Identification of ssDNA aptamers specific to clinical isolates of Streptococcus mutans strains with different cariogenicity.

Streptococcus mutans, a Gram-positive facultative anaerobic bacterium, is considered to be a major etiological factor for dental caries. In this study, plaques from dental enamel surfaces of caries-active and caries-free individuals were obtained and cultivated for S. mutans isolation. Morphology examination, biochemical characterization, and polymerase chain reaction were performed to identify S. mutans The cariogenicity of S. mutans strains isolated from clinical specimens was evaluated by testing the acidogenicity, aciduricity, extracellular polysaccharide production, and adhesion ability of the bacteria. Finally, subtractive SELEX (systematic evolution of ligands by exponential enrichment) technology targeting whole intact cells was used to screen for ssDNA aptamers specific to the strains with high cariogenicity. After nine rounds of subtractive SELEX, sufficient pool enrichment was achieved as shown by radioactive isotope analysis. The enriched pool was cloned and sequenced randomly, followed by MEME online and RNA structure software analysis of the sequences. Results from the flow cytometry indicated that aptamers H1, H16, H4, L1, L10, and H19 could discriminate highly cariogenic S. mutans strains from poorly cariogenic strains. Among these, Aptamer H19 had the strongest binding capacity with cariogenic S. mutans strains with a dissociation constant of 69.45 ± 38.53 nM. In conclusion, ssDNA aptamers specific to highly cariogenic clinical S. mutans strains were successfully obtained. These ssDNA aptamers might be used for the early diagnosis and treatment of dental caries.

Surgical Template to Minimize the Damage of Tooth Buds in Young Children With Mandibular Distraction Osteogenesis.

BACKGROUND: Mandibular distraction osteogenesis has become one of the most powerful reconstructive techniques for the treatment of mandibular deformities in young children. Damage to the tooth buds is often cited as a complication of it. The purpose of this study was to precisely design the osteotomy line and avoid the damage of tooth buds at the mandibular angle area with the help of surgical template. METHODS: Six patients aged from 6 to 10 years were selected in this study. Three-dimensional computed tomography data was put into Mimics software for preoperative planning the osteotomy line and the accurate placement of the distractor based on the exact position of tooth follicle. And then the surgical template was manufactured by a three-dimensional printer with rapid prototyping technique. The surgeons were guided to perform the osteotomy aided with the prefabricated template through an intraoral approach. Distraction began 7 days postoperation with a rate of 1 mm/d and the distractor was removed after a 6 to 8 months consolidation period. The tooth buds were observed through radiographs that were performed at 5 time intervals: before distraction, at the end of latency, at the end of distraction, at the end of consolidation and 2 years of the postoperation to remove the distractor. RESULTS: The average follow-up time is 24 months. Facial appearance and occlusal plane of all the young patients were greatly improved without complications. From the radiological observation, the tooth buds was intact after the surgery and the second molar normally erupted after removing the distractor. CONCLUSIONS: The surgical template was considered to be helpful in improving the surgical accuracy and avoiding the tooth buds damage during mandibular distraction osteogenesis.

pH-triggered polydopamine-decorated nanocellulose membranes for continuously selective separation of organic dyes.

Membrane separation technology has emerged as a powerful tool to separate organic dyes from industrial wastewater. However, continuously selective separation of organic dyes with similar molecular weight remains challenging. Herein, we presented a pH-triggered membrane composed of polydopamine-decorated tunicate-derived cellulose nanofibers (PDA@TCNFs) for selective separation of organic dyes. Such self-supporting membranes with nanoporous structure were fabricated by facile vacuum-assisted filtration of PDA@TCNF suspension. The incorporation of polydopamine not only enhanced the stability of the membranes, but also endowed membranes with excellent pH sensitivity, facilitating the continuously selective separation of organic dyes. These pH-triggered PDA@TCNF membranes could selectively separate Methyl Orange (MO) and Rhodamine B (RB) from the MO/RB mixed solution by switching the pH values. The continuously selective separation of the MO/RB mixed solution was demonstrated, where both MO and RB recovery ratios maintained at ∼99 % during 50 repeated cycles. This work provides a new strategy to develop a pH-triggered sustainable nanocellulose-based membrane for continuously selective separation of mixed dyes.

pH-responsive size-adjustable liposomes induce apoptosis of fibroblasts and macrophages for rheumatoid arthritis treatment.

In rheumatoid arthritis (RA), macrophages infiltrate joints, while fibroblast-like synovial cells proliferate abnormally, forming a barrier against drug delivery, which hinders effective drug delivery to joint focus. Here we firstly designed a pH-responsive size-adjustable nanoparticle, composed by methotrexate (MTX)-human serum albumin (HSA) complex coating with pH-responsive liposome (Lipo/MTX-HSA) for delivering drugs specifically to inflamed joints in acidic environments. We showed in vitro that the nanoparticles can induce mitochondrial dysfunction, promote apoptosis of fibroblast-like synoviocytes and macrophages, further reduce the secretion of inflammatory factors (TNF-α, IL-1ß, MMP-9), and regulate the inflammatory microenvironment. We also demonstrated similar effects in a rat model of arthritis, in which Lipo/MTX-HSA accumulated in arthritic joints, and at low pH, liposome phospholipid bilayer cleavage released small-sized MTX-HSA, which effectively reduced the number of fibroblast-synoviocytes and macrophages in joints, alleviated joint inflammation, and repaired bone erosion. These findings suggest that microenvironment-responsive size-adjustable nanoparticles show promise as a treatment against rheumatoid arthritis. STATEMENT OF SIGNIFICANCE: Abnormal proliferation of fibroblast synoviocytes poses a physical barrier to effective nanoparticle delivery. We designed size-adjustable nano-delivery systems by preparing liposomes with cholesterol hemisuccinate (CHEM), which were subsequently loaded with small-sized albumin nanoparticles encapsulating the cytotoxic drug MTX (MTX-HSA), termed Lipo/MTX-HSA. Upon tail vein injection, Lipo/MTX-HSA could be aggregated at the site of inflammation via the ELVIS effect in the inflamed joint microenvironment. Specifically, intracellular acidic pH-triggered dissociation of liposomes promoted the release of MTX-HSA, which was further targeted to fibroblasts or across fibroblasts to macrophages to exert anti-inflammatory effects. The results showed that liposomes with adjustable particle size achieved efficient drug delivery, penetration and retention in joint sites; the strategy exerted significant anti-inflammatory effects in the treatment of rheumatoid arthritis by inducing mitochondrial dysfunction to promote apoptosis in fibrosynoviocytes and macrophages.

Self-Report Amphiphilic Polymer-Based Drug Delivery System with ROS-Triggered Drug Release for Osteoarthritis Therapy.

The development of drug delivery systems with real-time cargo release monitoring capabilities is imperative for optimizing nanomedicine performance. Herein, we report an innovative self-reporting drug delivery platform based on a ROS-responsive random copolymer (P1) capable of visualizing cargo release kinetics via the activation of an integrated fluorophore. P1 was synthesized by copolymerization of pinacol boronate, PEG, and naphthalimide monomers to impart ROS-sensitivity, hydrophilicity, and fluorescence signaling, respectively. Detailed characterization verified that P1 self-assembles into 11 nm micelles with 10 µg mL-1 CMC and can encapsulate hydrophobic curcumin with 79% efficiency. Fluorescence assays demonstrated H2O2-triggered disassembly and curcumin release with concurrent polymer fluorescence turn-on. Both in vitro and in vivo studies validated the real-time visualization of drug release and ROS scavenging, as well as the therapeutic effect on osteoarthritis (OA). Overall, this nanotheranostic polymeric micelle system enables quantitative monitoring of drug release kinetics for enhanced treatment optimization across oxidative stress-related diseases.

Ppbv-level mid-infrared photoacoustic sensor for mouth alcohol test after consuming lychee fruits.

A ppbv-level mid-infrared photoacoustic spectroscopy sensor was developed for mouth alcohol tests. A compact CO2 laser with a sealed waveguide and integrated radio frequency (RF) power supply was used. The emission wavelength is ∼9.3 µm with a power of 10 W. A detection limit of ∼18 ppbv (1σ) for ethanol gas with an integration of 1 s was achieved. The sensor performed a linear dynamic range with an R square value of ∼0.999. A breath measurement experiment after consuming lychees was conducted. The photoacoustic signal amplitude decreased with the quality of lychee consumed, confirming the existence of residual alcohol in the mouth. During continuous measurement, the photoacoustic signal decreased in < 10 min when consuming 30 g lychee fruits, proving that the alcohol detected in exhaled breath originated from the oral cavity rather than the bloodstream. This work provided valuable information on the distinction of alcoholism and crime.

Longitudinal investigation of idiopathic osteosclerosis lesions of the jaws in a group of Chinese orthodontically-treated patients using digital panoramic radiography.

BACKGROUND/PURPOSE: Idiopathic osteosclerosis (IO) is an intraosseous lesion of asymptomatic, non-expansive, radiopaque. The study aimed to investigate the prevalence and morphometric parameters of IO in orthodontic patients and variations in longitudinal observations and to assess the relationship between IO and orthodontic treatment. MATERIALS AND METHODS: Five hundred and seventy-one orthodontically-treated patients were reviewed. A cross-sectional study was performed with the evaluated parameters, including the age and sex of patients, as well as the number, shape, location and morphometric data of IO observed in panoramic radiography. Long-term behaviour of IO and orthodontic tooth movement were also observed. Also, a control group was set up for comparisons. RESULTS: Sixty-eight (11.3%) patients had 78 lesions all in the mandible with premolar/molar preference and no sex predilection. Lesions were located more commonly at apical and separate sites related to teeth. A large majority of lesions enlarged in the 10-19 years old group, while most lesions had no change in the 30-39 years old group. Hindrances of tooth movement and external root resorption around IO were not found in affected patients. CONCLUSION: IO is labile lesion that may develop in early stages of life, with little change occurring once the affected individual is mature and being relatively stable in the middle stage of life. Our study supports the hypothesis that IO may be developmental anatomic variations of normal bone. However, no obvious association between IO and orthodontic treatment was found in patients, which may be due to the limitations of two-dimensional shooting of panoramic radiography and the sample size.

Can microplastics in offshore waters reflect plastic emissions from coastal regions?

Marine microplastic pollution is a major environmental challenge that threatens marine ecosystems and human health. Several models have been used to calculate and predict the theoretical amount of plastic waste discharged into the sea by coastal countries. Unlike earlier theoretical models of source discharge, we used the method of data normalisation to focus on the actual distribution of microplastics and their potential ecological risk in offshore surface waters. Our findings indicate that the average normalised abundance of microplastics in near-shore region of Bohai Sea was greater than the average normalised abundance of microplastics in the seas near the Yangtze River Delta urban agglomeration and the Pearl River Delta urban agglomeration. Moreover, the average amount of plastic waste discharged from terrestrial sources to the ocean per kilometre exhibited the following order: Bohai Rim urban agglomeration (150.90) (tonnes km-1) < the Pearl River Delta urban agglomeration (274.30) (tonnes km-1) < Yangtze River Delta urban agglomeration (577.44) (tonnes km-1). Further, the average microplastics abundance in offshore areas of different countries and the amount of plastic discharged per kilometre of the coastline were significantly negatively correlated, implying that microplastics were not necessarily abundant in coastal areas where large amounts of plastic are discharged into the sea. Hydrodynamic conditions had the greatest influence on the distribution of microplastics in offshore surface waters. The transport of nutrient salts from terrestrial areas to offshore waters was also influenced by hydrodynamics, with enrichment patterns in offshore areas exhibiting similar to those of microplastics. Therefore, when the offshore microplastic accumulation area overlapped with the nutrient salt enrichment zone, the health risk associated with the consumption of edible fish from offshore communities increased. In view of these findings, coastal countries must implement policies to reduce marine plastic waste emissions and develop management strategies based on their local pollution levels.

Effects of hydrogen rich water and pure water on periodontal inflammatory factor level, oxidative stress level and oral flora: a systematic review and meta-analysis.

Background: Hydrogen rich water (HRW) was used as an auxiliary treatment for periodontitis and peri-implantitis due to its good antioxidant properties. However, the stability of artificially added active hydrogen was far less than that of pure natural active hydrogen, which greatly reduced active hydrogen molecules number in HRW. Meanwhile, the effect of HRW was relatively slow. Finally, long-term drinking of HRW may cause abnormal liver function. Hence, this study sought to summarize and analyze the effects of HRW on oral inflammation and oral flora in various studies to determine whether HRW can be used to inhibit dental plaque formation and aliviate oral inflammation. Methods: Randomized controlled trials (RCTs) of HRW and pure water (PW) in the treatment of periodontal diseases published before March 2022 in the PubMed, Web of science, EMBASE, Cochrane, China Knowledge Resource Integrated, Wanfang, and Weipu databases were searched. Changes in the inflammatory factor levels, oxidative stress response, and oral flora were summarized and used as outcome indicators. The quality of included studies was assessed by Cochrane risk of bias assessment tool, and the standardized mean differences (SMD) and the 95% confidence intervals (CIs) were calculated using Review Manager 5.3. Results: In total, 17 studies, comprising 304 subjects, were included in this meta-analysis. Among them, 5 studies had a high risk of bias, and the rest had a certain risk of bias, thus, the total risk of bias was medium to low. The levels of interleukin (IL)-1ß (SMD =-0.73; 95% CI: -1.29 to -0.18; P=0.009), tumor necrosis factor alpha (SMD =-2.51; 95% CI: -3.56 to -1.46; P<0.00001), IL-6 (SMD =-1.31; 95% CI: -1.96 to -0.67; P<0.0001), 8-hydroxyguanosine (SMD =-1.61; 95% CI: -2.35 to -0.87; P<0.0001), and reactive oxygen metabolites (SMD =-0.49; 95% CI: -0.91 to -0.06; P=0.02) in the HRW group decreased significantly, while the glutathione peroxidase level increased (SMD =2.5; 95% CI: 1.85 to 3.15; P<0.00001). Additionally, HRW was shown to effectively inhibit oral pathogenic bacteria activity (SMD =-0.91; 95% CI: -1.16 to -0.66; P<0.00001). Conclusions: HRW effectively inhibits the inflammatory reaction, oxidative stress level, and bacterial proliferation activity in patients with periodontal disease.

Gene/paclitaxel co-delivering nanocarriers prepared by framework-induced self-assembly for the inhibition of highly drug-resistant tumors.

While drug resistance has been recognized as the main cause of unsuccessful chemotherapy, the efficient inhibition of highly drug-resistant tumors still remains a significant challenge, especially for in vivo treatments. Drug resistance has been associated with the high expression of the multi-drug resistance gene 1 (MDR1), which can encode an efflux transporter known as P-glycoprotein (P-gp) that is located in the cellular membrane. Therefore, the combined delivery of MDR1-inhibited genes and chemotherapeutic drugs is anticipated to enable the effective inhibition of drug-resistant tumors. Herein, highly paclitaxel (PTX)-resistant ovarian (OV) cancer with a drug resistance index reaching up to ~ 60 was chosen to evaluate the performance of an efficient gene/drug co-delivery nanocarrier. Inspired by the self-assembly that occurs in cells and exosomes, we designed a biomimetic lipid/dextran hybrid nanocarrier with a diameter of ~ 100 nm to enhance the endocytosis and the efficiency of drug/gene release within the cells. This nanocarrier was fabricated via the frame-guided self-assembly of lipid amphiphiles on the surfaces of redox-cleavable dextran-based nanogels. The anionic MDR1-siRNA and the hydrophobic drug PTX were respectively loaded into the cationic lipid shell and the hydrophobic internal core of the hybrid nanocarriers. MDR1-siRNA can knock down MDR1, promoting the accumulation of PTX in cells, and thus is expected to achieve an efficient inhibitory effect against highly PTX-resistant cancer cells. Both in vitro and in vivo studies revealed that this dual-delivery system significantly enhanced the therapeutic effect in comparison with that provided by a PTX-only system. Thus, the construction of gene/chemo co-delivered lipid/dextran nanocarriers provides a new strategy to inhibit highly drug-resistant tumors both in vitro and in vivo. In addition, this work will contribute toward the development of urgently needed tumor nanotherapy that is able to overcome drug resistance while also offering an unmatched range of effective therapeutic nanocarriers. STATEMENT OF SIGNIFICANCE: The biomimetic lipid/dextran hybrid nanocarrier with a diameter of ~ 100 nm, which was fabricated via the frame-guided self-assembly of lipid amphiphiles onto the surface of redox-cleavable dextran-based nanogels, provides a model carrier to co-deliver MDR1-siRNA and PTX.  The MDR1-siRNA/PTX co-loaded biomimetic lipid/dextran hybrid nanocarriers demonstrate good capability in overcoming the PTX-resistance in highly chemo-resistant human ovarian (OV) cancer cells both in vitro and in vivo.

Fluorescent Polysaccharide Nanogels for the Detection of Tumor Heterogeneity in Drug-Surviving Cancer Cells.

Tumor metastasis, recurrence, and drug resistance have been associated with tumor heterogeneity, and thus the identification of tumor heterogeneity has great significance in medicine. The approach provides a way to identify and isolate various cell subtypes from drug-surviving ovarian cancer cells, by synthesizing a series of polysaccharide nanogels and using them in flow cytometry analysis. The results show that the drug-surviving OVCAR-3 cells that are subjected to paclitaxel intervention comprise various cell subtypes, including drug-resistant and non-drug-resistant cell subtypes. Besides, there are significant differences between the drug-resistant cell subtype and non-drug-resistant cell subtype in terms of their migration and invasion behavior. In addition, the phenotype switch genes are detected by mRNA sequencing, and it is found that different subtypes show significant genetic differences with regard to their drug resistance, metastasis, and proliferation. In particular, modifying polysaccharide nanogels with lipids can promote the uptake of nanogels by drug-resistant cells, and thus the lipid modification can enhance the effectiveness of a chemotherapy drug carrier against drug-resistant cells. These studies reveal the heterogeneity of drug-surviving tumor cells, as well as the significant differences in drug-resistance, migration, and invasion capabilities of different subtypes, and demonstrate a way to overcome drug resistance.

Microplastics in a wind farm area: A case study at the Rudong Offshore Wind Farm, Yellow Sea, China.

Despite the rapid construction of offshore wind farms, the available information regarding the risks of this type of development in terms of emerging pollutants, particularly microplastics, is scarce. In this study, we quantified the level of microplastic pollution at an offshore wind farm in the Yellow Sea, China, in 2016. The abundance of microplastics was 0.330 ±â€¯0.278 items/m3 in the surface water and 2.58 ±â€¯1.14 items/g (dry) in the sediment. To the best of our knowledge, the level of microplastic pollution in our study area was slightly higher than that in coastal areas around the world. The microplastics detected in the surface waters and sediments were mainly fibrous (75.3% and 68.7%, respectively) and consisted of some granules and films. The microplastics in the samples might originate from garments or ropes via wastewater discharge. The abundance of plastic in the water and sediment samples collected from the wind farm area was lower than that in the samples collected from outside the wind farm area. The anthropogenic hydrodynamic effect was the main factor affecting the local distribution of microplastics. The presence of a wind farm could increase the bed shear stress during ebb tide, disturbing the bed sediment, facilitating its initiation and transport, and ultimately increasing the ease of washing away the microplastics adhered to the sediment. This study will serve as a reference for further studies of the distribution and migration of microplastics in coastal zones subjected to similar marine utilization.

[Changes in expressions of sRNA SpR19 and its potential target GroEL in Streptococcus mutans strains with different cariogenicity cultured under different pH conditions].

OBJECTIVE: To investigate the changes in the expression level of sRNA SpR19 and its potential target protein GroEL in clinical isolates of Streptococcus mutans with different cariogenicity exposed to different pH conditions and explore the possibility of using these molecules as biomarkers for assessing the cariogenicity of the bacteria. METHODS: The total RNAs were extracted from the clinical isolates of Streptococcus mutans with high (strain 17) and low cariogenicity (strain 5) for high-throughput sequencing for profiling of the differentially expressed sRNAs. The candidate sRNA, SpR19, was selected for further study on the basis of bioinformatics analysis considering the role of its potential target in the cariogenic process. The differential expression levels of SpR19 in the strains exposed to both pH5.5 and pH7 culture conditions were verified by quantitative real-time PCR. The expression of the potential target of SpR19, GroEL, was also investigated at both the protein and mRNA level using Western blotting and quantitative real-time PCR. RESULTS: Bioinformatic analysis suggested multiple potential target sites of SpR19 both in GroEL mRNA and in the upstream and downstream inter-genic regions. Under different pH conditions, the highly cariogenic strain 17 expressed consistently low levels of SpR19 as compared with the strain 5 with a low cariogenicity; GroEL showed a reverse expression pattern in the 2 strains. An inverse correlation was found between the expressions of SpR19 and GroEL. CONCLUSION: The highly cariogenic strain 17 expressed low levels of SpR19 and high levels of GroEL in both acidic and neutral culture conditions. SpR19 may negatively regulate the cariogenicity of Streptococcus mutants by targeting at GroEL.

A Practical Surgical Technique to Expose the Mental Nerve in Narrowing Genioplasty.

In this article, the authors present a practical surgical technique using the anatomical character of the inferior alveolar nerve to fully expose the mental nerve (MN) in narrowing genioplasty. During the procedure, a rectangular mandibular outer cortex adjacent to the mental foramen is removed before the osteotomy. The objective is to avoid the injury of the MN from the reciprocating saw or bur and offer abundant space for the surgical operation. The technique has a minimal learning curve and will be useful to plastic surgeons to minimize unintentional cutting or pulling injury to the MN in narrowing genioplasty.

[Horizontal transmission of Streptococcus mutans in caries-active preschool children].

OBJECTIVE: To analyze horizontal transmission patterns of Streptococcus mutans among caries-active preschool children for early interventions of dental caries. METHODS: Plaque samples obtained from 20 caries-active preschool children between 4 and 5 years of age were cultured under anaerobic conditions for isolating S. mutans, which were identified by morphological and biochemical analyses and PCR using primers homologous to the surface protein glucosyltransferase B (gtfB). The genotypes of the isolated S. mutans strains were determined by arbitrarily primed PCR (AP-PCR). RESULTS: Of the 200 S. mutans isolates obtained, 19 were excluded by biochemical analysis, and the remaining 181 isolates were identified as S. mutans by PCR with primers of gtfB, showing 37 different genotypes as identified by AP-PCR. Six children were found to carry S. mutans of a single genotype, 11 carried 2 genotypes, 2 had 3 genotypes, and 1 had 4 genotypes; 2 children from different classes were found to carry S. mutans of the same single genotype. CONCLUSION: We identified 37 genotypes of S. mutans in these caries-active preschool children, among whom horizontal transmissions of the strains were not found.

[Selection and identification of ssDNA aptamers specific to clinical isolates of Streptococcus mutans strains with different cariogenicity].

OBJECTIVE: To select and identify ssDNA aptamers specific to Streptococcus mutans strains with different cariogenicity isolated from clinical specimens. METHODS: Subtractive SELEX technology targeting the whole intact cells was used to screen for ssDNA aptamers specific to the clinical isolates Streptococcus mutans strains with different cariogenicity. Radioactive isotope, flow cytometry, gene cloning and sequencing, MEME online software and RNA structure analysis software were employed to analyze the first and secondary structures of the aptamers and identify the screened aptamers. RESULTS: Detection by radioactive isotope showed sufficient pool enrichment after 9 rounds of subtractive SELEX. Flow cytometry showed that the selected aptamers H1, H16, H4, L1, L10 and H19 were capable of binding specifically with highly cariogenic Streptococcus mutans strains but not with strains with a low cariogenicity. The aptamer H19 had the strongest binding capacity to highly cariogenic Streptococcus mutans strains, with a dissociation constant of 69.45∓38.53 nmol/L. CONCLUSION: We have obtained the ssDNA aptamers specific to the clinical isolates of highly cariogenic Streptococcus mutans strains.

[Preliminary screen of high cariogenicity Streptococcus mutans strains isolated from clinical specimens].

OBJECTIVE: To screen of high cariogenicity Streptococcus mutans (S. mutans) strains isolated from clinical specimens preliminary. METHODS: Acidogenicity, aciduricity, extracellular polysaccharide production and adhesion of 41 strains of S. mutans isolated from clinical specimens were investigated to screen high cariogenicity S. mutans strains. RESULTS: There were different cariogenicity among 41 strains of S. mutans, in which 3 strains of S. mutans had all high ability to produce extracellular polysaccharide, adhere to the saliva-coated hydroxyapatite, produce acid and tolerate acid, indicated there were 3 strains with high cariogenicity S. mutans strains isolated from clinical specimens. Another 3 strains of S. mutans with all low ability to produce extracellular polysaccharide, adhere to the saliva-coated hydroxyapatite, produce acid and tolerate acid indicated they were low cariogenicity S. mutans strains isolated from clinical specimens. CONCLUSION: We may have obtained high cariogenicity S. mutans strains isolated from clinical specimens.