaptive genetic diversity for conservation in a highly structured tree species.

PubMed

Rodríguez-Quilón, Isabel; Santos-Del-Blanco, Luis; Serra-Varela, María Jesús; Koskela, Jarkko; González-Martínez, Santiago C; Alía, Ricardo

2016-10-01

Preserving intraspecific genetic diversity is essential for long-term forest sustainability in a climate change scenario. Despite that, genetic information is largely neglected in conservation planning, and how conservation units should be defined is still heatedly debated. Here, we use maritime pine (Pinus pinaster Ait.), an outcrossing long-lived tree with a highly fragmented distribution in the Mediterranean biodiversity hotspot, to prove the importance of accounting for genetic variation, of both neutral molecular markers and quantitative traits, to define useful conservation units. Six gene pools associated to distinct evolutionary histories were identified within the species using 12 microsatellites and 266 single nucleotide polymorphisms (SNPs). In addition, height and survival standing variation, their genetic control, and plasticity were assessed in a multisite clonal common garden experiment (16 544 trees). We found high levels of quantitative genetic differentiation within previously defined neutral gene pools. Subsequent cluster analysis and post hoc trait distribution comparisons allowed us to define 10 genetically homogeneous population groups with high evolutionary potential. They constitute the minimum number of units to be represented in a maritime pine dynamic conservation program. Our results uphold that the identification of conservation units below the species level should account for key neutral and adaptive components of genetic diversity, especially in species with strong population structure and complex evolutionary histories. The environmental zonation approach currently used by the pan-European genetic conservation strategy for forest trees would be largely improved by gradually integrating molecular and quantitative trait information, as data become available. © 2016 by the Ecological Society of America.

Characterization of Foodborne Outbreaks of Salmonella enterica Serovar Enteritidis with Whole-Genome Sequencing Single Nucleotide Polymorphism-Based Analysis for Surveillance and Outbreak Detection.

PubMed

Taylor, Angela J; Lappi, Victoria; Wolfgang, William J; Lapierre, Pascal; Palumbo, Michael J; Medus, Carlota; Boxrud, David

2015-10-01

Salmonella enterica serovar Enteritidis is a significant cause of gastrointestinal illness in the United States; however, current molecular subtyping methods lack resolution for this highly clonal serovar. Advances in next-generation sequencing technologies have made it possible to examine whole-genome sequencing (WGS) as a potential molecular subtyping tool for outbreak detection and source trace back. Here, we conducted a retrospective analysis of S. Enteritidis isolates from seven epidemiologically confirmed foodborne outbreaks and sporadic isolates (not epidemiologically linked) to determine the utility of WGS to identify outbreaks. A collection of 55 epidemiologically characterized clinical and environmental S. Enteritidis isolates were sequenced. Single nucleotide polymorphism (SNP)-based cluster analysis of the S. Enteritidis genomes revealed well supported clades, with less than four-SNP pairwise diversity, that were concordant with epidemiologically defined outbreaks. Sporadic isolates were an average of 42.5 SNPs distant from the outbreak clusters. Isolates collected from the same patient over several weeks differed by only two SNPs. Our findings show that WGS provided greater resolution between outbreak, sporadic, and suspect isolates than the current gold standard subtyping method, pulsed-field gel electrophoresis (PFGE). Furthermore, results could be obtained in a time frame suitable for surveillance activities, supporting the use of WGS as an outbreak detection and characterization method for S. Enteritidis. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

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

PubMed Central

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

2017-01-01

Purpose 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. Methodology 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. Results 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. Conclusion 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. PMID:28945190

Multi-Virulence-Locus Sequence Typing of Staphylococcus lugdunensis Generates Results Consistent with a Clonal Population Structure and Is Reliable for Epidemiological Typing

PubMed Central

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

2014-01-01

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. PMID:25078912

Single-cell analysis of the fate of c-kit-positive bone marrow cells

NASA Astrophysics Data System (ADS)

Czarna, Anna; Sanada, Fumihiro; Matsuda, Alex; Kim, Junghyun; Signore, Sergio; Pereira, João D.; Sorrentino, Andrea; Kannappan, Ramaswamy; Cannatà, Antonio; Hosoda, Toru; Rota, Marcello; Crea, Filippo; Anversa, Piero; Leri, Annarosa

2017-10-01

The plasticity of c-kit-positive bone marrow cells (c-kit-BMCs) in tissues different from their organ of origin remains unclear. We tested the hypothesis that c-kit-BMCs are functionally heterogeneous and only a subgroup of these cells possesses cardiomyogenic potential. Population-based assays fall short of identifying the properties of individual stem cells, imposing on us the introduction of single cell-based approaches to track the fate of c-kit-BMCs in the injured heart; they included viral gene-tagging, multicolor clonal-marking and transcriptional profiling. Based on these strategies, we report that single mouse c-kit-BMCs expand clonally within the infarcted myocardium and differentiate into specialized cardiac cells. Newly-formed cardiomyocytes, endothelial cells, fibroblasts and c-kit-BMCs showed in their genome common sites of viral integration, providing strong evidence in favor of the plasticity of a subset of BMCs expressing the c-kit receptor. Similarly, individual c-kit-BMCs, which were infected with multicolor reporters and injected in infarcted hearts, formed cardiomyocytes and vascular cells organized in clusters of similarly colored cells. The uniform distribution of fluorescent proteins in groups of specialized cells documented the polyclonal nature of myocardial regeneration. The transcriptional profile of myogenic c-kit-BMCs and whole c-kit-BMCs was defined by RNA sequencing. Genes relevant for engraftment, survival, migration, and differentiation were enriched in myogenic c-kit-BMCs, a cell subtype which could not be assigned to a specific hematopoietic lineage. Collectively, our findings demonstrate that the bone marrow comprises a category of cardiomyogenic, vasculogenic and/or fibrogenic c-kit-positive cells and a category of c-kit-positive cells that retains an undifferentiated state within the damaged heart.

Genotyping and Source Tracking of Cronobacter sakazakii and C. malonaticus Isolates from Powdered Infant Formula and an Infant Formula Production Factory in China

PubMed Central

Fei, Peng; Man, Chaoxin; Lou, Binbin; Forsythe, Stephen J.; Chai, Yunlei; Li, Ran; Niu, Jieting

2015-01-01

Cronobacter spp. (formerly defined as Enterobacter sakazakii) are opportunistic bacterial pathogens of both infants and adults. In this study, we analyzed 70 Cronobacter isolates from powdered infant formula (PIF) and an infant formula production facility in China to determine possible contamination routes. The strains were profiled by multilocus sequence typing (MLST), pulsed-field gel electrophoresis (PFGE), PCR-based O-antigen serotyping, and ompA and rpoB sequence analyses. The isolates were primarily Cronobacter sakazakii (66/70) or Cronobacter malonaticus (4/70). The strains were divided into 38 pulsotypes (PTs) using PFGE and 19 sequence types (STs) by MLST. In contrast, rpoB and ompA sequence analyses divided the strains into 10 overlapping clusters each. PCR serotyping of the 66 C. sakazakii and 4 C. malonaticus strains resulted in the identification of four C. sakazakii serotypes (O1, O2, O4, and O7) and a single C. malonaticus serotype, O2. The dominant C. sakazakii sequence types from PIF and an infant formula production factory in China were C. sakazakii clonal complex 4 (CC4) (n = 19), ST1 (n = 14), and ST64 (n = 11). C. sakazakii CC4 is a clonal lineage strongly associated with neonatal meningitis. In the process of manufacturing PIF, the spray-drying, fluidized-bed-drying, and packing areas were the main areas with Cronobacter contamination. C. sakazakii strains with the same pulsotypes (PT3 and PT2) and sequence types (ST1 and ST64) were isolated both from processing equipment and from the PIF finished product. PMID:26048942

Genotyping and Source Tracking of Cronobacter sakazakii and C. malonaticus Isolates from Powdered Infant Formula and an Infant Formula Production Factory in China.

PubMed

Fei, Peng; Man, Chaoxin; Lou, Binbin; Forsythe, Stephen J; Chai, Yunlei; Li, Ran; Niu, Jieting; Jiang, Yujun

2015-08-15

Cronobacter spp. (formerly defined as Enterobacter sakazakii) are opportunistic bacterial pathogens of both infants and adults. In this study, we analyzed 70 Cronobacter isolates from powdered infant formula (PIF) and an infant formula production facility in China to determine possible contamination routes. The strains were profiled by multilocus sequence typing (MLST), pulsed-field gel electrophoresis (PFGE), PCR-based O-antigen serotyping, and ompA and rpoB sequence analyses. The isolates were primarily Cronobacter sakazakii (66/70) or Cronobacter malonaticus (4/70). The strains were divided into 38 pulsotypes (PTs) using PFGE and 19 sequence types (STs) by MLST. In contrast, rpoB and ompA sequence analyses divided the strains into 10 overlapping clusters each. PCR serotyping of the 66 C. sakazakii and 4 C. malonaticus strains resulted in the identification of four C. sakazakii serotypes (O1, O2, O4, and O7) and a single C. malonaticus serotype, O2. The dominant C. sakazakii sequence types from PIF and an infant formula production factory in China were C. sakazakii clonal complex 4 (CC4) (n = 19), ST1 (n = 14), and ST64 (n = 11). C. sakazakii CC4 is a clonal lineage strongly associated with neonatal meningitis. In the process of manufacturing PIF, the spray-drying, fluidized-bed-drying, and packing areas were the main areas with Cronobacter contamination. C. sakazakii strains with the same pulsotypes (PT3 and PT2) and sequence types (ST1 and ST64) were isolated both from processing equipment and from the PIF finished product. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Single-cell analysis of the fate of c-kit-positive bone marrow cells.

PubMed

Czarna, Anna; Sanada, Fumihiro; Matsuda, Alex; Kim, Junghyun; Signore, Sergio; Pereira, João D; Sorrentino, Andrea; Kannappan, Ramaswamy; Cannatà, Antonio; Hosoda, Toru; Rota, Marcello; Crea, Filippo; Anversa, Piero; Leri, Annarosa

2017-01-01

The plasticity of c-kit-positive bone marrow cells (c-kit-BMCs) in tissues different from their organ of origin remains unclear. We tested the hypothesis that c-kit-BMCs are functionally heterogeneous and only a subgroup of these cells possesses cardiomyogenic potential. Population-based assays fall short of identifying the properties of individual stem cells, imposing on us the introduction of single cell-based approaches to track the fate of c-kit-BMCs in the injured heart; they included viral gene-tagging, multicolor clonal-marking and transcriptional profiling. Based on these strategies, we report that single mouse c-kit-BMCs expand clonally within the infarcted myocardium and differentiate into specialized cardiac cells. Newly-formed cardiomyocytes, endothelial cells, fibroblasts and c-kit-BMCs showed in their genome common sites of viral integration, providing strong evidence in favor of the plasticity of a subset of BMCs expressing the c-kit receptor. Similarly, individual c-kit-BMCs, which were infected with multicolor reporters and injected in infarcted hearts, formed cardiomyocytes and vascular cells organized in clusters of similarly colored cells. The uniform distribution of fluorescent proteins in groups of specialized cells documented the polyclonal nature of myocardial regeneration. The transcriptional profile of myogenic c-kit-BMCs and whole c-kit-BMCs was defined by RNA sequencing. Genes relevant for engraftment, survival, migration, and differentiation were enriched in myogenic c-kit-BMCs, a cell subtype which could not be assigned to a specific hematopoietic lineage. Collectively, our findings demonstrate that the bone marrow comprises a category of cardiomyogenic, vasculogenic and/or fibrogenic c-kit-positive cells and a category of c-kit-positive cells that retains an undifferentiated state within the damaged heart.

SinCHet: a MATLAB toolbox for single cell heterogeneity analysis in cancer.

PubMed

Li, Jiannong; Smalley, Inna; Schell, Michael J; Smalley, Keiran S M; Chen, Y Ann

2017-09-15

Single-cell technologies allow characterization of transcriptomes and epigenomes for individual cells under different conditions and provide unprecedented resolution for researchers to investigate cellular heterogeneity in cancer. The SinCHet ( gle ell erogeneity) toolbox is developed in MATLAB and has a graphical user interface (GUI) for visualization and user interaction. It analyzes both continuous (e.g. mRNA expression) and binary omics data (e.g. discretized methylation data). The toolbox does not only quantify cellular heterogeneity using S hannon P rofile (SP) at different clonal resolutions but also detects heterogeneity differences using a D statistic between two populations. It is defined as the area under the P rofile of S hannon D ifference (PSD). This flexible tool provides a default clonal resolution using the change point of PSD detected by multivariate adaptive regression splines model; it also allows user-defined clonal resolutions for further investigation. This tool provides insights into emerging or disappearing clones between conditions, and enables the prioritization of biomarkers for follow-up experiments based on heterogeneity or marker differences between and/or within cell populations. The SinCHet software is freely available for non-profit academic use. The source code, example datasets, and the compiled package are available at http://labpages2.moffitt.org/chen/software/ . ann.chen@moffitt.org. Supplementary data are available at Bioinformatics online. © The Author(s) 2017. Published by Oxford University Press.

Evaluating the utility of syndromic surveillance algorithms for screening to detect potentially clonal hospital infection outbreaks

PubMed Central

Talbot, Thomas R; Schaffner, William; Bloch, Karen C; Daniels, Titus L; Miller, Randolph A

2011-01-01

Objective The authors evaluated algorithms commonly used in syndromic surveillance for use as screening tools to detect potentially clonal outbreaks for review by infection control practitioners. Design Study phase 1 applied four aberrancy detection algorithms (CUSUM, EWMA, space-time scan statistic, and WSARE) to retrospective microbiologic culture data, producing a list of past candidate outbreak clusters. In phase 2, four infectious disease physicians categorized the phase 1 algorithm-identified clusters to ascertain algorithm performance. In phase 3, project members combined the algorithms to create a unified screening system and conducted a retrospective pilot evaluation. Measurements The study calculated recall and precision for each algorithm, and created precision-recall curves for various methods of combining the algorithms into a unified screening tool. Results Individual algorithm recall and precision ranged from 0.21 to 0.31 and from 0.053 to 0.29, respectively. Few candidate outbreak clusters were identified by more than one algorithm. The best method of combining the algorithms yielded an area under the precision-recall curve of 0.553. The phase 3 combined system detected all infection control-confirmed outbreaks during the retrospective evaluation period. Limitations Lack of phase 2 reviewers' agreement indicates that subjective expert review was an imperfect gold standard. Less conservative filtering of culture results and alternate parameter selection for each algorithm might have improved algorithm performance. Conclusion Hospital outbreak detection presents different challenges than traditional syndromic surveillance. Nevertheless, algorithms developed for syndromic surveillance have potential to form the basis of a combined system that might perform clinically useful hospital outbreak screening. PMID:21606134

Cloning: plants – micropropagation/tissue culture

USDA-ARS?s Scientific Manuscript database

Clonal micropropagation is the multiplication of the buds and shoots that occur in leaf axils on a defined nutrient medium in an aseptic in vitro environment. The resulting shoots are either subdivided for continued multiplication or rooted and acclimatized to the greenhouse or field. Micropropagati...

Multiplexing clonality: combining RGB marking and genetic barcoding

PubMed Central

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

Processing-Dependent and Clonal Contamination Patterns of Listeria monocytogenes in the Cured Ham Food Chain Revealed by Genetic Analysis.

PubMed

Morganti, Marina; Scaltriti, Erika; Cozzolino, Paolo; Bolzoni, Luca; Casadei, Gabriele; Pierantoni, Marco; Foni, Emanuela; Pongolini, Stefano

2016-02-01

The quantitative and qualitative patterns of environmental contamination by Listeria monocytogenes were investigated in the production chain of dry-cured Parma ham. Standard arrays of surfaces were sampled in processing facilities during a single visit per plant in the three compartments of the food chain, i.e., ham production (19 plants) and postproduction, which was divided into deboning (43 plants) and slicing (25 plants) steps. The numbers of sampled surfaces were 384 in ham production, with 25 positive for L. monocytogenes, and 1,084 in postproduction, with 83 positives. Statistical analysis of the prevalence of contaminated surfaces showed that in ham production, contamination was higher at the beginning of processing and declined significantly toward the end, while in postproduction, prevalence rose toward the end of processing. Prevalence was higher in the deboning facilities than in slicing facilities and was dependent on the type of surface (floor/drainage > clothing > equipment). The qualitative pattern of contamination was investigated through an analysis of the survey isolates and a set of isolates derived from routine monitoring, including longitudinal isolations. Pulsed-field gel electrophoresis (PFGE) and whole-genome single-nucleotide polymorphism (SNP) analysis revealed a remarkable clonality of L. monocytogenes within plants, with the detection of 16 plant-specific clones out of 17 establishments with multiple isolates. Repeated detections of clonal isolates >6 months apart were also observed. Six was the maximum number of between-isolate differences in core SNPs observed within these clones. Based on the same six-SNP threshold, three clusters of clonal isolates, shared by six establishments, were also identified. The spread of L. monocytogenes within and between plants, as indicated by its clonal behavior, is a matter of concern for the hygienic management of establishments. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Cell signaling directing the formation and function of hemogenic endothelium during murine embryogenesis

USDA-ARS?s Scientific Manuscript database

During developmental hematopoiesis, multilineage hematopoietic progenitors are thought to derive from a subset of vascular endothelium. Herein, we define the phenotype of such hemogenic endothelial cells and demonstrate, on a clonal level, that they exhibit multilineage hematopoietic potential. Furt...

Multiplex CRISPR/Cas9-Based Genome Editing in Human Hematopoietic Stem Cells Models Clonal Hematopoiesis and Myeloid Neoplasia.

PubMed

Tothova, Zuzana; Krill-Burger, John M; Popova, Katerina D; Landers, Catherine C; Sievers, Quinlan L; Yudovich, David; Belizaire, Roger; Aster, Jon C; Morgan, Elizabeth A; Tsherniak, Aviad; Ebert, Benjamin L

2017-10-05

Hematologic malignancies are driven by combinations of genetic lesions that have been difficult to model in human cells. We used CRISPR/Cas9 genome engineering of primary adult and umbilical cord blood CD34 + human hematopoietic stem and progenitor cells (HSPCs), the cells of origin for myeloid pre-malignant and malignant diseases, followed by transplantation into immunodeficient mice to generate genetic models of clonal hematopoiesis and neoplasia. Human hematopoietic cells bearing mutations in combinations of genes, including cohesin complex genes, observed in myeloid malignancies generated immunophenotypically defined neoplastic clones capable of long-term, multi-lineage reconstitution and serial transplantation. Employing these models to investigate therapeutic efficacy, we found that TET2 and cohesin-mutated hematopoietic cells were sensitive to azacitidine treatment. These findings demonstrate the potential for generating genetically defined models of human myeloid diseases, and they are suitable for examining the biological consequences of somatic mutations and the testing of therapeutic agents. Copyright © 2017 Elsevier Inc. All rights reserved.

Clonal variation in proliferation rate of cultures of GPK cells.

PubMed

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.

Spatial Vulnerability: Bacterial Arrangements, Microcolonies, and Biofilms as Responses to Low Rather than High Phage Densities

PubMed Central

Abedon, Stephen T.

2012-01-01

The ability of bacteria to survive and propagate can be dramatically reduced upon exposure to lytic bacteriophages. Study of this impact, from a bacterium’s perspective, tends to focus on phage-bacterial interactions that are governed by mass action, such as can be observed within continuous flow or similarly planktonic ecosystems. Alternatively, bacterial molecular properties can be examined, such as specific phage‑resistance adaptations. In this study I address instead how limitations on bacterial movement, resulting in the formation of cellular arrangements, microcolonies, or biofilms, could increase the vulnerability of bacteria to phages. Principally: (1) Physically associated clonal groupings of bacteria can represent larger targets for phage adsorption than individual bacteria; and (2), due to a combination of proximity and similar phage susceptibility, individual bacteria should be especially vulnerable to phages infecting within the same clonal, bacterial grouping. Consistent with particle transport theory—the physics of movement within fluids—these considerations are suggestive that formation into arrangements, microcolonies, or biofilms could be either less profitable to bacteria when phage predation pressure is high or require more effective phage-resistance mechanisms than seen among bacteria not living within clonal clusters. I consider these ideas of bacterial ‘spatial vulnerability’ in part within a phage therapy context. PMID:22754643

Clostridium difficile PCR ribotypes 001 and 176 - the common denominator of C. difficile infection epidemiology in the Czech Republic, 2014.

PubMed

Krutova, Marcela; Matejkova, Jana; Kuijper, Ed J; Drevinek, Pavel; Nyc, Otakar

2016-07-21

In 2014, 18 hospitals in the Czech Republic participated in a survey of the incidence of Clostridium difficile infections (CDI) in the country. The mean CDI incidence was 6.1 (standard deviation (SD):7.2) cases per 10,000 patient bed-days and 37.8 cases (SD: 41.4) per 10,000 admissions. The mean CDI testing frequency was 39.5 tests (SD: 25.4) per 10,000 patient bed-days and 255.8 tests (SD: 164.0) per 10,000 admissions. A total of 774 C. difficile isolates were investigated, of which 225 (29%) belonged to PCR ribotype 176, and 184 isolates (24%) belonged to PCR ribotype 001. Multilocus variable-number tandem repeat analysis (MLVA) revealed 27 clonal complexes formed by 84% (190/225) of PCR ribotype 176 isolates, and 14 clonal complexes formed by 77% (141/184) of PCR ribotype 001 isolates. Clonal clusters of PCR ribotypes 176 and 001 were observed in 11 and 7 hospitals, respectively. Our data demonstrate the spread of two C. difficile PCR ribotypes within 18 hospitals in the Czech Republic, stressing the importance of standardising CDI testing protocols and implementing mandatory CDI surveillance in the country. This article is copyright of The Authors, 2016.

Clustering of Staphylococcus aureus bovine mastitis strains from regions of Central-Eastern Poland based on their biochemical and genetic characteristics.

PubMed

Puacz, E; Ilczyszyn, W M; Kosecka, M; Buda, A; Dudziak, W; Polakowska, K; Panz, T; Białecka, A; Kasprowicz, A; Lisowski, A; Krukowski, H; Cuteri, V; Międzobrodzki, J

2015-01-01

Staphylococcus aureus strains were isolated from mastitic milk of cows with infected mammary glands. The animals were living in 12 different farms near Lublin, in Central-Eastern Poland. A biochemical identification method based on enzymatic assay was performed, followed by haemolytic and proteolytic tests. PCR-RFLP targeted on the gap gene allowed the genetic identification of strains at the species level and verified phenotypic identification results. A molecular typing method using triplex PCR was performed to recognize the genetic similarity of the analyzed strains. DNA microarray hybridization (StaphyType, Alere Technologies) was used for detection of antibiotic resistance and virulence associated markers. The results obtained indicate high genetic similarity in strains isolated from the same sites. High genetic similarities were also detected between strains isolated from cows from different farms of the same region. A slightly lower similarity was noted however, in strains from various regions indicating that the strains are herd specific and that the cow's infections caused by S. aureus were of a clonal character. In 21 representative isolates selected for DNA-microarray testing, only fosfomycin (fosB) and penicillin resistance markers (blaZ, blaI, blaR) were detected. The presence of genes coding for haemolysins (lukF, lukS, hlgA, hla, hld, hlb), proteases (aur, sspA, sspB, sspP), enterotoxins (entA, entD, entG, entI, entJ, entM, entN, entO, entR, entU, egc-cluster), adhesins (icaA, icaC, icaD, bbp, clfA, clfB, fib, fnbA, map, vwb) or immune evasion proteins (scn, chp, sak) was common and, with exceptions, matched triplex PCR-defined clusters.

Genetic diversity of drug and multidrug-resistant Mycobacterium tuberculosis circulating in Veracruz, Mexico

PubMed Central

Munro-Rojas, Daniela; Fernandez-Morales, Esdras; Zarrabal-Meza, José; Martínez-Cazares, Ma. Teresa; Parissi-Crivelli, Aurora; Fuentes-Domínguez, Javier; Séraphin, Marie Nancy; Lauzardo, Michael; González-y-Merchand, Jorge Alberto; Rivera-Gutierrez, Sandra

2018-01-01

Background Mexico is one of the most important contributors of drug and multidrug-resistant tuberculosis in Latin America; however, knowledge of the genetic diversity of drug-resistant tuberculosis isolates is limited. Methods In this study, the genetic structure of 112 Mycobacterium tuberculosis strains from the southeastern Mexico was determined by spoligotyping and 24-loci MIRU-VNTRs. Findings The results show eight major lineages, the most of which was T1 (24%), followed by LAM (16%) and H (15%). A total of 29 (25%) isolates were identified as orphan. The most abundant SITs were SIT53/T1 and SIT42/LAM9 with 10 isolates each and SIT50/H3 with eight isolates. Fifty-two spoligotype patterns, twenty-seven clusters and ten clonal complexes were observed, demonstrating an important genetic diversity of drug and multidrug-resistant tuberculosis isolates in circulation and transmission level of these aggravated forms of tuberculosis. Being defined as orphan or as part of an orphan cluster, was a risk factor for multidrug resistant-tuberculosis (OR 2.5, IC 1.05–5.86 and OR 3.3, IC 1–11.03, respectively). Multiple correspondence analyses showed association of some clusters and SITs with specific geographical locations. Conclusions Our study provides one of the most detailed description of the genetic structure of drug and multidrug-resistant tuberculosis strains in southeast Mexico, establishing for the first time a baseline of the genotypes observed in resistant isolates circulating, however further studies are required to better elucidate the genetic structure of tuberculosis in region and the factors that could be participating in their dispersion. PMID:29543819

South American Plasmodium falciparum after the Malaria Eradication Era: Clonal Population Expansion and Survival of the Fittest Hybrids

PubMed Central

Griffing, Sean M.; Mixson-Hayden, Tonya; Sridaran, Sankar; Alam, Md Tauqeer; McCollum, Andrea M.; Cabezas, César; Marquiño Quezada, Wilmer; Barnwell, John W.; Macedo De Oliveira, Alexandre; Lucas, Carmen; Arrospide, Nancy; Escalante, Ananias A.; Bacon, David J.; Udhayakumar, Venkatachalam

2011-01-01

Malaria has reemerged in many regions where once it was nearly eliminated. Yet the source of these parasites, the process of repopulation, their population structure, and dynamics are ill defined. Peru was one of malaria eradication's successes, where Plasmodium falciparum was nearly eliminated for two decades. It reemerged in the 1990s. In the new era of malaria elimination, Peruvian P. falciparum is a model of malaria reinvasion. We investigated its population structure and drug resistance profiles. We hypothesized that only populations adapted to local ecological niches could expand and repopulate and originated as vestigial populations or recent introductions. We investigated the genetic structure (using microsatellites) and drug resistant genotypes of 220 parasites collected from patients immediately after peak epidemic expansion (1999–2000) from seven sites across the country. The majority of parasites could be grouped into five clonal lineages by networks and AMOVA. The distribution of clonal lineages and their drug sensitivity profiles suggested geographic structure. In 2001, artesunate combination therapy was introduced in Peru. We tested 62 parasites collected in 2006–2007 for changes in genetic structure. Clonal lineages had recombined under selection for the fittest parasites. Our findings illustrate that local adaptations in the post-eradication era have contributed to clonal lineage expansion. Within the shifting confluence of drug policy and malaria incidence, populations continue to evolve through genetic outcrossing influenced by antimalarial selection pressure. Understanding the population substructure of P. falciparum has implications for vaccine, drug, and epidemiologic studies, including monitoring malaria during and after the elimination phase. PMID:21949680

Tracking global changes induced in the CD4 T-cell receptor repertoire by immunization with a complex antigen using short stretches of CDR3 protein sequence.

PubMed

Thomas, Niclas; Best, Katharine; Cinelli, Mattia; Reich-Zeliger, Shlomit; Gal, Hilah; Shifrut, Eric; Madi, Asaf; Friedman, Nir; Shawe-Taylor, John; Chain, Benny

2014-11-15

The clonal theory of adaptive immunity proposes that immunological responses are encoded by increases in the frequency of lymphocytes carrying antigen-specific receptors. In this study, we measure the frequency of different T-cell receptors (TcR) in CD4 + T cell populations of mice immunized with a complex antigen, killed Mycobacterium tuberculosis, using high throughput parallel sequencing of the TcRβ chain. Our initial hypothesis that immunization would induce repertoire convergence proved to be incorrect, and therefore an alternative approach was developed that allows accurate stratification of TcR repertoires and provides novel insights into the nature of CD4 + T-cell receptor recognition. To track the changes induced by immunization within this heterogeneous repertoire, the sequence data were classified by counting the frequency of different clusters of short (3 or 4) continuous stretches of amino acids within the antigen binding complementarity determining region 3 (CDR3) repertoire of different mice. Both unsupervised (hierarchical clustering) and supervised (support vector machine) analyses of these different distributions of sequence clusters differentiated between immunized and unimmunized mice with 100% efficiency. The CD4 + TcR repertoires of mice 5 and 14 days postimmunization were clearly different from that of unimmunized mice but were not distinguishable from each other. However, the repertoires of mice 60 days postimmunization were distinct both from naive mice and the day 5/14 animals. Our results reinforce the remarkable diversity of the TcR repertoire, resulting in many diverse private TcRs contributing to the T-cell response even in genetically identical mice responding to the same antigen. However, specific motifs defined by short stretches of amino acids within the CDR3 region may determine TcR specificity and define a new approach to TcR sequence classification. The analysis was implemented in R and Python, and source code can be found in Supplementary Data. b.chain@ucl.ac.uk Supplementary data are available at Bioinformatics online. © The Author 2014. Published by Oxford University Press.

Synergistic cooperation promotes multicellular performance and unicellular free-rider persistence

PubMed Central

Driscoll, William W; Travisano, Michael

2017-01-01

The evolution of multicellular life requires cooperation among cells, which can be undermined by intra-group selection for selfishness. Theory predicts that selection to avoid non-cooperators limits social interactions among non-relatives, yet previous evolution experiments suggest that intra-group conflict is an outcome, rather than a driver, of incipient multicellular life cycles. Here we report the evolution of multicellularity via two distinct mechanisms of group formation in the unicellular budding yeast Kluyveromyces lactis. Cells remain permanently attached following mitosis, giving rise to clonal clusters (staying together); clusters then reversibly assemble into social groups (coming together). Coming together amplifies the benefits of multicellularity and allows social clusters to collectively outperform solitary clusters. However, cooperation among non-relatives also permits fast-growing unicellular lineages to ‘free-ride' during selection for increased size. Cooperation and competition for the benefits of multicellularity promote the stable coexistence of unicellular and multicellular genotypes, underscoring the importance of social and ecological context during the transition to multicellularity. PMID:28580966

Genotypic Diversity of Phytophthora cinnamomi and P. plurivora in Maryland's Nurseries and Mid-Atlantic Forests.

PubMed

Beaulieu, Justine; Ford, Blaine; Balci, Yilmaz

2017-06-01

Genetic diversity of two Phytophthora spp.-P. cinnamomi (102 isolates), commonly encountered in Maryland nurseries and forests in the Mid-Atlantic United States, and P. plurivora (186 isolates), a species common in nurseries-was characterized using amplified fragment length polymorphism. Expected heterozygosity and other indices suggested a lower level of diversity among P. cinnamomi than P. plurivora isolates. Hierarchical clustering showed P. cinnamomi isolates separated into four clusters, and two of the largest clusters were closely related, containing 80% of the isolates. In contrast, P. plurivora isolates separated into six clusters, one of which included approximately 40% of the isolates. P. plurivora isolates recovered from the environment (e.g., soil and water) were genotypically more diverse than those found causing lesions. For both species, isolate origin (forest versus nursery or among nurseries) was a significant factor of heterozygosity. Clonal groups existed within P. cinnamomi and P. plurivora and included isolates from both forest and nurseries, suggesting that a pathway from nurseries to forests or vice versa exists.

Plasmodium falciparum sulfadoxine resistance is geographically and genetically clustered within the DR Congo

PubMed Central

Taylor, Steve M.; Antonia, Alejandro L.; Parobek, Christian M.; Juliano, Jonathan J.; Janko, Mark; Emch, Michael; Alam, Md Tauqeer; Udhayakumar, Venkatachalam; Tshefu, Antoinette K.; Meshnick, Steven R.

2013-01-01

Understanding the spatial clustering of Plasmodium falciparum populations can assist efforts to contain drug-resistant parasites and maintain the efficacy of future drugs. We sequenced single nucleotide polymorphisms (SNPs) in the dihydropteroate synthase gene (dhps) associated with sulfadoxine resistance and 5 microsatellite loci flanking dhps in order to investigate the genetic backgrounds, genetic relatedness, and geographic clustering of falciparum parasites in the Democratic Republic of the Congo (DRC). Resistant haplotypes were clustered into subpopulations: one in the northeast DRC, and the other in the balance of the DRC. Network and clonal lineage analyses of the flanking microsatellites indicate that geographically-distinct mutant dhps haplotypes derive from separate lineages. The DRC is therefore a watershed for haplotypes associated with sulfadoxine resistance. Given the importance of central Africa as a corridor for the spread of antimalarial resistance, the identification of the mechanisms of this transit can inform future policies to contain drug-resistant parasite strains. PMID:23372922

Spanish multicenter study of the epidemiology and mechanisms of amoxicillin-clavulanate resistance in Escherichia coli.

PubMed

Ortega, Adriana; Oteo, Jesús; Aranzamendi-Zaldumbide, Maitane; Bartolomé, Rosa M; Bou, Germán; Cercenado, Emilia; Conejo, M Carmen; González-López, Juan José; Marín, Mercedes; Martínez-Martínez, Luis; Merino, María; Navarro, Ferran; Oliver, Antonio; Pascual, Alvaro; Rivera, Alba; Rodríguez-Baño, Jesús; Weber, Irene; Aracil, Belén; Campos, José

2012-07-01

We conducted a prospective multicenter study in Spain to characterize the mechanisms of resistance to amoxicillin-clavulanate (AMC) in Escherichia coli. Up to 44 AMC-resistant E. coli isolates (MIC ≥ 32/16 μg/ml) were collected at each of the seven participant hospitals. Resistance mechanisms were characterized by PCR and sequencing. Molecular epidemiology was studied by pulsed-field gel electrophoresis (PFGE) and by multilocus sequence typing. Overall AMC resistance was 9.3%. The resistance mechanisms detected in the 257 AMC-resistant isolates were OXA-1 production (26.1%), hyperproduction of penicillinase (22.6%), production of plasmidic AmpC (19.5%), hyperproduction of chromosomic AmpC (18.3%), and production of inhibitor-resistant TEM (IRT) (17.5%). The IRTs identified were TEM-40 (33.3%), TEM-30 (28.9%), TEM-33 (11.1%), TEM-32 (4.4%), TEM-34 (4.4%), TEM-35 (2.2%), TEM-54 (2.2%), TEM-76 (2.2%), TEM-79 (2.2%), and the new TEM-185 (8.8%). By PFGE, a high degree of genetic diversity was observed although two well-defined clusters were detected in the OXA-1-producing isolates: the C1 cluster consisting of 19 phylogroup A/sequence type 88 [ST88] isolates and the C2 cluster consisting of 19 phylogroup B2/ST131 isolates (16 of them producing CTX-M-15). Each of the clusters was detected in six different hospitals. In total, 21.8% of the isolates were serotype O25b/phylogroup B2 (O25b/B2). AMC resistance in E. coli is widespread in Spain at the hospital and community levels. A high prevalence of OXA-1 was found. Although resistant isolates were genetically diverse, clonality was linked to OXA-1-producing isolates of the STs 88 and 131. Dissemination of IRTs was frequent, and the epidemic O25b/B2/ST131 clone carried many different mechanisms of AMC resistance.

Evidence of substantial recombination among Trypanosoma cruzi II strains from Minas Gerais.

PubMed

Baptista, Rodrigo de Paula; D'Ávila, Daniella Alchaar; Segatto, Marcela; do Valle, Ítalo Faria; Franco, Glória Regina; Valadares, Helder Magno Silva; Gontijo, Eliane Dias; Galvão, Lúcia Maria da Cunha; Pena, Sérgio Danilo Junho; Chiari, Egler; Machado, Carlos Renato; Macedo, Andréa Mara

2014-03-01

Due to the scarcity of evidence of sexuality in Trypanosoma cruzi, the causative agent of Chagas disease, it has been general accepted that the parasite reproduction is essentially clonal with infrequent genetic recombination. This assumption is mainly supported by indirect evidence, such as Hardy-Weinberg imbalances, linkage disequilibrium and a strong correlation between independent sets of genetic markers of T. cruzi populations. However, because the analyzed populations are usually isolated from different geographic regions, the possibility of population substructuring as generating these genetic marker imbalances cannot be eliminated. To investigate this possibility, we firstly compared the allele frequencies and haplotype networks using seven different polymorphic loci (two from mitochondrial and five from different nuclear chromosomes) in two groups of TcII strains: one including isolates obtained from different regions in Latin America and the other including isolates obtained only from patients of the Minas Gerais State in Brazil. Our hypothesis was that if the population structure is essentially clonal, Hardy-Weinberg disequilibrium and a sharp association between the clusters generated by analyzing independent markers should be observed in both strain groups, independent of the geographic origin of the samples. The results demonstrated that the number of microsatellite loci in linkage disequilibrium decreased from 4 to 1 when only strains from Minas Gerais were analyzed. Moreover, we did not observed any correlation between the clusters when analyzing the nuclear and mitochondrial loci, suggesting independent inheritance of these markers among the Minas Gerais strains. Besides, using a second subset of five physically linked microsatellite loci and the Minas Gerais strains, we could also demonstrate evidence of homologous recombination roughly proportional to the relative distance among them. Taken together, our results do not support a clonal population structure for T. cruzi, particularly in TcII, which coexists in the same geographical area, suggesting that genetic exchanges among these strains may occur more frequently than initially expected. Copyright © 2013 Elsevier B.V. All rights reserved.

Prevalence, genetic relatedness and antibiotic resistance of hospital-acquired clostridium difficile PCR ribotype 018 strains.

PubMed

Seo, Mi-Ran; Kim, Jieun; Lee, Yangsoon; Lim, Dong-Gyun; Pai, Hyunjoo

2018-05-01

Clostridium difficile infection (CDI) is a major healthcare-associated infection. The aim of this study was to investigate the genetic relatedness of the endemic C. difficile PCR ribotype 018 strains in an institution and changes to their characteristics during a five-year period. A total of 207 isolates from inpatients at Hanyang University Hospital from 2009 to 2013 were analysed using multilocus variable-number tandem-repeat analysis (MLVA). Minimum inhibitory concentrations (MICs) of several antibiotics were determined. In total, 204 (98.6%) were genetically related, with a summed tandem-repeat distance (STRD) ≤ 10. Minimum-spanning-tree analysis identified 78 MLVA types, categorized into six clonal complexes (CCs). The largest cluster, CC-I, included 51 MLVA types from 148 isolates (71.5%) and the second largest cluster, CC-II, included 10 MLVA types from 36 isolates (17.4%). Resistance rates for antibiotics were: clindamycin (CLI), 97.6%; moxifloxacin (MXF), 98.6%; vancomycin (VAN), 1.4%; and rifaximin (RFX), 8.2%. All isolates were susceptible to piperacillin/tazobactam (TZP) and metronidazole (MTZ). Comparing the MICs of antibiotics for the isolates each year from 2009 to 2013, MICs of antibiotics that promote CDI, such as CLI, MXF, TZP and RFX, increased over the five-year period (P-value by Kruskal-Wallis test: < 0.0001, <0.0001, <0.0001, and <0.0001 respectively); however, MICs of VAN or MTZ, antibiotics for treatment of CDI, did not increase or decreased over the same time period (P-value by Kruskal-Wallis test: 0.166, <0.0001). C. difficile RT018 isolates in a tertiary hospital over a five-year period presented a close clonal relationship. MICs of antibiotics promoting CDI increased with this clonal expansion. Copyright © 2018 Elsevier B.V. and International Society of Chemotherapy. All rights reserved.

Whole Genome Sequencing Demonstrates Limited Transmission within Identified Mycobacterium tuberculosis Clusters in New South Wales, Australia

PubMed Central

Gurjav, Ulziijargal; Outhred, Alexander C.; Jelfs, Peter; McCallum, Nadine; Wang, Qinning; Hill-Cawthorne, Grant A.; Marais, Ben J.; Sintchenko, Vitali

2016-01-01

Australia has a low tuberculosis incidence rate with most cases occurring among recent immigrants. Given suboptimal cluster resolution achieved with 24-locus mycobacterium interspersed repetitive unit (MIRU-24) genotyping, the added value of whole genome sequencing was explored. MIRU-24 profiles of all Mycobacterium tuberculosis culture-confirmed tuberculosis cases diagnosed between 2009 and 2013 in New South Wales (NSW), Australia, were examined and clusters identified. The relatedness of cases within the largest MIRU-24 clusters was assessed using whole genome sequencing and phylogenetic analyses. Of 1841 culture-confirmed TB cases, 91.9% (1692/1841) had complete demographic and genotyping data. East-African Indian (474; 28.0%) and Beijing (470; 27.8%) lineage strains predominated. The overall rate of MIRU-24 clustering was 20.1% (340/1692) and was highest among Beijing lineage strains (35.7%; 168/470). One Beijing and three East-African Indian (EAI) clonal complexes were responsible for the majority of observed clusters. Whole genome sequencing of the 4 largest clusters (30 isolates) demonstrated diverse single nucleotide polymorphisms (SNPs) within identified clusters. All sequenced EAI strains and 70% of Beijing lineage strains clustered by MIRU-24 typing demonstrated distinct SNP profiles. The superior resolution provided by whole genome sequencing demonstrated limited M. tuberculosis transmission within NSW, even within identified MIRU-24 clusters. Routine whole genome sequencing could provide valuable public health guidance in low burden settings. PMID:27737005

Multilocus sequence type system for the plant pathogen Xylella fastidiosa and relative contributions of recombination and point mutation to clonal diversity.

PubMed

Scally, Mark; Schuenzel, Erin L; Stouthamer, Richard; Nunney, Leonard

2005-12-01

Multilocus sequence typing (MLST) identifies and groups bacterial strains based on DNA sequence data from (typically) seven housekeeping genes. MLST has also been employed to estimate the relative contributions of recombination and point mutation to clonal divergence. We applied MLST to the plant pathogen Xylella fastidiosa using an initial set of sequences for 10 loci (9.3 kb) of 25 strains from five different host plants, grapevine (PD strains), oleander (OLS strains), oak (OAK strains), almond (ALS strains), and peach (PP strains). An eBURST analysis identified six clonal complexes using the grouping criterion that each member must be identical to at least one other member at 7 or more of the 10 loci. These clonal complexes corresponded to previously identified phylogenetic clades; clonal complex 1 (CC1) (all PD strains plus two ALS strains) and CC2 (OLS strains) defined the X. fastidiosa subsp. fastidiosa and X. fastidiosa subsp. sandyi clades, while CC3 (ALS strains), CC4 (OAK strains), and CC5 (PP strains) were subclades of X. fastidiosa subsp. multiplex. CC6 (ALS strains) identified an X. fastidiosa subsp. multiplex-like group characterized by a high frequency of intersubspecific recombination. Compared to the recombination rate in other bacterial species, the recombination rate in X. fastidiosa is relatively low. Recombination between different alleles was estimated to give rise to 76% of the nucleotide changes and 31% of the allelic changes observed. The housekeeping loci holC, nuoL, leuA, gltT, cysG, petC, and lacF were chosen to form the basis of a public database for typing X. fastidiosa (www.mlst.net). These loci identified the same six clonal complexes using the strain grouping criterion of identity at five or more loci with at least one other member.

Multilocus sequence typing and pulsed-field gel electrophoresis analysis of Oenococcus oeni from different wine-producing regions of China.

PubMed

Wang, Tao; Li, Hua; Wang, Hua; Su, Jing

2015-04-16

The present study established a typing method with NotI-based pulsed-field gel electrophoresis (PFGE) and stress response gene schemed multilocus sequence typing (MLST) for 55 Oenococcus oeni strains isolated from six individual regions in China and two model strains PSU-1 (CP000411) and ATCC BAA-1163 (AAUV00000000). Seven stress response genes, cfa, clpL, clpP, ctsR, mleA, mleP and omrA, were selected for MLST testing, and positive selective pressure was detected for these genes. Furthermore, both methods separated the strains into two clusters. The PFGE clusters are correlated with the region, whereas the sequence types (STs) formed by the MLST confirm the two clusters identified by PFGE. In addition, the population structure was a mixture of evolutionary pathways, and the strains exhibited both clonal and panmictic characteristics. Copyright © 2015 Elsevier B.V. All rights reserved.

Characterization and virulence clustering analysis of extraintestinal pathogenic Escherichia coli isolated from swine in China.

PubMed

Zhu, Yinchu; Dong, Wenyang; Ma, Jiale; Yuan, Lvfeng; Hejair, Hassan M A; Pan, Zihao; Liu, Guangjin; Yao, Huochun

2017-04-08

Swine extraintestinal pathogenic Escherichia coli (ExPEC) is an important pathogen that leads to economic and welfare costs in the swine industry worldwide, and is occurring with increasing frequency in China. By far, various virulence factors have been recognized in ExPEC. Here, we investigated the virulence genotypes and clonal structure of collected strains to improve the knowledge of phylogenetic traits of porcine ExPECs in China. We isolated 64 Chinese porcine ExPEC strains from 2013 to 14 in China. By multiplex PCR, the distribution of isolates belonging to phylogenetic groups B1, B2, A and D was 9.4%, 10.9%, 57.8% and 21.9%, respectively. Nineteen virulence-related genes were detected by PCR assay; ompA, fimH, vat, traT and iutA were highly prevalent. Virulence-related genes were remarkably more prevalent in group B2 than in groups A, B1 and D; notably, usp, cnf1, hlyD, papA and ibeA were only found in group B2 strains. Genotyping analysis was performed and four clusters of strains (named I to IV) were identified. Cluster IV contained all isolates from group B2 and Cluster IV isolates had the strongest pathogenicity in a mouse infection model. As phylogenetic group B2 and D ExPEC isolates are generally considered virulent, multilocus sequence typing (MLST) analysis was performed for these isolates to further investigate genetic relationships. Two novel sequence types, ST5170 and ST5171, were discovered. Among the nine clonal complexes identified among our group B2 and D isolates, CC12 and CC95 have been indicated to have high zoonotic pathogenicity. The distinction between group B2 and non-B2 isolates in virulence and genotype accorded with MLST analysis. This study reveals significant genetic diversity among ExPEC isolates and helps us to better understand their pathogenesis. Importantly, our data suggest group B2 (Cluster IV) strains have the highest risk of causing animal disease and illustrate the correlation between genotype and virulence.

Genetic structure of populations of Legionella pneumophila.

PubMed Central

Selander, R K; McKinney, R M; Whittam, T S; Bibb, W F; Brenner, D J; Nolte, F S; Pattison, P E

1985-01-01

The genetic structure of populations of Legionella pneumophila was defined by an analysis of electrophoretically demonstrable allelic variation at structural genes encoding 22 enzymes in 292 isolates from clinical and environmental sources. Nineteen of the loci were polymorphic, and 62 distinctive electrophoretic types (ETs), representing multilocus genotypes, were identified. Principal coordinates and clustering analyses demonstrated that isolates received as L. pneumophila were a heterogeneous array of genotypes that included two previously undescribed species. For 50 ETs of L. pneumophila (strict sense), mean genetic diversity per locus was 0.312, and diversity was equivalent in ETs represented by isolates recovered from clinical sources and those collected from environmental sources. Cluster analysis revealed four major groups or lineages of ETs in L. pneumophila. Genetic diversity among ETs of the same serotype was, on average, 93% of that in the total sample of ETs. Isolates marked by particular patterns of reactivity to a panel of nine monoclonal antibodies were also genetically heterogeneous, mean diversity within patterns being about 75% of the total. Both Pontiac fever and the pneumonic form of legionellosis may be caused by isolates of the same ET. The genetic structure of L. pneumophila is clonal, and many clones apparently are worldwide in distribution. The fact that L. pneumophila is only 60% as variable as Escherichia coli raises the possibility that isolates recovered from clinical cases and man-made environments are a restricted subset of all clones in the species as a whole. PMID:4030689

Dissemination of clonally related multidrug-resistant Klebsiella pneumoniae in Ireland.

PubMed

Morris, D; O'Connor, M; Izdebski, R; Corcoran, M; Ludden, C E; McGrath, E; Buckley, V; Cryan, B; Gniadkowski, M; Cormican, M

2016-01-01

In October 2012, an outbreak of gentamicin-resistant, ciprofloxacin non-susceptible extended-spectrum β-lactamase (ESBL)-producing Klebsiella pneumoniae occurred in a neonatal intensive care unit in Ireland. In order to determine whether the outbreak strain was more widely dispersed in the country, 137 isolates of K. pneumoniae with this resistance phenotype collected from 17 hospitals throughout Ireland between January 2011 and July 2013 were examined. ESBL production was confirmed phenotypically and all isolates were screened for susceptibility to 19 antimicrobial agents and for the presence of genes encoding bla TEM, bla SHV, bla OXA, and bla CTX-M; 22 isolates were also screened for bla KPC, bla NDM, bla VIM, bla IMP and bla OXA-48 genes. All isolates harboured bla SHV and bla CTX-M and were resistant to ciprofloxacin, gentamicin, nalidixic acid, amoxicillin-clavulanate, and cefpodoxime; 15 were resistant to ertapenem, seven to meropenem and five isolates were confirmed as carbapenemase producers. Pulsed-field gel electrophoresis of all isolates identified 16 major clusters, with two clusters comprising 61% of the entire collection. Multilocus sequence typing of a subset of these isolates identified a novel type, ST1236, a single locus variant of ST48. Data suggest that two major clonal groups, ST1236/ST48 (CG43) and ST15/ST14 (CG15) have been circulating in Ireland since at least January 2011.

Recombination-Mediated Host Adaptation by Avian Staphylococcus aureus

PubMed Central

Murray, Susan; Pascoe, Ben; Méric, Guillaume; Mageiros, Leonardos; Yahara, Koji; Hitchings, Matthew D.; Friedmann, Yasmin; Wilkinson, Thomas S.; Gormley, Fraser J.; Mack, Dietrich; Bray, James E.; Lamble, Sarah; Bowden, Rory; Jolley, Keith A.; Maiden, Martin C.J.; Wendlandt, Sarah; Schwarz, Stefan; Corander, Jukka; Fitzgerald, J. Ross

2017-01-01

Staphylococcus aureus are globally disseminated among farmed chickens causing skeletal muscle infections, dermatitis, and septicaemia. The emergence of poultry-associated lineages has involved zoonotic transmission from humans to chickens but questions remain about the specific adaptations that promote proliferation of chicken pathogens. We characterized genetic variation in a population of genome-sequenced S. aureus isolates of poultry and human origin. Genealogical analysis identified a dominant poultry-associated sequence cluster within the CC5 clonal complex. Poultry and human CC5 isolates were significantly distinct from each other and more recombination events were detected in the poultry isolates. We identified 44 recombination events in 33 genes along the branch extending to the poultry-specific CC5 cluster, and 47 genes were found more often in CC5 poultry isolates compared with those from humans. Many of these gene sequences were common in chicken isolates from other clonal complexes suggesting horizontal gene transfer among poultry associated lineages. Consistent with functional predictions for putative poultry-associated genes, poultry isolates showed enhanced growth at 42 °C and greater erythrocyte lysis on chicken blood agar in comparison with human isolates. By combining phenotype information with evolutionary analyses of staphylococcal genomes, we provide evidence of adaptation, following a human-to-poultry host transition. This has important implications for the emergence and dissemination of new pathogenic clones associated with modern agriculture. PMID:28338786

Molecular Characterization of Streptococcus agalactiae Isolated from Bovine Mastitis in Eastern China

PubMed Central

Yang, Yongchun; Liu, Yinglong; Ding, Yunlei; Yi, Li; Ma, Zhe; Fan, Hongjie; Lu, Chengping

2013-01-01

One hundred and two Streptococcus agalactiae (group B streptococcus [GBS]) isolates were collected from dairy cattle with subclinical mastitis in Eastern China during 2011. Clonal groups were established by multilocus sequence typing (MLST) and pulsed-field gel electrophoresis (PFGE), respectively. Capsular polysaccharides (CPS), pilus and alpha-like-protein (Alp) family genes were also characterized by molecular techniques. MLST analysis revealed that these isolates were limited to three clonal groups and were clustered in six different lineages, i.e. ST (sequence type) 103, ST568, ST67, ST301, ST313 and ST570, of which ST568 and ST570 were new genotypes. PFGE analysis revealed this isolates were clustered in 27 PFGE types, of which, types 7, 8, 14, 15, 16, 18, 23 and 25 were the eight major types, comprising close to 70% (71/102) of all the isolates. The most prevalent sequence types were ST103 (58% isolates) and ST568 (31% isolates), comprising capsular genotype Ia isolates without any of the detected Alp genes, suggesting the appearance of novel genomic backgrounds of prevalent strains of bovine S. agalactiae. All the strains possessed the pilus island 2b (PI-2b) gene and the prevalent capsular genotypes were types Ia (89% isolates) and II (11% isolates), the conserved pilus type providing suitable data for the development of vaccines against mastitis caused by S. agalactiae. PMID:23874442

Molecular Epidemiology of Carbapenem-Resistant Acinetobacter baumannii Isolates in the Gulf Cooperation Council States: Dominance of OXA-23-Type Producers

PubMed Central

Sartor, Anna L.; Sidjabat, Hanna E.; Balkhy, Hanan H.; Walsh, Timothy R.; Al Johani, Sameera M.; AlJindan, Reem Y.; Alfaresi, Mubarak; Ibrahim, Emad; Al-Jardani, Amina; Al Salman, Jameela; Dashti, Ali A.; Johani, Khalid; Paterson, David L.

2015-01-01

The molecular epidemiology and mechanisms of resistance of carbapenem-resistant Acinetobacter baumannii (CRAB) were determined in hospitals in the states of the Cooperation Council for the Arab States of the Gulf (Gulf Cooperation Council [GCC]), namely, Saudi Arabia, United Arab Emirates, Oman, Qatar, Bahrain, and Kuwait. Isolates were subjected to PCR-based detection of antibiotic resistance genes and repetitive sequence-based PCR (rep-PCR) assessments of clonality. Selected isolates were subjected to multilocus sequence typing (MLST). We investigated 117 isolates resistant to carbapenem antibiotics (either imipenem or meropenem). All isolates were positive for OXA-51. The most common carbapenemases were the OXA-23-type, found in 107 isolates, followed by OXA-40-type (OXA-24-type), found in 5 isolates; 3 isolates carried the ISAba1 element upstream of blaOXA-51-type. No OXA-58-type, NDM-type, VIM-type, or IMP-type producers were detected. Multiple clones were detected with 16 clusters of clonally related CRAB. Some clusters involved hospitals in different states. MLST analysis of 15 representative isolates from different clusters identified seven different sequence types (ST195, ST208, ST229, ST436, ST450, ST452, and ST499), as well as three novel STs. The vast majority (84%) of the isolates in this study were associated with health care exposure. Awareness of multidrug-resistant organisms in GCC states has important implications for optimizing infection control practices; establishing antimicrobial stewardship programs within hospital, community, and agricultural settings; and emphasizing the need for establishing regional active surveillance systems. This will help to control the spread of CRAB in the Middle East and in hospitals accommodating transferred patients from this region. PMID:25568439

Molecular epidemiology of carbapenem-resistant Acinetobacter baumannii isolates in the Gulf Cooperation Council States: dominance of OXA-23-type producers.

PubMed

Zowawi, Hosam M; Sartor, Anna L; Sidjabat, Hanna E; Balkhy, Hanan H; Walsh, Timothy R; Al Johani, Sameera M; AlJindan, Reem Y; Alfaresi, Mubarak; Ibrahim, Emad; Al-Jardani, Amina; Al Salman, Jameela; Dashti, Ali A; Johani, Khalid; Paterson, David L

2015-03-01

The molecular epidemiology and mechanisms of resistance of carbapenem-resistant Acinetobacter baumannii (CRAB) were determined in hospitals in the states of the Cooperation Council for the Arab States of the Gulf (Gulf Cooperation Council [GCC]), namely, Saudi Arabia, United Arab Emirates, Oman, Qatar, Bahrain, and Kuwait. Isolates were subjected to PCR-based detection of antibiotic resistance genes and repetitive sequence-based PCR (rep-PCR) assessments of clonality. Selected isolates were subjected to multilocus sequence typing (MLST). We investigated 117 isolates resistant to carbapenem antibiotics (either imipenem or meropenem). All isolates were positive for OXA-51. The most common carbapenemases were the OXA-23-type, found in 107 isolates, followed by OXA-40-type (OXA-24-type), found in 5 isolates; 3 isolates carried the ISAba1 element upstream of blaOXA-51-type. No OXA-58-type, NDM-type, VIM-type, or IMP-type producers were detected. Multiple clones were detected with 16 clusters of clonally related CRAB. Some clusters involved hospitals in different states. MLST analysis of 15 representative isolates from different clusters identified seven different sequence types (ST195, ST208, ST229, ST436, ST450, ST452, and ST499), as well as three novel STs. The vast majority (84%) of the isolates in this study were associated with health care exposure. Awareness of multidrug-resistant organisms in GCC states has important implications for optimizing infection control practices; establishing antimicrobial stewardship programs within hospital, community, and agricultural settings; and emphasizing the need for establishing regional active surveillance systems. This will help to control the spread of CRAB in the Middle East and in hospitals accommodating transferred patients from this region. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Clonal emergence of Klebsiella pneumoniae ST14 co-producing OXA-48-type and NDM carbapenemases with high rate of colistin resistance in Dubai, United Arab Emirates.

PubMed

Moubareck, Carole Ayoub; Mouftah, Shaimaa F; Pál, Tibor; Ghazawi, Akela; Halat, Dalal H; Nabi, Anju; AlSharhan, Mouza A; AlDeesi, Zulfa O; Peters, Christabel C; Celiloglu, Handan; Sannegowda, Manjunath; Sarkis, Dolla K; Sonnevend, Ágnes

2018-03-09

Few studies have addressed the molecular epidemiology of carbapenem resistant Enterobacteriaceae (CRE) isolates in the Arabian Peninsula, and such investigations have been missing from Dubai, a major economical, tourism and medical centre of the region. The antibiotic susceptibility, the carbapenemase type produced, and the clonality of 89 CRE strains isolated in five major Dubai hospitals in June 2015 - June 2016 were determined. Thirty three percent of the collection of 70 K. pneumoniae, 13 E. coli and 6 other Enterobacteriaceae were extremely drug resistant, 27% were resistant to colistin, and 4.5% (four K. pneumoniae isolates) were resistant to all antibiotics tested. The colistin resistance rate in K. pneumoniae was 31.4%. None of the isolates carried mobile colistin resistance genes. Seventy-seven isolates produced carbapenemase: 53.3% OXA-48-like, 24.7% NDM, and 22.1% both OXA-48-like and NDM, respectively. PFGE clustered 50% of K. pneumoniae into a 35-membered group, which showed significant association with double carbapenemase production, with extreme drug resistance, and with being isolated from Emirati patients. Members of the cluster belonged to sequence type ST14. The rate of colistin resistance in K. pneumoniae ST14 was 37.1% vs. 27.1% of K. pneumoniae isolates outside of the cluster. Two of the panresistant K. pneumoniae isolates also belonged to ST14, whereas the other two were ST15 and ST231, respectively. In conclusion, beyond the overall high colistin resistance rate in CRE, the emergence of a highly resistant clone of K. pneumoniae ST14 in all Dubai hospitals investigated is a serious problem requiring immediate attention. Copyright © 2018. Published by Elsevier B.V.

Altered growth pattern, not altered growth per se, is the hallmark of early lesions preceding cancer development.

PubMed

Doratiotto, S; Marongiu, F; Faedda, S; Pani, P; Laconi, E

2009-01-01

Many human solid cancers arise from focal proliferative lesions that long precede the overt clinical appearance of the disease. The available evidence supports the notion that cancer precursor lesions are clonal in origin, and this notion forms the basis for most of the current theories on the pathogenesis of neoplastic disease. In contrast, far less attention has been devoted to the analysis of the phenotypic property that serves to define these focal lesions, i.e. their altered growth pattern. In fact, the latter is often considered a mere morphological by-product of clonal growth, with no specific relevance in the process. In the following study, evidence will be presented to support the concept that focal growth pattern is an inherent property of altered cells, independent of clonal growth; furthermore, it will be discussed how such a property, far from being merely descriptive, might indeed play a fundamental role in the sequence of events leading to the development of cancer. Within this paradigm, the earliest steps of neoplasia should be considered and analysed as defects in the mechanisms of tissue pattern formation.

Systemic Mastocytosis with Smoldering Multiple Myeloma: Report of a Case

PubMed Central

Garcia, Gwenalyn; Ying, Liu; Hurford, Matthew; Odaimi, Marcel

2016-01-01

Systemic mastocytosis (SM) is a disease characterized by a clonal infiltration of mast cells affecting various tissues of the body. It is grouped into six different subtypes according to the World Health Organization classification. It is called indolent systemic mastocytosis (ISM) when there is no evidence of end organ dysfunction, while the presence of end organ dysfunction defines aggressive systemic mastocytosis (ASM). When SM coexists with a clonal hematological disorder, it is classified as systemic mastocytosis with associated clonal hematological nonmast cell lineage disease (SM-AHNMD). Over 80% of SM-AHNMD cases involve disorders of the myeloid cell lines. To our knowledge, there are only 8 reported cases to date of SM associated with a plasma cell disorder. We report a patient with ISM who was found to have concomitant smoldering multiple myeloma. His disease later progressed to ASM. We discuss this rare association between SM and a plasma cell disorder, and potential common pathophysiologic mechanisms linking the two disorders will be reviewed. We also discuss prognostic factors in SM as well as the management options considered during the evolution of the patient's disease. PMID:27293930

Clonal Structure and Characterization of Staphylococcus aureus Strains from Invasive Infections in Paediatric Patients from South Poland: Association between Age, spa Types, Clonal Complexes, and Genetic Markers

PubMed Central

Ilczyszyn, Weronika M.; Sabat, Artur J.; Akkerboom, Viktoria; Szkarlat, Anna; Klepacka, Joanna; Sowa-Sierant, Iwona; Wasik, Barbara; Kosecka-Strojek, Maja; Buda, Aneta; Miedzobrodzki, Jacek; Friedrich, Alexander W.

2016-01-01

The aim of current study was to examine clonal structure and genetic profile of invasive Staphylococcus aureus isolates recovered from infants and children treated at the Jagiellonian University Children’s Hospital of Krakow, Poland. The 107 invasive S. aureus isolates, collected between February 2012 and August 2014, were analysed retrospectively. Antimicrobial susceptibility testing, spa typing and DNA microarray analysis were performed to determine clonal distribution, diversity and gene content in regard to patients characteristics. In total, 107 isolates were recovered from 88 patients with clinical symptoms of invasive bacterial infection. The final set of 92 non-duplicate samples included 38 MRSA isolates. Additionally, a set of 54 S. aureus isolates collected during epidemiological screening was genotyped and analysed. There were 72 healthcare-associated (HCA) and 20 community-onset (CO) infection events caused by 33 and 5 MRSA isolates, respectively. The majority of isolates were affiliated with the major European clonal complexes CC5 (t003, spa-CC 002), CC45 (spa-CC 015), CC7 or CC15 (t084, t091, spa-CC 084). Two epidemic clones (CC5-MRSA-II or CC45-MRSA-IV) dominated among MRSA isolates, while MSSA population contained 15 different CCs. The epidemiological screening isolates belonged to similar genetic lineages as those collected from invasive infection cases. The HCA infection events, spa types t003, t2642 or CC5 were significantly associated with infections occurring in neonates and children under 5 years of age. Moreover, carriage of several genetic markers, including erm(A), sea (N315), egc-cluster, chp was significantly higher in isolates obtained from children in this age group. The spa types t091 and t008 were underrepresented among patients aged 5 years or younger, whereas spa type t008, CC8 and presence of splE was associated with infection in children aged 10 years or older. The HCA-MRSA strains were most frequently found in children under 5 years, although the majority of invasive infections was associated with MSSA strains. Moreover, an association between age group of children from the study population and a specific strain genotype (spa type, clonal complex or genetic content) was observed among the patients. PMID:26992009

Trends and molecular mechanisms of antimicrobial resistance in clinical staphylococci isolated from companion animals over a 16 year period.

PubMed

Couto, Natacha; Monchique, Cláudia; Belas, Adriana; Marques, Cátia; Gama, Luís T; Pomba, Constança

2016-06-01

The objective of this study was to investigate the evolution of resistance to antimicrobials, corresponding mechanisms and molecular characteristics of Staphylococcus spp., between 1999 and 2014. Susceptibility to 38 antimicrobials was determined for 632 clinical staphylococcal isolates obtained from companion animals (dogs, cats, horses and other animals). Twenty antimicrobial resistance genes, including mecA and mecC, were screened by PCR. Methicillin-resistant staphylococci were characterized by spa (Staphylococcus aureus), SCCmec, MLST and PFGE typing. Statistical analyses were performed using SAS v9.3 and differences were considered relevant if P ≤ 0.05. The mecA gene was identified in 74 staphylococcal isolates (11.6%): 11 MRSA (40.7%), 40 methicillin-resistant Staphylococcus pseudintermedius (MRSP; 8.7%) and 23 methicillin-resistant CoNS (26.7%). Resistance to the majority of antimicrobials and the number of mecA-positive isolates increased significantly over time. Eighteen spa types were identified, including two new ones. MRSA isolates were divided into three PFGE clusters that included ST22-IV, ST105-II, ST398-V and ST5-VI. Most methicillin-resistant Staphylococcus epidermidis isolates were of clonal complex (CC) 5, including a new ST, and clustered in eight PFGE clusters. MRSP were grouped into five PFGE clusters and included ST45-NT, ST71-II-III, ST195-III, ST196-V, ST339-NT, ST342-IV and the new ST400-III. Methicillin-resistant Staphylococcus haemolyticus clustered in two PFGE clusters. The significant increase in antimicrobial-resistant and mecA-positive isolates in recent years is worrying. Furthermore, several isolates are MDR, which complicates antimicrobial treatment and increases the risk of transfer to humans or human isolates. Several clonal lineages of MRSA and methicillin-resistant S. epidermidis circulating in human hospitals and the community were found, suggesting that companion animals can become infected with and contribute to the dissemination of highly successful human clones. Urgent measures, such as determination of clinical breakpoints and guidelines for antimicrobial use, are needed. © The Author 2016. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

The Memory Cytotoxic T-Lymphocyte (CTL) Response to Human Cytomegalovirus Infection Contains Individual Peptide-Specific CTL Clones That Have Undergone Extensive Expansion In Vivo

PubMed Central

Weekes, Michael P.; Wills, Mark R.; Mynard, Kim; Carmichael, Andrew J.; Sissons, J. G. Patrick

1999-01-01

Human cytomegalovirus (HCMV)-specific CD8+ cytotoxic T lymphocytes (CTL) appear to play an important role in the control of virus replication and in protection against HCMV-related disease. We have previously reported high frequencies of memory CTL precursors (CTLp) specific to the HCMV tegument protein pp65 in the peripheral blood of healthy virus carriers. In some individuals, the CTL response to this protein is focused on only a single epitope, whereas in other virus carriers CTL recognized multiple epitopes which we identified by using synthetic peptides. We have analyzed the clonal composition of the memory CTL response to four of these pp65 epitopes by sequencing the T-cell receptors (TCR) of multiple independently derived epitope-specific CTL clones, which were derived by formal single-cell cloning or from clonal CTL microcultures. In all cases, we have observed a high degree of clonal focusing: the majority of CTL clones specific to a defined pp65 peptide from any one virus carrier use only one or two different TCRs at the level of the nucleotide sequence. Among virus carriers who have the same major histocompatibility complex (MHC) class I allele, we observed that CTL from different donors that recognize the same peptide-MHC complex often used the same Vβ segment, although other TCR gene segments and CDR3 length were not in general conserved. We have also examined the clonal composition of CTL specific to pp65 peptides in asymptomatic human immunodeficiency virus-infected individuals. We have observed a similarly focused peptide-specific CTL response. Thus, the large population of circulating HCMV peptide-specific memory CTLp in virus carriers in fact contains individual CTL clones that have undergone extensive clonal expansion in vivo. PMID:9971792

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

PubMed

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.

Molecular Characterization of Carbapenemase-Producing Escherichia coli and Klebsiella pneumoniae in the Countries of the Gulf Cooperation Council: Dominance of OXA-48 and NDM Producers

PubMed Central

Sartor, Anna L.; Balkhy, Hanan H.; Walsh, Timothy R.; Al Johani, Sameera M.; AlJindan, Reem Y.; Alfaresi, Mubarak; Ibrahim, Emad; Al-Jardani, Amina; Al-Abri, Seif; Al Salman, Jameela; Dashti, Ali A.; Kutbi, Abdullah H.; Schlebusch, Sanmarié; Sidjabat, Hanna E.; Paterson, David L.

2014-01-01

The molecular epidemiology and mechanisms of resistance of carbapenem-resistant Enterobacteriaceae (CRE) were determined in hospitals in the countries of the Gulf Cooperation Council (GCC), namely, Saudi Arabia, United Arab Emirates, Oman, Qatar, Bahrain, and Kuwait. Isolates were subjected to PCR-based detection of antibiotic-resistant genes and repetitive sequence-based PCR (rep-PCR) assessments of clonality. Sixty-two isolates which screened positive for potential carbapenemase production were assessed, and 45 were found to produce carbapenemase. The most common carbapenemases were of the OXA-48 (35 isolates) and NDM (16 isolates) types; 6 isolates were found to coproduce the OXA-48 and NDM types. No KPC-type, VIM-type, or IMP-type producers were detected. Multiple clones were detected with seven clusters of clonally related Klebsiella pneumoniae. Awareness of CRE in GCC countries has important implications for controlling the spread of CRE in the Middle East and in hospitals accommodating patients transferred from the region. PMID:24637692

Symplasmata are a clonal, conditional, and reversible type of bacterial multicellularity

DOE PAGES

Tecon, Robin; Leveau, Johan H. J.

2016-08-18

Microorganisms are capable of remarkable social behaviours, such as forming transient multicellular assemblages with properties and adaptive abilities exceeding those of individual cells. Here, we report on the formation and structure of genets known as symplasmata produced by Pantoea eucalypti bacteria. Each symplasmatum develops clonally and stochastically from a single bacterium into a membrane-delimited, capsule-embedded cluster of progeny cells and with a frequency that depends on temperature, pH, and nutrient availability. Transposon mutagenesis identified several gene products required for symplasmata formation, including master regulator LrhA, replication inhibitor CspD, polysaccharide transporter RfbX3, and autoinducer synthase PhzI. We also show that bacteriamore » inside symplasmata are shaped irregularly with punctuated cell-to-cell contacts, metabolically responsive to environmental stimuli, dispersal-ready, and transcriptionally reprogrammed to anticipate multiple alternative futures in terms of carbon source availability. In conclusion, the structured and conditionable nature of symplasmata offers exciting prospects towards a mechanistic understanding of multicellular behaviours and their ecological significance.« less

A Single-Cell Roadmap of Lineage Bifurcation in Human ESC Models of Embryonic Brain Development.

PubMed

Yao, Zizhen; Mich, John K; Ku, Sherman; Menon, Vilas; Krostag, Anne-Rachel; Martinez, Refugio A; Furchtgott, Leon; Mulholland, Heather; Bort, Susan; Fuqua, Margaret A; Gregor, Ben W; Hodge, Rebecca D; Jayabalu, Anu; May, Ryan C; Melton, Samuel; Nelson, Angelique M; Ngo, N Kiet; Shapovalova, Nadiya V; Shehata, Soraya I; Smith, Michael W; Tait, Leah J; Thompson, Carol L; Thomsen, Elliot R; Ye, Chaoyang; Glass, Ian A; Kaykas, Ajamete; Yao, Shuyuan; Phillips, John W; Grimley, Joshua S; Levi, Boaz P; Wang, Yanling; Ramanathan, Sharad

2017-01-05

During human brain development, multiple signaling pathways generate diverse cell types with varied regional identities. Here, we integrate single-cell RNA sequencing and clonal analyses to reveal lineage trees and molecular signals underlying early forebrain and mid/hindbrain cell differentiation from human embryonic stem cells (hESCs). Clustering single-cell transcriptomic data identified 41 distinct populations of progenitor, neuronal, and non-neural cells across our differentiation time course. Comparisons with primary mouse and human gene expression data demonstrated rostral and caudal progenitor and neuronal identities from early brain development. Bayesian analyses inferred a unified cell-type lineage tree that bifurcates between cortical and mid/hindbrain cell types. Two methods of clonal analyses confirmed these findings and further revealed the importance of Wnt/β-catenin signaling in controlling this lineage decision. Together, these findings provide a rich transcriptome-based lineage map for studying human brain development and modeling developmental disorders. Copyright © 2017 Elsevier Inc. All rights reserved.

Global distribution and epidemiologic associations of Escherichia coli clonal group A, 1998-2007.

PubMed

Johnson, James R; Menard, Megan E; Lauderdale, Tsai-Ling; Kosmidis, Chris; Gordon, David; Collignon, Peter; Maslow, Joel N; Andrasević, Arjana Tambić; Kuskowski, Michael A

2011-11-01

Escherichia coli clonal group A (CGA) was first reported in 2001 as an emerging multidrug-resistant extraintestinal pathogen. Because CGA has considerable implications for public health, we examined the trends of its global distribution, clinical associations, and temporal prevalence for the years 1998-2007. We characterized 2,210 E. coli extraintestinal clinical isolates from 32 centers on 6 continents by CGA status for comparison with trimethoprim/sulfamethoxazole (TMP/SMZ) phenotype, specimen type, inpatient/outpatient source, and adult/child host; we adjusted for clustering by center. CGA prevalence varied greatly by center and continent, was strongly associated with TMP/SMZ resistance but not with other epidemiologic variables, and exhibited no temporal prevalence trend. Our findings indicate that CGA is a prominent, primarily TMP/SMZ-resistant extraintestinal pathogen concentrated within the Western world, with considerable pathogenic versatility. The stable prevalence of CGA over time suggests full emergence by the late 1990s, followed by variable endemicity worldwide as an antimicrobial drug-resistant public health threat.

Population structure of Streptococcus oralis

PubMed Central

Do, Thuy; Jolley, Keith A.; Maiden, Martin C. J.; Gilbert, Steven C.; Clark, Douglas; Wade, William G.; Beighton, David

2009-01-01

Streptococcus oralis is a member of the normal human oral microbiota, capable of opportunistic pathogenicity; like related oral streptococci, it exhibits appreciable phenotypic and genetic variation. A multilocus sequence typing (MLST) scheme for S. oralis was developed and the resultant data analysed to examine the population structure of the species. Analysis of 113 isolates, confirmed as belonging to the S. oralis/mitis group by 16S rRNA gene sequencing, characterized the population as highly diverse and undergoing inter- and intra-species recombination with a probable clonal complex structure. ClonalFrame analysis of these S. oralis isolates along with examples of Streptococcus pneumoniae, Streptococcus mitis and Streptococcus pseudopneumoniae grouped the named species into distinct, coherent populations and did not support the clustering of S. pseudopneumoniae with S. mitis as reported previously using distance-based methods. Analysis of the individual loci suggested that this discrepancy was due to the possible hybrid nature of S. pseudopneumoniae. The data are available on the public MLST website (http://pubmlst.org/soralis/). PMID:19423627

Superclone Expansion, Long-Distance Clonal Dispersal and Local Genetic Structuring in the Coral Pocillopora damicornis Type β in Reunion Island, South Western Indian Ocean.

PubMed

Gélin, Pauline; Fauvelot, Cécile; Mehn, Vincent; Bureau, Sophie; Rouzé, Héloïse; Magalon, Hélène

2017-01-01

The scleractinian coral Pocillopora damicornis type β is known to present a mixed reproduction mode: through sexual reproduction, new genotypes are created, while asexual reproduction insures their propagation. In order to investigate the relative proportion of each reproduction mode in P. damicornis type β populations from Reunion Island, Indian Ocean, clonal propagation along the west coast was assessed through four sampling sites with increasing geographical distance between sites. Coral colonies were sampled either exhaustively, randomly or haphazardly within each site, and genotypic diversity was assessed using 13 microsatellite loci over a total of 510 P. damicornis type β determined a posteriori from their mtDNA haplotype (a 840 bp sequenced fragment of the Open Reading Frame). Overall, 47% of all the sampled colonies presented the same multi-locus genotype (MLG), a superclone, suggesting that asexual propagation is extremely important in Reunion Island. Within each site, numerous MLGs were shared by several colonies, suggesting local clonal propagation through fragmentation. Moreover, some of these MLGs were found to be shared among several sites located 40 km apart. While asexual reproduction by fragmentation seems unlikely over long distances, our results suggest a production of parthenogenetic larvae. Despite shared MLGs, two differentiated clusters were enclosed among populations of the west coast of Reunion Island, revealing the necessity to set up appropriate managing strategies at a local scale.

High-throughput analysis of spatio-temporal dynamics in Dictyostelium

PubMed Central

Sawai, Satoshi; Guan, Xiao-Juan; Kuspa, Adam; Cox, Edward C

2007-01-01

We demonstrate a time-lapse video approach that allows rapid examination of the spatio-temporal dynamics of Dictyostelium cell populations. Quantitative information was gathered by sampling life histories of more than 2,000 mutant clones from a large mutagenesis collection. Approximately 4% of the clonal lines showed a mutant phenotype at one stage. Many of these could be ordered by clustering into functional groups. The dataset allows one to search and retrieve movies on a gene-by-gene and phenotype-by-phenotype basis. PMID:17659086

Tuberculosis transmission of predominant genotypes of Mycobacterium tuberculosis in northern suburbs of Buenos Aires city region.

PubMed

Morcillo, N; Zumarraga, M; Imperiale, B; Di Giulio, B; Chirico, C; Kuriger, A; Alito, A; Kremer, K; Cataldi, A

2007-01-01

In 2003, the incidence of tuberculosis in Argentina showed an increase compared to 2002. The severe national crisis at the end of the 90s has probably strongly contributed to this situation. The goal of this work was to estimate the extent of the spread of the most predominant Mycobacterium tuberculosis strains and to assess the spread of predominant M. tuberculosis clusters as determined by spoligotyping and IS6110 RFLP. The study involved 590 pulmonary, smear-positive TB cases receiving medical attention at health centers and hospitals in Northern Buenos Aires (NBA) suburbs, from October 2001 to December 2002. From a total of 208 clinical isolates belonging to 6 major clusters, 63 (30.2%) isolates had identical spoligotyping and IS6110 RFLP pattern. Only 22.2% were shown to have epidemiological connections with another member of their respective cluster. In these major clusters, 30.2% of the 208 TB cases studied by both molecular techniques and contact tracing could be convincingly attributable to a recently acquired infection. This knowledge may be useful to assess the clonal distribution of predominant M. tuberculosis clusters in Argentina, which may make an impact on TB control strategies.

The multilocus sequence typing network: mlst.net.

PubMed

Aanensen, David M; Spratt, Brian G

2005-07-01

The unambiguous characterization of strains of a pathogen is crucial for addressing questions relating to its epidemiology, population and evolutionary biology. Multilocus sequence typing (MLST), which defines strains from the sequences at seven house-keeping loci, has become the method of choice for molecular typing of many bacterial and fungal pathogens (and non-pathogens), and MLST schemes and strain databases are available for a growing number of prokaryotic and eukaryotic organisms. Sequence data are ideal for strain characterization as they are unambiguous, meaning strains can readily be compared between laboratories via the Internet. Laboratories undertaking MLST can quickly progress from sequencing the seven gene fragments to characterizing their strains and relating them to those submitted by others and to the population as a whole. We provide the gateway to a number of MLST schemes, each of which contain a set of tools for the initial characterization of strains, and methods for relating query strains to other strains of the species, including clustering based on differences in allelic profiles, phylogenetic trees based on concatenated sequences, and a recently developed method (eBURST) for identifying clonal complexes within a species and displaying the overall structure of the population. This network of MLST websites is available at http://www.mlst.net.

Assay Design Affects the Interpretation of T-Cell Receptor Gamma Gene Rearrangements

PubMed Central

Cushman-Vokoun, Allison M.; Connealy, Solomon; Greiner, Timothy C.

2010-01-01

Interpretation of capillary electrophoresis results derived from multiplexed fluorochrome-labeled primer sets can be complicated by small peaks, which may be incorrectly interpreted as clonal T-cell receptor-γ gene rearrangements. In this report, different assay designs were used to illustrate how design may adversely affect specificity. Ten clinical cases, with subclonal peaks containing one of the two infrequently used joining genes, were identified with a tri-color, one-tube assay. The DNA was amplified with the same NED fluorochrome on all three joining primers, first combined (one-color assay) and then amplified separately using a single NED-labeled joining primer. The single primer assay design shows how insignificant peaks could easily be wrongly interpreted as clonal T-cell receptor-γ gene rearrangements. Next, the performance of the one-tube assay was compared with the two-tube BIOMED-2-based TCRG Gene Clonality Assay in a series of 44 cases. Whereas sensitivity was similar between the two methods (92.9% vs. 96.4%; P = 0.55), specificity was significantly less in the BIOMED-2 assay (87.5% vs. 56.3%; P = 0.049) when a 2× ratio was used to define clonality. Specificity was improved to 81.3% by the use of a 5× peak height ratio (P = 0.626). These findings illustrate how extra caution is needed in interpreting a design with multiple, separate distributions, which is more difficult to interpret than a single distribution assay. PMID:20959612

A dominant lineage of Mycoplasma bovis is associated with an increased number of severe mastitis cases in cattle.

PubMed

Bürki, Sibylle; Spergser, Joachim; Bodmer, Michèle; Pilo, Paola

2016-11-30

Mycoplasma bovis is the most frequent etiologic agent of bovine mycoplasmosis. It causes various diseases in bovines and considerable economic loss due to the lack of effective treatment or preventive measures such as vaccination. In contrast to the US, where M. bovis-mastitis has been reported for a long time, M. bovis infections in Switzerland and Austria were predominantly associated with pneumonia and subclinical mastitis. However, since 2007 the situation has changed with the emergence of severe M. bovis-associated mastitis cases in both countries. In order to evaluate the molecular epidemiology of the bacteria isolated from these infections, recent and old Swiss, along with recent Austrian M. bovis isolates were analyzed by a typing method displaying intermediate resolution of evolutionary relationships among isolates called Multi-Locus Sequence Typing (MLST). The analysis of Swiss and Austrian M. bovis isolates revealed two major lineages. Isolates collected since 2007 in both countries cluster in the lineage I including ST5, ST33, ST34, 36, and ST38-40 (clonal complex 1), while all Swiss isolates recovered before 2007 cluster in the lineage II comprising ST17 and ST35 (clonal complex 5). Further investigations are necessary to understand if lineage I has a higher predilection or virulence toward mammary gland cells than the old lineage or if other factors are involved in the increased number of severe mastitis cases. Copyright © 2016 Elsevier B.V. All rights reserved.

densityCut: an efficient and versatile topological approach for automatic clustering of biological data

PubMed Central

Ding, Jiarui; Shah, Sohrab; Condon, Anne

2016-01-01

Motivation: Many biological data processing problems can be formalized as clustering problems to partition data points into sensible and biologically interpretable groups. Results: This article introduces densityCut, a novel density-based clustering algorithm, which is both time- and space-efficient and proceeds as follows: densityCut first roughly estimates the densities of data points from a K-nearest neighbour graph and then refines the densities via a random walk. A cluster consists of points falling into the basin of attraction of an estimated mode of the underlining density function. A post-processing step merges clusters and generates a hierarchical cluster tree. The number of clusters is selected from the most stable clustering in the hierarchical cluster tree. Experimental results on ten synthetic benchmark datasets and two microarray gene expression datasets demonstrate that densityCut performs better than state-of-the-art algorithms for clustering biological datasets. For applications, we focus on the recent cancer mutation clustering and single cell data analyses, namely to cluster variant allele frequencies of somatic mutations to reveal clonal architectures of individual tumours, to cluster single-cell gene expression data to uncover cell population compositions, and to cluster single-cell mass cytometry data to detect communities of cells of the same functional states or types. densityCut performs better than competing algorithms and is scalable to large datasets. Availability and Implementation: Data and the densityCut R package is available from https://bitbucket.org/jerry00/densitycut_dev. Contact: condon@cs.ubc.ca or sshah@bccrc.ca or jiaruid@cs.ubc.ca Supplementary information: Supplementary data are available at Bioinformatics online. PMID:27153661

Generation of diversity in Streptococcus mutans genes demonstrated by MLST.

PubMed

Do, Thuy; Gilbert, Steven C; Clark, Douglas; Ali, Farida; Fatturi Parolo, Clarissa C; Maltz, Marisa; Russell, Roy R; Holbrook, Peter; Wade, William G; Beighton, David

2010-02-05

Streptococcus mutans, consisting of serotypes c, e, f and k, is an oral aciduric organism associated with the initiation and progression of dental caries. A total of 135 independent Streptococcus mutans strains from caries-free and caries-active subjects isolated from various geographical locations were examined in two versions of an MLST scheme consisting of either 6 housekeeping genes [accC (acetyl-CoA carboxylase biotin carboxylase subunit), gki (glucokinase), lepA (GTP-binding protein), recP (transketolase), sodA (superoxide dismutase), and tyrS (tyrosyl-tRNA synthetase)] or the housekeeping genes supplemented with 2 extracellular putative virulence genes [gtfB (glucosyltransferase B) and spaP (surface protein antigen I/II)] to increase sequence type diversity. The number of alleles found varied between 20 (lepA) and 37 (spaP). Overall, 121 sequence types (STs) were defined using the housekeeping genes alone and 122 with all genes. However pi, nucleotide diversity per site, was low for all loci being in the range 0.019-0.007. The virulence genes exhibited the greatest nucleotide diversity and the recombination/mutation ratio was 0.67 [95% confidence interval 0.3-1.15] compared to 8.3 [95% confidence interval 5.0-14.5] for the 6 concatenated housekeeping genes alone. The ML trees generated for individual MLST loci were significantly incongruent and not significantly different from random trees. Analysis using ClonalFrame indicated that the majority of isolates were singletons and no evidence for a clonal structure or evidence to support serotype c strains as the ancestral S. mutans strain was apparent. There was also no evidence of a geographical distribution of individual isolates or that particular isolate clusters were associated with caries. The overall low sequence diversity suggests that S. mutans is a newly emerged species which has not accumulated large numbers of mutations but those that have occurred have been shuffled as a consequence of intra-species recombination generating genotypes which can be readily distinguished by sequence analysis.

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

PubMed

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.

Phylogenetic relationships of Shiga toxin-producing Escherichia coli isolated from Peruvian children

PubMed Central

Contreras, C. A.; Ruiz, J.; Lacher, D. W.; Rivera, F. P.; Saenz, Y.; Chea-Woo, E.; Zavaleta, N.; Gil, A. I.; Lanata, C. F.; Huicho, L.; Maves, R. C.; Torres, C.; DebRoy, C.; Cleary, T. G.

2011-01-01

The aim of this study was to determine the prevalence, virulence factors (stx, eae, ehxA and astA) and phylogenetic relationships [PFGE and multilocus sequence typing (MLST)] of Shiga toxin-producing Escherichia coli (STEC) strains isolated from four previous cohort studies in 2212 Peruvian children aged <36 months. STEC prevalence was 0.4 % (14/3219) in diarrhoeal and 0.6 % (15/2695) in control samples. None of the infected children developed haemolytic uraemic syndrome (HUS) or other complications of STEC. stx1 was present in 83 % of strains, stx2 in 17 %, eae in 72 %, ehxA in 59 % and astA in 14 %. The most common serotype was O26 : H11 (14 %) and the most common seropathotype was B (45 %). The strains belonged mainly to phylogenetic group B1 (52 %). The distinct combinations of alleles across the seven MLST loci were used to define 13 sequence types among 19 STEC strains. PFGE typing of 20 STEC strains resulted in 19 pulsed-field patterns. Comparison of the patterns revealed 11 clusters (I–XI), each usually including strains belonging to different serotypes; one exception was cluster VI, which gathered exclusively seven strains of seropathotype B, clonal group enterohaemorrhagic E. coli (EHEC) 2 and phylogenetic group B1. In summary, STEC prevalence was low in Peruvian children with diarrhoea in the community setting. The strains were phylogenetically diverse and associated with mild infections. However, additional studies are needed in children with bloody diarrhoea and HUS. PMID:21292859

Antimicrobial susceptibility of Histophilus somni isolated from clinically affected cattle in Australia.

PubMed

Goldspink, Lauren K; Mollinger, Joanne L; Barnes, Tamsin S; Groves, Mitchell; Mahony, Timothy J; Gibson, Justine S

2015-02-01

This study investigated antimicrobial resistance traits, clonal relationships and epidemiology of Histophilus somni isolated from clinically affected cattle in Queensland and New South Wales, Australia. Isolates (n = 53) were subjected to antimicrobial susceptibility testing against six antimicrobial agents (ceftiofur, enrofloxacin, florfenicol, tetracycline, tilmicosin and tulathromycin) using disc diffusion and minimum inhibitory concentration (MIC) assays. Clonal relationships were assessed using repetitive sequence PCR and descriptive epidemiological analysis was performed. The H. somni isolates appeared to be geographically clonal, with 27/53 (47%) isolates grouping in one cluster from one Australian state. On the basis of disc diffusion, 34/53 (64%) isolates were susceptible to all antimicrobial agents tested; there was intermediate susceptibility to tulathromycin in 12 isolates, tilmicosin in seven isolates and resistance to tilmicosin in one isolate. Using MIC, all but one isolate was susceptible to all antimicrobial agents tested; the non-susceptible isolate was resistant to tetracycline, but this MIC result could not be compared to disc diffusion, since there are no interpretative guidelines for disc diffusion for H. somni against tetracycline. In this study, there was little evidence of antimicrobial resistance in H. somni isolates from Australian cattle. Disc diffusion susceptibility testing results were comparable to MIC results for most antimicrobial agents tested; however, results for isolates with intermediate susceptibility or resistance to tilmicosin and tulathromycin on disc diffusion should be interpreted with caution in the absence of MIC results. Copyright © 2014 Elsevier Ltd. All rights reserved.

Dissemination of the ST-103 clonal complex serogroup C meningococci in Salvador, Brazil.

PubMed

Cordeiro, Soraia Machado; Cardoso, Cristiane Wanderley; de Araújo, Lorena Galvão; Ribeiro, Luis Eduardo; Azevedo, Jailton; Silva, Rita de Cassia Vilasboas; Dos Reis, Mitermayer Galvão; Ko, Albert Icksang; Reis, Joice Neves

2018-01-01

Invasive meningococcal disease (IMD) is a major public health problem worldwide. An epidemic of serogroup C (NmC) IMD occurred in 2010 in the city of Salvador. In this study, we describe the antigenic and genetic characterization of meningococcal isolates collected from meningitis cases in Salvador from 2001 to 2012. Pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST) were performed for the analysis of IMD isolates. A total of 733 cases were identified, and the serogroup was determined for 391 (53.0%) of these. Most cases were caused by NmC (53%) or B (47%). The most prevalent strains were B:4,7:P1.19,15 (32.9%; 129/391) and C:23:P1.14-6 (28.6%; 112/391). Based on PFGE/MLST analysis, 71.3% (77/108 PFGE-tested isolates) clustered as two clones of sequence type ST-3779 and ST-3780, both belonging to the ST-103 clonal complex. ST-3779 has been detected in Salvador since 1996 and together with ST-3780 became predominant after 2005. There was a predominance of C:23:P1.14-6, ST-3779/3780 in Salvador during the period of 2007-2012, establishing a major clonal lineage, which remained in the community for a long time; this has serious implications for public health, particularly in terms of prevention and control strategies of IMD. Copyright © 2017 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.

Evolutionary perspectives on clonal reproduction in vertebrate animals

PubMed Central

Avise, John C.

2015-01-01

A synopsis is provided of different expressions of whole-animal vertebrate clonality (asexual organismal-level reproduction), both in the laboratory and in nature. For vertebrate taxa, such clonal phenomena include the following: human-mediated cloning via artificial nuclear transfer; intergenerational clonality in nature via parthenogenesis and gynogenesis; intergenerational hemiclonality via hybridogenesis and kleptogenesis; intragenerational clonality via polyembryony; and what in effect qualifies as clonal replication via self-fertilization and intense inbreeding by simultaneous hermaphrodites. Each of these clonal or quasi-clonal mechanisms is described, and its evolutionary genetic ramifications are addressed. By affording an atypical vantage on standard vertebrate reproduction, clonality offers fresh perspectives on the evolutionary and ecological significance of recombination-derived genetic variety. PMID:26195735

Clonal Integration Enhances the Performance of a Clonal Plant Species under Soil Alkalinity Stress

PubMed Central

Sun, Juanjuan; Chen, Jishan; Zhang, Yingjun

2015-01-01

Clonal plants have been shown to successfully survive in stressful environments, including salinity stress, drought and depleted nutrients through clonal integration between original and subsequent ramets. However, relatively little is known about whether clonal integration can enhance the performance of clonal plants under alkalinity stress. We investigated the effect of clonal integration on the performance of a typical rhizomatous clonal plant, Leymus chinensis, using a factorial experimental design with four levels of alkalinity and two levels of rhizome connection treatments, connected (allowing integration) and severed (preventing integration). Clonal integration was estimated by comparing physiological and biomass features between the rhizome-connected and rhizome-severed treatments. We found that rhizome-connected treatment increased the biomass, height and leaf water potential of subsequent ramets at highly alkalinity treatments but did not affect them at low alkalinity treatments. However, rhizome-connected treatment decreased the root biomass of subsequent ramets and did not influence the photosynthetic rates of subsequent ramets. The biomass of original ramets was reduced by rhizome-connected treatment at the highest alkalinity level. These results suggest that clonal integration can increase the performance of clonal plants under alkalinity stress. Rhizome-connected plants showed dramatically increased survival of buds with negative effects on root weight, indicating that clonal integration influenced the resource allocation pattern of clonal plants. A cost-benefit analysis based on biomass measures showed that original and subsequent ramets significantly benefited from clonal integration in highly alkalinity stress, indicating that clonal integration is an important adaptive strategy by which clonal plants could survive in local alkalinity soil. PMID:25790352

Tracking Genomic Cancer Evolution for Precision Medicine: The Lung TRACERx Study

PubMed Central

Jamal-Hanjani, Mariam; Hackshaw, Alan; Ngai, Yenting; Shaw, Jacqueline; Dive, Caroline; Quezada, Sergio; Middleton, Gary; de Bruin, Elza; Le Quesne, John; Shafi, Seema; Falzon, Mary; Horswell, Stuart; Blackhall, Fiona; Khan, Iftekhar; Janes, Sam; Nicolson, Marianne; Lawrence, David; Forster, Martin; Fennell, Dean; Lee, Siow-Ming; Lester, Jason; Kerr, Keith; Muller, Salli; Iles, Natasha; Smith, Sean; Murugaesu, Nirupa; Mitter, Richard; Salm, Max; Stuart, Aengus; Matthews, Nik; Adams, Haydn; Ahmad, Tanya; Attanoos, Richard; Bennett, Jonathan; Birkbak, Nicolai Juul; Booton, Richard; Brady, Ged; Buchan, Keith; Capitano, Arrigo; Chetty, Mahendran; Cobbold, Mark; Crosbie, Philip; Davies, Helen; Denison, Alan; Djearman, Madhav; Goldman, Jacki; Haswell, Tom; Joseph, Leena; Kornaszewska, Malgorzata; Krebs, Matthew; Langman, Gerald; MacKenzie, Mairead; Millar, Joy; Morgan, Bruno; Naidu, Babu; Nonaka, Daisuke; Peggs, Karl; Pritchard, Catrin; Remmen, Hardy; Rowan, Andrew; Shah, Rajesh; Smith, Elaine; Summers, Yvonne; Taylor, Magali; Veeriah, Selvaraju; Waller, David; Wilcox, Ben; Wilcox, Maggie; Woolhouse, Ian; McGranahan, Nicholas; Swanton, Charles

2014-01-01

The importance of intratumour genetic and functional heterogeneity is increasingly recognised as a driver of cancer progression and survival outcome. Understanding how tumour clonal heterogeneity impacts upon therapeutic outcome, however, is still an area of unmet clinical and scientific need. TRACERx (TRAcking non-small cell lung Cancer Evolution through therapy [Rx]), a prospective study of patients with primary non-small cell lung cancer (NSCLC), aims to define the evolutionary trajectories of lung cancer in both space and time through multiregion and longitudinal tumour sampling and genetic analysis. By following cancers from diagnosis to relapse, tracking the evolutionary trajectories of tumours in relation to therapeutic interventions, and determining the impact of clonal heterogeneity on clinical outcomes, TRACERx may help to identify novel therapeutic targets for NSCLC and may also serve as a model applicable to other cancer types. PMID:25003521

NetF-producing Clostridium perfringens: Clonality and plasmid pathogenicity loci analysis.

PubMed

Mehdizadeh Gohari, Iman; Kropinski, Andrew M; Weese, Scott J; Whitehead, Ashley E; Parreira, Valeria R; Boerlin, Patrick; Prescott, John F

2017-04-01

Clostridium perfringens is an important cause of foal necrotizing enteritis and canine acute hemorrhagic diarrhea. A major virulence determinant of the strains associated with these diseases appears to be a beta-sheet pore-forming toxin, NetF, encoded within a pathogenicity locus (NetF locus) on a large tcp-conjugative plasmid. Strains producing NetF also produce the putative toxin NetE, encoded within the same pathogenicity locus, as well as CPE enterotoxin and CPB2 on a second plasmid, and sometimes the putative toxin NetG within a pathogenicity locus (NetG locus) on another separate large conjugative plasmid. Previous genome sequences of two netF-positive C. perfringens showed that they both shared three similar plasmids, including the NetF/NetE and CPE/CPB2 toxins-encoding plasmids mentioned above and a putative bacteriocin-encoding plasmid. The main purpose of this study was to determine whether all NetF-producing strains share this common plasmid profile and whether their distinct NetF and CPE pathogenicity loci are conserved. To answer this question, 15 equine and 15 canine netF-positive isolates of C. perfringens were sequenced using Illumina Hiseq2000 technology. In addition, the clonal relationships among the NetF-producing strains were evaluated by core genome multilocus sequence typing (cgMLST). The data obtained showed that all NetF-producing strains have a common plasmid profile and that the defined pathogenicity loci on the plasmids are conserved in all these strains. cgMLST analysis showed that the NetF-producing C. perfringens strains belong to two distinct clonal complexes. The pNetG plasmid was absent from isolates of one of the clonal complexes, and there were minor but consistent differences in the NetF/NetE and CPE/CPB2 plasmids between the two clonal complexes. Copyright © 2017 Elsevier B.V. All rights reserved.

A space-for-time substitution reveals the long-term decline in genotypic diversity of a widespread salt marsh plant, Spartina alterniflora, over a span of 1500 years

USGS Publications Warehouse

Travis, S.E.; Hester, M.W.

2005-01-01

1. Clonal populations face a trade-off between sexual recruitment and vegetative growth and, once established, may undergo continuous declines in genotypic diversity if their sexual recruits make poor competitors. The geological history of delta formation in the Lower Mississippi River Valley was used to age eight 5. alterniflora marshes for use in a space-for-time substitution ranging over 1500 years, in order to determine the long-term effects of clonal growth on genotypic diversity in natural populations. 2. We also predicted that highly heterozygous clones are competitively superior, leading to an increase in the overall level of genetic diversity as a marsh ages and/or to an increasingly positive relationship between clone size and individual heterozygosity, and that the clumping of ramets within clones will occur over increasingly large distances as populations age, while the clumping of genetically related clones will become less pronounced as intraclonal competition begins to obscure the initial effects of localized seedling recruitment. 3. Using molecular markers to differentiate clones, we documented a decline in clonal richness at the rate of approximately 1% 100 years -1 that was accompanied for the first 300-500 years by an increase in the distance over which clumping of ramets within genets occurred. Older populations, in the 500-1500-year range, showed evidence of clone fragmentation. 4. The spatial clustering of kin was observed for only two marshes, and exhibited no clear relationship with marsh age. 5. Whereas the overall level of genetic diversity was consistent among marshes and showed no clear relationship with marsh age, the relationship between heterozygosity and individual clone size became increasingly pronounced within older marshes. 6. Our results suggest that under natural conditions S. alterniflora marshes will rarely reach ages sufficient for the loss of all clonal diversity, or for the effects of inbreeding and drift to pose a significant threat to population viability. ?? 2005 British Ecological Society.

Population structure of clinical Pseudomonas aeruginosa from West and Central African countries.

PubMed

Cholley, Pascal; Ka, Roughyatou; Guyeux, Christophe; Thouverez, Michelle; Guessennd, Nathalie; Ghebremedhin, Beniam; Frank, Thierry; Bertrand, Xavier; Hocquet, Didier

2014-01-01

Pseudomonas aeruginosa (PA) has a non-clonal, epidemic population with a few widely distributed and frequently encountered sequence types (STs) called 'high-risk clusters'. Clinical P. aeruginosa (clinPA) has been studied in all inhabited continents excepted in Africa, where a very few isolates have been analyzed. Here, we characterized a collection of clinPA isolates from four countries of West and Central Africa. 184 non-redundant isolates of clinPA from hospitals of Senegal, Ivory Coast, Nigeria, and Central African Republic were genotyped by MLST. We assessed their resistance level to antibiotics by agar diffusion and identified the extended-spectrum β-lactamases (ESBLs) and metallo-β-lactamases (MBLs) by sequencing. The population structure of the species was determined by a nucleotide-based analysis of the entire PA MLST database and further localized on the phylogenetic tree (i) the sequence types (STs) of the present collection, (ii) the STs by continents, (iii) ESBL- and MBL-producing STs from the MLST database. We found 80 distinct STs, of which 24 had no relationship with any known STs. 'High-risk' international clonal complexes (CC155, CC244, CC235) were frequently found in West and Central Africa. The five VIM-2-producing isolates belonged to CC233 and CC244. GES-1 and GES-9 enzymes were produced by one CC235 and one ST1469 isolate, respectively. We showed the spread of 'high-risk' international clonal complexes, often described as multidrug-resistant on other continents, with a fully susceptible phenotype. The MBL- and ESBL-producing STs were scattered throughout the phylogenetic tree and our data suggest a poor association between a continent and a specific phylogroup. ESBL- and MBL-encoding genes are borne by both successful international clonal complexes and distinct local STs in clinPA of West and Central Africa. Furthermore, our data suggest that the spread of a ST could be either due to its antibiotic resistance or to features independent from the resistance to antibiotics.

Proceedings from the National Cancer Institute's Second International Workshop on the Biology, Prevention, and Treatment of Relapse after Hematopoietic Stem Cell Transplantation: Part I. Biology of relapse after transplantation.

PubMed

Gress, Ronald E; Miller, Jeffrey S; Battiwalla, Minoo; Bishop, Michael R; Giralt, Sergio A; Hardy, Nancy M; Kröger, Nicolaus; Wayne, Alan S; Landau, Dan A; Wu, Catherine J

2013-11-01

In the National Cancer Institute's Second Workshop on the Biology, Prevention, and Treatment of Relapse after Hematopoietic Stem Cell Transplantation, the Scientific/Educational Session on the Biology of Relapse discussed recent advances in understanding some of the host-, disease-, and transplantation-related contributions to relapse, emphasizing concepts with potential therapeutic implications. Relapse after hematopoietic stem cell transplantation (HSCT) represents tumor escape, from the cytotoxic effects of the conditioning regimen and from immunologic control mediated by reconstituted lymphocyte populations. Factors influencing the biology of the therapeutic graft-versus-malignancy (GVM) effect-and relapse-include conditioning regimen effects on lymphocyte populations and homeostasis, immunologic niches, and the tumor microenvironment; reconstitution of lymphocyte populations and establishment of functional immune competence; and genetic heterogeneity within the malignancy defining potential for clonal escape. Recent developments in T cell and natural killer cell homeostasis and reconstitution are reviewed, with implications for prevention and treatment of relapse, as is the application of modern genome sequencing to defining the biologic basis of GVM, clonal escape, and relapse after HSCT. Published by Elsevier Inc.

Molecular characterization of carbapenemase-producing Escherichia coli and Klebsiella pneumoniae in the countries of the Gulf cooperation council: dominance of OXA-48 and NDM producers.

PubMed

Zowawi, Hosam M; Sartor, Anna L; Balkhy, Hanan H; Walsh, Timothy R; Al Johani, Sameera M; AlJindan, Reem Y; Alfaresi, Mubarak; Ibrahim, Emad; Al-Jardani, Amina; Al-Abri, Seif; Al Salman, Jameela; Dashti, Ali A; Kutbi, Abdullah H; Schlebusch, Sanmarié; Sidjabat, Hanna E; Paterson, David L

2014-06-01

The molecular epidemiology and mechanisms of resistance of carbapenem-resistant Enterobacteriaceae (CRE) were determined in hospitals in the countries of the Gulf Cooperation Council (GCC), namely, Saudi Arabia, United Arab Emirates, Oman, Qatar, Bahrain, and Kuwait. Isolates were subjected to PCR-based detection of antibiotic-resistant genes and repetitive sequence-based PCR (rep-PCR) assessments of clonality. Sixty-two isolates which screened positive for potential carbapenemase production were assessed, and 45 were found to produce carbapenemase. The most common carbapenemases were of the OXA-48 (35 isolates) and NDM (16 isolates) types; 6 isolates were found to coproduce the OXA-48 and NDM types. No KPC-type, VIM-type, or IMP-type producers were detected. Multiple clones were detected with seven clusters of clonally related Klebsiella pneumoniae. Awareness of CRE in GCC countries has important implications for controlling the spread of CRE in the Middle East and in hospitals accommodating patients transferred from the region. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

Spatial and Temporal Analysis of Populations of the Sudden Oak Death Pathogen in Oregon Forests.

PubMed

Kamvar, Z N; Larsen, M M; Kanaskie, A M; Hansen, E M; Grünwald, N J

2015-07-01

Sudden oak death caused by the oomycete Phytophthora ramorum was first discovered in California toward the end of the 20th century and subsequently emerged on tanoak forests in Oregon before its first detection in 2001 by aerial surveys. The Oregon Department of Forestry has since monitored the epidemic and sampled symptomatic tanoak trees from 2001 to the present. Populations sampled over this period were genotyped using microsatellites and studied to infer the population genetic history. To date, only the NA1 clonal lineage is established in this region, although three lineages exist on the North American west coast. The original introduction into the Joe Hall area eventually spread to several regions: mostly north but also east and southwest. A new introduction into Hunter Creek appears to correspond to a second introduction not clustering with the early introduction. Our data are best explained by both introductions originating from nursery populations in California or Oregon and resulting from two distinct introduction events. Continued vigilance and eradication of nursery populations of P. ramorum are important to avoid further emergence and potential introduction of other clonal lineages.

Phenotypic Variations in the Foliar Chemical Profile of Persea americana Mill. cv. Hass.

PubMed

García-Rodríguez, Yolanda Magdalena; Torres-Gurrola, Guadalupe; Meléndez-González, Claudio; Espinosa-García, Francisco J

2016-12-01

The Hass avocado tree Persea americana cv. Hass was derived from a single hybrid tree of P. americana var. drymifolia and P. americana var. guatemalensis, and it is propagated clonally by grafting. This cultivar is the most widely planted in the world but its profile of secondary metabolites has been studied rarely despite of its importance in plant protection. We illustrate the variability of the volatilome of mature leaves by describing the average chemical composition and the phenotypic variability found in 70 trees. Contrary to the uniformity expected in the Hass cultivar, high variability coefficients were found for most of the 36 detected foliar volatile compounds; furthermore we found six chemotypes grouping the foliar phenotypes of the sampled trees using hierarchical cluster analysis. About 48% of trees were grouped in one chemotype; five chemotypes grouped the remaining trees. The compounds that determined these chemotypes were: estragole, α-farnesene, β-caryophyllene, germacrene D, α-cubebene and eugenol. This striking variation in a cultivar propagated clonally is discussed in terms of somatic mutation. © 2016 Wiley-VHCA AG, Zurich, Switzerland.

Hotspot mutation panel testing reveals clonal evolution in a study of 265 paired primary and metastatic tumors.

PubMed

Goswami, Rashmi S; Patel, Keyur P; Singh, Rajesh R; Meric-Bernstam, Funda; Kopetz, E Scott; Subbiah, Vivek; Alvarez, Ricardo H; Davies, Michael A; Jabbar, Kausar J; Roy-Chowdhuri, Sinchita; Lazar, Alexander J; Medeiros, L Jeffrey; Broaddus, Russell R; Luthra, Rajyalakshmi; Routbort, Mark J

2015-06-01

We used a clinical next-generation sequencing (NGS) hotspot mutation panel to investigate clonal evolution in paired primary and metastatic tumors. A total of 265 primary and metastatic tumor pairs were sequenced using a 46-gene cancer mutation panel capable of detecting one or more single-nucleotide variants as well as small insertions/deletions. Mutations were tabulated together with tumor type and percentage, mutational variant frequency, time interval between onset of primary tumor and metastasis, and neoadjuvant therapy status. Of note, 227 of 265 (85.7%) tumor metastasis pairs showed identical mutation calls. Of the tumor pairs with identical mutation calls, 160 (60.4%) possessed defining somatic mutation signatures and 67 (25.3%) did not exhibit any somatic mutations. There were 38 (14.3%) cases that showed at least one novel mutation call between the primary and metastasis. Metastases were almost two times more likely to show novel mutations (n = 20, 7.5%) than primary tumors (n = 12, 4.5%). TP53 was the most common additionally mutated gene in metastatic lesions, followed by PIK3CA and SMAD4. PIK3CA mutations were more often associated with metastasis in colon carcinoma samples. Clinical NGS hotspot panels can be useful in analyzing clonal evolution within tumors as well as in determining subclonal mutations that can expand in future metastases. PIK3CA, SMAD4, and TP53 are most often involved in clonal divergence, providing potential targets that may help guide the clinical management of tumor progression or metastases. ©2015 American Association for Cancer Research.

A Livestock-Associated, Multidrug-Resistant, Methicillin-Resistant Staphylococcus aureus Clonal Complex 97 Lineage Spreading in Dairy Cattle and Pigs in Italy

PubMed Central

Feltrin, Fabiola; Alba, Patricia; Kraushaar, Britta; Ianzano, Angela; Argudín, María Angeles; Di Matteo, Paola; Porrero, María Concepción; Aarestrup, Frank M.; Butaye, Patrick; Franco, Alessia

2015-01-01

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 differences in MRSA and methicillin-susceptible S. aureus (MSSA) mainly of swine and bovine origins. Forty-seven CC97 isolates, 35 MRSA isolates, and 6 MSSA isolates from different Italian pig and cattle holdings; 5 pig MRSA isolates from Germany; and 1 human MSSA isolate from Spain were characterized 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 of the isolates were of SCCmec type V (SCCmec V), except for two German MRSA isolates (SCCmec III). Five main clusters were identified by PFGE, with the German isolates (clusters I and II) showing 60.5% similarity with the Italian isolates, most of which (68.1%) grouped into cluster V. All CC97 isolates were Panton-Valentine leukocidin (PVL) negative, and a few (n = 7) tested positive for sak or scn. All MRSA isolates were multidrug resistant (MDR), and the main features were erm(B)- or erm(C)-mediated (n = 18) macrolide-lincosamide-streptogramin B resistance, vga(A)-mediated (n = 37) pleuromutilin 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 of a MRSA lineage that originated in cattle as MSSA and needs further investigation. Measures should be implemented at the farm level to prevent spillover to humans in intensive farming areas. PMID:26590279

Dissemination of clonal Salmonella enterica serovar Typhimurium isolates causing salmonellosis in Mauritius.

PubMed

Issack, Mohammad I; Garcia-Migura, Lourdes; Ramsamy, Veemala D; Svendsen, Christina A; Pornruangwong, Srirat; Pulsrikarn, Chaiwat; Hendriksen, Rene S

2013-07-01

Salmonella enterica serotype Typhimurium is one of the leading causes of salmonellosis in Mauritius, where it has also been associated with outbreaks of foodborne illness. However, little is known about its molecular epidemiology in the country. This study was therefore undertaken to investigate the clonality and source of Salmonella Typhimurium in Mauritius by studying human, food, and poultry isolates by pulsed-field gel electrophoresis (PFGE) and antibiotic minimum inhibitory concentration determination. Forty-nine isolates collected between 2008 and 2011 were analyzed, including 25 stool isolates from foodborne illness outbreaks and sporadic gastroenteritis cases, four blood isolates, one postmortem colon isolate, 14 food isolates, and five poultry isolates. All isolates were pansusceptible to the 16 antibiotics tested, except for two isolates that were resistant to sulfamethoxazole and trimethoprim. Overall characterization of the isolates by PFGE digested with XbaI and BlnI resulted in eight different patterns. The largest of the clusters in the composite dataset consisted of 20 isolates, including two raw chicken isolates, four poultry isolates, and nine human stool isolates from two outbreaks. A second cluster consisted of 18 isolates, of which 12 originated from human blood and stool samples from both sporadic and outbreak cases. Six food isolates were also found in this cluster, including isolates from raw and grilled chicken, marlin mousse, and cooked pork. One poultry isolate had a closely related PFGE pattern. The results indicate that one clone of Salmonella Typhimurium found in poultry has been causing outbreaks of foodborne illness in Mauritius and another clone that has caused many cases of gastrointestinal illness and bacteremia in humans could also be linked to poultry. Thus, poultry appears to be a major reservoir for Salmonella Typhimurium in Mauritius. Initiating on-farm control strategies and measures against future dissemination may substantially reduce the number of cases of salmonellosis in the country.

Serogroup, virulence, and molecular traits of Vibrio parahaemolyticus isolated from clinical and cockle sources in northeastern Thailand.

PubMed

Mala, Wanida; Alam, Munirul; Angkititrakul, Sunpetch; Wongwajana, Suwin; Lulitanond, Viraphong; Huttayananont, Sriwanna; Kaewkes, Wanlop; Faksri, Kiatichai; Chomvarin, Chariya

2016-04-01

Vibrio parahaemolyticus is responsible for seafood-borne gastroenteritis worldwide. Isolates of V. parahaemolyticus from clinical samples (n=74) and cockles (Anadara granosa) (n=74) in Thailand were analyzed by serotyping, determination of virulence and related marker genes present, response to antimicrobial agents, and genetic relatedness. Serological analysis revealed 31 different serotypes, 10 of which occurred among both clinical and cockle samples. The clinical isolates commonly included the pandemic serogroup O3:K6, while a few of the cockle isolates exhibited likely pandemic serovariants such as O3:KUT and O4:KUT, but not O3:K6. The pandemic (orf8 gene-positive) strains were more frequently found among clinical isolates (78.4%) than cockle isolates (28.4%) (p<0.001). Likewise, the virulence and related marker genes were more commonly detected among clinical than cockle isolates; i.e., tdh gene (93.2% versus 29.7%), vcrD2 (97.3% versus 23.0%), vopB2 (89.2% versus 13.5%), vopT (98.6% versus 36.5%) (all p<0.001) and trh (10.8% versus 1.4%) (p<0.05). Pulsed-field gel electrophoresis of NotI-digested genomic DNA of 41 randomly selected V. parahaemolyticus isolates representing different serotypes produced 33 pulsotypes that formed 5 different clusters (clonal complexes) (A-E) in a dendrogram. Vibrio parahaemolyticus O3:K6 and likely related pandemic serotypes were especially common among the numerous clinical isolates in cluster C, suggesting a close clonal link among many of these isolates. Most clinical and cockle isolates were resistant to ampicillin. This study indicates that O3:K6 and its likely serovariants based on the PFGE clusters, are causative agents. Seafoods such as cockles potentially serve as a source of virulent V. parahaemolyticus, but further work is required to identify possible additional sources. Copyright © 2016. Published by Elsevier B.V.

Genomic dissection of the 1994 Cronobacter sakazakii outbreak in a French neonatal intensive care unit.

PubMed

Masood, Naqash; Moore, Karen; Farbos, Audrey; Paszkiewicz, Konrad; Dickins, Ben; McNally, Alan; Forsythe, Stephen

2015-10-05

Cronobacter sakazakii is a member of the genus Cronobacter that has frequently been isolated from powdered infant formula (PIF) and linked with rare but fatal neonatal infections such as meningitis and necrotising enterocolitis. The Cronobacter MLST scheme has reported over 400 sequence types and 42 clonal complexes; however C. sakazakii clonal complex 4 (CC4) has been linked strongly with neonatal infections, especially meningitis. There have been a number of reported Cronobacter outbreaks over the last three decades. The largest outbreak of C. sakazakii was in a neonatal intensive care unit (NICU) in France (1994) that lasted over 3 months and claimed the lives of three neonates. The present study used whole genome sequencing data of 26 isolates obtained from this outbreak to reveal their relatedness. This study is first of its kind to use whole genome sequencing data to analyse a Cronobacter outbreak. Whole genome sequencing data was generated for 26 C. sakazakii isolates on the Illumina MiSeq platform. The whole genome phylogeny was determined using Mugsy and RaxML. SNP calls were determined using SMALT and SAMtools, and filtered using VCFtools. The whole genome phylogeny suggested 3 distant clusters of C. sakazakii isolates were associated with the outbreak. SNP typing and phylogeny indicate the source of the C. sakazakii could have been from extrinsic contamination of reconstituted infant formula from the NICU environment and personnel. This pool of strains would have contributed to the prolonged duration of the outbreak, which was up to 3 months. Furthermore 3 neonates were co-infected with C. sakazakii from two different genotype clusters. The genomic investigation revealed the outbreak consisted of an heterogeneous population of C. sakazakii isolates. The source of the outbreak was not identified, but probably was due to environmental and personnel reservoirs resulting in extrinsic contamination of the neonatal feeds. It also indicated that C. sakazakii isolates from different genotype clusters have the ability to co-infect neonates.

Invasive alien plants benefit more from clonal integration in heterogeneous environments than natives.

PubMed

Wang, Yong-Jian; Müller-Schärer, Heinz; van Kleunen, Mark; Cai, Ai-Ming; Zhang, Ping; Yan, Rong; Dong, Bi-Cheng; Yu, Fei-Hai

2017-12-01

What confers invasive alien plants a competitive advantage over native plants remains open to debate. Many of the world's worst invasive alien plants are clonal and able to share resources within clones (clonal integration), particularly in heterogeneous environments. Here, we tested the hypothesis that clonal integration benefits invasive clonal plants more than natives and thus confers invasives a competitive advantage. We selected five congeneric and naturally co-occurring pairs of invasive alien and native clonal plants in China, and grew pairs of connected and disconnected ramets under heterogeneous light, soil nutrient and water conditions that are commonly encountered by alien plants during their invasion into new areas. Clonal integration increased biomass of all plants in all three heterogeneous resource environments. However, invasive plants benefited more from clonal integration than natives. Consequently, invasive plants produced more biomass than natives. Our results indicate that clonal integration may confer invasive alien clonal plants a competitive advantage over natives. Therefore, differences in the ability of clonal integration could potentially explain, at least partly, the invasion success of alien clonal plants in areas where resources are heterogeneously distributed. © 2017 The Authors. New Phytologist © 2017 New Phytologist Trust.

An invasive clonal plant benefits from clonal integration more than a co-occurring native plant in nutrient-patchy and competitive environments.

PubMed

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 from clonal integration more than co-occurring native J. repens, suggesting that the invasiveness of A. philoxeroides may be closely related to clonal integration in heterogeneous environments.

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

PubMed Central

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 from clonal integration more than co-occurring native J. repens, suggesting that the invasiveness of A. philoxeroides may be closely related to clonal integration in heterogeneous environments. PMID:24816849

Morphological and Inter Simple Sequence Repeat (ISSR) markers analyses of Corynespora cassiicola isolates from rubber plantations in Malaysia.

PubMed

Nghia, Nguyen Anh; Kadir, Jugah; Sunderasan, E; Puad Abdullah, Mohd; Malik, Adam; Napis, Suhaimi

2008-10-01

Morphological features and Inter Simple Sequence Repeat (ISSR) polymorphism were employed to analyse 21 Corynespora cassiicola isolates obtained from a number of Hevea clones grown in rubber plantations in Malaysia. The C. cassiicola isolates used in this study were collected from several states in Malaysia from 1998 to 2005. The morphology of the isolates was characteristic of that previously described for C. cassiicola. Variations in colony and conidial morphology were observed not only among isolates but also within a single isolate with no inclination to either clonal or geographical origin of the isolates. ISSR analysis delineated the isolates into two distinct clusters. The dendrogram created from UPGMA analysis based on Nei and Li's coefficient (calculated from the binary matrix data of 106 amplified DNA bands generated from 8 ISSR primers) showed that cluster 1 encompasses 12 isolates from the states of Johor and Selangor (this cluster was further split into 2 sub clusters (1A, 1B), sub cluster 1B consists of a unique isolate, CKT05D); while cluster 2 comprises of 9 isolates that were obtained from the other states. Detached leaf assay performed on selected Hevea clones showed that the pathogenicity of representative isolates from cluster 1 (with the exception of CKT05D) resembled that of race 1; and isolates in cluster 2 showed pathogenicity similar to race 2 of the fungus that was previously identified in Malaysia. The isolate CKT05D from sub cluster 1B showed pathogenicity dissimilar to either race 1 or race 2.

Multiple genotypes within aecial clusters in Puccinia graminis and Puccinia coronata: improved understanding of the biology of cereal rust fungi.

PubMed

Berlin, Anna; Samils, Berit; Andersson, Björn

2017-01-01

Cereal rust fungi ( Puccinia spp.) are among the most economically important plant pathogens. These fungi have a complex life cycle, including five spore stages and two hosts. They infect one grass host on which they reproduce clonally and cause the cereal rust diseases, while the alternate host is required for sexual reproduction. Although previous studies clearly demonstrate the importance of the alternate host in creating genetic diversity in cereal rust fungi, little is known about the amount of novel genotypes created in each successful completion of a sexual reproduction event. In this study, single sequence repeat markers were used to study the genotypic diversity within aecial clusters by genotyping individual aecial cups. Two common cereal rusts, Puccinia graminis causing stem rust and Puccinia coronata the causal agent of crown rust were investigated. We showed that under natural conditions, a single aecial cluster usually include several genotypes, either because a single pycnial cluster is fertilized by several different pycniospores, or because aecia within the cluster are derived from more than one fertilized adjoining pycnial cluster, or a combination of both. Our results imply that although sexual events in cereal rust fungi in most regions of the world are relatively rare, the events that occur may still significantly contribute to the genetic variation within the pathogen populations.

Fertility depression among cheese‐making Penicillium roqueforti strains suggests degeneration during domestication

PubMed Central

Ropars, Jeanne; Lo, Ying‐Chu; Dumas, Emilie; Snirc, Alodie; Begerow, Dominik; Rollnik, Tanja; Lacoste, Sandrine; Dupont, Joëlle; Giraud, Tatiana; López‐Villavicencio, Manuela

2016-01-01

Genetic differentiation occurs when gene flow is prevented, due to reproductive barriers or asexuality. Investigating the early barriers to gene flow is important for understanding the process of speciation. Here, we therefore investigated reproductive isolation between different genetic clusters of the fungus Penicillium roqueforti, used for maturing blue cheeses, and also occurring as food spoiler or in silage. We investigated premating and postmating fertility between and within three genetic clusters (two from cheese and one from other substrates), and we observed sexual structures under scanning electron microscopy. All intercluster types of crosses showed some fertility, suggesting that no intersterility has evolved between domesticated and wild populations despite adaptation to different environments and lack of gene flow. However, much lower fertility was found in crosses within the cheese clusters than within the noncheese cluster, suggesting reduced fertility of cheese strains, which may constitute a barrier to gene flow. Such degeneration may be due to bottlenecks during domestication and/or to the exclusive clonal replication of the strains in industry. This study shows that degeneration has occurred rapidly and independently in two lineages of a domesticated species. Altogether, these results inform on the processes and tempo of degeneration and speciation. PMID:27470007

Intraspecific genetic structure, divergence and high rates of clonality in an amphi-Atlantic starfish.

PubMed

Garcia-Cisneros, Alex; Palacín, Creu; Ventura, Carlos Renato Rezende; Feital, Barbara; Paiva, Paulo Cesar; Pérez-Portela, Rocío

2018-02-01

Intraspecific genetic diversity and divergence have a large influence on the adaption and evolutionary potential of species. The widely distributed starfish, Coscinasterias tenuispina, combines sexual reproduction with asexual reproduction via fission. Here we analyse the phylogeography of this starfish to reveal historical and contemporary processes driving its intraspecific genetic divergence. We further consider whether asexual reproduction is the most important method of propagation throughout the distribution range of this species. Our study included 326 individuals from 16 populations, covering most of the species' distribution range. A total of 12 nuclear microsatellite loci and sequences of the mitochondrial cytochrome c oxidase subunit I (COI) gene were analysed. COI and microsatellites were clustered in two isolated lineages: one found along the southwestern Atlantic and the other along the northeastern Atlantic and Mediterranean Sea. This suggests the existence of two different evolutionary units. Marine barriers along the European coast would be responsible for population clustering: the Almeria-Oran Front that limits the entrance of migrants from the Atlantic to the Mediterranean, and the Siculo-Tunisian strait that divides the two Mediterranean basins. The presence of identical genotypes was detected in all populations, although two monoclonal populations were found in two sites where annual mean temperatures and minimum values were the lowest. Our results based on microsatellite loci showed that intrapopulation genetic diversity was significantly affected by clonality whereas it had lower effect for the global phylogeography of the species, although still some impact on populations' genetic divergence could be observed between some populations. © 2017 John Wiley & Sons Ltd.

Telangiectatic focal nodular hyperplasia: a variant of hepatocellular adenoma.

PubMed

Paradis, Valerie; Benzekri, Asmae; Dargère, Delphine; Bièche, Ivan; Laurendeau, Ingrid; Vilgrain, Valerie; Belghiti, Jacques; Vidaud, Michel; Degott, Claude; Bedossa, Pierre

2004-05-01

"Telangiectatic focal nodular hyperplasia" designate atypical lesions considered as variants of focal nodular hyperplasia (FNH). However, because "telangiectatic FNH" share several morphologic patterns with hepatocellular adenomas, classification of such lesions deserve further clarification. Therefore, the aim of the present study was to reconsider the classification of telangiectatic FNH with the help of a molecular approach. Ten telangiectatic FNH, 6 typical FNH, and 6 hepatocellular adenomas were studied. DNA, RNA, and protein from each lesion were extracted. Clonality was assessed by the study of the X chromosome inactivation pattern (HUMARA assay). Angiopoietin (ANGPT-1 and ANGPT-2) mRNA, genes the expression of which is typically modified in FNH, were quantified by a real-time RT-PCR procedure. Protein profiles were analyzed by SELDI-TOF PROTEINCHIP (Cyphergen Biosystem, Inc., Fremont, CA) technology. Although all informative cases of FNH (5 of 6) and hepatocellular adenomas (6 of 6) were polyclonal and monoclonal, respectively, clonal analysis showed a nonrandom pattern of X chromosome inactivation consistent with a monoclonal lesion in 6 of 8 cases of telangiectatic FNH. The mean value of the ANGPT-1/ANGPT-2 mRNA ratio was 21.4 in FNH, 2.6 in adenomas, and 2.1 in telangiectatic FNH (P

Clustering of two genes putatively involved in cyanate detoxification evolved recently and independently in multiple fungal lineages.

PubMed

Elmore, M Holly; McGary, Kriston L; Wisecaver, Jennifer H; Slot, Jason C; Geiser, David M; Sink, Stacy; O'Donnell, Kerry; Rokas, Antonis

2015-02-06

Fungi that have the enzymes cyanase and carbonic anhydrase show a limited capacity to detoxify cyanate, a fungicide employed by both plants and humans. Here, we describe a novel two-gene cluster that comprises duplicated cyanase and carbonic anhydrase copies, which we name the CCA gene cluster, trace its evolution across Ascomycetes, and examine the evolutionary dynamics of its spread among lineages of the Fusarium oxysporum species complex (hereafter referred to as the FOSC), a cosmopolitan clade of purportedly clonal vascular wilt plant pathogens. Phylogenetic analysis of fungal cyanase and carbonic anhydrase genes reveals that the CCA gene cluster arose independently at least twice and is now present in three lineages, namely Cochliobolus lunatus, Oidiodendron maius, and the FOSC. Genome-wide surveys within the FOSC indicate that the CCA gene cluster varies in copy number across isolates, is always located on accessory chromosomes, and is absent in FOSC's closest relatives. Phylogenetic reconstruction of the CCA gene cluster in 163 FOSC strains from a wide variety of hosts suggests a recent history of rampant transfers between isolates. We hypothesize that the independent formation of the CCA gene cluster in different fungal lineages and its spread across FOSC strains may be associated with resistance to plant-produced cyanates or to use of cyanate fungicides in agriculture. © The Author(s) 2015. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

Selection of Phage Display Peptides Targeting Human Pluripotent Stem Cell-Derived Progenitor Cell Lines.

PubMed

Bignone, Paola A; Krupa, Rachel A; West, Michael D; Larocca, David

2016-01-01

The ability of human pluripotent stem cells (hPS) to both self-renew and differentiate into virtually any cell type makes them a promising source of cells for cell-based regenerative therapies. However, stem cell identity, purity, and scalability remain formidable challenges that need to be overcome for translation of pluripotent stem cell research into clinical applications. Directed differentiation from hPS cells is inefficient and residual contamination with pluripotent cells that have the potential to form tumors remains problematic. The derivation of scalable (self-renewing) embryonic progenitor stem cell lines offers a solution because they are well defined and clonally pure. Clonally pure progenitor stem cell lines also provide a means for identifying cell surface targeting reagents that are useful for identification, tracking, and repeated derivation of the corresponding progenitor stem cell types from additional hPS cell sources. Such stem cell targeting reagents can then be applied to the manufacture of genetically diverse banks of human embryonic progenitor cell lines for drug screening, disease modeling, and cell therapy. Here we present methods to identify human embryonic progenitor stem cell targeting peptides by selection of phage display libraries on clonal embryonic progenitor cell lines and demonstrate their use for targeting quantum dots (Qdots) for stem cell labeling.

Clonal analysis of lineage fate in native haematopoiesis.

PubMed

Rodriguez-Fraticelli, Alejo E; Wolock, Samuel L; Weinreb, Caleb S; Panero, Riccardo; Patel, Sachin H; Jankovic, Maja; Sun, Jianlong; Calogero, Raffaele A; Klein, Allon M; Camargo, Fernando D

2018-01-11

Haematopoiesis, the process of mature blood and immune cell production, is functionally organized as a hierarchy, with self-renewing haematopoietic stem cells and multipotent progenitor cells sitting at the very top. Multiple models have been proposed as to what the earliest lineage choices are in these primitive haematopoietic compartments, the cellular intermediates, and the resulting lineage trees that emerge from them. Given that the bulk of studies addressing lineage outcomes have been performed in the context of haematopoietic transplantation, current models of lineage branching are more likely to represent roadmaps of lineage potential than native fate. Here we use transposon tagging to clonally trace the fates of progenitors and stem cells in unperturbed haematopoiesis. Our results describe a distinct clonal roadmap in which the megakaryocyte lineage arises largely independently of other haematopoietic fates. Our data, combined with single-cell RNA sequencing, identify a functional hierarchy of unilineage- and oligolineage-producing clones within the multipotent progenitor population. Finally, our results demonstrate that traditionally defined long-term haematopoietic stem cells are a significant source of megakaryocyte-restricted progenitors, suggesting that the megakaryocyte lineage is the predominant native fate of long-term haematopoietic stem cells. Our study provides evidence for a substantially revised roadmap for unperturbed haematopoiesis, and highlights unique properties of multipotent progenitors and haematopoietic stem cells in situ.

Molecular epidemiology of clonal diploids: a quick overview and a short DIY (do it yourself) notice.

PubMed

De Meeûs, Thierry; Lehmann, Laurent; Balloux, François

2006-03-01

In this short review we report the basic notions needed for understanding the population genetics of clonal diploids. We focus on the consequences of clonality on the distribution of genetic diversity within individuals, between individuals and between populations. We then summarise how to detect clonality in mainly sexual populations, conversely, how to detect sexuality in mainly clonal populations and also how genetic differentiation between populations is affected by clonality in diploids. This information is then used for building recipes on how to analyse and interpret genetic polymorphism data in molecular epidemiology studies of clonal diploids.

CD3-CD4+ lymphoid variant of hypereosinophilic syndrome: nodal and extranodal histopathological and immunophenotypic features of a peripheral indolent clonal T-cell lymphoproliferative disorder.

PubMed

Lefèvre, Guillaume; Copin, Marie-Christine; Roumier, Christophe; Aubert, Hélène; Avenel-Audran, Martine; Grardel, Nathalie; Poulain, Stéphanie; Staumont-Sallé, Delphine; Seneschal, Julien; Salles, Gilles; Ghomari, Kamel; Terriou, Louis; Leclech, Christian; Morati-Hafsaoui, Chafika; Morschhauser, Franck; Lambotte, Olivier; Ackerman, Félix; Trauet, Jacques; Geffroy, Sandrine; Dumezy, Florent; Capron, Monique; Roche-Lestienne, Catherine; Taieb, Alain; Hatron, Pierre-Yves; Dubucquoi, Sylvain; Hachulla, Eric; Prin, Lionel; Labalette, Myriam; Launay, David; Preudhomme, Claude; Kahn, Jean-Emmanuel

2015-08-01

The CD3(-)CD4(+) lymphoid variant of hypereosinophilic syndrome is characterized by hypereosinophilia and clonal circulating CD3(-)CD4(+) T cells. Peripheral T-cell lymphoma has been described during this disease course, and we observed in our cohort of 23 patients 2 cases of angio-immunoblastic T-cell lymphoma. We focus here on histopathological (n=12 patients) and immunophenotypic (n=15) characteristics of CD3(-)CD4(+) lymphoid variant of hypereosinophilic syndrome. Atypical CD4(+) T cells lymphoid infiltrates were found in 10 of 12 CD3(-)CD4(+) L-HES patients, in lymph nodes (n=4 of 4 patients), in skin (n=9 of 9) and other extra-nodal tissues (gut, lacrymal gland, synovium). Lymph nodes displayed infiltrates limited to the interfollicular areas or even an effacement of nodal architecture, associated with proliferation of arborizing high endothelial venules and increased follicular dendritic cell meshwork. Analysis of 2 fresh skin samples confirmed the presence of CD3(-)CD4(+) T cells. Clonal T cells were detected in at least one tissue in 8 patients, including lymph nodes (n=4 of 4): the same clonal T cells were detected in blood and in at least one biopsy, with a maximum delay of 23 years between samples. In the majority of cases, circulating CD3(-)CD4(+) T cells were CD2(hi) (n=9 of 14), CD5(hi) (n=12 of 14), and CD7(-)(n=4 of 14) or CD7(low) (n=10 of 14). Angio-immunoblastic T-cell lymphoma can also present with CD3(-)CD4(+) T cells; despite other common histopathological and immunophenotypic features, CD10 expression and follicular helper T-cell markers were not detected in lymphoid variant of hypereosinophilic syndrome patients, except in both patients who developed angio-immunoblastic T-cell lymphoma, and only at T-cell lymphoma diagnosis. Taken together, persistence of tissular clonal T cells and histopathological features define CD3(-)CD4(+) lymphoid variant of hypereosinophilic syndrome as a peripheral indolent clonal T-cell lymphoproliferative disorder, which should not be confused with angio-immunoblastic T-cell lymphoma. Copyright© Ferrata Storti Foundation.

CD3−CD4+ lymphoid variant of hypereosinophilic syndrome: nodal and extranodal histopathological and immunophenotypic features of a peripheral indolent clonal T-cell lymphoproliferative disorder

PubMed Central

Lefèvre, Guillaume; Copin, Marie-Christine; Roumier, Christophe; Aubert, Hélène; Avenel-Audran, Martine; Grardel, Nathalie; Poulain, Stéphanie; Staumont-Sallé, Delphine; Seneschal, Julien; Salles, Gilles; Ghomari, Kamel; Terriou, Louis; Leclech, Christian; Morati-Hafsaoui, Chafika; Morschhauser, Franck; Lambotte, Olivier; Ackerman, Félix; Trauet, Jacques; Geffroy, Sandrine; Dumezy, Florent; Capron, Monique; Roche-Lestienne, Catherine; Taieb, Alain; Hatron, Pierre-Yves; Dubucquoi, Sylvain; Hachulla, Eric; Prin, Lionel; Labalette, Myriam; Launay, David; Preudhomme, Claude; Kahn, Jean-Emmanuel

2015-01-01

The CD3−CD4+ lymphoid variant of hypereosinophilic syndrome is characterized by hypereosinophilia and clonal circulating CD3−CD4+ T cells. Peripheral T-cell lymphoma has been described during this disease course, and we observed in our cohort of 23 patients 2 cases of angio-immunoblastic T-cell lymphoma. We focus here on histopathological (n=12 patients) and immunophenotypic (n=15) characteristics of CD3−CD4+ lymphoid variant of hypereosinophilic syndrome. Atypical CD4+ T cells lymphoid infiltrates were found in 10 of 12 CD3−CD4+ L-HES patients, in lymph nodes (n=4 of 4 patients), in skin (n=9 of 9) and other extra-nodal tissues (gut, lacrymal gland, synovium). Lymph nodes displayed infiltrates limited to the interfollicular areas or even an effacement of nodal architecture, associated with proliferation of arborizing high endothelial venules and increased follicular dendritic cell meshwork. Analysis of 2 fresh skin samples confirmed the presence of CD3−CD4+ T cells. Clonal T cells were detected in at least one tissue in 8 patients, including lymph nodes (n=4 of 4): the same clonal T cells were detected in blood and in at least one biopsy, with a maximum delay of 23 years between samples. In the majority of cases, circulating CD3−CD4+ T cells were CD2hi (n=9 of 14), CD5hi (n=12 of 14), and CD7−(n=4 of 14) or CD7low (n=10 of 14). Angio-immunoblastic T-cell lymphoma can also present with CD3−CD4+ T cells; despite other common histopathological and immunophenotypic features, CD10 expression and follicular helper T-cell markers were not detected in lymphoid variant of hypereosinophilic syndrome patients, except in both patients who developed angio-immunoblastic T-cell lymphoma, and only at T-cell lymphoma diagnosis. Taken together, persistence of tissular clonal T cells and histopathological features define CD3−CD4+ lymphoid variant of hypereosinophilic syndrome as a peripheral indolent clonal T-cell lymphoproliferative disorder, which should not be confused with angio-immunoblastic T-cell lymphoma. PMID:25682606

Clonal growth: invasion or stability? A comparative study of clonal architecture and diversity in native and introduced lineages of Phragmites australis (Poaceae).

PubMed

Douhovnikoff, Vladimir; Hazelton, Eric L G

2014-09-01

• The characteristics of clonal growth that are advantageous in invasive plants can also result in native plants' ability to resist invasion. In Maine, we compared the clonal architecture and diversity of an invasive lineage (introduced Phragmites) and a noninvasive lineage (native Phragmites) present in much of North America. This study is the first on stand-scale diversity using a sample size and systematic spatial-sampling scheme adequate for characterizing clonal structure in Phragmites. Our questions included: (1) Does the structure and extent of clonal growth suggest that the potential for clonal growth contributes to the invasiveness of the introduced lineage? (2) Is clonal growth common in the native lineage, acting as a possible source of ecological resistance and resilience?• Microsatellite markers were used to measure clonal sizes, architecture, and diversity within each lineage in stands within four marshes in Maine.• Clonal diversity measures indicated that clonal growth was significantly greater in stands of the native lineage than in the introduced. While lineage was a consistent predictor of clonal diversity relative ranking, the marsh location was a much stronger predictor of the absolute range of these values.• Our results indicate an important role for clonal growth in the space consolidation of native Phragmites and could explain why the introduced lineage, with stronger competitive traits, has not replaced the native where they co-occur. These results with regard to clone size, size distributions, singleton occurrence, and clonal architecture provide some evidence for stand development that follows a genotypic initial floristics model. © 2014 Botanical Society of America, Inc.

Recombinant transfer in the basic genome of E. coli

DOE PAGES

Dixit, Purushottam; Studier, F. William; Pang, Tin Yau; ...

2015-07-07

An approximation to the ~4-Mbp basic genome shared by 32 strains of E. coli representing six evolutionary groups has been derived and analyzed computationally. A multiple-alignment of the 32 complete genome sequences was filtered to remove mobile elements and identify the most reliable ~90% of the aligned length of each of the resulting 496 basic-genome pairs. Patterns of single bp mutations (SNPs) in aligned pairs distinguish clonally inherited regions from regions where either genome has acquired DNA fragments from diverged genomes by homologous recombination since their last common ancestor. Such recombinant transfer is pervasive across the basic genome, mostly betweenmore » genomes in the same evolutionary group, and generates many unique mosaic patterns. The six least-diverged genome-pairs have one or two recombinant transfers of length ~40–115 kbp (and few if any other transfers), each containing one or more gene clusters known to confer strong selective advantage in some environments. Moderately diverged genome pairs (0.4–1% SNPs) show mosaic patterns of interspersed clonal and recombinant regions of varying lengths throughout the basic genome, whereas more highly diverged pairs within an evolutionary group or pairs between evolutionary groups having >1.3% SNPs have few clonal matches longer than a few kbp. Many recombinant transfers appear to incorporate fragments of the entering DNA produced by restriction systems of the recipient cell. A simple computational model can closely fit the data. As a result, most recombinant transfers seem likely to be due to generalized transduction by co-evolving populations of phages, which could efficiently distribute variability throughout bacterial genomes.« less

Recombinant transfer in the basic genome of E. coli

DOE Office of Scientific and Technical Information (OSTI.GOV)

Dixit, Purushottam; Studier, F. William; Pang, Tin Yau

An approximation to the ~4-Mbp basic genome shared by 32 strains of E. coli representing six evolutionary groups has been derived and analyzed computationally. A multiple-alignment of the 32 complete genome sequences was filtered to remove mobile elements and identify the most reliable ~90% of the aligned length of each of the resulting 496 basic-genome pairs. Patterns of single bp mutations (SNPs) in aligned pairs distinguish clonally inherited regions from regions where either genome has acquired DNA fragments from diverged genomes by homologous recombination since their last common ancestor. Such recombinant transfer is pervasive across the basic genome, mostly betweenmore » genomes in the same evolutionary group, and generates many unique mosaic patterns. The six least-diverged genome-pairs have one or two recombinant transfers of length ~40–115 kbp (and few if any other transfers), each containing one or more gene clusters known to confer strong selective advantage in some environments. Moderately diverged genome pairs (0.4–1% SNPs) show mosaic patterns of interspersed clonal and recombinant regions of varying lengths throughout the basic genome, whereas more highly diverged pairs within an evolutionary group or pairs between evolutionary groups having >1.3% SNPs have few clonal matches longer than a few kbp. Many recombinant transfers appear to incorporate fragments of the entering DNA produced by restriction systems of the recipient cell. A simple computational model can closely fit the data. As a result, most recombinant transfers seem likely to be due to generalized transduction by co-evolving populations of phages, which could efficiently distribute variability throughout bacterial genomes.« less

Genomic Structural Variations Affecting Virulence During Clonal Expansion of Pseudomonas syringae pv. actinidiae Biovar 3 in Europe.

PubMed

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.

Cellular aging (the Hayflick limit) and species longevity: a unification model based on clonal succession.

PubMed

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.

Core genome conservation of Staphylococcus haemolyticus limits sequence based population structure analysis.

PubMed

Cavanagh, Jorunn Pauline; Klingenberg, Claus; Hanssen, Anne-Merethe; Fredheim, Elizabeth Aarag; Francois, Patrice; Schrenzel, Jacques; Flægstad, Trond; Sollid, Johanna Ericson

2012-06-01

The notoriously multi-resistant Staphylococcus haemolyticus is an emerging pathogen causing serious infections in immunocompromised patients. Defining the population structure is important to detect outbreaks and spread of antimicrobial resistant clones. Currently, the standard typing technique is pulsed-field gel electrophoresis (PFGE). In this study we describe novel molecular typing schemes for S. haemolyticus using multi locus sequence typing (MLST) and multi locus variable number of tandem repeats (VNTR) analysis. Seven housekeeping genes (MLST) and five VNTR loci (MLVF) were selected for the novel typing schemes. A panel of 45 human and veterinary S. haemolyticus isolates was investigated. The collection had diverse PFGE patterns (38 PFGE types) and was sampled over a 20 year-period from eight countries. MLST resolved 17 sequence types (Simpsons index of diversity [SID]=0.877) and MLVF resolved 14 repeat types (SID=0.831). We found a low sequence diversity. Phylogenetic analysis clustered the isolates in three (MLST) and one (MLVF) clonal complexes, respectively. Taken together, neither the MLST nor the MLVF scheme was suitable to resolve the population structure of this S. haemolyticus collection. Future MLVF and MLST schemes will benefit from addition of more variable core genome sequences identified by comparing different fully sequenced S. haemolyticus genomes. Copyright © 2012 Elsevier B.V. All rights reserved.

Molecular evidence of Burkholderia pseudomallei genotypes based on geographical distribution.

PubMed

Zulkefli, Noorfatin Jihan; Mariappan, Vanitha; Vellasamy, Kumutha Malar; Chong, Chun Wie; Thong, Kwai Lin; Ponnampalavanar, Sasheela; Vadivelu, Jamuna; Teh, Cindy Shuan Ju

2016-01-01

Background. Central intermediary metabolism (CIM) in bacteria is defined as a set of metabolic biochemical reactions within a cell, which is essential for the cell to survive in response to environmental perturbations. The genes associated with CIM are commonly found in both pathogenic and non-pathogenic strains. As these genes are involved in vital metabolic processes of bacteria, we explored the efficiency of the genes in genotypic characterization of Burkholderia pseudomallei isolates, compared with the established pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST) schemes. Methods. Nine previously sequenced B. pseudomallei isolates from Malaysia were characterized by PFGE, MLST and CIM genes. The isolates were later compared to the other 39 B. pseudomallei strains, retrieved from GenBank using both MLST and sequence analysis of CIM genes. UniFrac and hierachical clustering analyses were performed using the results generated by both MLST and sequence analysis of CIM genes. Results. Genetic relatedness of nine Malaysian B. pseudomallei isolates and the other 39 strains was investigated. The nine Malaysian isolates were subtyped into six PFGE profiles, four MLST profiles and five sequence types based on CIM genes alignment. All methods demonstrated the clonality of OB and CB as well as CMS and THE. However, PFGE showed less than 70% similarity between a pair of morphology variants, OS and OB. In contrast, OS was identical to the soil isolate, MARAN. To have a better understanding of the genetic diversity of B. pseudomallei worldwide, we further aligned the sequences of genes used in MLST and genes associated with CIM for the nine Malaysian isolates and 39 B. pseudomallei strains from NCBI database. Overall, based on the CIM genes, the strains were subtyped into 33 profiles where majority of the strains from Asian countries were clustered together. On the other hand, MLST resolved the isolates into 31 profiles which formed three clusters. Hierarchical clustering using UniFrac distance suggested that the isolates from Australia were genetically distinct from the Asian isolates. Nevertheless, statistical significant differences were detected between isolates from Malaysia, Thailand and Australia. Discussion. Overall, PFGE showed higher discriminative power in clustering the nine Malaysian B. pseudomallei isolates and indicated its suitability for localized epidemiological study. Compared to MLST, CIM genes showed higher resolution in distinguishing those non-related strains and better clustering of strains from different geographical regions. A closer genetic relatedness of Malaysian isolates with all Asian strains in comparison to Australian strains was observed. This finding was supported by UniFrac analysis which resulted in geographical segregation between Australia and the Asian countries.

Variability of Cutaneous Leishmaniasis Lesions Is Not Associated with Genetic Diversity of Leishmania tropica in Khyber Pakhtunkhwa Province of Pakistan.

PubMed

Khan, Nazma Habib; Llewellyn, Martin S; Schönian, Gabriele; Sutherland, Colin J

2017-11-01

Leishmania tropica is the causative agent of cutaneous leishmaniasis in Pakistan. Here, intraspecific diversity of L. tropica from northern Pakistan was investigated using multilocus microsatellite typing. Fourteen polymorphic microsatellite markers were typed in 34 recently collected L. tropica isolates from Pakistan along with 158 archival strains of diverse Afro-Eurasian origins. Previously published profiles for 145 strains of L. tropica originating from different regions of Africa, Central Asia, Iran, and Middle East were included for comparison. Six consistently well-supported genetic groups were resolved: 1) Asia, 2) Morroco A, 3) Namibia and Kenya A, 4) Kenya B/Tunisia and Galilee, 5) Morocco B, and 6) Middle East. Strains from northern Pakistan were assigned to Asian cluster except for three that were placed in a geographically distant genetic group; Morocco A. Lesion variability among these Pakistani strains was not associated with specific L. tropica genetic profile. Pakistani strains showed little genetic differentiation from strains of Iraq, Afghanistan, and Syria (F ST = 0.00-0.06); displayed evidence of modest genetic flow with India (F ST = 0.14). Furthermore, genetic structuring within these isolates was not geographically defined. Pak-Afghan cluster was in significant linkage disequilibrium (I A = 1.43), had low genetic diversity, and displayed comparatively higher heterozygosity (F IS = -0.62). Patterns of genetic diversity observed suggest dominance of a minimally diverse clonal lineage within northern Pakistan. This is surprising as a wide clinical spectrum was observed in patients, suggesting the importance of host and other factors. Further genotyping studies of L. tropica isolates displaying different clinical phenotypes are required to validate this potentially important observation.

Prevalence and characterisation of Staphylococcus aureus causing community-acquired skin and soft tissue infections on Java and Bali, Indonesia.

PubMed

Santosaningsih, Dewi; Santoso, Sanarto; Setijowati, Nanik; Rasyid, Harun A; Budayanti, Nyoman S; Suata, Ketut; Widhyatmoko, Dicky B; Purwono, Priyo B; Kuntaman, Kuntaman; Damayanti, Damayanti; Prakoeswa, Cita R S; Laurens, Mitchell; van Nierop, Josephine W I; Nanninga, Geraldine L; Oudenes, Neline; de Regt, Michelle; Snijders, Susan V; Verbrugh, Henri A; Severin, Juliëtte A

2018-01-01

To define the role of Staphylococcus aureus in community settings among patients with skin and soft tissue infections (SSTI) in Indonesia. Staphylococcus aureus were cultured from anterior nares, throat and wounds of 567 ambulatory patients presenting with SSTI. The mecA gene and genes encoding Panton-Valentine leukocidin (PVL; lukF-PV and lukS-PV) and exfoliative toxin (ET; eta and etb) were determined by PCR. Clonal relatedness among methicillin-resistant S. aureus (MRSA) and PVL-positive S. aureus was analysed using multilocus variable-number tandem-repeat analysis (MLVA) typing, and multilocus sequence typing (MLST) for a subset of isolates. Staphylococcal cassette chromosome mec (SCCmec) was determined for all MRSA isolates. Moreover, determinants for S. aureus SSTI, and PVL/ET-positive vs PVL/ET-negative S. aureus were assessed. Staphylococcus aureus were isolated from SSTI wounds of 257 (45.3%) patients, eight (3.1%) of these were MRSA. Genes encoding PVL and ETs were detected in 21.8% and 17.5% of methicillin-susceptible S. aureus (MSSA), respectively. PVL-positive MRSA was not detected. Nasopharyngeal S. aureus carriage was an independent determinant for S. aureus SSTI (odds ratio [OR] 1.8). Primary skin infection (OR 5.4) and previous antibiotic therapy (OR 3.5) were associated with PVL-positive MSSA. Primary skin infection (OR 2.2) was the only factor associated with ET-positive MSSA. MLVA typing revealed two more prevalent MSSA clusters. One ST1-MRSA-SCCmec type IV isolate and a cluster of ST239-MRSA-SCCmec type III were found. Community-acquired SSTI in Indonesia was frequently caused by PVL-positive MSSA, and the hospital-associated ST239-MRSA may have spread from the hospital into the community. © 2017 John Wiley & Sons Ltd.

Complex Routes of Nosocomial Vancomycin-Resistant Enterococcus faecium Transmission Revealed by Genome Sequencing.

PubMed

Raven, Kathy E; Gouliouris, Theodore; Brodrick, Hayley; Coll, Francesc; Brown, Nicholas M; Reynolds, Rosy; Reuter, Sandra; Török, M Estée; Parkhill, Julian; Peacock, Sharon J

2017-04-01

Vancomycin-resistant Enterococcus faecium (VREfm) is a leading cause of nosocomial infection. Here, we describe the utility of whole-genome sequencing in defining nosocomial VREfm transmission. A retrospective study at a single hospital in the United Kingdom identified 342 patients with E. faecium bloodstream infection over 7 years. Of these, 293 patients had a stored isolate and formed the basis for the study. The first stored isolate from each case was sequenced (200 VREfm [197 vanA, 2 vanB, and 1 isolate containing both vanA and vanB], 93 vancomycin-susceptible E. faecium) and epidemiological data were collected. Genomes were also available for E. faecium associated with bloodstream infections in 15 patients in neighboring hospitals, and 456 patients across the United Kingdom and Ireland. The majority of infections in the 293 patients were hospital-acquired (n = 249) or healthcare-associated (n = 42). Phylogenetic analysis showed that 291 of 293 isolates resided in a hospital-associated clade that contained numerous discrete clusters of closely related isolates, indicative of multiple introductions into the hospital followed by clonal expansion associated with transmission. Fine-scale analysis of 6 exemplar phylogenetic clusters containing isolates from 93 patients (32%) identified complex transmission routes that spanned numerous wards and years, extending beyond the detection of conventional infection control. These contained both vancomycin-resistant and -susceptible isolates. We also identified closely related isolates from patients at Cambridge University Hospitals NHS Foundation Trust and regional and national hospitals, suggesting interhospital transmission. These findings provide important insights for infection control practice and signpost areas for interventions. We conclude that sequencing represents a powerful tool for the enhanced surveillance and control of nosocomial E. faecium transmission and infection. © The Author 2017. Published by Oxford University Press for the Infectious Diseases Society of America.

Capsular profiling of the Cronobacter genus and the association of specific Cronobacter sakazakii and C. malonaticus capsule types with neonatal meningitis and necrotizing enterocolitis.

PubMed

Ogrodzki, P; Forsythe, S

2015-10-08

Cronobacter sakazakii and C. malonaticus can cause serious diseases especially in infants where they are associated with rare but fatal neonatal infections such as meningitis and necrotising enterocolitis. This study used 104 whole genome sequenced strains, covering all seven species in the genus, to analyse capsule associated clusters of genes involved in the biosynthesis of the O-antigen, colanic acid, bacterial cellulose, enterobacterial common antigen (ECA), and a previously uncharacterised K-antigen. Phylogeny of the gnd and galF genes flanking the O-antigen region enabled the defining of 38 subgroups which are potential serotypes. Two variants of the colanic acid synthesis gene cluster (CA1 and CA2) were found which differed with the absence of galE in CA2. Cellulose (bcs genes) were present in all species, but were absent in C. sakazakii sequence type (ST) 13 and clonal complex (CC) 100 strains. The ECA locus was found in all strains. The K-antigen capsular polysaccharide Region 1 (kpsEDCS) and Region 3 (kpsMT) genes were found in all Cronobacter strains. The highly variable Region 2 genes were assigned to 2 homology groups (K1 and K2). C. sakazakii and C. malonaticus isolates with capsular type [K2:CA2:Cell(+)] were associated with neonatal meningitis and necrotizing enterocolitis. Other capsular types were less associated with clinical infections. This study proposes a new capsular typing scheme which identifies a possible important virulence trait associated with severe neonatal infections. The various capsular polysaccharide structures warrant further investigation as they could be relevant to macrophage survival, desiccation resistance, environmental survival, and biofilm formation in the hospital environment, including neonatal enteral feeding tubes.

Defining the Role of the Environment in the Emergence and Persistence of vanA Vancomycin-Resistant Enterococcus (VRE) in an Intensive Care Unit: A Molecular Epidemiological Study.

PubMed

Lee, Andie S; White, Elizabeth; Monahan, Leigh G; Jensen, Slade O; Chan, Raymond; van Hal, Sebastiaan J

2018-06-01

OBJECTIVETo describe the transmission dynamics of the emergence and persistence of vanA vancomycin-resistant enterococcus (VRE) in an intensive care unit (ICU) using whole-genome sequencing of patient and environmental isolates.DESIGNRetrospective cohort study.SETTINGICU in a tertiary referral center.PARTICIPANTSPatients admitted to the ICU over an 11-month period.METHODS VanA VRE isolated from patients (n=31) were sequenced using the Illumina MiSeq platform. Environmental samples from bed spaces, equipment, and waste rooms were collected. All vanA VRE-positive environmental samples (n=14) were also sequenced. Data were collected regarding patient ward and bed movements.RESULTSThe 31 patient vanA VRE isolates were from screening (n=19), urine (n=4), bloodstream (n=3), skin/wound (n=3), and intra-abdominal (n=2) sources. The phylogeny from sequencing data confirmed several VRE clusters, with 1 group accounting for 38 of 45 isolates (84%). Within this cluster, cross-transmission was extensive and complex across the ICU. Directionality indicated that colonized patients contaminated environmental sites. Similarly, environmental sources not only led to patient colonization but also to infection. Notably, shared equipment acted as a conduit for transmission between different ICU areas. Infected patients, however, were not linked to further VRE transmission.CONCLUSIONSGenomic sequencing confirmed a predominantly clonal outbreak of VRE with complex transmission dynamics. The environmental reservoir, particularly from shared equipment, played a key role in ongoing VRE spread. This study provides evidence to support the use of multifaceted strategies, with an emphasis on measures to reduce bacterial burden in the environment, for successful VRE control.Infect Control Hosp Epidemiol 2018;39:668-675.

Spread of Plasmids Carrying Multiple GES Variants

PubMed Central

Cuzon, Gaelle; Bogaerts, Pierre; Bauraing, Caroline; Huang, Te-Din; Glupczynski, Youri

2016-01-01

Five GES-producing Enterobacteriaceae isolates that displayed an extended-spectrum β-lactamase (ESBL) phenotype harbored two GES variants: GES-7 ESBL and GES-6 carbapenemase. In all isolates, the two GES alleles were located on the same integron that was inserted into an 80-kb IncM1 self-conjugative plasmid. Whole-genome sequencing suggested in vivo horizontal gene transfer of the plasmid along with clonal diffusion of Enterobacter cloacae. To our knowledge, this is the first description in Europe of clustered Enterobacteriaceae isolates carrying two GES β-lactamases, of which one has extended activity toward carbapenems. PMID:27216071

VEB-1 Extended-Spectrum β-lactamase–producing Acinetobacter baumannii, France1

PubMed Central

Coignard, Bruno; Carbonne, Anne; Blanckaert, Karine; Bajolet, Odile; Bernet, Claude; Verdeil, Xavier; Astagneau, Pascal; Desenclos, Jean-Claude; Nordmann, Patrice

2006-01-01

VEB-1 extended-spectrum β-lactamase–producing Acinetobacter baumannii was responsible for an outbreak in hospitals in France. A national alert was triggered in September 2003 when 4 hospitals reported clusters of A. baumannii infection with similar susceptibility profiles. Case definitions and laboratory guidelines were disseminated, and prospective surveillance was implemented; strains were sent to a single laboratory for characterization and typing. From April 2003 through June 2004, 53 hospitals reported 290 cases of A. baumannii infection or colonization; 275 isolates were blaVEB-1-positive and clonally related. Cases were first reported in 5 districts of northern France, then in 10 other districts in 4 regions. Within a region, interhospital spread was associated with patient transfer. In northern France, investigation and control measures led to a reduction of reported cases after January 2004. The national alert enabled early control of new clusters, demonstrating the usefulness of early warning about antimicrobial drug resistance. PMID:16965700

Serotypes and Clonal Diversity of Streptococcus pneumoniae Causing Invasive Disease in the Era of PCV13 in Catalonia, Spain

PubMed Central

del Amo, Eva; Esteva, Cristina; Hernandez-Bou, Susanna; Galles, Carmen; Navarro, Marian; Sauca, Goretti; Diaz, Alvaro; Gassiot, Paula; Marti, Carmina; Larrosa, Nieves; Ciruela, Pilar; Jane, Mireia; Sá-Leão, Raquel; Muñoz-Almagro, Carmen

2016-01-01

The aim of this study was to study the serotypes and clonal diversity of pneumococci causing invasive pneumococcal disease in Catalonia, Spain, in the era of 13-valent pneumococcal conjugate vaccine (PCV13). In our region, this vaccine is only available in the private market and it is estimated a PCV13 vaccine coverage around 55% in children. A total of 1551 pneumococcal invasive isolates received between 2010 and 2013 in the Molecular Microbiology Department at Hospital Sant Joan de Déu, Barcelona, were included. Fifty-two serotypes and 249 clonal types—defined by MLST—were identified. The most common serotypes were serotype 1 (n = 182; 11.7%), 3 (n = 145; 9.3%), 19A (n = 137; 8.8%) and 7F (n = 122; 7.9%). Serotype 14 was the third most frequent serotype in children < 2 years (15 of 159 isolates). PCV7 serotypes maintained their proportion along the period of study, 16.6% in 2010 to 13.4% in 2013, whereas there was a significant proportional decrease in PCV13 serotypes, 65.3% in 2010 to 48.9% in 2013 (p<0.01). This decrease was mainly attributable to serotypes 19A and 7F. Serotype 12F achieved the third position in 2013 (n = 22, 6.4%). The most frequent clonal types found were ST306 (n = 154, 9.9%), ST191 (n = 111, 7.2%), ST989 (n = 85, 5.5%) and ST180 (n = 80, 5.2%). Despite their decrease, PCV13 serotypes continue to be a major cause of disease in Spain. These results emphasize the need for complete PCV13 vaccination. PMID:26953887

Ecological Consequences of Clonal Integration in Plants

PubMed Central

Liu, Fenghong; Liu, Jian; Dong, Ming

2016-01-01

Clonal plants are widespread throughout the plant kingdom and dominate in diverse habitats. Spatiotemporal heterogeneity of environment is pervasive at multiple scales, even at scales relevant to individual plants. Clonal integration refers to resource translocation and information communication among the ramets of clonal plants. Due to clonal integration, clonal plant species possess a series of peculiar attributes: plasticity in response to local and non-local conditions, labor division with organ specialization for acquiring locally abundant resources, foraging behavior by selective placement of ramets in resource-rich microhabitats, and avoidance of intraclonal competition. Clonal integration has very profound ecological consequences for clonal plants. It allows them to efficiently cope with environmental heterogeneity, by alleviating local resource shortages, buffering environmental stresses and disturbances, influencing competitive ability, increasing invasiveness, and altering species composition and invasibility at the community level. In this paper, we present a comprehensive review of research on the ecological consequences of plant clonal integration based on a large body of literature. We also attempt to propose perspectives for future research. PMID:27446093

United we stand, divided we fall: a meta-analysis of experiments on clonal integration and its relationship to invasiveness.

PubMed

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

2013-02-01

Many ecosystems are dominated by clonal plants. Among the most distinctive characteristics of clonal plants is their potential for clonal integration (i.e. the translocation of resources between interconnected ramets), suggesting that integration may play a role in their success. However, a general synthesis of effects of clonal integration on plant performance is lacking. We conducted a meta-analysis on the effects of clonal integration on biomass production and asexual reproduction of the whole clone, the recipient part (i.e. the part of a clone that imports resources) and the donor part (i.e. the part of a clone that exports resources). The final dataset contained 389 effect sizes from 84 studies covering 57 taxa. Overall, clonal integration increased performance of recipient parts without decreasing that of donor parts, and thus increased performance of whole clones. Among the studies and taxa considered, the benefits of clonal integration did not differ between two types of experimental approaches, between stoloniferous and rhizomatous growth forms, between directions of resource translocation (from younger to older ramet or vice versa), or among types of translocated resources (water, nutrients and carbohydrates). Clonal taxa with larger benefits of integration on whole-clone performance were not more invasive globally, but taxa in which recipient parts in unfavorable patches benefited more from integration were. Our results demonstrate general performance benefits of clonal integration, at least in the short term, and suggest that clonal integration contributes to the success of clonal plants.

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

PubMed

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.

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

PubMed

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 reserved.

Disturbance Is an Important Driver of Clonal Richness in Tropical Seagrasses

PubMed Central

McMahon, Kathryn M.; Evans, Richard D.; van Dijk, Kor-jent; Hernawan, Udhi; Kendrick, Gary A.; Lavery, Paul S.; Lowe, Ryan; Puotinen, Marji; Waycott, Michelle

2017-01-01

Clonality is common in many aquatic plant species, including seagrasses, where populations are maintained through a combination of asexual and sexual reproduction. One common measure used to describe the clonal structure of populations is clonal richness. Clonal richness is strongly dependent on the biological characteristics of the species, and how these interact with the environment but can also reflect evolutionary scale processes especially at the edge of species ranges. However, little is known about the spatial patterns and drivers of clonal richness in tropical seagrasses. This study assessed the spatial patterns of clonal richness in meadows of three tropical seagrass species, Thalassia hemprichii, Halodule uninervis, and Halophila ovalis, spanning a range of life-history strategies and spatial scales (2.5–4,711 km) in Indonesia and NW Australia. We further investigated the drivers of clonal richness using general additive mixed models for two of the species, H. uninervis and H. ovalis, over 8° latitude. No significant patterns were observed in clonal richness with latitude, yet disturbance combined with sea surface temperature strongly predicted spatial patterns of clonal richness. Sites with a high probability of cyclone disturbance had low clonal richness, whereas an intermediate probability of cyclone disturbance and the presence of dugong grazing combined with higher sea surface temperatures resulted in higher levels of clonal richness. We propose potential mechanisms for these patterns related to the recruitment and mortality rates of individuals as well as reproductive effort. Under a changing climate, increased severity of tropical cyclones and the decline in populations of mega-grazers have the potential to reduce clonal richness leading to less genetically diverse populations. PMID:29259609

Analysis of the mutations induced by conazole fungicides in vivo.

PubMed

Ross, Jeffrey A; Leavitt, Sharon A

2010-05-01

The mouse liver tumorigenic conazole fungicides triadimefon and propiconazole have previously been shown to be in vivo mouse liver mutagens in the Big Blue transgenic mutation assay when administered in feed at tumorigenic doses, whereas the non-tumorigenic conazole myclobutanil was not mutagenic. DNA sequencing of the mutants recovered from each treatment group as well as from animals receiving control diet was conducted to gain additional insight into the mode of action by which tumorigenic conazoles induce mutations. Relative dinucleotide mutabilities (RDMs) were calculated for each possible dinucleotide in each treatment group and then examined by multivariate statistical analysis techniques. Unsupervised hierarchical clustering analysis of RDM values segregated two independent control groups together, along with the non-tumorigen myclobutanil. The two tumorigenic conazoles clustered together in a distinct grouping. Partitioning around mediods of RDM values into two clusters also groups the triadimefon and propiconazole together in one cluster and the two control groups and myclobutanil together in a second cluster. Principal component analysis of these results identifies two components that account for 88.3% of the variability in the points. Taken together, these results are consistent with the hypothesis that propiconazole- and triadimefon-induced mutations do not represent clonal expansion of background mutations and support the hypothesis that they arise from the accumulation of reactive electrophilic metabolic intermediates within the liver in vivo.

Particular Distribution of Enterobacter cloacae Strains Isolated from Urinary Tract Infection within Clonal Complexes.

PubMed

Akbari, Majid; Bakhshi, Bita; Najar Peerayeh, Shahin

2016-01-01

Based on biochemical properties, Enterobacter cloacae represents a large complex of at least 13 variant species, subspecies, and genotypes that progressively identified as the most species causing hospital-acquired infections. The aim of this study was to determine the relevance between phylogenetically related strains within the E. cloacae complex and the frequency of urinary tract infection caused by them. A 268-bp fragment was obtained from hsp60 gene for 50 clinical E. cloacae isolates from urine cultures of inpatients that admitted to six hospitals in Tehran, Iran during December 2012 to November 2013. The 107 nucleotide sequences were analyzed and the evolutionary distances of sequences were computed and neighbor-joining tree was calculated. It showed that all of the genetic clusters have not an equal involvement in pathogenesis of urinary tract infections. Three superior clusters were found, together representing more than two third (80%) of the isolates (cluster VI with 25 members; clusters III and VIII with 9 and 6 members, respectively) and some genetic clusters were absent (IV, X, XII, and xiii), some of which are supposed to be associated with plants and no human infection has been reported. This study, for the first time, reports the unequal contribution of E. cloacae complex subspecies and clusters in urinary tract infections in Iran and together with studies from other countries suggest that the subspecies of E.hormaechei subsp. Oharae is the most prevalent E. cloacae complex subspecies regardless of country under study.

Genetic and morphological characterization of Cladobotryum species causing cobweb disease of mushrooms.

PubMed

McKay, G J; Egan, D; Morris, E; Scott, C; Brown, A E

1999-02-01

Cladobotryum dendroides (= Dactylium dendroides) has hitherto been regarded as the major causal agent of cobweb disease of the cultivated mushroom, Agaricus bisporus. Nucleotide sequence data for the internal transcribed spacer (ITS) regions of four Cladobotryum/Hypomyces species reported to be associated with cobweb disease, however, indicate that the most common pathogen is now C. mycophilum. This cobweb pathogen varies somewhat in conidial septation from published descriptions of C. mycophilum and lacks the distinctive colony odor. ITS sequencing revealed minor nucleotide variation which split isolates of the pathogen into three subgroups, two comprising isolates that were sensitive to methylbenzimidazole carbamate (MBC) fungicides and one comprising MBC-resistant isolates. The MBC-resistant isolates, which were only obtained from Ireland and Great Britain, clustered together strongly in randomly amplified polymorphic DNA (RAPD) PCR analysis, suggesting that they may be clonal. The MBC-sensitive isolates were more diverse. A RAPD fragment of 800 to 900 bp, containing a microsatellite and found in the MBC-resistant isolates, also indicated their clonal nature; the microsatellites of these isolates contained the same number of GA repeats. Smaller, polymorphic microsatellites, similarly comprising GA repeats, in the MBC-sensitive isolates in general correlated with their geographic origin.

Genetic and Morphological Characterization of Cladobotryum Species Causing Cobweb Disease of Mushrooms

PubMed Central

McKay, Gareth J.; Egan, Damian; Morris, Elizabeth; Scott, Carol; Brown, Averil E.

1999-01-01

Cladobotryum dendroides (= Dactylium dendroides) has hitherto been regarded as the major causal agent of cobweb disease of the cultivated mushroom, Agaricus bisporus. Nucleotide sequence data for the internal transcribed spacer (ITS) regions of four Cladobotryum/Hypomyces species reported to be associated with cobweb disease, however, indicate that the most common pathogen is now C. mycophilum. This cobweb pathogen varies somewhat in conidial septation from published descriptions of C. mycophilum and lacks the distinctive colony odor. ITS sequencing revealed minor nucleotide variation which split isolates of the pathogen into three subgroups, two comprising isolates that were sensitive to methylbenzimidazole carbamate (MBC) fungicides and one comprising MBC-resistant isolates. The MBC-resistant isolates, which were only obtained from Ireland and Great Britain, clustered together strongly in randomly amplified polymorphic DNA (RAPD) PCR analysis, suggesting that they may be clonal. The MBC-sensitive isolates were more diverse. A RAPD fragment of 800 to 900 bp, containing a microsatellite and found in the MBC-resistant isolates, also indicated their clonal nature; the microsatellites of these isolates contained the same number of GA repeats. Smaller, polymorphic microsatellites, similarly comprising GA repeats, in the MBC-sensitive isolates in general correlated with their geographic origin. PMID:9925589

Five Genes Encoding Surface-Exposed LPXTG Proteins Are Enriched in Hospital-Adapted Enterococcus faecium Clonal Complex 17 Isolates▿

PubMed Central

Hendrickx, Antoni P. A.; van Wamel, Willem J. B.; Posthuma, George; Bonten, Marc J. M.; Willems, Rob J. L.

2007-01-01

Most Enterococcus faecium isolates associated with hospital outbreaks and invasive infections belong to a distinct genetic subpopulation called clonal complex 17 (CC17). It has been postulated that the genetic evolution of CC17 involves the acquisition of various genes involved in antibiotic resistance, metabolic pathways, and virulence. To gain insight into additional genes that may have favored the rapid emergence of this nosocomial pathogen, we aimed to identify surface-exposed LPXTG cell wall-anchored proteins (CWAPs) specifically enriched in CC17 E. faecium. Using PCR and Southern and dot blot hybridizations, 131 E. faecium isolates (40 CC17 and 91 non-CC17) were screened for the presence of 22 putative CWAP genes identified from the E. faecium TX0016 genome. Five genes encoding LPXTG surface proteins were specifically enriched in E. faecium CC17 isolates. These five LPXTG surface protein genes were found in 28 to 40 (70 to 100%) of CC17 and in only 7 to 24 (8 to 26%) of non-CC17 isolates (P < 0.05). Three of these CWAP genes clustered together on the E. faecium TX0016 genome, which may comprise a novel enterococcal pathogenicity island covering E. faecium contig 609. Expression at the mRNA level was demonstrated, and immunotransmission electron microscopy revealed an association of the five LPXTG surface proteins with the cell wall. Minimal spanning tree analysis based on the presence and absence of 22 CWAP genes revealed grouping of all 40 CC17 strains together with 18 hospital-derived but evolutionary unrelated non-CC17 isolates in a distinct CWAP-enriched cluster, suggesting horizontal transfer of CWAP genes and a role of these CWAPs in hospital adaptation. PMID:17873043

Group B streptococci causing neonatal infections in barcelona are a stable clonal population: 18-year surveillance.

PubMed

Martins, E R; Andreu, A; Correia, P; Juncosa, T; Bosch, J; Ramirez, M; Melo-Cristino, J

2011-08-01

We analyzed 212 group B streptococci (GBS) from newborns with invasive infections in the area of Barcelona, Spain, between 1992 and 2009, with the aim of documenting changes in the prevalences of serotypes, antimicrobial resistance, and genetic lineages and evaluating their associations with either early-onset disease (EOD) or late-onset disease (LOD). Serotypes III (n = 118) and Ia (n = 47) together accounted for nearly 78% of the isolates. All isolates carried an alpha or alpha-like protein gene, and specific associations between genes and serotypes, such as serotype Ib and bca, serotype II and bca, serotype III and rib, and serotype V and alp3, reflected the presence of particular genetic lineages. Macrolide resistance (14.2%) was significantly associated with serotype V. Pulsed-field gel electrophoresis (PFGE) clustering was an excellent predictor of serotype and antibiotic resistance. The combination of PFGE and multilocus sequence typing revealed a large number of genetically distinct lineages. Still, specific lineages were dominant in our collection, particularly the serotype III/ST17/rib lineage, which had enhanced potential to cause LOD. Serotype Ia was concentrated in a single PFGE cluster composed of two genetic lineages: ST23/eps and ST24/bca. The ST24/bca sublineage of serotype Ia, which is found infrequently elsewhere, may be emerging as an important cause of neonatal invasive infections in the Mediterranean region. In spite of the introduction of prophylaxis, resulting in a pronounced decline in the frequency of EOD, the study revealed a remarkably stable clonal structure of GBS causing neonatal infections in Barcelona over a period of 18 years.

Group B Streptococci Causing Neonatal Infections in Barcelona Are a Stable Clonal Population: 18-Year Surveillance▿

PubMed Central

Martins, E. R.; Andreu, A.; Correia, P.; Juncosa, T.; Bosch, J.; Ramirez, M.; Melo-Cristino, J.

2011-01-01

We analyzed 212 group B streptococci (GBS) from newborns with invasive infections in the area of Barcelona, Spain, between 1992 and 2009, with the aim of documenting changes in the prevalences of serotypes, antimicrobial resistance, and genetic lineages and evaluating their associations with either early-onset disease (EOD) or late-onset disease (LOD). Serotypes III (n = 118) and Ia (n = 47) together accounted for nearly 78% of the isolates. All isolates carried an alpha or alpha-like protein gene, and specific associations between genes and serotypes, such as serotype Ib and bca, serotype II and bca, serotype III and rib, and serotype V and alp3, reflected the presence of particular genetic lineages. Macrolide resistance (14.2%) was significantly associated with serotype V. Pulsed-field gel electrophoresis (PFGE) clustering was an excellent predictor of serotype and antibiotic resistance. The combination of PFGE and multilocus sequence typing revealed a large number of genetically distinct lineages. Still, specific lineages were dominant in our collection, particularly the serotype III/ST17/rib lineage, which had enhanced potential to cause LOD. Serotype Ia was concentrated in a single PFGE cluster composed of two genetic lineages: ST23/eps and ST24/bca. The ST24/bca sublineage of serotype Ia, which is found infrequently elsewhere, may be emerging as an important cause of neonatal invasive infections in the Mediterranean region. In spite of the introduction of prophylaxis, resulting in a pronounced decline in the frequency of EOD, the study revealed a remarkably stable clonal structure of GBS causing neonatal infections in Barcelona over a period of 18 years. PMID:21697333

Molecular Typing and Virulence Gene Profiles of Enterotoxin Gene Cluster (egc)-Positive Staphylococcus aureus Isolates Obtained from Various Food and Clinical Specimens.

PubMed

Song, Minghui; Shi, Chunlei; Xu, Xuebing; Shi, Xianming

2016-11-01

The enterotoxin gene cluster (egc) has been proposed to contribute to the Staphylococcus aureus colonization, which highlights the need to evaluate genetic diversity and virulence gene profiles of the egc-positive population. Here, a total of 43 egc-positive isolates (16.2%) were identified from 266 S. aureus isolates that were obtained from various food and clinical specimens in Shanghai. Seven different egc profiles were found based on the polymerase chain reaction (PCR) result for egc genes. Then, these 43 egc-positive isolates were further typed by multilocus sequence typing, pulsed-field gel electrophoresis (PFGE), multiple-locus variable-number tandem-repeat analysis (MLVA), and accessory gene regulatory (agr) typing. It showed that the 43 egc-positive isolates displayed 17 sequence types, 28 PFGE patterns, 29 MLVA types, and 4 agr types, respectively. Among them, the dominant clonal lineage was CC5-agr II (48.84%). Thirty toxin and 20 adhesion-associated genes were detected by PCR in egc-positive isolates. Notably, invasive toxin genes showed a high prevalence, such as 76.7% for Panton-Valentine leukocidin encoding genes, 27.9% for sec, and 23.3% for tsst-1. Most of the examined adhesion-associated genes were found to be conserved (76.7-100%), whereas the fnbB gene was only found in 8 (18.6%) isolates. In addition, 33 toxin gene profiles and 13 adhesion gene profiles were identified, respectively. Our results imply that isolates belonging to the same clonal lineage harbored similar adhesion gene profiles but diverse toxin gene profiles. Overall, the high prevalence of invasive virulence genes increases the potential risk of egc-positive isolates in S. aureus infection.

Effects of Sublethal Fungicides on Mutation Rates and Genomic Variation in Fungal Plant Pathogen, Sclerotinia sclerotiorum.

PubMed

Amaradasa, B Sajeewa; Everhart, Sydney E

2016-01-01

Pathogen exposure to sublethal doses of fungicides may result in mutations that may represent an important and largely overlooked mechanism of introducing new genetic variation into strictly clonal populations, including acquisition of fungicide resistance. We tested this hypothesis using the clonal plant pathogen, Sclerotinia sclerotiorum. Nine susceptible isolates were exposed independently to five commercial fungicides with different modes of action: boscalid (respiration inhibitor), iprodione (unclear mode of action), thiophanate methyl (inhibition of microtubulin synthesis) and azoxystrobin and pyraclostrobin (quinone outside inhibitors). Mycelium of each isolate was inoculated onto a fungicide gradient and sub-cultured from the 50-100% inhibition zone for 12 generations and experiment repeated. Mutational changes were assessed for all isolates at six neutral microsatellite (SSR) loci and for a subset of isolates using amplified fragment length polymorphisms (AFLPs). SSR analysis showed 12 of 85 fungicide-exposed isolates had a total of 127 stepwise mutations with 42 insertions and 85 deletions. Most stepwise deletions were in iprodione- and azoxystrobin-exposed isolates (n = 40/85 each). Estimated mutation rates were 1.7 to 60-fold higher for mutated loci compared to that expected under neutral conditions. AFLP genotyping of 33 isolates (16 non-exposed control and 17 fungicide exposed) generated 602 polymorphic alleles. Cluster analysis with principal coordinate analysis (PCoA) and discriminant analysis of principal components (DAPC) identified fungicide-exposed isolates as a distinct group from non-exposed control isolates (PhiPT = 0.15, P = 0.001). Dendrograms based on neighbor-joining also supported allelic variation associated with fungicide-exposure. Fungicide sensitivity of isolates measured throughout both experiments did not show consistent trends. For example, eight isolates exposed to boscalid had higher EC50 values at the end of the experiment, and when repeated, only one isolate had higher EC50 while most isolates showed no difference. Results of this support the hypothesis that sublethal fungicide stress increases mutation rates in a largely clonal plant pathogen under in vitro conditions. Collectively, this work will aid our understanding how non-lethal fungicide exposure may affect genomic variation, which may be an important mechanism of novel trait emergence, adaptation, and evolution for clonal organisms.

Effects of Sublethal Fungicides on Mutation Rates and Genomic Variation in Fungal Plant Pathogen, Sclerotinia sclerotiorum

PubMed Central

Amaradasa, B. Sajeewa

2016-01-01

Pathogen exposure to sublethal doses of fungicides may result in mutations that may represent an important and largely overlooked mechanism of introducing new genetic variation into strictly clonal populations, including acquisition of fungicide resistance. We tested this hypothesis using the clonal plant pathogen, Sclerotinia sclerotiorum. Nine susceptible isolates were exposed independently to five commercial fungicides with different modes of action: boscalid (respiration inhibitor), iprodione (unclear mode of action), thiophanate methyl (inhibition of microtubulin synthesis) and azoxystrobin and pyraclostrobin (quinone outside inhibitors). Mycelium of each isolate was inoculated onto a fungicide gradient and sub-cultured from the 50–100% inhibition zone for 12 generations and experiment repeated. Mutational changes were assessed for all isolates at six neutral microsatellite (SSR) loci and for a subset of isolates using amplified fragment length polymorphisms (AFLPs). SSR analysis showed 12 of 85 fungicide-exposed isolates had a total of 127 stepwise mutations with 42 insertions and 85 deletions. Most stepwise deletions were in iprodione- and azoxystrobin-exposed isolates (n = 40/85 each). Estimated mutation rates were 1.7 to 60-fold higher for mutated loci compared to that expected under neutral conditions. AFLP genotyping of 33 isolates (16 non-exposed control and 17 fungicide exposed) generated 602 polymorphic alleles. Cluster analysis with principal coordinate analysis (PCoA) and discriminant analysis of principal components (DAPC) identified fungicide-exposed isolates as a distinct group from non-exposed control isolates (PhiPT = 0.15, P = 0.001). Dendrograms based on neighbor-joining also supported allelic variation associated with fungicide-exposure. Fungicide sensitivity of isolates measured throughout both experiments did not show consistent trends. For example, eight isolates exposed to boscalid had higher EC50 values at the end of the experiment, and when repeated, only one isolate had higher EC50 while most isolates showed no difference. Results of this support the hypothesis that sublethal fungicide stress increases mutation rates in a largely clonal plant pathogen under in vitro conditions. Collectively, this work will aid our understanding how non-lethal fungicide exposure may affect genomic variation, which may be an important mechanism of novel trait emergence, adaptation, and evolution for clonal organisms. PMID:27959950

Pandemic extra-intestinal pathogenic Escherichia coli (ExPEC) clonal group O6-B2-ST73 as a cause of avian colibacillosis in Brazil.

PubMed

Cunha, Marcos Paulo Vieira; Saidenberg, Andre Becker; Moreno, Andrea Micke; Ferreira, Antonio José Piantino; Vieira, Mônica Aparecida Midolli; Gomes, Tânia Aparecida Tardelli; Knöbl, Terezinha

2017-01-01

Extra-intestinal pathogenic Escherichia coli (ExPEC) represent an emerging pathogen, with pandemic strains increasingly involved in cases of urinary tract infections (UTIs), bacteremia, and meningitis. In addition to affecting humans, the avian pathotype of ExPEC, avian pathogenic E. coli (APEC), causes severe economic losses to the poultry industry. Several studies have revealed overlapping characteristics between APEC and human ExPEC, leading to the hypothesis of a zoonotic potential of poultry strains. However, the description of certain important pandemic clones, such as Sequence Type 73 (ST73), has not been reported in food sources. We characterized 27 temporally matched APEC strains from diverse poultry farms in Brazil belonging to the O6 serogroup because this serogroup is frequently described as a causal factor in UTI and septicemia in humans in Brazil and worldwide. The isolates were genotypically characterized by identifying ExPEC virulence factors, phylogenetically tested by phylogrouping and multilocus sequence type (MLST) analysis, and compared to determine their similarity employing the pulsed field gel electrophoresis (PFGE) technique. The strains harbored a large number of virulence determinants that are commonly described in uropathogenic E. coli (UPEC) and sepsis associated E. coli (SEPEC) strains and, to a lesser extent in neonatal meningitis associated E. coli (NMEC), such as pap (85%), sfa (100%), usp (100%), cnf1 (22%), kpsMTII (66%), hlyA (52%), and ibeA (4%). These isolates also yielded a low prevalence of some genes that are frequently described in APEC, such as iss (37%), tsh, ompT, and hlyF (8% each), and cvi/cva (0%). All strains were classified as part of the B2 phylogroup and sequence type 73 (ST73), with a cluster of 25 strains showing a clonal profile by PFGE. These results further suggest the zoonotic potential of some APEC clonal lineages and their possible role in the epidemiology of human ExPEC, in addition to providing the first description of the O6-B2-ST73 clonal group in poultry.

Intraspecific competition and light effect on reproduction of Ligularia virgaurea, an invasive native alpine grassland clonal herb

PubMed Central

Xie, Tian-peng; Zhang, Ge-fei; Zhao, Zhi-gang; Du, Guo-zhen; He, Gui-yong

2014-01-01

The relationship between sexual reproduction and clonal growth in clonal plants often shows up at the ramet level. However, only a few studies focus on the relationship at the genet level, which could finally account for evolution. The sexual reproduction and clonal growth of Ligularia virgaurea, a perennial herb widely distributed in the alpine grasslands of the Qinghai-Tibetan Plateau of China, were studied under different competition intensities and light conditions at the genet level through a potted experiment. The results showed that: (1) sexual reproduction did not depend on density or light, and increasing clonal growth with decreasing density and increasing light intensity indicated that intraspecific competition and light intensity may affect the clonal life history of L. virgaurea; (2) both sexual reproduction and clonal growth show a positive linear relationship with genet size under different densities and light conditions; (3) a threshold size is required for sexual reproduction and no evidence of a threshold size for clonal growth under different densities and light conditions; (4) light level affected the allocation of total biomass to clonal and sexual structures, with less allocation to clonal structures and more allocation to sexual structures in full sunlight than in shade; (5) light determined the onset of sexual reproduction, and the genets in the shade required a smaller threshold size for sexual reproduction to occur than the plants in full sunlight; and (6) no evidence was found of trade-offs between clonal growth and sexual reproduction under different densities and light conditions at the genet level, and the positive correlation between two reproductive modes indicated that these are two integrated processes. Clonal growth in this species may be viewed as a growth strategy that tends to maximize genet fitness. PMID:24683463

Population expansion, clonal growth, and specific differentiation patterns in primary cultures of hepatocytes induced by HGF/SF, EGF and TGF alpha in a chemically defined (HGM) medium

PubMed Central

1996-01-01

Mature adult parenchymal hepatocytes, typically of restricted capacity to proliferate in culture, can now enter into clonal growth under the influence of hepatocyte growth factor (scatter factor) (HGF/SF), epidermal growth factor (EGF), and transforming growth factor alpha (TGFalpha) in the presence of a new chemically defined medium (HGM). The expanding populations of hepatocytes lose expression of hepatocyte specific genes (albumin, cytochrome P450 IIB1), acquire expression of markers expressed by bile duct epithelium (cytokeratin 19), produce TGFalpha and acidic FGF and assume a very simplified morphologic phenotype by electron microscopy. A major change associated with this transition is the decrease in ratio between transcription factors C/EBPalpha and C/EBPbeta, as well as the emergence in the proliferating hepatocytes of transcription factors AP1, NFkappaB. The liver associated transcription factors HNFI, HNF3, and HNF4 are preserved throughout this process. After population expansion and clonal growth, the proliferating hepatocytes can return to mature hepatocyte phenotype in the presence of EHS gel (Matrigel). This includes complete restoration of electron microscopic structure and albumin expression. The hepatocyte cultures however can instead be induced to form acinar/ductular structures akin to bile ductules (in the presence of HGF/SF and type I collagen). These transformations affect the entire population of the hepatocytes and occur even when DNA synthesis is inhibited. Similar acinar/ductular structures are seen in embryonic liver when HGF/SF and its receptor are expressed at high levels. These findings strongly support the hypothesis that mature hepatocytes can function as or be a source of bipotential facultative hepatic stem cells (hepatoblasts). These studies also provide evidence for the growth factor and matrix signals that govern these complex phenotypic transitions of facultative stem cells which are crucial for recovery from acute and chronic liver injury. PMID:8601590

Microsatellites within the feline androgen receptor are suitable for X chromosome-linked clonality testing in archival material.

PubMed

Farwick, Nadine M; Klopfleisch, Robert; Gruber, Achim D; Weiss, Alexander Th A

2017-04-01

Objectives A hallmark of neoplasms is their origin from a single cell; that is, clonality. Many techniques have been developed in human medicine to utilise this feature of tumours for diagnostic purposes. One approach is X chromosome-linked clonality testing using polymorphisms of genes encoded by genes on the X chromosome. The aim of this study was to determine if the feline androgen receptor gene was suitable for X chromosome-linked clonality testing. Methods The feline androgen receptor gene was characterised and used to test clonality of feline lymphomas by PCR and polyacrylamide gel electrophoresis, using archival formalin-fixed, paraffin-embedded material. Results Clonality of the feline lymphomas under study was confirmed and the gene locus was shown to represent a suitable target in clonality testing. Conclusions and relevance Because there are some pitfalls of using X chromosome-linked clonality testing, further studies are necessary to establish this technique in the cat.

Genomic Structural Variations Affecting Virulence During Clonal Expansion of Pseudomonas syringae pv. actinidiae Biovar 3 in Europe

PubMed Central

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. PMID:29675009

Comparative phenotypic and genotypic analyses of Salmonella Rissen that originated from food animals in Thailand and United States.

PubMed

Pornsukarom, S; Patchanee, P; Erdman, M; Cray, P F; Wittum, T; Lee, J; Gebreyes, W A

2015-03-01

Salmonella enterica serovar Rissen has been recognized as one of the most common serovar among humans and pork production systems in different parts of the world, especially Asia. In the United States, this serovar caused outbreaks but its epidemiologic significance remains unknown. The objectives of this study were to compare the phenotypic (antimicrobial susceptibility) and genotypic attributes of Salmonella Rissen isolated in Thailand (Thai) and the United States (US). All the Thai isolates (n = 30) were recovered from swine faecal samples. The US isolates (n = 35) were recovered from swine faecal samples (n = 29), cattle (n = 2), chicken (n = 2), dog (n = 1) and a ready-to-eat product (n = 1). The antimicrobial susceptibility of isolates was determined using the Kirby-Bauer disk diffusion method with a panel of 12 antimicrobials. Pulse-field gel electrophoresis (PFGE) was used to determine the genotypic diversity of isolates. All Thai isolates showed multidrug resistance (MDR) with the most frequent antibiotic resistance shown against ampicillin (100%), sulfisoxazole (96.7%), tetracycline (93.3%), streptomycin (90%) and chloramphenicol (30%). About half of the isolates of USA origin were pan-susceptible and roughly 30% were resistant to only tetracycline (R-type: Te). Salmonella Rissen isolated from Thailand and the USA in this study were found to be clonally unrelated. Genotypic analyses indicated that isolates were clustered primarily based on the geographic origin implying the limited clonality among the strains. Clonal relatedness among different host species within the same geography (USA) was found. We found genotypic similarity in Thai and US isolates in few instances but with no epidemiological link. Further studies to assess propensity for increased inter-regional transmission and dissemination is warranted. © 2014 Blackwell Verlag GmbH.

Comparative Analysis of the Orphan CRISPR2 Locus in 242 Enterococcus faecalis Strains

PubMed Central

Hullahalli, Karthik; Rodrigues, Marinelle; Schmidt, Brendan D.; Li, Xiang; Bhardwaj, Pooja; Palmer, Kelli L.

2015-01-01

Clustered, Regularly Interspaced Short Palindromic Repeats and their associated Cas proteins (CRISPR-Cas) provide prokaryotes with a mechanism for defense against mobile genetic elements (MGEs). A CRISPR locus is a molecular memory of MGE encounters. It contains an array of short sequences, called spacers, that generally have sequence identity to MGEs. Three different CRISPR loci have been identified among strains of the opportunistic pathogen Enterococcus faecalis. CRISPR1 and CRISPR3 are associated with the cas genes necessary for blocking MGEs, but these loci are present in only a subset of E. faecalis strains. The orphan CRISPR2 lacks cas genes and is ubiquitous in E. faecalis, although its spacer content varies from strain to strain. Because CRISPR2 is a variable locus occurring in all E. faecalis, comparative analysis of CRISPR2 sequences may provide information about the clonality of E. faecalis strains. We examined CRISPR2 sequences from 228 E. faecalis genomes in relationship to subspecies phylogenetic lineages (sequence types; STs) determined by multilocus sequence typing (MLST), and to a genome phylogeny generated for a representative 71 genomes. We found that specific CRISPR2 sequences are associated with specific STs and with specific branches on the genome tree. To explore possible applications of CRISPR2 analysis, we evaluated 14 E. faecalis bloodstream isolates using CRISPR2 analysis and MLST. CRISPR2 analysis identified two groups of clonal strains among the 14 isolates, an assessment that was confirmed by MLST. CRISPR2 analysis was also used to accurately predict the ST of a subset of isolates. We conclude that CRISPR2 analysis, while not a replacement for MLST, is an inexpensive method to assess clonality among E. faecalis isolates, and can be used in conjunction with MLST to identify recombination events occurring between STs. PMID:26398194

Fine-Scale Genetic Structure of Monilinia fructicola During Brown Rot Epidemics Within Individual Peach Tree Canopies.

PubMed

Everhart, S E; Scherm, H

2015-04-01

The purpose of this study was to determine the fine-scale genetic structure of populations of the brown rot pathogen Monilinia fructicola within individual peach tree canopies to better understand within-tree plant pathogen diversity and to complement previous work on spatiotemporal development of brown rot disease at the canopy level. Across 3 years in a total of six trees, we monitored disease development, collected isolates from every M. fructicola symptom during the course of the season, and created high-resolution three-dimensional maps of all symptom and isolate locations within individual canopies using an electromagnetic digitizer. Each canopy population (65 to 173 isolates per tree) was characterized using a set of 13 microsatellite markers and analyzed for evidence of spatial genetic autocorrelation among isolates during the epidemic phase of the disease. Results showed high genetic diversity (average uh=0.529) and high genotypic diversity (average D=0.928) within canopies. The percentage of unique multilocus genotypes within trees was greater for blossom blight isolates (78.2%) than for fruit rot isolates (51.3%), indicating a greater contribution of clonal reproduction during the preharvest epidemic. For fruit rot isolates, between 54.2 and 81.7% of isolates were contained in one to four dominant clonal genotypes per tree having at least 10 members. All six fruit rot populations showed positive and significant spatial genetic autocorrelation for distance classes between 0.37 and 1.48 m. Despite high levels of within-tree pathogen diversity, the contribution of locally available inoculum combined with short-distance dispersal is likely the main factor generating clonal population foci and associated spatial genetic clustering within trees.

Characterization of Ocular Methicillin-Resistant Staphylococcus epidermidis Isolates Belonging Predominantly to Clonal Complex 2 Subcluster II

PubMed Central

Hofling-Lima, Ana Luisa; Pignatari, Antonio C. C.

2014-01-01

Staphylococcus epidermidis is an abundant member of the microbiota of the human skin and wet mucosa, which is commonly associated with sight-threatening infections in eyes with predisposing factors. Ocular S. epidermidis has become notorious because of its capability to form biofilms on different ocular devices and due to the evolving rates of antimicrobial resistance. In this study, the molecular epidemiology of 30 ocular methicillin-resistant S. epidermidis (MRSE) isolates was assessed using multilocus sequence typing (MLST). Antimicrobial resistance, accessory gene-regulator and staphylococcal cassette chromosome mec (SCCmec) types, biofilm formation, and the occurrence of biofilm-associated genes were correlated with MLST clonal complexes. Sequence types (STs) frequently found in the hospital setting were rarely found in our collection. Overall, 12 different STs were detected with a predominance of ST59 (30%), ST5 and ST6 (13.3% each). Most of the isolates (93.3%) belonged to the clonal complex 2 (CC2) and grouped mainly within subcluster CC2-II (92.9%). Isolates grouped within this subcluster were frequently biofilm producers (92.3%) with a higher occurrence of the aap (84.5%) and bhp (46.1%) genes compared to icaA (19.2%). SCCmec type IV (53.8%) was predominant within CC2-II strains, while 38.4% were nontypeable. In addition, CC2-II strains were frequently multidrug resistant (80.7%) and demonstrated to be particularly resistant to ciprofloxacin (80.8%), ofloxacin (77%), azithromycin (61.5%), and gentamicin (57.7%). Our findings demonstrate the predominance of a particular MRSE cluster causing ocular infections, which was associated with high rates of antimicrobial resistance and particularly the carriage of biofilm-related genes coding for proteinaceous factors implicated in biofilm accumulation. PMID:24523473

Clonal Spread of a Unique Strain of Macrolide-Resistant Mycoplasma Pneumoniae Within a Single Family in Italy.

PubMed

Chironna, Maria; Loconsole, Daniela; De Robertis, Anna Lisa; Morea, Anna; Scalini, Egidio; Quarto, Michele; Tafuri, Silvio; Germinario, Cinzia; Manzionna, Mariano

2016-03-01

Macrolide-resistant Mycoplasma pneumoniae (MR-MP) is an increasing problem worldwide. This study describes the clonal spread of a unique strain of MR-MP within a single family. On January 23, 2015, nasopharyngeal swabs and sputum samples were collected from the index case (a 9-year-old girl) in southern Italy. The patient had pneumonia and was initially treated with clarithromycin. MR-MP infection was suspected due to prolonged symptoms despite appropriate antibiotic therapy. Two further cases of pneumonia occurred in relatives (a 7-year-old cousin and the 36-year-old mother of the index case); therefore, respiratory samples were also collected from other family members. Sequence analysis identified mutations associated with resistance to macrolides. Both P1 major adhesion protein typing and multiple loci variable-number tandem repeat analysis (MLVA) typing were performed to assess the relatedness of the strains. The index case, the cousin, the mother, and another 4 family members (twin siblings of the index case, a 3-year-old cousin, and the grandmother) were positive for MR-MP. All strains harbored the mutation A2063G, had the same P1 subtype (1), and were MLVA (7/4/5/7/2) type Z. In addition, the index case's aunt (31 years of age and the probable source of infection) harbored an M pneumoniae strain with the same molecular profile; however, this strain was susceptible to macrolides. This cluster of MR-MP infection/carriage caused by a clonal strain suggests a high transmission rate within this family and highlights the need for increased awareness among clinicians regarding the circulation of MR-MP. Novel strategies for the treatment and prevention of M pneumoniae infections are required.

Molecular characterization of macrolide resistant Streptococcus pyogenes isolates from pharyngitis patients in Serbia.

PubMed

Opavski, Natasa; Gajic, Ina; Borek, Anna L; Obszańska, Katarzyna; Stanojevic, Maja; Lazarevic, Ivana; Ranin, Lazar; Sitkiewicz, Izabela; Mijac, Vera

2015-07-01

A steady increase in macrolide resistance in Streptococcus pyogenes, group A streptococci (GAS) was reported in Serbia during 2004-2009 (9.9%). However, there are no data on the molecular epidemiology of pharyngeal macrolide resistance GAS (MRGAS) isolates. Therefore, the aims of this first nationwide study were to examine the prevalence of macrolide resistance in Serbian GAS and to determine their resistance phenotypes, genotypes and clonal relationships. Overall 3893 non-duplicate pharyngeal S. pyogenes isolates from outpatients with GAS infection were collected throughout country during 2008 and 2009. Among 486 macrolide resistant pharyngeal isolates collected, 103 were further characterized. Macrolide resistance phenotypes and genotypes were determined by double-disk diffusion test and PCR, respectively. Strain relatedness was determined by emm typing, multilocus sequence typing (MLST), multilocus variable tandem repeat analysis (MLVA), phage profiling (PP) and virulence factor profiling (VFP). Overall, macrolide resistance among GAS isolates in Serbia was 12.5%. M phenotype was the most common (71.8%), followed by iMLS (18.4%) and cMLS (9.7%). Three clonal complexes--emm75/mefA/ST49, emm12/mefA/ST36 and emm77/ermA/tetO/ST63 comprised over 90% of the tested strains. Although MLVA, PP and VFP distinguished 10, 20 and 12 different patterns, respectively, cluster analysis disclosed only small differences between strains which belonged to the same emm/ST type. Our data indicate dominance of three major internationally widely disseminated macrolide resistant clones and a high genetic homogeneity among the Serbian MRGAS population. Continued surveillance of macrolide resistance and clonal composition in MRGAS in Serbia in future is necessary to determine stability of MRGAS clones and to guide therapy strategies. Copyright © 2015 Elsevier B.V. All rights reserved.

Diagnostic Utility of a Clonality Test for Lymphoproliferative Diseases in Koreans Using the BIOMED-2 PCR Assay

PubMed Central

Kim, Young; Choi, Yoo Duk; Choi, Chan

2013-01-01

Background A clonality test for immunoglobulin (IG) and T cell receptor (TCR) is a useful adjunctive method for the diagnosis of lymphoproliferative diseases (LPDs). Recently, the BIOMED-2 multiplex polymerase chain reaction (PCR) assay has been established as a standard method for assessing the clonality of LPDs. We tested clonality in LPDs in Koreans using the BIOMED-2 multiplex PCR and compared the results with those obtained in European, Taiwanese, and Thai participants. We also evaluated the usefulness of the test as an ancillary method for diagnosing LPDs. Methods Two hundred and nineteen specimens embedded in paraffin, including 78 B cell lymphomas, 80 T cell lymphomas and 61 cases of reactive lymphadenitis, were used for the clonality test. Results Mature B cell malignancies showed 95.7% clonality for IG, 2.9% co-existing clonality, and 4.3% polyclonality. Mature T cell malignancies exhibited 83.8% clonality for TCR, 8.1% co-existing clonality, and 16.2% polyclonality. Reactive lymphadenitis showed 93.4% polyclonality for IG and TCR. The majority of our results were similar to those obtained in Europeans. However, the clonality for IGK of B cell malignancies and TCRG of T cell malignancies was lower in Koreans than Europeans. Conclusions The BIOMED-2 multiplex PCR assay was a useful adjunctive method for diagnosing LPDs. PMID:24255634

Pre-leukemic clonal hematopoiesis and the risk of therapy-related myeloid neoplasms: a case-control study

PubMed Central

Takahashi, Koichi; Wang, Feng; Kantarjian, Hagop; Doss, Denaha; Khanna, Kanhav; Thompson, Erika; Zhao, Li; Patel, Keyur; Neelapu, Sattva; Gumbs, Curtis; Bueso-Ramos, Carlos; DiNardo, Courtney D; Colla, Simona; Ravandi, Farhad; Zhang, Jianhua; Huang, Xuelin; Wu, Xifeng; Samaniego, Felipe; Garcia-Manero, Guillermo; Andrew Futreal, P.

2017-01-01

Background Therapy-related myeloid neoplasms (t-MNs) are often fatal secondary malignancies. Risk factors for t-MNs are not well understood. Recent studies suggested that individuals with clonal hematopoiesis have higher risk of developing hematological malignancies. We hypothesized that cancer patients with clonal hematopoiesis have increased risk of developing t-MNs. Methods We conducted a retrospective case-control study to compare the prevalence of clonal hematopoiesis between patients who developed t-MNs (cases) and who did not develop t-MNs (control). For cases, we studied14 patients with various types of cancers who developed t-MNs and whose paired samples of t-MN bone marrow (BM) and peripheral blood (PB) that were previously obtained at the time of primary cancer diagnosis were available. Fifty four patients with lymphoma who received combination chemotherapy and did not develop t-MNs after at least 5 years of follow up were studied as a control. We performed molecular barcode sequencing of 32 genes on the pre-treatment PB samples to detect clonal hematopoiesis. For the t-MN cases, we also performed targeted gene sequencing on t-MN BM samples and investigated clonal evolution from clonal hematopoiesis to t-MNs. To confirm association between clonal hematopoiesis and t-MN development, we also analyzed prevalence of clonal hematopoiesis in a separate cohort of 74 patients with lymphoma. All of these patients were treated under the prospective randomized trial of frontline chemotherapy with cyclophosphamide, doxorubicin, vincristine, and prednisone (CHOP) with or without melatonin and 5 (7%) of them had developed t-MNs. Findings In 14 patients with t-MNs, we detected pre-leukemic mutations in 10 of their prior PB samples (71%). In control, clonal hematopoiesis was detected in 17 patients (31%), and the cumulative incidence of t-MNs at 5 years was significantly higher in patients with clonal hematopoiesis (30% [95% CI: 16% – 51%] vs. 7% [95% CI: 2% – 21%], P = 0.016). In the separate cohort, 5 patients (7%) developed t-MNs and 4 (80%) of them had clonal hematopoiesis. The cumulative incidence of t-MNs at 10 years was significantly higher in patients with clonal hematopoiesis (29% [95% CI: 8%–53%] vs. 0% [95% CI: 0%–0%], P = 0.0009). Multivariate Fine and Gray model showed that having clonal hematopoiesis significantly increased the risk of t-MN development (HR = 13.7, P = 0.013). Interpretation Pre-leukemic clonal hematopoiesis is frequently detected in patients with t-MNs at the time of their primary cancer diagnosis and before patients were exposed to chemotherapy/radiation therapy. Detection of clonal hematopoiesis significantly increased the risk of t-MN development in patients with lymphoma. These data suggest potential approaches of screening clonal hematopoiesis in cancer patients to identify patients at risk of t-MNs and warrants a validation in prospective trial investigating a role of clonal hematopoiesis as a predictive marker for t-MNs. PMID:27923552

Lentiviral and targeted cellular barcoding reveals ongoing clonal dynamics of cell lines in vitro and in vivo

PubMed Central

2014-01-01

Background Cell lines are often regarded as clonal, even though this simplifies what is known about mutagenesis, transformation and other processes that destabilize them over time. Monitoring these clonal dynamics is important for multiple areas of biomedical research, including stem cell and cancer biology. Tracking the contributions of individual cells to large populations, however, has been constrained by limitations in sensitivity and complexity. Results We utilize cellular barcoding methods to simultaneously track the clonal contributions of tens of thousands of cells. We demonstrate that even with optimal culturing conditions, common cell lines including HeLa, K562 and HEK-293 T exhibit ongoing clonal dynamics. Starting a population with a single clone diminishes but does not eradicate this phenomenon. Next, we compare lentiviral and zinc-finger nuclease barcode insertion approaches, finding that the zinc-finger nuclease protocol surprisingly results in reduced clonal diversity. We also document the expected reduction in clonal complexity when cells are challenged with genotoxic stress. Finally, we demonstrate that xenografts maintain clonal diversity to a greater extent than in vitro culturing of the human non-small-cell lung cancer cell line HCC827. Conclusions We demonstrate the feasibility of tracking and quantifying the clonal dynamics of entire cell populations within multiple cultured cell lines. Our results suggest that cell heterogeneity should be considered in the design and interpretation of in vitro culture experiments. Aside from clonal cell lines, we propose that cellular barcoding could prove valuable in modeling the clonal behavior of heterogeneous cell populations over time, including tumor populations treated with chemotherapeutic agents. PMID:24886633

Listeria monocytogenes sequence type 1 is predominant in ruminant rhombencephalitis

PubMed Central

Dreyer, Margaux; Aguilar-Bultet, Lisandra; Rupp, Sebastian; Guldimann, Claudia; Stephan, Roger; Schock, Alexandra; Otter, Arthur; Schüpbach, Gertraud; Brisse, Sylvain; Lecuit, Marc; Frey, Joachim; Oevermann, Anna

2016-01-01

Listeria (L.) monocytogenes is an opportunistic pathogen causing life-threatening infections in diverse mammalian species including humans and ruminants. As little is known on the link between strains and clinicopathological phenotypes, we studied potential strain-associated virulence and organ tropism in L. monocytogenes isolates from well-defined ruminant cases of clinical infections and the farm environment. The phylogeny of isolates and their virulence-associated genes were analyzed by multilocus sequence typing (MLST) and sequence analysis of virulence-associated genes. Additionally, a panel of representative isolates was subjected to in vitro infection assays. Our data suggest the environmental exposure of ruminants to a broad range of strains and yet the strong association of sequence type (ST) 1 from clonal complex (CC) 1 with rhombencephalitis, suggesting increased neurotropism of ST1 in ruminants, which is possibly related to its hypervirulence. This study emphasizes the importance of considering clonal background of L. monocytogenes isolates in surveillance, epidemiological investigation and disease control. PMID:27848981

Clonal dynamics of native haematopoiesis

PubMed Central

Sun, Jianlong; Ramos, Azucena; Chapman, Brad; Johnnidis, Jonathan B.; Le, Linda; Ho, Yu-Jui; Klein, Allon; Hofmann, Oliver; Camargo, Fernando D.

2015-01-01

It is currently thought that life-long blood cell production is driven by the action of a small number of multipotent haematopoietic stem cells. Evidence supporting this view has been largely acquired through the use of functional assays involving transplantation. However, whether these mechanisms also govern native non-transplant haematopoiesis is entirely unclear. Here we have established a novel experimental model in mice where cells can be uniquely and genetically labelled in situ to address this question. Using this approach, we have performed longitudinal analyses of clonal dynamics in adult mice that reveal unprecedented features of native haematopoiesis. In contrast to what occurs following transplantation, steady-state blood production is maintained by the successive recruitment of thousands of clones, each with a minimal contribution to mature progeny. Our results demonstrate that a large number of long-lived progenitors, rather than classically defined haematopoietic stem cells, are the main drivers of steady-state haematopoiesis during most of adulthood. Our results also have implications for understanding the cellular origin of haematopoietic disease. PMID:25296256

Clonal dynamics of native haematopoiesis.

PubMed

Sun, Jianlong; Ramos, Azucena; Chapman, Brad; Johnnidis, Jonathan B; Le, Linda; Ho, Yu-Jui; Klein, Allon; Hofmann, Oliver; Camargo, Fernando D

2014-10-16

It is currently thought that life-long blood cell production is driven by the action of a small number of multipotent haematopoietic stem cells. Evidence supporting this view has been largely acquired through the use of functional assays involving transplantation. However, whether these mechanisms also govern native non-transplant haematopoiesis is entirely unclear. Here we have established a novel experimental model in mice where cells can be uniquely and genetically labelled in situ to address this question. Using this approach, we have performed longitudinal analyses of clonal dynamics in adult mice that reveal unprecedented features of native haematopoiesis. In contrast to what occurs following transplantation, steady-state blood production is maintained by the successive recruitment of thousands of clones, each with a minimal contribution to mature progeny. Our results demonstrate that a large number of long-lived progenitors, rather than classically defined haematopoietic stem cells, are the main drivers of steady-state haematopoiesis during most of adulthood. Our results also have implications for understanding the cellular origin of haematopoietic disease.

Epigenetic Memory Underlies Cell-Autonomous Heterogeneous Behavior of Hematopoietic Stem Cells.

PubMed

Yu, Vionnie W C; Yusuf, Rushdia Z; Oki, Toshihiko; Wu, Juwell; Saez, Borja; Wang, Xin; Cook, Colleen; Baryawno, Ninib; Ziller, Michael J; Lee, Eunjung; Gu, Hongcang; Meissner, Alexander; Lin, Charles P; Kharchenko, Peter V; Scadden, David T

2016-11-17

Stem cells determine homeostasis and repair of many tissues and are increasingly recognized as functionally heterogeneous. To define the extent of-and molecular basis for-heterogeneity, we overlaid functional, transcriptional, and epigenetic attributes of hematopoietic stem cells (HSCs) at a clonal level using endogenous fluorescent tagging. Endogenous HSC had clone-specific functional attributes over time in vivo. The intra-clonal behaviors were highly stereotypic, conserved under the stress of transplantation, inflammation, and genotoxic injury, and associated with distinctive transcriptional, DNA methylation, and chromatin accessibility patterns. Further, HSC function corresponded to epigenetic configuration but not always to transcriptional state. Therefore, hematopoiesis under homeostatic and stress conditions represents the integrated action of highly heterogeneous clones of HSC with epigenetically scripted behaviors. This high degree of epigenetically driven cell autonomy among HSCs implies that refinement of the concepts of stem cell plasticity and of the stem cell niche is warranted. Copyright © 2016 Elsevier Inc. All rights reserved.

Human Staphylococcus aureus lineages among Zoological Park residents in Greece

PubMed Central

Drougka, E.; Foka, A.; Posantzis, D.; Giormezis, N.; Anastassiou, E.D.; Petinaki, E.; Spiliopoulou, I.

2015-01-01

Staphylococcus aureus is a part of the microbiota flora in many animal species. The clonal spread of S. aureus among animals and personnel in a Zoological Park was investigated. Samples were collected from colonized and infected sites among 32 mammals, 11 birds and eight humans. The genes mecA, mecC, lukF/lukS-PV (encoding Panton-Valentine leukocidin, PVL) and tst (toxic shock syndrome toxin-1) were investigated by PCR. Clones were defined by Multilocus Sequence Typing (MLST), spa type and Pulsed-Field Gel Electrophoresis (PFGE). Seven S. aureus isolates were recovered from four animals and one from an employee. All were mecA, mecC and tst–negative, whereas, one carried the PVL genes and was isolated from an infected Squirrel monkey. Clonal analysis revealed the occurrence of seven STs, eight PFGE and five spa types including ones of human origin. Even though a variety of genotypes were identified among S. aureus strains colonizing zoo park residents, our results indicate that colonization with human lineages has indeed occurred. PMID:26623381

Multilocus Sequence Types of Campylobacter jejuni Isolates from Different Sources in Eastern China.

PubMed

Zhang, Gong; Zhang, Xiaoyan; Hu, Yuanqing; Jiao, Xin-An; Huang, Jinlin

2015-09-01

Campylobacter jejuni is a major food-borne pathogen that causes human gastroenteritis in many developed countries. In our study, we applied multilocus sequence typing (MLST) technology to 167 C. jejuni isolates from diverse sources in Eastern China to examine their genetic diversity. MLST defined 94 sequence types (STs) belonging to 18 clonal complexes (CCs). Forty-five STs from 60 isolates (36%) and 22 alleles have not been previously documented in an international database. One hundred and two isolates, accounting for 61.1% of all isolates, belonged to eight clonal complexes. The eight major CCs were also the most common complexes from different sources. The most common ST type of isolates from human and food was ST-353. The dominant ST type in chicken and foods was ST-354. Among 21 STs that contained two or more different sources isolates, 15 STs contained human isolates and isolates from other sources, suggesting that potentially pathogenic strains are not restricted to specific lineages.

Whole-Genome Characterization of Epidemic Neisseria meningitidis Serogroup C and Resurgence of Serogroup W, Niger, 2015.

PubMed

Kretz, Cecilia B; Retchless, Adam C; Sidikou, Fati; Issaka, Bassira; Ousmane, Sani; Schwartz, Stephanie; Tate, Ashley H; Pana, Assimawè; Njanpop-Lafourcade, Berthe-Marie; Nzeyimana, Innocent; Nse, Ricardo Obama; Deghmane, Ala-Eddine; Hong, Eva; Brynildsrud, Ola Brønstad; Novak, Ryan T; Meyer, Sarah A; Oukem-Boyer, Odile Ouwe Missi; Ronveaux, Olivier; Caugant, Dominique A; Taha, Muhamed-Kheir; Wang, Xin

2016-10-01

In 2015, Niger reported the largest epidemic of Neisseria meningitidis serogroup C (NmC) meningitis in sub-Saharan Africa. The NmC epidemic coincided with serogroup W (NmW) cases during the epidemic season, resulting in a total of 9,367 meningococcal cases through June 2015. To clarify the phylogenetic association, genetic evolution, and antibiotic determinants of the meningococcal strains in Niger, we sequenced the genomes of 102 isolates from this epidemic, comprising 81 NmC and 21 NmW isolates. The genomes of 82 isolates were completed, and all 102 were included in the analysis. All NmC isolates had sequence type 10217, which caused the outbreaks in Nigeria during 2013-2014 and for which a clonal complex has not yet been defined. The NmC isolates from Niger were substantially different from other NmC isolates collected globally. All NmW isolates belonged to clonal complex 11 and were closely related to the isolates causing recent outbreaks in Africa.

Recent advances in understanding clonal haematopoiesis in aplastic anaemia

PubMed Central

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

2016-01-01

Summary 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. PMID:28107566

Recent advances in understanding clonal haematopoiesis in aplastic anaemia.

PubMed

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.

Non-cell-autonomous driving of tumour growth supports sub-clonal heterogeneity.

PubMed

Marusyk, Andriy; Tabassum, Doris P; Altrock, Philipp M; Almendro, Vanessa; Michor, Franziska; Polyak, Kornelia

2014-10-02

Cancers arise through a process of somatic evolution that can result in substantial sub-clonal heterogeneity within tumours. The mechanisms responsible for the coexistence of distinct sub-clones and the biological consequences of this coexistence remain poorly understood. Here we used a mouse xenograft model to investigate the impact of sub-clonal heterogeneity on tumour phenotypes and the competitive expansion of individual clones. We found that tumour growth can be driven by a minor cell subpopulation, which enhances the proliferation of all cells within a tumour by overcoming environmental constraints and yet can be outcompeted by faster proliferating competitors, resulting in tumour collapse. We developed a mathematical modelling framework to identify the rules underlying the generation of intra-tumour clonal heterogeneity. We found that non-cell-autonomous driving of tumour growth, together with clonal interference, stabilizes sub-clonal heterogeneity, thereby enabling inter-clonal interactions that can lead to new phenotypic traits.

Relative efficiency and sample size for cluster randomized trials with variable cluster sizes.

PubMed

You, Zhiying; Williams, O Dale; Aban, Inmaculada; Kabagambe, Edmond Kato; Tiwari, Hemant K; Cutter, Gary

2011-02-01

The statistical power of cluster randomized trials depends on two sample size components, the number of clusters per group and the numbers of individuals within clusters (cluster size). Variable cluster sizes are common and this variation alone may have significant impact on study power. Previous approaches have taken this into account by either adjusting total sample size using a designated design effect or adjusting the number of clusters according to an assessment of the relative efficiency of unequal versus equal cluster sizes. This article defines a relative efficiency of unequal versus equal cluster sizes using noncentrality parameters, investigates properties of this measure, and proposes an approach for adjusting the required sample size accordingly. We focus on comparing two groups with normally distributed outcomes using t-test, and use the noncentrality parameter to define the relative efficiency of unequal versus equal cluster sizes and show that statistical power depends only on this parameter for a given number of clusters. We calculate the sample size required for an unequal cluster sizes trial to have the same power as one with equal cluster sizes. Relative efficiency based on the noncentrality parameter is straightforward to calculate and easy to interpret. It connects the required mean cluster size directly to the required sample size with equal cluster sizes. Consequently, our approach first determines the sample size requirements with equal cluster sizes for a pre-specified study power and then calculates the required mean cluster size while keeping the number of clusters unchanged. Our approach allows adjustment in mean cluster size alone or simultaneous adjustment in mean cluster size and number of clusters, and is a flexible alternative to and a useful complement to existing methods. Comparison indicated that we have defined a relative efficiency that is greater than the relative efficiency in the literature under some conditions. Our measure of relative efficiency might be less than the measure in the literature under some conditions, underestimating the relative efficiency. The relative efficiency of unequal versus equal cluster sizes defined using the noncentrality parameter suggests a sample size approach that is a flexible alternative and a useful complement to existing methods.

Clonality Testing in Veterinary Medicine: A Review With Diagnostic Guidelines.

PubMed

Keller, S M; Vernau, W; Moore, P F

2016-07-01

The accurate distinction of reactive and neoplastic lymphoid proliferations can present challenges. Given the different prognoses and treatment strategies, a correct diagnosis is crucial. Molecular clonality assays assess rearranged lymphocyte antigen receptor gene diversity and can help differentiate reactive from neoplastic lymphoid proliferations. Molecular clonality assays are commonly used to assess atypical, mixed, or mature lymphoid proliferations; small tissue fragments that lack architecture; and fluid samples. In addition, clonality testing can be utilized to track neoplastic clones over time or across anatomic sites. Molecular clonality assays are not stand-alone tests but useful adjuncts that follow clinical, morphologic, and immunophenotypic assessment. Even though clonality testing provides valuable information in a variety of situations, the complexities and pitfalls of this method, as well as its dependency on the experience of the interpreter, are often understated. In addition, a lack of standardized terminology, laboratory practices, and interpretational guidelines hinders the reproducibility of clonality testing across laboratories in veterinary medicine. The objectives of this review are twofold. First, the review is intended to familiarize the diagnostic pathologist or interested clinician with the concepts, potential pitfalls, and limitations of clonality testing. Second, the review strives to provide a basis for future harmonization of clonality testing in veterinary medicine by providing diagnostic guidelines. © The Author(s) 2016.

Synthesis of clonality and polyploidy in vertebrate animals by hybridization between two sexual species.

PubMed

Choleva, Lukáš; Janko, Karel; De Gelas, Koen; Bohlen, Jörg; Šlechtová, Věra; Rábová, Marie; Ráb, Petr

2012-07-01

Because most clonal vertebrates have hybrid genomic constitutions, tight linkages are assumed among hybridization, clonality, and polyploidy. However, predictions about how these processes mechanistically relate during the switch from sexual to clonal reproduction have not been validated. Therefore, we performed a crossing experiment to test the hypothesis that interspecific hybridization per se initiated clonal diploid and triploid spined loaches (Cobitis) and their gynogenetic reproduction. We reared two F1 families resulting from the crossing of 14 pairs of two sexual species, and found their diploid hybrid constitution and a 1:1 sex ratio. While males were infertile, females produced unreduced nonrecombinant eggs (100%). Synthetic triploid females and males (96.3%) resulted in each of nine backcrossed families from eggs of synthesized diploid F1s fertilized by haploid sperm from sexual males. Five individuals (3.7%) from one backcross family were genetically identical to the somatic cells of the mother and originated via gynogenesis; the sperm of the sexual male only triggered clonal development of the egg. Our reconstruction of the evolutionary route from sexuality to clonality and polyploidy in these fish shows that clonality and gynogenesis may have been directly triggered by interspecific hybridization and that polyploidy is a consequence, not a cause, of clonality. © 2012 The Author(s).

Phenotypic profile of expanded NK cells in chronic lymphoproliferative disorders: a surrogate marker for NK-cell clonality

PubMed Central

Bárcena, Paloma; Jara-Acevedo, María; Tabernero, María Dolores; López, Antonio; Sánchez, María Luz; García-Montero, Andrés C.; Muñoz-García, Noemí; Vidriales, María Belén; Paiva, Artur; Lecrevisse, Quentin; Lima, Margarida; Langerak, Anton W.; Böttcher, Sebastian; van Dongen, Jacques J.M.

2015-01-01

Currently, the lack of a universal and specific marker of clonality hampers the diagnosis and classification of chronic expansions of natural killer (NK) cells. Here we investigated the utility of flow cytometric detection of aberrant/altered NK-cell phenotypes as a surrogate marker for clonality, in the diagnostic work-up of chronic lymphoproliferative disorders of NK cells (CLPD-NK). For this purpose, a large panel of markers was evaluated by multiparametric flow cytometry on peripheral blood (PB) CD56low NK cells from 60 patients, including 23 subjects with predefined clonal (n = 9) and polyclonal (n = 14) CD56low NK-cell expansions, and 37 with CLPD-NK of undetermined clonality; also, PB samples from 10 healthy adults were included. Clonality was established using the human androgen receptor (HUMARA) assay. Clonal NK cells were found to show decreased expression of CD7, CD11b and CD38, and higher CD2, CD94 and HLADR levels vs. normal NK cells, together with a restricted repertoire of expression of the CD158a, CD158b and CD161 killer-associated receptors. In turn, NK cells from both clonal and polyclonal CLPD-NK showed similar/overlapping phenotypic profiles, except for high and more homogeneous expression of CD94 and HLADR, which was restricted to clonal CLPD-NK. We conclude that the CD94hi/HLADR+ phenotypic profile proved to be a useful surrogate marker for NK-cell clonality. PMID:26556869

Natural and Chemotherapy-Induced Clonal Evolution of Tumors.

PubMed

Ibragimova, M K; Tsyganov, M M; Litviakov, N V

2017-04-01

Evolution and natural selection of tumoral clones in the process of transformation and the following carcinogenesis can be called natural clonal evolution. Its main driving factors are internal: genetic instability initiated by driver mutations and microenvironment, which enables selective pressure while forming the environment for cell transformation and their survival. We present our overview of contemporary research dealing with mechanisms of carcinogenesis in different localizations from precancerous pathologies to metastasis and relapse. It shows that natural clonal evolution establishes intratumoral heterogeneity and enables tumor progression. Tumors of monoclonal origin are of low-level intratumoral heterogeneity in the initial stages, and this increases with the size of the tumor. Tumors of polyclonal origin are of extremely high-level intratumoral heterogeneity in the initial stages and become more homogeneous when larger due to clonal expansion. In cases of chemotherapy-induced clonal evolution of a tumor, chemotherapy becomes the leading factor in treatment. The latest research shows that the impact of chemotherapy can radically increase the speed of clonal evolution and lead to new malignant and resistant clones that cause tumor metastasis. Another option of chemotherapy-induced clonal evolution is formation of a new dominant clone from a clone that was minor in the initial tumor and obtained free space due to elimination of sensitive clones by chemotherapy. As a result, in ~20% of cases, chemotherapy can stimulate metastasis and relapse of tumors due to clonal evolution. The conclusion of the overview formulates approaches to tumor treatment based on clonal evolution: in particular, precision therapy, prediction of metastasis stimulation in patients treated with chemotherapy, methods of genetic evaluation of chemotherapy efficiency and clonal-oriented treatment, and approaches to manipulating the clonal evolution of tumors are presented.

Multidisciplinary insight into clonal expansion of HTLV-1-infected cells in adult T-cell leukemia via modeling by deterministic finite automata coupled with high-throughput sequencing.

PubMed

Farmanbar, Amir; Firouzi, Sanaz; Park, Sung-Joon; Nakai, Kenta; Uchimaru, Kaoru; Watanabe, Toshiki

2017-01-31

Clonal expansion of leukemic cells leads to onset of adult T-cell leukemia (ATL), an aggressive lymphoid malignancy with a very poor prognosis. Infection with human T-cell leukemia virus type-1 (HTLV-1) is the direct cause of ATL onset, and integration of HTLV-1 into the human genome is essential for clonal expansion of leukemic cells. Therefore, monitoring clonal expansion of HTLV-1-infected cells via isolation of integration sites assists in analyzing infected individuals from early infection to the final stage of ATL development. However, because of the complex nature of clonal expansion, the underlying mechanisms have yet to be clarified. Combining computational/mathematical modeling with experimental and clinical data of integration site-based clonality analysis derived from next generation sequencing technologies provides an appropriate strategy to achieve a better understanding of ATL development. As a comprehensively interdisciplinary project, this study combined three main aspects: wet laboratory experiments, in silico analysis and empirical modeling. We analyzed clinical samples from HTLV-1-infected individuals with a broad range of proviral loads using a high-throughput methodology that enables isolation of HTLV-1 integration sites and accurate measurement of the size of infected clones. We categorized clones into four size groups, "very small", "small", "big", and "very big", based on the patterns of clonal growth and observed clone sizes. We propose an empirical formal model based on deterministic finite state automata (DFA) analysis of real clinical samples to illustrate patterns of clonal expansion. Through the developed model, we have translated biological data of clonal expansion into the formal language of mathematics and represented the observed clonality data with DFA. Our data suggest that combining experimental data (absolute size of clones) with DFA can describe the clonality status of patients. This kind of modeling provides a basic understanding as well as a unique perspective for clarifying the mechanisms of clonal expansion in ATL.

Identification of a role for the nuclear receptor EAR-2 in the maintenance of clonogenic status within the leukemia cell hierarchy

PubMed Central

Ichim, CV; Atkins, HL; Iscove, NN; Wells, RA

2016-01-01

Identification of genes that regulate clonogenicity of acute myelogenous leukemia (AML) cells is hindered by the difficulty of isolating pure populations of cells with defined proliferative abilities. By analyzing the growth of clonal siblings in low passage cultures of the cell line OCI/AML4 we resolved this heterogeneous population into strata of distinct clonogenic potential, permitting analysis of the transcriptional signature of single cells with defined proliferative abilities. By microarray analysis we showed that the expression of the orphan nuclear receptor EAR-2 (NR2F6) is greater in leukemia cells with extensive proliferative capacity than in those that have lost proliferative ability. EAR-2 is expressed highly in long-term hematopoietic stem cells, relative to short-term hematopoietic stem and progenitor cells, and is downregulated in AML cells after induction of differentiation. Exogenous expression of EAR-2 increased the growth of U937 cells and prevented the proliferative arrest associated with terminal differentiation, and blocked differentiation of U937 and 32Dcl3 cells. Conversely, silencing of EAR-2 by short-hairpin RNA initiated terminal differentiation of these cell lines. These data identify EAR-2 as an important factor in the regulation of clonogenicity and differentiation, and establish that analysis of clonal siblings allows the elucidation of differences in gene expression within the AML hierarchy. PMID:21637284

Use of the accessory genome for characterization and typing of Acinetobacter baumannii.

PubMed

Turton, Jane F; Baddal, Buket; Perry, Claire

2011-04-01

Outbreak strains of Acinetobacter baumannii are highly clonal, and cross-infection investigations can be difficult. We sought targets based on AbaR resistance islands and on other genes found in some, but not all, sequenced isolates of A. baumannii among a set of clinical isolates (n = 70) that included multiple representatives of a number of pulsed-field gel electrophoresis (PFGE)-defined types. These included representatives that varied in their profiles at two variable-number tandem repeat (VNTR) loci, which can provide discrimination within a PFGE cluster. Detection, or not, of each element sought provided some degree of discrimination among the set, with the presence or absence of genes coding for a phage terminase (ACICU_02185), a sialic acid synthase (ACICU_00080), a polysaccharide biosynthesis protein (AB57_0094), aphA1, bla(TEM), and integron-associated orfX (Kyoto Encyclopedia of Genes and Genomes [KEGG] no. K03830) proving the most helpful in discriminating between closely related isolates in our panel. The results support VNTR data in describing distinct populations of highly similar isolates. Such analysis, in combination with other typing methods, can inform epidemiological investigations and provide additional characterization of isolates. Most genotypes carrying bla(OXA-23-like) were PCR positive for a yeeA-bla(OXA-23) fragment found in an AbaR4-type island, suggesting that this is widespread.

Genetic Diversity and Domestication Footprints of Chinese Cherry [Cerasus pseudocerasus (Lindl.) G.Don] as Revealed by Nuclear Microsatellites

PubMed Central

Zhang, Jing; Chen, Tao; Wang, Yan; Chen, Qing; Sun, Bo; Luo, Ya; Zhang, Yong; Tang, Haoru; Wang, Xiaorong

2018-01-01

Chinese cherry [Cerasus pseudocerasus (Lindl.) G.Don] is a commercially important fruit crop in China, but its structure patterns and domestication history remain imprecise. To address these questions, we estimated the genetic structure and domestication history of Chinese cherry using 19 nuclear microsatellite markers and 650 representative accessions (including 118 Cerasus relatives) selected throughout their natural eco-geographical distributions. Our structure analyses detected no genetic contribution from Cerasus relatives to the evolution history of Chinese cherry. A separate genetic structure was detected in wild Chinese cherries and rough geographical structures were observed in cultivated Chinese cherries. One wild (wild Chinese cherry, WC) and two cultivated (cultivated Chinese cherry, CC1 and CC2) genetic clusters were defined. Our approximate Bayesian computation analyses supported an independent domestication history with two domestication events for CC1 and CC2, happening about 3900 and 2200 years ago, respectively. Moderate loss of genetic diversity, over 1000-year domestication bottlenecks and divergent domestication in fruit traits were also detected in cultivated Chinese cherries, which is highly correlated to long-term clonal propagation and different domestication trends and preferences. Our study is the first to comprehensively and systematically investigate the structure patterns and domestication history for Chinese cherry, providing important references for revealing the evolution and domestication history of perennial woody fruit trees. PMID:29535750

Globally diverse Toxoplasma gondii isolates comprise six major clades originating from a small number of distinct ancestral lineages

PubMed Central

Su, Chunlei; Khan, Asis; Zhou, Peng; Majumdar, Debashree; Ajzenberg, Daniel; Dardé, Marie-Laure; Zhu, Xing-Quan; Ajioka, James W.; Rosenthal, Benjamin M.; Dubey, Jitender P.; Sibley, L. David

2012-01-01

Marked phenotypic variation characterizes isolates of Toxoplasma gondii, a ubiquitous zoonotic parasite that serves as an important experimental model for studying apicomplexan parasites. Progress in identifying the heritable basis for clinically and epidemiologically significant differences requires a robust system for describing and interpreting evolutionary subdivisions in this prevalent pathogen. To develop such a system, we have examined more than 950 isolates collected from around the world and genotyped them using three independent sets of polymorphic DNA markers, sampling 30 loci distributed across all nuclear chromosomes as well as the plastid genome. Our studies reveal a biphasic pattern consisting of regions in the Northern Hemisphere where a few, highly clonal and abundant lineages predominate; elsewhere, and especially in portions of South America are characterized by a diverse assemblage of less common genotypes that show greater evidence of recombination. Clustering methods were used to organize the marked genetic diversity of 138 unique genotypes into 15 haplogroups that collectively define six major clades. Analysis of gene flow indicates that a small number of ancestral lineages gave rise to the existing diversity through a process of limited admixture. Identification of reference strains for these major groups should facilitate future studies on comparative genomics and identification of genes that control important biological phenotypes including pathogenesis and transmission. PMID:22431627

Anterior pituitary cells defective in the cell-autonomous factor, df, undergo cell lineage specification but not expansion.

PubMed

Gage, P J; Roller, M L; Saunders, T L; Scarlett, L M; Camper, S A

1996-01-01

The Ames dwarf mouse transmits a recessive mutation (df) resulting in a profound anterior pituitary hypocellularity due to a general lack of thyrotropes, somatotropes and lactotropes. These cell types are also dependent on the pituitary-specific transcription factor, Pit-1. We present evidence that expression of Pit-1 and limited commitment to these cells lineages occurs in df/df pituitaries. Thus, the crucial role of df may be in lineage-specific proliferation, rather than cytodifferentiation. The presence of all three Pit-1-dependent cell types in clonally derived clusters provides compelling evidence that these three lineages share a common, pluripotent precursor cell. Clusters containing different combinations of Pit-1-dependent cell types suggests that the Pit-1+ precursor cells choose from multiple developmental options during ontogeny. Characterization of df/df<-->+/+ chimeric mice demonstrated that df functions by a cell-autonomous mechanism. Therefore, df and Pit-1 are both cell-autonomous factors required for thyrotrope, somatotrope and lactotrope ontogeny, but their relative roles are different.

Microsatellite markers identify three lineages of Phytophthora ramorum in US nurseries, yet single lineages in US forest and European nursery populations.

PubMed

Ivors, K; Garbelotto, M; Vries, I D E; Ruyter-Spira, C; Te Hekkert, B; Rosenzweig, N; Bonants, P

2006-05-01

Analysis of 12 polymorphic simple sequence repeats identified in the genome sequence of Phytophthora ramorum, causal agent of 'sudden oak death', revealed genotypic diversity to be significantly higher in nurseries (91% of total) than in forests (18% of total). Our analysis identified only two closely related genotypes in US forests, while the genetic structure of populations from European nurseries was of intermediate complexity, including multiple, closely related genotypes. Multilocus analysis determined populations in US forests reproduce clonally and are likely descendants of a single introduced individual. The 151 isolates analysed clustered in three clades. US forest and European nursery isolates clustered into two distinct clades, while one isolate from a US nursery belonged to a third novel clade. The combined microsatellite, sequencing and morphological analyses suggest the three clades represent distinct evolutionary lineages. All three clades were identified in some US nurseries, emphasizing the role of commercial plant trade in the movement of this pathogen.

Extensive Clonality and Strong Differentiation in the Insular Pacific Tree Santalum insulare: Implications for its Conservation

PubMed Central

LHUILLIER, EMELINE; BUTAUD, JEAN-FRANÇOIS; BOUVET, JEAN-MARC

2006-01-01

• Background and Aims The impact of evolutionary forces on insular systems is particularly exacerbated by the remoteness of islands, strong founder effects, small population size and the influence of biotic and abiotic factors. Patterns of molecular diversity were analysed in an island system with Santalum insulare, a sandalwood species endemic to eastern Polynesia. The aims were to evaluate clonality and to study the genetic diversity and structure of this species, in order to understand the evolutionary process and to define a conservation strategy. • Methods Eight nuclear microsatellites were used to investigate clonality, genetic variation and structure of the French Polynesian sandalwood populations found on ten islands distributed over three archipelagos. • Key Results It was found that 58 % of the 384 trees analysed were clones. The real size of the populations is thus dramatically reduced, with sometimes only one genet producing ramets by root suckering. The diversity parameters were low for islands (nA = 1·5–5·0; HE = 0·28–0·49). No departure from Hardy–Weinberg proportion was observed except within Tahiti island, where a significant excess of homozygotes was noted in the highland population. Genetic structure was characterized by high levels of differentiation between archipelagos (27 % of the total variation) and islands (FST = 0·50). The neighbour-joining tree did not discriminate the three archipelagos but separated the Society archipelago from the other two. • Conclusions This study shows that clonality is a frequent phenomenon in S. insulare. The genetic diversity within populations is lower than the values assessed in species distributed on the mainland, as a consequence of insularity. But this can also be explained by the overexploitation of sandalwood. The differentiation between archipelagos and islands within archipelagos is very high because of the limited gene flow due to oceanic barriers. Delineation of evolutionary significant units and principles for population management are proposed based on this molecular analysis. PMID:16945947

Glioblastoma adaptation traced through decline of an IDH1 clonal driver and macro-evolution of a double-minute chromosome

PubMed Central

Favero, F.; McGranahan, N.; Salm, M.; Birkbak, N. J.; Sanborn, J. Z.; Benz, S. C.; Becq, J.; Peden, J. F.; Kingsbury, Z.; Grocok, R. J.; Humphray, S.; Bentley, D.; Spencer-Dene, B.; Gutteridge, A.; Brada, M.; Roger, S.; Dietrich, P.-Y.; Forshew, T.; Gerlinger, M.; Rowan, A.; Stamp, G.; Eklund, A. C.; Szallasi, Z.; Swanton, C.

2015-01-01

Background Glioblastoma (GBM) is the most common malignant brain cancer occurring in adults, and is associated with dismal outcome and few therapeutic options. GBM has been shown to predominantly disrupt three core pathways through somatic aberrations, rendering it ideal for precision medicine approaches. Methods We describe a 35-year-old female patient with recurrent GBM following surgical removal of the primary tumour, adjuvant treatment with temozolomide and a 3-year disease-free period. Rapid whole-genome sequencing (WGS) of three separate tumour regions at recurrence was carried out 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 in the primary, a known GBM driver event, was detectable at only very low frequency in the recurrent tumour. After sub-clonal diversification, evidence was found for a whole-genome doubling event and a translocation between the amplified regions of PDGFRA, KIT and CDK4, encoded within a double-minute chromosome also incorporating miR26a-2. The WGS analysis uncovered progressive evolution of the double-minute chromosome converging on the KIT/PDGFRA/PI3K/mTOR axis, superseding the IDH1 mutation in dominance in a mutually exclusive manner at recurrence, consequently the patient was treated with imatinib. Despite rapid sequencing and cancer genome-guided therapy against amplified oncogenes, the disease progressed, and the patient died shortly after. Conclusion This case sheds light on the dynamic evolution of a GBM tumour, defining the origins of the lethal sub-clone, the macro-evolutionary genomic events dominating the disease at recurrence and the loss of a clonal driver. Even in the era of rapid WGS analysis, cases such as this illustrate the significant hurdles for precision medicine success. PMID:25732040

2-Way k-Means as a Model for Microbiome Samples.

PubMed

Jackson, Weston J; Agarwal, Ipsita; Pe'er, Itsik

2017-01-01

Motivation . Microbiome sequencing allows defining clusters of samples with shared composition. However, this paradigm poorly accounts for samples whose composition is a mixture of cluster-characterizing ones and which therefore lie in between them in the cluster space. This paper addresses unsupervised learning of 2-way clusters. It defines a mixture model that allows 2-way cluster assignment and describes a variant of generalized k -means for learning such a model. We demonstrate applicability to microbial 16S rDNA sequencing data from the Human Vaginal Microbiome Project.

2-Way k-Means as a Model for Microbiome Samples

PubMed Central

2017-01-01

Motivation. Microbiome sequencing allows defining clusters of samples with shared composition. However, this paradigm poorly accounts for samples whose composition is a mixture of cluster-characterizing ones and which therefore lie in between them in the cluster space. This paper addresses unsupervised learning of 2-way clusters. It defines a mixture model that allows 2-way cluster assignment and describes a variant of generalized k-means for learning such a model. We demonstrate applicability to microbial 16S rDNA sequencing data from the Human Vaginal Microbiome Project. PMID:29177026

Selective Inhibition of Tumor Growth by Clonal NK Cells Expressing an ErbB2/HER2-Specific Chimeric Antigen Receptor

PubMed Central

Schönfeld, Kurt; Sahm, Christiane; Zhang, Congcong; Naundorf, Sonja; Brendel, Christian; Odendahl, Marcus; Nowakowska, Paulina; Bönig, Halvard; Köhl, Ulrike; Kloess, Stephan; Köhler, Sylvia; Holtgreve-Grez, Heidi; Jauch, Anna; Schmidt, Manfred; Schubert, Ralf; Kühlcke, Klaus; Seifried, Erhard; Klingemann, Hans G; Rieger, Michael A; Tonn, Torsten; Grez, Manuel; Wels, Winfried S

2015-01-01

Natural killer (NK) cells are an important effector cell type for adoptive cancer immunotherapy. Similar to T cells, NK cells can be modified to express chimeric antigen receptors (CARs) to enhance antitumor activity, but experience with CAR-engineered NK cells and their clinical development is still limited. Here, we redirected continuously expanding and clinically usable established human NK-92 cells to the tumor-associated ErbB2 (HER2) antigen. Following GMP-compliant procedures, we generated a stable clonal cell line expressing a humanized CAR based on ErbB2-specific antibody FRP5 harboring CD28 and CD3ζ signaling domains (CAR 5.28.z). These NK-92/5.28.z cells efficiently lysed ErbB2-expressing tumor cells in vitro and exhibited serial target cell killing. Specific recognition of tumor cells and antitumor activity were retained in vivo, resulting in selective enrichment of NK-92/5.28.z cells in orthotopic breast carcinoma xenografts, and reduction of pulmonary metastasis in a renal cell carcinoma model, respectively. γ-irradiation as a potential safety measure for clinical application prevented NK cell replication, while antitumor activity was preserved. Our data demonstrate that it is feasible to engineer CAR-expressing NK cells as a clonal, molecularly and functionally well-defined and continuously expandable cell therapeutic agent, and suggest NK-92/5.28.z cells as a promising candidate for use in adoptive cancer immunotherapy. PMID:25373520

Genetic homogeneity of Clostridium botulinum type A1 strains with unique toxin gene clusters.

PubMed

Raphael, Brian H; Luquez, Carolina; McCroskey, Loretta M; Joseph, Lavin A; Jacobson, Mark J; Johnson, Eric A; Maslanka, Susan E; Andreadis, Joanne D

2008-07-01

A group of five clonally related Clostridium botulinum type A strains isolated from different sources over a period of nearly 40 years harbored several conserved genetic properties. These strains contained a variant bont/A1 with five nucleotide polymorphisms compared to the gene in C. botulinum strain ATCC 3502. The strains also had a common toxin gene cluster composition (ha-/orfX+) similar to that associated with bont/A in type A strains containing an unexpressed bont/B [termed A(B) strains]. However, bont/B was not identified in the strains examined. Comparative genomic hybridization demonstrated identical genomic content among the strains relative to C. botulinum strain ATCC 3502. In addition, microarray data demonstrated the absence of several genes flanking the toxin gene cluster among the ha-/orfX+ A1 strains, suggesting the presence of genomic rearrangements with respect to this region compared to the C. botulinum ATCC 3502 strain. All five strains were shown to have identical flaA variable region nucleotide sequences. The pulsed-field gel electrophoresis patterns of the strains were indistinguishable when digested with SmaI, and a shift in the size of at least one band was observed in a single strain when digested with XhoI. These results demonstrate surprising genomic homogeneity among a cluster of unique C. botulinum type A strains of diverse origin.

Clonal analysis of human embryonic stem cell differentiation into teratomas.

PubMed

Blum, Barak; Benvenisty, Nissim

2007-08-01

Differentiation of human embryonic stem cells (HESCs) can be studied in vivo through the induction of teratomas in immune-deficient mice. Cells within the teratomas differentiate into all three embryonic germ layers. However, the exact nature of the proliferation and differentiation of HESCs within the teratoma is not fully characterized, and it is not clear whether the differentiation is cell autonomous or affected by neighboring cells. Here, we establish a genetic approach to study the clonality of differentiation in teratomas using a mixture of HESC lines. We first demonstrate, by means of 5-bromo-2'-deoxyuridine incorporation, that cell proliferation occurs throughout the teratoma, and that there are no clusters of undifferentiated-proliferating cells. Using a combination of laser capture microdissection and DNA fingerprinting analysis, we show that different cell lines contribute mutually to the same distinctive tissue structures. Further support for the nonclonal differentiation within the teratoma was achieved by fluorescence in situ hybridization analysis of sex chromosomes. We therefore suggest that in vivo differentiation of HESCs is polyclonal and, thus, may not be cell autonomous, stressing the need for a three-dimensional growth in order to achieve complex differentiation of HESCs. Disclosure of potential conflicts of interest is found at the end of this article.

Phenotypic plasticity and specialization in clonal versus non-clonal plants: A data synthesis

NASA Astrophysics Data System (ADS)

Fazlioglu, Fatih; Bonser, Stephen P.

2016-11-01

Reproductive strategies can be associated with ecological specialization and generalization. Clonal plants produce lineages adapted to the maternal habitat that can lead to specialization. However, clonal plants frequently display high phenotypic plasticity (e.g. clonal foraging for resources), factors linked to ecological generalization. Alternately, sexual reproduction can be associated with generalization via increasing genetic variation or specialization through rapid adaptive evolution. Moreover, specializing to high or low quality habitats can determine how phenotypic plasticity is expressed in plants. The specialization hypothesis predicts that specialization to good environments results in high performance trait plasticity and specialization to bad environments results in low performance trait plasticity. The interplay between reproductive strategies, phenotypic plasticity, and ecological specialization is important for understanding how plants adapt to variable environments. However, we currently have a poor understanding of these relationships. In this study, we addressed following questions: 1) Is there a relationship between phenotypic plasticity, specialization, and reproductive strategies in plants? 2) Do good habitat specialists express greater performance trait plasticity than bad habitat specialists? We searched the literature for studies examining plasticity for performance traits and functional traits in clonal and non-clonal plant species from different habitat types. We found that non-clonal (obligate sexual) plants expressed greater performance trait plasticity and functional trait plasticity than clonal plants. That is, non-clonal plants exhibited a specialist strategy where they perform well only in a limited range of habitats. Clonal plants expressed less performance loss across habitats and a more generalist strategy. In addition, specialization to good habitats did not result in greater performance trait plasticity. This result was contrary to the predictions of the specialization hypothesis. Overall, reproductive strategies are associated with ecological specialization or generalization through phenotypic plasticity. While specialization is common in plant populations, the evolution of specialization does not control the nature of phenotypic plasticity as predicted under the specialization hypothesis.

Demographic consequences of greater clonal than sexual reproduction in Dicentra canadensis.

PubMed

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 for resource investment in sexual reproduction at the individual level. However, chronic failure in sexual reproduction may exacerbate the imbalance between sexual and clonal reproduction and eventually lead to irreversible loss of sex in the population.

Extended phenotype and clinical subgroups in unilateral Meniere disease: A cross-sectional study with cluster analysis.

PubMed

Frejo, L; Martin-Sanz, E; Teggi, R; Trinidad, G; Soto-Varela, A; Santos-Perez, S; Manrique, R; Perez, N; Aran, I; Almeida-Branco, M S; Batuecas-Caletrio, A; Fraile, J; Espinosa-Sanchez, J M; Perez-Guillen, V; Perez-Garrigues, H; Oliva-Dominguez, M; Aleman, O; Benitez, J; Perez, P; Lopez-Escamez, J A

2017-12-01

To define clinical subgroups by cluster analysis in patients with unilateral Meniere disease (MD) and to compare them with the clinical subgroups found in bilateral MD. A cross-sectional study with a two-step cluster analysis. A tertiary referral multicenter study. Nine hundred and eighty-eight adult patients with unilateral MD. best predictors to define clinical subgroups with potential different aetiologies. We established five clusters in unilateral MD. Group 1 is the most frequently found, includes 53% of patients, and it is defined as the sporadic, classic MD without migraine and without autoimmune disorder (AD). Group 2 is found in 8% of patients, and it is defined by hearing loss, which antedates the vertigo episodes by months or years (delayed MD), without migraine or AD in most of cases. Group 3 involves 13% of patients, and it is considered familial MD, while group 4, which includes 15% of patients, is linked to the presence of migraine in all cases. Group 5 is found in 11% of patients and is defined by a comorbid AD. We found significant differences in the distribution of AD in clusters 3, 4 and 5 between patients with uni- and bilateral MD. Cluster analysis defines clinical subgroups in MD, and it extends the phenotype beyond audiovestibular symptoms. This classification will help to improve the phenotyping in MD and facilitate the selection of patients for randomised clinical trials. © 2017 John Wiley & Sons Ltd.

Subnanometer to nanometer transition metal CO oxidation catalysts

DOEpatents

Vajda, Stefan; Fortunelli, Alessandro; Yasumatsu, Hisato

2017-12-26

The present invention provides a catalyst defined in part by a conductive substrate; a film overlaying a surface of the substrate; and a plurality of metal clusters supported by the layer, wherein each cluster comprises between 8 and 11 atoms. Further provided is a catalyst defined in part by a conductive substrate; a layer overlaying a surface of the substrate; and a plurality of metal clusters supported by the layer, wherein each cluster comprises at least two metals.

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

PubMed Central

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

Clonal integration facilitates spread of Paspalum paspaloides from terrestrial to cadmium-contaminated aquatic habitats.

PubMed

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.

Clonal growth strategy, diversity and structure: A spatiotemporal response to sedimentation in tropical Cyperus papyrus swamps.

PubMed

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.

Clonal growth strategy, diversity and structure: A spatiotemporal response to sedimentation in tropical Cyperus papyrus swamps

PubMed Central

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. PMID:29338034

Detecting truly clonal alterations from multi-region profiling of tumours

PubMed Central

Werner, Benjamin; Traulsen, Arne; Sottoriva, Andrea; Dingli, David

2017-01-01

Modern cancer therapies aim at targeting tumour-specific alterations, such as mutations or neo-antigens, and maximal treatment efficacy requires that targeted alterations are present in all tumour cells. Currently, treatment decisions are based on one or a few samples per tumour, creating uncertainty on whether alterations found in those samples are actually present in all tumour cells. The probability of classifying clonal versus sub-clonal alterations from multi-region profiling of tumours depends on the earliest phylogenetic branching event during tumour growth. By analysing 181 samples from 10 renal carcinoma and 11 colorectal cancers we demonstrate that the information gain from additional sampling falls onto a simple universal curve. We found that in colorectal cancers, 30% of alterations identified as clonal with one biopsy proved sub-clonal when 8 samples were considered. The probability to overestimate clonal alterations fell below 1% in 7/11 patients with 8 samples per tumour. In renal cell carcinoma, 8 samples reduced the list of clonal alterations by 40% with respect to a single biopsy. The probability to overestimate clonal alterations remained as high as 92% in 7/10 renal cancer patients. Furthermore, treatment was associated with more unbalanced tumour phylogenetic trees, suggesting the need of denser sampling of tumours at relapse. PMID:28344344

Heterogeneous water supply affects growth and benefits of clonal integration between co-existing invasive and native Hydrocotyle species.

PubMed

Wang, Yong-Jian; Bai, Yun-Fei; Zeng, Shi-Qi; Yao, Bin; Wang, Wen; Luo, Fang-Li

2016-07-21

Spatial patchiness and temporal variability in water availability are common in nature under global climate change, which can remarkably influence adaptive responses of clonal plants, i.e. clonal integration (translocating resources between connected ramets). However, little is known about the effects of spatial patchiness and temporal heterogeneity in water on growth and clonal integration between congeneric invasive and native Hydrocotyle species. In a greenhouse experiment, we subjected severed or no severed (intact) fragments of Hydrocotyle vulgaris, a highly invasive species in China, and its co-existing, native congener H. sibthorpioides to different spatial patchiness (homogeneous and patchy) and temporal interval (low and high interval) in water supply. Clonal integration had significant positive effects on growth of both species. In the homogeneous water conditions, clonal integration greatly improved the growth in fragments of both species under low interval in water. However, in the patchy water conditions, clonal integration significantly increased growth in both ramets and fragments of H. vulgaris under high interval in water. Therefore, spatial patchiness and temporal interval in water altered the effects of clonal integration of both species, especially for H. vulgaris. The adaptation of H. vulgaris might lead to invasive growth and potential spread under the global water variability.

Detecting truly clonal alterations from multi-region profiling of tumours

NASA Astrophysics Data System (ADS)

Werner, Benjamin; Traulsen, Arne; Sottoriva, Andrea; Dingli, David

2017-03-01

Modern cancer therapies aim at targeting tumour-specific alterations, such as mutations or neo-antigens, and maximal treatment efficacy requires that targeted alterations are present in all tumour cells. Currently, treatment decisions are based on one or a few samples per tumour, creating uncertainty on whether alterations found in those samples are actually present in all tumour cells. The probability of classifying clonal versus sub-clonal alterations from multi-region profiling of tumours depends on the earliest phylogenetic branching event during tumour growth. By analysing 181 samples from 10 renal carcinoma and 11 colorectal cancers we demonstrate that the information gain from additional sampling falls onto a simple universal curve. We found that in colorectal cancers, 30% of alterations identified as clonal with one biopsy proved sub-clonal when 8 samples were considered. The probability to overestimate clonal alterations fell below 1% in 7/11 patients with 8 samples per tumour. In renal cell carcinoma, 8 samples reduced the list of clonal alterations by 40% with respect to a single biopsy. The probability to overestimate clonal alterations remained as high as 92% in 7/10 renal cancer patients. Furthermore, treatment was associated with more unbalanced tumour phylogenetic trees, suggesting the need of denser sampling of tumours at relapse.

Effects of Clonal Reproduction on Evolutionary Lag and Evolutionary Rescue.

PubMed

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.

Clonal sets of a binary relation

NASA Astrophysics Data System (ADS)

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.

Rise and fall of subclones from diagnosis to relapse in pediatric B-acute lymphoblastic leukaemia | Office of Cancer Genomics

Cancer.gov

There is incomplete understanding of genetic heterogeneity and clonal evolution during cancer progression. Here we use deep whole-exome sequencing to describe the clonal architecture and evolution of 20 pediatric B-acute lymphoblastic leukaemias from diagnosis to relapse. We show that clonal diversity is comparable at diagnosis and relapse and clonal survival from diagnosis to relapse is not associated with mutation burden.

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

PubMed Central

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

Kin Recognition in a Clonal Fish, Poecilia formosa

PubMed Central

Makowicz, Amber M.; Tiedemann, Ralph; Schlupp, Ingo

2016-01-01

Relatedness strongly influences social behaviors in a wide variety of species. For most species, the highest typical degree of relatedness is between full siblings with 50% shared genes. However, this is poorly understood in species with unusually high relatedness between individuals: clonal organisms. Although there has been some investigation into clonal invertebrates and yeast, nothing is known about kin selection in clonal vertebrates. We show that a clonal fish, the Amazon molly (Poecilia formosa), can distinguish between different clonal lineages, associating with genetically identical, sister clones, and use multiple sensory modalities. Also, they scale their aggressive behaviors according to the relatedness to other females: they are more aggressive to non-related clones. Our results demonstrate that even in species with very small genetic differences between individuals, kin recognition can be adaptive. Their discriminatory abilities and regulation of costly behaviors provides a powerful example of natural selection in species with limited genetic diversity. PMID:27483372

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

PubMed Central

Ramsköld, Daniel; Deng, Qiaolin; Johnsson, Per; Michaëlsson, Jakob; Frisén, Jonas; Sandberg, Rickard

2016-01-01

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 in vivo 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. Importantly, clonal aRME was detected but was surprisingly scarce (<1% of genes) and affected mainly the most low-expressed genes. Consequently, the overwhelming portion of 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. PMID:27668657

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

PubMed

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 (<1% of genes) and mainly affected the most weakly expressed genes. Consequently, the overwhelming majority of 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.

Clonality: an R package for testing clonal relatedness of two tumors from the same patient based on their genomic profiles.

PubMed

Ostrovnaya, Irina; Seshan, Venkatraman E; Olshen, Adam B; Begg, Colin B

2011-06-15

If a cancer patient develops multiple tumors, it is sometimes impossible to determine whether these tumors are independent or clonal based solely on pathological characteristics. Investigators have studied how to improve this diagnostic challenge by comparing the presence of loss of heterozygosity (LOH) at selected genetic locations of tumor samples, or by comparing genomewide copy number array profiles. We have previously developed statistical methodology to compare such genomic profiles for an evidence of clonality. We assembled the software for these tests in a new R package called 'Clonality'. For LOH profiles, the package contains significance tests. The analysis of copy number profiles includes a likelihood ratio statistic and reference distribution, as well as an option to produce various plots that summarize the results. Bioconductor (http://bioconductor.org/packages/release/bioc/html/Clonality.html) and http://www.mskcc.org/mskcc/html/13287.cfm.

Insights in Anaphylaxis and Clonal Mast Cell Disorders.

PubMed

González-de-Olano, David; Álvarez-Twose, Iván

2017-01-01

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].

Insights in Anaphylaxis and Clonal Mast Cell Disorders

PubMed Central

González-de-Olano, David; Álvarez-Twose, Iván

2017-01-01

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]. PMID:28740494

Genetic characterization of Hawaiian isolates of Plasmodium relictum reveals mixed-genotype infections

USGS Publications Warehouse

Jarvi, S.I.; Farias, M.E.M.; Atkinson, C.T.

2008-01-01

Background: The relatively recent introduction of a highly efficient mosquito vector and an avian pathogen (Plasmodium relictum) to an isolated island ecosystem with nai??ve, highly susceptible avian hosts provides a unique opportunity to investigate evolution of virulence in a natural system. Mixed infections can significantly contribute to the uncertainty in host-pathogen dynamics with direct impacts on virulence. Toward further understanding of how host-parasite and parasite-parasite relationships may impact virulence, this study characterizes within-host diversity of malaria parasite populations based on genetic analysis of the trap (thrombospondin-related anonymous protein) gene in isolates originating from Hawaii, Maui and Kauai Islands. Methods: A total of 397 clones were produced by nested PCR amplification and cloning of a 1664 bp fragment of the trap gene from two malarial isolates, K1 (Kauai) and KV115 (Hawaii) that have been used for experimental studies, and from additional isolates from wild birds on Kauai, Maui and Hawaii Islands. Diversity of clones was evaluated initially by RFLP-based screening, followed by complete sequencing of 33 selected clones. Results: RFLP analysis of trap revealed a minimum of 28 distinct RFLP haplotypes among the 397 clones from 18 birds. Multiple trap haplotypes were detected in every bird evaluated, with an average of 5.9 haplotypes per bird. Overall diversity did not differ between the experimental isolates, however, a greater number of unique haplotypes were detected in K1 than in KV115. We detected high levels of clonal diversity with clear delineation between isolates K1 and KV115 in a haplotype network. The patterns of within-host haplotype clustering are consistent with the possibility of a clonal genetic structure and rapid within-host mutation after infection. Conclusion: Avian malaria (P. relictum) and Avipoxvirus are the significant infectious diseases currently affecting the native Hawaiian avifauna. This study shows that clonal diversity of Hawaiian isolates of P. relictum is much higher than previously recognized. Mixed infections can significantly contribute to the uncertainty in host-pathogen dynamics with direct implications for host demographics, disease management strategies, and evolution of virulence. The results of this study indicate a widespread presence of multiple-genotype malaria infections with high clonal diversity in native birds of Hawaii, which when coupled with concurrent infection with Avipoxvirus, may significantly influence evolution of virulence. ?? 2008 Jarvi et al; licensee BioMed Central Ltd.

Novel immortal human cell lines reveal subpopulations in the nucleus pulposus

PubMed Central

2014-01-01

Introduction Relatively little is known about cellular subpopulations in the mature nucleus pulposus (NP). Detailed understanding of the ontogenetic, cellular and molecular characteristics of functional intervertebral disc (IVD) cell populations is pivotal to the successful development of cell replacement therapies and IVD regeneration. In this study, we aimed to investigate whether phenotypically distinct clonal cell lines representing different subpopulations in the human NP could be generated using immortalization strategies. Methods Nondegenerate healthy disc material (age range, 8 to 15 years) was obtained as surplus surgical material. Early passage NP monolayer cell cultures were initially characterized using a recently established NP marker set. NP cells were immortalized by simian virus 40 large T antigen (SV40LTag) and human telomerase reverse transcriptase expression. Immortalized cells were clonally expanded and characterized based on collagen type I, collagen type II, α1 (COL2A1), and SRY-box 9 (SOX9) protein expression profiles, as well as on expression of a subset of established in vivo NP cell lineage markers. Results A total of 54 immortal clones were generated. Profiling of a set of novel NP markers (CD24, CA12, PAX1, PTN, FOXF1 and KRT19 mRNA) in a representative set of subclones substantiated successful immortalization of multiple cellular subpopulations from primary isolates and confirmed their NP origin and/or phenotype. We were able to identify two predominant clonal NP subtypes based on their morphological characteristics and their ability to induce SOX9 and COL2A1 under conventional differentiation conditions. In addition, cluster of differentiation 24 (CD24)–negative NP responder clones formed spheroid structures in various culture systems, suggesting the preservation of a more immature phenotype compared to CD24-positive nonresponder clones. Conclusions Here we report the generation of clonal NP cell lines from nondegenerate human IVD tissue and present a detailed characterization of NP cellular subpopulations. Differential cell surface marker expression and divergent responses to differentiation conditions suggest that the NP subtypes may correspond to distinct maturation stages and represent distinct NP cell subpopulations. Hence, we provide evidence that the immortalization strategy that we applied is capable of detecting cell heterogeneity in the NP. Our cell lines yield novel insights into NP biology and provide promising new tools for studies of IVD development, cell function and disease. PMID:24972717

Novel immortal human cell lines reveal subpopulations in the nucleus pulposus.

PubMed

van den Akker, Guus G H; Surtel, Don A M; Cremers, Andy; Rodrigues-Pinto, Ricardo; Richardson, Stephen M; Hoyland, Judith A; van Rhijn, Lodewijk W; Welting, Tim J M; Voncken, Jan Willem

2014-06-27

Relatively little is known about cellular subpopulations in the mature nucleus pulposus (NP). Detailed understanding of the ontogenetic, cellular and molecular characteristics of functional intervertebral disc (IVD) cell populations is pivotal to the successful development of cell replacement therapies and IVD regeneration. In this study, we aimed to investigate whether phenotypically distinct clonal cell lines representing different subpopulations in the human NP could be generated using immortalization strategies. Nondegenerate healthy disc material (age range, 8 to 15 years) was obtained as surplus surgical material. Early passage NP monolayer cell cultures were initially characterized using a recently established NP marker set. NP cells were immortalized by simian virus 40 large T antigen (SV40LTag) and human telomerase reverse transcriptase expression. Immortalized cells were clonally expanded and characterized based on collagen type I, collagen type II, α1 (COL2A1), and SRY-box 9 (SOX9) protein expression profiles, as well as on expression of a subset of established in vivo NP cell lineage markers. A total of 54 immortal clones were generated. Profiling of a set of novel NP markers (CD24, CA12, PAX1, PTN, FOXF1 and KRT19 mRNA) in a representative set of subclones substantiated successful immortalization of multiple cellular subpopulations from primary isolates and confirmed their NP origin and/or phenotype. We were able to identify two predominant clonal NP subtypes based on their morphological characteristics and their ability to induce SOX9 and COL2A1 under conventional differentiation conditions. In addition, cluster of differentiation 24 (CD24)-negative NP responder clones formed spheroid structures in various culture systems, suggesting the preservation of a more immature phenotype compared to CD24-positive nonresponder clones. Here we report the generation of clonal NP cell lines from nondegenerate human IVD tissue and present a detailed characterization of NP cellular subpopulations. Differential cell surface marker expression and divergent responses to differentiation conditions suggest that the NP subtypes may correspond to distinct maturation stages and represent distinct NP cell subpopulations. Hence, we provide evidence that the immortalization strategy that we applied is capable of detecting cell heterogeneity in the NP. Our cell lines yield novel insights into NP biology and provide promising new tools for studies of IVD development, cell function and disease.

Preleukaemic clonal haemopoiesis and risk of therapy-related myeloid neoplasms: a case-control study.

PubMed

Takahashi, Koichi; Wang, Feng; Kantarjian, Hagop; Doss, Denaha; Khanna, Kanhav; Thompson, Erika; Zhao, Li; Patel, Keyur; Neelapu, Sattva; Gumbs, Curtis; Bueso-Ramos, Carlos; DiNardo, Courtney D; Colla, Simona; Ravandi, Farhad; Zhang, Jianhua; Huang, Xuelin; Wu, Xifeng; Samaniego, Felipe; Garcia-Manero, Guillermo; Futreal, P Andrew

2017-01-01

Therapy-related myeloid neoplasms are secondary malignancies that are often fatal, but their risk factors are not well understood. Evidence suggests that individuals with clonal haemopoiesis have increased risk of developing haematological malignancies. We aimed to identify whether patients with cancer who have clonal haemopoiesis are at an increased risk of developing therapy-related myeloid neoplasms. We did this retrospective case-control study to compare the prevalence of clonal haemopoiesis between patients treated for cancer who later developed therapy-related myeloid neoplasms (cases) and patients who did not develop these neoplasms (controls). All patients in both case and control groups were treated at MD Anderson Cancer Center (Houston, TX, USA) from 1997 to 2015. We used the institutional medical database to locate these patients. Patients were included as cases if they were treated for a primary cancer, subsequently developed therapy-related myeloid neoplasms, and had available paired samples of bone marrow from the time of therapy-related myeloid neoplasm diagnosis and peripheral blood from the time of primary cancer diagnosis. Patients were eligible for inclusion as age-matched controls if they were treated for lymphoma, received combination chemotherapy, and did not develop therapy-related myeloid neoplasms after at least 5 years of follow-up. We used molecular barcode sequencing of 32 genes on the pretreatment peripheral blood samples to detect clonal haemopoiesis. For cases, we also used targeted gene sequencing on bone marrow samples and investigated clonal evolution from clonal haemopoiesis to the development of therapy-related myeloid neoplasms. To further clarify the association between clonal haemopoiesis and therapy-related myeloid neoplasm development, we also analysed the prevalence of clonal haemopoiesis in an external cohort of patients with lymphoma who were treated in a randomised trial of front-line chemotherapy with cyclophosphamide, doxorubicin, vincristine, and prednisone, with or without melatonin. This trial was done at MD Anderson Cancer Center between 1999 and 2001 (protocol number 98-009). We identified 14 cases and 54 controls. Of the 14 cases, we detected clonal haemopoiesis in the peripheral blood samples of ten (71%) patients. We detected clonal haemopoiesis in 17 (31%) of the 54 controls. The cumulative incidence of therapy-related myeloid neoplasms in both cases and controls at 5 years was significantly higher in patients with clonal haemopoiesis (30%, 95% CI 16-51) than in those without (7%, 2-21; p=0·016). In the external cohort, five (7%) of 74 patients developed therapy-related myeloid neoplasms, of whom four (80%) had clonal haemopoiesis; 11 (16%) of 69 patients who did not develop therapy-related myeloid neoplasms had clonal haemopoiesis. In the external cohort, the cumulative incidence of therapy-related myeloid neoplasms at 10 years was significantly higher in patients with clonal haemopoiesis (29%, 95% CI 8-53) than in those without (0%, 0-0; p=0·0009). In a multivariate Fine and Gray model based on the external cohort, the presence of clonal haemopoiesis significantly increased the risk of therapy-related myeloid neoplasm development (hazard ratio 13·7, 95% CI 1·7-108·7; p=0·013). Preleukaemic clonal haemopoiesis is common in patients with therapy-related myeloid neoplasms at the time of their primary cancer diagnosis and before they have been exposed to treatment. Our results suggest that clonal haemopoiesis could be used as a predictive marker to identify patients with cancer who are at risk of developing therapy-related myeloid neoplasms. A prospective trial to validate this concept is warranted. Cancer Prevention Research Institute of Texas, Red and Charline McCombs Institute for the Early Detection and Treatment of Cancer, NIH through MD Anderson Cancer Center Support Grant, and the MD Anderson MDS & AML Moon Shots Program. Copyright © 2017 Elsevier Ltd. All rights reserved.

World Health Organization-defined eosinophilic disorders: 2017 update on diagnosis, risk stratification, and management.

PubMed

Gotlib, Jason

2017-11-01

The eosinophilias encompass a broad range of nonhematologic (secondary or reactive) and hematologic (primary, clonal) disorders with potential for end-organ damage. Hypereosinophilia has generally been defined as a peripheral blood eosinophil count greater than 1500/mm 3 and may be associated with tissue damage. After exclusion of secondary causes of eosinophilia, diagnostic evaluation of primary eosinophilias relies on a combination of morphologic review of the blood and marrow, standard cytogenetics, fluorescent in situ-hybridization, flow immunocytometry, and T-cell clonality assessment to detect histopathologic or clonal evidence for an acute or chronic myeloid or lymphoproliferative disorder. Disease prognosis relies on identifying the subtype of eosinophilia. After evaluation of secondary causes of eosinophilia, the 2016 World Health Organization endorses a semi-molecular classification scheme of disease subtypes which includes the major category "myeloid/lymphoid neoplasms with eosinophilia and rearrangement of PDGFRA, PDGFRB, or FGFR1 or with PCM1-JAK2," and the "MPN subtype, chronic eosinophilic leukemia, not otherwise specified" (CEL, NOS). Lymphocyte-variant hypereosinophilia is an aberrant T-cell clone-driven reactive eosinophila, and idiopathic hypereosinophilic syndrome (HES) is a diagnosis of exclusion. The goal of therapy is to mitigate eosinophil-mediated organ damage. For patients with milder forms of eosinophilia (e.g., < 1500/mm 3 ) without symptoms or signs of organ involvement, a watch and wait approach with close-follow-up may be undertaken. Identification of rearranged PDGFRA or PDGFRB is critical because of the exquisite responsiveness of these diseases to imatinib. Corticosteroids are first-line therapy for patients with lymphocyte-variant hypereosinophilia and HES. Hydroxyurea and interferon-alpha have demonstrated efficacy as initial treatment and steroid-refractory cases of HES. In addition to hydroxyurea, second line cytotoxic chemotherapy agents and hematopoietic cell transplant have been used for aggressive forms of HES and CEL with outcomes reported for limited numbers of patients. The use of antibodies against interleukin-5 (IL-5) (mepolizumab), the IL-5 receptor (benralizumab), and CD52 (alemtuzumab), as well as other targets on eosinophils remains an active area of investigation. © 2017 Wiley Periodicals, Inc.

CLONAL EVOLUTION IN CANCER

PubMed Central

Greaves, Mel; Maley, Carlo C.

2012-01-01

Cancers evolve by a reiterative process of clonal expansion, genetic diversification and clonal selection within the adaptive landscapes of tissue ecosystems. The dynamics are complex with highly variable patterns of genetic diversity and resultant clonal architecture. Therapeutic intervention may decimate cancer clones, and erode their habitats, but inadvertently provides potent selective pressure for the expansion of resistant variants. The inherently Darwinian character of cancer lies at the heart of therapeutic failure but perhaps also holds the key to more effective control. PMID:22258609

Biological and Clinical Implications of Clonal Heterogeneity and Clonal Evolution in Multiple Myeloma.

PubMed

Bianchi, Giada; Ghobrial, Irene M

Clonal heterogeneity and clonal evolution have emerged as critical concepts in the field of oncology over the past four decades, largely thanks to the implementation of novel technologies such as comparative genomic hybridization, whole genome/exome sequencing and epigenetic analysis. Along with the identification of cancer stem cells in the majority of neoplasia, the recognition of intertumor and intratumor variability has provided a novel perspective to understand the mechanisms behind tumor evolution and its implication in terms of treatment failure and cancer relapse or recurrence. First hypothesized over two decades ago, clonal heterogeneity and clonal evolution have been confirmed in multiple myeloma (MM), an incurable cancer of plasma cells, almost universally preceded by a pre-malignant conditioned named monoclonal gammopathy of undetermined significance (MGUS). The genetic events and molecular mechanisms underlying such evolution have been difficult to dissect. Moreover, while a role for the bone marrow microenvironment in supporting MM cell survival, proliferation and drug-resistance has been well established, whether it is directly involved in driving evolution from MGUS to MM is at present unclear. We present in this review a historical excursus on the concepts of clonal heterogeneity and clonal evolution in MM with a special emphasis on their role in the progression from MGUS to MM; the contribution of the microenvironment; and the clinical implications in terms of resistance to treatment and disease relapse/recurrence.

Biological and Clinical Implications of Clonal Heterogeneity and Clonal Evolution in Multiple Myeloma

PubMed Central

Bianchi, Giada; Ghobrial, Irene M.

2015-01-01

Clonal heterogeneity and clonal evolution have emerged as critical concepts in the field of oncology over the past four decades, largely thanks to the implementation of novel technologies such as comparative genomic hybridization, whole genome/exome sequencing and epigenetic analysis. Along with the identification of cancer stem cells in the majority of neoplasia, the recognition of intertumor and intratumor variability has provided a novel perspective to understand the mechanisms behind tumor evolution and its implication in terms of treatment failure and cancer relapse or recurrence. First hypothesized over two decades ago, clonal heterogeneity and clonal evolution have been confirmed in multiple myeloma (MM), an incurable cancer of plasma cells, almost universally preceded by a pre-malignant conditioned named monoclonal gammopathy of undetermined significance (MGUS). The genetic events and molecular mechanisms underlying such evolution have been difficult to dissect. Moreover, while a role for the bone marrow microenvironment in supporting MM cell survival, proliferation and drug-resistance has been well established, whether it is directly involved in driving evolution from MGUS to MM is at present unclear. We present in this review a historical excursus on the concepts of clonal heterogeneity and clonal evolution in MM with a special emphasis on their role in the progression from MGUS to MM; the contribution of the microenvironment; and the clinical implications in terms of resistance to treatment and disease relapse/recurrence. PMID:25705146

Strong but diverging clonality - climate relationships of different plant clades explain weak overall pattern across China.

PubMed

Ye, Duo; Liu, Guofang; Song, Yao-Bin; Cornwell, William K; Dong, Ming; Cornelissen, Johannes H C

2016-06-01

The clonal strategy should be relatively important in stressful environments (i.e. of low resource availability or harsh climate), e.g. in cold habitats. However, our understanding of the distribution pattern of clonality along environmental gradients is still far from universal. The weakness and inconsistency of overall clonality-climate relationships across taxa, as reported in previous studies, may be due to different phylogenetic lineages having fundamental differences in functional traits other than clonality determining their climate response. Thus, in this study we compared the clonality-climate relationships along a latitudinal gradient within and between different lineages at several taxonomic levels, including four major angiosperm lineages (Magnoliidae, Monocotyledoneae, Superrosidae and Superasteridae), orders and families. To this aim we used a species clonality dataset for 4015 vascular plant species in 545 terrestrial communities across China. Our results revealed clear predictive patterns of clonality proportion in relation to environmental gradients for the predominant representatives of each of the taxonomic levels above, but the relationships differed in shape and strength between the 4 major angiosperm lineages, between the 12 orders and between the 12 families. These different relationships canceled out one another when all lineages at a certain taxonomic level were pooled. Our findings highlight the importance of explicitly accounting for the functional or taxonomic scale for studying variation in plant ecological strategy across environmental gradients.

The clonal origin and clonal evolution of epithelial tumours

PubMed Central

Garcia, Sergio Britto; Novelli, Marco; Wright, Nicholas A

2000-01-01

While the origin of tumours, whether from one cell or many, has been a source of fascination for experimental oncologists for some time, in recent years there has been a veritable explosion of information about the clonal architecture of tumours and their antecedents, stimulated, in the main, by the ready accessibility of new molecular techniques. While most of these new results have apparently confirmed the monoclonal origin of human epithelial (and other) tumours, there are a significant number of studies in which this conclusion just cannot be made. Moreover, analysis of many articles show that the potential impact of such considerations as patch size and clonal evolution on determinations of clonality have largely been ignored, with the result that a number of these studies are confounded. However, the clonal architecture of preneoplastic lesions provide some interesting insights — many lesions which might have been hitherto regarded as hyperplasias are apparently clonal in derivation. If this is indeed true, it calls into some question our hopeful corollary that a monoclonal origin presages a neoplastic habitus. Finally, it is clear, for many reasons, that methods of analysis which involve the disaggregation of tissues, albeit microdissected, are far from ideal and we should be putting more effort into techniques where the clonal architecture of normal tissues, preneoplastic and preinvasive lesions and their derivative tumours can be directly visualized in situ. PMID:10762440

Consequences of clonality for sexual fitness: Clonal expansion enhances fitness under spatially restricted dispersal.

PubMed

Van Drunen, Wendy E; van Kleunen, Mark; Dorken, Marcel E

2015-07-21

Clonality is a pervasive feature of sessile organisms, but this form of asexual reproduction is thought to interfere with sexual fitness via the movement of gametes among the modules that comprise the clone. This within-clone movement of gametes is expected to reduce sexual fitness via mate limitation of male reproductive success and, in some cases, via the production of highly inbred (i.e., self-fertilized) offspring. However, clonality also results in the spatial expansion of the genetic individual (i.e., genet), and this should decrease distances gametes and sexually produced offspring must travel to avoid competing with other gametes and offspring from the same clone. The extent to which any negative effects of clonality on mating success might be offset by the positive effects of spatial expansion is poorly understood. Here, we develop spatially explicit models in which fitness was determined by the success of genets through their male and female sex functions. Our results indicate that clonality serves to increase sexual fitness when it is associated with the outward expansion of the genet. Our models further reveal that the main fitness benefit of clonal expansion might occur through the dispersal of offspring over a wider area compared with nonclonal phenotypes. We conclude that, instead of interfering with sexual reproduction, clonal expansion should often serve to enhance sexual fitness.

Assessing the genome level diversity of Listeria monocytogenes from contaminated ice cream and environmental samples linked to a listeriosis outbreak in the United States.

PubMed

Chen, Yi; Luo, Yan; Curry, Phillip; Timme, Ruth; Melka, David; Doyle, Matthew; Parish, Mickey; Hammack, Thomas S; Allard, Marc W; Brown, Eric W; Strain, Errol A

2017-01-01

A listeriosis outbreak in the United States implicated contaminated ice cream produced by one company, which operated 3 facilities. We performed single nucleotide polymorphism (SNP)-based whole genome sequencing (WGS) analysis on Listeria monocytogenes from food, environmental and clinical sources, identifying two clusters and a single branch, belonging to PCR serogroup IIb and genetic lineage I. WGS Cluster I, representing one outbreak strain, contained 82 food and environmental isolates from Facility I and 4 clinical isolates. These isolates differed by up to 29 SNPs, exhibited 9 pulsed-field gel electrophoresis (PFGE) profiles and multilocus sequence typing (MLST) sequence type (ST) 5 of clonal complex 5 (CC5). WGS Cluster II contained 51 food and environmental isolates from Facility II, 4 food isolates from Facility I and 5 clinical isolates. Among them the isolates from Facility II and clinical isolates formed a clade and represented another outbreak strain. Isolates in this clade differed by up to 29 SNPs, exhibited 3 PFGE profiles and ST5. The only isolate collected from Facility III belonged to singleton ST489, which was in a single branch separate from Clusters I and II, and was not associated with the outbreak. WGS analyses clustered together outbreak-associated isolates exhibiting multiple PFGE profiles, while differentiating them from epidemiologically unrelated isolates that exhibited outbreak PFGE profiles. The complete genome of a Cluster I isolate allowed the identification and analyses of putative prophages, revealing that Cluster I isolates differed by the gain or loss of three putative prophages, causing the banding pattern differences among all 3 AscI-PFGE profiles observed in Cluster I isolates. WGS data suggested that certain ice cream varieties and/or production lines might have contamination sources unique to them. The SNP-based analysis was able to distinguish CC5 as a group from non-CC5 isolates and differentiate among CC5 isolates from different outbreaks/incidents.

Assessing the genome level diversity of Listeria monocytogenes from contaminated ice cream and environmental samples linked to a listeriosis outbreak in the United States

PubMed Central

Chen, Yi; Luo, Yan; Curry, Phillip; Timme, Ruth; Melka, David; Doyle, Matthew; Parish, Mickey; Hammack, Thomas S.; Allard, Marc W.; Brown, Eric W.; Strain, Errol A.

2017-01-01

A listeriosis outbreak in the United States implicated contaminated ice cream produced by one company, which operated 3 facilities. We performed single nucleotide polymorphism (SNP)-based whole genome sequencing (WGS) analysis on Listeria monocytogenes from food, environmental and clinical sources, identifying two clusters and a single branch, belonging to PCR serogroup IIb and genetic lineage I. WGS Cluster I, representing one outbreak strain, contained 82 food and environmental isolates from Facility I and 4 clinical isolates. These isolates differed by up to 29 SNPs, exhibited 9 pulsed-field gel electrophoresis (PFGE) profiles and multilocus sequence typing (MLST) sequence type (ST) 5 of clonal complex 5 (CC5). WGS Cluster II contained 51 food and environmental isolates from Facility II, 4 food isolates from Facility I and 5 clinical isolates. Among them the isolates from Facility II and clinical isolates formed a clade and represented another outbreak strain. Isolates in this clade differed by up to 29 SNPs, exhibited 3 PFGE profiles and ST5. The only isolate collected from Facility III belonged to singleton ST489, which was in a single branch separate from Clusters I and II, and was not associated with the outbreak. WGS analyses clustered together outbreak-associated isolates exhibiting multiple PFGE profiles, while differentiating them from epidemiologically unrelated isolates that exhibited outbreak PFGE profiles. The complete genome of a Cluster I isolate allowed the identification and analyses of putative prophages, revealing that Cluster I isolates differed by the gain or loss of three putative prophages, causing the banding pattern differences among all 3 AscI-PFGE profiles observed in Cluster I isolates. WGS data suggested that certain ice cream varieties and/or production lines might have contamination sources unique to them. The SNP-based analysis was able to distinguish CC5 as a group from non-CC5 isolates and differentiate among CC5 isolates from different outbreaks/incidents. PMID:28166293

Characterization of a Large Cluster of Influenza A(H1N1)pdm09 Viruses Cross-Resistant to Oseltamivir and Peramivir during the 2013-2014 Influenza Season in Japan

PubMed Central

Takashita, Emi; Kiso, Maki; Fujisaki, Seiichiro; Yokoyama, Masaru; Nakamura, Kazuya; Shirakura, Masayuki; Sato, Hironori; Odagiri, Takato; Kawaoka, Yoshihiro

2015-01-01

Between September 2013 and July 2014, 2,482 influenza 2009 pandemic A(H1N1) [A(H1N1)pdm09] viruses were screened in Japan for the H275Y substitution in their neuraminidase (NA) protein, which confers cross-resistance to oseltamivir and peramivir. We found that a large cluster of the H275Y mutant virus was present prior to the main influenza season in Sapporo/Hokkaido, with the detection rate for this mutant virus reaching 29% in this area. Phylogenetic analysis suggested the clonal expansion of a single mutant virus in Sapporo/Hokkaido. To understand the reason for this large cluster, we examined the in vitro and in vivo properties of the mutant virus. We found that it grew well in cell culture, with growth comparable to that of the wild-type virus. The cluster virus also replicated well in the upper respiratory tract of ferrets and was transmitted efficiently between ferrets by way of respiratory droplets. Almost all recently circulating A(H1N1)pdm09 viruses, including the cluster virus, possessed two substitutions in NA, V241I and N369K, which are known to increase replication and transmission fitness. A structural analysis of NA predicted that a third substitution (N386K) in the NA of the cluster virus destabilized the mutant NA structure in the presence of the V241I and N369K substitutions. Our results suggest that the cluster virus retained viral fitness to spread among humans and, accordingly, caused the large cluster in Sapporo/Hokkaido. However, the mutant NA structure was less stable than that of the wild-type virus. Therefore, once the wild-type virus began to circulate in the community, the mutant virus could not compete and faded out. PMID:25691635

Multimorbidity and health-related quality of life (HRQoL) in a nationally representative population sample: implications of count versus cluster method for defining multimorbidity on HRQoL.

PubMed

Wang, Lili; Palmer, Andrew J; Cocker, Fiona; Sanderson, Kristy

2017-01-09

No universally accepted definition of multimorbidity (MM) exists, and implications of different definitions have not been explored. This study examined the performance of the count and cluster definitions of multimorbidity on the sociodemographic profile and health-related quality of life (HRQoL) in a general population. Data were derived from the nationally representative 2007 Australian National Survey of Mental Health and Wellbeing (n = 8841). The HRQoL scores were measured using the Assessment of Quality of Life (AQoL-4D) instrument. The simple count (2+ & 3+ conditions) and hierarchical cluster methods were used to define/identify clusters of multimorbidity. Linear regression was used to assess the associations between HRQoL and multimorbidity as defined by the different methods. The assessment of multimorbidity, which was defined using the count method, resulting in the prevalence of 26% (MM2+) and 10.1% (MM3+). Statistically significant clusters identified through hierarchical cluster analysis included heart or circulatory conditions (CVD)/arthritis (cluster-1, 9%) and major depressive disorder (MDD)/anxiety (cluster-2, 4%). A sensitivity analysis suggested that the stability of the clusters resulted from hierarchical clustering. The sociodemographic profiles were similar between MM2+, MM3+ and cluster-1, but were different from cluster-2. HRQoL was negatively associated with MM2+ (β: -0.18, SE: -0.01, p < 0.001), MM3+ (β: -0.23, SE: -0.02, p < 0.001), cluster-1 (β: -0.10, SE: 0.01, p < 0.001) and cluster-2 (β: -0.36, SE: 0.01, p < 0.001). Our findings confirm the existence of an inverse relationship between multimorbidity and HRQoL in the Australian population and indicate that the hierarchical clustering approach is validated when the outcome of interest is HRQoL from this head-to-head comparison. Moreover, a simple count fails to identify if there are specific conditions of interest that are driving poorer HRQoL. Researchers should exercise caution when selecting a definition of multimorbidity because it may significantly influence the study outcomes.

Somatic mutations contribute to genotypic diversity in sterile and fertile populations of the threatened shrub, Grevillea rhizomatosa (Proteaceae).

PubMed

Gross, C L; Nelson, Penelope A; Haddadchi, Azadeh; Fatemi, Mohammad

2012-02-01

Grevillea rhizomatosa is a spreading shrub which exhibits multiple breeding strategies within a narrow area in the fire-prone heathlands of eastern Australia. Reproductive strategies include self-compatibility, self-incompatibility and clonality (with and without sterility). The close proximity of contrasting breeding systems provides an opportunity to explore the evolution of sterility and to compare and contrast the origins of genotypic diversity (recombinant or somatic) against degrees of sexual expression. ISSR markers for 120 band positions (putative loci) were used to compare genetic diversity among five populations at a macro-scale of 5 m between samples (n = 244 shrubs), and at a micro-scale of nearest neighbours for all plants in five 25-m(2) quadrats with contrasting fertilities (n = 162 shrubs). Nearest-neighbour sampling included several clusters of connected ramets. Matrix incompatibility (MIC) analyses were used to evaluate the relative contribution of recombination and somatic mutation to genotype diversity. High levels of genotypic diversity were found in all populations regardless of fertilities (fertile populations, G/N ≥ 0·94; sterile populations, G/N ≥ 0·97) and most sterile populations had a unique genetic profile. Somatic mutations were detected along connected ramets in ten out of 42 ramet clusters. MIC analyses showed that somatic mutations have contributed to diversity in all populations and particularly so in sterile populations. Somatic mutations contribute significantly to gene diversity in sterile populations of Grevillea rhizomatosa, the accumulation of which is the likely cause of male and female sterility. High levels of genetic diversity therefore may not always be synonymous with sexual fitness and genetic health. We hypothesize that frequent fires drive selection for clonal reproduction, at the cost of flowering such that sexual functions are not maintained through selection, and the build-up of somatic mutations in meristems results in high genotype diversity at the cost of pollen and ovule fertilities.

Discriminating micropathogen lineages and their reticulate evolution through graph theory-based network analysis: the case of Trypanosoma cruzi, the agent of Chagas disease.

PubMed

Arnaud-Haond, Sophie; Moalic, Yann; Barnabé, Christian; Ayala, Francisco José; Tibayrenc, Michel

2014-01-01

Micropathogens (viruses, bacteria, fungi, parasitic protozoa) share a common trait, which is partial clonality, with wide variance in the respective influence of clonality and sexual recombination on the dynamics and evolution of taxa. The discrimination of distinct lineages and the reconstruction of their phylogenetic history are key information to infer their biomedical properties. However, the phylogenetic picture is often clouded by occasional events of recombination across divergent lineages, limiting the relevance of classical phylogenetic analysis and dichotomic trees. We have applied a network analysis based on graph theory to illustrate the relationships among genotypes of Trypanosoma cruzi, the parasitic protozoan responsible for Chagas disease, to identify major lineages and to unravel their past history of divergence and possible recombination events. At the scale of T. cruzi subspecific diversity, graph theory-based networks applied to 22 isoenzyme loci (262 distinct Multi-Locus-Enzyme-Electrophoresis -MLEE) and 19 microsatellite loci (66 Multi-Locus-Genotypes -MLG) fully confirms the high clustering of genotypes into major lineages or "near-clades". The release of the dichotomic constraint associated with phylogenetic reconstruction usually applied to Multilocus data allows identifying putative hybrids and their parental lineages. Reticulate topology suggests a slightly different history for some of the main "near-clades", and a possibly more complex origin for the putative hybrids than hitherto proposed. Finally the sub-network of the near-clade T. cruzi I (28 MLG) shows a clustering subdivision into three differentiated lesser near-clades ("Russian doll pattern"), which confirms the hypothesis recently proposed by other investigators. The present study broadens and clarifies the hypotheses previously obtained from classical markers on the same sets of data, which demonstrates the added value of this approach. This underlines the potential of graph theory-based network analysis for describing the nature and relationships of major pathogens, thereby opening stimulating prospects to unravel the organization, dynamics and history of major micropathogen lineages.

Identification of chronic rhinosinusitis phenotypes using cluster analysis.

PubMed

Soler, Zachary M; Hyer, J Madison; Ramakrishnan, Viswanathan; Smith, Timothy L; Mace, Jess; Rudmik, Luke; Schlosser, Rodney J

2015-05-01

Current clinical classifications of chronic rhinosinusitis (CRS) have been largely defined based upon preconceived notions of factors thought to be important, such as polyp or eosinophil status. Unfortunately, these classification systems have little correlation with symptom severity or treatment outcomes. Unsupervised clustering can be used to identify phenotypic subgroups of CRS patients, describe clinical differences in these clusters and define simple algorithms for classification. A multi-institutional, prospective study of 382 patients with CRS who had failed initial medical therapy completed the Sino-Nasal Outcome Test (SNOT-22), Rhinosinusitis Disability Index (RSDI), Medical Outcomes Study Short Form-12 (SF-12), Pittsburgh Sleep Quality Index (PSQI), and Patient Health Questionnaire (PHQ-2). Objective measures of CRS severity included Brief Smell Identification Test (B-SIT), CT, and endoscopy scoring. All variables were reduced and unsupervised hierarchical clustering was performed. After clusters were defined, variations in medication usage were analyzed. Discriminant analysis was performed to develop a simplified, clinically useful algorithm for clustering. Clustering was largely determined by age, severity of patient reported outcome measures, depression, and fibromyalgia. CT and endoscopy varied somewhat among clusters. Traditional clinical measures, including polyp/atopic status, prior surgery, B-SIT and asthma, did not vary among clusters. A simplified algorithm based upon productivity loss, SNOT-22 score, and age predicted clustering with 89% accuracy. Medication usage among clusters did vary significantly. A simplified algorithm based upon hierarchical clustering is able to classify CRS patients and predict medication usage. Further studies are warranted to determine if such clustering predicts treatment outcomes. © 2015 ARS-AAOA, LLC.

Enforced Clonality Confers a Fitness Advantage

PubMed Central

Martínková, Jana; Klimešová, Jitka

2016-01-01

In largely clonal plants, splitting of a maternal plant into potentially independent plants (ramets) is usually spontaneous; however, such fragmentation also occurs in otherwise non-clonal species due to application of external force. This process might play an important yet largely overlooked role for otherwise non-clonal plants by providing a mechanism to regenerate after disturbance. Here, in a 5-year garden experiment on two short-lived, otherwise non-clonal species, Barbarea vulgaris and Barbarea stricta, we compared the fitness of plants fragmented by simulated disturbance (“enforced ramets”) both with plants that contemporaneously originate in seed and with individuals unscathed by the disturbance event. Because the ability to regrow from fragments is related to plant age and stored reserves, we compared the effects of disturbance applied during three different ontogenetic stages of the plants. In B. vulgaris, enforced ramet fitness was higher than the measured fitness values of both uninjured plants and plants established from seed after the disturbance. This advantage decreased with increasing plant age at the time of fragmentation. In B. stricta, enforced ramet fitness was lower than or similar to fitness of uninjured plants and plants grown from seed. Our results likely reflect the habitat preferences of the study species, as B. vulgaris occurs in anthropogenic, disturbed habitats where body fragmentation is more probable and enforced clonality thus more advantageous than in the more natural habitats preferred by B. stricta. Generalizing from our results, we see that increased fitness yielded by enforced clonality would confer an evolutionary advantage in the face of disturbance, especially in habitats where a seed bank has not been formed, e.g., during invasion or colonization. Our results thus imply that enforced clonality should be taken into account when studying population dynamics and life strategies of otherwise non-clonal species in disturbed habitats. PMID:26858732

Differential influence of clonal integration on morphological and growth responses to light in two invasive herbs.

PubMed

Xu, Cheng-Yuan; Schooler, Shon S; Van Klinken, Rieks D

2012-01-01

In contrast to seeds, high sensitivity of vegetative fragments to unfavourable environments may limit the expansion of clonal invasive plants. However, clonal integration promotes the establishment of propagules in less suitable habitats and may facilitate the expansion of clonal invaders into intact native communities. Here, we examine the influence of clonal integration on the morphology and growth of ramets in two invasive plants, Alternanthera philoxeroides and Phyla canescens, under varying light conditions. In a greenhouse experiment, branches, connected ramets and severed ramets of the same mother plant were exposed under full sun and 85% shade and their morphological and growth responses were assessed. The influence of clonal integration on the light reaction norm (connection×light interaction) of daughter ramets was species-specific. For A. philoxeroides, clonal integration evened out the light response (total biomass, leaf mass per area, and stem number, diameter and length) displayed in severed ramets, but these connection×light interactions were largely absent for P. canescens. Nevertheless, for both species, clonal integration overwhelmed light effect in promoting the growth of juvenile ramets during early development. Also, vertical growth, as an apparent shade acclimation response, was more prevalent in severed ramets than in connected ramets. Finally, unrooted branches displayed smaller organ size and slower growth than connected ramets, but the pattern of light reaction was similar, suggesting mother plants invest in daughter ramets prior to their own branches. Clonal integration modifies light reaction norms of morphological and growth traits in a species-specific manner for A. philoxeroides and P. canescens, but it improves the establishment of juvenile ramets of both species in light-limiting environments by promoting their growth during early development. This factor may be partially responsible for their ability to successfully colonize native plant communities.

Low incidence of clonality in cold water corals revealed through the novel use of standardized protocol adapted to deep sea sampling

USGS Publications Warehouse

Becheler, Ronan; Cassone, Anne-Laure; Noel, Philippe; Mouchel, Olivier; Morrison, Cheryl L.; Arnaud-Haond, Sophie

2017-01-01

Sampling in the deep sea is a technical challenge, which has hindered the acquisition of robust datasets that are necessary to determine the fine-grained biological patterns and processes that may shape genetic diversity. Estimates of the extent of clonality in deep-sea species, despite the importance of clonality in shaping the local dynamics and evolutionary trajectories, have been largely obscured by such limitations. Cold-water coral reefs along European margins are formed mainly by two reef-building species, Lophelia pertusa and Madrepora oculata. Here we present a fine-grained analysis of the genotypic and genetic composition of reefs occurring in the Bay of Biscay, based on an innovative deep-sea sampling protocol. This strategy was designed to be standardized, random, and allowed the georeferencing of all sampled colonies. Clonal lineages discriminated through their Multi-Locus Genotypes (MLG) at 6–7 microsatellite markers could thus be mapped to assess the level of clonality and the spatial spread of clonal lineages. High values of clonal richness were observed for both species across all sites suggesting a limited occurrence of clonality, which likely originated through fragmentation. Additionally, spatial autocorrelation analysis underlined the possible occurrence of fine-grained genetic structure in several populations of both L. pertusa and M. oculata. The two cold-water coral species examined had contrasting patterns of connectivity among canyons, with among-canyon genetic structuring detected in M. oculata, whereas L. pertusa was panmictic at the canyon scale. This study exemplifies that a standardized, random and georeferenced sampling strategy, while challenging, can be applied in the deep sea, and associated benefits outlined here include improved estimates of fine grained patterns of clonality and dispersal that are comparable across sites and among species.

Propagule Pressure, Habitat Conditions and Clonal Integration Influence the Establishment and Growth of an Invasive Clonal Plant, Alternanthera philoxeroides.

PubMed

You, Wen-Hua; Han, Cui-Min; Fang, Long-Xiang; Du, Dao-Lin

2016-01-01

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.

Low incidence of clonality in cold water corals revealed through the novel use of a standardized protocol adapted to deep sea sampling

NASA Astrophysics Data System (ADS)

Becheler, Ronan; Cassone, Anne-Laure; Noël, Philippe; Mouchel, Olivier; Morrison, Cheryl L.; Arnaud-Haond, Sophie

2017-11-01

Sampling in the deep sea is a technical challenge, which has hindered the acquisition of robust datasets that are necessary to determine the fine-grained biological patterns and processes that may shape genetic diversity. Estimates of the extent of clonality in deep-sea species, despite the importance of clonality in shaping the local dynamics and evolutionary trajectories, have been largely obscured by such limitations. Cold-water coral reefs along European margins are formed mainly by two reef-building species, Lophelia pertusa and Madrepora oculata. Here we present a fine-grained analysis of the genotypic and genetic composition of reefs occurring in the Bay of Biscay, based on an innovative deep-sea sampling protocol. This strategy was designed to be standardized, random, and allowed the georeferencing of all sampled colonies. Clonal lineages discriminated through their Multi-Locus Genotypes (MLG) at 6-7 microsatellite markers could thus be mapped to assess the level of clonality and the spatial spread of clonal lineages. High values of clonal richness were observed for both species across all sites suggesting a limited occurrence of clonality, which likely originated through fragmentation. Additionally, spatial autocorrelation analysis underlined the possible occurrence of fine-grained genetic structure in several populations of both L. pertusa and M. oculata. The two cold-water coral species examined had contrasting patterns of connectivity among canyons, with among-canyon genetic structuring detected in M. oculata, whereas L. pertusa was panmictic at the canyon scale. This study exemplifies that a standardized, random and georeferenced sampling strategy, while challenging, can be applied in the deep sea, and associated benefits outlined here include improved estimates of fine grained patterns of clonality and dispersal that are comparable across sites and among species.

Cutibacterium acnes molecular typing: time to standardize the method.

PubMed

Dagnelie, M-A; Khammari, A; Dréno, B; Corvec, S

2018-03-12

The Gram-positive, anaerobic/aerotolerant bacterium Cutibacterium acnes is a commensal of healthy human skin; it is subdivided into six main phylogenetic groups or phylotypes: IA1, IA2, IB, IC, II and III. To decipher how far specific subgroups of C. acnes are involved in disease physiopathology, different molecular typing methods have been developed to identify these subgroups: i.e. phylotypes, clonal complexes, and types defined by single-locus sequence typing (SLST). However, as several molecular typing methods have been developed over the last decade, it has become a difficult task to compare the results from one article to another. Based on the scientific literature, the aim of this narrative review is to propose a standardized method to perform molecular typing of C. acnes, according to the degree of resolution needed (phylotypes, clonal complexes, or SLST types). We discuss the existing different typing methods from a critical point of view, emphasizing their advantages and drawbacks, and we identify the most frequently used methods. We propose a consensus algorithm according to the needed phylogeny resolution level. We first propose to use multiplex PCR for phylotype identification, MLST9 for clonal complex determination, and SLST for phylogeny investigation including numerous isolates. There is an obvious need to create a consensus about molecular typing methods for C. acnes. This standardization will facilitate the comparison of results between one article and another, and also the interpretation of clinical data. Copyright © 2018 European Society of Clinical Microbiology and Infectious Diseases. Published by Elsevier Ltd. All rights reserved.

Combination of t(4;14), del(17p13), del(1p32) and 1q21 gain FISH probes identifies clonal heterogeneity and enhances the detection of adverse cytogenetic profiles in 233 newly diagnosed multiple myeloma.

PubMed

Smol, Thomas; Dufour, Annika; Tricot, Sabine; Wemeau, Mathieu; Stalnikiewicz, Laure; Bernardi, Franck; Terré, Christine; Ducourneau, Benoît; Bisiau, Hervé; Daudignon, Agnès

2017-01-01

Our aim was to set the FISH combination of del(17p13), t(4;14), 1q21 gain and del(1p32), four adverse cytogenetic factors rarely evaluated together, and compare our technical thresholds with those defined in the literature. Two hundred thirty-three patients with MM at diagnosis were studied using FISH to target 4 unfavorable cytogenetic abnormalities: 17p13 deletion, t(4;14) translocation, 1p32 deletion and 1q21 gain. Technical thresholds were determined for each probe using isolated CD138-expressing PC from patients without MM. The FISH analysis identified abnormalities in 79.0% of patients. Del(17p13) was detected in 15.0% of cases, t(4;14) in 11.5%, 1q21 gain in 37.8% and del(1p32) in 8.7%. Adding 1p32/1q21 FISH probes has enabled us to identify adverse cytogenetic profiles in 39.0% of patients without del(17p13) or t(4;14). Clonal heterogeneity was observed in 51.1% of patients as well as an increase in the number of adverse abnormalities when related clones were greater than or equal to 2 (85.1% against 45.6%). FISH allowed detecting accumulation of adverse abnormalities and clonal heterogeneity in MM with a combination of 4 probes. The impacts of these two parameters need to be evaluated, and could be included in future cytogenetic classifications.

Whole genome sequencing analyses of Listeria monocytogenes that persisted in a milkshake machine for a year and caused illnesses in Washington State.

PubMed

Li, Zhen; Pérez-Osorio, Ailyn; Wang, Yu; Eckmann, Kaye; Glover, William A; Allard, Marc W; Brown, Eric W; Chen, Yi

2017-06-15

In 2015, in addition to a United States multistate outbreak linked to contaminated ice cream, another outbreak linked to ice cream was reported in the Pacific Northwest of the United States. It was a hospital-acquired outbreak linked to milkshakes, made from contaminated ice cream mixes and milkshake maker, served to patients. Here we performed multiple analyses on isolates associated with this outbreak: pulsed-field gel electrophoresis (PFGE), whole genome single nucleotide polymorphism (SNP) analysis, species-specific core genome multilocus sequence typing (cgMLST), lineage-specific cgMLST and whole genome-specific MLST (wgsMLST)/outbreak-specific cgMLST. We also analyzed the prophages and virulence genes. The outbreak isolates belonged to sequence type 1038, clonal complex 101, genetic lineage II. There were no pre-mature stop codons in inlA. Isolates contained Listeria Pathogenicity Island 1 and multiple internalins. PFGE and multiple whole genome sequencing (WGS) analyses all clustered together food, environmental and clinical isolates when compared to outgroup from the same clonal complex, which supported the finding that L. monocytogenes likely persisted in the soft serve ice cream/milkshake maker from November 2014 to November 2015 and caused 3 illnesses, and that the outbreak strain was transmitted between two ice cream production facilities. The whole genome SNP analysis, one of the two species-specific cgMLST, the lineage II-specific cgMLST and the wgsMLST/outbreak-specific cgMLST showed that L. monocytogenes cells persistent in the milkshake maker for a year formed a unique clade inside the outbreak cluster. This clustering was consistent with the cleaning practice after the outbreak was initially recognized in late 2014 and early 2015. Putative prophages were conserved among prophage-containing isolates. The loss of a putative prophage in two isolates resulted in the loss of the AscI restriction site in the prophage, which contributed to their AscI-PFGE banding pattern differences from other isolates. The high resolution of WGS analyses allowed the differentiation of epidemiologically unrelated isolates, as well as the elucidation of the microevolution and persistence of isolates within the scope of one outbreak. We applied a wgsMLST scheme which is essentially the outbreak-specific cgMLST. This scheme can be combined with lineage-specific cgMLST and species-specific cgMLST to maximize the resolution of WGS.

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

PubMed

Sanou, Adama; Tarnagda, Zekiba; Kanyala, Estelle; Zingué, Dezemon; Nouctara, Moumini; Ganamé, Zakaria; Combary, Adjima; Hien, Hervé; Dembele, Mathurin; Kabore, Antoinette; Meda, Nicolas; Van de Perre, Philippe; Neveu, Dorine; Bañuls, Anne Laure; Godreuil, Sylvain

2014-10-01

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

Effects of complex life cycles on genetic diversity: cyclical parthenogenesis.

PubMed

Rouger, R; Reichel, K; Malrieu, F; Masson, J P; Stoeckel, S

2016-11-01

Neutral patterns of population genetic diversity in species with complex life cycles are difficult to anticipate. Cyclical parthenogenesis (CP), in which organisms undergo several rounds of clonal reproduction followed by a sexual event, is one such life cycle. Many species, including crop pests (aphids), human parasites (trematodes) or models used in evolutionary science (Daphnia), are cyclical parthenogens. It is therefore crucial to understand the impact of such a life cycle on neutral genetic diversity. In this paper, we describe distributions of genetic diversity under conditions of CP with various clonal phase lengths. Using a Markov chain model of CP for a single locus and individual-based simulations for two loci, our analysis first demonstrates that strong departures from full sexuality are observed after only a few generations of clonality. The convergence towards predictions made under conditions of full clonality during the clonal phase depends on the balance between mutations and genetic drift. Second, the sexual event of CP usually resets the genetic diversity at a single locus towards predictions made under full sexuality. However, this single recombination event is insufficient to reshuffle gametic phases towards full-sexuality predictions. Finally, for similar levels of clonality, CP and acyclic partial clonality (wherein a fixed proportion of individuals are clonally produced within each generation) differentially affect the distribution of genetic diversity. Overall, this work provides solid predictions of neutral genetic diversity that may serve as a null model in detecting the action of common evolutionary or demographic processes in cyclical parthenogens (for example, selection or bottlenecks).

Clonal plasticity and diversity facilitates the adaptation of Rhododendron aureum Georgi to alpine environment

PubMed Central

Wang, Xiaolong; Zhao, Wei; Li, Lin; You, Jian; Ni, Biao

2018-01-01

Four small oval populations and five large intensive populations of Rhododendron aureum growing at the alpine in Changbai Mountain (China) were studied in two types of habitat (in the tundra and in Betula ermanii forest). Identification and delimitation of genets were inferred from excavation in small populations and from amplified fragment length polymorphism (AFLP) markers by the standardized sampling design in large populations. Clonal architecture and clonal diversity were then estimated. For the four small populations, they were monoclonal, the spacer length (18.6 ± 5.6 in tundra, 29.7 ± 9.7 in Betula ermanii forest, P < 0.05) was shorter and branching intensity (136.7 ± 32.9 in tundra, 43.4 ± 12.3 in Betula ermanii forest, P < 0.05) was higher in the tundra than that in Betula ermanii forest. For the five large populations, they were composed of multiple genets with high level of clonal diversity (Simpson’s index D = 0.84, clonal richness R = 0.25, Fager's evenness E = 0.85); the spatial distribution of genets showed that the clonal growth strategy of R. aureum exhibits both guerilla and phalanx. Our results indicate that the clonal plasticity of R. aureum could enhance exploitation of resource heterogeneity and in turn greatly contribute to maintenance or improvement of fitness and the high clonal diversity of R. aureum increase the evolutionary rates to adapt the harsh alpine environment in Changbai Mountain. PMID:29746526

Biomass Allocation of Stoloniferous and Rhizomatous Plant in Response to Resource Availability: A Phylogenetic Meta-Analysis

PubMed Central

Xie, Xiu-Fang; Hu, Yu-Kun; Pan, Xu; Liu, Feng-Hong; Song, Yao-Bin; Dong, Ming

2016-01-01

Resource allocation to different functions is central in life-history theory. Plasticity of functional traits allows clonal plants to regulate their resource allocation to meet changing environments. In this study, biomass allocation traits of clonal plants were categorized into absolute biomass for vegetative growth vs. for reproduction, and their relative ratios based on a data set including 115 species and derived from 139 published literatures. We examined general pattern of biomass allocation of clonal plants in response to availabilities of resource (e.g., light, nutrients, and water) using phylogenetic meta-analysis. We also tested whether the pattern differed among clonal organ types (stolon vs. rhizome). Overall, we found that stoloniferous plants were more sensitive to light intensity than rhizomatous plants, preferentially allocating biomass to vegetative growth, aboveground part and clonal reproduction under shaded conditions. Under nutrient- and water-poor condition, rhizomatous plants were constrained more by ontogeny than by resource availability, preferentially allocating biomass to belowground part. Biomass allocation between belowground and aboveground part of clonal plants generally supported the optimal allocation theory. No general pattern of trade-off was found between growth and reproduction, and neither between sexual and clonal reproduction. Using phylogenetic meta-analysis can avoid possible confounding effects of phylogeny on the results. Our results shown the optimal allocation theory explained a general trend, which the clonal plants are able to plastically regulate their biomass allocation, to cope with changing resource availability, at least in stoloniferous and rhizomatous plants. PMID:27200071

Clonal propagation of eucalyptus in Brazilian nurseries

Treesearch

Ken McNabb; Natal Goncalves; Jose Goncalves

2002-01-01

Brazil has established extensive Eucalyptus plantations to support a growing forest products industry. During the past 25 years, the country has been a pioneer in developing clonal propagation systems to regenerate these highly productive plantations. Original clonal selections optimized disease resistance, coppicing ability, and volume growth, while recent priorities...

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

USDA-ARS?s Scientific Manuscript database

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. ...

SNP-based differentiation of Phytophthora infestans clonal lineages using locked nucleic acid probes and high resolution melt analysis

USDA-ARS?s Scientific Manuscript database

Phytophthora infestans, the cause of the devastating late blight disease of potato and tomato, exhibits a clonal reproductive lifestyle in North America. Phenotypes such as fungicide sensitivity and host preference are conserved among individuals within clonal lineages, while substantial phenotypic ...

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

USDA-ARS?s Scientific Manuscript database

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...

Chromosome aberrations of clonal origin are present in astronauts' blood lymphocytes.

PubMed

George, K; Durante, M; Willingham, V; Cucinotta, F A

2004-01-01

Radiation-induced chromosome translocations remain in peripheral blood cells over many years, and can potentially be used to measure retrospective doses or prolonged low-dose rate exposures. However, several recent studies have indicated that some individuals possess clones of cells with balanced chromosome abnormalities, which can result in an overestimation of damage and, therefore, influence the accuracy of dose calculations. We carefully examined the patterns of chromosome damage found in the blood lymphocytes of twelve astronauts, and also applied statistical methods to screen for the presence of potential clones. Cells with clonal aberrations were identified in three of the twelve individuals. These clonal cells were present in samples collected both before and after space flight, and yields are higher than previously reported for healthy individuals in this age range (40-52 years of age). The frequency of clonal damage appears to be even greater in chromosomes prematurely condensed in interphase, when compared with equivalent analysis in metaphase cells. The individuals with clonal aberrations were followed-up over several months and the yields of all clones decreased during this period. Since clonal aberrations may be associated with increased risk of tumorigenesis, it is important to accurately identify cells containing clonal rearrangements for risk assessment as well as biodosimetry. Copyright 2003 S. Karger AG, Basel

Chromosome aberrations of clonal origin are present in astronauts' blood lymphocytes

NASA Technical Reports Server (NTRS)

George, K.; Durante, M.; Willingham, V.; Cucinotta, F. A.

2004-01-01

Radiation-induced chromosome translocations remain in peripheral blood cells over many years, and can potentially be used to measure retrospective doses or prolonged low-dose rate exposures. However, several recent studies have indicated that some individuals possess clones of cells with balanced chromosome abnormalities, which can result in an overestimation of damage and, therefore, influence the accuracy of dose calculations. We carefully examined the patterns of chromosome damage found in the blood lymphocytes of twelve astronauts, and also applied statistical methods to screen for the presence of potential clones. Cells with clonal aberrations were identified in three of the twelve individuals. These clonal cells were present in samples collected both before and after space flight, and yields are higher than previously reported for healthy individuals in this age range (40-52 years of age). The frequency of clonal damage appears to be even greater in chromosomes prematurely condensed in interphase, when compared with equivalent analysis in metaphase cells. The individuals with clonal aberrations were followed-up over several months and the yields of all clones decreased during this period. Since clonal aberrations may be associated with increased risk of tumorigenesis, it is important to accurately identify cells containing clonal rearrangements for risk assessment as well as biodosimetry. Copyright 2003 S. Karger AG, Basel.

Clinical implications of somatic mutations in aplastic anemia and myelodysplastic syndrome in genomic age.

PubMed

Maciejewski, Jaroslaw P; Balasubramanian, Suresh K

2017-12-08

Recent technological advances in genomics have led to the discovery of new somatic mutations and have brought deeper insights into clonal diversity. This discovery has changed not only the understanding of disease mechanisms but also the diagnostics and clinical management of bone marrow failure. The clinical applications of genomics include enhancement of current prognostic schemas, prediction of sensitivity or refractoriness to treatments, and conceptualization and selective application of targeted therapies. However, beyond these traditional clinical aspects, complex hierarchical clonal architecture has been uncovered and linked to the current concepts of leukemogenesis and stem cell biology. Detection of clonal mutations, otherwise typical of myelodysplastic syndrome, in the course of aplastic anemia (AA) and paroxysmal nocturnal hemoglobinuria has led to new pathogenic concepts in these conditions and created a new link between AA and its clonal complications, such as post-AA and paroxysmal nocturnal hemoglobinuria. Distinctions among founder vs subclonal mutations, types of clonal evolution (linear or branching), and biological features of individual mutations (sweeping, persistent, or vanishing) will allow for better predictions of the biologic impact they impart in individual cases. As clonal markers, mutations can be used for monitoring clonal dynamics of the stem cell compartment during physiologic aging, disease processes, and leukemic evolution. © 2016 by The American Society of Hematology. All rights reserved.

Three-gene identity coefficients demonstrate that clonal reproduction promotes inbreeding and spatial relatedness in yellow-cedar, Callitropsis nootkatensis.

PubMed

Thompson, Stacey Lee; Bérubé, Yanik; Bruneau, Anne; Ritland, Kermit

2008-10-01

Asexual reproduction has the potential to promote population structuring through matings between clones as well as through limited dispersal of related progeny. Here we present an application of three-gene identity coefficients that tests whether clonal reproduction promotes inbreeding and spatial relatedness within populations. With this method, the first two genes are sampled to estimate pairwise relatedness or inbreeding, whereas the third gene is sampled from either a clone or a sexually derived individual. If three-gene coefficients are significantly greater for clones than nonclones, then clonality contributes excessively to genetic structure. First, we describe an estimator of three-gene identity and briefly evaluate its properties. We then use this estimator to test the effect of clonality on the genetic structure within populations of yellow-cedar (Callitropsis nootkatensis) using a molecular marker survey. Five microsatellite loci were genotyped for 485 trees sampled from nine populations. Our three-gene analyses show that clonal ramets promote inbreeding and spatial structure in most populations. Among-population correlations between clonal extent and genetic structure generally support these trends, yet with less statistical significance. Clones appear to contribute to genetic structure through the limited dispersal of offspring from replicated ramets of the same clonal genet, whereas this structure is likely maintained by mating among these relatives.

Four Main Virotypes among Extended-Spectrum-β-Lactamase-Producing Isolates of Escherichia coli O25b:H4-B2-ST131: Bacterial, Epidemiological, and Clinical Characteristics

PubMed Central

Mora, Azucena; Mamani, Rosalia; López, Cecilia; Blanco, Miguel; Dahbi, Ghizlane; Herrera, Alexandra; Marzoa, Juan; Fernández, Val; de la Cruz, Fernando; Martínez-Martínez, Luis; Alonso, María Pilar; Nicolas-Chanoine, Marie-Hélène; Johnson, James R.; Johnston, Brian; López-Cerero, Lorena; Pascual, Álvaro; Rodríguez-Baño, Jesús

2013-01-01

A total of 1,021 extended-spectrum-β-lactamase-producing Escherichia coli (ESBLEC) isolates obtained in 2006 during a Spanish national survey conducted in 44 hospitals were analyzed for the presence of the O25b:H4-B2-ST131 (sequence type 131) clonal group. Overall, 195 (19%) O25b-ST131 isolates were detected, with prevalence rates ranging from 0% to 52% per hospital. Molecular characterization of 130 representative O25b-ST131 isolates showed that 96 (74%) were positive for CTX-M-15, 15 (12%) for CTX-M-14, 9 (7%) for SHV-12, 6 (5%) for CTX-M-9, 5 (4%) for CTX-M-32, and 1 (0.7%) each for CTX-M-3 and the new ESBL enzyme CTX-M-103. The 130 O25b-ST131 isolates exhibited relatively high virulence scores (mean, 14.4 virulence genes). Although the virulence profiles of the O25b-ST131 isolates were fairly homogeneous, they could be classified into four main virotypes based on the presence or absence of four distinctive virulence genes: virotypes A (22%) (afa FM955459 positive, iroN negative, ibeA negative, sat positive or negative), B (31%) (afa FM955459 negative, iroN positive, ibeA negative, sat positive or negative), C (32%) (afa FM955459 negative, iroN negative, ibeA negative, sat positive), and D (13%) (afa FM955459 negative, iroN positive or negative, ibeA positive, sat positive or negative). The four virotypes were also identified in other countries, with virotype C being overrepresented internationally. Correspondingly, an analysis of XbaI macrorestriction profiles revealed four major clusters, which were largely virotype specific. Certain epidemiological and clinical features corresponded with the virotype. Statistically significant virotype-specific associations included, for virotype B, older age and a lower frequency of infection (versus colonization), for virotype C, a higher frequency of infection, and for virotype D, younger age and community-acquired infections. In isolates of the O25b:H4-B2-ST131 clonal group, these findings uniquely define four main virotypes, which are internationally distributed, correspond with pulsed-field gel electrophoresis (PFGE) profiles, and exhibit distinctive clinical-epidemiological associations. PMID:23926164

Novel type of VanB2 teicoplanin-resistant hospital-associated Enterococcus faecium.

PubMed

Santona, Antonella; Paglietti, Bianca; Al-Qahtani, Ahmed A; Bohol, Marie Fe F; Senok, Abiola; Deligios, Massimo; Rubino, Salvatore; Al-Ahdal, Mohammed N

2014-08-01

Seven high-risk clones of vancomycin-resistant Enterococcus faecium (VREF) belonging to clonal complex 17 were identified using multilocus sequence typing (MLST) among clinical isolates from Saudi Arabia. Among these isolates, a new hospital-associated sequence type (ST795), VanB(2)-type teicoplanin-resistant strain was detected. Its unusual phenotype resulted from a new combination of mutations in the ddl, vanS and vanW genes, which confirmed the trend of evolution in VanB-type resistance. Furthermore, characteristics of adaptation and persistence in the hospital environment of ST795 were emphasised by the presence of genes and clusters recognised to be specific for hospital-associated VREF. Copyright © 2014 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved.

Pathovars of Pseudomonas syringae Causing Bacterial Brown Spot and Halo Blight in Phaseolus vulgaris L. Are Distinguishable by Ribotyping

PubMed Central

González, Ana J.; Landeras, Elena; Mendoza, M. Carmen

2000-01-01

Ribotyping was evaluated as a method to differentiate between Pseudomonas syringae pv. phaseolicola and pv. syringae strains causing bacterial brown spot and halo blight diseases in Phaseolus vulgaris L. Ribotyping, with restriction enzymes BglI and SalI and using the Escherichia coli rrnB operon as the probe, differentiated 11 and 14 ribotypes, respectively, and a combination of data from both procedures yielded 19 combined ribotypes. Cluster analysis of the combined ribotypes differentiated the pathovars phaseolicola and syringae, as well as different clonal lineages within these pathovars. The potential of ribotyping to screen for correlations between lineages and factors such as geographical region and/or bean varieties is also reported. PMID:10653764

A new group of cosmopolitan bacteriophages induce a carrier state in the pandemic strain of Vibrio parahaemolyticus.

PubMed

Bastías, Roberto; Higuera, Gastón; Sierralta, Walter; Espejo, Romilio T

2010-04-01

A clonal population of pathogenic Vibrio parahaemolyticus O3 : K6 serovar has spread in coastal waters, causing outbreaks worldwide since 1996. Bacteriophage infection is one of the main factors affecting bacterial strain concentration in the ocean. We studied the occurrence and properties of phages infecting this V. parahaemolyticus pandemic strain in coastal waters. Analysing 143 samples, phages were found in 13. All isolates clustered in a closely related group of podophages with at least 90% nucleotide sequence identity in three essential genes, despite distant geographical origins. These bacteriophages were able to multiply on the V. parahaemolyticus pandemic strain, but the impact on host concentration and subsequent growth was negligible. Infected bacteria continued producing the phage but were not lysogenized. The phage genome of prototype strain VP93 is 43 931 nucleotides and contains 337 bp direct terminal repeats at both ends. VP93 is the first non-Pseudomonas phage related to the PhiKMV-like subgroup of the T7 supergroup. The lack of a major effect on host growth suggests that these phages exert little control on the propagation of the pandemic strain in the environment. This form of phage growth can be modelled if phage-sensitive and -resistant cells that convert to each other with a high frequency are present in clonal cultures of pandemic V. parahaemolyticus.

Genetic Diversity and Population Structure of Vibrio cholerae

PubMed Central

Beltrán, Pilar; Delgado, Gabriela; Navarro, Armando; Trujillo, Francisca; Selander, Robert K.; Cravioto, Alejandro

1999-01-01

Multilocus enzyme electrophoresis (MLEE) of 397 Vibrio cholerae isolates, including 143 serogroup reference strains and 244 strains from Mexico and Guatemala, identified 279 electrophoretic types (ETs) distributed in two major divisions (I and II). Linkage disequilibrium was demonstrated in both divisions and in subdivision Ic of division I but not in subdivision Ia, which includes 76% of the ETs. Despite this evidence of relatively frequent recombination, clonal lineages may persist for periods of time measured in at least decades. In addition to the pandemic clones of serogroups O1 and O139, which form a tight cluster of four ETs in subdivision Ia, MLEE analysis identified numerous apparent clonal lineages of non-O1 strains with intercontinental distributions. A clone of serogroup O37 that demonstrated epidemic potential in the 1960s is closely related to the pandemic O1/O139 clones, but the nontoxigenic O1 Inaba El Tor reference strain is not. A strain of serogroup O22, which has been identified as the most likely donor of exogenous rfb region DNA to the O1 progenitor of the O139 clone, is distantly related to the O1/O139 clones. The close evolutionary relationships of the O1, O139, and O37 epidemic clones indicates that new cholera clones are likely to arise by the modification of a lineage that is already epidemic or is closely related to such a clone. PMID:9986816

Genetic diversity and population structure of Vibrio cholerae.

PubMed

Beltrán, P; Delgado, G; Navarro, A; Trujillo, F; Selander, R K; Cravioto, A

1999-03-01

Multilocus enzyme electrophoresis (MLEE) of 397 Vibrio cholerae isolates, including 143 serogroup reference strains and 244 strains from Mexico and Guatemala, identified 279 electrophoretic types (ETs) distributed in two major divisions (I and II). Linkage disequilibrium was demonstrated in both divisions and in subdivision Ic of division I but not in subdivision Ia, which includes 76% of the ETs. Despite this evidence of relatively frequent recombination, clonal lineages may persist for periods of time measured in at least decades. In addition to the pandemic clones of serogroups O1 and O139, which form a tight cluster of four ETs in subdivision Ia, MLEE analysis identified numerous apparent clonal lineages of non-O1 strains with intercontinental distributions. A clone of serogroup O37 that demonstrated epidemic potential in the 1960s is closely related to the pandemic O1/O139 clones, but the nontoxigenic O1 Inaba El Tor reference strain is not. A strain of serogroup O22, which has been identified as the most likely donor of exogenous rfb region DNA to the O1 progenitor of the O139 clone, is distantly related to the O1/O139 clones. The close evolutionary relationships of the O1, O139, and O37 epidemic clones indicates that new cholera clones are likely to arise by the modification of a lineage that is already epidemic or is closely related to such a clone.

Distribution of antimicrobial resistance determinants, virulence-associated factors and clustered regularly interspaced palindromic repeats loci in isolates of Enterococcus faecalis from various settings and genetic lineages.

PubMed

Gawryszewska, Iwona; Malinowska, Katarzyna; Kuch, Alicja; Chrobak-Chmiel, Dorota; Trokenheim, Lucja Laniewska-; Hryniewicz, Waleria; Sadowy, Ewa

2017-03-01

Enterococcus faecalis represents an important factor of hospital-associated infections (HAIs). The knowledge on its evolution from a commensal to an opportunistic pathogen is still limited; thus, we performed a study to characterise distribution of factors that may contribute to this adaptation. Using a collection obtained from various settings (hospitalised patients, community carriers, animals, fresh food, sewage, water), we investigated differences in antimicrobial susceptibility, distribution of antimicrobial resistance genes, virulence-associated determinants and phenotypes, and CRISPR loci in the context of the clonal relatedness of isolates. Bayesian Analysis of Population Structure revealed the presence of three major groups; two subgroups comprised almost exclusively HAI isolates, belonging to previously proposed enterococcal high-risk clonal complexes (HiRECCs) 6 and 28. Isolates of these two subgroups were significantly enriched in antimicrobial resistance genes, presumably produced a polysaccharide capsule and often carried the aggregation substance asa1; distribution of other virulence-associated genes, such as esp and cyl, formation of a biofilm and gelatinase production were more variable. Moreover, both subgroups showed a low prevalence of CRISPR-Cas 1 and 3 and presence of small CRISPR2 variants. Our study confirms the importance of HiRECCs in the population of E. faecalis and their confinement to the hospital settings. © FEMS 2017. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Multilocus sequence typing analyses of Clostridium perfringens type A strains harboring tpeL and netB genes.

PubMed

Nakano, V; Ignacio, A; Llanco, L; Bueris, V; Sircili, M P; Avila-Campos, M J

2017-04-01

Clostridium perfringens is an anaerobic bacterium ubiquitous in various environments, especially in soil and the gastrointestinal tract of healthy humans and animals. In this study, multilocus sequence typing protocol was used to investigate genotypic relationships among 40 C. perfringens strains isolated from humans and broiler chicken with necrotic enteritis [NE]. The results indicated a few clonal populations, mainly observed in human strains, with 32.5% of all strains associated with one of three clonal complexes and 30 sequences types. The CC-1 cluster showed an interesting and unexpected result because it contained seven strains [six from animals and one of human origin]. Detection assays for toxin genes tpeL and netB were also performed. The netB gene was only observed in 7.5% of the strains from healthy human. The toxin gene tpeL was detected in 22.5% of the C. perfringens strains isolated from three individuals and in six broilers with NE. Our study describes the role of some C. perfringens strains of human origin acting as reservoirs of virulence genes and sources of infection. In addition, the strains of human and animal origin were found to be genetically distinct but phylogenetically close, and the human strains showed more diversity than the animal strains. Copyright © 2017 Elsevier Ltd. All rights reserved.

Characterization of carbapenem nonsusceptible Pseudomonas aeruginosa in Denmark: a nationwide, prospective study.

PubMed

Hansen, Frank; Johansen, Helle Krogh; Østergaard, Claus; Arpi, Magnus; Hansen, Dennis Schrøder; Littauer, Pia; Holm, Anette; Heltberg, Ole; Schumacher, Helga; Fuursted, Kurt; Lykke, Mari-Ann Domar; Tønning, Birgitte; Hammerum, Anette M; Justesen, Ulrik Stenz

2014-02-01

From January 1st 2011 through June 30th 2011, 116 nonreplicate, noncystic fibrosis-related Pseudomonas aeruginosa isolates with reduced carbapenem susceptibility were collected from 12 out of 13 Danish departments of clinical microbiology. The presence of acquired β-lactamases was assessed with combination tablet-diffusion methodology and polymerase chain reaction. In addition, antimicrobial susceptibility testing, an efflux pump inhibitor assay, and pulsed-field gel electrophoresis (PFGE) were performed. Isolates producing acquired β-lactamases were further investigated by serotyping and multi locus sequence typing. Eight isolates produced the metallo-β-lactamase (MBL) VIM-2, and one isolate produced OXA-10 and VEB-1-like extended-spectrum beta-lactamase (ESBL). Phenotypic indications of derepressed AmpC and efflux pump were seen in 56 and 43 isolates, respectively. Overall, the results indicate that mutational factors related to permeability--often combined with derepressed, chromosomal AmpC--is the main factor behind carbapenem nonsusceptibility in Danish P. aeruginosa isolates. The ESBL producer and all the VIM producers belonged to international clones. PFGE revealed that most of the isolates were unrelated, but clonal spread was seen; the 116 isolates distributed in 97 PFGE types, with the largest cluster consisting of 4 isolates (including three isolates from the same hospital with 100% similarity). Thirty-two isolates were pair-wise related, while the remaining isolates were clonally unrelated, as were all nine ESBL/MBL producers.

Distribution of antimicrobial resistance determinants, virulence-associated factors and clustered regularly interspaced palindromic repeats loci in isolates of Enterococcus faecalis from various settings and genetic lineages

PubMed Central

Gawryszewska, Iwona; Malinowska, Katarzyna; Kuch, Alicja; Chrobak-Chmiel, Dorota; Trokenheim, Łucja Łaniewska-; Hryniewicz, Waleria; Sadowy, Ewa

2017-01-01

Abstract Enterococcus faecalis represents an important factor of hospital-associated infections (HAIs). The knowledge on its evolution from a commensal to an opportunistic pathogen is still limited; thus, we performed a study to characterise distribution of factors that may contribute to this adaptation. Using a collection obtained from various settings (hospitalised patients, community carriers, animals, fresh food, sewage, water), we investigated differences in antimicrobial susceptibility, distribution of antimicrobial resistance genes, virulence-associated determinants and phenotypes, and CRISPR loci in the context of the clonal relatedness of isolates. Bayesian Analysis of Population Structure revealed the presence of three major groups; two subgroups comprised almost exclusively HAI isolates, belonging to previously proposed enterococcal high-risk clonal complexes (HiRECCs) 6 and 28. Isolates of these two subgroups were significantly enriched in antimicrobial resistance genes, presumably produced a polysaccharide capsule and often carried the aggregation substance asa1; distribution of other virulence-associated genes, such as esp and cyl, formation of a biofilm and gelatinase production were more variable. Moreover, both subgroups showed a low prevalence of CRISPR-Cas 1 and 3 and presence of small CRISPR2 variants. Our study confirms the importance of HiRECCs in the population of E. faecalis and their confinement to the hospital settings. PMID:28334141

A module located at a chromosomal integration hot spot is responsible for the multidrug resistance of a reference strain from Escherichia coli clonal group A.

PubMed

Lescat, Mathilde; Calteau, Alexandra; Hoede, Claire; Barbe, Valérie; Touchon, Marie; Rocha, Eduardo; Tenaillon, Olivier; Médigue, Claudine; Johnson, James R; Denamur, Erick

2009-06-01

Escherichia coli clonal group A (CGA) commonly exhibits a distinctive multidrug antimicrobial resistance phenotype-i.e., resistance to ampicillin, chloramphenicol, streptomycin, sulfonamides, tetracycline, and trimethoprim (ACSSuTTp)-and has accounted for up to 50% of trimethoprim-sulfamethoxazole-resistant E. coli urinary tract infections in some locales. Annotation of the whole-genome sequencing of UMN026, a reference CGA strain, clarified the genetic basis for this strain's ACSSuTTp antimicrobial resistance phenotype. Most of the responsible genes were clustered in a unique 23-kbp chromosomal region, designated the genomic resistance module (GRM), which occurred within a 105-kbp genomic island situated at the leuX tRNA. The GRM is characterized by numerous remnants of mobilization and rearrangement events suggesting multiple horizontal transfers. Additionally, comparative genomic analysis of the leuX tRNA genomic island in 14 sequenced E. coli genomes showed that this region is a hot spot of integration, with the presence/absence of specific subregions being uncorrelated with either the phylogenetic group or the pathotype. Our data illustrate the importance of whole-genome sequencing in the detection of genetic elements involved in antimicrobial resistance. Additionally, this is the first documentation of the bla(TEM) and dhfrVII genes in a chromosomal location in E. coli strains.

Multiple locus VNTR analysis highlights that geographical clustering and distribution of Dichelobacter nodosus, the causal agent of footrot in sheep, correlates with inter-country movements☆

PubMed Central

Russell, Claire L.; Smith, Edward M.; Calvo-Bado, Leonides A.; Green, Laura E.; Wellington, Elizabeth M.H.; Medley, Graham F.; Moore, Lynda J.; Grogono-Thomas, Rosemary

2014-01-01

Dichelobacter nodosus is a Gram-negative, anaerobic bacterium and the causal agent of footrot in sheep. Multiple locus variable number tandem repeat (VNTR) analysis (MLVA) is a portable technique that involves the identification and enumeration of polymorphic tandem repeats across the genome. The aims of this study were to develop an MLVA scheme for D. nodosus suitable for use as a molecular typing tool, and to apply it to a global collection of isolates. Seventy-seven isolates selected from regions with a long history of footrot (GB, Australia) and regions where footrot has recently been reported (India, Scandinavia), were characterised. From an initial 61 potential VNTR regions, four loci were identified as usable and in combination had the attributes required of a typing method for use in bacterial epidemiology: high discriminatory power (D > 0.95), typeability and reproducibility. Results from the analysis indicate that D. nodosus appears to have evolved via recombinational exchanges and clonal diversification. This has resulted in some clonal complexes that contain isolates from multiple countries and continents; and others that contain isolates from a single geographic location (country or region). The distribution of alleles between countries matches historical accounts of sheep movements, suggesting that the MLVA technique is sufficiently specific and sensitive for an epidemiological investigation of the global distribution of D. nodosus. PMID:23748018

Lineage tracing reveals multipotent stem cells maintain human adenomas and the pattern of clonal expansion in tumor evolution

PubMed Central

Humphries, Adam; Cereser, Biancastella; Gay, Laura J.; Miller, Daniel S. J.; Das, Bibek; Gutteridge, Alice; Elia, George; Nye, Emma; Jeffery, Rosemary; Poulsom, Richard; Novelli, Marco R.; Rodriguez-Justo, Manuel; McDonald, Stuart A. C.; Wright, Nicholas A.; Graham, Trevor A.

2013-01-01

The genetic and morphological development of colorectal cancer is a paradigm for tumorigenesis. However, the dynamics of clonal evolution underpinning carcinogenesis remain poorly understood. Here we identify multipotential stem cells within human colorectal adenomas and use methylation patterns of nonexpressed genes to characterize clonal evolution. Numerous individual crypts from six colonic adenomas and a hyperplastic polyp were microdissected and characterized for genetic lesions. Clones deficient in cytochrome c oxidase (CCO−) were identified by histochemical staining followed by mtDNA sequencing. Topographical maps of clone locations were constructed using a combination of these data. Multilineage differentiation within clones was demonstrated by immunofluorescence. Methylation patterns of adenomatous crypts were determined by clonal bisulphite sequencing; methylation pattern diversity was compared with a mathematical model to infer to clonal dynamics. Individual adenomatous crypts were clonal for mtDNA mutations and contained both mucin-secreting and neuroendocrine cells, demonstrating that the crypt contained a multipotent stem cell. The intracrypt methylation pattern was consistent with the crypts containing multiple competing stem cells. Adenomas were epigenetically diverse populations, suggesting that they were relatively mitotically old populations. Intratumor clones typically showed less diversity in methylation pattern than the tumor as a whole. Mathematical modeling suggested that recent clonal sweeps encompassing the whole adenoma had not occurred. Adenomatous crypts within human tumors contain actively dividing stem cells. Adenomas appeared to be relatively mitotically old populations, pocketed with occasional newly generated subclones that were the result of recent rapid clonal expansion. Relative stasis and occasional rapid subclone growth may characterize colorectal tumorigenesis. PMID:23766371

Lineage tracing reveals multipotent stem cells maintain human adenomas and the pattern of clonal expansion in tumor evolution.

PubMed

Humphries, Adam; Cereser, Biancastella; Gay, Laura J; Miller, Daniel S J; Das, Bibek; Gutteridge, Alice; Elia, George; Nye, Emma; Jeffery, Rosemary; Poulsom, Richard; Novelli, Marco R; Rodriguez-Justo, Manuel; McDonald, Stuart A C; Wright, Nicholas A; Graham, Trevor A

2013-07-02

The genetic and morphological development of colorectal cancer is a paradigm for tumorigenesis. However, the dynamics of clonal evolution underpinning carcinogenesis remain poorly understood. Here we identify multipotential stem cells within human colorectal adenomas and use methylation patterns of nonexpressed genes to characterize clonal evolution. Numerous individual crypts from six colonic adenomas and a hyperplastic polyp were microdissected and characterized for genetic lesions. Clones deficient in cytochrome c oxidase (CCO(-)) were identified by histochemical staining followed by mtDNA sequencing. Topographical maps of clone locations were constructed using a combination of these data. Multilineage differentiation within clones was demonstrated by immunofluorescence. Methylation patterns of adenomatous crypts were determined by clonal bisulphite sequencing; methylation pattern diversity was compared with a mathematical model to infer to clonal dynamics. Individual adenomatous crypts were clonal for mtDNA mutations and contained both mucin-secreting and neuroendocrine cells, demonstrating that the crypt contained a multipotent stem cell. The intracrypt methylation pattern was consistent with the crypts containing multiple competing stem cells. Adenomas were epigenetically diverse populations, suggesting that they were relatively mitotically old populations. Intratumor clones typically showed less diversity in methylation pattern than the tumor as a whole. Mathematical modeling suggested that recent clonal sweeps encompassing the whole adenoma had not occurred. Adenomatous crypts within human tumors contain actively dividing stem cells. Adenomas appeared to be relatively mitotically old populations, pocketed with occasional newly generated subclones that were the result of recent rapid clonal expansion. Relative stasis and occasional rapid subclone growth may characterize colorectal tumorigenesis.

Effects of complex life cycles on genetic diversity: cyclical parthenogenesis

PubMed Central

Rouger, R; Reichel, K; Malrieu, F; Masson, J P; Stoeckel, S

2016-01-01

Neutral patterns of population genetic diversity in species with complex life cycles are difficult to anticipate. Cyclical parthenogenesis (CP), in which organisms undergo several rounds of clonal reproduction followed by a sexual event, is one such life cycle. Many species, including crop pests (aphids), human parasites (trematodes) or models used in evolutionary science (Daphnia), are cyclical parthenogens. It is therefore crucial to understand the impact of such a life cycle on neutral genetic diversity. In this paper, we describe distributions of genetic diversity under conditions of CP with various clonal phase lengths. Using a Markov chain model of CP for a single locus and individual-based simulations for two loci, our analysis first demonstrates that strong departures from full sexuality are observed after only a few generations of clonality. The convergence towards predictions made under conditions of full clonality during the clonal phase depends on the balance between mutations and genetic drift. Second, the sexual event of CP usually resets the genetic diversity at a single locus towards predictions made under full sexuality. However, this single recombination event is insufficient to reshuffle gametic phases towards full-sexuality predictions. Finally, for similar levels of clonality, CP and acyclic partial clonality (wherein a fixed proportion of individuals are clonally produced within each generation) differentially affect the distribution of genetic diversity. Overall, this work provides solid predictions of neutral genetic diversity that may serve as a null model in detecting the action of common evolutionary or demographic processes in cyclical parthenogens (for example, selection or bottlenecks). PMID:27436524

Glioblastoma adaptation traced through decline of an IDH1 clonal driver and macro-evolution of a double-minute chromosome.

PubMed

Favero, F; McGranahan, N; Salm, M; Birkbak, N J; Sanborn, J Z; Benz, S C; Becq, J; Peden, J F; Kingsbury, Z; Grocok, R J; Humphray, S; Bentley, D; Spencer-Dene, B; Gutteridge, A; Brada, M; Roger, S; Dietrich, P-Y; Forshew, T; Gerlinger, M; Rowan, A; Stamp, G; Eklund, A C; Szallasi, Z; Swanton, C

2015-05-01

Glioblastoma (GBM) is the most common malignant brain cancer occurring in adults, and is associated with dismal outcome and few therapeutic options. GBM has been shown to predominantly disrupt three core pathways through somatic aberrations, rendering it ideal for precision medicine approaches. We describe a 35-year-old female patient with recurrent GBM following surgical removal of the primary tumour, adjuvant treatment with temozolomide and a 3-year disease-free period. Rapid whole-genome sequencing (WGS) of three separate tumour regions at recurrence was carried out and interpreted relative to WGS of two regions of the primary tumour. 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 in the primary, a known GBM driver event, was detectable at only very low frequency in the recurrent tumour. After sub-clonal diversification, evidence was found for a whole-genome doubling event and a translocation between the amplified regions of PDGFRA, KIT and CDK4, encoded within a double-minute chromosome also incorporating miR26a-2. The WGS analysis uncovered progressive evolution of the double-minute chromosome converging on the KIT/PDGFRA/PI3K/mTOR axis, superseding the IDH1 mutation in dominance in a mutually exclusive manner at recurrence, consequently the patient was treated with imatinib. Despite rapid sequencing and cancer genome-guided therapy against amplified oncogenes, the disease progressed, and the patient died shortly after. This case sheds light on the dynamic evolution of a GBM tumour, defining the origins of the lethal sub-clone, the macro-evolutionary genomic events dominating the disease at recurrence and the loss of a clonal driver. Even in the era of rapid WGS analysis, cases such as this illustrate the significant hurdles for precision medicine success. © The Author 2015. Published by Oxford University Press on behalf of the European Society for Medical Oncology.

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

Treesearch

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...

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

Treesearch

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...

GACD: Integrated Software for Genetic Analysis in Clonal F1 and Double Cross Populations.

PubMed

Zhang, Luyan; Meng, Lei; Wu, Wencheng; Wang, Jiankang

2015-01-01

Clonal species are common among plants. Clonal F1 progenies are derived from the hybridization between 2 heterozygous clones. In self- and cross-pollinated species, double crosses can be made from 4 inbred lines. A clonal F1 population can be viewed as a double cross population when the linkage phase is determined. The software package GACD (Genetic Analysis of Clonal F1 and Double cross) is freely available public software, capable of building high-density linkage maps and mapping quantitative trait loci (QTL) in clonal F1 and double cross populations. Three functionalities are integrated in GACD version 1.0: binning of redundant markers (BIN); linkage map construction (CDM); and QTL mapping (CDQ). Output of BIN can be directly used as input of CDM. After adding the phenotypic data, the output of CDM can be used as input of CDQ. Thus, GACD acts as a pipeline for genetic analysis. GACD and example datasets are freely available from www.isbreeding.net. © The American Genetic Association. 2015. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Quantitative stability of hematopoietic stem and progenitor cell clonal output in rhesus macaques receiving transplants

PubMed Central

Koelle, Samson J.

2017-01-01

Autologous transplantation of hematopoietic stem and progenitor cells lentivirally labeled with unique oligonucleotide barcodes flanked by sequencing primer targets enables quantitative assessment of the self-renewal and differentiation patterns of these cells in a myeloablative rhesus macaque model. Compared with other approaches to clonal tracking, this approach is highly quantitative and reproducible. We documented stable multipotent long-term hematopoietic clonal output of monocytes, granulocytes, B cells, and T cells from a polyclonal pool of hematopoietic stem and progenitor cells in 4 macaques observed for up to 49 months posttransplantation. A broad range of clonal behaviors characterized by contribution level and biases toward certain cell types were extremely stable over time. Correlations between granulocyte and monocyte clonalities were greatest, followed by correlations between these cell types and B cells. We also detected quantitative expansion of T cell–biased clones consistent with an adaptive immune response. In contrast to recent data from a nonquantitative murine model, there was little evidence for clonal succession after initial hematopoietic reconstitution. These findings have important implications for human hematopoiesis, given the similarities between macaque and human physiologies. PMID:28087539

[Comparison of clonal architecture between two divergent Leymus chinensis types in Songnen grassland].

PubMed

He, Nianpeng; Wu, Ling; Zhou, Daowei

2004-12-01

This paper studied the clonal architecture of two divergent Leymus chinensis types (grey-green type and yellow-green type) in Songnen grassland, and compared their internode length, spacer length, interbranching length, interbranching angle, and ramet population density and height under the same habitat. The results showed that there was no significant difference in these clonal characteristics except spacer length and ramet population density between the two types of L. chinensis, and yellow-green type, with less spacer length and more ramet density than grey-green type, should be more adaptable to the resourceful habitat. Moreover, the V-indices of the clonal architecture of two divergent L. chinensis types were all close to 1, and the difference was not significant. Therefore, both of the two types belonged to typical guerilla clonal plant.

Diagnostic value of immunoglobulin κ light chain gene rearrangement analysis in B-cell lymphomas.

PubMed

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

2015-03-01

Analysis of the immunoglobulin κ light chain (IGK) gene is an alternative method for B-cell clonality assessment in the diagnosis of mature B-cell proliferations in which the detection of clonal immunoglobulin heavy chain (IGH) gene rearrangements fails. The aim of the present study was to evaluate the added value of standardized BIOMED-2 assay for the detection of clonal IGK gene rearrangements in the diagnostic setting of suspected B-cell lymphomas. With this purpose, 92 specimens from 80 patients with the final diagnosis of mature B-cell lymphoma (37 specimens), mature T-cell lymphoma (26 specimens) and reactive lymphoid proliferation (29 specimens) were analyzed for B-cell clonality. B-cell clonality analysis was performed using the BIOMED-2 IGH and IGK gene clonality assays. The determined sensitivity of the IGK assay was 67.6%, while the determined sensitivity of the IGH assay was 75.7%. The sensitivity of combined IGH+IGK assay was 81.1%. The determined specificity of the IGK assay was 96.2% in the group of T-cell lymphomas and 96.6% in the group of reactive lesions. The determined specificity of the IGH assay was 84.6% in the group of lymphomas and 86.2% in the group of reactive lesions. The comparison of GeneScan (GS) and heteroduplex pretreatment-polyacrylamide gel electrophoresis (HD-PAGE) methods for the analysis of IGK gene rearrangements showed a higher efficacy of GS analysis in a series of 27 B-cell lymphomas analyzed by both methods. In the present study, we demonstrated that by applying the combined IGH+IGK clonality assay the overall detection rate of B-cell clonality was increased by 5.4%. Thus, we confirmed the added value of the standardized BIOMED-2 IGK assay for assessment of B-cell clonality in suspected B-cell lymphomas with inconclusive clinical and cyto/histological diagnosis.

The maintenance of sex: Ronald Fisher meets the Red Queen.

PubMed

Green, David; Mason, Chris

2013-08-21

Sex in higher diploids carries a two-fold cost of males that should reduce its fitness relative to cloning, and result in its extinction. Instead, sex is widespread and clonal species face early obsolescence. One possible reason is that sex is an adaptation that allows organisms to respond more effectively to endless changes in their environment. The purpose of this study was to model mutation and selection in a diploid organism in an evolving environment and ascertain their support for sex. We used a computational approach to model finite populations where a haploid environment subjects a diploid host to endlessly evolving change. Evolution in both populations is primarily through adoption of novel advantageous mutations within a large allele space. Sex outcompetes cloning by two complementary mechanisms. First, sexual diploids adopt advantageous homozygous mutations more rapidly than clonal ones under conditions of lag load (the gap between the actual adaptation of the diploid population and its theoretical optimum). This rate advantage can offset the higher fecundity of cloning. Second, a relative advantage to sex emerges where populations are significantly polymorphic, because clonal polymorphism runs the risk of clonal interference caused by selection on numerous lines of similar adaptation. This interference extends allele lifetime and reduces the rate of adaptation. Sex abolishes the interference, making selection faster and elevating population fitness. Differences in adaptation between sexual and clonal populations increase markedly with the number of loci under selection, the rate of mutation in the host, and a rapidly evolving environment. Clonal interference in these circumstances leads to conditions where the greater fecundity of clones is unable to offset their poor adaptation. Sexual and clonal populations then either co-exist, or sex emerges as the more stable evolutionary strategy. Sex can out-compete clones in a rapidly evolving environment, such as that characterized by pathogens, where clonal interference reduces the adaptation of clonal populations and clones adopt advantageous mutations more slowly. Since all organisms carry parasitic loads, the model is of potentially general applicability.

Longevity of clonal plants: why it matters and how to measure it

PubMed Central

de Witte, Lucienne C.; Stöcklin, Jürg

2010-01-01

Background Species' life-history and population dynamics are strongly shaped by the longevity of individuals, but life span is one of the least accessible demographic traits, particularly in clonal plants. Continuous vegetative reproduction of genets enables persistence despite low or no sexual reproduction, affecting genet turnover rates and population stability. Therefore, the longevity of clonal plants is of considerable biological interest, but remains relatively poorly known. Scope Here, we critically review the present knowledge on the longevity of clonal plants and discuss its importance for population persistence. Direct life-span measurements such as growth-ring analysis in woody plants are relatively easy to take, although, for many clonal plants, these methods are not adequate due to the variable growth pattern of ramets and difficult genet identification. Recently, indirect methods have been introduced in which genet size and annual shoot increments are used to estimate genet age. These methods, often based on molecular techniques, allow the investigation of genet size and age structure of whole populations, a crucial issue for understanding their viability and persistence. However, indirect estimates of clonal longevity are impeded because the process of ageing in clonal plants is still poorly understood and because their size and age are not always well correlated. Alternative estimators for genet life span such as somatic mutations have recently been suggested. Conclusions Empirical knowledge on the longevity of clonal species has increased considerably in the last few years. Maximum age estimates are an indicator of population persistence, but are not sufficient to evaluate turnover rates and the ability of long-lived clonal plants to enhance community stability and ecosystem resilience. In order to understand the dynamics of populations it will be necessary to measure genet size and age structure, not only life spans of single individuals, and to use such data for modelling of genet dynamics. PMID:20880935

Cluster Differences Scaling with a Within-Clusters Loss Component and a Fuzzy Successive Approximation Strategy To Avoid Local Minima.

ERIC Educational Resources Information Center

Heiser, Willem J.; And Others

1997-01-01

The least squares loss function of cluster differences scaling, originally defined only on residuals of pairs allocated to different clusters, is extended with a loss component for pairs allocated to the same cluster. Findings show that this makes the method equivalent to multidimensional scaling with cluster constraints on the coordinates. (SLD)

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

PubMed

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.

Cluster-distinguishing genotypic and phenotypic diversity of carbapenem-resistant Gram-negative bacteria in solid-organ transplantation patients: a comparative study.

PubMed

Karampatakis, Theodoros; Geladari, Anastasia; Politi, Lida; Antachopoulos, Charalampos; Iosifidis, Elias; Tsiatsiou, Olga; Karyoti, Aggeliki; Papanikolaou, Vasileios; Tsakris, Athanassios; Roilides, Emmanuel

2017-07-31

Solid-organ transplant recipients may display high rates of colonization and/or infection by multidrug-resistant bacteria. We analysed and compared the phenotypic and genotypic diversity of carbapenem-resistant (CR) strains of Klebsiella pneumoniae, Pseudomonas aeruginosa and Acinetobacter baumannii isolated from patients in the Solid Organ Transplantation department of our hospital. Between March 2012 and August 2013, 56 CR strains from various biological fluids underwent antimicrobial susceptibility testing with VITEK 2, molecular analysis by PCR amplification and genotypic analysis with pulsed-field gel electrophoresis (PFGE). They were clustered according to antimicrobial drug susceptibility and genotypic profiles. Diversity analyses were performed by calculating Simpson's diversity index and applying computed rarefaction curves.Results/Key findings. Among K. pneumoniae, KP-producers predominated (57.1 %). VIM and OXA-23 carbapenemases prevailed among P. aeruginosa and A. baumannii (89.4 and 88.9 %, respectively). KPC-producing K. pneumoniae and OXA-23 A. baumannii were assigned in single PFGE pulsotypes. VIM-producing P. aeruginosa generated multiple pulsotypes. CR K. pneumoniae strains displayed phenotypic diversity in tigecycline, colistin (CS), amikacin (AMK), gentamicin (GEN) and co-trimoxazole (SXT) (16 clusters); P. aeruginosa displayed phenotypic diversity in cefepime (FEP), ceftazidime, aztreonam, piperacillin, piperacillin-tazobactam, AMK, GEN and CS (9 clusters); and A. baumannii displayed phenotypic diversity in AMK, GEN, SXT, FEP, tobramycin and rifampicin (8 clusters). The Simpson diversity indices for the interpretative phenotype and PFGE analysis were 0.89 and 0.6, respectively, for K. pneumoniae strains (P<0.001); 0.77 and 0.6 for P. aeruginosa (P=0.22); and 0.86 and 0.19 for A. baumannii (P=0.004). The presence of different antimicrobial susceptibility profiles does not preclude the possibility that two CR K. pneumoniae or A. baumannii isolates are clonally related.

Emergence and genomic diversification of a virulent serogroup W:ST-2881(CC175) Neisseria meningitidis clone in the African meningitis belt.

PubMed

Lamelas, Araceli; Hauser, Julia; Dangy, Jean-Pierre; Hamid, Abdul-Wahab M; Röltgen, Katharina; Abdul Sater, Mohamad R; Hodgson, Abraham; Sie, Ali; Junghanss, Thomas; Harris, Simon R; Parkhill, Julian; Bentley, Stephen D; Pluschke, Gerd

2017-08-01

Countries of the African 'meningitis belt' are susceptible to meningococcal meningitis outbreaks. While in the past major epidemics have been primarily caused by serogroup A meningococci, W strains are currently responsible for most of the cases. After an epidemic in Mecca in 2000, W:ST-11 strains have caused many outbreaks worldwide. An unrelated W:ST-2881 clone was described for the first time in 2002, with the first meningitis cases caused by these bacteria reported in 2003. Here we describe results of a comparative whole-genome analysis of 74 W:ST-2881 strains isolated within the framework of two longitudinal colonization and disease studies conducted in Ghana and Burkina Faso. Genomic data indicate that the W:ST-2881 clone has emerged from Y:ST-175(CC175) bacteria by capsule switching. The circulating W:ST-2881 populations were composed of a variety of closely related but distinct genomic variants with no systematic differences between colonization and disease isolates. Two distinct and geographically clustered phylogenetic clonal variants were identified in Burkina Faso and a third in Ghana. On the basis of the presence or absence of 17 recombination fragments, the Ghanaian variant could be differentiated into five clusters. All 25 Ghanaian disease isolates clustered together with 23 out of 40 Ghanaian isolates associated with carriage within one cluster, indicating that W:ST-2881 clusters differ in virulence. More than half of the genes affected by horizontal gene transfer encoded proteins of the 'cell envelope' and the 'transport/binding protein' categories, which indicates that exchange of non-capsular antigens plays an important role in immune evasion.

Detection of clonal aberrations by cytogenetic analysis after different culture methods and by FISH in 129 patients with Chronic Lymphocytic Leukemia.

PubMed

Jenderny, Jutta; Goldmann, Claudia; Thede, Rebekka; Ebrecht, Monika; Korioth, Frank

2014-01-01

There are only a few cytogenetic analysis (CA) studies that directly compare the novel cultivation technique using immunostimulatory CpG-oligonucleotide DSP30/interleukin-2 (DSP30/IL2) with other culture methods. Therefore, parallel cultures of peripheral blood of 129 chronic lymphocytic leukemia (CLL) patients were set up in unstimulated cultures, in the presence of pokeweed medium (PWM), and with DSP30/IL2. Furthermore, CA results were compared with data obtained by FISH. Clonal aberrations were observed by CA in 6% of the cases in unstimulated cultures, in 27% of the cases with PWM, and in 40% of the cases with DSP30/IL2. Some clonal aberrations were detected by CA only with one culture method. Using 3 different culture methods, clonal aberrations were detected in 41% of the cases by CA and in 71% of the cases by FISH. Altogether, 78% of the cases exhibited clonal aberrations discovered by CA and FISH. Also, CA detected clonal aberrations not targeted by FISH in 7% of the cases, and FISH identified clonal aberrations not detected by CA in 36% of the cases. Our study demonstrates that the combined use of CA with different culture methods together with FISH increases our knowledge of the genetic complexity and heterogeneity in CLL pathogenesis. © 2014 S. Karger AG, Basel.

Evolution of Tumor Clones in Adult Acute Lymphoblastic Leukemia.

PubMed

Smirnova, S Yu; Sidorova, Yu V; Ryzhikova, N V; Sychevskaya, K A; Parovichnikova, E N; Sudarikov, A B

2016-01-01

Clonal instability of a tumor cell population in acute lymphoblastic leukemia (ALL) may complicate the monitoring of a minimal residual disease (MRD) by means of patient-specific targets identified at the disease onset. Most of the data concerning the possible instability of rearranged clonal TCR and IG genes during disease recurrence were obtained for ALL in children. The appropriate features of adult ALL, which are known to differ from those of childhood ALL in certain biological characteristics and prognosis, remain insufficiently studied. The aim of this study was to assess the stability of IG and TCR gene rearrangements in adult ALL. Rearrangements were identified according to the BIOMED-2 protocol (PCR followed by fragment analysis). Mismatch in clonal rearrangements at onset and relapse was identified in 83% of patients, indicating clonal instability during treatment. Clonal evolution and diversity of IG and TCR gene rearrangements may be one of the tumor progression mechanisms. New rearrangements may emerge due to residual VDJ-recombinase activity in tumor cells. Also, many clonal IG and TCR gene rearrangements may be present at different levels at a diagnosis, but less abundant clones may be "invisible" due to limited detection sensitivity. Later, major clones may disappear in the course of chemotherapy, while others may proliferate. Investigation of clonal evolution and heterogeneity in ALL and their impact on the treatment efficacy will contribute to the identification of new prognostic factors and the development of therapeutic approaches.

Plant Clonal Integration Mediates the Horizontal Redistribution of Soil Resources, Benefiting Neighboring Plants.

PubMed

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.

Standardizing the Nomenclature for Clonal Lineages of the Sudden Oak Death Pathogen, Phytophthora ramorum

USDA-ARS?s Scientific Manuscript database

Phytophthora ramorum, the causal agent of sudden oak death and ramorum blight, is known to exist as three distinct clonal lineages based on a range of molecular marker systems. However, in the recent literature there exists no consensus on naming of lineages. Here we name clonal lineages of P. ramor...

Investigation of glutathione-derived electrostatic and hydrogen-bonding interactions and their role in defining Grx5 [2Fe-2S] cluster optical spectra and transfer chemistry.

PubMed

Sen, Sambuddha; Bonfio, Claudia; Mansy, Sheref S; Cowan, J A

2018-03-01

Human glutaredoxin 5 (Grx5) is one of the core components of the Isc (iron-sulfur cluster) assembly and trafficking machinery, and serves as an intermediary cluster carrier, putatively delivering cluster from the Isu scaffold protein to target proteins. The tripeptide glutathione is intimately involved in this role, providing cysteinyl coordination to the iron center of the Grx5-bound [2Fe-2S] cluster. Grx5 has a well-defined glutathione-binding pocket with protein amino acid residues providing many ionic and hydrogen binding contacts to the bound glutathione. In this report, we investigated the importance of these interactions in cluster chirality and exchange reactivity by systematically perturbing the crucial contacts by use of natural and non-natural amino acid substitutions to disrupt the binding contacts from both the protein and glutathione. Native Grx5 could be reconstituted with all of the glutathione analogs used, as well as other thiol ligands, such as DTT or L-cysteine, by in vitro chemical reconstitution, and the holo proteins were found to transfer [2Fe-2S] cluster to apo ferredoxin 1 at comparable rates. However, the circular dichroism spectra of these derivatives displayed prominent differences that reflect perturbations in local cluster chirality. These studies provided a detailed molecular understanding of glutathione-protein interactions in holo Grx5 that define both cluster spectroscopy and exchange chemistry.

Jumping translocations of 1q12 in multiple myeloma: a novel mechanism for deletion of 17p in cytogenetically defined high-risk disease.

PubMed

Sawyer, Jeffrey R; Tian, Erming; Heuck, Christoph J; Epstein, Joshua; Johann, Donald J; Swanson, Charles M; Lukacs, Janet L; Johnson, Marian; Binz, Regina; Boast, Angela; Sammartino, Gael; Usmani, Saad; Zangari, Maurizio; Waheed, Sarah; van Rhee, Frits; Barlogie, Bart

2014-04-17

Multiple myeloma (MM) is a B-cell malignancy driven in part by increasing copy number alterations (CNAs) during disease progression. Prognostically significant CNAs accumulate during clonal evolution and include gains of 1q21 and deletions of 17p, among others. Unfortunately, the mechanisms underlying the accumulation of CNAs and resulting subclonal heterogeneity in high-risk MM are poorly understood. To investigate the impact of jumping translocations of 1q12 (JT1q12) on receptor chromosomes (RCs) and subsequent clonal evolution, we analyzed specimens from 86 patients selected for unbalanced 1q12 aberrations by G-banding. Utilizing spectral karyotyping and locus-specific fluorescence in situ hybridization, we identified 10 patients with unexpected focal amplifications of an RC that subsequently translocated as part of a sequential JT1q12 to one or more additional RCs. Four patients exhibited amplification and translocation of 8q24 (MYC), 3 showed amplification of 16q11, and 1 each displayed amplification of 18q21.3 (BCL2), 18q23, or 4p16 (FGFR3). Unexpectedly, in 6 of 14 patients with the combination of the t(4;14) and deletion of 17p, we identified the loss of 17p as resulting from a JT1q12. Here, we provide evidence that the JT1q12 is a mechanism for the simultaneous gain of 1q21 and deletion of 17p in cytogenetically defined high-risk disease.

Revealing hidden clonal complexity in Mycobacterium tuberculosis infection by qualitative and quantitative improvement of sampling.

PubMed

Pérez-Lago, L; Palacios, J J; Herranz, M; Ruiz Serrano, M J; Bouza, E; García-de-Viedma, D

2015-02-01

The analysis of microevolution events, its functional relevance and impact on molecular epidemiology strategies, constitutes one of the most challenging aspects of the study of clonal complexity in infection by Mycobacterium tuberculosis. In this study, we retrospectively evaluated whether two improved sampling schemes could provide access to the clonal complexity that is undetected by the current standards (analysis of one isolate from one sputum). We evaluated in 48 patients the analysis by mycobacterial interspersed repetitive unit-variable number tandem repeat of M. tuberculosis isolates cultured from bronchial aspirate (BAS) or bronchoalveolar lavage (BAL) and, in another 16 cases, the analysis of a higher number of isolates from independent sputum samples. Analysis of the isolates from BAS/BAL specimens revealed clonal complexity in a very high proportion of cases (5/48); in most of these cases, complexity was not detected when the isolates from sputum samples were analysed. Systematic analysis of isolates from multiple sputum samples also improved the detection of clonal complexity. We found coexisting clonal variants in two of 16 cases that would have gone undetected in the analysis of the isolate from a single sputum specimen. Our results suggest that analysis of isolates from BAS/BAL specimens is highly efficient for recording the true clonal composition of M. tuberculosis in the lungs. When these samples are not available, we recommend increasing the number of isolates from independent sputum specimens, because they might not harbour the same pool of bacteria. Our data suggest that the degree of clonal complexity in tuberculosis has been underestimated because of the deficiencies inherent in a simplified procedure. Copyright © 2014 European Society of Clinical Microbiology and Infectious Diseases. Published by Elsevier Ltd. All rights reserved.

VDJ-Seq: Deep Sequencing Analysis of Rearranged Immunoglobulin Heavy Chain Gene to Reveal Clonal Evolution Patterns of B Cell Lymphoma.

PubMed

Jiang, Yanwen; Nie, Kui; Redmond, David; Melnick, Ari M; Tam, Wayne; Elemento, Olivier

2015-12-28

Understanding tumor clonality is critical to understanding the mechanisms involved in tumorigenesis and disease progression. In addition, understanding the clonal composition changes that occur within a tumor in response to certain micro-environment or treatments may lead to the design of more sophisticated and effective approaches to eradicate tumor cells. However, tracking tumor clonal sub-populations has been challenging due to the lack of distinguishable markers. To address this problem, a VDJ-seq protocol was created to trace the clonal evolution patterns of diffuse large B cell lymphoma (DLBCL) relapse by exploiting VDJ recombination and somatic hypermutation (SHM), two unique features of B cell lymphomas. In this protocol, Next-Generation sequencing (NGS) libraries with indexing potential were constructed from amplified rearranged immunoglobulin heavy chain (IgH) VDJ region from pairs of primary diagnosis and relapse DLBCL samples. On average more than half million VDJ sequences per sample were obtained after sequencing, which contain both VDJ rearrangement and SHM information. In addition, customized bioinformatics pipelines were developed to fully utilize sequence information for the characterization of IgH-VDJ repertoire within these samples. Furthermore, the pipeline allows the reconstruction and comparison of the clonal architecture of individual tumors, which enables the examination of the clonal heterogeneity within the diagnosis tumors and deduction of clonal evolution patterns between diagnosis and relapse tumor pairs. When applying this analysis to several diagnosis-relapse pairs, we uncovered key evidence that multiple distinctive tumor evolutionary patterns could lead to DLBCL relapse. Additionally, this approach can be expanded into other clinical aspects, such as identification of minimal residual disease, monitoring relapse progress and treatment response, and investigation of immune repertoires in non-lymphoma contexts.

Reconstruction of cartilage with clonal mesenchymal stem cell-acellular dermal matrix in cartilage defect model in nonhuman primates.

PubMed

Ma, Anlun; Jiang, Li; Song, Lijun; Hu, Yanxin; Dun, Hao; Daloze, Pierre; Yu, Yonglin; Jiang, Jianyuan; Zafarullah, Muhammad; Chen, Huifang

2013-07-01

Articular cartilage defects are commonly associated with trauma, inflammation and osteoarthritis. Mesenchymal stem cell (MSC)-based therapy is a promising novel approach for repairing articular cartilage. Direct intra-articular injection of uncommitted MSCs does not regenerate high-quality cartilage. This study explored utilization of a new three-dimensional, selected chondrogenic clonal MSC-loaded monkey acellular dermal matrix (MSC-ADM) scaffold to repair damaged cartilage in an experimental model of knee joint cartilage defect in Cynomolgus monkeys. MSCs were characterized for cell size, cell yield, phenotypes, proliferation and chondrogenic differentiation capacity. Chondrogenic differentiation assays were performed at different MSC passages by sulfated glycosaminoglycans (sGAG), collagen, and fluorescence activated cell sorter (FACS) analysis. Selected chondrogenic clonal MSCs were seeded onto ADM scaffold with the sandwich model and MSC-loaded ADM grafts were analyzed by confocal microscopy and scanning electron microscopy. Cartilage defects were treated with normal saline, clonal MSCs and clonal MSC-ADM grafts, respectively. The clinical parameters, and histological and immunohistochemical examinations were evaluated at weeks 8, 16, 24 post-treatment, respectively. Polyclonal and clonal MSCs could differentiate into the chondrogenic lineage after stimulation with suitable chondrogenic factors. They expressed mesenchymal markers and were negative for hematopoietic markers. Articular cartilage defects were considerably improved and repaired by selected chondrogenic clonal MSC-based treatment, particularly, in MSC-ADM-treated group. The histological scores in MSC-ADM-treated group were consistently higher than those of other groups. Our results suggest that selected chondrogenic clonal MSC-loaded ADM grafts could improve the cartilage lesions in Cynomolgus monkey model, which may be applicable for repairing similar human cartilage defects. Copyright © 2013 Elsevier B.V. All rights reserved.

Serotyping of Toxoplasma gondii in Cats (Felis domesticus) Reveals Predominance of Type II Infections in Germany

PubMed Central

Maksimov, Pavlo; Zerweck, Johannes; Dubey, Jitender P.; Pantchev, Nikola; Frey, Caroline F.; Maksimov, Aline; Reimer, Ulf; Schutkowski, Mike; Hosseininejad, Morteza; Ziller, Mario; Conraths, Franz J.; Schares, Gereon

2013-01-01

Background Cats are definitive hosts of Toxoplasma gondii and play an essential role in the epidemiology of this parasite. The study aims at clarifying whether cats are able to develop specific antibodies against different clonal types of T. gondii and to determine by serotyping the T. gondii clonal types prevailing in cats as intermediate hosts in Germany. Methodology To establish a peptide-microarray serotyping test, we identified 24 suitable peptides using serological T. gondii positive (n=21) and negative cat sera (n=52). To determine the clonal type-specific antibody response of cats in Germany, 86 field sera from T. gondii seropositive naturally infected cats were tested. In addition, we analyzed the antibody response in cats experimentally infected with non-canonical T. gondii types (n=7). Findings Positive cat reference sera reacted predominantly with peptides harbouring amino acid sequences specific for the clonal T. gondii type the cats were infected with. When the array was applied to field sera from Germany, 98.8% (85/86) of naturally-infected cats recognized similar peptide patterns as T. gondii type II reference sera and showed the strongest reaction intensities with clonal type II-specific peptides. In addition, naturally infected cats recognized type II-specific peptides significantly more frequently than peptides of other type-specificities. Cats infected with non-canonical types showed the strongest reactivity with peptides presenting amino-acid sequences specific for both, type I and type III. Conclusions Cats are able to mount a clonal type-specific antibody response against T. gondii. Serotyping revealed for most seropositive field sera patterns resembling those observed after clonal type II-T. gondii infection. This finding is in accord with our previous results on the occurrence of T. gondii clonal types in oocysts shed by cats in Germany. PMID:24244652

T-cell stimuli independently sum to regulate an inherited clonal division fate

PubMed Central

Marchingo, J. M.; Prevedello, G.; Kan, A.; Heinzel, S.; Hodgkin, P. D.; Duffy, K. R.

2016-01-01

In the presence of antigen and costimulation, T cells undergo a characteristic response of expansion, cessation and contraction. Previous studies have revealed that population-level reproducibility is a consequence of multiple clones exhibiting considerable disparity in burst size, highlighting the requirement for single-cell information in understanding T-cell fate regulation. Here we show that individual T-cell clones resulting from controlled stimulation in vitro are strongly lineage imprinted with highly correlated expansion fates. Progeny from clonal families cease dividing in the same or adjacent generations, with inter-clonal variation producing burst-size diversity. The effects of costimulatory signals on individual clones sum together with stochastic independence; therefore, the net effect across multiple clones produces consistent, but heterogeneous population responses. These data demonstrate that substantial clonal heterogeneity arises through differences in experience of clonal progenitors, either through stochastic antigen interaction or by differences in initial receptor sensitivities. PMID:27869196

Multifocal clonal evolution characterized using circulating tumour DNA in a case of metastatic breast cancer

PubMed Central

Murtaza, Muhammed; Dawson, Sarah-Jane; Pogrebniak, Katherine; Rueda, Oscar M.; Provenzano, Elena; Grant, John; Chin, Suet-Feung; Tsui, Dana W. Y.; Marass, Francesco; Gale, Davina; Ali, H. Raza; Shah, Pankti; Contente-Cuomo, Tania; Farahani, Hossein; Shumansky, Karey; Kingsbury, Zoya; Humphray, Sean; Bentley, David; Shah, Sohrab P.; Wallis, Matthew; Rosenfeld, Nitzan; Caldas, Carlos

2015-01-01

Circulating tumour DNA analysis can be used to track tumour burden and analyse cancer genomes non-invasively but the extent to which it represents metastatic heterogeneity is unknown. Here we follow a patient with metastatic ER-positive and HER2-positive breast cancer receiving two lines of targeted therapy over 3 years. We characterize genomic architecture and infer clonal evolution in eight tumour biopsies and nine plasma samples collected over 1,193 days of clinical follow-up using exome and targeted amplicon sequencing. Mutation levels in the plasma samples reflect the clonal hierarchy inferred from sequencing of tumour biopsies. Serial changes in circulating levels of sub-clonal private mutations correlate with different treatment responses between metastatic sites. This comparison of biopsy and plasma samples in a single patient with metastatic breast cancer shows that circulating tumour DNA can allow real-time sampling of multifocal clonal evolution. PMID:26530965

Clonal evolution of chemotherapy-resistant urothelial carcinoma.

PubMed

Faltas, Bishoy M; Prandi, Davide; Tagawa, Scott T; Molina, Ana M; Nanus, David M; Sternberg, Cora; Rosenberg, Jonathan; Mosquera, Juan Miguel; Robinson, Brian; Elemento, Olivier; Sboner, Andrea; Beltran, Himisha; Demichelis, Francesca; Rubin, Mark A

2016-12-01

Chemotherapy-resistant urothelial carcinoma has no uniformly curative therapy. Understanding how selective pressure from chemotherapy directs the evolution of urothelial carcinoma and shapes its clonal architecture is a central biological question with clinical implications. To address this question, we performed whole-exome sequencing and clonality analysis of 72 urothelial carcinoma samples, including 16 matched sets of primary and advanced tumors prospectively collected before and after chemotherapy. Our analysis provided several insights: (i) chemotherapy-treated urothelial carcinoma is characterized by intra-patient mutational heterogeneity, and the majority of mutations are not shared; (ii) both branching evolution and metastatic spread are very early events in the natural history of urothelial carcinoma; (iii) chemotherapy-treated urothelial carcinoma is enriched with clonal mutations involving L1 cell adhesion molecule (L1CAM) and integrin signaling pathways; and (iv) APOBEC-induced mutagenesis is clonally enriched in chemotherapy-treated urothelial carcinoma and continues to shape the evolution of urothelial carcinoma throughout its lifetime.

Multifocal clonal evolution characterized using circulating tumour DNA in a case of metastatic breast cancer.

PubMed

Murtaza, Muhammed; Dawson, Sarah-Jane; Pogrebniak, Katherine; Rueda, Oscar M; Provenzano, Elena; Grant, John; Chin, Suet-Feung; Tsui, Dana W Y; Marass, Francesco; Gale, Davina; Ali, H Raza; Shah, Pankti; Contente-Cuomo, Tania; Farahani, Hossein; Shumansky, Karey; Kingsbury, Zoya; Humphray, Sean; Bentley, David; Shah, Sohrab P; Wallis, Matthew; Rosenfeld, Nitzan; Caldas, Carlos

2015-11-04

Circulating tumour DNA analysis can be used to track tumour burden and analyse cancer genomes non-invasively but the extent to which it represents metastatic heterogeneity is unknown. Here we follow a patient with metastatic ER-positive and HER2-positive breast cancer receiving two lines of targeted therapy over 3 years. We characterize genomic architecture and infer clonal evolution in eight tumour biopsies and nine plasma samples collected over 1,193 days of clinical follow-up using exome and targeted amplicon sequencing. Mutation levels in the plasma samples reflect the clonal hierarchy inferred from sequencing of tumour biopsies. Serial changes in circulating levels of sub-clonal private mutations correlate with different treatment responses between metastatic sites. This comparison of biopsy and plasma samples in a single patient with metastatic breast cancer shows that circulating tumour DNA can allow real-time sampling of multifocal clonal evolution.

Clonal Evolution of Chemotherapy-resistant Urothelial Carcinoma

PubMed Central

Faltas, Bishoy M.; Prandi, Davide; Tagawa, Scott T.; Molina, Ana M.; Nanus, David M.; Sternberg, Cora; Rosenberg, Jonathan; Mosquera, Juan Miguel; Robinson, Brian; Elemento, Olivier; Sboner, Andrea; Beltran, Himisha; Demichelis, Francesca; Rubin, Mark A.

2017-01-01

Chemotherapy-resistant urothelial carcinoma (UC) has no uniformly curative therapy. Understanding how selective pressure from chemotherapy directs UC’s evolution and shapes its clonal architecture is a central biological question with clinical implications. To address this question, we performed whole-exome sequencing and clonality analysis of 72 UCs including 16 matched sets of primary and advanced tumors prospectively collected before and after chemotherapy. Our analysis provided several insights: (i) chemotherapy-treated UC is characterized by intra-patient mutational heterogeneity and the majority of mutations are not shared, (ii) both branching evolution and metastatic spread are very early events in the natural history of UC; (iii) chemotherapy-treated UC is enriched with clonal mutations involving L1-cell adhesion molecule (L1CAM) and integrin signaling pathways; (iv) APOBEC induced-mutagenesis is clonally-enriched in chemotherapy-treated UC and continues to shape UC’s evolution throughout its lifetime. PMID:27749842

Epigenetic Memory as a Basis for Intelligent Behavior in Clonal Plants.

PubMed

Latzel, Vít; Rendina González, Alejandra P; Rosenthal, Jonathan

2016-01-01

Environmentally induced epigenetic change enables plants to remember past environmental interactions. If this memory capability is exploited to prepare plants for future challenges, it can provide a basis for highly sophisticated behavior, considered intelligent by some. Against the backdrop of an overview of plant intelligence, we hypothesize: (1) that the capability of plants to engage in such intelligent behavior increases with the additional level of complexity afforded by clonality, and; (2) that more faithful inheritance of epigenetic information in clonal plants, in conjunction with information exchange and coordination between connected ramets, is likely to enable especially advanced intelligent behavior in this group. We therefore further hypothesize that this behavior provides ecological and evolutionary advantages to clonal plants, possibly explaining, at least in part, their widespread success. Finally, we suggest avenues of inquiry to enable assessing intelligent behavior and the role of epigenetic memory in clonal species.

Tracking the Evolution of Non-Small-Cell Lung Cancer.

PubMed

Jamal-Hanjani, Mariam; Wilson, Gareth A; McGranahan, Nicholas; Birkbak, Nicolai J; Watkins, Thomas B K; Veeriah, Selvaraju; Shafi, Seema; Johnson, Diana H; Mitter, Richard; Rosenthal, Rachel; Salm, Max; Horswell, Stuart; Escudero, Mickael; Matthews, Nik; Rowan, Andrew; Chambers, Tim; Moore, David A; Turajlic, Samra; Xu, Hang; Lee, Siow-Ming; Forster, Martin D; Ahmad, Tanya; Hiley, Crispin T; Abbosh, Christopher; Falzon, Mary; Borg, Elaine; Marafioti, Teresa; Lawrence, David; Hayward, Martin; Kolvekar, Shyam; Panagiotopoulos, Nikolaos; Janes, Sam M; Thakrar, Ricky; Ahmed, Asia; Blackhall, Fiona; Summers, Yvonne; Shah, Rajesh; Joseph, Leena; Quinn, Anne M; Crosbie, Phil A; Naidu, Babu; Middleton, Gary; Langman, Gerald; Trotter, Simon; Nicolson, Marianne; Remmen, Hardy; Kerr, Keith; Chetty, Mahendran; Gomersall, Lesley; Fennell, Dean A; Nakas, Apostolos; Rathinam, Sridhar; Anand, Girija; Khan, Sajid; Russell, Peter; Ezhil, Veni; Ismail, Babikir; Irvin-Sellers, Melanie; Prakash, Vineet; Lester, Jason F; Kornaszewska, Malgorzata; Attanoos, Richard; Adams, Haydn; Davies, Helen; Dentro, Stefan; Taniere, Philippe; O'Sullivan, Brendan; Lowe, Helen L; Hartley, John A; Iles, Natasha; Bell, Harriet; Ngai, Yenting; Shaw, Jacqui A; Herrero, Javier; Szallasi, Zoltan; Schwarz, Roland F; Stewart, Aengus; Quezada, Sergio A; Le Quesne, John; Van Loo, Peter; Dive, Caroline; Hackshaw, Allan; Swanton, Charles

2017-06-01

Among patients with non-small-cell lung cancer (NSCLC), data on intratumor heterogeneity and cancer genome evolution have been limited to small retrospective cohorts. We wanted to prospectively investigate intratumor heterogeneity in relation to clinical outcome and to determine the clonal nature of driver events and evolutionary processes in early-stage NSCLC. In this prospective cohort study, we performed multiregion whole-exome sequencing on 100 early-stage NSCLC tumors that had been resected before systemic therapy. We sequenced and analyzed 327 tumor regions to define evolutionary histories, obtain a census of clonal and subclonal events, and assess the relationship between intratumor heterogeneity and recurrence-free survival. We observed widespread intratumor heterogeneity for both somatic copy-number alterations and mutations. Driver mutations in EGFR, MET, BRAF, and TP53 were almost always clonal. However, heterogeneous driver alterations that occurred later in evolution were found in more than 75% of the tumors and were common in PIK3CA and NF1 and in genes that are involved in chromatin modification and DNA damage response and repair. Genome doubling and ongoing dynamic chromosomal instability were associated with intratumor heterogeneity and resulted in parallel evolution of driver somatic copy-number alterations, including amplifications in CDK4, FOXA1, and BCL11A. Elevated copy-number heterogeneity was associated with an increased risk of recurrence or death (hazard ratio, 4.9; P=4.4×10 -4 ), which remained significant in multivariate analysis. Intratumor heterogeneity mediated through chromosome instability was associated with an increased risk of recurrence or death, a finding that supports the potential value of chromosome instability as a prognostic predictor. (Funded by Cancer Research UK and others; TRACERx ClinicalTrials.gov number, NCT01888601 .).

Clonal evolution in paired endometrial intraepithelial neoplasia/atypical hyperplasia and endometrioid adenocarcinoma.

PubMed

Russo, Mariano; Broach, James; Sheldon, Kathryn; Houser, Kenneth R; Liu, Dajiang J; Kesterson, Joshua; Phaeton, Rebecca; Hossler, Carrie; Hempel, Nadine; Baker, Maria; Newell, Jordan M; Zaino, Richard; Warrick, Joshua I

2017-09-01

Endometrial intraepithelial neoplasia (EIN) and atypical endometrial hyperplasia (AH) are histomorphologically defined precursors to endometrioid adenocarcinoma, which are unified as EIN/AH by the World Health Organization. EIN/AH harbors a constellation of molecular alterations similar to those found in endometrioid adenocarcinoma. However, the process of clonal evolution from EIN/AH to carcinoma is poorly characterized. To investigate, we performed next-generation sequencing, copy number alteration (CNA) analysis, and immunohistochemistry for mismatch repair protein expression on EIN/AH and endometrioid adenocarcinoma samples from 6 hysterectomy cases with spatially distinct EIN/AH and carcinoma. In evaluating all samples, EIN/AH and carcinoma did not differ in mutational burden, CNA burden, or specific genes mutated (all P>.1). All paired EIN/AH and carcinoma samples shared at least one identical somatic mutation, frequently in PI(3)K pathway members. Large CNAs (>10 genes in length) were identified in 83% of cases; paired EIN/AH and carcinoma samples shared at least one identical CNA in these cases. Mismatch repair protein expression matched in all paired EIN/AH and carcinoma samples. All paired EIN/AH and carcinoma samples had identical The Cancer Genome Atlas subtype, with 3 classified as "copy number low endometrioid" and 3 classified as "microsatellite instability hypermutated." Although paired EIN/AH and carcinoma samples were clonal, private mutations (ie, present in only one sample) were identified in EIN/AH and carcinoma in all cases, frequently in established cancer-driving genes. These findings indicate that EIN/AH gives rise to endometrioid adenocarcinoma by a complex process of subclone evolution, not a linear accumulation of molecular events. Copyright © 2017 Elsevier Inc. All rights reserved.

Mutational landscape of gastric adenocarcinoma in Chinese: implications for prognosis and therapy.

PubMed

Chen, Kexin; Yang, Da; Li, Xiangchun; Sun, Baocun; Song, Fengju; Cao, Wenfeng; Brat, Daniel J; Gao, Zhibo; Li, Haixin; Liang, Han; Zhao, Yanrui; Zheng, Hong; Li, Miao; Buckner, Jan; Patterson, Scott D; Ye, Xiang; Reinhard, Christoph; Bhathena, Anahita; Joshi, Deepa; Mischel, Paul S; Croce, Carlo M; Wang, Yi Michael; Raghavakaimal, Sreekumar; Li, Hui; Lu, Xin; Pan, Yang; Chang, Han; Ba, Sujuan; Luo, Longhai; Cavenee, Webster K; Zhang, Wei; Hao, Xishan

2015-01-27

Gastric cancer (GC) is a highly heterogeneous disease. To identify potential clinically actionable therapeutic targets that may inform individualized treatment strategies, we performed whole-exome sequencing on 78 GCs of differing histologies and anatomic locations, as well as whole-genome sequencing on two GC cases, each with three primary tumors and two matching lymph node metastases. The data showed two distinct GC subtypes with either high-clonality (HiC) or low-clonality (LoC). The HiC subtype of intratumoral heterogeneity was associated with older age, TP53 (tumor protein P53) mutation, enriched C > G transition, and significantly shorter survival, whereas the LoC subtype was associated with younger age, ARID1A (AT rich interactive domain 1A) mutation, and significantly longer survival. Phylogenetic tree analysis of whole-genome sequencing data from multiple samples of two patients supported the clonal evolution of GC metastasis and revealed the accumulation of genetic defects that necessitate combination therapeutics. The most recurrently mutated genes, which were validated in a separate cohort of 216 cases by targeted sequencing, were members of the homologous recombination DNA repair, Wnt, and PI3K-ERBB pathways. Notably, the drugable NRG1 (neuregulin-1) and ERBB4 (V-Erb-B2 avian erythroblastic leukemia viral oncogene homolog 4) ligand-receptor pair were mutated in 10% of GC cases. Mutations of the BRCA2 (breast cancer 2, early onset) gene, found in 8% of our cohort and validated in The Cancer Genome Atlas GC cohort, were associated with significantly longer survivals. These data define distinct clinicogenetic forms of GC in the Chinese population that are characterized by specific mutation sets that can be investigated for efficacy of single and combination therapies.

The cacao pathogen Moniliophthora roreri (Marasmiaceae) possesses biallelic A and B mating loci but reproduces clonally

PubMed Central

Díaz-Valderrama, J R; Aime, M C

2016-01-01

The cacao pathogen Moniliophthora roreri belongs to the mushroom-forming family Marasmiaceae, but it has never been observed to produce a fruiting body, which calls to question its capacity for sexual reproduction. In this study, we identified potential A (HD1 and HD2) and B (pheromone precursors and pheromone receptors) mating genes in M. roreri. A PCR-based method was subsequently devised to determine the mating type for a set of 47 isolates from across the geographic range of the fungus. We developed and generated an 11-marker microsatellite set and conducted association and linkage disequilibrium (standardized index of association, IAs) analyses. We also performed an ancestral reconstruction analysis to show that the ancestor of M. roreri is predicted to be heterothallic and tetrapolar, which together with sliding window analyses support that the A and B mating loci are likely unlinked and follow a tetrapolar organization within the genome. The A locus is composed of a pair of HD1 and HD2 genes, whereas the B locus consists of a paired pheromone precursor, Mr_Ph4, and receptor, STE3_Mr4. Two A and B alleles but only two mating types were identified. Association analyses divided isolates into two well-defined genetically distinct groups that correlate with their mating type; IAs values show high linkage disequilibrium as is expected in clonal reproduction. Interestingly, both mating types were found in South American isolates but only one mating type was found in Central American isolates, supporting a prior hypothesis of clonal dissemination throughout Central America after a single or very few introductions of the fungus from South America. PMID:26932308

The cacao pathogen Moniliophthora roreri (Marasmiaceae) possesses biallelic A and B mating loci but reproduces clonally.

PubMed

Díaz-Valderrama, J R; Aime, M C

2016-06-01

The cacao pathogen Moniliophthora roreri belongs to the mushroom-forming family Marasmiaceae, but it has never been observed to produce a fruiting body, which calls to question its capacity for sexual reproduction. In this study, we identified potential A (HD1 and HD2) and B (pheromone precursors and pheromone receptors) mating genes in M. roreri. A PCR-based method was subsequently devised to determine the mating type for a set of 47 isolates from across the geographic range of the fungus. We developed and generated an 11-marker microsatellite set and conducted association and linkage disequilibrium (standardized index of association, IA(s)) analyses. We also performed an ancestral reconstruction analysis to show that the ancestor of M. roreri is predicted to be heterothallic and tetrapolar, which together with sliding window analyses support that the A and B mating loci are likely unlinked and follow a tetrapolar organization within the genome. The A locus is composed of a pair of HD1 and HD2 genes, whereas the B locus consists of a paired pheromone precursor, Mr_Ph4, and receptor, STE3_Mr4. Two A and B alleles but only two mating types were identified. Association analyses divided isolates into two well-defined genetically distinct groups that correlate with their mating type; IA(s) values show high linkage disequilibrium as is expected in clonal reproduction. Interestingly, both mating types were found in South American isolates but only one mating type was found in Central American isolates, supporting a prior hypothesis of clonal dissemination throughout Central America after a single or very few introductions of the fungus from South America.

Rise and fall of outbreak-specific clone inside endemic pulsotype of Salmonella 4,[5],12:i:-; insights from high-resolution molecular surveillance in Emilia-Romagna, Italy, 2012 to 2015.

PubMed

Morganti, Marina; Bolzoni, Luca; Scaltriti, Erika; Casadei, Gabriele; Carra, Elena; Rossi, Laura; Gherardi, Paola; Faccini, Fabio; Arrigoni, Norma; Sacchi, Anna Rita; Delledonne, Marco; Pongolini, Stefano

2018-03-01

Background and aimEpidemiology of human non-typhoid salmonellosis is characterised by recurrent emergence of new clones of the pathogen over time. Some clonal lines of Salmonella have shaped epidemiology of the disease at global level, as happened for serotype Enteritidis or, more recently, for Salmonella 4,[5],12:i:-, a monophasic variant of serotype Typhimurium. The same clonal behaviour is recognisable at sub-serotype level where single outbreaks or more generalised epidemics are attributable to defined clones. The aim of this study was to understand the dynamics of a clone of Salmonella 4,[5],12:i:- over a 3-year period (2012-15) in a province of Northern Italy where the clone caused a large outbreak in 2013. Furthermore, the role of candidate outbreak sources was investigated and the accuracy of multilocus variable-number tandem repeat analysis (MLVA) was evaluated. Methods: we retrospectively investigated the outbreak through whole genome sequencing (WGS) and further monitored the outbreak clone for 2 years after its conclusion. Results: The study showed the transient nature of the clone in the population, possibly as a consequence of its occasional expansion in a food-processing facility. We demonstrated that important weaknesses characterise conventional typing methods applied to clonal pathogens such as Salmonella 4,[5],12:i:-, namely lack of accuracy for MLVA and inadequate resolution power for PFGE to be reliably used for clone tracking. Conclusions : The study provided evidence for the remarkable prevention potential of whole genome sequencing used as a routine tool in systems that integrate human, food and animal surveillance.

Cell plasticity and heterogeneity in cancer.

PubMed

Marjanovic, Nemanja D; Weinberg, Robert A; Chaffer, Christine L

2013-01-01

Heterogeneity within a given cancer arises from diverse cell types recruited to the tumor and from genetic and/or epigenetic differences amongst the cancer cells themselves. These factors conspire to create a disease with various phenotypes. There are 2 established models of cancer development and progression to metastatic disease. These are the clonal evolution and cancer stem cell models. The clonal evolution theory suggests that successive mutations accumulating in a given cell generate clonal outgrowths that thrive in response to microenvironmental selection pressures, dictating the phenotype of the tumor. The alternative cancer stem cell (CSC) model suggests that cancer cells with similar genetic backgrounds can be hierarchically organized according to their tumorigenic potential. Accordingly, CSCs reside at the apex of the hierarchy and are thought to possess the majority of a cancer's tumor-initiating and metastatic ability. A defining feature of this model is its apparent unidirectional nature, whereby CSCs undergo symmetric division to replenish the CSC pool and irreversible asymmetric division to generate daughter cells (non-CSCs) with low tumorigenic potential. However, evolving evidence supports a new model of tumorigenicity, in which considerable plasticity exists between the non-CSC and CSC compartments, such that non-CSCs can reacquire a CSC phenotype. These findings suggest that some tumors may adhere to a plastic CSC model, in which bidirectional conversions are common and essential components of tumorigenicity. Accumulating evidence surrounding the plasticity of cancer cells, in particular, suggests that aggressive CSCs can be created de novo within a tumor. Given the current focus on therapeutic targeting of CSCs, we discuss the implications of non-CSC-to-CSC conversions on the development of future therapies. © 2012 American Association for Clinical Chemistry

Partial reconstitution of humoral immunity and fewer infections in patients with chronic lymphocytic leukemia treated with ibrutinib.

PubMed

Sun, Clare; Tian, Xin; Lee, Yuh Shan; Gunti, Sreenivasulu; Lipsky, Andrew; Herman, Sarah E M; Salem, Dalia; Stetler-Stevenson, Maryalice; Yuan, Constance; Kardava, Lela; Moir, Susan; Maric, Irina; Valdez, Janet; Soto, Susan; Marti, Gerald E; Farooqui, Mohammed Z; Notkins, Abner L; Wiestner, Adrian; Aue, Georg

2015-11-05

Chronic lymphocytic leukemia (CLL) is characterized by immune dysregulation, often including hypogammaglobulinemia, which contributes to a high rate of infections and morbidity. Ibrutinib, a covalent inhibitor of Bruton tyrosine kinase (BTK), inhibits B-cell receptor signaling and is an effective, US Food and Drug Administration (FDA)-approved treatment of CLL. Inactivating germline mutations in BTK cause a severe B-cell defect and agammaglobulinemia. Therefore, we assessed the impact of ibrutinib on immunoglobulin levels, normal B cells, and infection rate in patients with CLL treated with single-agent ibrutinib on a phase 2 investigator-initiated trial. Consistent with previous reports, immunoglobulin G (IgG) levels remained stable during the first 6 months on treatment, but decreased thereafter. In contrast, there were a transient increase in IgM and a sustained increase in IgA (median increase 45% at 12 months, P < .0001). To distinguish the effects on clonal B cells from normal B cells, we measured serum free light chains (FLCs). In κ-clonal CLL cases, clonal (κ) FLCs were elevated at baseline and normalized by 6 months. Nonclonal (λ) FLCs, which were often depressed at baseline, increased, suggesting the recovery of normal B cells. Consistently, we observed normal B-cell precursors in the bone marrow and an increase in normal B-cell numbers in the peripheral blood. Patients with superior immune reconstitution, as defined by an increase in serum IgA of ≥50% from baseline to 12 months, had a significantly lower rate of infections (P = .03). These data indicate that ibrutinib allows for a clinically meaningful recovery of humoral immune function in patients with CLL. This trial was registered at www.clinicaltrials.gov as #NCT015007330.

Partial reconstitution of humoral immunity and fewer infections in patients with chronic lymphocytic leukemia treated with ibrutinib

PubMed Central

Sun, Clare; Tian, Xin; Lee, Yuh Shan; Gunti, Sreenivasulu; Lipsky, Andrew; Herman, Sarah E. M.; Salem, Dalia; Stetler-Stevenson, Maryalice; Yuan, Constance; Kardava, Lela; Moir, Susan; Maric, Irina; Valdez, Janet; Soto, Susan; Marti, Gerald E.; Farooqui, Mohammed Z.; Notkins, Abner L.; Aue, Georg

2015-01-01

Chronic lymphocytic leukemia (CLL) is characterized by immune dysregulation, often including hypogammaglobulinemia, which contributes to a high rate of infections and morbidity. Ibrutinib, a covalent inhibitor of Bruton tyrosine kinase (BTK), inhibits B-cell receptor signaling and is an effective, US Food and Drug Administration (FDA)-approved treatment of CLL. Inactivating germline mutations in BTK cause a severe B-cell defect and agammaglobulinemia. Therefore, we assessed the impact of ibrutinib on immunoglobulin levels, normal B cells, and infection rate in patients with CLL treated with single-agent ibrutinib on a phase 2 investigator-initiated trial. Consistent with previous reports, immunoglobulin G (IgG) levels remained stable during the first 6 months on treatment, but decreased thereafter. In contrast, there were a transient increase in IgM and a sustained increase in IgA (median increase 45% at 12 months, P < .0001). To distinguish the effects on clonal B cells from normal B cells, we measured serum free light chains (FLCs). In κ-clonal CLL cases, clonal (κ) FLCs were elevated at baseline and normalized by 6 months. Nonclonal (λ) FLCs, which were often depressed at baseline, increased, suggesting the recovery of normal B cells. Consistently, we observed normal B-cell precursors in the bone marrow and an increase in normal B-cell numbers in the peripheral blood. Patients with superior immune reconstitution, as defined by an increase in serum IgA of ≥50% from baseline to 12 months, had a significantly lower rate of infections (P = .03). These data indicate that ibrutinib allows for a clinically meaningful recovery of humoral immune function in patients with CLL. This trial was registered at www.clinicaltrials.gov as #NCT015007330. PMID:26337493

The clonal distribution and diversity of extraintestinal Escherichia coli isolates vary according to patient characteristics.

PubMed

Banerjee, Ritu; Johnston, Brian; Lohse, Christine; Chattopadhyay, Sujay; Tchesnokova, Veronika; Sokurenko, Evgeni V; Johnson, James R

2013-12-01

The clonal distribution of Escherichia coli across an unselected population in the current era of widespread antimicrobial resistance is incompletely defined. In this study, we used a newly described clonal typing strategy based on sequencing of fumC and fimH (i.e., CH typing) to infer multilocus sequence types (STs) for 299 consecutive, nonduplicate extraintestinal E. coli isolates from all cultures submitted to Olmsted County, MN, laboratories in February and March 2011 and then compared STs with epidemiological data. Forty-seven different STs were identified, most commonly ST131 (27%), ST95 (11%), ST73 (8%), ST127 (6%), and ST69 (5%). Isolates from these five STs comprised two-thirds of health care-associated (HA) isolates but only half of community-associated (CA) isolates. ST131 was represented overwhelmingly (88%) by a single recently expanded H30 subclone, which was the most extensively antimicrobial-resistant subclone overall and was especially predominant in HA infections and among adults >50 years old. In contrast, among patients 11 to 50 years old, ST69, -95, and -73 were more common. Because of the preponderance of the H30 subclone of ST131, ST diversity was lower among HA than CA isolates, and among antimicrobial-resistant than antimicrobial-susceptible isolates, which otherwise had similar ST distributions. In conclusion, in this U.S. Midwest region, the distribution and diversity of STs among extraintestinal E. coli clinical isolates vary by patient age, type of infection, and resistance phenotype. ST131 predominates among young children and the elderly, HA infections, and antimicrobial-resistant isolates, whereas other well-known pathogenic lineages are more common among adolescents and young adults, CA infections, and antimicrobial-susceptible isolates.

Co-colonization and clonal diversity of methicillin-sensitive and methicillin-resistant Staphylococcus aureus in sows.

PubMed

Fetsch, Alexandra; Roesler, Uwe; Kraushaar, Britta; Friese, Anika

2016-03-15

Methicillin-susceptible Staphylococcus (S.) aureus (MSSA) and methicillin-resistant S. aureus (MRSA) are colonizers of skin and mucosa. In humans, MSSA and MRSA compete for colonization space in the anterior nares of pig farmers; however, it was also shown that MSSA/MRSA co-colonization is common and one clone can be found rather than differing types of MSSA and MRSA. We investigated the colonization and clonality of both, MSSA and MRSA in pigs over a longer time. Eighteen sows were nasally sampled three times every ten weeks. Additionally, environmental samples were taken. Samples were investigated for MSSA and MRSA, respectively. The spa type was defined from up to five MRSA and MSSA isolates found per sample and sampling time; selected isolates were further investigated by microarray. Three sows (16.7%) were completely negative for MSSA and MRSA. Twelve pigs (66.7%) were irregularly positive for both, MSSA and MRSA over the time, whereas seven out of them (38.9%) were simultaneously colonized. CC398 (t034, t011) MRSA and CC9 (t337, t1430, and t13816) MSSA associated spa types were exclusively found. In 44.4% (n=8) of sows up to two different types of MSSA were present at the same time and sample. Strains of the same clonal lineage showed a high genetic identity despite their origin. Highly identic clones were present in sows and their environment. As conclusion, MSSA/MRSA may not exclude each other in the anterior nares of pigs. Pigs may also carry different clones at the same time. Copyright © 2016 Elsevier B.V. All rights reserved.

Genetic characterization of Hawaiian isolates of Plasmodium relictum reveals mixed-genotype infections

PubMed Central

Jarvi, Susan I; Farias, Margaret EM; Atkinson, Carter T

2008-01-01

Background The relatively recent introduction of a highly efficient mosquito vector and an avian pathogen (Plasmodium relictum) to an isolated island ecosystem with naïve, highly susceptible avian hosts provides a unique opportunity to investigate evolution of virulence in a natural system. Mixed infections can significantly contribute to the uncertainty in host-pathogen dynamics with direct impacts on virulence. Toward further understanding of how host-parasite and parasite-parasite relationships may impact virulence, this study characterizes within-host diversity of malaria parasite populations based on genetic analysis of the trap (thrombospondin-related anonymous protein) gene in isolates originating from Hawaii, Maui and Kauai Islands. Methods A total of 397 clones were produced by nested PCR amplification and cloning of a 1664 bp fragment of the trap gene from two malarial isolates, K1 (Kauai) and KV115 (Hawaii) that have been used for experimental studies, and from additional isolates from wild birds on Kauai, Maui and Hawaii Islands. Diversity of clones was evaluated initially by RFLP-based screening, followed by complete sequencing of 33 selected clones. Results RFLP analysis of trap revealed a minimum of 28 distinct RFLP haplotypes among the 397 clones from 18 birds. Multiple trap haplotypes were detected in every bird evaluated, with an average of 5.9 haplotypes per bird. Overall diversity did not differ between the experimental isolates, however, a greater number of unique haplotypes were detected in K1 than in KV115. We detected high levels of clonal diversity with clear delineation between isolates K1 and KV115 in a haplotype network. The patterns of within-host haplotype clustering are consistent with the possibility of a clonal genetic structure and rapid within-host mutation after infection. Conclusion Avian malaria (P. relictum) and Avipoxvirus are the significant infectious diseases currently affecting the native Hawaiian avifauna. This study shows that clonal diversity of Hawaiian isolates of P. relictum is much higher than previously recognized. Mixed infections can significantly contribute to the uncertainty in host-pathogen dynamics with direct implications for host demographics, disease management strategies, and evolution of virulence. The results of this study indicate a widespread presence of multiple-genotype malaria infections with high clonal diversity in native birds of Hawaii, which when coupled with concurrent infection with Avipoxvirus, may significantly influence evolution of virulence. Reviewers This article was reviewed by Joseph Schall (nominated by Laura Landweber), Daniel Jeffares (nominated by Anthony Poole) and Susan Perkins (nominated by Eugene Koonin). PMID:18578879

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

Treesearch

Daniel Manter; Everett Hansen; Jennifer. Parke

2010-01-01

Phytophthora ramorum populations are clonal and consist of three clonal lineages: EU1 is the only lineage found in Europe with a few isolated nursery infections in the USA; NA1 is associated with natural infestations in California and Oregon as well as some nursery infections in North America, and NA2 has a limited distribution and has only...

Marker-based estimates reveal significant non-additive effects in clonally propagated cassava (Manihot esculenta): implications for the prediction of total genetic value and the selection of varieties

USDA-ARS?s Scientific Manuscript database

In clonally propagated crops, non-additive genetic effects can be effectively exploited by the identification of superior genetic individuals as varieties. Cassava (Manihot esculenta Crantz) is a clonally propagated staple food crop that feeds hundreds of millions. We quantified the amount and natur...

Routine conventional karyotyping of lymphoma staging bone marrow samples does not contribute clinically relevant information.

PubMed

Nardi, Valentina; Pulluqi, Olja; Abramson, Jeremy S; Dal Cin, Paola; Hasserjian, Robert P

2015-06-01

Bone marrow (BM) evaluation is an important part of lymphoma staging, which guides patient management. Although positive staging marrow is defined as morphologically identifiable disease, such samples often also include flow cytometric analysis and conventional karyotyping. Cytogenetic analysis is a labor-intensive and costly procedure and its utility in this setting is uncertain. We retrospectively reviewed pathological reports of 526 staging marrow specimens in which conventional karyotyping had been performed. All samples originated from a single institution from patients with previously untreated Hodgkin and non-Hodgkin lymphomas presenting in an extramedullary site. Cytogenetic analysis revealed clonal abnormalities in only eight marrow samples (1.5%), all of which were positive for lymphoma by morphologic evaluation. Flow cytometry showed a small clonal lymphoid population in three of the 443 morphologically negative marrow samples (0.7%). Conventional karyotyping is rarely positive in lymphoma staging marrow samples and, in our cohort, the BM karyotype did not contribute clinically relevant information in the vast majority of cases. Our findings suggest that karyotyping should not be performed routinely on BM samples taken to stage previously diagnosed extramedullary lymphomas unless there is pathological evidence of BM involvement by lymphoma. © 2015 Wiley Periodicals, Inc.

Translational Implications of Tumor Heterogeneity

PubMed Central

Jamal-Hanjani, Mariam; Quezada, Sergio A.; Larkin, James; Swanton, Charles

2015-01-01

Advances in next-generation sequencing and bioinformatics have led to an unprecedented view of the cancer genome and its evolution. Genomic studies have demonstrated the complex and heterogeneous clonal landscape of tumors of different origins, and the potential impact of intratumor heterogeneity on treatment response and resistance, cancer progression and the risk of disease relapse. However, the significance of subclonal mutations, in particular mutations in driver genes, and their evolution through time and their dynamics in response to cancer therapies, is yet to be determined. The necessary tools are now available to prospectively determine whether clonal heterogeneity can be used as a biomarker of clinical outcome, and to what extent subclonal somatic alterations might influence clinical outcome. Studies that employ longitudinal tissue sampling, integrating both genomic and clinical data, have the potential to reveal the subclonal composition and track the evolution of tumors in order to address these questions, and to begin to define the breadth of genetic diversity in different tumor types, and its relevance to patient outcome. Such studies may provide further evidence for novel drug resistance mechanisms informing novel combinatorial, adaptive and tumour immune-therapies placed within the context of tumor evolution. PMID:25770293

The genomic landscape of acute lymphoblastic leukemia in children and young adults.

PubMed

Mullighan, Charles G

2014-12-05

Our understanding of the genetic basis of childhood acute lymphoblastic leukemia (ALL) has been greatly advanced by genomic profiling and sequencing studies. These efforts have characterized the genetic basis of recently described and poorly understood subtypes of ALL, including early T-cell precursor ALL, Philadelphia chromosome-like (Ph-like) ALL, and ALL with intrachromosomal amplification of chromosome 21, and have identified several rational therapeutic targets in high-risk ALL, notably ABL1-class and JAK-STAT inhibitors in Ph-like ALL. Deep sequencing studies are also refining our understanding of the genetic basis of clonal heterogeneity and relapse. These studies have elucidated the nature of clonal evolution during disease progression and identified genetic changes that confer resistance to specific therapeutic agents, including CREBBP and NT5C2. Genomic profiling has also identified common and rare inherited genetic variants that influence the risk of developing leukemia. These efforts are now being extended to ALL in adolescents and adults with the goal of fully defining the genetic landscape of ALL to further improve treatment outcomes in high-risk populations. © 2014 by The American Society of Hematology. All rights reserved.

Advances in the translational genomics of neuroblastoma

PubMed Central

Bosse, Kristopher R.; Maris, John M.

2015-01-01

Neuroblastoma is an embryonal malignancy that commonly affects young children and is remarkably heterogenous in its malignant potential. Recently, the genetic basis of neuroblastoma has come into focus, which has catalyzed not only a more comprehensive understanding of neuroblastoma tumorigenesis, but has also revealed novel oncogenic vulnerabilities that are being leveraged therapeutically. Neuroblastoma is a model pediatric solid tumor in its use of recurrent genomic alterations, such as high-level MYCN amplification, for risk stratification. Given the relative paucity of recurrent activating somatic point mutations or gene fusions in primary neuroblastoma tumors studied at initial diagnosis, innovative treatment approaches beyond small molecules targeting mutated or dysregulated kinases will be required moving forward to achieve noticeable improvements in overall patient survival. However, the clonally acquired, oncogenic aberrations in relapsed neuroblastomas are currently being defined and may offer an opportunity to improve patient outcomes with molecularly targeted therapy directed towards aberrantly regulated pathways in relapsed disease. This review will summarize the current state of knowledge of neuroblastoma genetics and genomics, highlighting the improved prognostication and potential therapeutic opportunities that have arisen from recent advances in understanding germline predisposition, recurrent segmental chromosomal alterations, somatic point mutations and translocations, and clonal evolution in relapsed neuroblastoma. PMID:26539795

Biofilm formation, antibiotic susceptibility and RAPD genotypes in Pseudomonas aeruginosa clinical strains isolated from single centre intensive care unit patients.

PubMed

Vaněrková, Martina; Mališová, Barbora; Kotásková, Iva; Holá, Veronika; Růžička, Filip; Freiberger, Tomáš

2017-11-01

The aim of this study was to analyse genotypes, antimicrobial susceptibility patterns and serotypes in Pseudomonas aeruginosa clinical strains, including the clonal dissemination of particular strains throughout various intensive care units in one medical centre. Using random amplified polymorphic DNA (RAPD-PCR) and P. aeruginosa antisera, 22 different genotypes and 8 serotypes were defined among 103 isolates from 48 patients. No direct association between P. aeruginosa strain genotypes and serotypes was observed. RAPD typing in strains with the same serotype revealed different genotypes and, on the contrary, most strains with a different serotype displayed the same amplification pattern. The resulting banding patterns showed a high degree of genetic heterogeneity among all isolates from the patients examined, suggesting a non-clonal relationship between isolates from these patients. A higher degree of antibiotic resistance and stronger biofilm production in common genotypes compared to rare ones and genetic homogeneity of the most resistant strains indicated the role of antibiotic pressure in acquiring resistant and more virulent strains in our hospital. In conclusion, genetic characterisation of P. aeruginosa strains using RAPD method was shown to be more accurate in epidemiological analyses than phenotyping.

A genotyping protocol for multiple tissue types from the polyploid tree species Sequoia sempervirens (Cupressaceae)1

PubMed Central

Narayan, Lakshmi; Dodd, Richard S.; O’Hara, Kevin L.

2015-01-01

Premise of the study: Identifying clonal lineages in asexually reproducing plants using microsatellite markers is complicated by the possibility of nonidentical genotypes from the same clonal lineage due to somatic mutations, null alleles, and scoring errors. We developed and tested a clonal identification protocol that is robust to these issues for the asexually reproducing hexaploid tree species coast redwood (Sequoia sempervirens). Methods: Microsatellite data from four previously published and two newly developed primers were scored using a modified protocol, and clones were identified using Bruvo genetic distances. The effectiveness of this clonal identification protocol was assessed using simulations and by genotyping a test set of paired samples of different tissue types from the same trees. Results: Data from simulations showed that our protocol allowed us to accurately identify clonal lineages. Multiple test samples from the same trees were identified correctly, although certain tissue type pairs had larger genetic distances on average. Discussion: The methods described in this paper will allow for the accurate identification of coast redwood clones, facilitating future studies of the reproductive ecology of this species. The techniques used in this paper can be applied to studies of other clonal organisms as well. PMID:25798341

A genotyping protocol for multiple tissue types from the polyploid tree species Sequoia sempervirens (Cupressaceae).

PubMed

Narayan, Lakshmi; Dodd, Richard S; O'Hara, Kevin L

2015-03-01

Identifying clonal lineages in asexually reproducing plants using microsatellite markers is complicated by the possibility of nonidentical genotypes from the same clonal lineage due to somatic mutations, null alleles, and scoring errors. We developed and tested a clonal identification protocol that is robust to these issues for the asexually reproducing hexaploid tree species coast redwood (Sequoia sempervirens). Microsatellite data from four previously published and two newly developed primers were scored using a modified protocol, and clones were identified using Bruvo genetic distances. The effectiveness of this clonal identification protocol was assessed using simulations and by genotyping a test set of paired samples of different tissue types from the same trees. Data from simulations showed that our protocol allowed us to accurately identify clonal lineages. Multiple test samples from the same trees were identified correctly, although certain tissue type pairs had larger genetic distances on average. The methods described in this paper will allow for the accurate identification of coast redwood clones, facilitating future studies of the reproductive ecology of this species. The techniques used in this paper can be applied to studies of other clonal organisms as well.

Advances for Studying Clonal Evolution in Cancer

PubMed Central

Raphael, Benjamin J.; Chen, Feng; Wendl, Michael C.

2013-01-01

The “clonal evolution” model of cancer emerged and “evolved” amid ongoing advances in technology, especially in recent years during which next generation sequencing instruments have provided ever higher resolution pictures of the genetic changes in cancer cells and heterogeneity in tumors. It has become increasingly clear that clonal evolution is not a single sequential process, but instead frequently involves simultaneous evolution of multiple subclones that co-exist because they are of similar fitness or are spatially separated. Co-evolution of subclones also occurs when they complement each other’s survival advantages. Recent studies have also shown that clonal evolution is highly heterogeneous: different individual tumors of the same type may undergo very different paths of clonal evolution. New methodological advancements, including deep digital sequencing of a mixed tumor population, single cell sequencing, and the development of more sophisticated computational tools, will continue to shape and reshape the models of clonal evolution. In turn, these will provide both an improved framework for the understanding of cancer progression and a guide for treatment strategies aimed at the elimination of all, rather than just some, of the cancer cells within a patient. PMID:23353056

Advances for studying clonal evolution in cancer.

PubMed

Ding, Li; Raphael, Benjamin J; Chen, Feng; Wendl, Michael C

2013-11-01

The "clonal evolution" model of cancer emerged and "evolved" amid ongoing advances in technology, especially in recent years during which next generation sequencing instruments have provided ever higher resolution pictures of the genetic changes in cancer cells and heterogeneity in tumors. It has become increasingly clear that clonal evolution is not a single sequential process, but instead frequently involves simultaneous evolution of multiple subclones that co-exist because they are of similar fitness or are spatially separated. Co-evolution of subclones also occurs when they complement each other's survival advantages. Recent studies have also shown that clonal evolution is highly heterogeneous: different individual tumors of the same type may undergo very different paths of clonal evolution. New methodological advancements, including deep digital sequencing of a mixed tumor population, single cell sequencing, and the development of more sophisticated computational tools, will continue to shape and reshape the models of clonal evolution. In turn, these will provide both an improved framework for the understanding of cancer progression and a guide for treatment strategies aimed at the elimination of all, rather than just some, of the cancer cells within a patient. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

Clonal integration supports the expansion from terrestrial to aquatic environments of the amphibious stoloniferous herb Alternanthera philoxeroides.

PubMed

Wang, N; Yu, F-H; Li, P-X; He, W-M; Liu, J; Yu, G-L; Song, Y-B; Dong, M

2009-05-01

Effects of clonal integration on land plants have been extensively studied, but little is known about the role in amphibious plants that expand from terrestrial to aquatic conditions. We simulated expansion from terrestrial to aquatic habitats in the amphibious stoloniferous alien invasive alligator weed (Alternanthera philoxeroides) by growing basal ramets of clonal fragments in soils connected (allowing integration) or disconnected (preventing integration) to the apical ramets of the same fragments submerged in water to a depth of 0, 5, 10 or 15 cm. Clonal integration significantly increased growth and clonal reproduction of the apical ramets, but decreased both of these characteristics in basal ramets. Consequently, integration did not affect the performance of whole clonal fragments. We propose that alligator weed possesses a double-edged mechanism during population expansion: apical ramets in aquatic habitats can increase growth through connected basal parts in terrestrial habitats; however, once stolon connections with apical ramets are lost by external disturbance, the basal ramets in terrestrial habitats increase stolon and ramet production for rapid spreading. This may contribute greatly to the invasiveness of alligator weed and also make it very adaptable to habitats with heavy disturbance and/or highly heterogeneous resource supply.

Evolution of oesophageal adenocarcinoma from metaplastic columnar epithelium without goblet cells in Barrett's oesophagus.

PubMed

Lavery, Danielle L; Martinez, Pierre; Gay, Laura J; Cereser, Biancastella; Novelli, Marco R; Rodriguez-Justo, Manuel; Meijer, Sybren L; Graham, Trevor A; McDonald, Stuart A C; Wright, Nicholas A; Jansen, Marnix

2016-06-01

Barrett's oesophagus commonly presents as a patchwork of columnar metaplasia with and without goblet cells in the distal oesophagus. The presence of metaplastic columnar epithelium with goblet cells on oesophageal biopsy is a marker of cancer progression risk, but it is unclear whether clonal expansion and progression in Barrett's oesophagus is exclusive to columnar epithelium with goblet cells. We developed a novel method to trace the clonal ancestry of an oesophageal adenocarcinoma across an entire Barrett's segment. Clonal expansions in Barrett's mucosa were identified using cytochrome c oxidase enzyme histochemistry. Somatic mutations were identified through mitochondrial DNA sequencing and single gland whole exome sequencing. By tracing the clonal origin of an oesophageal adenocarcinoma across an entire Barrett's segment through a combination of histopathological spatial mapping and clonal ordering, we find that this cancer developed from a premalignant clonal expansion in non-dysplastic ('cardia-type') columnar metaplasia without goblet cells. Our data demonstrate the premalignant potential of metaplastic columnar epithelium without goblet cells in the context of Barrett's oesophagus. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/

Cost of resistance to trematodes in freshwater snail populations with low clonal diversity.

PubMed

Dagan, Yael; Kosman, Evsey; Ben-Ami, Frida

2017-12-13

The persistence of high genetic variability in natural populations garners considerable interest among ecologists and evolutionary biologists. One proposed hypothesis for the maintenance of high levels of genetic diversity relies on frequency-dependent selection imposed by parasites on host populations (Red Queen hypothesis). A complementary hypothesis suggests that a trade-off between fitness costs associated with tolerance to stress factors and fitness costs associated with resistance to parasites is responsible for the maintenance of host genetic diversity. The present study investigated whether host resistance to parasites is traded off with tolerance to environmental stress factors (high/low temperatures, high salinity), by comparing populations of the freshwater snail Melanoides tuberculata with low vs. high clonal diversity. Since polyclonal populations were found to be more parasitized than populations with low clonal diversity, we expected them to be tolerant to environmental stress factors. We found that clonal diversity explained most of the variation in snail survival under high temperature, thereby suggesting that tolerance to high temperatures of clonally diverse populations is higher than that of populations with low clonal diversity. Our results suggest that resistance to parasites may come at a cost of reduced tolerance to certain environmental stress factors.

Genetic Dissection of Clonally Inherited Genomes of Poeciliopsis. I. Linkage Analysis and Preliminary Assessment of Deleterious Gene Loads

PubMed Central

Leslie, James F.; Vrijenhoek, Robert C.

1978-01-01

Theoretical considerations suggest that a high load of deleterious mutations should accumulate in asexual genomes. An ideal system for testing this hypothesis occurs in the hybrid all-female fish Poeciliopsis monacha-lucida. The hybrid genotype is retained between generations by an oogenetic process that transmits only a nonrecombinant haploid monacha genome to their ova. The hybrid genotype is re-established in nature by fertilization of these monacha eggs with sperm from a sexual species, P. lucida. The unique reproductive mechanism of these hybrids allows the genetic dissection of the clonal monacha genome by forced matings with males of P. monacha. The resultant F1 hybrids and their backcross progeny were examined to determine the amount and kinds of genetic changes that might have occurred in two clonal monacha genomes.—Using six allozyme markers, four similar linkage groups were identified in each clonal genome. Segregation and assortment at these loci revealed no apparent differences between monacha genomes from sexually and clonally reproducing species. Mortality of F1 and backcross progeny revealed differences between the two clonal genomes, suggesting that deleterious genes may accumulate in genomes sheltered from recombination. PMID:17248875

The presence of both bone sialoprotein-binding protein gene and collagen adhesin gene as a typical virulence trait of the major epidemic cluster in isolates from orthopedic implant infections.

PubMed

Campoccia, Davide; Speziale, Pietro; Ravaioli, Stefano; Cangini, Ilaria; Rindi, Simonetta; Pirini, Valter; Montanaro, Lucio; Arciola, Carla Renata

2009-12-01

Staphylococcus aureus is a major, highly clonal, pathogen causing implant infections. This study aimed at investigating the diverse distribution of bacterial adhesins in most prevalent S. aureus strain types causing orthopaedic implant infections. 200 S. aureus isolates, categorized into ribogroups by automated ribotyping, i.e. rDNA restriction fragment length polymorphism analysis, were screened for the presence of a panel of adhesins genes. Within the collection of isolates, automated ribotyping detected 98 distinct ribogroups. For many ribogroups, characteristic tandem genes arrangements could be identified. In the predominant S. aureus cluster, enlisting 27 isolates, the bbp gene encoding bone sialoprotein-binding protein appeared a typical virulence trait, found in 93% of the isolates. Conversely, the bbp gene was identified in just 10% of the remaining isolates of the collection. In this cluster, co-presence of bbp with the cna gene encoding collagen adhesin was a pattern consistently observed. These findings indicate a crucial role of both these adhesins, able to bind the most abundant bone proteins, in the pathogenesis of orthopaedic implant infections, there where biomaterials interface bone tissues. This study suggests that specific adhesins may synergistically act in the onset of implant infections and that anti-adhesin strategies should be targeted to adhesins conjointly present.

spa Typing and Multilocus Sequence Typing Show Comparable Performance in a Macroepidemiologic Study of Staphylococcus aureus in the United States

PubMed Central

O'Hara, F. Patrick; Suaya, Jose A.; Ray, G. Thomas; Baxter, Roger; Brown, Megan L.; Mera, Robertino M.; Close, Nicole M.; Thomas, Elizabeth

2016-01-01

A number of molecular typing methods have been developed for characterization of Staphylococcus aureus isolates. The utility of these systems depends on the nature of the investigation for which they are used. We compared two commonly used methods of molecular typing, multilocus sequence typing (MLST) (and its clustering algorithm, Based Upon Related Sequence Type [BURST]) with the staphylococcal protein A (spa) typing (and its clustering algorithm, Based Upon Repeat Pattern [BURP]), to assess the utility of these methods for macroepidemiology and evolutionary studies of S. aureus in the United States. We typed a total of 366 clinical isolates of S. aureus by these methods and evaluated indices of diversity and concordance values. Our results show that, when combined with the BURP clustering algorithm to delineate clonal lineages, spa typing produces results that are highly comparable with those produced by MLST/BURST. Therefore, spa typing is appropriate for use in macroepidemiology and evolutionary studies and, given its lower implementation cost, this method appears to be more efficient. The findings are robust and are consistent across different settings, patient ages, and specimen sources. Our results also support a model in which the methicillin-resistant S. aureus (MRSA) population in the United States comprises two major lineages (USA300 and USA100), which each consist of closely related variants. PMID:26669861

spa Typing and Multilocus Sequence Typing Show Comparable Performance in a Macroepidemiologic Study of Staphylococcus aureus in the United States.

PubMed

O'Hara, F Patrick; Suaya, Jose A; Ray, G Thomas; Baxter, Roger; Brown, Megan L; Mera, Robertino M; Close, Nicole M; Thomas, Elizabeth; Amrine-Madsen, Heather

2016-01-01

A number of molecular typing methods have been developed for characterization of Staphylococcus aureus isolates. The utility of these systems depends on the nature of the investigation for which they are used. We compared two commonly used methods of molecular typing, multilocus sequence typing (MLST) (and its clustering algorithm, Based Upon Related Sequence Type [BURST]) with the staphylococcal protein A (spa) typing (and its clustering algorithm, Based Upon Repeat Pattern [BURP]), to assess the utility of these methods for macroepidemiology and evolutionary studies of S. aureus in the United States. We typed a total of 366 clinical isolates of S. aureus by these methods and evaluated indices of diversity and concordance values. Our results show that, when combined with the BURP clustering algorithm to delineate clonal lineages, spa typing produces results that are highly comparable with those produced by MLST/BURST. Therefore, spa typing is appropriate for use in macroepidemiology and evolutionary studies and, given its lower implementation cost, this method appears to be more efficient. The findings are robust and are consistent across different settings, patient ages, and specimen sources. Our results also support a model in which the methicillin-resistant S. aureus (MRSA) population in the United States comprises two major lineages (USA300 and USA100), which each consist of closely related variants.

Application of pulsed-field gel electrophoresis to characterise and trace the prevalence of Enterobacter sakazakii in an infant formula processing facility.

PubMed

Mullane, N R; Whyte, P; Wall, P G; Quinn, T; Fanning, S

2007-05-01

Enterobacter sakazakii (E. sakazakii) contamination of powdered infant formula (PIF) and its processing environment was monitored between April 2005 and March 2006. The purpose of the monitoring programme was to locate points of contamination, investigate clonal persistence, and identify possible dissemination routes along the processing chain. A total of 80 E. sakazakii isolates were recovered from the manufacturing facility. The overall frequency of isolation of E. sakazakii in intermediate and final product was 2.5%, while specific locations in the processing environment were contaminated at frequencies up to 31%. All E. sakazakii isolates were characterised by pulsed-field gel electrophoresis (PFGE). XbaI macrorestriction digests yielded 19 unique pulse-types that could be grouped into 6 clusters of between 5 and 32 isolates. The formation of large clusters was consistent with the presence of a number of clones in the manufacturing environment. While the majority of isolates were of environmental origin (72.5%), no cluster was confined to one specific location and indistinguishable PFGE profiles were generated from isolates cultured from the manufacturing environment, sampling points along the processing chain and from intermediate and final product. These findings suggest that the manufacturing environment serves as a key route for sporadic contamination of PIF. These data will support the development of efficient intervention measures contributing to the reduction of E. sakazakii in the PIF processing chain.

Improved clonality detection in B-cell lymphoma using a semi-nested modification of the BIOMED-2 PCR assay for IGH rearrangement: A paraffin-embedded tissue study.

PubMed

Sakamoto, Yuma; Masaki, Ayako; Aoyama, Satsuki; Han, Shusen; Saida, Kosuke; Fujii, Kana; Takino, Hisashi; Murase, Takayuki; Iida, Shinsuke; Inagaki, Hiroshi

2017-09-01

The BIOMED-2 PCR protocol for targeting the IGH gene is widely employed for detecting clonality in B-cell malignancies. Unfortunately, the detection of clonality with this method is not very sensitive when paraffin sections are used as a DNA source. To increase the sensitivity, we devised a semi-nested modification of a JH consensus primer. The clonality detection rates of three assays were compared: the standard BIOMED-2, BIOMED-2 assay followed by BIOMED-2 re-amplification, and BIOMED-2 assay followed by semi-nested BIOMED-2. We tested more than 100 cases using paraffin-embedded tissues of various B-cell lymphomas, and found that the clonality detection rates with the above three assays were 63.9%, 79.6%, and 88.0%, respectively. While BIOMED-2 re-amplification was significantly more sensitive than the standard BIOMED-2, the semi-nested BIOMED-2 was significantly more sensitive than both the standard BIOMED-2 and BIOMED-2 re-amplification. An increase in sensitivity was observed in all lymphoma subtypes examined. In conclusion, tumor clonality may be detected in nearly 90% of B-cell lymphoma cases with semi-nested BIOMED-2. This ancillary assay may be useful when the standard BIOMED-2 fails to detect clonality in histopathologically suspected B-cell lymphomas. © 2017 Japanese Society of Pathology and John Wiley & Sons Australia, Ltd.

Purification of Bone Marrow Clonal Cells from Patients with Myelodysplastic Syndrome via IGF-IR

PubMed Central

He, Qi; Chang, Chun-Kang; Xu, Feng; Zhang, Qing-Xia; Shi, Wen-Hui; Li, Xiao

2015-01-01

Malignant clonal cells purification can greatly benefit basic and clinical studies in myelodysplastic syndrome (MDS). In this study, we investigated the potential of using type 1 insulin-like growth factor receptor (IGF-IR) as a marker for purification of malignant bone marrow clonal cells from patients with MDS. The average percentage of IGF-IR expression in CD34+ bone marrow cells among 15 normal controls was 4.5%, 70% of which also express the erythroid lineage marker CD235a. This indicates that IGF-IR mainly express in erythropoiesis. The expression of IGF-IR in CD34+ cells of 55 MDS patients was significantly higher than that of cells from the normal controls (54.0 vs. 4.5%). Based on the pattern of IGF-IR expression in MDS patients and normal controls, sorting of IGF-IR-positive and removal of CD235a-positive erythroid lineage cells with combination of FISH detection were performed on MDS samples with chromosomal abnormalities. The percentage of malignant clonal cells significantly increased after sorting. The enrichment effect was more significant in clonal cells with a previous percentage lower than 50%. This enrichment effect was present in samples from patients with +8, 5q-/-5, 20q-/-20 or 7q-/-7 chromosomal abnormalities. These data suggest that IGF-IR can be used as a marker for MDS bone marrow clonal cells and using flow cytometry for positive IGF-IR sorting may effectively purify MDS clonal cells. PMID:26469401

Distributed controller clustering in software defined networks.

PubMed

Abdelaziz, Ahmed; Fong, Ang Tan; Gani, Abdullah; Garba, Usman; Khan, Suleman; Akhunzada, Adnan; Talebian, Hamid; Choo, Kim-Kwang Raymond

2017-01-01

Software Defined Networking (SDN) is an emerging promising paradigm for network management because of its centralized network intelligence. However, the centralized control architecture of the software-defined networks (SDNs) brings novel challenges of reliability, scalability, fault tolerance and interoperability. In this paper, we proposed a novel clustered distributed controller architecture in the real setting of SDNs. The distributed cluster implementation comprises of multiple popular SDN controllers. The proposed mechanism is evaluated using a real world network topology running on top of an emulated SDN environment. The result shows that the proposed distributed controller clustering mechanism is able to significantly reduce the average latency from 8.1% to 1.6%, the packet loss from 5.22% to 4.15%, compared to distributed controller without clustering running on HP Virtual Application Network (VAN) SDN and Open Network Operating System (ONOS) controllers respectively. Moreover, proposed method also shows reasonable CPU utilization results. Furthermore, the proposed mechanism makes possible to handle unexpected load fluctuations while maintaining a continuous network operation, even when there is a controller failure. The paper is a potential contribution stepping towards addressing the issues of reliability, scalability, fault tolerance, and inter-operability.

Height-age and height-diameter relationships for monocultures and mixtures of eastern cottonwood clones

Treesearch

Steven A. Knowe; G. Sam Foster; Randall J. Rousseau; Warren L Nance

1998-01-01

Data from an eastern cottonwood clonal mixing study in Mississippi and Kentucky, USA, were used to test the effects of planting locations and genetics (clonal proportions) on height-age and height-d.b.h. functions. Planting locations, which accounted for 5.6 percent of the variation in observed dominant height growth (p = 0.0001), were more important than clonal...

Standardizing the nomenclature for clonal lineages of the sudden oak death pathogen, Phytophthora ramorum

Treesearch

N.J. Grünwald; E.M. Goss; K. Ivors; M. Garbelotto; F.N. Martin; S. Prospero; E. Hansen; P.J.M. Bonants; R.C. Hamelin; G. Chastagner; S. Werres; D.M. Rizzo; G. Abad; P. Beales; G.J. Bilodeau; C.L. Blomquist; C. Brasier; S.C. Brière; A. Chandelier; J.M. Davidson; S. Denman; M. Elliott; S.J. Frankel; E.M. Goheen; H. de Gruyter; K. Heungens; D. James; A. Kanaskie; M.G. McWilliams; W. Man in ' t Veld; E. Moralejo; N.K. Osterbauer; M.E. Palm; J.L. Parke; A.M. Perez Sierra; S.F. Shamoun; N. Shishkoff; P.W. Tooley; A.M. Vettraino; J. Webber; T.L. Widmer

2009-01-01

Phytophthora ramorum, the causal agent of sudden oak death and ramorum blight, is known to exist as three distinct clonal lineages which can only be distinguished by performing molecular marker-based analyses. However, in the recent literature there exists no consensus on naming of these lineages. Here we propose a system for naming clonal lineages of P. ramorum based...

Defining clusters in APT reconstructions of ODS steels.

PubMed

Williams, Ceri A; Haley, Daniel; Marquis, Emmanuelle A; Smith, George D W; Moody, Michael P

2013-09-01

Oxide nanoclusters in a consolidated Fe-14Cr-2W-0.3Ti-0.3Y₂O₃ ODS steel and in the alloy powder after mechanical alloying (but before consolidation) are investigated by atom probe tomography (APT). The maximum separation method is a standard method to define and characterise clusters from within APT data, but this work shows that the extent of clustering between the two materials is sufficiently different that the nanoclusters in the mechanically alloyed powder and in the consolidated material cannot be compared directly using the same cluster selection parameters. As the cluster selection parameters influence the size and composition of the clusters significantly, a procedure to optimise the input parameters for the maximum separation method is proposed by sweeping the d(max) and N(min) parameter space. By applying this method of cluster parameter selection combined with a 'matrix correction' to account for trajectory aberrations, differences in the oxide nanoclusters can then be reliably quantified. Copyright © 2012 Elsevier B.V. All rights reserved.

Structure and functional dynamics of the mitochondrial Fe/S cluster synthesis complex.

PubMed

Boniecki, Michal T; Freibert, Sven A; Mühlenhoff, Ulrich; Lill, Roland; Cygler, Miroslaw

2017-11-03

Iron-sulfur (Fe/S) clusters are essential protein cofactors crucial for many cellular functions including DNA maintenance, protein translation, and energy conversion. De novo Fe/S cluster synthesis occurs on the mitochondrial scaffold protein ISCU and requires cysteine desulfurase NFS1, ferredoxin, frataxin, and the small factors ISD11 and ACP (acyl carrier protein). Both the mechanism of Fe/S cluster synthesis and function of ISD11-ACP are poorly understood. Here, we present crystal structures of three different NFS1-ISD11-ACP complexes with and without ISCU, and we use SAXS analyses to define the 3D architecture of the complete mitochondrial Fe/S cluster biosynthetic complex. Our structural and biochemical studies provide mechanistic insights into Fe/S cluster synthesis at the catalytic center defined by the active-site Cys of NFS1 and conserved Cys, Asp, and His residues of ISCU. We assign specific regulatory rather than catalytic roles to ISD11-ACP that link Fe/S cluster synthesis with mitochondrial lipid synthesis and cellular energy status.

Beyond society: the evolution of organismality

PubMed Central

Queller, David C.; Strassmann, Joan E.

2009-01-01

The evolution of organismality is a social process. All organisms originated from groups of simpler units that now show high cooperation among the parts and are nearly free of conflicts. We suggest that this near-unanimous cooperation be taken as the defining trait of organisms. Consistency then requires that we accept some unconventional organisms, including some social insect colonies, some microbial groups and viruses, a few sexual partnerships and a number of mutualistic associations. Whether we call these organisms or not, a major task is to explain such cooperative entities, and our survey suggests that many of the traits commonly used to define organisms are not essential. These non-essential traits include physical contiguity, indivisibility, clonality or high relatedness, development from a single cell, short-term and long-term genetic cotransmission, germ–soma separation and membership in the same species. PMID:19805423

Cross-infections with Pseudomonas aeruginosa in patients with cystic fibrosis attending the Warsaw Centre.

PubMed

Milczewska, Justyna; Wołkowicz, Tomasz; Zacharczuk, Katarzyna; Kwiatkowska, Monika

2015-01-01

1. To assess the prevalence of cross-infections with P. aeruginosa in order to evaluate the epidemiological situation of this infection in patients with cystic fibrosis attending our centre; 2. To correlate the clinical features of the patients carrying a potentially transmissible strain with the entire study group in order to determine the risk factors and possible effects of its acquisition. 170 Pseudomonas aeruginosa strains obtained from the respiratory tract of 75 cystic fibrosis patients attending the Warsaw Centre in 2011 and 2012 were typed using restriction enzyme analysis-pulsed field gel electrophoresis (Spe I restriction enzyme was used). Simultaneously, the information concerning contacts between patients, as well as several clinical data regarding the course of the disease were collected. Twenty four clusters of strains were detected. The main cluster included 49 isolates derived from 21 patients. The other detected clusters included 2 to 12 isolates derived from 1 to 7 patients. Three clusters comprised the isolates derived from three pairs of siblings. There were 15 clusters containing 2 to 7 strains belonging to the same patient. The remaining 24 patients were infected with their own strains, not fitting any clonal group. Several clinical parameters showed that the 21 patients whose strains constituted the main cluster, were in worse clinical condition than the other patients in the study group. Moreover, the total duration of their hospitalizations in order to perform intravenous antibiotic treatment was longer. 1. Frequent hospitalizations of CF patients with a more severe course of the disease seem to be a risk factor of cross-infections with P. aeruginosa. 2. Intensification of measures to prevent cross-infection, such as hygienic precautions, patient segregation, introduction of home intravenous antibiotic therapy programme, as well as further education of patients and their parents should lead to the improvement of the epidemiological situation in our centre. .

VizieR Online Data Catalog: Coma clusters and filaments galaxies FIR survey (Fuller+, 2016)

NASA Astrophysics Data System (ADS)

Fuller, C.; Davies, J. I.; Smith, M. W. L.; Valiante, E.; Eales, S.; Bourne, N.; Dunne, L.; Dye, S.; Furlanetto, C.; Ibar, E.; Ivison, R.; Maddox, S.; Sansom, A.; Michalowski, M. J.; Davis, T.

2017-05-01

We have undertaken a Herschel FIR survey of the Coma cluster and the galaxy filament it resides within. Our survey covers an area of ~150 deg2 observed in five bands at 100, 160, 250, 350 and 500um. We have used the SDSS spectroscopic survey to define an area and redshift selected sample of 744 Coma cluster galaxies - the CCC. For comparison, we also define in a similar way a sample of 951 galaxies in the connecting filament - the CFC. (2 data files).

Clonality in myeloproliferative disorders: Analysis by means of polymerase chain reaction

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gilliland, D.G.; Blanchard, K.L.; Levy, J.

1991-08-01

The myeloproliferative syndromes are acquired disorders of hematopoiesis that provide insights into the transition from somatic cell mutation to neoplasia. The clonal origin of specific blood cells can be assessed in patients with X chromosome-linked polymorphisms, taking advantage of random inactivation of the X chromosome. The authors have adapted the PCR for determination of clonality on as few as 100 cells, including individual colonies grown in culture. Amplifying a polymorphic portion of the X chromosome-linked phosphoglycerate kinase (PGK) gene after selective digestion of the active X chromosome with a methylation-sensitive restriction enzyme gave results fully concordant with standard Southern blottingmore » of DNA samples form normal (polyclonal) polymorphonuclear cells (PMN) as well as clonal PMN from patients with myelodysplastic syndrome and polycythemia vera (PCV). They have used this technique to demonstrate heterogeneity of lineage involvement in patients with PCV. The same clinical phenotype may arise from clonal proliferation of different hematopoietic progenitors.« less

Clonal Evaluation of Prostate Cancer by ERG/SPINK1 Status to Improve Prognosis Prediction

DTIC Science & Technology

2017-12-01

meaning that most men with prostate cancer have multiple, genetically distinct cancers. Pathologists cannot assess clonality by routine microscopic...Hence, in this proposal we utilized dual ERG/SPINK1 immunohistochemistry (IHC)—as a readout of clonal, mutually exclusive molecular subtypes—to assess...multiclonal (also referred to as multifocal), meaning that more than 80% of men with prostate cancer actually have multiple, genetically distinct

Environmental colonization and onward clonal transmission of carbapenem-resistant Acinetobacter baumannii (CRAB) in a medical intensive care unit: the case for environmental hygiene.

PubMed

Ng, Deborah H L; Marimuthu, Kalisvar; Lee, Jia Jun; Khong, Wei Xin; Ng, Oon Tek; Zhang, Wei; Poh, Bee Fong; Rao, Pooja; Raj, Maya Devi Rajinder; Ang, Brenda; De, Partha Pratim

2018-01-01

In May 2015, we noticed an increase in carbapenem-resistant Acinetobacter baumannii (CRAB) infections in the Medical Intensive Care Unit (MICU). To investigate this, we studied the extent of environmental contamination and subsequent onward clonal transmission of CRAB. We conducted a one-day point prevalence screening (PPS) of the patients and environment in the MICU. We screened patients using endotracheal tube aspirates and swabs from nares, axillae, groin, rectum, wounds, and exit sites of drains. We collected environmental samples from patients' rooms and environment outside the patients' rooms. CRAB isolates from the PPS and clinical samples over the subsequent one month were studied for genetic relatedness by whole genome sequencing (WGS). We collected 34 samples from seven patients and 244 samples from the environment. On the day of PPS, we identified 8 CRAB carriers: 3 who screened positive and 5 previously known clinical infections. We detected environmental contamination in nearly two-thirds of the rooms housing patients with CRAB. WGS demonstrated genetic clustering of isolates within rooms but not across rooms. We analysed 4 CRAB isolates from clinical samples following the PPS. One genetically-related CRAB was identified in the respiratory sample of a patient with nosocomial pneumonia, who was admitted to the MICU five days after the PPS. The extensive environmental colonization of CRAB by patients highlights the importance of environmental hygiene. The transmission dynamics of CRAB needs further investigation.

CRISPR Associated Diversity within a Population of Sulfolobus islandicus

PubMed Central

Held, Nicole L.; Herrera, Alfa; Cadillo-Quiroz, Hinsby; Whitaker, Rachel J.

2010-01-01

Background Predator-prey models for virus-host interactions predict that viruses will cause oscillations of microbial host densities due to an arms race between resistance and virulence. A new form of microbial resistance, CRISPRs (clustered regularly interspaced short palindromic repeats) are a rapidly evolving, sequence-specific immunity mechanism in which a short piece of invading viral DNA is inserted into the host's chromosome, thereby rendering the host resistant to further infection. Few studies have linked this form of resistance to population dynamics in natural microbial populations. Methodology/Principal Findings We examined sequence diversity in 39 strains of the archeaon Sulfolobus islandicus from a single, isolated hot spring from Kamchatka, Russia to determine the effects of CRISPR immunity on microbial population dynamics. First, multiple housekeeping genetic markers identify a large clonal group of identical genotypes coexisting with a diverse set of rare genotypes. Second, the sequence-specific CRISPR spacer arrays split the large group of isolates into two very different groups and reveal extensive diversity and no evidence for dominance of a single clone within the population. Conclusions/Significance The evenness of resistance genotypes found within this population of S. islandicus is indicative of a lack of strain dominance, in contrast to the prediction for a resistant strain in a simple predator-prey interaction. Based on evidence for the independent acquisition of resistant sequences, we hypothesize that CRISPR mediated clonal interference between resistant strains promotes and maintains diversity in this natural population. PMID:20927396

Spread of a Major Clone of Salmonella enterica Serotype Enteritidis in Poultry and in Salmonellosis Outbreaks in Southern Brazil.

PubMed

Borges, Karen Apellanis; Furian, Thales Quedi; de Souza, Sara Neves; Tondo, Eduardo César; Streck, André Felipe; Salle, Carlos Tadeu Pippi; de Souza Moraes, Hamilton Luiz; do Nascimento, Vladimir Pinheiro

2017-01-01

Salmonella spp. are among the most important agents of foodborne diseases all over the world. Human Salmonella outbreaks are often associated with the consumption of poultry products (meat and eggs), and one of the most prevalent serotypes associated with these products is Salmonella Enteritidis. Brazil is one of the most important poultry exporters in the world. In southern Brazil, three closely related clones of Salmonella Enteritidis have been responsible for the majority of foodborne Salmonella outbreaks over the past decade. However, until now, there has been little information regarding the clonal relationship among the Brazilian Salmonella strains of avian origin and those involved in foodborne outbreaks. Therefore, the aim of the present study was to complete the molecular characterization of Salmonella Enteritidis strains isolated from poultry and food sources involved in Salmonella outbreaks. PCR ribotyping was performed to discriminate the strains into different ribotype profiles according to the banding pattern amplification. This technique was able to differentiate the Salmonella Enteritidis strains into two banding patterns: R2 and R4. R2 accounted for 98.7% of the strains. DNA sequencing of the 600-bp fragment, present in all ribotypes, was applied to confirm this result. The sequences generated showed high levels of similarity, ranging from 99.7 to 100%, and were grouped into a single cluster. These results suggest that there is a clonal relationship among the Salmonella Enteritidis strains responsible for several salmonellosis outbreaks and the strains collected from poultry sources.

Clonal structure of Streptococcus sanguinis strains isolated from endocarditis cases and the oral cavity.

PubMed

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.

A Monomorphic Haplotype of Chromosome Ia Is Associated with Widespread Success in Clonal and Nonclonal Populations of Toxoplasma gondii

PubMed Central

Khan, Asis; Miller, Natalie; Roos, David S.; Dubey, J. P.; Ajzenberg, Daniel; Dardé, Marie Laure; Ajioka, James W.; Rosenthal, Benjamin; Sibley, L. David

2011-01-01

ABSTRACT Toxoplasma gondii is a common parasite of animals that also causes a zoonotic infection in humans. Previous studies have revealed a strongly clonal population structure that is shared between North America and Europe, while South American strains show greater genetic diversity and evidence of sexual recombination. The common inheritance of a monomorphic version of chromosome Ia (referred to as ChrIa*) among three clonal lineages from North America and Europe suggests that inheritance of this chromosome might underlie their recent clonal expansion. To further examine the diversity and distribution of ChrIa, we have analyzed additional strains with greater geographic diversity. Our findings reveal that the same haplotype of ChrIa* is found in the clonal lineages from North America and Europe and in older lineages in South America, where sexual recombination is more common. Although lineages from all three continents harbor the same conserved ChrIa* haplotype, strains from North America and Europe are genetically separate from those in South America, and these respective geographic regions show limited evidence of recent mixing. Genome-wide, array-based profiling of polymorphisms provided evidence for an ancestral flow from particular older southern lineages that gave rise to the clonal lineages now dominant in the north. Collectively, these data indicate that ChrIa* is widespread among nonclonal strains in South America and has more recently been associated with clonal expansion of specific lineages in North America and Europe. These findings have significant implications for the spread of genetic loci influencing transmission and virulence in pathogen populations. PMID:22068979

Improved clonality detection in Hodgkin lymphoma using a semi-nested modification of the BIOMED-2 PCR assay for IGH and IGK rearrangements: A paraffin-embedded tissue study.

PubMed

Han, Shusen; Masaki, Ayako; Sakamoto, Yuma; Takino, Hisashi; Murase, Takayuki; Iida, Shinsuke; Inagaki, Hiroshi

2018-05-01

The BIOMED-2 PCR protocols targeting IGH and IGK genes may be useful for detecting clonality in Hodgkin lymphoma (HL). The clonality detection rates, however, have not been very high with these methods using paraffin-embedded tumor sections. We previously described the usefulness of the semi-nested BIOMED-2 IGH assay in B-cell malignancies. In this study, we devised a novel semi-nested BIOMED-2 IGK assay. Employing 58 cases of classical HL, we carried out the standard BIOMED-2, BIOMED-2 followed by BIOMED-2 re-amplification, and BIOMED-2 followed by semi-nested BIOMED-2, all targeting IGH and IGK, using paraffin-embedded tissues. In both IGH and IGK assays, semi-nested assays yielded significantly higher clonality detection rates than the standard assays and re-amplification assays. Clonality was detected in 13/58 (22.4%) classical HL cases using the standard IGH/IGK assays while it was detected in 38/58 (65.5%) cases using semi-nested IGH/IGK assays. The detection rates were not associated with the HL subtypes, CD30-positive cell density, CD20-positive cell density, or Epstein-Barr virus (EBV) positivity. In conclusion, tumor clonality was detected in nearly two-thirds of classical HL cases using semi-nested BIOMED-2 IGH/IGK assays using paraffin tumor sections. These semi-nested assays may be useful when the standard IGH/IGK assays fail to detect clonality in histopathologically suspected HLs. © 2018 Japanese Society of Pathology and John Wiley & Sons Australia, Ltd.

Usefullness of IGH/TCR PCR studies in lymphoproliferative disorders with inconclusive clonality by flow cytometry.

PubMed

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.

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

PubMed

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.

Environmental and genetic correlates of allocation to sexual reproduction in the circumpolar plant Bistorta vivipara.

PubMed

Bills, John W; Roalson, Eric H; Busch, Jeremiah W; Eidesen, Pernille B

2015-07-01

• Sexual reproduction often requires more energy and time than clonal reproduction. In marginal arctic conditions, species that can reproduce both sexually and clonally dominate. Plants with this capacity may thrive because they can alter reproduction depending on environmental conditions. Bistorta vivipara is a circumpolar herb that predominately reproduces clonally, but certain environmental conditions promote higher investment in flowers (and possible sexual reproduction). Despite largely reproducing clonally, the herb has high levels of genetic variation, and the processes underlying this paradoxical pattern of variation remain unclear. Here we identified environmental factors associated with sexual investment and examined whether sexual reproduction is associated with higher levels of genetic variation.• We sampled 20 populations of B. vivipara across the high Arctic archipelago of Svalbard. In each population, we measured reproductive traits, environmental variables, and collected samples for genetic analyses. These samples permitted hypotheses to be tested regarding sexual investment and ecological and genetic correlates.• Increased soil nitrogen and organic matter content and decreased elevation were positively associated with investment in flowers. Increased investment in flowers significantly correlated with more genotypes per population. Linkage disequilibrium was consistent with predominant clonality, but several populations showed higher genetic variation and lower differentiation than expected. There was no geographical genetic structure.• In B. vivipara, sexual investment is positively associated with habitat quality. Bistorta vivipara predominantly reproduces clonally, but occasional outcrossing, efficient clonal reproduction, and dispersal by bulbils can explain the considerable genetic variation and weak genetic structure in B. vivipara. © 2015 Botanical Society of America, Inc.

Applying a Resources Framework to Analysis of the Force and Motion Conceptual Evaluation

ERIC Educational Resources Information Center

Smith, Trevor I.; Wittman, Michael C.

2008-01-01

We suggest one redefinition of common clusters of questions used to analyze student responses on the Force and Motion Conceptual Evaluation. Our goal is to propose a methodology that moves beyond an analysis of student learning defined by correct responses, either on the overall test or on clusters of questions defined solely by content. We use…

A subtype based analysis of urological chronic pelvic pain syndrome in men.

PubMed

Davis, Seth N P; Binik, Yitzchak M; Amsel, Rhonda; Carrier, Serge

2013-07-01

The current conceptualization of urological chronic pelvic pain syndrome in men recognizes a wide variety of pain, psychosocial, sexual and urological symptoms and markers that may contribute to decreased quality of life. Unfortunately, this syndrome is difficult to clearly define and treat due to heterogeneous symptom profiles. We systematically describe these heterogeneous symptoms and investigated whether they could be subtyped into distinct syndromes. A total of 171 men diagnosed with urological chronic pelvic pain syndrome completed validated questionnaires, a structured genital pain interview, digital pain threshold testing and urological assessment. Pain interview results are systematically presented as descriptive information. We used k-means cluster analysis to define subtypes. Seven homogenous, distinct clusters were defined, each with a remarkably different symptom presentation. These clusters were described and related to previous hypotheses of urological chronic pelvic pain syndrome etiology. These clusters may represent distinct subtypes of urological chronic pelvic pain syndrome that can be used to guide treatment more effectively. Defining subtypes may also improve our understanding of the underlying mechanisms of urological chronic pelvic pain syndrome. Copyright © 2013 American Urological Association Education and Research, Inc. Published by Elsevier Inc. All rights reserved.

Clonal relationship and differentiation among Mycobacterium abscessus isolates as determined using the semiautomated repetitive extragenic palindromic sequence PCR-based DiversiLab system.

PubMed

Mougari, Faiza; Raskine, Laurent; Ferroni, Agnes; Marcon, Estelle; Sermet-Gaudelus, Isabelle; Veziris, Nicolas; Heym, Beate; Gaillard, Jean-Louis; Nassif, Xavier; Cambau, Emmanuelle

2014-06-01

Mycobacterium abscessus is a rapidly growing mycobacterium that causes respiratory tract infections in predisposed patients, such as those with cystic fibrosis and nosocomial skin and soft tissue infections. In order to investigate the clonal relationships between the strains causing epidemic episodes, we evaluated the discriminatory power of the semiautomated DiversiLab (DL) repetitive extragenic palindromic sequence PCR (REP-PCR) test for M. abscessus genotyping. Since M. abscessus was shown to be composed of subspecies (M. abscessus subsp. massiliense, M. abscessus subsp. bolletii, and M. abscessus subsp. abscessus), we also evaluated the ability of this technique to differentiate subspecies. The technique was applied to two collections of clinical isolates, (i) 83 M. abscessus original isolates (43 M. abscessus subsp. abscessus, 12 M. abscessus subsp. bolletii, and 28 M. abscessus subsp. massiliense) from infected patients and (ii) 35 repeated isolates obtained over 1 year from four cystic fibrosis patients. The DL REP-PCR test was standardized for DNA extraction, DNA amplification, and electrophoresis pattern comparisons. Among the isolates from distinct patients, 53/83 (62%) isolates showed a specific pattern, and 30 were distributed in 11 clusters and 6 patterns, with 2 to 4 isolates per pattern. The clusters and patterns did not fully correlate with multilocus sequence typing (MLST) analysis results. This revealed a high genomic diversity between patients, with a discriminatory power of 98% (Simpson's diversity index). However, since some isolates shared identical patterns, this raises the question of whether it is due to transmission between patients or a common reservoir. Multiple isolates from the same patient showed identical patterns, except for one patient infected by two strains. Between the M. abscessus subspecies, the indexes were <70%, indicating that the DL REP-PCR test is not an accurate tool for identifying organisms to the subspecies level. REP-PCR appears to be a rapid genotyping method that is useful for investigating epidemics of M. abscessus infections. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

Tracking the global dispersal of a cosmopolitan insect pest, the peach potato aphid.

PubMed

Margaritopoulos, John T; Kasprowicz, Louise; Malloch, Gaynor L; Fenton, Brian

2009-05-11

Global commerce and human transportation are responsible for the range expansion of various insect pests such as the plant sucking aphids. High resolution DNA markers provide the opportunity to examine the genetic structure of aphid populations, identify aphid genotypes and infer their evolutionary history and routes of expansion which is of value in developing management strategies. One of the most widespread aphid species is the peach-potato aphid Myzus persicae, which is considered as a serious pest on various crops in many parts of the world. The present study examined the genetic variation of this aphid at a world scale and then related this to distribution patterns. In particular, 197 aphid parthenogenetic lineages from around the world were analysed with six microsatellite loci. Bayesian clustering and admixture analysis split the aphid genotypes into three genetic clusters: European M. persicae persicae, New Zealand M. persicae persicae and Global M. persicae nicotianae. This partition was supported by FST and genetic distance analyses. The results showed two further points, a possible connection between genotypes found in the UK and New Zealand and globalization of nicotianae associated with colonisation of regions where tobacco is not cultivated. In addition, we report the presence of geographically widespread clones and for the first time the presence of a nicotianae genotype in the Old and New World. Lastly, heterozygote deficiency was detected in some sexual and asexual populations. The study revealed important genetic variation among the aphid populations we examined and this was partitioned according to region and host-plant. Clonal selection and gene flow between sexual and asexual lineages are important factors shaping the genetic structure of the aphid populations. In addition, the results reflected the globalization of two subspecies of M. persicae with successful clones being spread at various scales throughout the world. A subspecies appears to result from direct selection on tobacco plants. This information highlights the ultimate ability of a polyphagous aphid species to generate and maintain ecologically successful gene combinations through clonal propagation and the role of human transportation and global commerce for expanding their range.

Tracking the global dispersal of a cosmopolitan insect pest, the peach potato aphid

PubMed Central

Margaritopoulos, John T; Kasprowicz, Louise; Malloch, Gaynor L; Fenton, Brian

2009-01-01

Background Global commerce and human transportation are responsible for the range expansion of various insect pests such as the plant sucking aphids. High resolution DNA markers provide the opportunity to examine the genetic structure of aphid populations, identify aphid genotypes and infer their evolutionary history and routes of expansion which is of value in developing management strategies. One of the most widespread aphid species is the peach-potato aphid Myzus persicae, which is considered as a serious pest on various crops in many parts of the world. The present study examined the genetic variation of this aphid at a world scale and then related this to distribution patterns. In particular, 197 aphid parthenogenetic lineages from around the world were analysed with six microsatellite loci. Results Bayesian clustering and admixture analysis split the aphid genotypes into three genetic clusters: European M. persicae persicae, New Zealand M. persicae persicae and Global M. persicae nicotianae. This partition was supported by FST and genetic distance analyses. The results showed two further points, a possible connection between genotypes found in the UK and New Zealand and globalization of nicotianae associated with colonisation of regions where tobacco is not cultivated. In addition, we report the presence of geographically widespread clones and for the first time the presence of a nicotianae genotype in the Old and New World. Lastly, heterozygote deficiency was detected in some sexual and asexual populations. Conclusion The study revealed important genetic variation among the aphid populations we examined and this was partitioned according to region and host-plant. Clonal selection and gene flow between sexual and asexual lineages are important factors shaping the genetic structure of the aphid populations. In addition, the results reflected the globalization of two subspecies of M. persicae with successful clones being spread at various scales throughout the world. A subspecies appears to result from direct selection on tobacco plants. This information highlights the ultimate ability of a polyphagous aphid species to generate and maintain ecologically successful gene combinations through clonal propagation and the role of human transportation and global commerce for expanding their range. PMID:19432979

Occurrence of the Plasmid-Mediated Fluoroquinolone Resistance qepA1 Gene in Two Clonal Clinical Isolates of CTX-M-15-Producing Escherichia coli from Algeria.

PubMed

Yanat, Betitera; Dali Yahia, Radia; Yazi, Leila; Machuca, Jesús; Díaz-De-Alba, Paula; Touati, Abdelaziz; Pascual, Álvaro; Rodríguez-Martínez, José-Manuel

2017-06-01

QepA is a plasmid-mediated quinolone resistance determinant of low prevalence described worldwide, mainly in Enterobacteriaceae. This study describes, for the first time in Algeria, two clonally related, QepA-producing Escherichia coli clinical isolates positive for CTX-M-15. The clonal spread of these multidrug-resistant isolates is a major public health concern.

Successful Immunosuppressive Therapy for Severe Infectious Mononucleosis in a Patient with Clonal Proliferation of EBV-infected CD8-positive Cells.

PubMed

Hosoi, Hiroki; Sonoki, Takashi; Murata, Shogo; Mushino, Toshiki; Kuriyama, Kodai; Nishikawa, Akinori; Hanaoka, Nobuyoshi; Ohshima, Koichi; Imadome, Ken-Ichi; Nakakuma, Hideki

2015-01-01

A 30-year-old woman was diagnosed with severe infectious mononucleosis (IM). The Epstein-Barr virus (EBV) had infected both CD19- and CD8-positive cells, and clonal proliferation of EBV-infected cells and T-cells was detected. Although we suspected malignant lymphoma, her condition improved following immunosuppressive therapy. A similar case was recently reported; therefore, this case is the second case of IM with EBV-infected CD8-positive cells and clonal proliferation of EBV-infected cells. Our results demonstrate that the clonal proliferation of EBV-infected cells is not always an indication for chemotherapy in the primary infection phase and that monitoring the EBV viral load is useful for therapeutic decision-making.

Spatial Competition: Roughening of an Experimental Interface.

PubMed

Allstadt, Andrew J; Newman, Jonathan A; Walter, Jonathan A; Korniss, G; Caraco, Thomas

2016-07-28

Limited dispersal distance generates spatial aggregation. Intraspecific interactions are then concentrated within clusters, and between-species interactions occur near cluster boundaries. Spread of a locally dispersing invader can become motion of an interface between the invading and resident species, and spatial competition will produce variation in the extent of invasive advance along the interface. Kinetic roughening theory offers a framework for quantifying the development of these fluctuations, which may structure the interface as a self-affine fractal, and so induce a series of temporal and spatial scaling relationships. For most clonal plants, advance should become spatially correlated along the interface, and width of the interface (where invader and resident compete directly) should increase as a power function of time. Once roughening equilibrates, interface width and the relative location of the most advanced invader should each scale with interface length. We tested these predictions by letting white clover (Trifolium repens) invade ryegrass (Lolium perenne). The spatial correlation of clover growth developed as anticipated by kinetic roughening theory, and both interface width and the most advanced invader's lead scaled with front length. However, the scaling exponents differed from those predicted by recent simulation studies, likely due to clover's growth morphology.

Spatial Competition: Roughening of an Experimental Interface

PubMed Central

Allstadt, Andrew J.; Newman, Jonathan A.; Walter, Jonathan A.; Korniss, G.; Caraco, Thomas

2016-01-01

Limited dispersal distance generates spatial aggregation. Intraspecific interactions are then concentrated within clusters, and between-species interactions occur near cluster boundaries. Spread of a locally dispersing invader can become motion of an interface between the invading and resident species, and spatial competition will produce variation in the extent of invasive advance along the interface. Kinetic roughening theory offers a framework for quantifying the development of these fluctuations, which may structure the interface as a self-affine fractal, and so induce a series of temporal and spatial scaling relationships. For most clonal plants, advance should become spatially correlated along the interface, and width of the interface (where invader and resident compete directly) should increase as a power function of time. Once roughening equilibrates, interface width and the relative location of the most advanced invader should each scale with interface length. We tested these predictions by letting white clover (Trifolium repens) invade ryegrass (Lolium perenne). The spatial correlation of clover growth developed as anticipated by kinetic roughening theory, and both interface width and the most advanced invader’s lead scaled with front length. However, the scaling exponents differed from those predicted by recent simulation studies, likely due to clover’s growth morphology. PMID:27465518

Genetic relationships within the genus Prevotella analyzed by multilocus enzyme electrophoresis and DNA-DNA hybridization.

PubMed

Combe, M L; Pons, J L

1999-12-01

The genetic diversity and relationships within the genus Prevotella were studied by analyzing twenty-five strains by multilocus enzyme electrophoresis (MLEE) at nine metabolic enzyme loci and DNA-DNA hybridization. MLEE revealed a high genetic diversity with 25 electrophoretic types (ETs) for the 25 strains studied, a mean number of alleles per enzyme locus of 6.8 and a mean genetic diversity per locus of 0.786. The index of association described by Maynard Smith et al. (1993) revealed a clonal structure within the genus Prevotella. A dendrogram generated by cluster analysis of a matrix of ETs showed that species like P. bivia, P. buccae, P. oris, P. oralis, P. nigrescens, and P. denticola form clusters that are consistent with DNA homologies. However, strains identified as P. melaninogenica or P. loescheii by DNA-DNA hybridization did not constitute distinct subpopulations in MLEE. MLEE analysis demonstrated its high power in differentiating closely related strains. It provides an alternative to 16S rRNA analysis for the study of phylogenetic relationships within the genus Prevotella, especially for differentiating strains with high DNA homology or high rRNA homology.

Metabolomic analysis of pancreatic β-cell insulin release in response to glucose.

PubMed

Huang, Mei; Joseph, Jamie W

2012-01-01

Defining the key metabolic pathways that are important for fuel-regulated insulin secretion is critical to providing a complete picture of how nutrients regulate insulin secretion. We have performed a detailed metabolomics study of the clonal β-cell line 832/13 using a gas chromatography-mass spectrometer (GC-MS) to investigate potential coupling factors that link metabolic pathways to insulin secretion. Mid-polar and polar metabolites, extracted from the 832/13 β-cells, were derivatized and then run on a GC/MS to identify and quantify metabolite concentrations. Three hundred fifty-five out of 527 chromatographic peaks could be identified as metabolites by our metabolomic platform. These identified metabolites allowed us to perform a systematic analysis of key pathways involved in glucose-stimulated insulin secretion (GSIS). Of these metabolites, 41 were consistently identified as biomarker for GSIS by orthogonal partial least-squares (OPLS). Most of the identified metabolites are from common metabolic pathways including glycolytic, sorbitol-aldose reductase pathway, pentose phosphate pathway, and the TCA cycle suggesting these pathways play an important role in GSIS. Lipids and related products were also shown to contribute to the clustering of high glucose sample groups. Amino acids lysine, tyrosine, alanine and serine were upregulated by glucose whereas aspartic acid was downregulated by glucose suggesting these amino acids might play a key role in GSIS. In summary, a coordinated signaling cascade elicited by glucose metabolism in pancreatic β-cells is revealed by our metabolomics platform providing a new conceptual framework for future research and/or drug discovery.

Analysis of the type II-A CRISPR-Cas system of Streptococcus agalactiae reveals distinctive features according to genetic lineages

PubMed Central

Lier, Clément; Baticle, Elodie; Horvath, Philippe; Haguenoer, Eve; Valentin, Anne-Sophie; Glaser, Philippe; Mereghetti, Laurent; Lanotte, Philippe

2015-01-01

CRISPR-Cas systems (clustered regularly interspaced short palindromic repeats/CRISPR-associated proteins) are found in 90% of archaea and about 40% of bacteria. In this original system, CRISPR arrays comprise short, almost unique sequences called spacers that are interspersed with conserved palindromic repeats. These systems play a role in adaptive immunity and participate to fight non-self DNA such as integrative and conjugative elements, plasmids, and phages. In Streptococcus agalactiae, a bacterium implicated in colonization and infections in humans since the 1960s, two CRISPR-Cas systems have been described. A type II-A system, characterized by proteins Cas9, Cas1, Cas2, and Csn2, is ubiquitous, and a type I–C system, with the Cas8c signature protein, is present in about 20% of the isolates. Unlike type I–C, which appears to be non-functional, type II-A appears fully functional. Here we studied type II-A CRISPR-cas loci from 126 human isolates of S. agalactiae belonging to different clonal complexes that represent the diversity of the species and that have been implicated in colonization or infection. The CRISPR-cas locus was analyzed both at spacer and repeat levels. Major distinctive features were identified according to the phylogenetic lineages previously defined by multilocus sequence typing, especially for the sequence type (ST) 17, which is considered hypervirulent. Among other idiosyncrasies, ST-17 shows a significantly lower number of spacers in comparison with other lineages. This characteristic could reflect the peculiar virulence or colonization specificities of this lineage. PMID:26124774

A comparison of IQ and memory cluster solutions in moderate and severe pediatric traumatic brain injury.

PubMed

Thaler, Nicholas S; Terranova, Jennifer; Turner, Alisa; Mayfield, Joan; Allen, Daniel N

2015-01-01

Recent studies have examined heterogeneous neuropsychological outcomes in childhood traumatic brain injury (TBI) using cluster analysis. These studies have identified homogeneous subgroups based on tests of IQ, memory, and other cognitive abilities that show some degree of association with specific cognitive, emotional, and behavioral outcomes, and have demonstrated that the clusters derived for children with TBI are different from those observed in normal populations. However, the extent to which these subgroups are stable across abilities has not been examined, and this has significant implications for the generalizability and clinical utility of TBI clusters. The current study addressed this by comparing IQ and memory profiles of 137 children who sustained moderate-to-severe TBI. Cluster analysis of IQ and memory scores indicated that a four-cluster solution was optimal for the IQ scores and a five-cluster solution was optimal for the memory scores. Three clusters on each battery differed primarily by level of performance, while the others had pattern variations. Cross-plotting the clusters across respective IQ and memory test scores indicated that clusters defined by level were generally stable, while clusters defined by pattern differed. Notably, children with slower processing speed exhibited low-average to below-average performance on memory indexes. These results provide some support for the stability of previously identified memory and IQ clusters and provide information about the relationship between IQ and memory in children with TBI.

Application of a Dot Blot Hybridization Platform to Assess Streptococcus uberis Population Structure in Dairy Herds

PubMed Central

Albuquerque, Pedro; Ribeiro, Niza; Almeida, Alexandre; Panschin, Irena; Porfirio, Afonso; Vales, Marta; Diniz, Francisca; Madeira, Helena; Tavares, Fernando

2017-01-01

Streptococcus uberis is considered one of the most important pathogens associated with bovine mastitis. While traditionally acknowledged as an environmental pathogen, S. uberis has been shown to adopt a contagious epidemiological pattern in several dairy herds. Since different control strategies are employed depending on the mode of transmission, in-depth studies of S. uberis populations are essential to determine the best practices to control this pathogen. In this work, we optimized and validated a dot blot platform, combined with automatic image analysis, to rapidly assess the population structure of infective S. uberis, and evaluated its efficiency when compared to multilocus sequence analysis (MLSA) genotyping. Two dairy herds with prevalent S. uberis infections were followed in a 6 month period, in order to collect and characterize isolates from cows with persistent infections. These herds, located in Portugal (Barcelos and Maia regions), had similar management practices, with the herd from Barcelos being smaller and having a better milking parlor management, since infected cow segregation was immediate. A total of 54 S. uberis isolates were obtained from 24 different cows from the two herds. To overcome operator-dependent analysis of the dot blots and increase the technique's consistency and reliability, the hybridization signals were converted into probability values, with average probabilities higher than 0.5 being considered positive results. These data allowed to confirm the isolates' identity as S. uberis using taxa-specific markers and to determine the presence of virulence- and antibiotic resistance-related genes. In addition, MLSA allowed to disclose the most prevalent S. uberis clonal lineages in both herds. Seven different clusters were identified, with Barcelos showing a high clonal diversity and Maia a dominant lineage infecting most cows, suggesting distinct epidemiological patterns, with S. uberis displaying an environmental or contagious transmission pattern depending on the herd. Overall, this work showed the utility of dot blot and MLSA to characterize population structure and epidemiological patterns of mastitis-causing S. uberis. This approach allowed to disclose prevalent virulence patterns and clonal lineages of S. uberis in two distinct herds, and gain insights on the impact of herd management practices on pathogen population structure. PMID:28174566

Temporal Fluctuation of Multidrug Resistant Salmonella Typhi Haplotypes in the Mekong River Delta Region of Vietnam

PubMed Central

Chau, Tran Thuy; Duy, Pham Thanh; La, Tran Thi Phi; Hoang, Nguyen Van Minh; Nga, Tran Vu Thieu; Campbell, James I.; Manh, Bui Huu; Vinh Chau, Nguyen Van; Hien, Tran Tinh; Farrar, Jeremy; Dougan, Gordon; Baker, Stephen

2011-01-01

Background Typhoid fever remains a public health problem in Vietnam, with a significant burden in the Mekong River delta region. Typhoid fever is caused by the bacterial pathogen Salmonella enterica serovar Typhi (S. Typhi), which is frequently multidrug resistant with reduced susceptibility to fluoroquinolone-based drugs, the first choice for the treatment of typhoid fever. We used a GoldenGate (Illumina) assay to type 1,500 single nucleotide polymorphisms (SNPs) and analyse the genetic variation of S. Typhi isolated from 267 typhoid fever patients in the Mekong delta region participating in a randomized trial conducted between 2004 and 2005. Principal Findings The population of S. Typhi circulating during the study was highly clonal, with 91% of isolates belonging to a single clonal complex of the S. Typhi H58 haplogroup. The patterns of disease were consistent with the presence of an endemic haplotype H58-C and a localised outbreak of S. Typhi haplotype H58-E2 in 2004. H58-E2-associated typhoid fever cases exhibited evidence of significant geo-spatial clustering along the Sông H u branch of the Mekong River. Multidrug resistance was common in the established clone H58-C but not in the outbreak clone H58-E2, however all H58 S. Typhi were nalidixic acid resistant and carried a Ser83Phe amino acid substitution in the gyrA gene. Significance The H58 haplogroup dominates S. Typhi populations in other endemic areas, but the population described here was more homogeneous than previously examined populations, and the dominant clonal complex (H58-C, -E1, -E2) observed in this study has not been detected outside Vietnam. IncHI1 plasmid-bearing S. Typhi H58-C was endemic during the study period whilst H58-E2, which rarely carried the plasmid, was only transient, suggesting a selective advantage for the plasmid. These data add insight into the outbreak dynamics and local molecular epidemiology of S. Typhi in southern Vietnam. PMID:21245916

BRILIA: Integrated Tool for High-Throughput Annotation and Lineage Tree Assembly of B-Cell Repertoires.

PubMed

Lee, Donald W; Khavrutskii, Ilja V; Wallqvist, Anders; Bavari, Sina; Cooper, Christopher L; Chaudhury, Sidhartha

2016-01-01

The somatic diversity of antigen-recognizing B-cell receptors (BCRs) arises from Variable (V), Diversity (D), and Joining (J) (VDJ) recombination and somatic hypermutation (SHM) during B-cell development and affinity maturation. The VDJ junction of the BCR heavy chain forms the highly variable complementarity determining region 3 (CDR3), which plays a critical role in antigen specificity and binding affinity. Tracking the selection and mutation of the CDR3 can be useful in characterizing humoral responses to infection and vaccination. Although tens to hundreds of thousands of unique BCR genes within an expressed B-cell repertoire can now be resolved with high-throughput sequencing, tracking SHMs is still challenging because existing annotation methods are often limited by poor annotation coverage, inconsistent SHM identification across the VDJ junction, or lack of B-cell lineage data. Here, we present B-cell repertoire inductive lineage and immunosequence annotator (BRILIA), an algorithm that leverages repertoire-wide sequencing data to globally improve the VDJ annotation coverage, lineage tree assembly, and SHM identification. On benchmark tests against simulated human and mouse BCR repertoires, BRILIA correctly annotated germline and clonally expanded sequences with 94 and 70% accuracy, respectively, and it has a 90% SHM-positive prediction rate in the CDR3 of heavily mutated sequences; these are substantial improvements over existing methods. We used BRILIA to process BCR sequences obtained from splenic germinal center B cells extracted from C57BL/6 mice. BRILIA returned robust B-cell lineage trees and yielded SHM patterns that are consistent across the VDJ junction and agree with known biological mechanisms of SHM. By contrast, existing BCR annotation tools, which do not account for repertoire-wide clonal relationships, systematically underestimated both the size of clonally related B-cell clusters and yielded inconsistent SHM frequencies. We demonstrate BRILIA's utility in B-cell repertoire studies related to VDJ gene usage, mechanisms for adenosine mutations, and SHM hot spot motifs. Furthermore, we show that the complete gene usage annotation and SHM identification across the entire CDR3 are essential for studying the B-cell affinity maturation process through immunosequencing methods.

Genomic profiling of dedifferentiated liposarcoma compared to matched well-differentiated liposarcoma reveals higher genomic complexity and a common origin

PubMed Central

Beird, Hannah C.; Wu, Chia-Chin; Ingram, Davis R.; Wang, Wei-Lien; Alimohamed, Asrar; Gumbs, Curtis; Little, Latasha; Song, Xingzhi; Feig, Barry W.; Roland, Christina L.; Zhang, Jianhua; Benjamin, Robert S.; Hwu, Patrick; Lazar, Alexander J.; Futreal, P. Andrew; Somaiah, Neeta

2018-01-01

Well-differentiated (WD) liposarcoma is a low-grade mesenchymal tumor with features of mature adipocytes and high propensity for local recurrence. Often, WD patients present with or later progress to a higher-grade nonlipogenic form known as dedifferentiated (DD) liposarcoma. These DD tumors behave more aggressively and can metastasize. Both WD and DD liposarcomas harbor neochromosomes formed from amplifications and rearrangements of Chr 12q that encode oncogenes (MDM2, CDK4, and YEATS2) and adipocytic differentiation factors (HMGA2 and CPM). However, genomic changes associated with progression from WD to DD have not been well-defined. Therefore, we selected patients with matched WD and DD tumors for extensive genomic profiling in order to understand their clonal relationships and to delineate any defining alterations for each entity. Exome and transcriptomic sequencing was performed for 17 patients with both WD and DD diagnoses. Somatic point and copy-number alterations were integrated with transcriptional analyses to determine subtype-associated genomic features and pathways. The results were, on average, that only 8.3% of somatic mutations in WD liposarcoma were shared with their cognate DD component. DD tumors had higher numbers of somatic copy-number losses, amplifications involving Chr 12q, and fusion transcripts than WD tumors. HMGA2 and CPM rearrangements occur more frequently in DD components. The shared somatic mutations indicate a clonal origin for matched WD and DD tumors and show early divergence with ongoing genomic instability due to continual generation and selection of neochromosomes. Stochastic generation and subsequent expression of fusion transcripts from the neochromosome that involve adipogenesis genes such as HMGA2 and CPM may influence the differentiation state of the subsequent tumor. PMID:29610390

Genetic Heterogeneity in Therapy-Naïve Synchronous Primary Breast Cancers and Their Metastases.

PubMed

Ng, Charlotte K Y; Bidard, Francois-Clement; Piscuoglio, Salvatore; Geyer, Felipe C; Lim, Raymond S; de Bruijn, Ino; Shen, Ronglai; Pareja, Fresia; Berman, Samuel H; Wang, Lu; Pierga, Jean-Yves; Vincent-Salomon, Anne; Viale, Agnes; Norton, Larry; Sigal, Brigitte; Weigelt, Britta; Cottu, Paul; Reis-Filho, Jorge S

2017-08-01

Purpose: Paired primary breast cancers and metachronous metastases after adjuvant treatment are reported to differ in their clonal composition and genetic alterations, but it is unclear whether these differences stem from the selective pressures of the metastatic process, the systemic therapies, or both. We sought to define the repertoire of genetic alterations in breast cancer patients with de novo metastatic disease who had not received local or systemic therapy. Experimental Design: Up to two anatomically distinct core biopsies of primary breast cancers and synchronous distant metastases from nine patients who presented with metastatic disease were subjected to high-depth whole-exome sequencing. Mutations, copy number alterations and their cancer cell fractions, and mutation signatures were defined using state-of-the-art bioinformatics methods. All mutations identified were validated with orthogonal methods. Results: Genomic differences were observed between primary and metastatic deposits, with a median of 60% (range 6%-95%) of shared somatic mutations. Although mutations in known driver genes including TP53, PIK3CA , and GATA3 were preferentially clonal in both sites, primary breast cancers and their synchronous metastases displayed spatial intratumor heterogeneity. Likely pathogenic mutations affecting epithelial-to-mesenchymal transition-related genes, including SMAD4, TCF7L2 , and TCF4 ( ITF2 ), were found to be restricted to or enriched in the metastatic lesions. Mutational signatures of trunk mutations differed from those of mutations enriched in the primary tumor or the metastasis in six cases. Conclusions: Synchronous primary breast cancers and metastases differ in their repertoire of somatic genetic alterations even in the absence of systemic therapy. Mutational signature shifts might contribute to spatial intratumor genetic heterogeneity. Clin Cancer Res; 23(15); 4402-15. ©2017 AACR . ©2017 American Association for Cancer Research.

A Refined Methodology for Defining Plant Communities Using Postagricultural Data from the Neotropics

PubMed Central

Myster, Randall W.

2012-01-01

How best to define and quantify plant communities was investigated using long-term plot data sampled from a recovering pasture in Puerto Rico and abandoned sugarcane and banana plantations in Ecuador. Significant positive associations between pairs of old field species were first computed and then clustered together into larger and larger species groups. I found that (1) no pasture or plantation had more than 5% of the possible significant positive associations, (2) clustering metrics showed groups of species participating in similar clusters among the five pasture/plantations over a gradient of decreasing association strength, and (3) there was evidence for repeatable communities—especially after banana cultivation—suggesting that past crops not only persist after abandonment but also form significant associations with invading plants. I then showed how the clustering hierarchy could be used to decide if any two pasture/plantation plots were in the same community, that is, to define old field communities. Finally, I suggested a similar procedure could be used for any plant community where the mechanisms and tolerances of species form the “cohesion” that produces clustering, making plant communities different than random assemblages of species. PMID:22536137

Distributed controller clustering in software defined networks

PubMed Central

Gani, Abdullah; Akhunzada, Adnan; Talebian, Hamid; Choo, Kim-Kwang Raymond

2017-01-01

Software Defined Networking (SDN) is an emerging promising paradigm for network management because of its centralized network intelligence. However, the centralized control architecture of the software-defined networks (SDNs) brings novel challenges of reliability, scalability, fault tolerance and interoperability. In this paper, we proposed a novel clustered distributed controller architecture in the real setting of SDNs. The distributed cluster implementation comprises of multiple popular SDN controllers. The proposed mechanism is evaluated using a real world network topology running on top of an emulated SDN environment. The result shows that the proposed distributed controller clustering mechanism is able to significantly reduce the average latency from 8.1% to 1.6%, the packet loss from 5.22% to 4.15%, compared to distributed controller without clustering running on HP Virtual Application Network (VAN) SDN and Open Network Operating System (ONOS) controllers respectively. Moreover, proposed method also shows reasonable CPU utilization results. Furthermore, the proposed mechanism makes possible to handle unexpected load fluctuations while maintaining a continuous network operation, even when there is a controller failure. The paper is a potential contribution stepping towards addressing the issues of reliability, scalability, fault tolerance, and inter-operability. PMID:28384312

CLImAT-HET: detecting subclonal copy number alterations and loss of heterozygosity in heterogeneous tumor samples from whole-genome sequencing data.

PubMed

Yu, Zhenhua; Li, Ao; Wang, Minghui

2017-03-15

Copy number alterations (CNA) and loss of heterozygosity (LOH) represent a large proportion of genetic structural variations of cancer genomes. These aberrations are continuously accumulated during the procedure of clonal evolution and patterned by phylogenetic branching. This invariably results in the emergence of multiple cell populations with distinct complement of mutational landscapes in tumor sample. With the advent of next-generation sequencing technology, inference of subclonal populations has become one of the focused interests in cancer-associated studies, and is usually based on the assessment of combinations of somatic single-nucleotide variations (SNV), CNA and LOH. However, cancer samples often have several inherent issues, such as contamination of normal stroma, tumor aneuploidy and intra-tumor heterogeneity. Addressing these critical issues is imperative for accurate profiling of clonal architecture. We present CLImAT-HET, a computational method designed for capturing clonal diversity in the CNA/LOH dimensions by taking into account the intra-tumor heterogeneity issue, in the case where a reference or matched normal sample is absent. The algorithm quantitatively represents the clonal identification problem using a factorial hidden Markov model, and takes an integrated analysis of read counts and allele frequency data. It is able to infer subclonal CNA and LOH events as well as the fraction of cells harboring each event. The results on simulated datasets indicate that CLImAT-HET has high power to identify CNA/LOH segments, it achieves an average accuracy of 0.87. It can also accurately infer proportion of each clonal population with an overall Pearson correlation coefficient of 0.99 and a mean absolute error of 0.02. CLImAT-HET shows significant advantages when compared with other existing methods. Application of CLImAT-HET to 5 primary triple negative breast cancer samples demonstrates its ability to capture clonal diversity in the CAN/LOH dimensions. It detects two clonal populations in one sample, and three clonal populations in one other sample. CLImAT-HET, a novel algorithm is introduced to infer CNA/LOH segments from heterogeneous tumor samples. We demonstrate CLImAT-HET's ability to accurately recover clonal compositions using tumor WGS data without a match normal sample.

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.

PubMed

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 Elsevier GmbH. All rights reserved.

Disentangling giant component and finite cluster contributions in sparse random matrix spectra.

PubMed

Kühn, Reimer

2016-04-01

We describe a method for disentangling giant component and finite cluster contributions to sparse random matrix spectra, using sparse symmetric random matrices defined on Erdős-Rényi graphs as an example and test bed. Our methods apply to sparse matrices defined in terms of arbitrary graphs in the configuration model class, as long as they have finite mean degree.

Detection of clonal evolution in hematopoietic malignancies by combining comparative genomic hybridization and single nucleotide polymorphism arrays.

PubMed

Hartmann, Luise; Stephenson, Christine F; Verkamp, Stephanie R; Johnson, Krystal R; Burnworth, Bettina; Hammock, Kelle; Brodersen, Lisa Eidenschink; de Baca, Monica E; Wells, Denise A; Loken, Michael R; Zehentner, Barbara K

2014-12-01

Array comparative genomic hybridization (aCGH) has become a powerful tool for analyzing hematopoietic neoplasms and identifying genome-wide copy number changes in a single assay. aCGH also has superior resolution compared with fluorescence in situ hybridization (FISH) or conventional cytogenetics. Integration of single nucleotide polymorphism (SNP) probes with microarray analysis allows additional identification of acquired uniparental disomy, a copy neutral aberration with known potential to contribute to tumor pathogenesis. However, a limitation of microarray analysis has been the inability to detect clonal heterogeneity in a sample. This study comprised 16 samples (acute myeloid leukemia, myelodysplastic syndrome, chronic lymphocytic leukemia, plasma cell neoplasm) with complex cytogenetic features and evidence of clonal evolution. We used an integrated manual peak reassignment approach combining analysis of aCGH and SNP microarray data for characterization of subclonal abnormalities. We compared array findings with results obtained from conventional cytogenetic and FISH studies. Clonal heterogeneity was detected in 13 of 16 samples by microarray on the basis of log2 values. Use of the manual peak reassignment analysis approach improved resolution of the sample's clonal composition and genetic heterogeneity in 10 of 13 (77%) patients. Moreover, in 3 patients, clonal disease progression was revealed by array analysis that was not evident by cytogenetic or FISH studies. Genetic abnormalities originating from separate clonal subpopulations can be identified and further characterized by combining aCGH and SNP hybridization results from 1 integrated microarray chip by use of the manual peak reassignment technique. Its clinical utility in comparison to conventional cytogenetic or FISH studies is demonstrated. © 2014 American Association for Clinical Chemistry.

Facilitation of amphibious habit by physiological integration in the clonal, perennial, climbing herb Ipomoea aquatica.

PubMed

Lin, Hui-Feng; Alpert, Peter; Zhang, Qian; Yu, Fei-Hai

2018-03-15

Physiological integration of connected ramets of clonal plants can increase clonal performance when ramets grow in contrasting microenvironments within a habitat. In amphibious clonal species, integration of ramets in different habitats, terrestrial and aquatic, is possible. This may increase performance of amphibious clones, especially under eutrophic conditions. To test this, clonal fragments consisting of two ramets of the amphibious, perennial, climbing herb Ipomoea aquatica connected by a stem were placed such that the proximal ramet was rooted in a simulated riparian community of four other species, while the distal ramet extended into a simulated aquatic habitat with open water and sediment. The connection between ramets was either left intact or severed, and 0, 5, or 25mg N L -1 was added to the aquatic habitat to simulate different degrees of eutrophication. Without added N, fragments in which the original ramets were left connected accumulated two times more total mass than fragments in which the ramets were disconnected from one another. The positive effect of connection increased two-fold with increasing N. These results were consistent with the hypotheses that physiological integration between connected terrestrial and aquatic ramets can increase clonal performance in plants and that this effect can be greater when the aquatic ramet is richer in nutrients. Connection reduced root to shoot ratio in terrestrial ramets, but increased it in aquatic ones, suggesting that physiological integration induced a division of labor in which terrestrial ramets specialized for light acquisition and aquatic ramets specialized for acquisition of nutrients. This provides the first report of increase in clonal performance and induction of division of labor due to physiological integration between ramets in different habitats. Copyright © 2017 Elsevier B.V. All rights reserved.

Effects of Cu Pollution on the Expansion of an Amphibious Clonal Herb in Aquatic-Terrestrial Ecotones.

PubMed

Xu, Liang; Zhou, Zhen-Feng

2016-01-01

Physiological integration can enhance the performance of clonal plants in aquatic and terrestrial heterogeneous habitats and associated ecotones. Similar to nutrients, pollutants may be transported among connected ramets via physiological integration. Few studies have examined the expansion of amphibious clonal plants from terrestrial to aquatic environments, particularly when the local water supply is polluted with heavy metals. A greenhouse experiment was conducted using the amphibious plant Alternanthera philoxeroides to determine whether Cu can spread among clonal plants and examine the corresponding effects of this pollution on the expansion of clonal plants in aquatic-terrestrial ecotones. Ramets from the same clonal fragments were rooted in unpolluted soil and polluted water at five different levels. The responses of the ramets in terrestrial and aquatic habitats were quantified via traits associated with growth, morphology and Cu accumulation. The results indicated that ramets in soil and water significantly differed in nearly all of these traits. The expansion of populations from terrestrial to polluted aquatic habitats was facilitated by stem elongation rather than new ramet production. The accumulated Cu in polluted ramets can be horizontally transported to other ramets in soil via connected stolons. In terms of clonal growth patterns, variations in Cu pollution intensity were negatively correlated with variations in the morphological and growth traits of ramets in polluted aquatic habitats and unpolluted soil. We concluded that Cu ions are distributed among the clones and accumulated in different ramet tissues in heterogeneous habitats. Therefore, we suggest that Cu pollution of aquatic-terrestrial ecotones, especially at high levels, can affect the growth and expansion of the whole clones because Cu ions are shared between integrated ramets.

Ultra-sensitive Sequencing Identifies High Prevalence of Clonal Hematopoiesis-Associated Mutations throughout Adult Life.

PubMed

Acuna-Hidalgo, Rocio; Sengul, Hilal; Steehouwer, Marloes; van de Vorst, Maartje; Vermeulen, Sita H; Kiemeney, Lambertus A L M; Veltman, Joris A; Gilissen, Christian; Hoischen, Alexander

2017-07-06

Clonal hematopoiesis results from somatic mutations in hematopoietic stem cells, which give an advantage to mutant cells, driving their clonal expansion and potentially leading to leukemia. The acquisition of clonal hematopoiesis-driver mutations (CHDMs) occurs with normal aging and these mutations have been detected in more than 10% of individuals ≥65 years. We aimed to examine the prevalence and characteristics of CHDMs throughout adult life. We developed a targeted re-sequencing assay combining high-throughput with ultra-high sensitivity based on single-molecule molecular inversion probes (smMIPs). Using smMIPs, we screened more than 100 loci for CHDMs in more than 2,000 blood DNA samples from population controls between 20 and 69 years of age. Loci screened included 40 regions known to drive clonal hematopoiesis when mutated and 64 novel candidate loci. We identified 224 somatic mutations throughout our cohort, of which 216 were coding mutations in known driver genes (DNMT3A, JAK2, GNAS, TET2, and ASXL1), including 196 point mutations and 20 indels. Our assay's improved sensitivity allowed us to detect mutations with variant allele frequencies as low as 0.001. CHDMs were identified in more than 20% of individuals 60 to 69 years of age and in 3% of individuals 20 to 29 years of age, approximately double the previously reported prevalence despite screening a limited set of loci. Our findings support the occurrence of clonal hematopoiesis-associated mutations as a widespread mechanism linked with aging, suggesting that mosaicism as a result of clonal evolution of cells harboring somatic mutations is a universal mechanism occurring at all ages in healthy humans. Copyright © 2017 American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.

The human urothelium consists of multiple clonal units, each maintained by a stem cell.

PubMed

Gaisa, Nadine T; Graham, Trevor A; McDonald, Stuart A C; Cañadillas-Lopez, Sagrario; Poulsom, Richard; Heidenreich, Axel; Jakse, Gerhard; Tadrous, Paul J; Knuechel, Ruth; Wright, Nicholas A

2011-10-01

Little is known about the clonal architecture of human urothelium. It is likely that urothelial stem cells reside within the basal epithelial layer, yet lineage tracing from a single stem cell as a means to show the presence of a urothelial stem cell has never been performed. Here, we identify clonally related cell areas within human bladder mucosa in order to visualize epithelial fields maintained by a single founder/stem cell. Sixteen frozen cystectomy specimens were serially sectioned. Patches of cells deficient for the mitochondrially encoded enzyme cytochrome c oxidase (CCO) were identified using dual-colour enzyme histochemistry. To show that these patches represent clonal proliferations, small CCO-proficient and -deficient areas were individually laser-capture microdissected and the entire mitochondrial genome (mtDNA) in each area was PCR amplified and sequenced to identify mtDNA mutations. Immunohistochemistry was performed for the different cell layers of the urothelium and adjacent mesenchyme. CCO-deficient patches could be observed in normal urothelium of all cystectomy specimens. The two-dimensional length of these negative patches varied from 2-3 cells (about 30 µm) to 4.7 mm. Each cell area within a CCO-deficient patch contained an identical somatic mtDNA mutation, indicating that the patch was a clonal unit. Patches contained all the mature cell differentiation stages present in the urothelium, suggesting the presence of a stem cell. Our results demonstrate that the normal mucosa of human bladder contains stem cell-derived clonal units that actively replenish the urothelium during ageing. The size of the clonal unit attributable to each stem cell was broadly distributed, suggesting replacement of one stem cell clone by another. Copyright © 2011 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Genet-specific DNA methylation probabilities detected in a spatial epigenetic analysis of a clonal plant population.

PubMed

Araki, Kiwako S; Kubo, Takuya; Kudoh, Hiroshi

2017-01-01

In sessile organisms such as plants, spatial genetic structures of populations show long-lasting patterns. These structures have been analyzed across diverse taxa to understand the processes that determine the genetic makeup of organismal populations. For many sessile organisms that mainly propagate via clonal spread, epigenetic status can vary between clonal individuals in the absence of genetic changes. However, fewer previous studies have explored the epigenetic properties in comparison to the genetic properties of natural plant populations. Here, we report the simultaneous evaluation of the spatial structure of genetic and epigenetic variation in a natural population of the clonal plant Cardamine leucantha. We applied a hierarchical Bayesian model to evaluate the effects of membership of a genet (a group of individuals clonally derived from a single seed) and vegetation cover on the epigenetic variation between ramets (clonal plants that are physiologically independent individuals). We sampled 332 ramets in a 20 m × 20 m study plot that contained 137 genets (identified using eight SSR markers). We detected epigenetic variation in DNA methylation at 24 methylation-sensitive amplified fragment length polymorphism (MS-AFLP) loci. There were significant genet effects at all 24 MS-AFLP loci in the distribution of subepiloci. Vegetation cover had no statistically significant effect on variation in the majority of MS-AFLP loci. The spatial aggregation of epigenetic variation is therefore largely explained by the aggregation of ramets that belong to the same genets. By applying hierarchical Bayesian analyses, we successfully identified a number of genet-specific changes in epigenetic status within a natural plant population in a complex context, where genotypes and environmental factors are unevenly distributed. This finding suggests that it requires further studies on the spatial epigenetic structure of natural populations of diverse organisms, particularly for sessile clonal species.

Effects of Cu Pollution on the Expansion of an Amphibious Clonal Herb in Aquatic-Terrestrial Ecotones

PubMed Central

Zhou, Zhen-Feng

2016-01-01

Physiological integration can enhance the performance of clonal plants in aquatic and terrestrial heterogeneous habitats and associated ecotones. Similar to nutrients, pollutants may be transported among connected ramets via physiological integration. Few studies have examined the expansion of amphibious clonal plants from terrestrial to aquatic environments, particularly when the local water supply is polluted with heavy metals. A greenhouse experiment was conducted using the amphibious plant Alternanthera philoxeroides to determine whether Cu can spread among clonal plants and examine the corresponding effects of this pollution on the expansion of clonal plants in aquatic-terrestrial ecotones. Ramets from the same clonal fragments were rooted in unpolluted soil and polluted water at five different levels. The responses of the ramets in terrestrial and aquatic habitats were quantified via traits associated with growth, morphology and Cu accumulation. The results indicated that ramets in soil and water significantly differed in nearly all of these traits. The expansion of populations from terrestrial to polluted aquatic habitats was facilitated by stem elongation rather than new ramet production. The accumulated Cu in polluted ramets can be horizontally transported to other ramets in soil via connected stolons. In terms of clonal growth patterns, variations in Cu pollution intensity were negatively correlated with variations in the morphological and growth traits of ramets in polluted aquatic habitats and unpolluted soil. We concluded that Cu ions are distributed among the clones and accumulated in different ramet tissues in heterogeneous habitats. Therefore, we suggest that Cu pollution of aquatic-terrestrial ecotones, especially at high levels, can affect the growth and expansion of the whole clones because Cu ions are shared between integrated ramets. PMID:27736932

Genomic analyses of dominant U.S. clonal lineages of Phytophthora infestans reveals a shared common ancestry for clonal lineages US11 and US18 and a lack of recently shared ancestry among all other U.S. lineages

USDA-ARS?s Scientific Manuscript database

The populations of the potato and tomato late blight pathogen, Phytophthora infestans, in the US are well known for emerging repeatedly as novel clonal lineages. These successions of dominant clones have historically been named US1-US24, in order of appearance, since their first characterization usi...

Clonal origins and parallel evolution of regionally synchronous colorectal adenoma and carcinoma.

PubMed

Kim, Tae-Min; An, Chang Hyeok; Rhee, Je-Keun; Jung, Seung-Hyun; Lee, Sung Hak; Baek, In-Pyo; Kim, Min Sung; Lee, Sug Hyung; Chung, Yeun-Jun

2015-09-29

Although the colorectal adenoma-to-carcinoma sequence represents a classical cancer progression model, the evolution of the mutational landscape underlying this model is not fully understood. In this study, we analyzed eight synchronous pairs of colorectal high-grade adenomas and carcinomas, four microsatellite-unstable (MSU) and four-stable (MSS) pairs, using whole-exome sequencing. In the MSU adenoma-carcinoma pairs, we observed no subclonal mutations in adenomas that became fixed in paired carcinomas, suggesting a 'parallel' evolution of synchronous adenoma-to-carcinoma, rather than a 'stepwise' evolution. The abundance of indel (in MSU and MSS pairs) and microsatellite instability (in MSU pairs) was noted in the later adenoma- or carcinoma-specific mutations, indicating that the mutational processes and functional constraints operative in early and late colorectal carcinogenesis are different. All MSU cases exhibited clonal, truncating mutations in ACVR2A, TGFBR2, and DNA mismatch repair genes, but none were present in APC or KRAS. In three MSS pairs, both APC and KRAS mutations were identified as both early and clonal events, often accompanying clonal copy number changes. An MSS case uniquely exhibited clonal ERBB2 amplification, followed by APC and TP53 mutations as carcinoma-specific events. Along with the previously unrecognized clonal origins of synchronous colorectal adenoma-carcinoma pairs, our study revealed that the preferred sequence of mutational events during colorectal carcinogenesis can be context-dependent.

[Lymphocytic Clonal Expansion in Adult Patients with Epstein-Barr Virus-Associated Lymphoproliferative Disease].

PubMed

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.

Emergence of Clonal Hematopoiesis in the Majority of Patients with Acquired Aplastic Anemia

PubMed Central

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

2015-01-01

Acquired aplastic anemia (aAA) is a non-malignant 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 in 22 patients. We found somatic mutations in sixteen patients (72.7%) with a median disease duration of 1 year; twelve (66.7%) were patients with pediatriconset aAA. Fifty-eight mutations in 51 unique genes were primarily in pathways of immunity and transcriptional regulation. Most frequently mutated was PIGA, with 7 mutations. Only two mutations were in genes recurrently-mutated in MDS. Two patients had oligoclonal loss of HLA alleles, linking immune escape to clone emergence. Two patients had activating mutations in key signaling pathways (STAT5B(p.N642H), 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 non-neoplastic blood disorder. Future prospective studies of clonal hematopoiesis in aAA will be critical for understanding outcomes, and for designing personalized treatment strategies. PMID:25800665

Aldosterone induces clonal β-cell failure through glucocorticoid receptor

PubMed Central

Chen, Fang; Liu, Jia; Wang, Yanyang; Wu, Tijun; Shan, Wei; Zhu, Yunxia; Han, Xiao

2015-01-01

Aldosterone excess causes insulin resistance in peripheral tissues and directly impairs the function of clonal β-cell. The aim of this study was to investigate the molecular mechanisms involved in the aldosterone-induced impairment of clonal β-cells. As expected, aldosterone induced apoptosis and β-cell dysfunction, including impairment of insulin synthesis and secretion, which were reversed by Glucocorticoid receptor (GR) antagonists or GR-specific siRNA. However, mineralocorticoid receptor (MR) antagonists or MR-specific siRNA had no effect on impairment of clonal β-cells induced by aldosterone. Besides, aldosterone significantly decreased expression and activity of MafA, while activated JNK and p38 MAPK in a GR-dependent manner. In addition, JNK inhibitors (SP600125) and/or p38 inhibitors (SB203580) could abolish the effect of aldosterone on MafA expression and activity. Importantly, overexpression of JNK1 or p38 reversed the protective effect of a GR antagonist on the decrease of MafA expression and activity. Furthermore, aldosterone inhibits MafA expression at the transcriptional and post-transcriptional level through activation of JNK and p38, respectively. Consequently, overexpression of MafA increased synthesis and secretion of insulin, and decreased apoptosis in clonal β-cells exposed to aldosterone. These findings identified aldosterone as an inducer of clonal β-cell failure that operates through the GR-MAPK-MafA signaling pathway. PMID:26287126

The association between polyploidy and clonal reproduction in diploid and tetraploid Chamerion angustifolium.

PubMed

Baldwin, Sarah J; Husband, Brian C

2013-04-01

Clonal reproduction is associated with the incidence of polyploidy in flowering plants. This pattern may arise through selection for increased clonality in polyploids compared to diploids to reduce mixed-ploidy mating. Here, we test whether clonal reproduction is greater in tetraploid than diploid populations of the mixed-ploidy plant, Chamerion angustifolium, through an analysis of the size and spatial distribution of clones in natural populations using AFLP genotyping and a comparison of root bud production in a greenhouse study. Natural tetraploid populations (N = 5) had significantly more AFLP genotypes (x¯ = 10.8) than diploid populations (x¯ = 6.0). Tetraploid populations tended to have fewer ramets per genotype and fewer genotypes with >1 ramet. In a spatial autocorrelation analysis, ramets within genotypes were more spatially aggregated in diploid populations than in tetraploid populations. In the greenhouse, tetraploids allocated 90.4% more dry mass to root buds than diploids, but tetraploids produced no more root buds and 44% fewer root buds per unit root mass than diploids. Our results indicate that clonal reproduction is significant in most populations, but tetraploid populations are not more clonal than diploids, nor are their clones more spatially aggregated. As a result, tetraploids may be less sheltered from mixed-ploidy mating and diploids more exposed to inbreeding, the balance of which could influence the establishment of tetraploids in diploid populations. © 2013 Blackwell Publishing Ltd.

Clonal reproduction in fungi

PubMed Central

Taylor, John W.; Hann-Soden, Christopher; Branco, Sara; Sylvain, Iman; Ellison, Christopher E.

2015-01-01

Research over the past two decades shows that both recombination and clonality are likely to contribute to the reproduction of all fungi. This view of fungi is different from the historical and still commonly held view that a large fraction of fungi are exclusively clonal and that some fungi have been exclusively clonal for hundreds of millions of years. Here, we first will consider how these two historical views have changed. Then we will examine the impact on fungal research of the concept of restrained recombination [Tibayrenc M, Ayala FJ (2012) Proc Natl Acad Sci USA 109 (48):E3305–E3313]. Using animal and human pathogenic fungi, we examine extrinsic restraints on recombination associated with bottlenecks in genetic variation caused by geographic dispersal and extrinsic restraints caused by shifts in reproductive mode associated with either disease transmission or hybridization. Using species of the model yeast Saccharomyces and the model filamentous fungus Neurospora, we examine intrinsic restraints on recombination associated with mating systems that range from strictly clonal at one extreme to fully outbreeding at the other and those that lie between, including selfing and inbreeding. We also consider the effect of nomenclature on perception of reproductive mode and a means of comparing the relative impact of clonality and recombination on fungal populations. Last, we consider a recent hypothesis suggesting that fungi thought to have the most severe intrinsic constraints on recombination actually may have the fewest. PMID:26195774

Concurrent Effects of Sediment Accretion and Nutrient Availability on the Clonal Growth Strategy of Carex brevicuspis-A Wetland Sedge That Produces Both Spreading and Clumping Ramets

PubMed Central

Chen, Xinsheng; Liao, Yulin; Xie, Yonghong; Li, Feng; Deng, Zhengmiao; Hou, Zhiyong; Wu, Chao

2017-01-01

Clonal plants producing both clumping and spreading ramets can adjust their growth forms in response to resource heterogeneity or environmental stress. They might produce clumping ramets to retain favorable patches, or produce spreading ramets to escape from stress-affected patches. This study aimed to investigate the rarely reported concurrent effects of sediment accretion and nutrient enrichment, which often occur simultaneously in lacustrine wetlands, on the vegetative propagation and clonal growth forms of Carex brevicuspis C.B. Clarke by conducting a factorial experiment of sediment burial and nutrient addition. Biomass accumulation, new ramet and rhizome numbers, and ramet length of C. brevicuspis were not affected at moderate burial, but were significantly lower after deep burial. Similarly, nutrient enrichment increased the growth and vegetative propagation of C. brevicuspis up to moderate sediment burial, but not after deep burial. Sediment accretion increased the proportion of spreading ramets produced by C. brevicuspis, whereas nutrient addition had no effect on the clonal growth forms. Our results indicated that the plasticity of clonal growth forms is an effective strategy used by plants to acclimate to moderate sediment accretion. Nutrient enrichment did not influence the clonal growth forms of C. brevicuspis and could not facilitate its acclimation to heavy sedimentation condition. PMID:29021805

Molecular Characteristics of Mantle Cell Lymphoma Presenting with Clonal Plasma Cell Component

PubMed Central

Visco, Carlo; Hoeller, Sylvia; Malik, Jeffrey T.; Xu-Monette, Zijun Y.; Wiggins, Michele L.; Liu, Jessica; Sanger, Warren G.; Liu, Zhongfeng; Chang, Julie; Ranheim, Erik A.; Gradowski, Joel F.; Serrrano, Sergio; Wang, Huan-You; Liu, Qingquan; Dave, Sandeep; Olsen, Brian; Gascoyne, Randy D.; Campo, Elias; Swerdlow, Steven H.; Chan, Wing C.; Tzankov, Alexander; Young, Ken H.

2011-01-01

The normal counterparts of mantle cell lymphoma (MCL) are naïve quiescent B-cells that have not been processed through the germinal center (GC). For this reason, while lymphomas arising from GC or post-GC B-cells often exhibit plasmacytic differentiation, MCL rarely presents with plasmacytic features. Seven cases of MCL with a monotypic plasma cell (PC) population were collected from six centers and studied by immunohistochemistry, FICTION (Fluorescence immunophenotyping and Interphase Cytogenetics as a Tool for the Investigation of Neoplasms), capillary gel electrophoresis, and restriction fragment length polymorphism of immunoglobulin heavy chain analysis (RFLP/IgH) of microdissections of each of the MCL and PC populations to assess their clonal relationship. Clinical presentation was rather unusual compared to typical MCL, with two cases arising from extranodal soft-tissues of the head. All MCL cases were morphologically and immunohistochemically typical, bearing the t(11;14)(q13;q32). In all cases PC populations were clonal. In 5 of the 7 cases, the MCL and PC clones showed identical restriction fragments, indicating a common clonal origin of the neoplastic populations. The two cases with clonal diversity denoted the coexistence of two different tumors in a composite lymphoma/plasma cell neoplasm. Our findings suggest that MCL can present with a PC component that is often clonally related to the lymphoma, representing a rare but unique biological variant of this tumor. PMID:21263238

Integration Site and Clonal Expansion in Human Chronic Retroviral Infection and Gene Therapy

PubMed Central

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

Global analysis of saliva as a source of bacterial genes for insights into human population structure and migration studies.

PubMed

Henne, Karsten; Li, Jing; Stoneking, Mark; Kessler, Olga; Schilling, Hildegard; Sonanini, Anne; Conrads, Georg; Horz, Hans-Peter

2014-08-22

The genetic diversity of the human microbiome holds great potential for shedding light on the history of our ancestors. Helicobacter pylori is the most prominent example as its analysis allowed a fine-scale resolution of past migration patterns including some that could not be distinguished using human genetic markers. However studies of H. pylori require stomach biopsies, which severely limits the number of samples that can be analysed. By focussing on the house-keeping gene gdh (coding for the glucose-6-phosphate dehydrogenase), on the virulence gene gtf (coding for the glucosyltransferase) of mitis-streptococci and on the 16S-23S rRNA internal transcribed spacer (ITS) region of the Fusobacterium nucleatum/periodonticum-group we here tested the hypothesis that bacterial genes from human saliva have the potential for distinguishing human populations. Analysis of 10 individuals from each of seven geographic regions, encompassing Africa, Asia and Europe, revealed that the genes gdh and ITS exhibited the highest number of polymorphic sites (59% and 79%, respectively) and most OTUs (defined at 99% identity) were unique to a given country. In contrast, the gene gtf had the lowest number of polymorphic sites (21%), and most OTUs were shared among countries. Most of the variation in the gdh and ITS genes was explained by the high clonal diversity within individuals (around 80%) followed by inter-individual variation of around 20%, leaving the geographic region as providing virtually no source of sequence variation. Conversely, for gtf the variation within individuals accounted for 32%, between individuals for 57% and among geographic regions for 11%. This geographic signature persisted upon extension of the analysis to four additional locations from the American continent. Pearson correlation analysis, pairwise Fst-cluster analysis as well as UniFrac analyses consistently supported a tree structure in which the European countries clustered tightly together and branched with American countries and South Africa, to the exclusion of Asian countries and the Congo. This study shows that saliva harbours protein-coding bacterial genes that are geographically structured, and which could potentially be used for addressing previously unresolved human migration events.

Tumor morphology and phenotypic evolution driven by selective pressure from the microenvironment.

PubMed

Anderson, Alexander R A; Weaver, Alissa M; Cummings, Peter T; Quaranta, Vito

2006-12-01

Emergence of invasive behavior in cancer is life-threatening, yet ill-defined due to its multifactorial nature. We present a multiscale mathematical model of cancer invasion, which considers cellular and microenvironmental factors simultaneously and interactively. Unexpectedly, the model simulations predict that harsh tumor microenvironment conditions (e.g., hypoxia, heterogenous extracellular matrix) exert a dramatic selective force on the tumor, which grows as an invasive mass with fingering margins, dominated by a few clones with aggressive traits. In contrast, mild microenvironment conditions (e.g., normoxia, homogeneous matrix) allow clones with similar aggressive traits to coexist with less aggressive phenotypes in a heterogeneous tumor mass with smooth, noninvasive margins. Thus, the genetic make-up of a cancer cell may realize its invasive potential through a clonal evolution process driven by definable microenvironmental selective forces. Our mathematical model provides a theoretical/experimental framework to quantitatively characterize this selective pressure for invasion and test ways to eliminate it.

The Profile-Query Relationship.

ERIC Educational Resources Information Center

Shepherd, Michael A.; Phillips, W. J.

1986-01-01

Defines relationship between user profile and user query in terms of relationship between clusters of documents retrieved by each, and explores the expression of cluster similarity and cluster overlap as linear functions of similarity existing between original pairs of profiles and queries, given the desired retrieval threshold. (23 references)…

Collective Functionality through Bacterial Individuality

NASA Astrophysics Data System (ADS)

Ackermann, Martin

According to the conventional view, the properties of an organism are a product of nature and nurture - of its genes and the environment it lives in. Recent experiments with unicellular organisms have challenged this view: several molecular mechanisms generate phenotypic variation independently of environmental signals, leading to variation in clonal groups. My presentation will focus on the causes and consequences of this microbial individuality. Using examples from bacterial genetic model systems, I will first discuss different molecular and cellular mechanisms that give rise to bacterial individuality. Then, I will discuss the consequences of individuality, and focus on how phenotypic variation in clonal populations of bacteria can promote interactions between individuals, lead to the division of labor, and allow clonal groups of bacteria to cope with environmental uncertainty. Variation between individuals thus provides clonal groups with collective functionality.

Molecular epidemiology of invasive Candida albicans at a tertiary hospital in northern Taiwan from 2003 to 2011.

PubMed

Wang, Shao-Hung; Shen, Mandy; Lin, Hsin-Chieh; Sun, Pei-Lun; Lo, Hsiu-Jung; Lu, Jang-Jih

2015-11-01

Candida albicans is a common cause of bloodstream fungal infections in hospitalized patients. To investigate its epidemiology, multilocus sequence typing (MLST) was performed on 285 C. albicans bloodstream isolates from patients in Chang Gung Memorial Hospital at Linkou (CGMHL), Taiwan from 2003 to 2011. Among these isolates, the three major diploid sequence types (DSTs) were 693, 659, and 443 with 19, 16, and 13 isolates, respectively. The 179 DSTs were classified into 16 clades by unweighted pair-group method using arithmetic averages (UPGMA). The major ones were clades 1, 4, 3, and 17 (54, 49, 31, and 31 isolates, respectively). Further analyses with eBURST clustered the 285 isolates into 28 clonal complexes (CC). The most common complexes were CC8, CC20, and CC9. DST 693 that had the highest number of isolates was determined to be the cluster founder of CC20, which belonged to clade 3. So far, 33 isolates worldwide including 29 from Taiwan and 4 from Korea, are CC20, suggesting that CC20 is an Asian cluster. Two fluconazole-resistant isolates belonging to CC12 and CC19 were detected. All other CGMHL isolates were susceptible to 5-flucytosine, amphotericin B, anidulfungin, caspofungin, fluconazole, itraconazole, micafungin, posaconazole, and voriconazole. However, CC20 isolates exhibited significantly lower susceptibility to fluconazole. In conclusion, the 285 CGMHL C. albicans isolates displayed geographically clustering with Asian isolates, and most of them are susceptible to common antifungal drugs. Isolates of DST 693, a Taiwanese major genotype belonging to MLST clade 3, were more resistant to fluconazole than other isolates. © The Author 2015. Published by Oxford University Press on behalf of The International Society for Human and Animal Mycology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

CRISPR/Cas9-Mediated Mutagenesis of Human Pluripotent Stem Cells in Defined Xeno-Free E8 Medium.

PubMed

Soh, Chew-Li; Huangfu, Danwei

2017-01-01

The recent advent of engineered nucleases including the CRISPR/Cas9 system has greatly facilitated genome manipulation in human pluripotent stem cells (hPSCs). In addition to facilitating hPSC-based disease studies, the application of genome engineering in hPSCs has also opened up new avenues for cell replacement therapy. To improve consistency and reproducibility of hPSC-based studies, and to meet the safety and regulatory requirements for clinical translation, it is necessary to use a defined, xeno-free cell culture system. This chapter describes protocols for CRISPR/Cas9 genome editing in an inducible Cas9 hPSC-based system, using cells cultured in chemically defined, xeno-free E8 Medium on a recombinant human vitronectin substrate. We detail procedures for the design and transfection of CRISPR guide RNAs, colony selection, and the expansion and validation of clonal mutant lines, all within this fully defined culture condition. These methods may be applied to a wide range of genome-engineering applications in hPSCs, including those that utilize different types of site-specific nucleases such as zinc finger nucleases (ZFNs) and TALENs, and form a closer step towards clinical utility of these cells.

Clonal reticulohistiocytosis of the skin and bone marrow associated with systemic mastocytosis and acute myeloid leukaemia.

PubMed

Fusco, Nicola; Bonometti, Arturo; Augello, Claudia; Fabris, Sonia; Boiocchi, Leonardo; Fiori, Stefano; Morotti, Denise; Fracchiolla, Nicola; Berti, Emilio; Gianelli, Umberto

2017-05-01

The aims of this study were to define whether diffuse cutaneous reticulohistiocytosis could be underpinned by somatic genetic alterations and represent a precursor of more aggressive forms of disease. A 59-year-old man with diffuse cutaneous reticulohistiocytosis experienced bone marrow localization of the disease, with associated systemic mastocytosis and acute myeloid leukaemia. Cytogenetic analyses of the bone marrow aspirate revealed the presence of a derivative chromosome giving rise to a partial trisomy of chromosome 1q and a partial monosomy of chromosome 9q. Therefore, we characterized the cutaneous lesions before and after chemotherapy by using an integrative approach combining histopathology, electron microscopy, and fluorescence in-situ hybridization. Histologically, the skin lesions belonged to the spectrum of diffuse cutaneous reticulohistiocytoses, as confirmed by immunohistochemistry and ultrastructural analyses. Fluorescence in-situ hybridization in the skin nodules confirmed the presence of the genetic alterations previously detected in the bone marrow. Here, we provide circumstantial evidence to suggest that at least a subset of cutaneous reticulohistiocytoses harbour clonal molecular alterations. Furthermore, we confirm that these lesions have the potential to arise in the setting of concurrent haematological disorders. In this hypothesis-generating study, two possible tumorigenesis models are proposed. © 2017 John Wiley & Sons Ltd.

Subclones dominate at MDS progression following allogeneic hematopoietic cell transplant

PubMed Central

Jacoby, Meagan A.; Duncavage, Eric J.; Chang, Gue Su; Miller, Christopher A.; Shao, Jin; Elliott, Kevin; Robinson, Joshua; Fulton, Robert S.; Fronick, Catrina C.; O’Laughlin, Michelle; Heath, Sharon E.; Welch, John S.; Link, Daniel C.; DiPersio, John F.; Westervelt, Peter; Ley, Timothy J.; Graubert, Timothy A.; Walter, Matthew J.

2018-01-01

Allogeneic hematopoietic cell transplantation (alloHCT) is a potentially curative treatment for myelodysplastic syndromes (MDS), but patients who relapse after transplant have poor outcomes. In order to understand the contribution of tumor clonal evolution to disease progression,we applied exome and error-corrected targeted sequencing coupled with copy number analysis to comprehensively define changes in the clonal architecture of MDS in response to therapy using 51 serially acquired tumor samples from 9 patients who progressed after an alloHCT. We show that small subclones before alloHCT can drive progression after alloHCT. Notably, at least one subclone expanded or emerged at progression in all patients. Newly acquired structural variants (SVs) were present in an emergent/expanding subclone in 8 of 9 patients at progression, implicating the acquisition of SVs as important late subclonal progression events. In addition, pretransplant therapy with azacitidine likely influenced the mutation spectrum and evolution of emergent subclones after alloHCT. Although subclone evolution is common, founding clone mutations are always present at progression and could be detected in the bone marrow as early as 30 and/or 100 days after alloHCT in 6 of 8 (75%) patients, often prior to clinical progression. In conclusion, MDS progression after alloHCT is characterized by subclonal expansion and evolution, which can be influenced by pretransplant therapy. PMID:29515031

Clonal and molecular analysis of the prospective anterior neural boundary in the mouse embryo

PubMed Central

Cajal, Marieke; Lawson, Kirstie A.; Hill, Bill; Moreau, Anne; Rao, Jianguo; Ross, Allyson; Collignon, Jérôme; Camus, Anne

2012-01-01

In the mouse embryo the anterior ectoderm undergoes extensive growth and morphogenesis to form the forebrain and cephalic non-neural ectoderm. We traced descendants of single ectoderm cells to study cell fate choice and cell behaviour at late gastrulation. In addition, we provide a comprehensive spatiotemporal atlas of anterior gene expression at stages crucial for anterior ectoderm regionalisation and neural plate formation. Our results show that, at late gastrulation stage, expression patterns of anterior ectoderm genes overlap significantly and correlate with areas of distinct prospective fates but do not define lineages. The fate map delineates a rostral limit to forebrain contribution. However, no early subdivision of the presumptive forebrain territory can be detected. Lineage analysis at single-cell resolution revealed that precursors of the anterior neural ridge (ANR), a signalling centre involved in forebrain development and patterning, are clonally related to neural ectoderm. The prospective ANR and the forebrain neuroectoderm arise from cells scattered within the same broad area of anterior ectoderm. This study establishes that although the segregation between non-neural and neural precursors in the anterior midline ectoderm is not complete at late gastrulation stage, this tissue already harbours elements of regionalisation that prefigure the later organisation of the head. PMID:22186731

Label-free isolation and deposition of single bacterial cells from heterogeneous samples for clonal culturing

NASA Astrophysics Data System (ADS)

Riba, J.; Gleichmann, T.; Zimmermann, S.; Zengerle, R.; Koltay, P.

2016-09-01

The isolation and analysis of single prokaryotic cells down to 1 μm and less in size poses a special challenge and requires micro-engineered devices to handle volumes in the picoliter to nanoliter range. Here, an advanced Single-Cell Printer (SCP) was applied for automated and label-free isolation and deposition of bacterial cells encapsulated in 35 pl droplets by inkjet-like printing. To achieve this, dispenser chips to generate micro droplets have been fabricated with nozzles 20 μm in size. Further, the magnification of the optical system used for cell detection was increased. Redesign of the optical path allows for collision-free addressing of any flat substrate since no compartment protrudes below the nozzle of the dispenser chip anymore. The improved system allows for deterministic isolation of individual bacterial cells. A single-cell printing efficiency of 93% was obtained as shown by printing fluorescent labeled E. coli. A 96-well plate filled with growth medium is inoculated with single bacteria cells on average within about 8 min. Finally, individual bacterial cells from a heterogeneous sample of E. coli and E. faecalis were isolated for clonal culturing directly on agar plates in user-defined array geometry.

CYP76M7 Is an ent-Cassadiene C11α-Hydroxylase Defining a Second Multifunctional Diterpenoid Biosynthetic Gene Cluster in Rice[W][OA

PubMed Central

Swaminathan, Sivakumar; Morrone, Dana; Wang, Qiang; Fulton, D. Bruce; Peters, Reuben J.

2009-01-01

Biosynthetic gene clusters are common in microbial organisms, but rare in plants, raising questions regarding the evolutionary forces that drive their assembly in multicellular eukaryotes. Here, we characterize the biochemical function of a rice (Oryza sativa) cytochrome P450 monooxygenase, CYP76M7, which seems to act in the production of antifungal phytocassanes and defines a second diterpenoid biosynthetic gene cluster in rice. This cluster is uniquely multifunctional, containing enzymatic genes involved in the production of two distinct sets of phytoalexins, the antifungal phytocassanes and antibacterial oryzalides/oryzadiones, with the corresponding genes being subject to distinct transcriptional regulation. The lack of uniform coregulation of the genes within this multifunctional cluster suggests that this was not a primary driving force in its assembly. However, the cluster is dedicated to specialized metabolism, as all genes in the cluster are involved in phytoalexin metabolism. We hypothesize that this dedication to specialized metabolism led to the assembly of the corresponding biosynthetic gene cluster. Consistent with this hypothesis, molecular phylogenetic comparison demonstrates that the two rice diterpenoid biosynthetic gene clusters have undergone independent elaboration to their present-day forms, indicating continued evolutionary pressure for coclustering of enzymatic genes encoding components of related biosynthetic pathways. PMID:19825834

Genetic relatedness of selected clinical Vibrio cholerae O139 isolates from the southern coastal area of China over a 20-year period.

PubMed

Li, B S; Xiao, Y; Wang, D C; Tan, H L; Ke, B X; He, D M; Ke, C W; Zhang, Y H

2016-09-01

Vibrio cholerae O139 emerged as a causative agent of epidemic cholera in 1992 in India and Bangladesh, and was subsequently reported in China in 1993. The genetic relatedness and molecular characteristics of V. cholerae O139 in Guangdong Province, located in the southern coastal area of China, remains undetermined. In this study, we investigated 136 clinical V. cholerae O139 isolates from 1993 to 2013 in Guangdong. By conventional PCR, 123 (90·4%) isolates were positive for ctxB, ace and zot. Sequencing of the positive amplicons indicated 113 (91·7%) isolates possessed the El Tor allele of ctxB (genotype 3); seven carried the classical ctxB type (genotype 1) and three harboured a novel ctxB type (genotype 5). With respect to tcpA, 123 (90·4%) isolates were positive for the El Tor allele. In addition, pulsed-field gel electrophoresis (with NotI digestion) differentiated the isolates into clusters A and B. Cluster A contained seven of the non-toxigenic isolates from 1998 to 2000; another six non-toxigenic isolates (from 1998 and 2007) and all of the toxigenic isolates formed cluster B. Our results suggest that over a 20-year period, the predominant O139 clinical isolates have maintained a relatively tight clonal structure, although some genetic variance and shift has occurred. Our data highlight the persistence of toxigenic V. cholerae O139 in clinical settings in the southern coastal area of China.

MicroRNA-106b~25 cluster is upregulated in relapsed MLL-rearranged pediatric acute myeloid leukemia

PubMed Central

Verboon, Lonneke J.; Obulkasim, Askar; de Rooij, Jasmijn D.E.; Katsman, Jenny E.; Sonneveld, Edwin; Baruchel, André; Trka, Jan; Reinhardt, Dirk; Pieters, Rob; Cloos, Jacqueline; Kaspers, Gertjan J.L.; Klusmann, Jan-Henning; Zwaan, Christian Michel; Fornerod, Maarten; van den Heuvel-Eibrink, Marry M.

2016-01-01

The most important reason for therapy failure in pediatric acute myeloid leukemia (AML) is relapse. In order to identify miRNAs that contribute to the clonal evolution towards relapse in pediatric AML, miRNA expression profiling of 127 de novo pediatric AML cases were used. In the diagnostic phase, no miRNA signatures could be identified that were predictive for relapse occurrence, in a large pediatric cohort, nor in a nested mixed lineage leukemia (MLL)-rearranged pediatric cohort. AML with MLL- rearrangements are found in 15-20% of all pediatric AML samples, and reveal a relapse rate up to 50% for certain translocation partner subgroups. Therefore, microRNA expression profiling of six paired initial diagnosis-relapse MLL-rearranged pediatric AML samples (test cohort) and additional eight paired initial diagnosis-relapse samples with MLL-rearrangements (validation cohort) was performed. A list of 53 differentially expressed miRNAs was identified of which the miR-106b~25 cluster, located in intron 13 of MCM7, was the most prominent. These differentially expressed miRNAs however could not predict a relapse in de novo AML samples with MLL-rearrangements at diagnosis. Furthermore, higher mRNA expression of both MCM7 and its upstream regulator E2F1 was found in relapse samples with MLL-rearrangements. In conclusion, we identified the miR-106b~25 cluster to be upregulated in relapse pediatric AML with MLL-rearrangements. PMID:27351222

Seizure clustering.

PubMed

Haut, Sheryl R

2006-02-01

Seizure clusters, also known as repetitive or serial seizures, occur commonly in epilepsy. Clustering implies that the occurrence of one seizure may influence the probability of a subsequent seizure; thus, the investigation of the clustering phenomenon yields insights into both specific mechanisms of seizure clustering and more general concepts of seizure occurrence. Seizure clustering has been defined clinically as a number of seizures per unit time and, statistically, as a deviation from a random distribution, or interseizure interval dependence. This review explores the pathophysiology, epidemiology, and clinical implications of clustering, as well as other periodic patterns of seizure occurrence. Risk factors for experiencing clusters and potential precipitants of clustering are also addressed.

Genetic Considerations for the Restoration of Smooth Cordgrass (Spartina alterniflora) Within Its Native Range

USGS Publications Warehouse

Travis, Steven E.; Proffitt, C. Edward; Edwards, Keith R.

2006-01-01

In order to remain viable over many generations, plant populations require the ability to respond adaptively to a changing environment. Such adaptive potential is directly controlled by underlying genetic variation, which can be measured in terms of both heterozygosity at the individual level and clonal, or genotypic diversity at the population level. This report summarizes research relating to the importance of genetic diversity in the restoration of salt marsh smooth cordgrass, Spartina alterniflora, a dominant member of low elevation intertidal marshes throughout the northern Gulf of Mexico and Atlantic Coasts of North America. Recent research has indicated that S. alterniflora is a partially clonal species characterized by the recruitment of seedlings exclusively during the initial colonization phase of population establishment. A major consequence of this finding is that clonal diversity generally peaks rather early in the development of a restored marsh, depending on the rate of natural immigration and/or the clonal diversity of planting units, and then undergoes a steady decline over geological time spans because of stochastic mortality and intraspecific competition. Low levels of clonal diversity resulting from restricted immigration or clonally depauperate planting materials in turn places strict limits on opportunities for outcrossing in a species known to suffer from severe inbreeding depression. Low clonal diversity may further lead to declining levels of heterozyosity of individual clones, which directly affects competitive ability. In addition, the planting of genetically diverse plant materials should take into account the genetic and adaptive differentiation that takes place when plant populations are widely separated in space and/or dwell under varying sets of environmental conditions. Thus, steps should be taken to ensure that S. alterniflora clones developed for restorative plantings are both genetically diverse and sufficiently pre-adapted to environmental conditions at the proposed restoration site. This can be achieved by growing plant materials collected from local sources and by either taking care to maintain relatively high levels of clonal diversity or by planting clones at sufficiently low densities that they will not quickly grow to monopolize a restoration site without first producing several generations of sexual recruits through crosses with nearby native populations.

Understanding ligand effects in gold clusters using mass spectrometry

DOE Office of Scientific and Technical Information (OSTI.GOV)

Johnson, Grant E.; Laskin, Julia

This review summarizes recent research on the influence of phosphine ligands on the size, stability, and reactivity of gold clusters synthesized in solution. Sub-nanometer clusters exhibit size- and composition-dependent properties that are unique from those of larger nanoparticles. The highly tunable properties of clusters and their high surface-to-volume ratio make them promising candidates for a variety of technological applications. However, because “each-atom-counts” toward defining cluster properties it is critically important to develop robust synthesis methods to efficiently prepare clusters of predetermined size. For decades phosphines have been known to direct the size-selected synthesis of gold clusters. Despite the preparation ofmore » numerous species it is still not understood how different functional groups at phosphine centers affect the size and properties of gold clusters. Using electrospray ionization mass spectrometry (ESI-MS) it is possible to characterize the effect of ligand substitution on the distribution of clusters formed in solution at defined reaction conditions. In addition, ligand exchange reactions on preformed clusters may be monitored using ESI-MS. Collision induced dissociation (CID) may also be employed to obtain qualitative insight into the fragmentation of mixed ligand clusters and the relative binding energies of differently substituted phosphines. Quantitative ligand binding energies and cluster stability may be determined employing surface induced dissociation (SID) in a custom-built Fourier transform ion cyclotron resonance mass spectrometer (FT-ICR-MS). Rice-Ramsperger-Kassel-Marcus (RRKM) based modeling of the SID data allows dissociation energies and entropy values to be extracted that may be compared with the results of high-level theoretical calculations. The charge reduction and reactivity of atomically precise gold clusters, including partially ligated species generated in the gas-phase by in source CID, on well-defined surfaces may be explored using ion soft landing (SL) in a custom-built instrument combined with in situ time of flight secondary ion mass spectrometry (TOF-SIMS). Jointly, this multipronged experimental approach allows characterization of the full spectrum of relevant phenomena including cluster synthesis, ligand exchange, thermochemistry, surface immobilization, and reactivity. The fundamental insights obtained from this work will facilitate the directed synthesis of gold clusters with predetermined size and properties for specific applications.« less

Personalising care of adults with asthma from Asia: a modified e-Dephi consensus study to inform management tailored to attitude and control profiles.

PubMed

Chisholm, Alison; Price, David B; Pinnock, Hilary; Lee, Tan Tze; Roa, Camilo; Cho, Sang-Heon; David-Wang, Aileen; Wong, Gary; van der Molen, Thys; Ryan, Dermot; Castillo-Carandang, Nina; Yong, Yee Vern

2017-01-05

REALISE Asia-an online questionnaire-based study of Asian asthma patients-identified five patient clusters defined in terms of their control status and attitude towards their asthma (categorised as: 'Well-adjusted and at least partly controlled'; 'In denial about symptoms'; 'Tolerating with poor control'; 'Adrift and poorly controlled'; 'Worried with multiple symptoms'). We developed consensus recommendations for tailoring management of these attitudinal-control clusters. An expert panel undertook a three-round electronic Delphi (e-Delphi): Round 1: panellists received descriptions of the attitudinal-control clusters and provided free text recommendations for their assessment and management. Round 2: panellists prioritised Round 1 recommendations and met (or joined a teleconference) to consolidate the recommendations. Round 3: panellists voted and prioritised the remaining recommendations. Consensus was defined as Round 3 recommendations endorsed by >50% of panellists. Highest priority recommendations were those receiving the highest score. The multidisciplinary panellists (9 clinicians, 1 pharmacist and 1 health social scientist; 7 from Asia) identified consensus recommendations for all clusters. Recommended pharmacological (e.g., step-up/down; self-management; simplified regimen) and non-pharmacological approaches (e.g., trigger management, education, social support; inhaler technique) varied substantially according to each cluster's attitude to asthma and associated psychosocial drivers of behaviour. The attitudinal-control clusters defined by REALISE Asia resonated with the international panel. Consensus was reached on appropriate tailored management approaches for all clusters. Summarised and incorporated into a structured management pathway, these recommendations could facilitate personalised care. Generalisability of these patient clusters should be assessed in other socio-economic, cultural and literacy groups and nationalities in Asia.

Accurate age determinations of several nearby open clusters containing magnetic Ap stars

NASA Astrophysics Data System (ADS)

Silaj, J.; Landstreet, J. D.

2014-06-01

Context. To study the time evolution of magnetic fields, chemical abundance peculiarities, and other characteristics of magnetic Ap and Bp stars during their main sequence lives, a sample of these stars in open clusters has been obtained, as such stars can be assumed to have the same ages as the clusters to which they belong. However, in exploring age determinations in the literature, we find a large dispersion among different age determinations, even for bright, nearby clusters. Aims: Our aim is to obtain ages that are as accurate as possible for the seven nearby open clusters α Per, Coma Ber, IC 2602, NGC 2232, NGC 2451A, NGC 2516, and NGC 6475, each of which contains at least one magnetic Ap or Bp star. Simultaneously, we test the current calibrations of Te and luminosity for the Ap/Bp star members, and identify clearly blue stragglers in the clusters studied. Methods: We explore the possibility that isochrone fitting in the theoretical Hertzsprung-Russell diagram (i.e. log (L/L⊙) vs. log Te), rather than in the conventional colour-magnitude diagram, can provide more precise and accurate cluster ages, with well-defined uncertainties. Results: Well-defined ages are found for all the clusters studied. For the nearby clusters studied, the derived ages are not very sensitive to the small uncertainties in distance, reddening, membership, metallicity, or choice of isochrones. Our age determinations are all within the range of previously determined values, but the associated uncertainties are considerably smaller than the spread in recent age determinations from the literature. Furthermore, examination of proper motions and HR diagrams confirms that the Ap stars identified in these clusters are members, and that the presently accepted temperature scale and bolometric corrections for Ap stars are approximately correct. We show that in these theoretical HR diagrams blue stragglers are particularly easy to identify. Conclusions: Constructing the theoretical HR diagram of a nearby open cluster makes possible an accurate age determination, with well defined uncertainty. This diagnostic of a cluster also provides a useful tool for studying unusual stars such as Ap stars and blue stragglers. Table 3 is available in electronic form at http://www.aanda.org

A partial list of southern clusters of galaxies

NASA Technical Reports Server (NTRS)

Quintana, H.; White, R. A.

1990-01-01

An inspection of 34 SRC/ESO J southern sky fields is the basis of the present list of clusters of galaxies and their approximate classifications in terms of cluster concentration, defined independently of richness and shape-symmetry. Where possible, an estimate of the cluster morphological population is provided. The Bautz-Morgan classification was applied using a strict comparison with clusters on the Palomar Sky Survey. Magnitudes were estimated on the basis of galaxies with photoelectric or photographic magnitudes.

Orbit Clustering Based on Transfer Cost

NASA Technical Reports Server (NTRS)

Gustafson, Eric D.; Arrieta-Camacho, Juan J.; Petropoulos, Anastassios E.

2013-01-01

We propose using cluster analysis to perform quick screening for combinatorial global optimization problems. The key missing component currently preventing cluster analysis from use in this context is the lack of a useable metric function that defines the cost to transfer between two orbits. We study several proposed metrics and clustering algorithms, including k-means and the expectation maximization algorithm. We also show that proven heuristic methods such as the Q-law can be modified to work with cluster analysis.

Finding and testing network communities by lumped Markov chains.

PubMed

Piccardi, Carlo

2011-01-01

Identifying communities (or clusters), namely groups of nodes with comparatively strong internal connectivity, is a fundamental task for deeply understanding the structure and function of a network. Yet, there is a lack of formal criteria for defining communities and for testing their significance. We propose a sharp definition that is based on a quality threshold. By means of a lumped Markov chain model of a random walker, a quality measure called "persistence probability" is associated to a cluster, which is then defined as an "α-community" if such a probability is not smaller than α. Consistently, a partition composed of α-communities is an "α-partition." These definitions turn out to be very effective for finding and testing communities. If a set of candidate partitions is available, setting the desired α-level allows one to immediately select the α-partition with the finest decomposition. Simultaneously, the persistence probabilities quantify the quality of each single community. Given its ability in individually assessing each single cluster, this approach can also disclose single well-defined communities even in networks that overall do not possess a definite clusterized structure.

Genetic Heterogeneity and Clonal Evolution of Tumor Cells and their Impact on Precision Cancer Medicine.

PubMed

Sabaawy, Hatem E

2013-11-18

The efficacy of targeted therapies in leukemias and solid tumors depends upon the accurate detection and sustained targeting of initial and evolving driver mutations and/or aberrations in cancer cells. Tumor clonal evolution of the diverse populations of cancer cells during cancer progression contributes to the longitudinal variations of clonal, morphological, anatomical, and molecular heterogeneity of tumors. Moreover, drug-resistant subclones present at initiation of therapy or emerging as a result of targeted therapies represent major challenges for achieving success of personalized therapies in providing meaningful improvement in cancer survival rates. Here, I briefly portray tumor cell clonal evolution at the cellular and molecular levels, and present the multiple types of genetic heterogeneity in tumors, with a focus on their impact on the implementation of personalized or precision cancer medicine.

Acute megakaryoblastic leukemia after transient myeloproliferative disorder with clonal karyotype evolution in a phenotypically normal neonate.

PubMed

Polski, Jacek M; Galambos, Csaba; Gale, Gordon B; Dunphy, Cherie H; Evans, H Lance; Batanian, Jacqueline R

2002-01-01

We report a case of transient myeloproliferative disorder (TMD) in a neonate without features of Down syndrome (DS) with clonal karyotype evolution, after apparent spontaneous resolution of TMD, but eventually progressing to acute megakaryoblastic leukemia (AMKL). The patient had petechiae, thrombocytopenia, and blastemia. Trisomy 21 with a satellited Y chromosome (Yqs) was found in proliferating blasts. A stimulated peripheral blood culture confirmed the constitutional origin of the Yqs, but did not reveal the presence of any trisomic 21 cell. By the age of 3 months, clonal chromosome evolution in the form of an interstitial deletion of the long-arm of chromosome 13 [del(13)(q13q31)] was detected along with trisomy 21 in unstimulated bone marrow cultures. However, remission was achieved without treatment at the age of 4 months. Trisomy 21 and del(13)(q13q31) were not identified in either cytogenetics or fluorescence in situ hybridization studies at that time. The child was asymptomatic until the age of 20 months when anemia and thrombocytopenia prompted a bone marrow biopsy, revealing changes consistent with AMKL. The remission proceeded by clonal karyotype evolution in a neonate with TMD demonstrates that clonal karyotype evolution does not indicate an immediately progressive disease. However, the development of AMKL after TMD in this case illustrates the increased risk for leukemia in TMD cases, even without DS. The gradual clonal evolution of the blasts in our patient suggests that "multiple hits" oncogenesis applies to TMD progression to acute leukemia.

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

PubMed

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.

Spatial Genetic Structure and Clonal Diversity in an Alpine Population of Salix herbacea (Salicaceae)

PubMed Central

Reisch, Christoph; Schurm, Sophia; Poschlod, Peter

2007-01-01

Background and Aims Many alpine plant species combine clonal and sexual reproduction to minimize the risks of flowering and seed production in high mountain regions. The spatial genetic structure and diversity of these alpine species is strongly affected by different clonal strategies (phalanx or guerrilla) and the proportion of generative and vegetative reproduction. Methods The clonal structure of the alpine plant species Salix herbacea was investigated in a 3 × 3 m plot of an alpine meadow using microsatellite (simple sequence repeat; SSR) analysis. The data obtained were compared with the results of a random amplified polymorphic DNA (RAPD) analysis. Key Results SSR analysis, based on three loci and 16 alleles, revealed 24 different genotypes and a proportion of distinguishable genotypes of 0·18. Six SSR clones were found consisting of at least five samples, 17 clones consisting of more than two samples and seven single genotypes. Mean clone size comprising at least five samples was 0·96 m2, and spatial autocorrelation analysis showed strong similarity of samples up to 130 cm. RAPD analysis revealed a higher level of clonal diversity but a comparable number of larger clones and a similar spatial structure. Conclusions The spatial genetic structure as well as the occurrence of single genotypes revealed in this study suggests both clonal and sexual propagation and repeated seedling recruitment in established populations of S. herbacea and is thus suggestive of a relaxed phalanx strategy. PMID:17242040

Flower-deficient mice have reduced susceptibility to skin papilloma formation

PubMed Central

Petrova, Evgeniya; López-Gay, Jesús M.; Rhiner, Christa; Moreno, Eduardo

2012-01-01

SUMMARY Skin papillomas arise as a result of clonal expansion of mutant cells. It has been proposed that the expansion of pretumoral cell clones is propelled not only by the increased proliferation capacity of mutant cells, but also by active cell selection. Previous studies in Drosophila describe a clonal selection process mediated by the Flower (Fwe) protein, whereby cells that express certain Fwe isoforms are recognized and forced to undergo apoptosis. It was further shown that knock down of fwe expression in Drosophila can prevent the clonal expansion of dMyc-overexpressing pretumoral cells. Here, we study the function of the single predicted mouse homolog of Drosophila Fwe, referred to as mFwe, by clonal overexpression of mFwe isoforms in Drosophila and by analyzing mFwe knock-out mice. We show that clonal overexpression of certain mFwe isoforms in Drosophila also triggers non-autonomous cell death, suggesting that Fwe function is evolutionarily conserved. Although mFwe-deficient mice display a normal phenotype, they develop a significantly lower number of skin papillomas upon exposure to DMBA/TPA two-stage skin carcinogenesis than do treated wild-type and mFwe heterozygous mice. Furthermore, mFwe expression is higher in papillomas and the papilloma-surrounding skin of treated wild-type mice compared with the skin of untreated wild-type mice. Thus, we propose that skin papilloma cells take advantage of mFwe activity to facilitate their clonal expansion. PMID:22362363

Fire coral clones demonstrate phenotypic plasticity among reef habitats.

PubMed

Dubé, Caroline E; Boissin, Emilie; Maynard, Jeffrey A; Planes, Serge

2017-08-01

Clonal populations are often characterized by reduced levels of genotypic diversity, which can translate into lower numbers of functional phenotypes, both of which impede adaptation. Study of partially clonal animals enables examination of the environmental settings under which clonal reproduction is favoured. Here, we gathered genotypic and phenotypic information from 3,651 georeferenced colonies of the fire coral Millepora platyphylla in five habitats with different hydrodynamic regimes in Moorea, French Polynesia. In the upper slope where waves break, most colonies grew as vertical sheets ("sheet tree") making them more vulnerable to fragmentation. Nearly all fire corals in the other habitats are encrusting or massive. The M. platyphylla population is highly clonal (80% of the colonies are clones), while characterized by the highest genotype diversity ever documented for terrestrial or marine populations (1,064 genotypes). The proportion of clones varies greatly among habitats (≥58%-97%) and clones (328 clonal lineages) are distributed perpendicularly from the reef crest, perfectly aligned with wave energy. There are six clonal lineages with clones dispersed in at least two adjacent habitats that strongly demonstrate phenotypic plasticity. Eighty per cent of the colonies in these lineages are "sheet tree" on the upper slope, while 80%-100% are encrusting or massive on the mid slope and back reef. This is a unique example of phenotypic plasticity among reef-building coral clones as corals typically have wave-tolerant growth forms in high-energy reef areas. © 2017 John Wiley & Sons Ltd.

A Public Health Model for the Molecular Surveillance of HIV Transmission in San Diego, California

PubMed Central

May, Susanne; Tweeten, Samantha; Drumright, Lydia; Pacold, Mary E.; Kosakovsky Pond, Sergei L.; Pesano, Rick L.; Lie, Yolanda S.; Richman, Douglas D.; Frost, Simon D.W.; Woelk, Christopher H.; Little, Susan J.

2009-01-01

Background Current public health efforts often use molecular technologies to identify and contain communicable disease networks, but not for HIV. Here, we investigate how molecular epidemiology can be used to identify highly-related HIV networks within a population and how voluntary contact tracing of sexual partners can be used to selectively target these networks. Methods We evaluated the use of HIV-1 pol sequences obtained from participants of a community-recruited cohort (n=268) and a primary infection research cohort (n=369) to define highly related transmission clusters and the use of contact tracing to link other individuals (n=36) within these clusters. The presence of transmitted drug resistance was interpreted from the pol sequences (Calibrated Population Resistance v3.0). Results Phylogenetic clustering was conservatively defined when the genetic distance between any two pol sequences was <1%, which identified 34 distinct transmission clusters within the combined community-recruited and primary infection research cohorts containing 160 individuals. Although sequences from the epidemiologically-linked partners represented approximately 5% of the total sequences, they clustered with 60% of the sequences that clustered from the combined cohorts (O.R. 21.7; p=<0.01). Major resistance to at least one class of antiretroviral medication was found in 19% of clustering sequences. Conclusions Phylogenetic methods can be used to identify individuals who are within highly related transmission groups, and contact tracing of epidemiologically-linked partners of recently infected individuals can be used to link into previously-defined transmission groups. These methods could be used to implement selectively targeted prevention interventions. PMID:19098493

Commentary on: "Clonal evolution of chemotherapy-resistant urothelial carcinoma." Faltas BM, Prandi D, Tagawa ST, Molina AM, Nanus DM, Sternberg C, Rosenberg J, Mosquera JM, Robinson B, Elemento O, Sboner A, Beltran H, Demichelis F, Rubin MA.: Nat Genet. 2016 Oct 17. http://dx.doi.org/10.1038/ng.3692.

PubMed

Lee, Byron H

2017-09-01

Chemotherapy-resistant urothelial carcinoma has no uniformly curative therapy. Understanding how selective pressure from chemotherapy directs the evolution of urothelial carcinoma and shapes its clonal architecture is a central biological question with clinical implications. To address this question, we performed whole-exome sequencing and clonality analysis of 72 urothelial carcinoma samples, including 16 matched sets of primary and advanced tumors prospectively collected before and after chemotherapy. Our analysis provided several insights that are as follows: (1) chemotherapy-treated urothelial carcinoma is characterized by intrapatient mutational heterogeneity, and most mutations are not shared; (2) both branching evolution and metastatic spread are very early events in the natural history of urothelial carcinoma; (3) chemotherapy-treated urothelial carcinoma is enriched with clonal mutations involving L1 cell-adhesion molecule and integrin signaling pathways; and (4) APOBEC-induced mutagenesis is clonally enriched in chemotherapy-treated urothelial carcinoma and continues to shape the evolution of urothelial carcinoma throughout its lifetime. Copyright © 2017 Elsevier Inc. All rights reserved.

[Clonal relationship and detection of blaKPC gene in strains of Klebsiella pneumoniae resistant to carbapenems, at a hospital in Venezuela].

PubMed

Martínez, Dianny; Araque, Yasmina; Roduifo, Hectorina; Caña, Luisa; García, José; Gonzáiez, Diorelis; Rodríguez, Lucy; De Donato, Marcos

2016-10-01

In order to study the clonal relationship and blaKPC gene detection in clinical isolates of Klebsiella pneumoniae resistant to carbapenems, we analyzed 22 clinical isolates of K. pneumoniae with resistance to imipenem and/ or meropenem, isolated in the laboratory of bacteriology at the University Hospital "Antonio Patricio de Alcalá" (HUAPA) from the Cumana city, Sucre state, Venezuela, for a period of five consecutive years. Susceptibility to different antimicrobials was determined, and the presence of carbapenemases was detected by modified Hodge method, phenyl boronic acid synergy and combination discs. blaKPC gene detection was conducted by polymerase chain reaction and the clonal relationship was determined by pulsed field electrophoresis. High rates of antimicrobial resistance were found, five strains were negative, at least one phenotypic method, and all carried the blaKPC gene. Clonal spread was observed only in the intensive care unit (ICU), while in other services, polyclonality was found. We concluded that blaKPC gene is present in K. pneumoniae strains resistant to carbapenems isolated in the HUAPA and clonal spread it was only in the ICU.