Junhyung Park

Detection and Genotyping of Mycobacterium Species from Clinical Isolates and Specimens by Oligonucleotide Array

ABSTRACT Identification of pathogenic Mycobacterium species is important for a successful diagnosis of mycobacteriosis. The purpose of this study was to develop an oligonucleotide array which could detect and differentiate mycobacteria to the species level by using the internal transcribed spacer (ITS) sequence. Using a genus-specific probe and 20 species-specific probes including two M. avium-intracellulare complex (MAC)-specific probes, we have developed an ITS-based oligonucleotide array for the rapid and reliable detection and discrimination of M. tuberculosis, MAC, M. fortuitum, M. chelonae, M. abscessus, M. kansasii, M. gordonae, M. scrofulaceum, M. szulgai, M. vaccae, M. xenopi, M. terrae, M. flavescens, M. smegmatis, M. malmoense, M. simiae, M. marinum, M. ulcerans, M. gastri, and M. leprae. All mycobacteria were hybridized with a genus-specific probe (PAN-03) for detection of the genus Mycobacterium. Mycobacterial species were expected to show a unique hybridization pattern with species-specific probes, except for M. marinum and M. ulcerans, which were not differentiated by ITS-based probe. Among the species-specific probes, two kinds of species-specific probes were designed for MAC in which there were many subspecies. The performance of the oligonucleotide array assay was demonstrated by using 46 reference strains, 149 clinical isolates, and 155 clinical specimens. The complete procedure (DNA extraction, PCR, DNA hybridization, and scanning) was carried out in 4.5 h. Our results indicated that the oligonucleotide array is useful for the identification and discrimination of mycobacteria from clinical isolates and specimens in an ordinary clinical laboratory.

Antimicrobial peptides in the centipede Scolopendra subspinipes mutilans

The centipede Scolopendra subspinipes mutilans is an environmentally beneficial and medically important arthropod species. Although this species is increasingly applied as a reliable source of new antimicrobial peptides, the transcriptome of this species is a prerequisite for more rational selection of antimicrobial peptides. In this report, we isolated total RNA from the whole body of adult centipedes, S. subspinipes mutilans, that were nonimmunized and immunized against Escherichia coli, and we generated a total of 77,063 pooled contigs and singletons using high-throughput sequencing. To screen putative antimicrobial peptides, in silico analyses of the S. subspinipes mutilans transcriptome were performed based on the physicochemical evidence of length, charge, isoelectric point, and in vitro and in vivo aggregation scores together with the existence of continuous antimicrobial peptide stretches. Moreover, we excluded some transcripts that showed similarity with both previously known antimicrobial peptides and the human proteome, had a proteolytic cleavage site, and had downregulated expression compared with the nonimmunized sample. As a result, we selected 17 transcripts and tested their antimicrobial activity with a radial diffusion assay. Among them, ten synthetic peptides experimentally showed antimicrobial activity against microbes and no toxicity to mouse erythrocytes. Our results provide not only a useful set of antimicrobial peptide candidates and an efficient strategy for novel antimicrobial peptide development but also the transcriptome data of a big centipede as a valuable resource.

Transcriptome Analysis Revealed Changes of Multiple Genes Involved in Haliotis discus hannai Innate Immunity during Vibrio parahemolyticus Infection

Abalone (Haliotis discus hannai) is one of the most valuable marine aquatic species in Korea, Japan and China. Tremendous exposure to bacterial infection is common in aquaculture environment, especially by Vibrio sp. infections. It’s therefore necessary and urgent to understand the mechanism of H. discus hannai host defense against Vibrio parahemolyticus infection. However studies on its immune system are hindered by the lack of genomic resources. In the present study, we sequenced the transcriptome of control and bacterial challenged H. discus hannai tissues. Totally, 138 MB of reference transcriptome were obtained from de novo assembly of 34 GB clean bases from ten different libraries and annotated with the biological terms (GO and KEGG). A total of 10,575 transcripts exhibiting the differentially expression at least one pair of comparison and the functional annotations highlight genes related to immune response, cell adhesion, immune regulators, redox molecules and mitochondrial coding genes. Mostly, these groups of genes were dominated in hemocytes compared to other tissues. This work is a prerequisite for the identification of those physiological traits controlling H. discus hannai ability to survive against Vibrio infection.

A novel semantic framework for toxicogenomics

Complex biological data stored in bioinformatics databases often require additional knowledge to specify and constrain the values held in a particular database. One method of capturing knowledge within bioinformatics applications and databases is the use of ontologies. Existing ontologies can model components of environmental risk and corresponding events, but cannot synthesize them or describe the broader framework of ideas used to conceptualize and study environmental risk. Environmental Risk Ontology (ERO) aims to synthesize and describe a framework for investigating environmental risk. ERO is intended as a metadata framework for data and literature curation and data mining. In this review, we summarize and describe the basic public databases available for toxicogenomics and present a novel approach that uses a semantic knowledge base — which also organizes the literature in a growing, comprehensive bibliographic database — concerning environmental risk and toxicogenomics.

Decision supporting frame to estimate chronic exposure suspicion to VOC chemicals using mixed statistical model

In this paper, we examine the model for a chemical exposure decision support algorithm. Our purpose is to suggest the model frame to describe possibility of exposure with low-dose VOC chemicals for long time under normal circumstances at working place. Forensic rhetoric terms, non-exclusion exposure suspicion (NES) and exclusion exposure suspicion (EES), were defined and various statistical methods were combined basis of Bayesian approach. Decisiontree (DT) methods of linear discriminant analysis (LDA), quadratic discriminant analysis (QDA), and naïve Bayes model were evaluated to classify 3 VOCs (toluene, xylene, and ehtybenzene) by means of the results of urinary test, gene expression and methylation expression experiments. Overall procedure is conducted by leave-one-out cross-validation that error rate of NES resulted in 11%.

SpiroESTdb: a transcriptome database and online tool for sparganum expressed sequences tags

BackgroundSparganum (plerocercoid of Spirometra erinacei) is a parasite that possesses the remarkable ability to survive by successfully modifying its physiology and morphology to suit various hosts and can be found in various tissues, even the nervous system. However, surprisingly little is known about the molecular function of genes that are expressed during the course of the parasite life cycle. To begin to decipher the molecular processes underlying gene function, we constructed a database of expressed sequence tags (ESTs) generated from sparganum.FindingsSpiroESTdb is a web-based information resource that is built upon the annotation and curation of 5,655 ESTs data. SpiroESTdb provides an integrated platform for expressed sequence data, expression dynamics, functional genes, genetic markers including single nucleotide polymorphisms and tandem repeats, gene ontology and KEGG pathway information. Moreover, SpiroESTdb supports easy access to gene pages, such as (i) curation and query forms, (ii) insilico expression profiling and (iii) BLAST search tools. Comprehensive descriptions of the sparganum content of all sequenced data are available, including summary reports. The contents of SpiroESTdb can be viewed and downloaded from the web (http://pathod.cdc.go.kr/spiroestdb).ConclusionsThis integrative web-based database of sequence data, functional annotations and expression profiling data will serve as a useful tool to help understand and expand the characterization of parasitic infections. It can also be used to identify potential industrial drug targets and vaccine candidate genes.

Transcriptome profiling and in silico analysis of the antimicrobial peptides of the grasshopper Oxya chinensis sinuosa.

Antimicrobial peptides/proteins (AMPs) are present in all types of organisms, from microbes and plants to vertebrates and invertebrates such as insects. The grasshopper Oxya chinensis sinuosa is an insect species that is widely consumed around the world for its broad medicinal value. However, the lack of available genetic information for this species is an obstacle to understanding the full potential of its AMPs. Analysis of the O. chinensis sinuosa transcriptome and expression profile is essential for extending the available genetic information resources. In this study, we determined the whole-body transcriptome of O. chinensis sinuosa and analyzed the potential AMPs induced by bacterial immunization. A high-throughput RNA-Seq approach generated 94,348 contigs and 66,555 unigenes. Of these unigenes, 36,032 (54.14%) matched known proteins in the NCBI database in a BLAST search. Functional analysis demonstrated that 38,219 unigenes were clustered into 5,499 gene ontology terms. In addition, 26 cDNAs encoding novel AMPs were identified by an in silico approach using public databases. Our transcriptome dataset and AMP profile greatly improve our understanding of O. chinensis sinuosa genetics and provide a huge number of gene sequences for further study, including genes of known importance and genes of unknown function.

Semantic data integration for toxicogenomic laboratory experiment management systems

Mircoarray technology leads rapid screening of differential expressed gene (DEG) from various kinds of chemical exposes. Using toxicogenomics for the risk assessment, various and heterogeneous data are contributed to each step, such as genome sequence, genotype, gene expression, phenotype, disease information etc. Accordingly ontology-based knowledge representations could prove to be successful in providing the semantics for the relationships of the drugs to a wide body of target information, a standardized annotation, integration and exchange of data. To derive actual roles of the DEGs, it is essentially required to construct interactions among DEGs and to link the known information of diseases. We depict reconstruction of semantic relationship among chemical, disease, and DEGs by using omics-data and laboratory experiment raw data in constructed toxicogenomic meta database. Omics- and experimental data are able to be easily uploaded and connected to the already constructed data network. This semantic data integration may represent the chemical-specific marker and target disease by integrated toxicogenomic data including complex expression profiles and experimental raw data. We expect that this system shows early promise in helping bridge the gap between pathophysiological processes and their molecular determinants.

Web-based Research Assistant Tools for Analysis of Microbial Diversity

The study of available genotypes (biodiversity analysis) in bacterial communities is of growing importance in several fields such as ecology, environmental technology, clinical diagnostics, etc. These culture-independent genotyping techniques, especially amplifying 16S rRNA genes, attempt to overcome some shortcomings of conventional cultivation method. Biodiversity analysis based on molecular technique were laborious for base-calling chromatogram, trimming primer sites, correcting strand directions, electing representative operation taxonomic units (OTU), etc. Also, biologists wanted intuitively to confirm results of the above processes. For making up these demands, we developed the web application based on Folder-Process-Filter (FPF) modeling with correspondence to classical Model-View-Controller model. The model of web application leads to keep virtues of simplicity and directness for development and management of the stepwise web interfaces. The web application was developed in Perl and CGI on Linux workstation. It can be freely accessed from http://home.pusan.ac.kr/～genome/tools/rat.htm.

Transcriptome analysis of olive flounder (Paralichthys olivaceus) head kidney infected with moderate and high virulent strains of infectious viral hemorrhagic septicaemia virus (VHSV)

&NA; Olive flounder (Paralichthys olivaceus) is one of the most valuable marine aquatic species in South Korea and faces tremendous exposure to the viral hemorrhagic septicemia virus (VHSV). Given the growing importance of flounder, it is therefore essential to understand the host defense of P. olivaceus against VHSV infection, but studies on its immune mechanism are hindered by the lack of genomic resources. In this study, the P. olivaceus was infected with disease‐causing VHSV isolates, ADC‐VHS2012‐11 and ADC‐VHS2014‐5 which showed moderate virulent (20% mortality) and high virulent (65% mortality), in order to investigate the effect of difference in pathogenicity in head kidney during 1, 3, 7 days of post‐infection using Illumina sequencing. After removing low‐quality sequences, we obtained 144,933,160 high quality reads from thirty‐six libraries which were further assembled into 53,384 unigenes with an average length of 563 bp with a range of 200 to 9605 bp. Transcriptome annotation revealed that 30,475 unigenes with a cut‐off e‐value of 10−5 were functionally annotated. In total, 10,046 unigenes were clustered into 26 functional categories by searching against the eggNOG database, and 22,233 unigenes to 52 GO terms. In addition, 12,985 unigenes were grouped into 387 KEGG pathways. Among the 13,270 differently expressed genes, 6578 and 6692 were differentially expressed only in moderate and high virulent, respectively. Based on our sequence analysis, many candidate genes with fundamental roles in innate immune system including, pattern recognition receptors (TLRs & RLRs), Mx, complement proteins, lectins, and cytokines (chemokines, IFN, IRF, IL, TRF) were differentially expressed. Furthermore, GO enrichment analysis for these genes revealed gene response to defense response to virus, apoptotic process and transcription factor activity. In summary, this study identifies several putative immune pathways and candidate genes deserving further investigation in the context of novel gene discovery, gene expression and regulation studies and lays the foundation for fish immunology especially in P. olivaceus against VHSV. HighlightsFirst RNA‐seq based transcriptome of moderate and high virulent VHSV‐infected P. olivaceus head kidney.The assembly contig length is more compared to the other olive flounder transcriptome published so far.Genes involved in innate immune response were showed significantly differentially upregulated during day 1 of high virulent and day 3 of moderate virulent.This de novo transcriptome provides useful information for evaluating the molecular mechanisms underlying host‐virus interaction.