Effects of different periodontal treatments in changing the prevalence and levels of Archaea present in the subgingival biofilm of subjects with periodontitis: A secondary analysis from a randomized controlled clinical trial.

OBJECTIVE: The aim of this randomized double-blind and placebo-controlled study was to assess if periodontal treatment with or without systemic antibiotic would change the mean level of Archaea. METHODS: Fifty-nine (59) subjects were randomly assigned to receive scaling and root planing (SRP) alone or combined with metronidazole (MTZ; 400 mg/TID) or either with MTZ and amoxicillin (AMX; 500 mg/TID) for 14 days. Clinical and microbiological examinations were performed at baseline and at 6 months post-SRP. Six subgingival plaque samples per subject were analysed for the presence and levels of Archaea using quantitative polymerase chain reaction. RESULTS: Scaling and root planing alone or combined with MTZ or MTZ + AMX significantly reduced the prevalence of subjects colonized by Archaea at 6 months post-therapy, without significant differences among groups (P > .05). Both therapies led to a statistically significant decrease in the mean percentage of sites colonized by Archaea (P < .05). The MTZ and MTZ + AMX group had a significantly lower mean number of sites colonized by Archaea and lower levels of these micro-organisms at sites with probing depth ≥5 mm at 6 months compared with SRP group (P < .05). CONCLUSION: Periodontal treatments including adjunctive MTZ or MTZ + AMX are more effective than mechanical treatment alone in reducing the levels and prevalence of sites colonized by Archaea in subjects with chronic periodontitis.

The combined and individual impact of diabetes and smoking on key subgingival periodontal pathogens in patients with chronic periodontitis.

BACKGROUND AND OBJECTIVE: Comprehension of the similarities and differences in the composition of the subgingival microbiota of patients with diabetes mellitus (DM), smokers or smokers with DM is an important step in developing therapies specific for these groups at risk for periodontitis. Therefore, the aim of this study was to compare the combined and individual effects of DM and smoking on the levels and prevalence of key subgingival periodontal pathogens in patients with chronic periodontitis. MATERIAL AND METHODS: One hundred patients with generalized chronic periodontitis were allocated into one of the following groups: DM (n = 25, non-smokers with type 2 DM); S (n = 25, non-diabetic smokers); SDM (n = 25, smokers with type 2 DM); and control (n = 25, non-diabetic non-smokers). Two subgingival biofilm samples from healthy sites (probing depth and clinical attachment level ≤3 mm and no bleeding) and 2 from diseased sites (probing depth and clinical attachment level ≥5 mm and bleeding on probing) were analyzed by quantitative polymerase chain reaction for Porphyromonas gingivalis, Tannerella forsythia, Treponema denticola, Eubacterium nodatum, Parvimonas micra, Fusobacterium nucleatum ssp. and Prevotella intermedia. RESULTS: There were no differences among groups in the mean counts of the bacterial species studied, considering all sampled sites (healthy plus diseased sites). There were also no differences among groups regarding the prevalence of any bacteria species in healthy and diseased sites (P > .05). The mean P. micra count was significantly higher in the healthy sites of both smoking groups, than in those of the control group (P < .05). CONCLUSION: The subgingival levels and prevalence of the bacterial species studied are not significantly different in subjects with chronic periodontitis presenting DM, smokers or smokers with DM. In addition, DM and smoking, jointly and individually, do not considerably affect the subgingival levels of target periodontal pathogens in patients with chronic periodontitis.

Diabetes may affect the expression of matrix metalloproteinases and their inhibitors more than smoking in chronic periodontitis.

BACKGROUND AND OBJECTIVE: No previous study has directly compared the levels of matrix metalloproteinases (MMPs) and tissue inhibitor of metalloproteinases (TIMPs) between smokers and individuals with diabetes mellitus (DM) with periodontitis. Therefore, the aim of this study was to evaluate the gene expression of MMP-1, MMP-2, MMP-8, MMP-9, TIMP-1 and TIMP-2 in tissues with chronic periodontitis (ChP) of smokers and individuals with type 2 DM. MATERIAL AND METHODS: Gingival biopsies were harvested from: non-smokers and non-diabetic individuals with ChP (n = 18) (ChP group); non-diabetic smokers (≥ 10 cigarettes per day for at least the past 5 years) with ChP (n = 18) (SChP group); non-smoking individuals with type 2 diabetes (glycated hemoglobin levels ≥ 7.5%) and ChP (n = 18) (DMChP group). The tissue levels of mRNA of MMP-1, MMP-2, MMP-8, MMP-9, TIMP-1 and TIMP-2 were evaluated by quantitative real-time polymerase chain reaction. RESULTS: The MMP-8 expression was the lowest in the ChP group (p < 0.05). The DMChP group presented increased mRNA levels of MMP-2 and MMP-9, when compared to the SChP group (p < 0.05). MMP-1 expression and the MMP-1/TIMP-1, MMP-2/TIMP-1, MMP-8/TIMP-1, MMP-9/TIMP-1, MMP-1/TIMP-2 and MMP-2/TIMP-2 ratios were higher in the DMChP group than in the ChP and SChP groups (p < 0.05). The DMChP group presented lower mRNA levels of TIMP-1 than the ChP group (p < 0.05). The MMP-8/TIMP-2 ratio was the highest in the SChP group (p < 0.05). CONCLUSION: Uncontrolled type 2 DM upregulates the ratio of MMP/TIMPs in sites with ChP more than smoking, which may contribute to a greater extracellular matrix degradation and periodontal breakdown in DM-related periodontitis.

Investigation of ferromagnetic resonance and magnetoresistance in anti-spin ice structures.

In this work, we report experimental and theoretical investigations performed in anti-spin ice structures, composed by square lattice of elongated antidots, patterned in nickel thin film. The magnetic vortex crystal state was obtained by micromagnetic simulation as the ground state magnetization, which arises due to the magnetic stray field at the antidot edges inducing chirality in the magnetization of platters among antidots. Ferromagnetic resonance (FMR) and magnetoresistance (MR) measurements were utilized to investigate the vortex crystal magnetization dynamics and magnetoelectric response. By using FMR, it was possible to detect the spin wave modes and vortex crystal resonance, in good agreement with dynamic micromagnetic simulation results. The vortex crystal magnetization configuration and its response to the external magnetic field, were used to explain the isotropic MR behaviour observed.

Amoxicillin Plus Metronidazole Therapy for Patients with Periodontitis and Type 2 Diabetes: A 2-year Randomized Controlled Trial.

The aim of this study was to assess the changes occurring in subgingival biofilm composition and in the periodontal clinical parameters of subjects with periodontitis and type 2 diabetes mellitus (DM) treated by means of scaling and root planing (SRP) only or combined with systemic metronidazole (MTZ) and amoxicillin (AMX). Fifty-eight subjects were randomly assigned to receive SRP only (n = 29) or with MTZ (400 mg/thrice a day [TID]) and AMX (500 mg/TID) (n = 29) for 14 d. Six subgingival plaque samples/subject were analyzed by checkerboard DNA-DNA hybridization for 40 bacterial species at baseline and 3 mo, 1 y, and 2 y posttherapy. At 2 y posttherapy, the antibiotic-treated group harbored lower mean proportions (5.5%) of red complex pathogens than the control group (12.1%) (P < 0.05). The proportions of the Actinomyces species remained stable in the antibiotic group but showed a statistically significant reduction in the control group from 1 to 2 y in subjects achieving a low risk clinical profile for future disease progression (i.e., ≤4 sites with probing depth [PD] ≥5 mm). The test group also had a lower mean number of sites with PD ≥5 mm (3.5 ± 3.4) and a higher percentage of subjects reaching the low risk clinical profile (76%) than the control group (14.7 ± 13.1 and 22%, respectively) (P < 0.05) at 2 y posttreatment. MTZ + AMX intake was the only significant predictor of subjects achieving the low risk at 2 y (odds ratio, 20.9; P = 0.0000). In conclusion, the results of this study showed that the adjunctive use of MTZ + AMX improves the microbiological and clinical outcomes of SRP in the treatment of subjects with generalized chronic periodontitis and type 2 DM up to 2 y (ClinicalTrials.gov NCT02135952).

Levels of Candidate Periodontal Pathogens in Subgingival Biofilm.

In recent years, several new periodontal taxa have been associated with the etiology of periodontitis. A recent systematic review provides further support for the pathogenic role of 17 species/phylotypes. Thus, the aim of this study was to assess the prevalence and levels of these species in subjects with generalized chronic periodontitis (GChP; n = 30), generalized aggressive periodontitis (GAgP; n = 30), and periodontal health (PH; n = 30). All subjects underwent clinical and microbiological assessment. Nine subgingival plaque samples were collected from each subject and analyzed for their content of 20 bacterial species/phylotypes through the RNA-oligonucleotide quantification technique. Subjects from the GChP and GAgP groups presented the highest mean values for all clinical parameters in comparison with the PH group (P < 0.05). Subjects with GChP and GAgP showed significantly higher mean levels of Bacteroidetes sp. human oral taxon (HOT) 274, Fretibacterium sp. HOT 360, and TM7 sp. HOT 356 phylotypes, as well as higher mean levels of Filifactor alocis, Fretibacterium fastidiosum, Porphyromonas gingivalis, Tannerella forsythia, and Selenomonas sputigena species than PH subjects (P < 0.05). GAgP subjects presented higher mean levels of TM7 sp. HOT 356 and F. alocis than GChP subjects (P < 0.05). A significantly higher mean prevalence of Bacteroidales sp. HOT 274, Desulfobulbus sp. HOT 041, Fretibacterium sp. HOT 360, and Fretibacterium sp. HOT 362 was found in subjects with GChP and GAgP than in PH subjects. Mean levels of P. gingivalis (r = 0.68), T. forsythia (r = 0.62), F. alocis (r = 0.51, P = 0.001), and Fretibacterium sp. HOT 360 (r = 0.41) were correlated with pocket depth (P < 0.001). In conclusion, Bacteroidales sp. HOT 274, Desulfobulbus sp. HOT 041, Fretibacterium sp. HOT 360, Fretibacterium sp. HOT 362, and TM7 sp. HOT 356 phylotypes, in addition to F. alocis, F. fastidiosum, and S. sputigena, seem to be associated with periodontitis, and their role in periodontal pathogenesis should be further investigated.

Could cytokine levels in the peri-implant crevicular fluid be used to distinguish between healthy implants and implants with peri-implantitis? A systematic review.

BACKGROUND AND OBJECTIVE: Despite investigative efforts to identify the levels of different types of cytokines in the peri-implant crevicular fluid (PICF), the efficacy of these biomarkers in assisting the diagnosis of peri-implantitis is still undetermined. This systematic review aimed to answer the following question: "Could cytokine levels in the PICF be used to distinguish between healthy implants and implants with peri-implantitis?" MATERIAL AND METHODS: This review was conducted and reported in accordance with the PRISMA statement. The MEDLINE and EMBASE databases were searched from 1990 up to and including March 2015, using MeSH terms and other keywords. Additional publications were searched using a hand search of reference lists of relevant studies. Titles and abstracts were screened and papers that fulfilled eligibility criteria were assessed. RESULTS: Out of 1212 titles, 18 studies reporting the levels of nine different cytokines were included. Proinflammatory cytokines [interleukin (IL)-1ß, IL-6, IL-12, IL-17 and tumor necrosis factor-α) were the cytokines studied most commonly, followed by anti-inflammatory cytokines (IL-4 and IL-10), osteoclastogenesis-related cytokines (RANKL) and chemokines (IL-8). Nine studies reported statistically significantly higher levels of proinflammatory cytokines in the PICF of implants with peri-implantitis than in the PICF of healthy implants. Most studies did not find any significant differences in the PICF levels of anti-inflammatory cytokines and RANKL between healthy implants and implants with peri-implantitis. IL-8 was the only chemokine studied and its levels did not differ significantly between healthy and diseased implants. The studies differed greatly in the manner in which they reported the results (e.g. concentrations or total amounts) and in the exclusion of confounders, such as smoking. CONCLUSION: The results of this systematic review indicate moderate evidence in the literature to support that implants with peri-implantitis present higher levels of proinflammatory cytokines in the PICF than do healthy implants. Evidence regarding the PICF levels of anti-inflammatory cytokines, osteoclastogenesis-related cytokines and chemokines as possible predictors of peri-implantitis is too limited.

Subgingival bacterial community profiles in HIV-infected Brazilian adults with chronic periodontitis.

BACKGROUND AND OBJECTIVE: To compare the subgingival microbial diversity between non-HIV-infected and HIV-infected individuals with chronic periodontitis using denaturing gradient gel electrophoresis (DGGE). MATERIAL AND METHODS: Thirty-two patients were selected: 11 were HIV-infected and 21 were non-HIV-infected, and all had chronic periodontitis. Periodontal measurements included probing depth, clinical attachment level, visible supragingival biofilm and bleeding on probing. Subgingival biofilm samples were collected from periodontal sites (50% with probing depth ≤ 4 mm and 50% with probing depth ≥ 5 mm) and whole-genomic-amplified DNA was obtained. The DNA samples were subjected to amplification of a 16S rRNA gene fragment using universal bacterial primers, followed by DGGE analysis of the amplified gene sequences. RESULTS: The non-HIV-infected group presented higher mean full-mouth visible supragingival biofilm (p = 0.004), bleeding on probing (p = 0.006), probing depth (p < 0.001) and clinical attachment level (p = 0.001) in comparison with the HIV-infected group. DGGE analysis revealed 81 distinct bands from all 33 individuals. Banding profiles revealed a higher diversity of the bacterial communities in the subgingival biofilm of HIV-infected patients with chronic periodontitis. Moreover, cluster and principal component analyses demonstrated that the bacterial community profiles differed between these two conditions. High interindividual and intra-individual variability in banding profiles were observed for both groups. CONCLUSION: HIV-infected patients with chronic periodontitis present greater subgingival microbial diversity. In addition, the bacterial communities associated with HIV-infected and non-HIV-infected individuals are different in structure.

Evaluation of human and microbial DNA content in subgingival plaque samples collected by paper points or curette.

Host DNA may adversely affect metagenomic studies focusing on the prokaryotic microbiota. This study compared the levels of host DNA in subgingival plaque collected by paper points and curette, using quantitative PCR. Lower proportions of host DNA and higher proportions of bacterial DNA were recovered from samples collected with curettes.

Subgingival bacterial recolonization after scaling and root planing in smokers with chronic periodontitis.

BACKGROUND: The aim of this study was to compare subgingival bacterial recolonization patterns after scaling and root planing in current smokers and non-smokers. METHODS: 15 smokers and 15 non-smokers with chronic periodontitis received scaling and root planing in six visits lasting one hour each, over a period of 21 days. Clinical monitoring was performed at baseline and 180 days, and microbiological monitoring was performed at baseline, immediately after scaling and root planing (Day 0) and at 42, 63 and 180 days post-therapy. Subgingival plaque samples were analysed by checkerboard DNA-DNA hybridization. RESULTS: An improvement in clinical condition was observed for smokers and non-smokers; however, non-smokers showed a greater reduction in mean clinical attachment level in intermediate sites in comparison with smokers (p < 0.05). At Day 0, there was a significant reduction in the mean counts of the three pathogens from the red complex, Eubacterium nodatum and Parvimonas micra only in non-smokers (p < 0.05). There was a significant increase in the proportion of host-compatible species in non-smokers and smokers from baseline to 180 days post-therapy (p < 0.05). However, a significant decrease in the pathogenic species was observed only in non-smokers. CONCLUSIONS: Smokers were more susceptible to the re-establishment of a pathogenic subgingival biofilm than non-smokers.

Do subjects with aggressive and chronic periodontitis exhibit a different cytokine/chemokine profile in the gingival crevicular fluid? A systematic review.

BACKGROUND AND OBJECTIVE: Microbiological and immunological hypotheses have been raised to explain the differences in the clinical manifestations of aggressive periodontitis and chronic periodontitis. However, studies comparing the cytokine/chemokine profiles in gingival crevicular fluid between these two clinical conditions have so far not been compiled. This systematic review aimed to answer the following question: "Do subjects with aggressive periodontitis and chronic periodontitis have a different profile of cytokines/chemokines in the gingival crevicular fluid?" MATERIAL AND METHODS: An electronic database search of MEDLINE/PubMed and Embase was performed from 1990 up to and including August 2013, using MeSH terms and other keywords. Titles and abstracts were screened and the papers that satisfied eligibility criteria were assessed. RESULTS: Of 1954 titles, 17 studies reporting the levels of 21 different cytokines/chemokines were included. Most studies did not find any significant differences in the gingival crevicular fluid levels of cytokines/chemokines between aggressive periodontitis and chronic periodontitis. Some studies demonstrated that the levels of specific proinflammatory and anti-inflammatory cytokines/chemokines were higher (n = 5) and lower (n = 3), respectively, in aggressive periodontitis than in chronic periodontitis. The studies differed in the manner in which they reported the results (e.g. concentrations or total amounts). It was not clear in some studies whether the sample sites from both groups were matched for disease severity. Some studies did not take into account confounders, such as smoking. CONCLUSION: The current weight of evidence is not sufficient to prove that there are distinct gingival crevicular fluid cytokine/chemokine profiles for patients with aggressive periodontitis and chronic periodontitis.

Full-mouth scaling and root planing in type 2 diabetic subjects: one-year microbiological outcomes.

BACKGROUND: This study evaluated the microbiological effects of full-mouth (FM) and partial-mouth (PM) scaling and root planing (SRP) in type 2 diabetic subjects with chronic periodontitis (ChP), up to 12 months. METHODS: Thirty-four type 2 diabetic subjects with ChP received either FMSRP (n = 17), in two sessions within two consecutive days, or PMSRP (n = 17) in four sessions within 21 days. Six subgingival biofilm samples per subject were analysed by checkerboard DNA-DNA hybridization for 40 bacterial species at baseline, 3 and 12 months. RESULTS: Both therapies significantly reduced the levels of the red complex species up to 12 months (p < 0.05). The levels of three putative pathogens from the orange complex were significantly reduced in the FMSRP group, whereas a single orange complex species was significantly decreased in the PMSRP group (p < 0.05). Furthermore, the proportions of the host-compatible Actinomyces species were significantly increased in both groups at 3 and 12 months. No significant differences were observed between groups for the counts and proportions of the individual species and the proportions of microbial complexes at any time point (p > 0.05). CONCLUSIONS: There were no differences in the bacterial species evaluated after FMSRP and PMSRP in the treatment of type 2 diabetic subjects with ChP, up to 12 months.

Newly identified pathogens associated with periodontitis: a systematic review.

There is substantial evidence supporting the role of certain oral bacteria species in the onset and progression of periodontitis. Nevertheless, results of independent-culture diagnostic methods introduced about a decade ago have pointed to the existence of new periodontal pathogens. However, the data of these studies have not been evaluated together, which may generate some misunderstanding on the actual role of these microorganisms in the etiology of periodontitis. The aim of this systematic review was to determine the current weight of evidence for newly identified periodontal pathogens based on the results of "association" studies. This review was conducted and reported in accordance with the PRISMA statement. The MEDLINE, EMBASE, and Cochrane databases were searched up to September 2013 for studies (1) comparing microbial data of subgingival plaque samples collected from subjects with periodontitis and periodontal health and (2) evaluating at least 1 microorganism other than the already-known periodontal pathogens. From 1,450 papers identified, 41 studies were eligible. The data were extracted and registered in predefined piloted forms. The results suggested that there is moderate evidence in the literature to support the association of 17 species or phylotypes from the phyla Bacteroidetes, Candidatus Saccharibacteria, Firmicutes, Proteobacteria, Spirochaetes, and Synergistetes. The phylum Candidatus Saccharibacteria and the Archaea domain also seem to have an association with disease. These data point out the importance of previously unidentified species in the etiology of periodontitis and might guide future investigations on the actual role of these suspected new pathogens in the onset and progression of this infection.

Reduction in prevalence of Archaea after periodontal therapy in subjects with generalized aggressive periodontitis.

BACKGROUND: There is evidence of a possible relationship between Archaea and periodontal disease; however, to date few studies have assessed the changes in prevalence of this domain after periodontal therapy. The aim of this randomized double-blind and placebo-controlled study was to assess if periodontal treatment with or without systemic antibiotic would change the prevalence of Archaea after periodontal therapy. METHODS: Thirty subjects were randomly assigned to receive scaling and root planing (SRP) alone or combined with metronidazole (MTZ) + amoxicillin (AMX) for 14 days. Clinical and microbiological examinations were performed at baseline and at six months post-SRP. Nine subgingival plaque samples per subject were analysed for the presence of Archaea. RESULTS: SRP alone or combined with MTZ + AMX significantly reduced the prevalence of subjects colonized by Archaea at six months post-therapy. However, no significant differences between treatment groups were observed (p > 0.05). Both therapies led to a statistically significant decrease in the mean percentage of sites colonized by Archaea (p < 0.05). A negative Spearman correlation was observed between the presence of Archaea and the mean clinical attachment gain at six months post-therapy (r(2) = -0.61; 95% CI -0.80- -0.31; p = 0.003). CONCLUSIONS: SRP alone or combined with MTZ + AMX provides a similar reduction in the prevalence of Archaea in the subgingival biofilm of subjects with generalized aggressive periodontitis.

Levels of Selenomonas species in generalized aggressive periodontitis.

BACKGROUND AND OBJECTIVE: To compare the levels of Selenomonas sputigena and uncultivated/unrecognized Selenomonas species in subgingival biofilms from periodontally healthy subjects and from subjects with generalized aggressive periodontitis. MATERIAL AND METHODS: Fifteen periodontally healthy subjects and 15 subjects with generalized aggressive periodontitis were recruited and their clinical periodontal parameters were evaluated. Nine subgingival plaque samples were collected from each subject and all were individually analyzed for the levels of 10 bacterial taxa, including cultured and uncultivated/unrecognized microorganisms, using the RNA-oligonucleotide quantification technique. Between-group differences in the levels of the test taxa were determined using the Mann-Whitney U-test. RESULTS: Subjects with generalized aggressive periodontitis showed significantly higher mean counts of Porphyromonas gingivalis, S. sputigena and the Mitsuokella sp. Human Oral Taxon (HOT) 131 (previously described as Selenomonas sp. oral clone CS002), while higher mean counts of Actinomyces gerencseriae and Streptococcus sanguinis were found in periodontally healthy subjects (p < 0.01). Selenomonas sp. HOT 146 was only detected in the generalized aggressive periodontitis group. In the generalized aggressive periodontitis group, the levels of P. gingivalis and S. sputigena were higher in deep sites (probing depth ≥ 5 mm) than in shallow sites (probing depth ≤ 3 mm) (p < 0.01). Furthermore, in subjects with generalized aggressive periodontitis, sites with probing depth of ≤ 3 mm harbored higher levels of these two species than sites with the same probing depth in periodontally healthy subjects. There were positive correlations between probing depth and the levels of P. gingivalis (r = 0.77; p < 0.01), S. sputigena (r = 0.60; p < 0.01) and Selenomonas dianae (previously described as Selenomonas sp. oral clone EW076) (r = 0.42, p < 0.05). CONCLUSION: S. sputigena and Mitsuokella sp. HOT 131 may be associated with the pathogenesis of generalized aggressive periodontitis, and their role in the onset and progression of this infection should be investigated further.

A preliminary study on the FAM5C expression in generalized chronic periodontitis.

OBJECTIVE: The Family with sequence similarity 5 member C (FAM5C) has been suggested to contribute in aggressive periodontitis. However, there is no data regarding its role in chronic periodontitis. The aim of this study was to evaluate the FAM5C expression in chronic periodontitis and to study association of FAM5C with key immunoinflammatory markers. MATERIAL AND METHODS: Gingival biopsies were harvested from periodontally healthy subjects (n = 10) and chronic periodontitis subjects (n = 15). The levels of mRNA of FAM5C, interleukin (IL)-17, IL-6, IL-23, IL-10, IL-4, interferon-c, toll-like receptor (TLR)-2, TLR-4, osteoprotegerin (OPG), receptor activator of NF-κB ligand (RANKL), tumor necrosis factor (TNF)-a, transforming growth factor-b, transcription factor forkhead box p3, and transcription factor orphan nuclear receptor C2 were evaluated by real-time polymerase chain reaction. RESULTS: FAM5C mRNA levels were not different between periodontally healthy and diseased tissues (P > 0.05). Gene expressions of IL-17, TNF-a, OPG, RANKL, TLR-2, and TLR-4 were higher in periodontitis, when compared to periodontally healthy sites (P < 0.05), while no differences between groups were observed for the other genes evaluated (P > 0.05). There were no correlations between the gene expression of FAM5C and the other immunoinflammatory markers (P > 0.05). CONCLUSION: Within the limits of this study, it seems that FAM5C expression does not contribute to chronic periodontitis.

Identification and isolation of full-length cDNA sequences by sequencing and analysis of expressed sequence tags from guarana (Paullinia cupana).

The current intense production of biological data, generated by sequencing techniques, has created an ever-growing volume of unanalyzed data. We reevaluated data produced by the guarana (Paullinia cupana) transcriptome sequencing project to identify cDNA clones with complete coding sequences (full-length clones) and complete sequences of genes of biotechnological interest, contributing to the knowledge of biological characteristics of this organism. We analyzed 15,490 ESTs of guarana in search of clones with complete coding regions. A total of 12,402 sequences were analyzed using BLAST, and 4697 full-length clones were identified, responsible for the production of 2297 different proteins. Eighty-four clones were identified as full-length for N-methyltransferase and 18 were sequenced in both directions to obtain the complete genome sequence, and confirm the search made in silico for full-length clones. Phylogenetic analyses were made with the complete genome sequences of three clones, which showed only 0.017% dissimilarity; these are phylogenetically close to the caffeine synthase of Theobroma cacao. The search for full-length clones allowed the identification of numerous clones that had the complete coding region, demonstrating this to be an efficient and useful tool in the process of biological data mining. The sequencing of the complete coding region of identified full-length clones corroborated the data from the in silico search, strengthening its efficiency and utility.

Prevalence and microbiological diversity of Archaea in peri-implantitis subjects by 16S ribosomal RNA clonal analysis.

BACKGROUND AND OBJECTIVE: This study evaluated the prevalence and the molecular diversity of Archaea in the subgingival biofilm samples of subjects with peri-implantitis. MATERIAL AND METHODS: Fifty subjects were assigned into two groups: Control (n = 25), consisting of subjects with healthy implants; and Test (n = 25), consisting of subjects with peri-implantitis sites, as well as a healthy implant. In the Test group, subgingival biofilm samples were taken from the deepest sites of the diseased implant. In both groups, subgingival biofilm was collected from one site with a healthy implant and from one site with a periodontally healthy tooth. DNA was extracted and the 16S ribosomal RNA gene was amplified with universal primer pairs for Archaea. Amplified genes were cloned and sequenced, and the phylotypes were identified by comparison with known 16S ribosomal RNA sequences. RESULTS: In the Control group, Archaea were detected in two and three sites of the implant and the tooth, respectively. In the Test group, Archaea were detected in 12, 4 and 2 sites of diseased implants, healthy implants and teeth, respectively. Diseased implants presented a significantly higher prevalence of Archaea in comparison with healthy implants and natural teeth, irrespective of group. Over 90% of the clone libraries were formed by Methanobrevibacter oralis, which was detected in both groups. Methanobacterium congelense/curvum was detected in four subjects from the Test group and in two subjects from the Control group. CONCLUSION: Although M. oralis was the main species of Archaea associated with both healthy and diseased implant sites, the data indicated an increased prevalence of Archaea in peri-implantitis sites, and their role in pathogenesis should be further investigated.

Microbiological diversity of generalized aggressive periodontitis by 16S rRNA clonal analysis.

BACKGROUND/AIM: The purpose of this study was to determine the bacterial diversity in the subgingival plaque of subjects with generalized aggressive periodontitis by using culture-independent molecular methods based on 16S ribosomal DNA cloning. METHODS: Samples from 10 subjects with generalized aggressive periodontitis were selected. DNA was extracted and the 16S rRNA gene was amplified with the universal primer pairs 9F and 1525R. Amplified genes were cloned, sequenced, and identified by comparison with known 16S rRNA sequences. RESULTS: One hundred and ten species were identified from 10 subjects and 1007 clones were sequenced. Of these, 70 species were most prevalent. Fifty-seven percent of the clone (40 taxa) sequences represented phylotypes for which no cultivated isolates have been reported. Several species of Selenomonas and Streptococcus were found at high prevalence and proportion in all subjects. Overall, 50% of the clone libraries were formed by these two genera. Selenomonas sputigena, the species most commonly detected, was found in nine of 10 subjects. Other species of Selenomonas were often present at high levels, including S. noxia, Selenomonas sp. EW084, Selenomonas sp. EW076, Selenomonas FT050, Selenomonas sp. P2PA_80, and Selenomonas sp. strain GAA14. The classical putative periodontal pathogens, such as, Aggregatibacter actinomycetemcomitans, was below the limit of detection and was not detected. CONCLUSION: These data suggest that other species, notably species of Selenomonas, may be associated with disease in generalized aggressive periodontitis subjects.