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July 7, 2019  |  

Nanoarrays on passivated aluminum surface for site-specific immobilization of biomolecules

The rapid development of biosensing platforms for highly sensitive and specific detection raises the desire of precise localization of biomolecules onto various material surfaces. Aluminum has been strategically employed in the biosensor system due to its compatibility with CMOS technology and its optical and electrical properties such as prominent propagation of surface plasmons. Herein, we present an adaptable method for preparation of carbon nanoarrays on aluminum surface passivated with poly(vinylphosphonic acid) (PVPA). The carbon nanoarrays were defined by means of electron beam induced deposition (EBID) and they were employed to realize site-specific immobilization of target biomolecules. To demonstrate the concept, selective streptavidin/neutravidin immobilization on the carbon nanoarrays was achieved through protein physisorption with a significantly high contrast of the carbon domains over the surrounding PVPA-modified aluminum surface. By adjusting the fabrication parameters, local protein densities could be varied on similarly sized nanodomains in a parallel process. Moreover, localization of single 40 nm biotinylated beads was achieved by loading them on the neutravidin-decorated nanoarrays. As a further demonstration, DNA polymerase with a streptavidin tag was bound to the biotin-beads that were immobilized on the nanoarrays and in situ rolling circle amplification (RCA) was subsequently performed. The observation of organized DNA arrays synthesized by RCA verified the nanoscale localization of the enzyme with retained biological activity. Hence, the presented approach could provide a flexible and universal avenue to precise localizing various biomolecules on aluminum surface for potential biosensor and bioelectronic applications.

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July 7, 2019  |  

Complete genome sequence of soil actinobacteria Streptomyces cavourensis TJ430.

A new actinobacteria Streptomyces cavourensis TJ430 was isolated from the mountain soil collected from the southwest of China. In previous study, TJ430 showed striking bactericidal activities and strong ability of antibiotic production. Here, we report complete genome of this bacterium, consisting of 7.6?Mb linear chromosome and 0.2?Mb plasmids. It was predicted 6450 genes in chromosome and 225 genes in plasmids, as well as 12 gene islands in chromosome. Abundant genes have predicted functions in antibiotic metabolism and stress resistance. A whole-genome comparison of S. cavourensis TJ430, S. coelicolor A3(2), and S. lividans 66 indicates that TJ430 has a relatively high degree of strain specificity. The 16S rRNA phylogenetic tree shows the high identities (99.79%) of TJ430 with S. cavourensis DSM40300. TJ430 is a new and rare Streptomyces species, and analysis of its genome helps us to better understand primary metabolism mechanism of this isolate, as well as the evolutionary biology.© 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

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July 7, 2019  |  

Near-complete genome sequence of Ralstonia solanacearum T523, a phylotype I tomato phytopathogen isolated from the Philippines.

Ralstonia solanacearum strain T523 is the major phytopathogen causing tomato bacterial wilt in the Philippines. Here, we report the complete chromosome and draft megaplasmid genomes with predicted gene inventories supporting rhizo- sphere processes, extensive plant virulence effectors, and the production of bioac- tive signaling metabolites, such as ralstonin, micacocidin, and homoserine lactone.

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July 7, 2019  |  

Complete genome sequence of the multidrug-resistant neonatal meningitis Escherichia coli serotype O75:H5:K1 strain mcjchv-1 (NMEC-O75).

Neonatal meningitis Escherichia coli (NMEC) is the second leading cause of neonatal bacterial meningitis worldwide. We report the genome sequence of the multidrug-resistant NMEC serotype O75:H5:K1 strain mcjchv-1, which resulted in an infant’s death. The O75 serogroup is rare among NMEC isolates; therefore, this strain is considered an emergent pathogen.

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July 7, 2019  |  

New variant of multidrug-resistant Salmonella enterica serovar Typhimurium associated with invasive disease in immunocompromised patients in Vietnam.

Nontyphoidal Salmonella (NTS), particularly Salmonella enterica serovar Typhimurium, is among the leading etiologic agents of bacterial enterocolitis globally and a well-characterized cause of invasive disease (iNTS) in sub-Saharan Africa. In contrast, S Typhimurium is poorly defined in Southeast Asia, a known hot spot for zoonotic disease with a recently described burden of iNTS disease. Here, we aimed to add insight into the epidemiology and potential impact of zoonotic transfer and antimicrobial resistance (AMR) in S Typhimurium associated with iNTS and enterocolitis in Vietnam. We performed whole-genome sequencing and phylogenetic reconstruction on 85 human (enterocolitis, carriage, and iNTS) and 113 animal S Typhimurium isolates isolated in Vietnam. We found limited evidence for the zoonotic transmission of S Typhimurium. However, we describe a chain of events where a pandemic monophasic variant of S Typhimurium (serovar I:4,[5],12:i:- sequence type 34 [ST34]) has been introduced into Vietnam, reacquired a phase 2 flagellum, and acquired an IncHI2 multidrug-resistant plasmid. Notably, these novel biphasic ST34 S Typhimurium variants were significantly associated with iNTS in Vietnamese HIV-infected patients. Our study represents the first characterization of novel iNTS organisms isolated outside sub-Saharan Africa and outlines a new pathway for the emergence of alternative Salmonella variants into susceptible human populations.IMPORTANCESalmonella Typhimurium is a major diarrheal pathogen and associated with invasive nontyphoid Salmonella (iNTS) disease in vulnerable populations. We present the first characterization of iNTS organisms in Southeast Asia and describe a different evolutionary trajectory from that of organisms causing iNTS in sub-Saharan Africa. In Vietnam, the globally distributed monophasic variant of Salmonella Typhimurium, the serovar I:4,[5],12:i:- ST34 clone, has reacquired a phase 2 flagellum and gained a multidrug-resistant plasmid to become associated with iNTS disease in HIV-infected patients. We document distinct communities of S Typhimurium and I:4,[5],12:i:- in animals and humans in Vietnam, despite the greater mixing of these host populations here. These data highlight the importance of whole-genome sequencing surveillance in a One Health context in understanding the evolution and spread of resistant bacterial infections. Copyright © 2018 Mather et al.

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July 7, 2019  |  

Overview of the germline and expressed repertoires of the TRB genes in Sus scrofa.

The a/ß T cell receptor (TR) is a complex heterodimer that recognizes antigenic peptides and binds to major histocompatibility complex (MH) molecules. Both a and ß chains are encoded by different genes localized on two distinct chromosomal loci: TRA and TRB. The present study employed the recent release of the swine genome assembly to define the genomic organization of the TRB locus. According to the sequencing data, the pig TRB locus spans approximately 400 kb of genomic DNA and consists of 38 TRBV genes belonging to 24 subgroups located upstream of three in tandem TRBD-J-C clusters, which are followed by a TRBV gene in an inverted transcriptional orientation. Comparative analysis confirms that the general organization of the TRB locus is similar among mammalian species, but the number of germline TRBV genes varies greatly even between species belonging to the same order, determining the diversity and specificity of the immune response. However, sequence analysis of the TRB locus also suggests the presence of blocks of conserved homology in the genomic region across mammals. Furthermore, by analysing a public cDNA collection, we identified the usage pattern of the TRBV, TRBD, and TRBJ genes in the adult pig TRB repertoire, and we noted that the expressed TRBV repertoire seems to be broader and more diverse than the germline repertoire, in line with the presence of a high level of TRBV gene polymorphisms. Because the nucleotide differences seems to be principally concentrated in the CDR2 region, it is reasonable to presume that most T cell ß-chain diversity can be related to polymorphisms in pig MH molecules. Domestic pigs represent a valuable animal model as they are even more anatomically, genetically and physiologically similar to humans than are mice. Therefore, present knowledge on the genomic organization of the pig TRB locus allows the collection of increased information on the basic aspects of the porcine immune system and contributes to filling the gaps left by rodent models.

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July 7, 2019  |  

Allele-level KIR genotyping of more than a million samples: Workflow, algorithm, and observations.

The killer-cell immunoglobulin-like receptor (KIR) genes regulate natural killer cell activity, influencing predisposition to immune mediated disease, and affecting hematopoietic stem cell transplantation (HSCT) outcome. Owing to the complexity of the KIR locus, with extensive gene copy number variation (CNV) and allelic diversity, high-resolution characterization of KIR has so far been applied only to relatively small cohorts. Here, we present a comprehensive high-throughput KIR genotyping approach based on next generation sequencing. Through PCR amplification of specific exons, our approach delivers both copy numbers of the individual genes and allelic information for every KIR gene. Ten-fold replicate analysis of a set of 190 samples revealed a precision of 99.9%. Genotyping of an independent set of 360 samples resulted in an accuracy of more than 99% taking into account consistent copy number prediction. We applied the workflow to genotype 1.8 million stem cell donor registry samples. We report on the observed KIR allele diversity and relative abundance of alleles based on a subset of more than 300,000 samples. Furthermore, we identified more than 2,000 previously unreported KIR variants repeatedly in independent samples, underscoring the large diversity of the KIR region that awaits discovery. This cost-efficient high-resolution KIR genotyping approach is now applied to samples of volunteers registering as potential donors for HSCT. This will facilitate the utilization of KIR as additional selection criterion to improve unrelated donor stem cell transplantation outcome. In addition, the approach may serve studies requiring high-resolution KIR genotyping, like population genetics and disease association studies.

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July 7, 2019  |  

Reference genes for RT-qPCR normalisation in different tissues, developmental stages and stress conditions of Hypericum perforatum

Hypericum perforatum is a widely known medicinal herb used mostly as a remedy for depression because of its abundant secondary metabolites. Quantitative real-time PCR (qRT-PCR) is an optimized method for the efficient and reliable quantification of gene expression studies. In general, reference genes are used in qRT-PCR analysis because of their known or suspected housekeeping roles. However, their expression level cannot be assumed to remain stable under all possible experimental conditions. Thus, the identification of high quality reference genes is very necessary for the interpretation of qRT-PCR data. In this study, we investigated the expression of fourteen candidate genes, including nine housekeeping genes and five potential candidate genes. Additionally, the HpHYP1 gene, belonging to the PR-10 family associated with stress control, was used for validation of the candidate reference genes. Three programs were applied to evaluate the gene expression stability across four different plant tissues, three developmental stages and a set of abiotic stress and hormonal treatments. The candidate genes showed a wide range of Ct values in all samples, indicating that they are differentially expressed. Integrating all of the algorithms and evaluations, ACT2 and TUB-ß were the most stable combination overall and for different developmental stages samples. Moreover, ACT2 and EF1-a were considered to be the two most applicable reference genes for different tissues and for stress samples. Majority of the conventional housekeeping genes exhibited better than the potential reference genes. The obtained results will contribute to improving credibility of standardization and quantification of transcription levels in future expression research of H. perforatum.

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