BACKGROUND: Next-generation sequencing (NGS) can detect many more microorganisms of a microbiome than traditional methods. This study aimed to analyze the vaginal microbiomes of Korean women by using NGS that included bacteria and other microorganisms. The NGS results were compared with the results of other assays, and NGS was evaluated for its feasibility for predicting vaginitis. METHODS: In total, 89 vaginal swab specimens were collected. Microscopic examinations of Gram staining and microbiological cultures were conducted on 67 specimens. NGS was performed with GS junior system on all of the vaginal specimens for the 16S rRNA, internal transcribed spacer (ITS), and Tvk genes to detect bacteria, fungi, and Trichomonas vaginalis. In addition, DNA probe assays of the Candida spp., Gardnerella vaginalis, and Trichomonas vaginalis were performed. Various predictors of diversity that were obtained from the NGS data were analyzed to predict vaginitis. RESULTS: ITS sequences were obtained in most of the specimens (56.2%). The compositions of the intermediate and vaginitis Nugent score groups were similar to each other but differed from the composition of the normal score group. The fraction of the Lactobacillus spp. showed the highest area under the curve value (0.8559) in ROC curve analysis. The NGS and DNA probe assay results showed good agreement (range, 86.2-89.7%). CONCLUSIONS: Fungi as well as bacteria should be considered for the investigation of vaginal microbiome. The intermediate and vaginitis Nugent score groups were indistinguishable in NGS. NGS is a promising diagnostic tool of the vaginal microbiome and vaginitis, although some problems need to be resolved.
Area Under Curve ; Bacteria/*genetics/isolation & purification ; Bacterial Proteins/genetics ; Candida/*genetics/isolation & purification ; Female ; Fungal Proteins/genetics ; Gardnerella vaginalis/genetics/isolation & purification ; High-Throughput Nucleotide Sequencing ; Humans ; *Microbiota ; RNA, Ribosomal, 16S/chemistry/genetics/metabolism ; ROC Curve ; Sequence Analysis, DNA ; Trichomonas vaginalis/genetics/isolation & purification ; Vagina/*microbiology ; Vaginitis/*diagnosis/microbiology

Direct tissue imaging mass spectrometry (IMS) by matrix-assisted laser desorption ionization and time-of-flight (MALDI-TOF) mass spectrometry has become increasingly important in biology and medicine, because this technology can detect the relative abundance and spatial distribution of interesting proteins in tissues. Five thyroid cancer samples, along with normal tissue, were sliced and transferred onto conductive glass slides. After laser scanning by MALDI-TOF equipped with a smart beam laser, images were created for individual masses and proteins were classified at 200-microm spatial resolution. Based on the spatial distribution, region-specific proteins on a tumor lesion could be identified by protein extraction from tumor tissue and analysis using liquid chromatography with tandem mass spectrometry (LC-MS/MS). Using all the spectral data at each spot, various intensities of a specific peak were detected in the tumor and normal regions of the thyroid. Differences in the molecular weights of expressed proteins between tumor and normal regions were analyzed using unsupervised and supervised clustering. To verify the presence of discovered proteins through IMS, we identified ribosomal protein P2, which is specific for cancer. We have demonstrated the feasibility of IMS as a useful tool for the analysis of tissue sections, and identified the tumor-specific protein ribosomal protein P2.
Aged ; Amino Acid Sequence ; Biological Markers/*analysis ; Carcinoma/*diagnosis/metabolism/pathology ; Chromatography, High Pressure Liquid ; Cluster Analysis ; Female ; Humans ; Image Processing, Computer-Assisted ; Male ; Middle Aged ; Molecular Sequence Data ; Molecular Weight ; Phosphoproteins/analysis/metabolism ; Proteome/analysis ; Proteomics ; Reproducibility of Results ; Ribosomal Proteins/analysis/metabolism ; *Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization ; *Tandem Mass Spectrometry ; Thyroid Gland/metabolism/pathology ; Thyroid Neoplasms/*diagnosis/metabolism/pathology

OBJECTIVETo establish an animal model of gastric cancer by long-term infection of Helicobacter pylori (H.pylori) and to elucidate the pathogenesis by proteomics analysis.

METHODSFifty male Mongolian gerbils (4-5 week-old and weighted 60-100 g) were infected with H.pylori and the gastric tissues were obtained after the infection at 3, 6, 12 and 24 months. Histological changes were evaluated by H-E staining of the gastric tissue sections. Detection of H.pylori was performed by in-vitro culture of fresh gastric tissue samples, PCR amplification of H.pylori 16s rRNA and localization by silver staining. In addition, proteins extracted from gastric tissue samples were subjected to two-dimensional electrophoresis (2-DE) at various infection time points. Protein spots with increased quantity over the course of H.pylori infection were selected and analyzed by LC-MS/MS. Finally, differentially expressed proteins between human gastric cancer tissue samples and lymph nodes were analyzed by real-time RT-PCR.

RESULTSColonization of H.pylori was observed in gastric tissue of gerbils as early as 3 months after H.pylori infection, and persisted till 24 months. Pathological examination of infected animals showed various histological changes including acute gastritis, atrophic gastritis, intestinal metaplasia and gastric carcinoma. Seventy-eight differentially expressed proteins were identified by proteomics analysis, among which 36 proteins were up-regulated and 42 were down-regulated. Analyzed by LC-MS/MS, ten proteins were identified, including lactate dehydrogenase, ATP synthase, fatty acid-binding protein, COX5B, peroxiredoxin-4, peroxide reductase, transgelin, succinyl-CoA ligase, keratin and protein disulfide-isomerase A2, among which transgelin, ATP synthase and lactate dehydrogenase were highly expressed in human gastric carcinoma and lymph nodes.

CONCLUSIONSH.pylori infection induces the expression of transgelin, ATP synthase and lactate dehydrogenase, implying possible roles in the pathogenesis of gastric diseases including cancer.

Animals ; Disease Models, Animal ; Gastritis ; microbiology ; pathology ; Gerbillinae ; Helicobacter Infections ; complications ; metabolism ; Helicobacter pylori ; genetics ; Humans ; L-Lactate Dehydrogenase ; metabolism ; Male ; Metaplasia ; Microfilament Proteins ; metabolism ; Muscle Proteins ; metabolism ; Proteomics ; Proton-Translocating ATPases ; metabolism ; RNA, Ribosomal, 16S ; analysis ; Stomach Neoplasms ; metabolism ; microbiology ; Tandem Mass Spectrometry

The in vivo assembly of ribosomal subunits is a highly complex process, with a tight coordination between protein assembly and rRNA maturation events, such as folding and processing of rRNA precursors, as well as modifications of selected bases. In the cell, a large number of factors are required to ensure the efficiency and fidelity of subunit production. Here we characterize the immature 30S subunits accumulated in a factor-null Escherichia coli strain (∆rsgA∆rbfA). The immature 30S subunits isolated with varying salt concentrations in the buffer system show interesting differences on both protein composition and structure. Specifically, intermediates derived under the two contrasting salt conditions (high and low) likely reflect two distinctive assembly stages, the relatively early and late stages of the 3' domain assembly, respectively. Detailed structural analysis demonstrates a mechanistic coupling between the maturation of the 5' end of the 17S rRNA and the assembly of the 30S head domain, and attributes a unique role of S5 in coordinating these two events. Furthermore, our structural results likely reveal the location of the unprocessed terminal sequences of the 17S rRNA, and suggest that the maturation events of the 17S rRNA could be employed as quality control mechanisms on subunit production and protein translation.
Cryoelectron Microscopy ; Escherichia coli ; metabolism ; Escherichia coli Proteins ; genetics ; metabolism ; GTP Phosphohydrolases ; genetics ; metabolism ; Mass Spectrometry ; Protein Structure, Secondary ; Protein Structure, Tertiary ; RNA, Ribosomal ; analysis ; metabolism ; Ribosomal Proteins ; chemistry ; genetics ; metabolism ; Ribosome Subunits, Small, Bacterial ; chemistry ; metabolism ; ultrastructure ; Salts ; chemistry

OBJECTIVES: Streptococcus mutans (S. mutans) is the major causative bacteria in dental caries. Xylitol is an effective anticarious natural sugar substitute by inhibiting the virulence of S. mutans. However, long-term xylitol consumption leads to the emergence of the xylitol-resistant S. mutans (XR). The aim of this study is to analyze the difference of gene expression profile of xylitol-sensitive S. mutans (XS) and XR in 0.5% glucose containing TYE media, using a DNA chip. METHODS: S. mutans KCTC3065 was maintained in 0.5% glucose and 1% xylitol containing TYE media, during 30 days at 37degrees C 10% CO2 to form XR. The same procedures without xylitol were repeated for the formation of XS. Both XS and XR were cultured in 0.5% glucose with or without 1% xylitol containing TYE media overnight and total RNA was extracted. RNA from XS was labeled with Cy-3 dye as control, and XR were labeled with Cy-5 as references. DNA chip was hybridized for 18-20 h at 42degrees C. RESULTS: A total of 277 genes of DNA chip data were significantly increased or decreased in XR. There is a total of 174 XR up-regulated genes in 0.5% glucose and 1% xylitol containing TYE media, and a total of 103 down-regulated genes. For compare with results of DNA chip, 11 in up-regulated genes and 10 in down-regulated were verified by RT-PCR. The most abundant increased genes in XR were related to cell envelope, cellular processes, DNA metabolism, transcription, and protein folding and stabilization. The decreased genes in XR were related to amino acid biosynthesis, toxin production and resistance, energy metabolism, ribosomal proteins synthesis, and signal transduction. CONCLUSIONS: These results indicate that the difference of gene expression profile of XS and XR may be in existence. In particular, results of this study for XR up-regulated genes have a lot of similarities with the already published xylitol-related researches and other functional studies.
Bacteria ; Chimera ; Dental Caries ; DNA ; Energy Metabolism ; Gene Expression ; Glucose ; Oligonucleotide Array Sequence Analysis ; Protein Folding ; Ribosomal Proteins ; RNA ; Streptococcus ; Streptococcus mutans ; Sweetening Agents ; Transcriptome ; Xylitol

OBJECTIVETo study differential expression profiles of ribosomal protein (RP) genes in healthy subjects and ulcerative colitis (UC) patients of Pi-asthenic syndrome (PAS) and of dampness-heat syndrome (DHS), thus providing experimental bases for " Pi as the source of qi and blood" theory from the view of protein synthesis.

METHODSRP genes arrays were made. The mucous membrane of colon was detected in four UC patients of PAS (UC-PAS), four UC patients of DHS (UC-DHS), and four healthy subjects (N), and data analyzed using BRB-TOOL Software Package (3.9). Bioinformatics analyses were conducted in differential genes.

RESULTSLow-density RP gene chips were successfully produced, including 77 RP genes and two RP like genes (RPL26-like1 and RPL7-like1). There were twelve differential genes between UC (PAS+DHS) and N, all of which were down-regulated genes. There were nineteen differential genes between UC-DHS and N, all of which showed down-regulating tendency. There were three differential genes between UC-PAS and N, all of which were down-regulated genes. There were six differential genes between UC-PAS and UC-DHS, all of which were up-regulated genes. Cluster analysis showed that normal and UC samples of this chip can be classified according to gene expression profiles, and UC-PAS and UC-DHS can be classified by clustering. Various differential genes had a common transcription regulatory factor.

CONCLUSIONSRP genes arrays were successfully produced. RP gene expressions were down-regulated in UC-PAS and UC-DHS. Corresponding gene expression profiles were shown in N, UC-PAS and UC-DHS.

Adult ; Aged ; Case-Control Studies ; Colitis, Ulcerative ; diagnosis ; genetics ; metabolism ; Female ; Gene Expression Profiling ; Humans ; Intestinal Mucosa ; metabolism ; Male ; Medicine, Chinese Traditional ; Middle Aged ; Oligonucleotide Array Sequence Analysis ; Ribosomal Proteins ; genetics ; metabolism ; Transcriptome ; Young Adult

We identified 6 sucrose-fermenting Vibrio vulnificus strains and examined their virulence characteristics. They were all encapsulated, motile, capable of producing toxins and utilizing transferrin-bound iron, cytotoxic to cultured cells, and virulent enough to kill mice. They could be definitely identified only by genetic identification methods such as PCR, and not by conventional culture-based identification methods such as API 20E (bioMerieux, France). These results indicate that it is essential to adopt genetic approaches as early as possible in order to avoid misdiagnosis of such strains, especially in clinical situations.
Animals ; Bacterial Proteins/genetics ; Fermentation ; Mice ; RNA, Ribosomal, 16S/genetics ; Sequence Analysis, DNA ; Sucrose/*metabolism ; Vibrio vulnificus/genetics/growth & development/*pathogenicity ; Virulence

Nocardia farcinica is an emerging pathogen in immunocompromised hosts. Even though several species of Nocardia have been reported as causative pathogens of catheter-related blood stream infections (CRBSI), CRBSI caused by N. farcinica has not been reported. A 70-yr-old man with a tunneled central venous catheter (CVC) for home parenteral nutrition was admitted with fever for two days. Norcardia species was isolated from the blood through CVC and peripheral bloods and identified to N. farcinica by 16S rRNA and rpoB gene sequence analyses. This report emphasizes the rapid and correct identification of causative agents in infectious diseases in the selection of antimicrobial agents and the consideration of catheter removal.
Aged ; Bacterial Proteins/genetics/metabolism ; Blood/microbiology ; Catheter-Related Infections/*diagnosis/microbiology/nursing ; Catheterization, Central Venous/*adverse effects ; Humans ; Immunocompromised Host ; Male ; Nocardia/classification/genetics/*isolation & purification ; Nocardia Infections/*diagnosis ; Parenteral Nutrition/nursing ; Polymerase Chain Reaction ; RNA, Ribosomal, 16S/genetics/metabolism ; Sequence Analysis, DNA

Protozoan ciliates are a group of unicellular eukaryotes. The special characteristics of stop codons usage in termination of protein biosynthesis in ciliates cells makes them an ideal model to study the mechanism of stop codon recognition of polypeptides release factors. To localize the functional positions of biomolecules in ciliates cell, we constructed a macronuclear artificial chromosome containing a gene encoding red fluorescence protein (EoMAC_R) based on the structural characteristics of ciliates chromosome. Three factors, L11, eRF1a, and eRF3 that are involved in termination process of protein synthesis were colocalized in Euplotes octocarinatus cells by using novel EoMAC_R and the previously constructed EoMAC_G. The results indicated that protein synthesis mainly occurred inside the "C" shape macronucleus, suggesting that EoMAC could be a useful tool for localizing biomolecules in ciliates cell.
Chromosomes, Artificial ; Codon, Terminator ; metabolism ; Euplotes ; chemistry ; Peptide Termination Factors ; analysis ; genetics ; metabolism ; Peptides ; metabolism ; Protein Biosynthesis ; genetics ; Protozoan Proteins ; analysis ; genetics ; Ribosomal Proteins ; analysis ; genetics

BACKGROUNDThis study was undertaken to obtain differentially expressed genes related to human glioma by cDNA microarray and the characterization of a novel full-length gene.

METHODSTotal RNA was extracted form human glioma and normal brain tissue, and mRNA was used as a probe. The results of hybridization procedure were scanned with the computer system. The gene named 507E08 cone was subsequently analyzed by northern blot, bioinformatic approach, and protein expression.

RESULTSFifteen differentially expressed genes were obtained from human glioma by hybridization and scanning for four times. Northern blot analysis confirmed that the 507E08 clone was low expressed in human brain tissue and over expressed in human glioma tissues. The analysis of BLASTn and BLASTx showed that the 507E08 clone was a novel full-length gene, which codes 203 amino acid of protein and is called human ribosomal protein 14.22 gene. The nucleotide sequence had been submitted to the GenBank with the accession number of AF329277. After expression in E. coli., protein yielded a major band of apparent molecular mass 22 kDa on an SDS-PAGE gel.

CONCLUSIONScDNA microarray technology can be successfully used to identify differentially expressed genes. The novel full-length gene of human ribosomal protein 13.22 may be correlated with the development of human glioma.

Amino Acid Sequence ; Base Sequence ; Blotting, Northern ; Cloning, Molecular ; DNA, Complementary ; chemistry ; genetics ; Electrophoresis, Polyacrylamide Gel ; Escherichia coli ; genetics ; Glioma ; genetics ; pathology ; Humans ; Molecular Sequence Data ; Oligonucleotide Array Sequence Analysis ; RNA, Messenger ; genetics ; metabolism ; Recombinant Proteins ; isolation & purification ; metabolism ; Ribosomal Proteins ; genetics ; metabolism ; Sequence Analysis, DNA