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In most Japanese populations of dojo loach (Misgurnus anguillicaudatus), gonochoristic diploids of genetically diversified groups (A and B, further subdivided into B1 and B2) are present, whereas unisexual clonal lineages inhabit certain localities in the Hokkaido and Ishikawa Prefectures in Japan. Through a series of genetic studies including DNA markers, the clonal loaches were deemed to originate from a hybridization event(s) between the A and B1 groups. However, combined analyses with other DNA markers are needed to identify each genetic group. In this study, we improved the PCR-restriction fragment length polymorphism (RFLP) analysis of the recombination activating gene 1 (RAG1) gene using digestion with two restriction enzymes, PvuII and StuI. The improved RAG1-RFLP analysis showed different fragment patterns for each group: two fragments (245 and 198 bp) for group A, three fragments (198, 147, and 98 bp) for group B1, and a single fragment (443 bp) for group B2. The clonal loaches exhibited four fragments (245, 198, 147, and 98 bp) derived from both groups A and B1. Moreover, the DNA markers were able to detect two different hybrid genotypes (A x B2 and B1 x B2). Thus, the improved RAG1-RFLP markers allowed for quick and accurate group identification of the dojo loaches.

Toll-like receptor 3 (SpTLR3) from Schizothorax prenanti (S. prenanti) was cloned and identified, and the tissue distribution of the SpTLR3 gene was examined in this study. Moreover, the relative mRNA expression levels of myeloid differentiation factor 88 gene (SpMyD88) and seven TLR genes (SpTLR2, SpTLR3, SpTLR4, SpTLR18, SpTLR22-1, SpTLR22-2 and SpTLR22-3) from S. prenanti after lipopolysaccharide (LPS) challenge were analyzed through quantitative real-time polymerase chain reaction (qRT-PCR). The full length of SpTLR3 gene is 3097 bp, and complete coding sequence (CDS) is 2715 bp, which encodes 904 amino acids. The SpTLR3 amino acid sequence shared 43.94–100% identity with TLR3 sequences from other vertebrates; SpTLR3 was expressed in all eight tissues examined; and the highest level appeared in the liver, which was significantly higher than in all other tissues (p < 0.05), followed by the levels in the heart and muscles. LPS significantly up-regulated all eight genes in the S. prenanti tissues at 12 or 24 h (p < 0.05). Compared with the PBS control group, no significant transcripts changes were found in SpTLR2 or SpTLR3 at 12 h after LPS induction, but they were significantly up-regulated at 24 h (p < 0.001). The most abundant transcripts were found in the head kidney SpTLR22 genes after 24 h LPS induction, with high to low levels, which were SpTLR22-1 (564-fold), SpTLR22-3 (508-fold) and SpTLR22-2 (351-fold). Among these eight genes, the expression level of SpTLR4 was the least up-regulated. Overall, SpTLR4 in the head kidney was involved in the antibacterial immune response earlier, and the level was increased at 12 h with extreme significance after LPS stimulation (p < 0.001), while the other seven genes were the most significantly up-regulated at 24 h post injection. Taken together, the results suggest that SpMyD88, SpTLR2, SpTLR3, SpTLR4, SpTLR18, SpTLR22-1, SpTLR22-2 and SpTLR22-3 participate in an innate immune response stimulated by LPS, and the response intensity of the genes was organ-specific, with differing kinetics. Our findings will contribute to a more complete understanding of the roles of these TLR genes in antibacterial immunity.

Made available in DSpace on 2022-04-29T08:40:48Z (GMT). No. of bitstreams: 0 Previous issue date: 2022-04-01 We evaluated differential expression of genes in leaf and xylem tissues for three Eucalyptus clones in the field using Illumina sequencing, under four contrasting fertilization regimes: a control combining nitrogen (N), phosphorus (P), and potassium (K) and three regimes with N, K, and P deficiency. The field results showed significantly better performance with a control fertilizing regime for height and circumference at 14 months, but no differences between clones. The number of up and down regulated DEG (differentially expressed genes) in pairwise clone comparisons was around 5900 for leaf and 6900 for xylem at FDR < 0.01. With fertilization treatment comparisons, DEG were only observed for N deficiency versus control with 45 up and down-regulated DEG for leaf and 1022 for xylem. The number of DEGs between fertilizer deficiency treatment and control varied greatly within each clone showing important clone by fertilization interaction. Gene ontology analysis showed that a great number of genes were related to stress, transport, and transcription factors. The co-expression analysis showed some significant correlations between complex network gene expression and tree growth induced by fertilization regime. For example, the co-expression of a 54 DEG network showed a significant correlation with tree height (0.84, P value: 1e−04) and DBH (0.89, P value: 7e−06) in the case of N deficiency versus control. Our results suggest that gene expression levels between different fertilization treatments and clones can provide a basis for future research on gene function in Eucalyptus under nutrient stress with the perspective of new development in Eucalyptus breeding. Departamento de Ciências Químicas E Biológicas Instituto de Biociências UNESP - Univ Estadual Paulista, SP Instituto de Biotecnologia (IBTEC) UNESP - Univ Estadual Paulista Alameda das Tecomarias s/n. Chácara Capão Bonito, SP AGAP Univ Montpellier CIRAD INRA and Montpellier SupAgro UMR AGAP DGD-RS CIRAD, Avenue Val de Montferrand FCA (Faculdade de Ciências Agronômicas Departamento de Ciência Florestal Solos e Ambiente UNESP Univ Estadual Paulista “Júlio de Mesquita Filho” Eco&Sols Univ Montpellier CIRAD INRA IRD Montpellier SupAgro UMR Ecoa&Sols CIRAD, 2 Place Viala Departamento de Ciências Químicas E Biológicas Instituto de Biociências UNESP - Univ Estadual Paulista, SP Instituto de Biotecnologia (IBTEC) UNESP - Univ Estadual Paulista Alameda das Tecomarias s/n. Chácara Capão Bonito, SP FCA (Faculdade de Ciências Agronômicas Departamento de Ciência Florestal Solos e Ambiente UNESP Univ Estadual Paulista “Júlio de Mesquita Filho”

Morphology and ultrastructure of pollen grains were studied in 13 Oblaĉinska sour cherry clones in three years using scanning electron microscopy (SEM). All studied clones had isopolar, radially symmetric and tricolpate pollen grains. Length and width of pollen grains varied in a range of 50.03-55.97 μm and 25.16-28.37 μm respectively. Pollen shape in all the studied clones was identified as prolate. The smallest colpus length was found in the clone 8 (43.73 μm), and the highest in the clone 3 (49.16 μm). The highest colpus and mesocolpium width had the clone 3 (1.73 μm; 14.87 μm) and the lowest had the clone 7 (1.46 μm; 13.78 μm). All studied clones had striate exine ornamentation. Number of ridges per 100 μm2 of the exine surface was the highest in the clone 11 (16.7) and lowest in the clone 2 (14.1). Ridge and furrow width ranged from 0.46 to 0.61 μm and from 0.45 to 0.59 μm, respectively. The clones have been classified into three clusters based on all studied properties. Pollen grains examination by SEM indicated that several morphological parameters (pollen size and exine characteristics) can be used to distinguish Oblaĉinska sour cherry clones. [Project of the Serbian Ministry of Education, Science and Technological Development, Grant no. TR 31063]

Let τn be a type of algebras in which all operation symbols have arity n, for a fixed n ≥ 1. For 0 < r ≤ n, this paper introduces a special kind of n-ary terms of type τn called K*(n, r)-full terms. The set of all K*(n, r)-full terms of type τn is closed under the superposition operation Sn; hence forms a clone denoted by cloneK*(n,r)(τn). We prove that cloneK* (n,r)(τn) is a Menger algebra of rank n. We study K*(n, r)-full hypersubstitutions and the related K*(n, r)-full closed identities and K*(n, r)-full closed varieties. A connection between identities in cloneK* (n,r)(τn) and K* (n, r)-full closed identities is established. The results obtained generalize the results of Denecke and Jampachon [K. Denecke and P. Jampachon, Clones of full terms, Algebra and Discrete Math. 4 (2004) 1–11].

Wheat blast, caused byMagnaporthe oryzae(Syn.Pyricularia oryzae), was recently reported in Africa for the first time. To determine the relationship between African wheat blast isolates from Zambia with other populations from South America and Bangladesh, we analyzed the genotyping data recently reported by Tembo et al. (2021). The dataset consists of 84 single nucleotide polymorphisms (SNPs) obtained by multiplex amplicon sequencing from 186 isolates amended with data from 67 isolates mined from publicly available genome sequences. We observed that the Zambian isolates (N=13, 2018-2020) are identical for the 84 SNPs to isolates from Bangladesh (N=81, 2016-2020) and one genotype from South America (Bolivia, N=1, 2012). We conclude that the "B71" clonal lineage that emerged in South East Asia in 2016 and traces its origins to South America is now established in Zambia. Further analyses using whole genome sequences are needed to validate this conclusion. These findings callforurgent action, including a mitigation plan to prevent further spread.

[Background] Cancer is a rapidly evolving, multifactorial disease that accumulates numerous genetic and epigenetic alterations. This results in molecular and phenotypic heterogeneity within the tumor, the complexity of which is further amplified through specific interactions between cancer cells. We aimed to dissect the molecular mechanisms underlying the cooperation between different clones., [Methods] We produced clonal cell lines derived from the MDA-MB-231 breast cancer cell line, using the UbC-StarTrack system, which allowed tracking of multiple clones by color: GFP C3, mKO E10 and Sapphire D7. Characterization of these clones was performed by growth rate, cell metabolic activity, wound healing, invasion assays and genetic and epigenetic arrays. Tumorigenicity was tested by orthotopic and intravenous injections. Clonal cooperation was evaluated by medium complementation, co-culture and co-injection assays., [Results] Characterization of these clones in vitro revealed clear genetic and epigenetic differences that affected growth rate, cell metabolic activity, morphology and cytokine expression among cell lines. In vivo, all clonal cell lines were able to form tumors; however, injection of an equal mix of the different clones led to tumors with very few mKO E10 cells. Additionally, the mKO E10 clonal cell line showed a significant inability to form lung metastases. These results confirm that even in stable cell lines heterogeneity is present. In vitro, the complementation of growth medium with medium or exosomes from parental or clonal cell lines increased the growth rate of the other clones. Complementation assays, co-growth and co-injection of mKO E10 and GFP C3 clonal cell lines increased the efficiency of invasion and migration., [Conclusions] These findings support a model where interplay between clones confers aggressiveness, and which may allow identification of the factors involved in cellular communication that could play a role in clonal cooperation and thus represent new targets for preventing tumor progression., SRYC acknowledges support from Fondo de Investigaciones Sanitarias (FIS; PI17/02247 and PI14/01320), Centro de Investigación Biomédica en Red de Cáncer (CIBERONC; CB16/12/00363) and Generalitat de Catalunya (AGAUR; 2017 SGR 1799 and 2014 SGR 1131). AMP is funded by a FP7 Marie Sklodowska-Curie COFUND program under Grant Agreement n° 267128 (INCOMED program). ADL is funded by a “Juan Rodés” contract (JR17/00016) from ISCIII.

Somatic mutations in oncogene and tumor suppressor genes accumulate in healthy tissues throughout life and delineate clones with limited expansion. Lifestyle‐related toxic insults increase the size and number of these clones that participate to tissue aging. Their identification has blurred the boundaries between clonal expansion and malignant tumor and has drawn more attention to the role of the host environment in tumor emergence and progression. Local tissue factors such as disrupted cell interactions and stromal cell senescence combine with systemic and distant alterations to initiate the reiterative process of clonal expansion, multilayer intrinsic diversification and clonal selection that eventually characterize overt tumor evolution. In turn, tumors remodel their close and distant environments, establishing positive feedback loops that contribute to disease progression. Strategies emerge to preserve the tumor suppressive properties of healthy tissue landscapes and delay age‐induced changes that eventually lead to cancer.

Abiotic stress affects metabolic processes in plants and restricts plant growth and development. In this experiment, Caucasian clover (Trifolium ambiguum M. Bieb.) was used as a material, and the CDS of TaMYC2, which is involved in regulating the response to abiotic stress, was cloned. The CDS of TaMYC2 was 726 bp in length and encoded 241 amino acids. The protein encoded by TaMYC2 was determined to be unstable, be highly hydrophilic, and contain 23 phosphorylation sites. Subcellular localization results showed that TaMYC2 was localized in the nucleus. TaMYC2 responded to salt, alkali, cold, and drought stress and could be induced by IAA, GA3, and MeJA. By analyzing the gene expression and antioxidant enzyme activity in plants before and after stress, we found that drought and cold stress could induce the expression of TaMYC2 and increase the antioxidant enzyme activity. TaMYC2 could also induce the expression of ROS scavenging-related and stress-responsive genes and increase the activity of antioxidant enzymes, thus improving the ability of plants to resist stress. The results of this experiment provide references for subsequent in-depth exploration of both the function of TaMYC2 in and the molecular mechanism underlying the resistance of Caucasian clover.

The emergence of carbapenemase-producing Enterobacteriaceae (CPE) is a critical concern worldwide. In Italy, CPE isolates are very frequent, with the KPC enzyme types strongly predominant whereas the New Delhi metallo-β-lactamase (NDM) enzymes are extremely rare. Here we report the first detection of NDM-5-producing Escherichia coli sequence type 167 (ST167) isolates from two patients with urinary tract infection (Ec001 and Ec002 from urines), including one with colonisation (Ec003 from faeces) admitted to the same hospital 2 months apart in 2017. Minimum inhibitory concentrations (MICs) were determined by broth microdilution. The carbapenemase type was identified both by phenotypic and genotypic methods. Isolate genotypes were investigated by phylogenetic typing, multilocus sequence typing (MLST) and pulsed-field gel electrophoresis (PFGE). Next-generation sequencing (NGS) was used to obtain complete sequences of plasmids. The three E. coli isolates carried the blaNDM-5 gene, shared the same resistance phenotype and belonged to ST167. By PFGE, isolates showed the same profile, suggesting that they were the same strain. NGS revealed that the blaNDM-5 gene was located on a 99-kb multireplicon plasmid (designed pNDM-5-IT) with a peculiar scaffold constituted by four replicons of the IncF type (FIA, FIB and two copies of the FII replicon). pNDM-5-IT plasmid harboured multiple resistance and virulence determinants, including the arginine deaminase (ADI) cluster never found associated with plasmids before. Since NDM-5-producing E. coli ST167 has been regarded as a successful epidemic clone in China, the emergence of such a clone carrying a plasmid associated both with multiresistance and virulence could be a public-health threat.

Three multistate outbreaks between 2014 and 2016, involving case patients in and outside the United States, were linked to stone fruit, caramel apples, and packaged leafy green salad contaminated with Listeria monocytogenes singleton sequence type 382 (ST382), a serotype IVb-v1 clone with limited genomic divergence. Isolates from these outbreaks and other ST382 isolates not associated with these outbreaks were analyzed by whole-genome sequencing (WGS) analysis. The primary differences among ST382 strains were single nucleotide polymorphisms (SNPs). WGS analysis differentiated ST382 from a clonal complex 1 outbreak strain co-contaminating the caramel apples. WGS clustered food, environmental, and clinical isolates within each outbreak, and also differentiated among the three outbreak strains and epidemiologically unrelated ST382 isolates, which were indistinguishable by pulsed-field gel electrophoresis. ST382 appeared to be an emerging clone that began to diverge from its ancestor approximately 32 years before 2016. We estimated that there was 1.29 nucleotide substitution per genome (2.94 Mbp) per year for this clone.

The prediction of clonal genetic value for yield is challenging in oil palm (Elaeis guineensis Jacq.). Currently, clonal selection involves two stages of phenotypic selection (PS): ortet preselection on traits with sufficient heritability among a small number of individuals in the best crosses in progeny tests, and final selection on performance in clonal trials. The present study evaluated the efficiency of genomic selection (GS) for clonal selection. The training set comprised almost 300 Deli × La Me crosses phenotyped for eight palm oil yield components and the validation set 42 Deli × La Me ortets. Genotyping-by-sequencing (GBS) revealed 15,054 single nucleotide polymorphisms (SNP). The effects of the SNP dataset (density and percentage of missing data) and two GS modeling approaches, ignoring (ASGM) and considering (PSAM) the parental origin of alleles, were assessed. The results showed prediction accuracies ranging from 0.08 to 0.70 for ortet candidates without data records, depending on trait, SNP dataset and modeling. ASGM was better (on average slightly more accurate, less sensitive to SNP dataset and simpler), although PSAM appeared interesting for a few traits. With ASGM, the number of SNPs had to reach 7,000, while the percentage of missing data per SNP was of secondary importance, and GS prediction accuracies were higher than those of PS for most of the traits. Finally, this makes possible two practical applications of GS, that will increase genetic progress by improving ortet preselection before clonal trials: (1) preselection at the mature stage on all yield components jointly using ortet genotypes and phenotypes, and (2) genomic preselection on more yield components than PS, among a large population of the best possible crosses at nursery stage.

Many germ cells are eliminated during development, long before oogenesis or spermatogenesis. In mouse fetal testes, the majority of germ cell apoptosis coincides with the onset of male differentiation, suggesting coordination of these processes. We studied fetal germ-cell fates and discovered that both apoptosis and differentiation initiate in clonally related clusters. Lineage tracing confirmed that germ cells die as clones independent of intercellular bridges, suggesting that shared intrinsic properties are apoptotic determinants. We identified transcriptional heterogeneity among fetal germ cells that included an apoptosis-susceptible population characterized by failure to differentiate, whereas successful differentiation to prospermatogonia occurred through the expression of epigenetically regulated genes, including LINE1. Our results indicate that the fetal germ-cell fate is based on discrete cell-heritable identities. Elevated DNA methylation in the apoptosis-susceptible subpopulation supports our hypothesis that earlier errors in germ-cell epigenetic reprogramming derail differentiation in cellular progeny, leading to fetal apoptotic selection that ultimately improves the gamete quality.

Genetic improvement of crop Water Use Efficiency (WUE) is a general goal because the increasing water scarcity and the trend to a more sustainable agriculture. For grapevines, this subject is relevant and need an urgent response because their wide distribution in semi-arid areas. New cultivars are difficult to introduce in viticulture due to the narrow dependency of consumer appreciation often linked to a certain particular wine taste. Clones of reputed cultivars would presumably be more accepted but little is known on the intra-cultivar genetic variability of the WUE. The present work compares, on the basis of two field assays, the variability of intrinsic water use efficiency (WUE) in a large collection of cultivars in contrast with a collection of clones of Tempranillo cultivar. The results show that clonal variability of WUE was around 80% of the inter-cultivar, thus providing a first assessment on the opportunity for clonal selection by WUE. Plotting the WUE data against stem water potential or stomatal conductance it was possible to identify cultivars and clones out of the confidence intervals of this linear regression thus with significantly higher and lower WUE values. The present results contribute to open the expectative for a genetic improvement of grapevine WUE, This work was performed with financial support from the Spanish Ministry of Science and Technology (project AGL2014-30408-C04-01), from INIA (project RTA2013-00068-C03-03), and a pre-doctoral fellowship BES-2015-073331.

Fluorescence expression tools for stable and innocuous whole mycoplasma cell labelling have been developed. A Tn4001-derivative mini-transposon affording unmarked, stable mutagenesis in mycoplasmas was modified to allow the constitutive, high-level expression of mCherry, mKO2 and mNeonGreen. These tools were used to introduce the respective fluorescent proteins as chromosomal tags in the phylogenetically distant species Mycoplasma mycoides subsp. mycoides and Mycoplasma bovis. The production, selection and characterisation of fluorescent clones were straightforward and resulted in the unprecedented observation of red and green fluorescent mycoplasma colonies in the two species, with no apparent cytotoxicity. Equivalent fluorescence expression levels were quantified by flow cytometry in both species, suggesting that these tools can be broadly applied in mycoplasmas. A macrophage infection assay was performed to assess the usefulness of mNeonGreen-expressing strains for monitoring mycoplasma infections, and notably cell invasion. The presence of fluorescent mycoplasmas inside live phagocytic cells was detected and quantified by flow cytometry and corroborated by confocal microscopy, which allowed the identification of individual mycoplasmas in the cytoplasm of infected cells. The fluorescence expression tools developed in this study are suitable for host-pathogen interaction studies and offer innumerable perspectives for the functional analysis of mycoplasmas both in vitro and in vivo.

Streptococcus pyogenes (Group A Streptococcus, GAS) is a strictly human bacterial pathogen. Since the mid-1980s, GAS M1T1 clone has been the most prevalent and globally disseminated serotype and is the culprit causing invasive and severe streptococcal infections, urging a better understanding of the emergence of hypervirulent M1T1 clone from an evolutionary perspective. This review highlights the molecular and evolutionary events leading to pandemic M1T1 strains, and discusses the pressure driving the genetic acquisition of novel virulence genes and the selection of hypervirulent isolates in host. By understanding the evolutionary selection and pressures that select and shape the pandemic M1T1 clone, we could potentially develop new therapeutic strategies to tackle challenges when dealing with the globally disseminated M1T1 GAS clone.

Objectives: Genomic characterization of the internationally spread sequence type (ST) 16 carbapenemresistant Klebsiella pneumoniae. Methods: The complete genomes of three carbapenem producing ST16 K. pneumoniae from Italian patients were analysed by single-nucleotide polymorphism-based phylogeny, core genome multilocus sequence typing, resistance, plasmid, and virulence content and compared with ten genomes of ST16 strains isolated in other countries. Plasmids carrying blaNDM-1 or blaOXA-232 carbapenemase genes were assembled and sequences were analysed. Results: The internationally spread ST16 K. pneumoniae clone showed variability in terms of distribution of NDM-1 and OXA-232 type carbapenemases. In some ST16 strains, up to six plasmids can be simultaneously present in the same cell, including ColE-like plasmids carrying blaOXA-232 and IncF plasmids carrying blaNDM-1. The differences observed in plasmid, resistance, and virulence content and core genome suggested that there is not a unique, highly conserved ST16 clone, but instead different variants of this lineage circulate worldwide. Conclusions: The ST16 K. pneumoniae clone has spread worldwide and may become a high-risk clone. (c) 2018 Published by Elsevier Ltd on behalf of European Society of Clinical Microbiology and Infectious Diseases.

South American leaf blight (SALB) is the worst impairment for rubber tree cultivation in the Amazon. Hevea brasiliensis clones produce high latex yields but most are SALB-susceptible (SS). So, resistance has been searched in related species. We selected defense related genes among those differentially expressed while contrasting SALB-resistant (SR) and susceptible clones from three Hevea species. SS clones over-expressed pathways responsive to salicylic (SA) and also jasmonic acid, thus entering a self-harmful cycle. SR clones displayed tightly coordinated SA-responsive and stress-control pathways. Polymorphism in the selected gene sequences supported SS × SR clone divergence. Made available in DSpace on 2020-05-28T04:43:25Z (GMT). No. of bitstreams: 1 Differential-expression-and-structural.pdf: 10306207 bytes, checksum: dbc6db81b21fc50c716e2e5cfc1ad556 (MD5) Previous issue date: 2020

WOS: 000443307900005, PubMed ID: 30155366, Background. Extraintestinal pathogenic Escherichia coli (ExPEC) is an important bacterium and responsible for many bloodstream infections, including urinary tract infections and even fatal bacteremia. The aim of this research was to investigate whether ExPEC strains isolated from Turkish blood cultures have a relationship between 16S rRNA based phylogenetic clusters and antibiotic resistance profiles, virulence factors or clonal lineages. Methods. Phenotypically identified ExPEC blood culture isolates (n = 104) were included in this study. The 16S rRNA partial sequence analysis was performed for genotypic identification of ExPEC isolates. Antibiotic susceptibility and Extended Spectrum beta-Lactamase testing of isolates were performed. Phylogenetic classification (A, B1, B2 and D), Multi Locus Sequence Typing analysis and virulence-associated genes were investigated. Results. Based on 16S rRNA partial sequence analysis, 97 out of 104 (93.26%) ExPEC isolates were confirmed as E. coli. Ampicillin (74.22%) and cefuroxime axetil (65.97%) resistances had the highest frequencies among the ExPEC isolates. In terms of phylogenetic classification of ExPEC, D (38.14%, 37/97) was the most prevalent group after A (29.89%, 29/97), B2 (20.61%, 20/97), and B1 (11.34%, 11/97). The sequence types of the 20 ExPEC isolates belonging to the B2 phylogenetic group were analyzed by Multi Locus Sequence Typing. Ten isolates out of 20 (50.0%) were identified as ST131. The other STs were ST95 (n=1), ST14 (n=1), ST10 (n=1), ST69 (n=1), ST1722 (n=2), ST141 (n=1), ST88 (n=1), ST80 (n=1), and ST998 (n=1). Of the ST131 strains, six (60%, 6/10) represented serogroup O25. The most common virulence factor genes were serum resistance factor gene, traT (55.7%) aerobactin siderophore receptor and yersiniabactin encoding genes iutA (45.3%) and fyuA (50.5%), respectively. In addition, PAI (41.2%), iroN (23.7%), hlyA (15.4%), kpsII (13.4%), ompT (13.4%), papG (12.4%), iss (9.3%), cnf1 (7.2%), ibeA (2.06%), and sfaS (2.06%) genes were present in the ExPEC isolates. Conclusion. The 16S rRNA-based phylogenetic relationship tree analysis showed that a large cluster was present among 97 ExPEC isolates along with related reference strains. There were 21 main clusters with 32 closely related subclusters. Based on our findings, different clonal lineages of ExPEC can display different antibiotic susceptibilities and virulence properties. We also concluded that virulence factors were not distributed depending on phylogenetic groups (A, B1, B2, and D). The ExPEC isolates belonging to the same phylogenetic group and sequence type could display different resistance and virulence characteristics., Scientific Research Unit of the Istanbul University [FUA-2016-20386, BYP-2016-2305], This study was supported by The Scientific Research Unit of the Istanbul University with project ID: FUA-2016-20386 and ID: BYP-2016-2305. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Embriões clones interespécie vêm sendo produzidos por diferentes grupos de pesquisa, com relativo sucesso em algumas espécies. Oócitos bovinos maturados in vitro e enucleados por micromanipulação foram utilizados em três experimentos como citoplasma receptor na transferência nuclear de fibroblastos ovinos, caprinos e suínos. Os fibroblastos foram cultivados até a terceira passagem antes de serem congelados e utilizados. A eletrofusão foi induzida pela aplicação de um pulso de 20 V durante 45 ms. A ativação foi realizada com 5 mM de ionomicina e 2 mM de 6DMAP. Embriões NTSC bovinos, NTSCi caprinos e ovinos foram cultivados em meio SOF, e embriões NTSCi suínos foram cultivados em NCSU23. As taxas de fusão dos complexos reconstruídos com células bovinas não diferiram daquelas observadas com células ovinas (88,2%), caprinas (74,1%) e suínas (79,4%). As taxas de clivagem nos grupos NTSCi ovino (60,3%), caprino (68,4%) e suína (57,1%) não diferiram dos grupos controles NTSC bovino. A taxa de blastocisto observada nos embriões NTSCi ovinos (10,3%) foi semelhante à taxa observada no grupo NTSC bovino (12,7%). No grupo NTSCi caprino, 5,3% dos embriões chegaram ao estádio de blastocisto, enquanto que no grupo NTSCi suíno não houve desenvolvimento até o estádio de blastocisto. O citoplasma bovino foi capaz de suportar o desenvolvimento de embriões NTSCi até o estádio de blastocisto utilizando-se núcleo de fibroblatos ovinos e caprinos. Interspecies embryo clones have been produced by research groups with relative success in some species. Bovine oocytes matured in vitro and enucleated by micromanipulation were used in three experiments as recipeient cytoplasm in nuclear transfer of ovine, caprine and porcine fibroblasts. The fibroblasts were cultivated until the third passage before being frozen and used. The electrofusion was induced by an application of a 20V pulse during 45 ms. The activation was done with 5 mM ionomycin and subsequently 2 mM 6DMAP. NTSC bovine embryos, NTSCi caprine and ovine embryos were cultivated in SOF medium and NTSCi porcine embryos were cultivated in NCSU23 medium. The fusion rates of the reconstructed complexes with bovine cells did not differ from those observed with ovine cells (88.2%), caprine cells (74.1%) and porcine cells (79.4%). The cleavage rates in ovine (60.3%), caprine (68.4%) and porcine (57.1%) NTSCi groups did not differ from the control group NTSC bovine. The blastocyst rate observed in the group of NTSCi ovine embryos (10.3%) was similar to the group of NTSC bovine embryos (12.7%). In NTSCi caprine embryos, 5.3% of the embryos developed up to the blastocyst stage, while in the NTSCi porcine group there was no development up to the blastocyst stage. In conclusion, the bovine cytoplasm was able to support the embryo development in NTSCi up to the blastocyst stage using ovine and caprine fibroblasts as donor cells.

Understanding the mechanisms of drug resistance in Plasmodium vivax, the parasite that causes the most widespread form of human malaria, is complicated by the lack of a suitable long-term cell culture system for this parasite. In contrast to P. falciparum, which can be more readily manipulated in the laboratory, insights about parasite biology need to be inferred from human studies. Here we analyze the genomes of parasites within 10 human P. vivax infections from the Peruvian Amazon. Using next-generation sequencing we show that some P. vivax infections analyzed from the region are likely polyclonal. Despite their polyclonality we observe limited parasite genetic diversity by showing that three or fewer haplotypes comprise 94% of the examined genomes, suggesting the recent introduction of parasites into this geographic region. In contrast we find more than three haplotypes in putative drug-resistance genes, including the gene encoding dihydrofolate reductase-thymidylate synthase and the P. vivax multidrug resistance associated transporter, suggesting that resistance mutations have arisen independently. Additionally, several drug-resistance genes are located in genomic regions with evidence of increased copy number. Our data suggest that whole genome sequencing of malaria parasites from patients may provide more insight about the evolution of drug resistance than genetic linkage or association studies, especially in geographical regions with limited parasite genetic diversity.

High-throughput molecular screening is required to investigate the origin and diffusion of antimicrobial resistance in pathogen outbreaks. The most frequent cause of human infection is Escherichia coli, which is dominated by sequence type 131 (ST131), a set of rapidly radiating pandemic clones. The highly infectious clades of ST131 originated firstly by a mutation enhancing virulence and adhesion. Secondly, single-nucleotide polymorphisms occurred enabling fluoroquinolone-resistance, which is near-fixed in all ST131. Thirdly, broader resistance through beta-lactamases has been gained and lost frequently, symptomatic of conflicting environmental selective effects. This flexible approach to gene exchange is worrying and supports the proposition that ST131 will develop an even wider range of plasmid and chromosomal elements promoting antimicrobial resistance. To stymie ST131, deep genome sequencing is required to understand the origin, evolution and spread of antimicrobial resistance genes. Phylogenetic methods that decipher past events can predict future patterns of virulence and transmission based on genetic signatures of adaptation and gene exchange. Both the effect of partial antimicrobial exposure and cell dormancy caused by variation in gene expression may accelerate the development of resistance. High-throughput sequencing can decode measurable evolution of cell populations within patients associated with systems-wide changes in gene expression during treatments. A multi-faceted approach can enhance assessment of antimicrobial resistance in E. coli ST131 by examining transmission dynamics between hosts to achieve a goal of pre-empting resistance before it emerges by optimising antimicrobial treatment protocols., 18 pages, 1 figure

Kava (Piper methysticum) is a major cash crop in the Pacific. The aim of this study was to assess genetic variation among 103 accessions of kava using SSRs and DArTs. Genetic structure was determined using clustering analyses (WPGMA) and principal coordinate analyses (PCA). Thirteen SSR primers and 75 DArT markers were found polymorphic, and the two types of markers generated similar clustering patterns. Genetic distances ranged from 0 to 0.65 with an average of 0.24 using SSRs and from 0 to 0.64 with an average of 0.24 using DArT. Eleven genotypes were identified with SSR while 28 genotypes were identified with DArT markers. By combining the two sets of markers, a total of only 30 distinct genotypes were observed. In the Vanuatu archipelago, noble cultivars originating from different islands clustered together within a very narrow genetic base despite their diversity of morphotypes. SSR and DArT fingerprints allowed the identification of kava cultivars unsuitable for consumption, so called two-days, and clearly differentiated the wild types classified as P. methysticum var. wichmannii from the cultivars as var. methysticum. Molecular data reveals that all noble cultivars evolved by the predominance of clonal selection. Although they are represented by clearly distinct morphotypes, these cultivars are genetically vulnerable and their potential to adapt to forthcoming changes is limited. These newly developed markers provide high resolution and will be useful for kava diversity analyses and quality assessment.

The fungal pathogen Cochliobolus carbonum (anamorph, Bipolaris zeicola) causes Northern Leaf Spot, leading to a ubiquitous and devastating foliar disease of corn in Yunnan Province, China. Asexual spores (conidia) play a major role in both epidemics and pathogenesis of Northern Leaf Spot, but the molecular mechanism of conidiation in C. carbonum has remained elusive. Here, using a map-based cloning strategy, we cloned a single dominant gene, designated as BZcon1 (for Bipolaris zeicola conidiation), which encodes a predicted unknown protein containing 402 amino acids, with two common conserved SANT/Myb domains in N-terminal. The BZcon1 knockout mutant completely lost the capability to produce conidiophores and conidia but displayed no effect on hyphal growth and sexual reproduction. The introduced BZcon1 gene fully complemented the BZcon1 null mutation, restoring the capability for sporulation. These data suggested that the BZcon1 gene is essential for the conidiation of C. carbonum.

Genomes of 82 Acinetobacter baumannii global clones 1 (GC1) and 2 (GC2) isolates were sequenced and different forms of the locus predicted to direct synthesis of the outer core (OC) of the lipooligosaccharide were identified. OCL1 was in all GC2 genomes, whereas GC1 isolates carried OCL1, OCL3 or a new locus, OCL5. Three mutants in which an insertion sequence (ISAba1 or ISAba23) interrupted OCL1 were identified. Isolates with OCL1 intact produced only lipooligosaccharide, while the mutants produced lipooligosaccharide of reduced molecular weight. Thus, the assignment of the OC locus as that responsible for the synthesis of the OC is correct.

The complete coding sequence of Wild Argali ISG15 cDNA was generated by rapid amplification of cDNA ends. The ISG15 cDNA was 642 bp with an open reading frame of 474 bp, which encoded a 17.47 kDa protein composed of 157 amino acids. Its amino acid sequence shared 97.9%, 80.8%, 91.4%, 94.3%, 78.3% identity with those of ISG15cDNA from Ovis aries (accession no. NM001009735.1), Capra hircus (accession no. HQ329186.1), Bos taurus (accession no. BC102318.1), Bubalus bubalis (accession no. HM543269.1), and Sus scrofa (accession no. EU647216.1), respectively. The entire coding sequence was inserted into the pET-28a vector and expressed in E. coli. The recombinant protein corresponded to the expected molecular mass of 25 kDa as judged by SDS-PAGE, and it was detected in the bacterial inclusion bodies. The expressed protein could be purified by Ni(2+) chelate affinity chromatography and the results from the lymphocyte proliferation test showed that the product could stimulate lymphocyte proliferation very well (p0.05), which further confirmed its biological activity.

The availability of genomic data in the last decade relating to different aphid species has allowed the analysis of the genomic variability occurring among such species, whereas intra-specific variability has hitherto very largely been neglected. In order to analyse the intra-genomic variability in the peach potato aphid, Myzus persicae, comparative analyses were performed revealing several clone-specific gene duplications, together with numerous deletions/rearrangements. Our comparative approach also allowed us to evaluate the synteny existing between the two M. persicae clones tested and between the peach potato aphid and the pea aphid, Acyrthosiphon pisum. Even if part of the observed rearrangements are related to a low quality of some assembled contigs and/or to the high number of contigs present in these aphid genomes, our evidence reveals that aphid clones are genetically more different than expected. These results suggest that the choice of performing genomes sequencing combining different biotypes/populations, as revealed in the case of the soybean aphid, Aphis glycines, is unlikely to be very informative in aphids. Interestingly, it is possible that the holocentric nature of aphid chromosomes favours genome rearrangements that can be successively inherited transgenerationally via the aphid&rsquo, s apomictic (parthenogenetic) mode of reproduction. Lastly, we evaluated the structure of the cluster of genes coding for the five histones (H1, H2A, H2B, H3 and H4) in order to better understand the quality of the two M. persicae genomes and thereby to improve our knowledge of this functionally important gene family.

Due to the misuse of chemical pesticides, small green leafhoppers (Empoasca onukii Matsuda) have developed resistance to pesticides, thereby posing a serious problem to the tea industry. Glutathione S-transferases (GSTs) are an important family of enzymes that are involved in pesticide resistance in Empoasca onukii Matsuda. Empoasca onukii GST sigma 1 (EoGSTs1, GenBank: MK443501) is a member of the GST family. In this study, the full-length cDNA of EoGSTs1 was cloned by reverse transcription polymerase chain reaction (qPCR), and its taxonomic identity was examined. Furthermore, we performed bioinformatics and phylogenetic analyses of the gene and structural and functional domain prediction of the protein. The results demonstrate that EoGSTS1 belongs to the Sigma family of GSTs; the full-length EoGSTs1 cDNA is 841 bp with a 624-bp coding region that encodes a 23.68932-kDa protein containing 207 amino acids. The theoretical isoelectric point (IEP) was calculated to be 6.00. Phylogenetic analysis indicates that EoGSTS1 is closely related to the Sub psaltriayangi subfamily of the Cicadoidea superfamily in order Hemiptera, whereas it is distantly related to Periplaneta americana of order Blattodea. Amino acid sequence alignment of EoGSTS1 and GSTs from four other insects of order Hemiptera revealed protein sequence conservation. Tertiary structure analysis and structural domain functional predictions of the protein revealed that EoGSTS1 contains nine α helices and two β sheets with one conserved GST domain. The results of enzyme activity assay showed that recombinant EoGSTs1 (rEoGSTs1) protein had catalytic activity for substrate 1-chloro-2,4-dinitrobenzene (CDNB) and exhibited the highest activity at pH 7 and 25 °C. The Michaelis constant Km of rEoGSTs1 protein was 0.07782 ± 0.01990 mmol/L, and the maximum reaction rate Vmax was 12.15 ± 1.673 µmol/min⋅mg. Our study clarified the taxonomic identity of small green leafhopper EoGSTs1 and revealed some properties of the gene and its encoded protein sequence. According to the catalytic activity of the rEoGSTs1 enzyme on the model substrate CDNB, we infer that it functions in the degradation of exogenous substances.

Obtaining high-quality sequence continuity of complex regions of recent segmental duplication remains one of the major challenges of finishing genome assemblies. In the human and mouse genomes, this was achieved by targeting large-insert clones using costly and laborious capillary-based sequencing approaches. Sanger shotgun sequencing of clone inserts, however, has now been largely abandoned, leaving most of these regions unresolved in newer genome assemblies generated primarily by next-generation sequencing hybrid approaches. Here we show that it is possible to resolve regions that are complex in a genome-wide context but simple in isolation for a fraction of the time and cost of traditional methods using long-read single molecule, real-time (SMRT) sequencing and assembly technology from Pacific Biosciences (PacBio). We sequenced and assembled BAC clones corresponding to a 1.3-Mbp complex region of chromosome 17q21.31, demonstrating 99.994% identity to Sanger assemblies of the same clones. We targeted 44 differences using Illumina sequencing and find that PacBio and Sanger assemblies share a comparable number of validated variants, albeit with different sequence context biases. Finally, we targeted a poorly assembled 766-kbp duplicated region of the chimpanzee genome and resolved the structure and organization for a fraction of the cost and time of traditional finishing approaches. Our data suggest a straightforward path for upgrading genomes to a higher quality finished state. © 2014 Huddleston et al.