Highly Sensitive Assay of Methyltransferase Activity Based on an Autonomous Concatenated DNA Circuit.

Methyltransferase-involved DNA methylation is one of the most important epigenetic processes, making the ultrasensitive MTase assay highly desirable in clinical diagnosis as well as biomedical research. Traditional single-stage amplification means often achieve linear amplification that might not fulfill the increasing demands for detecting trace amount of target. It is desirable to construct multistage cascaded amplifiers that allow for enhanced signal amplifications. Herein, a powerful nonenzymatic MTase-sensing platform is successfully engineered based on a two-layered DNA circuit, in which the upstream catalytic hairpin assembly (CHA) circuit successively generates DNA product that could be used to activate the downstream hybridization chain reaction (HCR) circuit, resulting in the generation of a dramatically amplified fluorescence signal. In the absence of M.SssI MTase, HpaII endonuclease could specifically recognize the auxiliary hairpin substrate and then catalytically cleave the corresponding recognition site, releasing a DNA fragment that triggers the CHA-HCR-mediated FRET transduction. Yet the M.SssI-methylated hairpin substrate could not be cleaved by HpaII enzyme, and thus prohibits the CHA-HCR-mediated FRET generation, providing a substantial signal difference with that of MTase-absent system. Taking advantage of the high specificity of multiple-guaranteed recognitions of MTase/endonuclease and the synergistic amplification features of concatenated CHA-HCR circuit, this method enables an ultrasensitive detection of MTase and its inhibitors in serum and E. coli cells. Furthermore, the rationally assembled CHA-HCR also allows for probing other different biotransformations through a facile design of the corresponding substrates. It is anticipated that the infinite layer of multilayered DNA circuit could further improve the signal gain of the system for accurately detecting other important biomarkers, and thus holds great promise for cancerous treatment and biomedical research.

Rapid urease test (RUT) for evaluation of urease activity in oral bacteria in vitro and in supragingival dental plaque ex vivo.

BACKGROUND: Urease is an enzyme produced by plaque bacteria hydrolysing urea from saliva and gingival exudate into ammonia in order to regulate the pH in the dental biofilm. The aim of this study was to assess the urease activity among oral bacterial species by using the rapid urease test (RUT) in a micro-plate format and to examine whether this test could be used for measuring the urease activity in site-specific supragingival dental plaque samples ex vivo. METHODS: The RUT test is based on 2% urea in peptone broth solution and with phenol red at pH 6.0. Oral bacterial species were tested for their urease activity using 100 µl of RUT test solution in the well of a micro-plate to which a 1 µl amount of cells collected after growth on blood agar plates or in broth, were added. The color change was determined after 15, 30 min, and 1 and 2 h. The reaction was graded in a 4-graded scale (none, weak, medium, strong). Ex vivo evaluation of dental plaque urease activity was tested in supragingival 1 µl plaque samples collected from 4 interproximal sites of front teeth and molars in 18 adult volunteers. The color reaction was read after 1 h in room temperature and scored as in the in vitro test. RESULTS: The strongest activity was registered for Staphylococcus epidermidis, Helicobacter pylori, Campylobacter ureolyticus and some strains of Haemophilus parainfluenzae, while known ureolytic species such as Streptococcus salivarius and Actinomyces naeslundii showed a weaker, variable and strain-dependent activity. Temperature had minor influence on the RUT reaction. The interproximal supragingival dental plaque between the lower central incisors (site 31/41) showed significantly higher scores compared to between the upper central incisors (site 11/21), between the upper left first molar and second premolar (site 26/25) and between the lower right second premolar and molar (site 45/46). CONCLUSION: The rapid urease test (RUT) in a micro-plate format can be used as a simple and rapid method to test urease activity in bacterial strains in vitro and as a chair-side method for testing urease activity in site-specific supragingival plaque samples ex vivo.

An oligodeoxyribonucleotide containing 5-formyl-2'-deoxycytidine (fC) at the CpG site forms a covalent complex with DNA cytosine-5 methyltransferases (DNMTs).

5-Methylcytosine (mC) is known to induce epigenetic changes. Ten-eleven translocation (TET) enzymes produce the further oxidized 5-substituted cytosine derivatives, 5-formylcytosine (fC) and 5-carboxylcytosine (caC). However, their roles are unclear thus far. Here, we synthesized oligodeoxyribonucleotides (ODNs) containing 5-formyl-2'-deoxycytidine and examined their interactions with DNA cytosine-5 methyltransferase (DNMT). We found that the ODN sequence containing fCpG formed a covalent complex with both bacterial and mouse recombinant DNMTs in the absence of any cofactors. The covalent bonding with DNMT suggests that the fCpG sequence in DNA may play a role in epigenetic regulation.

TEM-1-encoding small plasmids impose dissimilar fitness costs on Haemophilus influenzae and Haemophilus parainfluenzae.

Only two beta-lactamases, TEM-1 and ROB-1, have been observed in Haemophilus influenzae, while four different TEM but no ROB enzymes have been found in Haemophilus parainfluenzae. In order to investigate the mechanisms behind the dissemination of small beta-lactamase-encoding plasmids in H. influenzae and H. parainfluenzae, we assessed the fitness cost of three TEM-1- (pPN223, pA1209, pA1606), one TEM-15- (pSF3) and one ROB-1-bearing (pB1000) plasmid when expressed in either bacterial species. All plasmids were stable in H. influenzae and H. parainfluenzae except pB1000, which showed on average (sample mean) 76% curing in H. parainfluenzae after 5  days of subculture. Competition assays between isogenic strains with and without plasmid showed no competitive disadvantage of pPN223 and pA1606 in H. influenzae, or of pA1209 in H. parainfluenzae. In contrast, pSF3 and pB1000 were associated with significant competitive disadvantages in both species. Some of the competitive disadvantages may be related to differences in plasmid copy number and mRNA expression of the beta-lactamase genes, as revealed by quantitative PCR analysis. In conclusion, plasmids encoding TEM beta-lactamases isolated from H. influenzae and H. parainfluenzae can be stably transferred between species. The fast curing of pB1000 in H. parainfluenzae observed in this study correlates to the fact that ROB-1 has never been reported for this species. TEM-1-encoding plasmids are associated with the lowest level of fitness cost, but different TEM-1 plasmids confer different levels of fitness cost on the two hosts.

Characterization of extended-spectrum beta-lactamase-producing isolates of Haemophilus parainfluenzae.

OBJECTIVES: To characterize the beta-lactam resistance mechanisms of two clinical isolates of cefotaxime-resistant Haemophilus parainfluenzae recovered from patients in South Africa. METHODS: The relatedness of isolates and plasmids was assessed using PFGE and restriction enzyme analysis, respectively. Plasmid-mediated and chromosomally integrated bla(TEM) genes and ftsI genes were sequenced, and the plasmid-mediated bla(TEM-15) was used to transform a range of control organisms. RESULTS: The two isolates were found to be unique according to PFGE, but had an identical 3.7 kb plasmid encoding a TEM-15 beta-lactamase. Both isolates also had substitutions in penicillin binding protein 3 (PBP3) consistent with substitutions known to exist in beta-lactamase-negative ampicillin-resistant (BLNAR) strains of Haemophilus influenzae. The cefotaxime MICs for control strains of H. influenzae, H. parainfluenzae and BLNAR H. influenzae transformed with the plasmid-mediated bla(TEM-15) were 1.0, 1.0 and 4.0 mg/L, respectively, compared with 16.0 and 8.0 mg/L, respectively, for the two parent H. parainfluenzae. CONCLUSIONS: The high-level cefotaxime resistance in the H. parainfluenzae isolates was due to a combination of a plasmid-mediated TEM-15 extended-spectrum beta-lactamase with altered PBP3 probably contributing. Other contributing resistance mechanisms could not be excluded.