With fast development and wide applications of next generation sequencing (NGS), genomic sequence information is within reach to various research fields. Three benchtop NGS instruments are now available. The 454 GS Junior (Roche), Ion PGM (Life Technologies) and MiSeq (Illumina) are laser-printer sized and offer modest set-up and running costs. By reviewing 2 studies that compared the performance of these instruments, the major characteristics of each benchtop platforms are compared to enable direct comparisons. The 454 GS Junior generated the longest reads and most contiguous assemblies but had the lowest throughput. The Ion Torrent PGM had the highest throughput and fastest run time. The MiSeq had the highest throughput per run and lowest error rates. The Ion Torrent PGM and 454 GS Junior both produced homopolymer-associated indel errors. Although all the platforms allow multiplexing of samples, details of experimental design, library preparation and data analysis may constrain the options. The features of the platforms provide opportunities both to conduct groundbreaking studies and to waste money. Thus, careful considerations should be made before purchasing or using any of them.
Research Design ; Running ; Statistics as Topic

Vast array of microbes colonize to each anatomical environment of human body. Culture based methods are important in investigating the microbial structure, but they are extremely biased in their evaluation of microbial diversity by selecting particular population of microbiota. Recent advance in molecular technology has allowed sophisticated analysis of complex human microbiota by culture-independent methods. Here, we will discuss features of tools for human microbiota studies including Roche-454 and Illumina platform. We will also briefly discuss features of some strategies that are commonly applied to these platforms including 16S rRNA targeting and shotgun sequencing. New platforms such as PacBio and Oxford Nanopore are also introduced.
Bias (Epidemiology) ; Colon ; Human Body ; Humans ; Metagenome ; Nanopores

Diabetes mellitus (DM) is a complex disease with various systemic and oral complications including periodontitis. Periodontitis is a disease that leads to destruction of the soft and hard tissues of periodontium, which can result in periodontal bone loss and tooth loss. Although the etiology for periodontitis is bacterial plaque, the host immune response also mediates the destruction of periodontal tissues. DM is related to the development, progression and severity of periodontitis. There are several factors potentially contributing to the development of periodontitis in DM patients: 1) altered immune function, 2) hyperglycemia and advanced glycation end products, and 3) altered lipid metabolism. With limited number of studies, the potential mechanisms involved in the development of DM in periodontitis have also been suggested. DM clearly increases the risk of periodontitis and biological mechanisms have been illucidated. Less clear is the impact of periodontitis on the development of DM. It is possible that periodontitis may serve as initiators or propagators of insulin resistance in a way similar to obesity, thereby aggravating glycemic control.
Alveolar Bone Loss ; Diabetes Mellitus ; Glycosylation End Products, Advanced ; Humans ; Hyperglycemia ; Insulin Resistance ; Lipid Metabolism ; Obesity ; Periodontal Diseases* ; Periodontitis ; Periodontium ; Tooth Loss

In the gingival tissues of patients with periodontitis, inflammatory responses are mediated by a wide variety of genes. In this study, we screened for differentially expressed genes (DEGs) in periodontitis compared with normal tissue using an annealing control primer (ACP) system. By ACP RT-PCR analysis, we obtained about 160 amplicons, 8 of which were found to be differentially expressed. DEGs in patients with periodontitis were thus successfully and reliably identified by the ACP-based RT PCR technique. The DEGs identified in the screen may also enhance our understanding of the pathogenesis of periodontitis.
Humans ; Periodontitis ; Polymerase Chain Reaction

Periodontitis is a disease that leads to destruction of the soft and hard tissues of periodontium, which can result in periodontal bone loss and tooth loss in severe cases. Atherosclerosis is a disease characterized by artery wall thickening as a result of invasion and accumulation of foam cells. Epidemiologic studies have suggested the association with periodontitis and atherosclerosis. Periodontopathogens are frequently found in atheroma plaque. The possible mechanisms for systemic dissemination of oral bacteria have been suggested: 1) direct translocation of bacteria from dental plaque to systemic circulation through transcellular mechanism or by physical perturbations of the gingiva, 2) indirect dissemination to distant sites via survival in immune cells including macrophages and dendritic cells. There are several mechanisms by which oral bacteria may contribute to atherosclerosis development: 1) activation of innate immune response, 2) mediators activated by oral bacteria and 3) involvement of cytokines and heat shock proteins from oral bacteria. Thus, better understanding the role of periodontitis in atherosclerosis may be the key to improve the prevention and treatment of atherosclerosis.
Alveolar Bone Loss ; Arteries ; Atherosclerosis* ; Bacteria ; Cytokines ; Dendritic Cells ; Dental Plaque ; Foam Cells ; Gingiva ; Heat-Shock Proteins ; Immunity, Innate ; Macrophages ; Periodontal Diseases* ; Periodontitis ; Periodontium ; Plaque, Atherosclerotic ; Tooth Loss

In the oral cavity, complex microbial community is shaped by various host and environmental factors. Extensive literature describing the oral microbiome in the context of oral health and disease is available. Advances in DNA sequencing technologies and data analysis have drastically improved the analysis of the oral microbiome. For microbiome study, bacterial 16S ribosomal RNA gene amplification and sequencing is often employed owing to the cost-effective and fast nature of the method. In this review, practical considerations for performing a microbiome study, including experimental design, molecular analysis technology, and general data analysis, will be discussed.

BACKGROUND: There have been several reports describing the capability of ginseng extracts as an adjuvant. In this study, we tested if ginsan, a polysaccharide extracted from Panax ginseng, was effective in enhancing antibody response to orally delivered Salmonella antigen. METHODS: Ginsan was treated before oral salmonella antigen administration. Salmonella specific antibody was determined by ELISA. mRNA expression was determined by RT-PCR. Cell migration was determined by confocal microscopy and flow cytometry. COX expression was detected by western blot. RESULTS: Ginsan treatment before oral Salmonella antigen delivery significantly increased both secretory and serum antibody production. Ginsan increased the expression of COX in the Peyer's patches. Various genes were screened and we found that CCL3 mRNA expression was increased in the Peyer's patch. Ginsan increased dendritic cells in the Peyer's patch and newly migrated dendritic cells were mostly found in the subepithelial dome region. When COX inhibitors were treated, the expression of CCL3 was reduced. COX inhibitor also antagonized both the migration of dendritic cells and the humoral immune response against oral Salmonella antigen. CONCLUSION: Ginsan effectively enhances the humoral immune response to orally delivered antigen, mediated by CCL3 via COX. Ginsan may serve as a potent vaccine suppliment for oral immunization.
Antibody Formation ; Blotting, Western ; Cell Movement ; Dendritic Cells ; Enzyme-Linked Immunosorbent Assay ; Flow Cytometry ; Immunity, Humoral ; Immunization ; Microscopy, Confocal ; Panax ; Peyer's Patches ; Polysaccharides ; Prostaglandin-Endoperoxide Synthases ; RNA, Messenger ; Salmonella

Aggregatibacter actinomycetemcomitans is an important pathogen in the development of localized aggressive periodontitis. Lipopolysaccharide (LPS) is a virulent factor of periodontal pathogens that contributes to alveolar bone loss and connective tissue degradation in periodontal disease. Our present study was designed to investigate the cytokine expression and signaling pathways regulated by A. actinomycetemcomitans LPS (Aa LPS). Cytokine gene expression profiling in RAW 264.7 cells was performed by microarray analyses. The cytokine mRNA and protein levels and related signaling pathways induced by Aa LPS were measured by RT-PCR, ELISA and western blotting. Microarray results showed that Aa LPS strongly induced the expression of NF-kappaB, NF-kappaB-related genes, inflammatory cytokines, TNF-alpha and IL-1beta in RAW 264.7 cells. NF-kappaB inhibitor pretreatment significantly reduced the levels of TNF-alpha and IL-1beta mRNA and protein. In addition, the Aa LPS-induced TNF-alpha and IL-1beta expression was inhibited by p38/JNK MAP kinase inhibitor pretreatment. These results show that Aa LPS stimulates TNF-alpha and IL-1beta expression through NF-kappaB and p38/JNK activation in RAW 264.7 cells, suggesting the essential role of this pathway in the pathogenesis of localized aggressive periodontitis.
Aggregatibacter actinomycetemcomitans* ; Aggressive Periodontitis ; Alveolar Bone Loss ; Blotting, Western ; Connective Tissue ; Cytokines ; Enzyme-Linked Immunosorbent Assay ; Gene Expression Profiling ; NF-kappa B ; Periodontal Diseases ; Phosphotransferases ; RNA, Messenger ; Tumor Necrosis Factor-alpha*

Xylitol is a five-carbon sugar alcohol that inhibits the growth of oral streptococci, including Streptococcus mutans. In this study, we tested xylitol sensitivity among the oral streptococci. We also compared nucleotide homology of putative fructose phosphotransferase system (PTS) and xylitol sensitivity, since xylitol is transported via the fructose PTS. Among the tested Streptococci, S. pneumonia showed the highest resistance to xylitol while S. gordonii and S. sanguinis showed the most sensitive growth inhibition. These streptococci could be grouped according to their xylitol sensitivity. S. mutans and S. salivarius showed similar bacterial growth inhibition by xylitol. S. mitis, S. oralis, S. pneumonia, S. intermedius and S. anginosus showed relatively low sensitivity to xylitol. When the genetic homologies of five fructose PTSs were compared among the tested streptococci, closely related streptococci showed similar sensitivity to xylitol. Taken together, fructose PTSs may mediate the sensitivity to xylitol in oral streptococci.
Fructose ; Pneumonia ; Streptococcus ; Streptococcus mutans ; Xylitol*

The presence of distinct bacterial species is found to be dependent on age, diet, and disease. We compared the detection rate of several oral bacterial strains in a cohort of 36 subjects including healthy volunteers, periodontal patients, and oral cancer patients. Gargling samples were obtained from these subjects from which DNA was then extracted. Specific primers for 29 bacterial species were used for PCR detection. In the oral cancer patients, Capnocytophaga ochracea, Gemella morbillorum, and Streptococcus salivarius were detected more frequently compared with the healthy volunteers and periodontitis patients. Fusobacterium nucleatum/polymorphym and Prevotella nigrescens were significantly less prevalent in oral cancer patients than the other groups. In periodontitis patients, Porphyromonas gingivalis and Treponema denticola were more frequently found compared with the healthy volunteers. In the healthy volunteer group, Peptostreptococcus anaerobius was more frequently found than the other groups. The detection rate of several oral bacterial species was thus found to differ between healthy volunteers, periodontitis patients and oral cancer patients.
Capnocytophaga ; Cohort Studies ; Diet ; DNA ; Fusobacterium ; Gemella ; Healthy Volunteers* ; Humans ; Microbiota* ; Mouth Neoplasms* ; Peptostreptococcus ; Periodontitis* ; Polymerase Chain Reaction ; Porphyromonas gingivalis ; Prevotella nigrescens ; Streptococcus ; Treponema denticola