The run-on oligomer filament enzyme mechanism of SgrAI: Part 2. Kinetic modeling of the full DNA cleavage pathway.

Filament or run-on oligomer formation by enzymes is now recognized as a widespread phenomenon with potentially unique enzyme regulatory properties and biological roles. SgrAI is an allosteric type II restriction endonuclease that forms run-on oligomeric filaments with activated DNA cleavage activity and altered DNA sequence specificity. In this two-part work, we measure individual steps in the run-on oligomer filament mechanism to address specific questions of cooperativity, trapping, filament growth mechanisms, and sequestration of activity using fluorophore-labeled DNA, kinetic FRET measurements, and reaction modeling with global data fitting. The final models and rate constants show that the assembly step involving association of SgrAI-DNA complexes into the run-on oligomer filament is relatively slow (3-4 orders of magnitude slower than diffusion limited) and rate-limiting at low to moderate concentrations of SgrAI-DNA. The disassembly step involving dissociation of complexes of SgrAI-DNA from each other in the run-on oligomer filament is the next slowest step but is fast enough to limit the residence time of any one copy of SgrAI or DNA within the dynamic filament. Further, the rate constant for DNA cleavage is found to be 4 orders of magnitude faster in the run-on oligomer filament than in isolated SgrAI-DNA complexes and faster than dissociation of SgrAI-DNA complexes from the run-on oligomer filament, making the reaction efficient in that each association into the filament likely leads to DNA cleavage before filament dissociation.

The run-on oligomer filament enzyme mechanism of SgrAI: Part 1. Assembly kinetics of the run-on oligomer filament.

Filament or run-on oligomer formation by metabolic enzymes is now recognized as a widespread phenomenon having potentially unique enzyme regulatory properties and biological roles, and its dysfunction is implicated in human diseases such as cancer, diabetes, and developmental disorders. SgrAI is a bacterial allosteric type II restriction endonuclease that binds to invading phage DNA, may protect the host DNA from off-target cleavage activity, and forms run-on oligomeric filaments with enhanced DNA-cleavage activity and altered DNA sequence specificity. However, the mechanisms of SgrAI filament growth, cooperativity in filament formation, sequestration of enzyme activity, and advantages over other filament mechanisms remain unknown. In this first of a two-part series, we developed methods and models to derive association and dissociation rate constants of DNA-bound SgrAI in run-on oligomers and addressed the specific questions of cooperativity and filament growth mechanisms. We show that the derived rate constants are consistent with the run-on oligomer sizes determined by EM analysis and are most consistent with a noncooperative growth mode of the run-on oligomer. These models and methods are extended in the accompanying article to include the full DNA-cleavage pathway and address specific questions related to the run-on oligomer mechanism including the sequestration of DNA-cleavage activity and trapping of products.

Oestrogen receptor Rsa I gene polymorphism in osteoporosis periodontitis patients with or without dental fluorosis.

Background & objectives: : There is a paucity of information on association between dental fluorosis, osteoporosis and periodontitis. The aim of this pilot study was to evaluate oestrogen receptor (ER). Rsa 1: gene polymorphism in osteoporosis periodontitis patients with and without dental fluorosis. Methods: : Twenty one primary osteoporotic patients suffering from periodontitis with dental fluorosis and 20 primary osteoporotic patients suffering from periodontitis without dental fluorosis participated in this study. Periodontitis was diagnosed based on age, gender T-scores using clinical parameters such as plaque scores, gingival bleeding scores and probing pocket depth, clinical attachment level (CAL) and severity of dental fluorosis. DNA was genotyped at the RsaI RFLP (in exon 5) inside the ER gene to study ER Rsa I gene polymorphism in osteoporosis periodontitis patients with and without dental fluorosis. Results: : Patients with dental fluorosis had higher degree of osteoporosis than those without fluorosis. CAL was significantly higher (P <0.05) in those with dental fluorosis compared with those without. Rr heterozygote (21.95%) was observed in patients without fluorosis whereas RR mutant homozygote was absent in both the groups. Rr wild homozygote type was seen more in the patients with fluorosis (51.21%). Significant differences were found in distribution of these genotypes between patients with and without dental fluorosis. Interpretation & conclusions: : This preliminary study showed the presence of ER I gene polymorphism in osteoporosis periodontitis patients without dental fluorosis. Further studies with large sample size are needed to confirm the association shown in this preliminary study.

Vitamin D Receptor TaqI Gene Polymorphism and Dental Caries in Czech Children.

AIM: We analyzed the VDR TaqI (rs731236) gene polymorphism in children with and those without dental caries. METHODS: A total of 388 subjects, 153 caries-free (with decayed/missing/filled teeth [DMFT] = 0) and 235 children with dental caries (DMFT ≥1), were genotyped by the TaqMan method. RESULTS: Although no significant differences in VDR TaqI allele and genotype frequencies between caries-free and caries-affected children were detected, a significant association between this polymorphism and gingivitis was found (p < 0.05). CONCLUSIONS: In contrast to previous studies from China and Turkey, the VDR TaqI gene variant cannot be used as a marker for identification of Czech children with increased dental caries risk.

Sequence-Independent Cloning and Post-Translational Modification of Repetitive Protein Polymers through Sortase and Sfp-Mediated Enzymatic Ligation.

Repetitive protein-based polymers are important for many applications in biotechnology and biomaterials development. Here we describe the sequential additive ligation of highly repetitive DNA sequences, their assembly into genes encoding protein-polymers with precisely tunable lengths and compositions, and their end-specific post-translational modification with organic dyes and fluorescent protein domains. Our new Golden Gate-based cloning approach relies on incorporation of only type IIS BsaI restriction enzyme recognition sites using PCR, which allowed us to install ybbR-peptide tags, Sortase c-tags, and cysteine residues onto either end of the repetitive gene polymers without leaving residual cloning scars. The assembled genes were expressed in Escherichia coli and purified using inverse transition cycling (ITC). Characterization by cloud point spectrophotometry, and denaturing polyacrylamide gel electrophoresis with fluorescence detection confirmed successful phosphopantetheinyl transferase (Sfp)-mediated post-translational N-terminal labeling of the protein-polymers with a coenzyme A-647 dye (CoA-647) and simultaneous sortase-mediated C-terminal labeling with a GFP domain containing an N-terminal GG-motif in a one-pot reaction. In a further demonstration, we installed an N-terminal cysteine residue into an elastin-like polypeptide (ELP) that was subsequently conjugated to a single chain poly(ethylene glycol)-maleimide (PEG-maleimide) synthetic polymer, noticeably shifting the ELP cloud point. The ability to straightforwardly assemble repetitive DNA sequences encoding ELPs of precisely tunable length and to post-translationally modify them specifically at the N- and C- termini provides a versatile platform for the design and production of multifunctional smart protein-polymeric materials.

Locked nucleic acid couples with Fok I nucleases to target and cleave hepatitis B virus's gene in vitro.

Hepatitis B virus (HBV) is a dented double-stranded DNA virus. After infecting human hepatic cells, it forms cccDNA that replicates persistently and integrates randomly into the host's genome during the process of reserve transcription. On average, in each cell with chronic HBV infection, there are about 33 copies of cccDNA with a half of 35-57 days, which can be difficult to eradicate. A new strategy is to inhibit HBV transcription by using locked nucleic acid (LNA). Besides, cleaving HBV genome by targeted genome editing technologies could potentially cure patients. In this study, we explored new genome editing tools for HBV treatment. Based on LNA's ability to form triple helix by binding to duplex DNA, its stability towards nuclease and polymerase, and its sensitivity to single base mismatches, we designed LNA-modified oligonucleotides as DNA binding domain to effectively increase the specificity of target gene recognition. Meanwhile, by utilizing the small molecular weight and dimerization dependent activity of nuclease Fok I, we used Fok I recombinant dimer protein as DNA cleavage domain. Here, we established a method by chemical coupling of LNA-oligonucleotide with Fok I cleavage domain, and also validated the targeted cleavage of HBV genes with our new tools in vitro. These results provide new possibilities for future in vivo anti-virus gene therapy with high specificity and no integration risk.

The Role of Vitamin D Receptor Polymorphisms on Dental Caries.

OBJECTIVE: To determine the association between the ApaI, FokI, Cdx2 and TaqI polymorphisms of vitamin D receptor (VDR) gene in caries-active (high-moderate) and caries-free children. STUDY DESIGN: A hundred and fifty children (75 males, 75 females, mean age: 10.19 ± 1.61 years) were included in the study. The subjects were divided into three groups as high caries risk group (DMFT, dft>4)(n=55), moderate caries risk group (DMFT, dft=1-4)(n=57) and caries-free group (n=38). From each individual, blood samples were collected and DNA was extracted. The VDR gene was genotyped for the polymorphisms ApaI, FokI, Cdx2 and TaqI using polymerase chain reaction and restriction fragment length polymorphism methods. All data were analyzed by chi-square test, Fisher's exact test and t test. RESULTS: There was statistically significant difference in the frequency of TaqI genotypes (tt) between caries-active and caries-free children (p=0.029). No statistically significant differences were detected between ApaI, FokI, Cdx2 genotypes and dental caries. CONCLUSION: In the future, VDR gene polymorphisms may be used as a marker for the identification of patients with high caries risk.

A Modified Shuttle Plasmid Facilitates Expression of a Flavin Mononucleotide-Based Fluorescent Protein in Treponema denticola ATCC 35405.

Oral pathogens, including Treponema denticola, initiate the dysregulation of tissue homeostasis that characterizes periodontitis. However, progress of research on the roles of T. denticola in microbe-host interactions and signaling, microbial communities, microbial physiology, and molecular evolution has been hampered by limitations in genetic methodologies. This is typified by an extremely low transformation efficiency and inability to transform the most widely studied T. denticola strain with shuttle plasmids. Previous studies have suggested that robust restriction-modification (R-M) systems in T. denticola contributed to these problems. To facilitate further molecular genetic analysis of T. denticola behavior, we optimized existing protocols such that shuttle plasmid transformation efficiency was increased by >100-fold over prior reports. Here, we report routine transformation of T. denticola ATCC 35405 with shuttle plasmids, independently of both plasmid methylation status and activity of the type II restriction endonuclease encoded by TDE0911. To validate the utility of this methodological advance, we demonstrated expression and activity in T. denticola of a flavin mononucleotide-based fluorescent protein (FbFP) that is active under anoxic conditions. Addition of routine plasmid-based fluorescence labeling to the Treponema toolset will enable more-rigorous and -detailed studies of the behavior of this organism.

Secretion of recombinant archeal lipase mediated by SVP2 signal peptide in Escherichia coli and its optimization by response surface methodology.

Towards the targeting of recombinant Thermoanaerobacter thermohydrosulfuricus lipase (TtL) for secretion into the culture medium of Escherichia coli, we have investigated a combination of the archeal lipase gene with a Salinovibrio metalloprotease (SVP2) signal peptide sequence. The SVP2 signal peptide has shown all necessary features of a leader sequence for high level secretion of a recombinant target protein in E. coli. Two sets of primers were designed for amplification of the corresponding gene fragments by PCR. Firstly, the PCR product of the TtL gene with designed restriction sites of SacI and HindIII was cloned into pQE-80L plasmid, named as pQE80L-TtL. Afterwards, the amplified fragment of SVP2 signal peptide with EcoRI and SacI restriction sites was also cloned into pQE80L-TtL and the final construct pQE-STL was obtained. A study on the extracellular expression of recombinant STL revealed that most of the enzyme activity was located in the periplasmic space. Glycine and Triton X-100 were investigated to determine whether the leakage of recombinant STL from the outer membrane was promoted, and it was revealed that glycine has a positive effect. Statistical media optimization design was then applied to optimize the effect of seven factors including glycine, Triton X-100, IPTG, yeast extract concentration, incubation time, induction time, and temperature on the extracellular expression of STL. The optimum conditions for the secretion of the lipase was obtained by incubating recombinant E. coli BL21 cells in the medium supplemented by 1.27% glycine and 24h of incubation in the presence of 0.2mM IPTG concentration.

Novel polymer linkers for single molecule AFM force spectroscopy.

Flexible polymer linkers play an important role in various imaging and probing techniques that require surface immobilization, including atomic force microscopy (AFM). In AFM force spectroscopy, polymer linkers are necessary for the covalent attachment of molecules of interest to the AFM tip and the surface. The polymer linkers tether the molecules and provide their proper orientation in probing experiments. Additionally, the linkers separate specific interactions from nonspecific short-range adhesion and serve as a reference point for the quantitative analysis of single molecule probing events. In this report, we present our results on the synthesis and testing of a novel polymer linker and the identification of a number of potential applications for its use in AFM force spectroscopy experiments. The synthesis of the linker is based on the well-developed phosphoramidate (PA) chemistry that allows the routine synthesis of linkers with predetermined lengths and PA composition. These linkers are homogeneous in length and can be terminated with various functional groups. PA linkers with different functional groups were synthesized and tested in experimental systems utilizing different immobilization chemistries. We probed interactions between complementary DNA oligonucleotides; DNA and protein complexes formed by the site-specific binding protein SfiI; and interactions between amyloid peptide (Aß42). The results of the AFM force spectroscopy experiments validated the feasibility of the proposed approach for the linker design and synthesis. Furthermore, the properties of the tether (length, functional groups) can be adjusted to meet the specific requirements for different force spectroscopy experiments and system characteristics, suggesting that it could be used for a large number of various applications.

Evaluation of vitamin D receptor (VDR) gene polymorphisms (FokI, TaqI and ApaI) in a family with dentinogenesis imperfecta.

Dentinogenesis imperfecta Type II (DGI-II) is a condition inherited as an autosomal dominant trait and characterized by abnormal dentine structure affecting both the primary and secondary dentitions. The genetic etiology of the disease still remains unclear, suggesting a genetically heterogeneous background. The aim of this study is to manifest briefly DGI-II and to investigate the association between BsmI, TaqI and FokI polymorphisms of Vitamin D receptor (VDR) gene and dentinogenesis imperfecta type II in a Turkish family by PCR-RFLP methodology. The affected mother and her two affected daughters were bb for BsmI polymorphism, whereas her unaffected son and her husband were Bb for the same polymorphism. One of the affected children was tt, the rest of the family were Tt for TaqI polymorphism, and all of the enrolled subjects were FF for FokI polymorphism. As a conclusion, BsmI polymorphism bb seems to be associated with (DGI-II), but should be examined in larger numbers in order to be considered as a risk factor.

Effects of long-term use of antiretroviral therapy on the prevalence of oral Epstein-Barr virus.

BACKGROUND: The objectives of this study were to determine (i) the prevalence of oral Epstein-Barr virus (EBV) in HIV-infected subjects compared to non-HIV controls and (ii) the effects of long-term use of antiretroviral therapy (ART) on the prevalence of oral EBV. METHODS: A cross-sectional study was performed in HIV-infected subjects with and without ART, and non-HIV individuals. DNA in saliva samples was extracted and used as a template to detect EBV BamH1W and EBNA1 by quantitative polymerase chain reaction. Student t-test and ANOVA test were performed to determine the prevalence rates among groups. RESULTS: Forty-nine HIV-infected subjects: 37 on ART (age range 23-54 year, mean 37 year), 12 not on ART (age range 20-40 year, mean 31 year), and 20 non-HIV controls (age range 19-53 year, mean 31 year) were enrolled. The numbers of EBV BamH1W in saliva were found to be significantly higher in HIV-infected subjects than non-HIV controls (80% vs. 20%, mean = 12118 vs. 134 copies/10(5) cells, P < 0.001). HIV-infected subjects who were on ART had significantly lower numbers of EBV BamH1W than those who were not (mean = 4102 vs. 138613 copies/10(5) cells, P = 0.011). The numbers were significantly lower in those who received long-term ART compared with short-term (mean = 1401 vs. 11124 copies/10(5) cells, P = 0.034). No significant difference was observed between the groups when using EBNA1 primers. CONCLUSIONS: Prevalence of oral EBV was significantly higher in HIV-infected subjects than non-HIV-controls. The numbers of the virus were significantly decreased by ART. Long-term use of ART did not increase oral EBV.

Alveolar bone level is not associated with vitamin D receptor gene polymorphism and bone density in mandible.

The objective of this study was to determine, using digital panoramic radiographs, whether the bone level at the alveolar crest is related to the mandibular bone density and/or to vitamin D receptor (VDR) gene polymorphisms. We analyzed 319 digital panoramic radiographs from the same number of patients. Alveolar bone level was expressed as percentage of root length. The mandibular cortical width index was calculated as a measure of mandibular bone density, and, in 72 randomly selected cases, the haplotype of the VDR gene (BsmL) was determined by polymerase chain reaction. Alveolar bone level was not related to the mandibular cortical width index (p = 0.568) or VDR gene expression (p = 0.575). Bone loss was greater in smokers than in non-smokers (p = 0.036), and the mandibular cortical width index was higher in males (p = 0.04), the older age group (p = 0.032), and in those with more teeth (p = 0.01). Multivariate analysis confirmed the association between these variables and alveolar bone loss. Alveolar bone loss showed no significant relationship with the mandibular bone density evaluated on digital panoramic radiographs or with VDR genotype (BsmL) in Caucasian females and males aged under 47 years.

Analysis of genotypic variation in genes associated with virulence in Aggregatibacter actinomycetemcomitans clinical isolates.

BACKGROUND AND OBJECTIVE: Although certain serotypes of Aggregatibacter actinomycetemcomitans are associated more with aggressive periodontitis than are other serotypes, the correlation between distinct lineages and virulence traits in this species is poorly understood. This study aimed to evaluate the polymorphism of genes encoding putative virulence factors of clinical isolates, and to correlate these findings with A. actinomycetemcomitans serotypes, genotypes and periodontal status of the hosts. MATERIAL AND METHODS: Twenty-six clinical isolates from diverse geographic populations with different periodontal conditions were evaluated. Genotyping was performed using pulse-field gel electrophoresis. Polymorphisms in the genes encoding leukotoxin, Aae, ApaH and determinants for serotype-specific O polysaccharide were investigated. RESULTS: The isolates were classified into serotypes a-f, and exhibited three apaH genotypes, five aae alleles and 25 macrorestriction profiles. Two serotype b isolates (7.7%), obtained from Brazilian patients with aggressive periodontitis, were associated with the highly leukotoxic genotype; these isolates showed identical fingerprint patterns and aae and apaH genotypes. Serotype c, obtained from various periodontal conditions, was the most prevalent among Brazilian isolates, and isolates were distributed in two aae alleles, but formed a genetically distinct group based on apaH analysis. Cluster analysis showed a close relationship between fingerprinting genotypes and serotypes/apaH genotypes, but not with aae genotypes. CONCLUSION: Apart from the deletion in the ltx promoter region, no disease-associated markers were identified. Non-JP2-like strains recovered from individuals with periodontal disease exhibited considerable genetic variation regarding aae/apaH genotypes, serotypes and XhoI DNA fingerprints.

BsmI, TaqI, ApaI, and FokI polymorphisms in the vitamin D receptor gene and periodontitis: a meta-analysis of 15 studies including 1338 cases and 1302 controls.

AIM: A meta-analysis was conducted in order to investigate the potential association of vitamin D receptor (VDR) gene polymorphisms with susceptibility to aggressive and chronic periodontal disease. MATERIAL AND METHODS: A database search yielded a total of 15 studies involving 1338 cases and 1302 controls. Four polymorphisms were included in the meta-analysis: VDR TaqI (rs731236), VDR BsmI (rs1544410), VDR FokI (rs2228570), and VDR ApaI (rs7975232). Odds ratios (ORs) along with their 95% confidence intervals (CIs) were computed to compare the distribution of alleles and genotypes between cases and controls. RESULTS AND CONCLUSIONS: The combined results based on all studies showed that (1) chronic periodontitis cases had a significantly lower frequency of bb genotype of BsmI [OR=0.63, 95% CI=0.42, 0.94; p=0.02] in Asians; (2) chronic periodontitis cases had a significantly higher frequency of AA genotype of ApaI (OR=2.20, 95% CI=1.39, 3.48; p<0.001) in Asians; (3) chronic periodontitis cases had a weak significantly higher frequency of TT genotype of TaqI (OR=1.86, 95% CI=1.002, 3.46; p=0.049) in Asians. After Bonferroni's correction, we found that in Asians chronic periodontitis cases still had a significantly higher frequency of AA genotype of ApaI. No significant difference was found in any genotype of FokI. No association was found for all the VDR gene polymorphisms examined as far as the aggressive form of the disease is concerned. Future studies need to focus on the possible biological consequences and mechanisms of the VDR genetic variants. The current findings confirm that VDR gene is a candidate gene for periodontitis.

The effects of estrogen receptor α polymorphism on the prevalence of symptomatic temporomandibular disorders.

PURPOSE: The aim of this study is to identify any association between variants of the polymorphic estrogen receptor gene and various symptoms of temporomandibular disorder (TMD) including pain in the temporomandibular joint and masticatory muscles, joint crepitus, limited range of jaw movement, and bone changes in the condylar head. PATIENTS AND METHODS: Seventy-four patients with TMD were selected according to the Research Diagnostic Criteria for TMD for the study group. Sixty-four patients without TMD were selected as the control group. Genomic DNA was extracted from the epithelial layer of buccal mucosa. After amplification by polymerase chain reaction, direct haplotyping was undertaken to study the restriction fragment length polymorphism of PvuII and XbaI for the α estrogen receptor. Genomic prevalences in each of the symptom categories were analyzed by use of the χ(2) test. RESULTS: The haplotypes PX, Px, and px constituted 23.0%, 18.9%, and 58.1%, respectively, of the total α estrogen receptor alleles in the study group. The haplotype Px was found to be relatively more prevalent in subjects who had mouth opening limitation, and the haplotype PX was more prevalent in those patients with condylar head bone changes. However, neither of these observations carried statistical significance. CONCLUSION: Although certain symptoms of TMD were found to have a relatively higher prevalence of one form or another of the estrogen receptor allele, no haplotype was confirmed to be a significant marker of TMD risk.

[FBAT analysis of polymorphisms of vitamin D receptor (Taq I and Fok I) in aggressive periodontitis families].

OBJECTIVE: To analyze the correlation between polymorphisms of the vitamin D receptor gene (Taq I and Fok I) and the aggressive periodontitis by FBAT method. METHODS: 93 AgP nuclear families including 93 probands and their 155 relatives were recruited. The genotype frequency and polymorphism for VDR for the patients and their pedigree were detected by using polymerase chain reaction (PCR) combined with restriction fragment length polymorphism (RFLP). RESULTS: The frequency of allele T and t accounted for 94.6 % and 5.4 % in the total populations. No tt genotype were detected. The fathers of probands carried more allele t than the mothers(9.8% vs 1.6%, P=0.005). The frequency of allele F and f accounted for 57.1 % and 42.9 % in the total populations. The result of family based associated test (FBAT) including additive model, dominant model and recessive model showed that different alleles of Taq I and Fok I had no correlation with the onset of AgP(P>0.05). CONCLUSION: This was the first time to detect the VDR gene polymorphisms in Chinese families. The result of FBAT analysis can not show the correlation between vitamin D receptor gene polymorphisms (Taq I and Fok I) and the onset of AgP.

ATP-powered molecular recognition to engineer transient multivalency and self-sorting 4D hierarchical systems.

Biological systems organize multiple hierarchical structures in parallel, and create dynamic assemblies and functions by energy dissipation. In contrast, emerging artificial non-equilibrium self-assembling systems have remained relatively simplistic concerning hierarchical design, and non-equilibrium multi-component systems are uncharted territory. Here we report a modular DNA toolbox allowing to program transient non-equilibrium multicomponent systems across hierarchical length scales by introducing chemically fueled molecular recognition orchestrated by reaction networks of concurrent ATP-powered ligation and cleavage of freely programmable DNA building blocks. Going across hierarchical levels, we demonstrate transient side-chain functionalized nucleic acid polymers, and further introduce the concept of transient cooperative multivalency as a key to bridge length scales to pioneer fuel-driven encapsulation, self-assembly of colloids, and non-equilibrium transient narcissistic colloidal self-sorting on a systems level. The fully programmable and functionalizable DNA components pave the way to design chemically fueled 4D (3 space, 1 time) molecular multicomponent systems and autonomous materials.

Assessment of intraradicular bacterial composition by terminal restriction fragment length polymorphism analysis.

BACKGROUND: The aim of the study was to assess the bacterial community structures associated with endodontic infections using terminal restriction fragment length polymorphism (T-RFLP), and to investigate the correlation of whole community profiles with the manifestation of particular clinical features. METHODS: Intraradicular samples were collected from 34 subjects and classified into three study groups based on the observed clinical symptoms: acute (n = 16), sub-acute (n = 8), and asymptomatic (n = 10). Genomic DNA was extracted from each sample, submitted to polymerase chain reaction using a fluorescently labeled 16S ribosomal DNA forward primer, and digested with two tetrameric endonucleases (HhaI and MspI). The terminal restriction fragments (T-RFs) were subsequently discriminated in an automated DNA sequencer, and the results were filtered using a statistics-based criterion. RESULTS: Totals of 138 (HhaI) and 145 (MspI) unique T-RFs were detected (means 13.1 and 11.9) and there was high inter-subject variability in the bacterial assemblages. Odds-ratio analysis unveiled the existence of higher order groups of positively associated T-RFs, restating the concept that intricate ecological relationships may take place in the root canal space. A significantly greater T-RF prevalence was detected in acute cases, suggesting a straight correlation between species richness and spontaneous pain. CONCLUSION: Overall, no T-RFLP profile representing a specific bacterial consortium could be associated with the manifestation of symptoms of endodontic origin.

T-RFLP-based mcrA gene analysis of methanogenic archaea in association with oral infections and evidence of a novel Methanobrevibacter phylotype.

INTRODUCTION: Increasing evidence suggests a role for methanogenic archaea (methanogens) in human health and disease via syntrophic interactions with bacteria. Here we assessed the prevalence and distribution of methanogens and possible associations with bacteria in oral biofilms. METHODS: Forty-four periodontal and 32 endodontic samples from necrotic teeth with radiographic evidence of apical periodontitis were analysed. Terminal restriction fragment length polymorphism analysis based on the mcrA gene, specific to methanogens, was applied. The prevalence and amounts of methanogens in endodontic samples were compared with those of Porphyromonas gingivalis, Prevotella intermedia, Tannerella forsythia, Treponema spp. and Synergistes spp. based on real-time quantitative polymerase chain reactions. RESULTS: Besides dominance of the mcrA gene corresponding to Methanobrevibacter oralis, one mcrA gene type, for which no cultivated member has been reported previously, was identified in five periodontal samples and one endodontic sample. Rates of non-synonymous vs. synonymous nucleotide substitutions suggest that this mcrA gene type codes for a functionally active methyl-coenzyme M reductase. Methanobrevibacter smithii, the prominent methanogen in the human gut system, was not detected. Mean proportions of methanogens were comparable to Synergistes spp. ranging from 0.5 to 1.0% of the total microbial community. Treponema spp. dominated with a mean proportion of 10%, while the mean proportions of the other endodontic pathogens were below 0.1%. A positive association between methanogens and Synergistes spp. was found. CONCLUSION: Our data provide evidence of a novel, as yet uncultured methanogenic phylotype in association with oral infections, and indicate possible interactions between methanogens and Synergistes spp., the nature of which deserves further investigation.