Optimizing Desmostachya bipinnata-derived platinum nanoparticles for enhanced antibacterial and biofilm reduction.

This study presents the green synthesis and comprehensive characterization of platinum nanoparticles (PtNPs) using Desmostachya bipinnata (Db) extract, incorporated into two innovative mouthwash formulations (MW1 and MW2). UV-Vis spectroscopy confirmed the successful synthesis of PtNPs, with distinct absorption peaks between 250 and 600 nm. Fourier-transform infrared (FTIR) spectroscopy identified hydroxyl and carbonyl functional groups, critical for the bioreduction and stabilization of PtNPs. High-resolution transmission electron microscopy (HR-TEM) revealed uniformly dispersed, spherical nanoparticles with a size range of 10-20 nm, while dynamic light scattering (DLS) confirmed a hydrodynamic diameter of 10-30 nm and a low polydispersity index (PDI) of 0.238, indicating excellent stability. Both formulations exhibited robust antimicrobial, antibiofilm, and anti-plaque properties, with MW2 showing superior efficacy, particularly against Staphylococcus aureus and Escherichia coli, as well as a notable 70 % reduction in biofilm formation and a 60 % plaque reduction within 2 h of treatment. The study underscores the potential of Desmostachya bipinnata-derived PtNPs as a promising alternative to conventional mouthwash, offering enhanced antimicrobial efficacy, biofilm disruption, and plaque prevention, alongside excellent stability and biocompatibility for oral healthcare applications.

The anti-adherence activity and bactericidal effect of GO against Streptococcus mutans from Iraqi dental patients.

The high rate of microbes and their biological activity in the patient's mouth is a concern in the domains of dental caries and periodontal disease. The study aimed to shed light on the relationship between graphene oxide's nanoparticles (nGOs) antimicrobial properties and the growth of dental pathogenic bacteria. The forty swab samples were frequently collected from the patient's cavity mouth between November 2019 and January 2020, from patients who visited dentist clinics in Baghdad by taking swabs from mouth cavities with various dental caries with two age groups (5-17) and (18-60) from male and female to streaking them on Brain-Heart Infusion (BHI) agar, then identified by re-streaking on Mitis Salivarius Bacitracin (MSB) agar. All isolates were confirmed as Streptococcus mutans after API 20 Strep method. As well as the Colony Forming Units (CFU) were then determined after diluting the bacterial cell suspensions to obtain cell samples containing 1.5 × 108 CFU/ ml. The collagen-binding adhesin (cnm) and glucosyltransferases (gtf) of S. mutans genes were identified using polymerase chain reaction (PCR) method before and after exposure to the nGOs, which were prepared in different pulse laser energy (500, 600, and 700 mJ) with presence and absence of the magnetic field, and the data have been analyzing. After counting the CFU, the nGOs shows high effectiveness inhibiting the growth of S. mutans. This research provides definitive answers about the relationship between nGOs, antibacterial caries, and periodontal disease.

Genetic variation in the glucosyltransferase-B gene of Streptococcus mutans and its relationship with caries experience in children from Córdoba, Argentina, and with strains from other countries.

BACKGROUND: Caries is a worldwide distributed oral disease of multifactorial nature, with Streptococcus mutans being the most commonly isolated bacterial agent. The glycosyltransferases of this bacterium would play an essential role in the aetiology and pathogenesis of caries. AIM: We explored how the glucosyltransferase-B (gtf-B) gene variability of S. mutans from children in central Argentina correlated with their caries experience and how these strains were genetically related to those of other countries. DESIGN: Dental examinations were performed on 59 children; dmft and DMFT indexes were calculated. From stimulated saliva, S. mutans was grown and counted (CFU/mL). From bacterial DNA, the gtf-B gene was amplified and sequenced. Alleles were identified and their genealogical relationships established. Clinical, microbiological, and genetic variables were correlated with caries experience. Our sequences were included in a matrix with those from 16 countries (n = 358); genealogical relationships among alleles were obtained. Population genetic analyses were performed for countries with >20 sequences. RESULTS: The mean dmft + DMFT was 6.45. Twenty-two gtf-B alleles were identified here, which showed low genetic differentiation in the network. Caries experience was correlated with CFU/mL, but not with allele variation. Low differentiation was found among the 70 alleles recovered from the 358 sequences and among the countries analyzed. CONCLUSION: In this study, caries experience in children was correlated with the number of CFU/mL of S. mutans but not with the gtf-B gene variability. Combined genetic analyses of worldwide strains support the theory that this bacterium experienced population expansions, probably associated with agriculture development and/or food industrialization.

Enhancement of cariogenic virulence properties of dental plaque in asthmatics.

OBJECTIVE: The aim of this study was to investigate the caries risk of asthmatics in relation to acidogenicity and the expression of caries-related genes in dental plaque. METHODS: A case-control study composed of 38 asthmatics (cases) and 22 controls with an age range from 6 to 60 years. Characteristics of asthma, use of medications, oral hygiene practices and dietary habits assessed by questionnaires and interviews. The dental plaque maturity evaluated using GC Tri Plaque ID Gel TM. The expression of brpA, gtfB, gbpB, ldh, luxS and spaP genes analyzed using real-time PCR. RESULTS: Asthmatics had a higher percentage of mature and acidogenic plaque than immature plaque. In contrast, immature plaque was more evident in controls. Acidogenic plaque commonly occurred in patients using 1 or a combination of two medications. High frequency in meals and sweets were found in asthmatics. Real-time PCR revealed that the expression of spaP, gtfB, gbpB, ldh, brpA and luxS were enhanced in asthmatics compared with the control group. CONCLUSION: An increase in acidogenic and mature plaque is found in asthmatics. The expression of spaP, gtfB, gbpB, ldh, brpA and luxS in dental plaque are upregulated in asthmatics.

Structural elucidation and functional characteristics of novel potential prebiotics produced from Limosilactobacillus reuteri N1 GtfB-treated maize starches.

The recently characterized Limosilactobacillus reuteri N1 GtfB (LrN1 GtfB) from glycoside hydrolase family 70 is a novel 4,6-α-glucanotransferase acting on starch/maltooligosaccharides with high enzyme activity and soluble protein yield (in heterogenous system). In this study, the influence of the treatment by LrN1 GtfB on the fine structure and functional characteristics of three maize starches were furtherly investigated and elucidated. Due to the treatment of LrN1 GtfB, the starch molecules were transformed into reuterans containing linear and branched (α1 â†’ 6) linkages with notably smaller molecular weight and shorter chain length. Moreover, the (α1 â†’ 6) linkage ratios in the GtfB-modified high-amylose maize starch (GHMS)/normal maize starch (GNMS)/waxy maize starch (GWMS) increased by 18.3 %/12.6 %/9.0 % as compared to their corresponding controls. In vitro digestibility experiment revealed that the resistant starch content of GHMS, GNMS and GWMS increased by 16 %, 18 % and 25 % as compared to the starch substrates. Furthermore, the butyric acid yielded from GHMS, GNMS and GWMS in the in vitro fermentation experiments were 1.4, 1.5 and 1.4 times higher than those of commercial galactose oligosaccharides. These results indicated that the highly-branched short-clustered reuteran synthesized by LrN1 GtfB might serve as novel potential prebiotics, and provide insights for the synthesis of promising prebiotic dietary fiber from starch.

Identification of a novel starch-converting GtfB enzyme from the Fructilactobacillus sanfranciscensis TMW11304 to reduce the viscoelasticity and retrogradation of tapioca starch.

Starch-converting α-glucanotransferases are efficient enzymatic toolkits for the biosynthesis of diverse α-glucans, which hold vast application potential in the food industry. In this work, we identified a novel GtfB protein from Fructilactobacillus sanfranciscensis TMW11304 (FsTMW11304 GtfB) in NCBI. Although this enzyme was highly conserved in motifs I-IV with those isomalto-maltopolysaccharides (IMMPs)-producing GtfB α-glucanotransferases, it possessed distinct deletions and mutations in two crucial loops shaping the active site. Hence, unlike those GtfB enzymes, FsTMW11304 GtfB not only exhibited excellent 4,6-α-glucanotransferase activity on amylose to generate atypically low-molecular-weight IMMPs with consecutive linear (α1 â†’ 6) linkages up to 48 %, but also held good capability towards branched substrates. Besides, compared with the control, the treatment by FsTMW11304 GtfB reduced the storage/loss modulus of granular and gelatinized tapioca starches (TS) by 12.0 %/17.9 % and 91.4 %/82.9 %, respectively, indicating that the rigidity of the gel structure was attenuated to different degrees in the two reaction systems. Furthermore, the setback viscosity observed in the gelatinized TS modified by FsTMW11304 GtfB was only 5 % of that observed in the control group, suggesting the short-term anti-retrogradation property has been substantially improved. Thus, FsTMW11304 GtfB represents a meaningful addition to the α-glucanotransferases in GH70 family, which expands the repertoire of diverse α-glucans synthesized from starch and facilitates the understanding of the structure-function relationship of the GtfB α-glucanotransferases.

Tailor-Made α-Glucans by Engineering the Processivity of α-Glucanotransferases via Tunnel-Cleft Active Center Interconversions.

The function of polysaccharides is intimately associated with their size, which is largely determined by the processivity of transferases responsible for their synthesis. A tunnel active center architecture has been recognized as a key factor that governs processivity of several glycoside hydrolases (GHs), e.g., cellulases and chitinases. Similar tunnel architecture is also observed in the Limosilactobacillus reuteri 121 GtfB (Lr121 GtfB) α-glucanotransferase from the GH70 family. The molecular element underpinning processivity of these transglucosylases remains underexplored. Here, we report the synthesis of the smallest (α1 → 4)-α-glucan interspersed with linear and branched (α1 → 6) linkages by a novel 4,6-α-glucanotransferase from L. reuteri N1 (LrN1 GtfB) with an open-clefted active center instead of the tunnel structure. Notably, the loop swapping engineering of LrN1 GtfB and Lr121 GtfB based on their crystal structures clarified the impact of the loop-mediated tunnel/cleft structure at the donor subsites -2 to -3 on processivity of these α-glucanotransferases, enabling the tailoring of both product sizes and substrate preferences. This study provides unprecedented insights into the processivity determinants and evolutionary diversification of GH70 α-glucanotransferases and offers a simple route for engineering starch-converting α-glucanotransferases to generate diverse α-glucans for different biotechnological applications.

N1019D Mutant of Limosilactobacillus reuteri 121 4,6-α-Glucanotransferase GtfB Significantly Improved Catalytic Activity.

Limosilactobacillus reuteri 121 4,6-α-glucanotransferase GtfB (Lr 121 GtfB), belonging to glycoside hydrolase family 70 (GH70), synthesizes linear isomalto/malto polysaccharides having (α1→6) linkages attached to the nonreducing ends of (α1→4) linked maltose oligosaccharide segments using starch or maltodextrin as a substrate. Since Lr 121 GtfB has low catalytic activity and efficiency, it leads to substrate regeneration and reduced substrate utilization. In this study, we superimposed the crystal structure of Lr 121 GtfB-ΔNΔV with that of L. reuteri NCC 2613 GtfB-ΔNΔV (Lr 2613 GtfB-ΔNΔV) to identify the acceptor binding subsites +1 to +3 and constructed five single-residue mutants and a random mutagenesis of N1019. Compared with the wild-type, N1019D Lr 121 GtfB-ΔN did not alter the product specificity, increased the catalytic activity and efficiency by 420 and 590%, respectively, and maintained >80% relative activity in the pH 3.5-6.5 interval. The findings will contribute to the industrial application of Lr 121 GtfB and provide new solutions for starch synthesis of higher value derivatives.

Exploring a GtfB-Type 4,6-α-Glucanotransferase to Synthesize the (α1 → 6) Linkages in Linear Chain and Branching Points from Amylose and Enhance the Functional Property of Granular Corn Starches.

Starch-converting α-glucanotransferases of glycoside hydrolase family 70 (GH70) are promising enzymatic tools for the production of diverse α-glucans with (potential) commercial applications in food and health and as biomaterials. In this study, a novel GtfB enzyme from Weissella confusa MBF8-1 was screened in the National Center for Biotechnology Information (NCBI) nonredundant protein database. The enzyme (named WcMBF8-1 GtfB) displayed high conservation in motifs I-IV with other GtfB enzymes but possessed unique variations in several substrate-binding residues. Structural characterizations of its α-glucan products revealed that WcMBF8-1 GtfB exhibited an atypical 4,6-α-glucanotransferase activity and was capable of catalyzing, by cleaving off (α1 → 4)-linkages in starch-like substrates and the synthesis of linear (α1 → 6) linkages and (α1 → 4,6) branching points. The product specificity enlarges the diversity of α-glucans and facilitates recognition of the determinants of the linkage specificity in GtfB enzymes. Furthermore, the contents of slowly digestible starch and resistant starch of granular corn starches, modified by WcMBF8-1 GtfB, increased by 6.7%, which suggested the potential value for the utilization of WcMBF8-1 GtfB to prepare "clean-label" starch ingredients with improved functional attributes.

Dietary Immunoglobulin Y by Targeting Both GbpB and GtfB Enhances the Anticaries Effect in Rats.

OBJECTIVE: The purpose of this work was to develop an anti-CAT-SYI immunoglobulin Y (IgY) antibody that targeted both GtfB (glucosyltransferase B) and GbpB (glucan-binding protein B) and test its anticaries properties in rats. METHODS: A new CAT-SYI fusion gene was created utilising functional DNA fragments from the GtfB and GbpB genes. The recombinant antigens, comprising the fused CAT-SYI antigen, GtfB, and GbpB, were expressed and purified using a prokaryotic expression and purification system. The purified recombinant antigens were utilised to immunise laying hens against particular IgY production. The biological activities of these particular IgY antibodies were then assessed both in vitro and in vivo, including their capacity to suppress biofilm formation and tooth caries. RESULTS: Results indicated that these produced IgY antibodies demonstrated a high antibody titer (>0.1 µg/mL) and could precisely recognise and bind to their respective antigens. Furthermore, it was discovered that these specific IgY antibodies successfully bind to Streptococcus mutans and significantly reduce biofilm development. After 8 weeks of ingesting antigen-specific IgY meals, comprising anti-GtfB IgY and anti-GbpB IgY, the severity of dental caries was dramatically reduced in S mutans-infected Sprague-Dawley rats (P < .01). Anti-CAT-SYI IgY therapy significantly reduced tooth cavities by 89.0% in vivo (P < .05) compared to other treatment groups. CONCLUSIONS: The anti-CAT-SYI IgY, a multitarget antibody that targets both GtfB and GbpB, displayed excellent inhibitory effects against S mutans, making it a promising targeted method with improved anticaries efficacy and significant application opportunities.

Structural and property characterization of low-molecular-weight novel reuterans synthesized from pea starch by Limosilactobacillus reuteri N1 GtfB with 4,6-α-glucanotransferase II activity.

Promising novel α-glucanotransferases with starch-converting activity have recently emerged from the CAZy GH70 GtfB subfamily. In this study, we thoroughly investigated and elucidated the impact of the newly characterized 4,6-α-glucanotransferase II Limosilactobacillus reuteri N1 GtfB (LrN1 GtfB), which was capable of synthesizing linear (α1 â†’ 6) and branched (α1 â†’ 4,6) linkages, on the fine structure, rheology, and retrogradation properties of pea starch (PS). The results revealed that as the reaction time increased, the total (α1 â†’ 6) linkages in linear chains and branching points of PS increased from 5.6 % to 18.7 %, the molecular weight decreased from 7.3 × 106 g/mol to 7.4 × 104 g/mol, and the percentage of short chains (DP ≤ 12) increased from 47.4 % to 92.7 %, thereby producing low-molecular-weight, short-clustered novel reuterans with new (α1 â†’ 6) linkages in both linear chains and branches. Additionally, LrN1 GtfB-modified PS exhibited lower storage/loss modulus and weaker creep property, indicating a significant attenuation of the strength and rigidity of the modified gel structure. Moreover, products derived from pea starch and LrN1 GtfB exhibited notably low retrogradation properties. These findings provide insights into the potential application of GtfB-type α-glucanotransferases in starch-based products, thereby producing unique-structured α-glucans with versatile properties from starch.

Insights into the Structure-Function Relationship of GH70 GtfB α-Glucanotransferases from the Crystal Structure and Molecular Dynamic Simulation of a Newly Characterized Limosilactobacillus reuteri N1 GtfB Enzyme.

α-Glucanotransferases of the CAZy family GH70 convert starch-derived donors to industrially important α-glucans. Here, we describe characteristics of a novel GtfB-type 4,6-α-glucanotransferase of high enzyme activity (60.8 U mg-1) from Limosilactobacillus reuteri N1 (LrN1 GtfB), which produces surprisingly large quantities of soluble protein in heterologous expression (173 mg pure protein per L of culture) and synthesizes the reuteran-like α-glucan with (α1 → 6) linkages in linear chains and branch points. Protein structural analysis of LrN1 GtfB revealed the potential crucial residues at subsites -2∼+2, particularly H265, Y214, and R302, in the active center as well as previously unidentified surface binding sites. Furthermore, molecular dynamic simulations have provided unprecedented insights into linkage specificity hallmarks of the enzyme. Therefore, LrN1 GtfB represents a potent enzymatic tool for starch conversion, and this study promotes our knowledge on the structure-function relationship of GH70 GtfB α-glucanotransferases, which might facilitate the production of tailored α-glucans by enzyme engineering in future.

Multiple approaches of loop region modification for thermostability improvement of 4,6-α-glucanotransferase from Limosilactobacillus fermentum NCC 3057.

4,6-α-glucanotransferase (4,6-α-GT), as a member of the glycoside hydrolase 70 (GH70) family, converts starch/maltooligosaccharides into α,1-6 bond-containing α-glucan and possesses potential applications in food, medical and related industries but does not satisfy the high-temperature requirement due to its poor thermostability. In this study, a 4,6-α-GT (ΔGtfB) from Limosilactobacillus fermentum NCC 3057 was used as a model enzyme to improve its thermostability. The loops of ΔGtfB as the target region were optimized using directed evolution, sequence alignment, and computer-aided design. A total of 11 positive mutants were obtained and iteratively combined to obtain a combined mutant CM9, with high resistance to temperature (50 °C). The activity of mutant CM9 was 2.08-fold the activity of the wild type, accompanied by a 5 °C higher optimal temperature, a 5.76 °C higher melting point (Tm, 59.46 °C), and an 11.95-fold longer half-life time (t1/2). The results showed that most of the polar residues in the loop region of ΔGtfB are mutated into rigid proline residues. Molecular dynamics simulation demonstrated that the root mean square fluctuation of CM9 significantly decreased by "Breathing" movement reduction of the loop region. This study provides a new strategy for improving the thermostability of 4,6-α-GT through rational loop region modification.

The catalytic domains of Streptococcus mutans glucosyltransferases: a structural analysis.

Streptococcus mutans, found in the human oral cavity, is a significant contributor to the pathogenesis of dental caries. This bacterium expresses three genetically distinct types of glucosyltransferases named GtfB (GTF-I), GtfC (GTF-SI) and GtfD (GTF-S) that play critical roles in the development of dental plaque. The catalytic domains of GtfB, GtfC and GtfD contain conserved active-site residues for the overall enzymatic activity that relate to hydrolytic glycosidic cleavage of sucrose to glucose and fructose, release of fructose and generation of a glycosyl-enzyme intermediate in the reducing end. In a subsequent transglycosylation step, the glucosyl moiety is transferred to the nonreducing end of an acceptor to form a growing glucan polymer chain made up of glucose molecules. It has been proposed that both sucrose breakdown and glucan synthesis occur in the same active site of the catalytic domain, although the active site does not appear to be large enough to accommodate both functions. These three enzymes belong to glycoside hydrolase family 70 (GH70), which shows homology to glycoside hydrolase family 13 (GH13). GtfC synthesizes both soluble and insoluble glucans (α-1,3 and α-1,6 glycosidic linkages), while GtfB and GtfD synthesize only insoluble or soluble glucans, respectively. Here, crystal structures of the catalytic domains of GtfB and GtfD are reported. These structures are compared with previously determined structures of the catalytic domain of GtfC. With this work, apo structures and inhibitor-complex structures with acarbose are now available for the catalytic domains of GtfC and GtfB. The structure of GtfC with maltose allows further identification and comparison of active-site residues. A model of sucrose binding to GtfB is also included. The new structure of the catalytic domain of GtfD affords a structural comparison of the three S. mutans glycosyltransferases. Unfortunately, the catalytic domain of GtfD is not complete since crystallization resulted in the structure of a truncated protein lacking approximately 200 N-terminal residues of domain IV.

A study on genetic and mutans streptococcal transmissibility of dental caries.

Background: Dental caries is characterized by an interplay between environmental and genetic factors. Aim: The aim of this study was to analyse the transmissibilities of high caries risk chromosomal loci at 5q 12.1-13.3 and low caries risk chromosomal loci at 13q31.1 and Streptococcus mutans (S. mutans) in family units. Materials: This prospective cohort study was performed on 56 families grouped into four: (a) Group I: 18 families of children with caries affected primary teeth; (b) Group II: 21 families of children with caries in permanent teeth; (c) Group III: 6 families of children with no caries in primary teeth and (d) Group IV: 12 families of children with no caries in permanent teeth. Blood, saliva and plaque samples were collected from consenting study participants. Isolated DNAs were subjected to polymerase chain reactions using suitable primers. Data collected was analysed with ANOVA and Chi-squared test. Results: Wide expression of chromosome loci 5q12.1-13.3 was obtained in both blood and saliva samples. For chromosome loci 13q31.1, no expression was found in saliva samples, hence indicating its local absence. For the GtfB expression, transmissibility was common for a single band expressing S. mutans. Conclusion: This study reflects upon newer findings in the field of genetic research on dental caries.

Lactobacillus Acidophilus-Derived Biosurfactant Effect on GTFB and GTFC Expression Level in Streptococcus Mutans Biofilm Cells.

Streptococcus mutans (S. mutans), harboring biofilm formation, considered as a main aetiological factor of dental caries. Gtf genes play an important role in S. mutans biofilm formation. The purpose of this study was to investigate the effect of Lactobacillus acidophilus-derived biosurfactant on S. mutans biofilm formation and gtfB/C expression level (S. mutans standard strain ATCC35668 and isolated S. mutans strain (22) from dental plaque). The Lactobacillus acidophilus (L. acidophilus) DSM 20079 was selected as a probiotic strain to produce biosurfactant. The FTIR analysis of its biosurfactant showed that it appears to have a protein-like component. Due to the release of such biosurfactants, L. acidophilus was able to interfere in the adhesion and biofilm formation of the S. mutans to glass slide. It also could make streptococcal chains shorter. Using realtime RT-PCR quantitation method made it clear that gtfB and gtfC gene expression were decreased in the presence of L. acidophilus-derived biosurfactant fraction. Several properties of S. mutans cells (the surface properties, biofilm formation, adhesion ability and gene expression) were changed after L. acidophilus- derived biosurfactant treatment. It is also concluded that biosurfacant treatment can provide an optional way to control biofilm development. On the basis of our findings, we can suggest that the prepared biosurfactant may interfere with adhesion processes of S. mutans to teeth surfaces, provided additional evaluation produce satisfactory results.

Insights into Broad-Specificity Starch Modification from the Crystal Structure of Limosilactobacillus Reuteri NCC 2613 4,6-α-Glucanotransferase GtfB.

GtfB-type α-glucanotransferase enzymes from glycoside hydrolase family 70 (GH70) convert starch substrates into α-glucans that are of interest as food ingredients with a low glycemic index. Characterization of several GtfBs showed that they differ in product- and substrate specificity, especially with regard to branching, but structural information is limited to a single GtfB, preferring mostly linear starches and featuring a tunneled binding groove. Here, we present the second crystal structure of a 4,6-α-glucanotransferase (Limosilactobacillus reuteri NCC 2613) and an improved homology model of a 4,3-α-glucanotransferase GtfB (L. fermentum NCC 2970) and show that they are able to convert both linear and branched starch substrates. Compared to the previously described GtfB structure, these two enzymes feature a much more open binding groove, reminiscent of and evolutionary closer to starch-converting GH13 α-amylases. Sequence analysis of 287 putative GtfBs suggests that only 20% of them are similarly "open" and thus suitable as broad-specificity starch-converting enzymes.

The effects of bimodal action of photodynamic and photothermal therapy on antimicrobial and shear bond strength properties of orthodontic composite containing nano-graphene oxide.

BACKGROUND: White spot lesions are a common adverse effect of fixed orthodontic treatment and represent the main challenge to achieving esthetic appearance. The purpose of the current study was to evaluate physico-mechanical and antimicrobial potency of orthodontic composite (OC; Transbond XT) containing nano-structured graphene oxide (nGO) (OC-nGO) as a novel composite following photodynamic therapy (PDT) and photothermal therapy (PTT) against Streptococcus mutans. MATERIALS AND METHODS: Following preparation of OC-nGO, shear bond strength (SBS) and adhesive remnant index (ARI) of the test OC-nGO (containing 1, 2, 5, and 10% wt. nGO) were measured using a universal testing machine and stereomicroscope, respectively. The antimicrobial activities of test OC contained different concentrations of nGO were determined by disk agar diffusion (DAD), biofilm formation inhibition, and eluted components assays. After continuously rinsed in the aging process (up to 180 days), the antimicrobial activity of OC-nGO containing the highest concentration of nGO which had simultaneously the highest antimicrobial activity and SBS value were determined by DAD, biofilm formation, metabolic activity, and gtfB gene expression assays following photo-activation using diode laser irradiation against S. mutans. Data were analyzed using One-way Analysis of Variance (ANOVA). The Bonferroni post hoc test was used for comparison between the experimental groups. The significant difference was considered at P values < 0.05. RESULTS: OC with 5% wt. nGO showed simultaneously the highest SBS value (10.64 ± 2.76 MPa, P < 0.05) an antimicrobial and anti-biofilm activities. The OC-nGO in all test concentrations of nGO had ARI scores as same as control group (Transbond XT without the nGO) (P < 0.05). In microbial biofilm formation and gene expression assays, the reduction of photothermal disinfection and anti-virulence activities of the 5% wt. OC-nGO against test bacterium was associated with the time of aging process, so they were reduced significantly up to day 150. Diode laser irradiated 5% wt. OC-nGO suppressed 15.6 and 8.1-fold gtfB mRNA expressions in the biofilm growth of the S. mutans at days 120 and 150 of rinsing (P < 0.05). Microbial biofilm formation and gtfB gene expression in S. mutans at day 180 following PAD had a high level of similarity with OOC as the control group. 5% wt. OC-nGO following photo-activation was not colonized by the S. mutans at day 90 and significant suppressed 91.98% and 76.37% of S. mutans biofilm formation at day 120 and 150, respectively (both P < 0.05). From day 120 onwards, metabolic activity was progressively increased on laser-irradiated 5% wt. OC-nGO discs compared to the control group (OC alone). Photo-activated OC-nGO containing 5% wt. nGO suppressed 86.94% and 46.82% metabolic activity of the S. mutans at days 120 and 150 of rinsing (both P < 0.05). CONCLUSIONS: Our data support that the photo-activated 5% wt. OC-nGO can serve as an orthodontic composite/adhesive additive to control cariogenic bacterial biofilms.

Wheat Starch with Low Retrogradation Properties Produced by Modification of the GtfB Enzyme 4,6-α-Glucanotransferase from Streptococcus thermophilus.

A GtfB enzyme 4,6-α-glucanotransferase from Streptococcus thermophilus lacking 761 N-terminal amino acids was heterologously expressed in Escherichia coli. Purified S. thermophilus GtfB showed transglycosylation activities toward starch, resulting in branch points of (α1→6)-glycosidic linkages plus linear chains of (α1→4)-glycosidic linkages. After wheat starch was modified at a rate of 0.1 g/mL by 1-4 U/g starch GtfB at pH 6.0 and 40 °C for 1 h, the weight-averaged molecular weight decreased from 1.70 × 107 g/mol to 1.21 × 106 to 3.41 × 106 g/mol, the amylose content decreased from 22.07 to 16.34-17.11%, and that of amylopectin long-branch chains decreased from 26.4 to 10.25-15.64% ( P < 0.05). After the GtfB-modified wheat starches were gelatinized and stored at 4 °C for 1-2 weeks, their endothermic enthalpies were significantly lower than that of the control sample ( P < 0.05), indicating low retrogradation rates.

Real-time quantitative polymerase chain reaction (QPCR) for the identification and quantification of streptococcus mutans in saliva and dental biofilm in children / Técnica de reacción de polimerasa en cadena (QPCR) en tiempo real para la identificación y cuantificación de streptococcus mutans en saliva y biopelícula dentaria de niños

ABSTRACT Introduction: the objective of this study was to use real-time qPCR to identify and quantify the Streptococcus mutans species in samples of saliva and dental biofilm. Methods: 27 children were randomly chosen with the following criteria: 8 years of age, low socio-economic levels, residing in the northern metropolitan area of Santiago de Chile; they were asked to attend an appointment while fasting with no teeth brushing for at least 12 hours, in order to collect non-stimulated saliva and a pool of supragingival dental biofilm of all the mesio-vestibular sides of anterior and posterior teeth. The amount of S. mutans in the samples was quantified by qPCR using primers that amplify a fragment of the gtfB gene of S. mutans. Results: the amplification showed 98% efficiency with a fluorescence of 3.36 cycles. The melting curve presented a single maximum at the same temperature for all samples. Conclusion: the methodology allows the specific identification and quantification of gene gtfB of S. mutans in saliva and dental biofilm in a quick and reliable manner, contributing to the identification of individual cariogenic risk.

RESUMEN. Introducción: el objetivo del presente estudio consistió en implementar la técnica de qPCR en tiempo real para identificar y cuantificar la especie Streptococcus mutans en muestras de saliva y biopelícula dentaria. Métodos: se seleccionaron al azar 27 niños de 8 años de edad, de nivel socio-económico bajo del área norte de la región metropolitana de Santiago de Chile, que se citaron en ayunas y sin cepillado durante al menos 12 horas, para colectar saliva no estimulada y un pool de biopelícula dentaria supragingival de todas las caras mesio-vestibulares de dientes anteriores y posteriores. Se cuantificó la cantidad de S. mutans en las muestras mediante qPCR empleando partidores que amplifican un fragmento del gen gtfB de S. mutans. Resultados: la amplificación presentó 98% de eficiencia con delta de fluorescencia de 3,36 ciclos. La curva de fusión (melting) presentó un solo máximo a una misma temperatura para todas las muestras. Conclusión: la metodología permite la identificación y cuantificación específica del gen gtfB de S. mutans en muestras de saliva y biopelícula dentaria, de forma rápida y exacta, aportando a la determinación del riesgo cariogénico individual.