Nicotine Regulates Streptococcus mutans Extracellular Polysaccharide and Related Protein Expression

poster abstractStreptococcus mutans, a gram-positive facultatively anaerobic bacterium, is considered as the primary contributor to caries due to its high acidogenicity and aciduricity. Smoking is one of the risk factors of periodontal disease and dental caries. Nicotine is one of the alkaloid pharmacologically active agents in tobacco. Previous studies indicated nicotine stimulated S. mutans biofilm formation and metabolism. However, the detailed mechanism is still unknown. Thus, the aim of this study is to investigate how nicotine facilitates S. mutans biofilm formation focused on extracellular polysaccharide synthesis. S. mutans UA159 (ATCC 700610) was used in the present study. Confocal laser scanning microscopy (CLSM) was used to investigate the effect of 0, 1, 2 and 4 mg/ml nicotine on 24 h S. mutans biofilm extracellular polysaccharide (EPS) expression (red fluorescentlabeled) and nucleic acid expression (green fluorescent-labeled). Western blot assays were used to investigate the effect of 0, 1, 2 and 4 mg/ml nicotine on the expression of glucosyltransferase (Gtfs), glucan-binding protein A (Gbp-A) and Gbp-B in 24 h S. mutans biofilm cells. CLSM results indicated nicotine increased both EPS and nucleic acid, and the ratio of EPS/nucleic acid was also increased. It implied EPS synthesis in single S. mutans cells was stimulated by nicotine treatment. Biofilm thickness was thicker in nicotine-treated groups than the non-treated group. Western blot assay results indicated that nicotine stimulated GtfC, Gbp-A and Gbp-B expression, but decreased GtfB expression. In conclusion, nicotine stimulates S. mutans cell proliferation and EPS synthesis partially by increasing GtfC, Gbp-A and Gbp-B

Effect of Green Tea on Streptococcus mutans Metabolic Activity, Planktonic Growth, and Biofilm Activity in the Presence of Nicotine

poster abstractStreptococcus mutans is the main bacterial cause of dental caries, and it has been proven by previous research that its growth is affected by various concentrations of nicotine and other agents. The amount of S. mutans in the mouth is directly proportional to the number of dental cavities. Studies have shown that smokers have an increased amount of caries, much of which is due to the low concentrations of nicotine the mouth is exposed to. It is known that S. mutans thrives in low-moderate concentrations of nicotine, and that nicotine is a promoting agent for S. mutans. S. mutans has also been proven as a contributor to atherosclerosis, resulting from dental plaque entering the bloodstream. Green Tea is a commonly consumed beverage, which has been known to reduce the number of dental cavities. Previous research has concluded that green tea contains polyphenols, which have antimicrobial effects, including an inhibitory effect on S. mutans. The objective of this research is to observe how green tea affects S. mutans metabolic activity, as well as biofilm and planktonic growth, in the presence of nicotine. The experiments compared S. mutans treated with nicotine concentrations (0-8 mg/ml), and S. mutans treated with a 2.5 g/200 mL concentration of Sencha Jade Reserve Japanese green tea in conjunction with the various nicotine concentrations. The assays were performed in a microtiter plate; the XTT and biofilm assays measured absorbance, and the planktonic assay measured kinetic growth. The experiments conclude that green tea has an inhibitory effect on nicotine-treated S. mutans metabolic activity and planktonic growth, with higher concentrations of green tea inhibiting more effectively. It was also concluded that green tea increases biofilm formation. These conclusions provide evidence of the inhibitory effect green tea has on nicotine-treated S. mutans, and may indicate a way to reduce the incidence of caries and atherosclerosis

EFFECT OF TOBACCO-TREATED MG63 OSTEOBLAST ON HUMAN PULP CELLS

poster abstractObjective: The objective of this study is to determine the effects of to-bacco products on protein concentration and growth of MG63 osteoblasts and the effects of the bacterial cells and culture supernatants on human pulp cells. The study was designed to observe the effects of P.gingivalis grown in four different tobacco solutions such as CSC (cigarette smoked condensate), nicotine (chewing tobacco), and DST (dissolvable smokeless tobacco) strips, and in the media control only without tobacco products. Methods: MG63 os-teoblast was grown in BHI-YE (Bacteria Heart Infusion-Yeast Extract) and hemin-vitamin K. In addition, MG63 osteoblast was grown in BHI-Y-E con-taining nicotine, CSC, and DST. Human pulp cells were grown in media con-taining BGS (Bovine Growth Serum) and washed. The pulp cell cultures will be assayed for cytotoxicity and the supernatants will be assayed for cyto-kines and MMP expression. Results: The protein assays was performed us-ing a microplate spectrophometer and SoftMax Pro 5.2, and we observed that nicotine and DST treated cells had significantly less protein than control cells, however, CSC treated cells had significantly more protein. The undilut-ed control had significantly less protein than the tobacco-treated superna-tants. Conclusion: Based on the previous experiments, we speculate that the additional protein in the undiluted CSC cells and tobacco-treated super-natant may stimulate more effect on human pulp cells than the control, nico-tine or DST treated cells or the control supernatant

Effect of nicotine on cariogenic virulence of Streptococcus mutans

Nicotine has well-documented effects on the growth and colonization of Streptococcus mutans. This study attempts to investigate the effects of nicotine on pathogenic factors of S. mutans, such as the effect on biofilm formation and viability, expression of pathogenic genes, and metabolites of S. mutans. The results demonstrated that addition of nicotine did not significantly influence the viability of S. mutans cells. The biofilms became increasingly compact as the concentrations of nicotine increased. The expression of virulence genes, such as ldh and phosphotransferase system (PTS)-associated genes, was upregulated, and nlmC was upregulated significantly, while ftf was downregulated. The lactate concentration of S. mutans grown in 1 mg/mL of nicotine was increased up to twofold over either biofilm or planktonic cells grown without nicotine. Changes in the metabolites involved in central carbon metabolism from sucrose indicated that most selected metabolites were detectable and influenced by increased concentrations of nicotine. This study demonstrated that nicotine can influence the pathogenicity of S. mutans and may lead to increased dental caries through the production of more lactate and the upregulation of virulence genes

EFFECTS OF NICOTINE ON QUORUM SENSING IN STREPTOCOCCUS MUTANS

poster abstractStreptococcus mutans is gram-positive cocci, facultative anaerobic bacterium commonly found in the human oral cavity, and significantly contributes to caries. The quorum sensing system plays an important role in microbial cell-cell interactions that leads to development of dental plaque. S. mutans produces glucosyltransferases together with the glucan binding proteins in order to facilitate the adherence of glucans to the tooth surface, thereby forming biofilms. Moreover, previous studies indicate that in the presence or absence of sucrose, a cell surface protein called antigen I/II plays an important role in the adherence process of S. mutans. There is a relationship between smoking and dental caries, in which the smoking population has a higher incidence of dental caries than a non-smoking population. Nicotine is one of the most important components in tobacco. In this study we investigated the effects of nicotine on quorum sensing in S. mutans. An S. mutans wild type UA159 and its knockout mutants defective in comC, comD and comE were used to investigate planktonic cell growth, the biofilm formation and biofilm metabolism at different concentrations of nicotine (0-32 mg/ml). The effects of nicotine on quorum sensing for S. mutans’ biofilm formation was evaluated using sucrose-dependent and sucrose-independent assays. The results indicate that S. mutans UA159 and its knockout mutants had no substantial differences in planktonic cell growth. In the presence of sucrose, the comC mutant was unable to produce biofilms, whereas the biofilm formation and biofilm metabolism of the comD and comE mutants were enhanced with increased nicotine concentration as with UA159 up to 8 mg/mL of nicotine. However, in the absence of sucrose, it was observed that the ComC mutant formed biofilms relatively similar to UA159. Biofilm formation in the comD and comE mutants was also enhanced with the increase of nicotine concentration up to 4 and 2 mg/mL, respectively. The results suggest that nicotine enhances the adherence process in S. mutans by antigen I/II through the comDE signaling pathway. However, the fact that the comC mutant produced biofilm in the absence of sucrose, but not in the presence of sucrose, indicates that the activity of antigen I/II is activated through different cell-cell signaling pathways depending on the amount of sucrose present

Characterization of transcriptome dynamics during watermelon fruit development: sequencing, assembly, annotation and gene expression profiles

<p>Abstract</p> <p>Background</p> <p>Cultivated watermelon [<it>Citrullus lanatus </it>(Thunb.) Matsum. & Nakai var. <it>lanatus</it>] is an important agriculture crop world-wide. The fruit of watermelon undergoes distinct stages of development with dramatic changes in its size, color, sweetness, texture and aroma. In order to better understand the genetic and molecular basis of these changes and significantly expand the watermelon transcript catalog, we have selected four critical stages of watermelon fruit development and used Roche/454 next-generation sequencing technology to generate a large expressed sequence tag (EST) dataset and a comprehensive transcriptome profile for watermelon fruit flesh tissues.</p> <p>Results</p> <p>We performed half Roche/454 GS-FLX run for each of the four watermelon fruit developmental stages (immature white, white-pink flesh, red flesh and over-ripe) and obtained 577,023 high quality ESTs with an average length of 302.8 bp. <it>De novo </it>assembly of these ESTs together with 11,786 watermelon ESTs collected from GenBank produced 75,068 unigenes with a total length of approximately 31.8 Mb. Overall 54.9% of the unigenes showed significant similarities to known sequences in GenBank non-redundant (nr) protein database and around two-thirds of them matched proteins of cucumber, the most closely-related species with a sequenced genome. The unigenes were further assigned with gene ontology (GO) terms and mapped to biochemical pathways. More than 5,000 SSRs were identified from the EST collection. Furthermore we carried out digital gene expression analysis of these ESTs and identified 3,023 genes that were differentially expressed during watermelon fruit development and ripening, which provided novel insights into watermelon fruit biology and a comprehensive resource of candidate genes for future functional analysis. We then generated profiles of several interesting metabolites that are important to fruit quality including pigmentation and sweetness. Integrative analysis of metabolite and digital gene expression profiles helped elucidating molecular mechanisms governing these important quality-related traits during watermelon fruit development.</p> <p>Conclusion</p> <p>We have generated a large collection of watermelon ESTs, which represents a significant expansion of the current transcript catalog of watermelon and a valuable resource for future studies on the genomics of watermelon and other closely-related species. Digital expression analysis of this EST collection allowed us to identify a large set of genes that were differentially expressed during watermelon fruit development and ripening, which provide a rich source of candidates for future functional analysis and represent a valuable increase in our knowledge base of watermelon fruit biology.</p

Analysis of expressed sequence tags generated from full-length enriched cDNA libraries of melon

Abstract Background Melon (Cucumis melo), an economically important vegetable crop, belongs to the Cucurbitaceae family which includes several other important crops such as watermelon, cucumber, and pumpkin. It has served as a model system for sex determination and vascular biology studies. However, genomic resources currently available for melon are limited. Result We constructed eleven full-length enriched and four standard cDNA libraries from fruits, flowers, leaves, roots, cotyledons, and calluses of four different melon genotypes, and generated 71,577 and 22,179 ESTs from full-length enriched and standard cDNA libraries, respectively. These ESTs, together with ~35,000 ESTs available in public domains, were assembled into 24,444 unigenes, which were extensively annotated by comparing their sequences to different protein and functional domain databases, assigning them Gene Ontology (GO) terms, and mapping them onto metabolic pathways. Comparative analysis of melon unigenes and other plant genomes revealed that 75% to 85% of melon unigenes had homologs in other dicot plants, while approximately 70% had homologs in monocot plants. The analysis also identified 6,972 gene families that were conserved across dicot and monocot plants, and 181, 1,192, and 220 gene families specific to fleshy fruit-bearing plants, the Cucurbitaceae family, and melon, respectively. Digital expression analysis identified a total of 175 tissue-specific genes, which provides a valuable gene sequence resource for future genomics and functional studies. Furthermore, we identified 4,068 simple sequence repeats (SSRs) and 3,073 single nucleotide polymorphisms (SNPs) in the melon EST collection. Finally, we obtained a total of 1,382 melon full-length transcripts through the analysis of full-length enriched cDNA clones that were sequenced from both ends. Analysis of these full-length transcripts indicated that sizes of melon 5' and 3' UTRs were similar to those of tomato, but longer than many other dicot plants. Codon usages of melon full-length transcripts were largely similar to those of Arabidopsis coding sequences. Conclusion The collection of melon ESTs generated from full-length enriched and standard cDNA libraries is expected to play significant roles in annotating the melon genome. The ESTs and associated analysis results will be useful resources for gene discovery, functional analysis, marker-assisted breeding of melon and closely related species, comparative genomic studies and for gaining insights into gene expression patterns.This work was supported by Research Grant Award No. IS-4223-09C from BARD, the United States-Israel Binational Agricultural Research and Development Fund, and by SNC Laboratoire ASL, de Ruiter Seeds B.V., Enza Zaden B.V., Gautier Semences S.A., Nunhems B.V., Rijk Zwaan B.V., Sakata Seed Inc, Semillas Fitó S.A., Seminis Vegetable Seeds Inc, Syngenta Seeds B.V., Takii and Company Ltd, Vilmorin and Cie S.A. and Zeraim Gedera Ltd (all of them as part of the support to ICuGI). CC was supported by CNRS ERL 8196.Peer Reviewe

Nicotine Regulates Streptococcus mutans Extracellular Polysaccharide and Related Protein Expression

poster abstractStreptococcus mutans, a gram-positive facultatively anaerobic bacterium, is considered as the primary contributor to caries due to its high acidogenicity and aciduricity. Smoking is one of the risk factors of periodontal disease and dental caries. Nicotine is one of the alkaloid pharmacologically active agents in tobacco. Previous studies indicated nicotine stimulated S. mutans biofilm formation and metabolism. However, the detailed mechanism is still unknown. Thus, the aim of this study is to investigate how nicotine facilitates S. mutans biofilm formation focused on extracellular polysaccharide synthesis. S. mutans UA159 (ATCC 700610) was used in the present study. Confocal laser scanning microscopy (CLSM) was used to investigate the effect of 0, 1, 2 and 4 mg/ml nicotine on 24 h S. mutans biofilm extracellular polysaccharide (EPS) expression (red fluorescentlabeled) and nucleic acid expression (green fluorescent-labeled). Western blot assays were used to investigate the effect of 0, 1, 2 and 4 mg/ml nicotine on the expression of glucosyltransferase (Gtfs), glucan-binding protein A (Gbp-A) and Gbp-B in 24 h S. mutans biofilm cells. CLSM results indicated nicotine increased both EPS and nucleic acid, and the ratio of EPS/nucleic acid was also increased. It implied EPS synthesis in single S. mutans cells was stimulated by nicotine treatment. Biofilm thickness was thicker in nicotine-treated groups than the non-treated group. Western blot assay results indicated that nicotine stimulated GtfC, Gbp-A and Gbp-B expression, but decreased GtfB expression. In conclusion, nicotine stimulates S. mutans cell proliferation and EPS synthesis partially by increasing GtfC, Gbp-A and Gbp-B