Production improvement of an antioxidant in cariogenic Streptococcus mutans UA140.

AIMS: This study aimed to improve the production of mutantioxidin, an antioxidant encoded by a biosynthetic gene cluster (mao) in Streptococcus mutans UA140, through a series of optimization methods. METHOD AND RESULTS: Through the construction of mao knockout strain S. mutans UA140∆mao, we identified mutantioxidin as the antioxidant encoded by mao and verified its antioxidant activity through a reactive oxygen species (ROS) tolerance assay. By optimizing the culture medium and fermentation time, 72 h of fermentation in chemically defined medium (CDM) medium was determined as the optimal fermentation conditions. Based on two promoters commonly used in Streptococcus (ldhp and xylS1p), eight promoter refactoring strains were constructed, nevertheless all showed impaired antioxidant production. In-frame deletion and complementation experiments demonstrated the positive regulatory role of mao1 and mao2, on mao. Afterward, the mao1 and mao2, overexpression strain S. mutans UA140/pDL278:: mao1mao2, were constructed, in which the production of mutantioxidin was improved significantly. CONCLUSIONS: In this study, through a combination of varied strategies such as optimization of fermentation conditions and overexpression of regulatory genes, production of mutantioxidin was increased by 10.5 times ultimately.

Comparative Microbial Profiles of Caries and Black Extrinsic Tooth Stain in Primary Dentition.

Extrinsic black tooth stain (BS) is a common oral disease associated with lower caries experience in preschool children, although the microbiotic features contributing to the low risk of caries in this group remain elusive. In this study, we aimed at identifying the dominant bacteria in dental plaque to indicate the incidence of caries in the primary dentition. Subjects were divided into 3 groups based on the clinical examination: group CF, children without pigment who had no caries lesions or restorations (n = 18); group CS, children who were diagnosed with severe early childhood caries (n = 17); and group BS, children with pigment (black extrinsic stain) without caries or restorations (n = 15). The total microbial genomic DNA was extracted and subjected to bacterial 16S ribosomal RNA gene sequencing using an Illumina HiSeq platform. The differential dominant bacteria were determined using Wilcoxon rank-sum testing and linear discriminant analysis effect size (LEfSe). Co-occurrence network analysis was performed using sparse correlations for compositional data, calculation and functional features were predicted using PICRUSt. Interestingly, our results showed that the relative abundance of Pseudopropionibacterium, Actinomyces, Rothia, and Cardiobacterium was from high to low and that of Porphyromonas was low to high in the BS, CF, and CS groups, consistent with the clinical incidence of caries in the 3 groups. Moreover, an increased level of Selenomonas_3, Fusobacterium, and Leptotrichia was associated with high caries prevalence. We found that the interactions among genera in the BS and CS plaque communities are less complex than those in the CF communities at the taxon level. Functional features, including cofactor and vitamin metabolism, glycan biosynthesis and metabolism, and translation, significantly increased in caries plaque samples. These bacterial competition- and commensalism-induced changes in microbiota would result in a change of their symbiotic function, finally affecting the balance of oral microflora.

Insight into sludge dewatering by advanced oxidation using persulfate as oxidant and Fe2+ as activator: Performance, mechanism and extracellular polymers and heavy metals behaviors.

This study established a Fe2+/persulfate oxidation system to dewater sludge in WWTPs. Dewatering performance, persulfate consumption and the variations of sludge pH, TN and TP during dewatering process were monitored. EPS and ζ-potential behaviors for ameliorating sludge dewatering was investigated. Transformation, leaching toxicity and environmental risk of heavy metals in sludge during dewatering were determined. Results demonstrated that after treated by Fe2+/persulfate oxidation system with 0.6 mmol/g-VS of persulfate at Fe2+/persulfate molar ratio 0.6, WC decreased to 53.5% and SCST increased to 4.15, which implied an excellent improvement of sludge dewatering. The fast persulfate consumption, the decrease of sludge pH and the increase of TN illustrated the positive effects of Fe2+ in activating persulfate and the decomposition of EPS by the activation products, SO4•- and •OH. Another product (Fe3+) generated during persulfate activation could decrease the content of phosphorus-containing matter (released from EPS decomposition) through the precipitation reaction with PO43-. The decrease of TOC and UV-254 happened in HPO-A, HPO-N and TPI-A organic substance of EPS (mainly contained in TB-EPS fraction) indicated that the destruction of hydrophobic organic matter of EPS would stimulate the release of bound water, which was beneficial to dewater sludge. The largest protein loss in TB-EPS (from 24.5 to 10.7 mg/L) indicated that the effective decomposition of TB-EPS could significantly ameliorate sludge dewatering. The increase of ζ-potential indicated the degradation of organic matter in EPS with negative charge. To sum up, the destruction of protein-like substances in hydrophobic organic matter of TB-EPS was the main mechanism for improving sludge dewatering by Fe2+/persulfate oxidation system. 3D-EEM fluorescence spectroscopy analysis proved that these protein-like substances were mainly tryptophan protein and humic acid. Moreover, due to the disruption of EPS, the contents of heavy metals in sludge, and their leaching toxicity and environmental risk were reduced. Therefore, Fe2+/persulfate oxidation system has potential and application prospects to improve sludge dewatering and optimize sludge management in WWTPs.

The adsorption behavior of metals in aqueous solution by microplastics effected by UV radiation.

Microplastics are considered as the carrier to heavy metals in the environment. But the sorption ability of microplastics influenced by photo-aging is remaining unclear. In the present study, the sorption of two kinds of metal ions (Cu2+ and Zn2+) in the aqueous solution by both the virgin and aged microplastics was investigated. Polyethylene terephthalate (PET) debris, one of the typical kinds of microplastics was chosen in this study. Photo-aging of microplastics in environment was simulated using UV radiation in the laboratory. Date analysis indicated that the aged microplastics had higher adsorption capacity of heavy metals than original ones. This could be related to the increased surface area and oxygen containing function appeared in the surface of aged microplastics after UV radiation. When prolonging the time of radiation, the enhanced adsorption capacities of microplastics appeared for Cu2+ and Zn2+. These results showed a great interaction between the aging degree of plastics and sorption capacity to heavy metals. Meanwhile, external conditions including temperature and pH value were also showed great influence to the adsorption behavior.

Association between obstructive sleep apnoea and chronic periodontitis: a population-based study.

AIM: This study set out to explore the association between obstructive sleep apnoea (OSA) and prior chronic periodontitis (CP) by utilizing a population-based data set with a case-control design in Taiwan. MATERIALS AND METHODS: We included 7673 subjects with OSA as cases, and randomly selected 21,963 subjects without a history of OSA as controls. We performed conditional logistic regression analyses to explore the association between OSA and having been previously diagnosed with CP. RESULTS: There was a significant difference in the prevalence of prior CP between cases and controls (33.8% versus 22.6%, p < 0.001). Conditional logistic regression analysis suggested that the odds ratio of prior CP for cases was 1.75 (95% CI = 1.68-1.88) times greater than that of controls after adjusting for monthly income and geographical location, as well as hypertension, diabetes, coronary heart disease, hyperlipidaemia, obesity, tobacco use disorder, and chronic obstructive pulmonary disease and alcohol abuse. CONCLUSION: Our study detected an association between OSA and a prior diagnosis of CP.

Constructed wetlands as neglected fixed source of microplastics and antibiotic resistance genes in natural water bodies?

The pollution status and the harm caused by microplastics and antibiotic resistance genes (ARGs) in aquatic ecosystems have been a growing concern. The presence of microplastics could accelerate the transfer and spread of ARGs. Before sewage reaches natural water bodies, microplastics and ARGs need to be eliminated through specific processes. Constructed wetlands are currently an effective and environmentally friendly wastewater treatment process. Research has shown significant effectiveness in removing microplastics and ARGs. Microplastics and ARGs can be removed through processes such as adsorption, capture, adhesion, and biodegradation. However, long-term continuous operation could lead to constructed wetlands becoming significant reservoirs of microplastics and ARGs. Inflow loads and seasonal variations in constructed wetlands may result in the reintroduction of persistent microplastics and ARGs into the receiving water body, establishing the constructed wetland as a continuous source of these pollutants in the receiving water body. The key to the widespread application of constructed wetlands lies in solving this challenging problem. Therefore, although constructed wetlands serve as a green strategy for removing microplastics and ARGs, there are still many gaps in our knowledge. Based on the current accumulation of microplastics and ARGs in constructed wetlands, this paper summarizes the removal of microplastics and ARGs in existing constructed wetlands and explores the interaction between them. Additionally, it proposes suggestions for optimizing the process and improving the reliability of monitoring microplastics and ARGs in sewage.

A quantitative method for the measurement of membrane affinity by polydiacetylene-based colorimetric assay.

The measurement of membrane affinity is an important early screening step during drug discovery. However, classical methods for membrane affinity measurement are tedious and difficult to implement in high-throughput screening. This article describes a quantitative method for the measurement of membrane affinity by colorimetric assay based on polydiacetylene (PDA) sensors. Prepared lipid/PDA chromatic vesicles were used to model cell membranes. By measuring the colorimetric response of the chromatic vesicles when drug-membrane interactions occurred, membrane affinity constant K(b) could be calculated using a simple quantitative model. Under optimized preparation conditions, the calculated log(K(b)) values exhibited an in-batch relative standard deviation (RSD) of less than 4% and a between-batch RSD of less than 8% for all three reference compounds. The logarithm of K(b) of the six ß-blockers exhibited excellent linear correlation with the logarithm of the liposome/water partition coefficient (K(m)) with R(2)=0.9793. For neutral compounds, the log(K(b)) of n-fatty alcohols correlated with the logarithm of the n-octanol/water partition coefficient (K(oct)) with a linear correlation coefficient R(2)=0.9833. This work provides a simple, convenient, and reproducible method for the rapid measurement of membrane affinity and presents important implications for high-throughput screening.

Streptococcus mutans suppresses filamentous growth of Candida albicans through secreting mutanocyclin, an unacylated tetramic acid.

Fungi and bacteria often co-exist and physically or chemically interact with each other in their natural niches. This inter-kingdom species interaction is exemplified by the gram-positive bacterial pathogen Streptococcus mutans and opportunistic fungal pathogen Candida albicans, which co-exist in the human mouth. It has been demonstrated that the dynamic interaction between these two species plays a critical role in their virulence and biofilm development. In this study, we discovered that S. mutans represses filamentous development and virulence in C. albicans through secreting a secondary metabolite, mutanocyclin (a tetramic acid). Mutanocyclin functions by regulating the PKA catabolic subunit Tpk2 and its preferential binding target Sfl1. Inactivation of Tpk2 in C. albicans results in an increased sensitivity to mutanocyclin, whereas overexpression of Tpk2 leads to an increased resistance. Dysfunction of SFL1 and its downstream target genes overrides the hyphal growth defect caused by mutanocyclin. Further investigation demonstrates that three glycosylphosphatidylinositol (GPI)-anchored proteins (Spr1, Hyr4, and Iff8), associated with cell wall biogenesis and remodeling, and a set of filamentous regulators also contribute to the mutanocyclin response. We propose that both transcriptional regulation and cell wall composition contribute to mutanocyclin-mediated filamentous inhibition. This repressive effect of mutanocyclin could function as a natural regulator of filamentous development in C. albicans.

A 4arm-PEG macromolecule crosslinked chitosan hydrogels as antibacterial wound dressing.

In order to develop better wound dressings, a novel chitosan hydrogel (Cn-Nm gel) was designed and fabricated by using aldehyde-4-arm polyethylene glycol (4r-PEG-CHO) to crosslink the chitosan dissolved in alkaline solution, amino-4-arm polyethylene glycol (4r-PEG-NH2) was chosen as the additive simultaneously. The special dissolution technique and macromolecular crosslinking structure endows the Cn-Nm gels with better performance than that of gels prepared by acid dissolving method with micromolecule crosslinker. First, Cn-Nm gels own strong toughness with 500 kPa tensile strength and 1000% elongation, about 400% swelling ratio and fast water absorption rate. Second, about 300 kPa adhesive strength and strippability between the gels and skin is achieved. More importantly, Cn-Nm gels show nearly 100% antibacterial rate towards Escherichia coli and Staphylococcus aureus. Excellent biocompatibility is also proved by the mouse fibroblasts tests. All of the performance makes this developed chitosan-based gel be the potential candidate as a wound dressing.

Optimizing cardiac CT angiography minimum detectable difference via Taguchi's dynamic algorithm, a V-shaped line gauge, and three PMMA phantoms.

BACKGROUND: Radiologists widely use the minimum detectable difference (MDD) concept for inspecting the imaging quality and quantify the spatial resolution of scans. OBJECTIVE: This study adopted Taguchi's dynamic algorithm to optimize the MDD of cardiac CT angiography (CTA) using a V-shaped line gauge and three PMMA phantoms (50, 70, and 90 kg). METHODS: The phantoms were customized in compliance with the ICRU-48 report, whereas the V-shaped line gauge was indigenous to solidify the cardiac CTA scan image quality by two adjacent peaks along the V-shaped slit. Accordingly, the six factors A-F assigned in this study were A (kVp), B (mAs), C (CT pitch), D (FOV), E (iDose), and F (reconstruction filter). Since each factor could have two or three levels, eighteen groups of factor combinations were organized according to Taguchi's dynamic algorithm. Three welltrained radiologists ranked the CTA scan images three times for three different phantoms. Thus, 27 (3 × 3 × 3) ranked scores were summed and averaged to imply the integrated performance of one specific group, and eventually, 18 groups of CTA scan images were analyzed. The unique signal-to-noise ratio (S/N, dB) and sensitivity in the dynamic algorithm were calculated to reveal the true contribution of assigned factors and clarify the situation in routine CTA diagnosis. RESULTS: Minimizing the cross-interactions among factors, the optimal factor combination was found to be as follows: A (100 kVp), B (600 mAs), C (pitch 0.200 mm), D (FOV 280 mm), E (iDose 5), and F (filter XCA). The respective MDD values were 2.15, 2.32, and 1.87 mm for 50, 70, and 90 kg phantoms, respectively. The MDD of the 90 kg phantom had the most precise spatial resolution, while that of the 70 kg phantom was the worst. CONCLUSION: The Taguchi static and dynamic optimization algorithms were compared, and the latter's superiority was substantiated.

Dynamic Control Strategy to Produce Riboflavin with Lignocellulose Hydrolysate in the Thermophile Geobacillus thermoglucosidasius.

Efficient utilization of both glucose and xylose, the two most abundant sugars in biomass hydrolysates, is one of the main objectives of biofermentation with lignocellulosic materials. The utilization of xylose is commonly inhibited by glucose, which is known as glucose catabolite repression (GCR). Here, we report a GCR-based dynamic control (GCR-DC) strategy aiming at better co-utilization of glucose and xylose, by decoupling the cell growth and biosynthesis of riboflavin as a product. Using the thermophilic strain Geobacillus thermoglucosidasius DSM 2542 as a host, we constructed additional riboflavin biosynthetic pathways that were activated by xylose but not glucose. The engineered strains showed a two-stage fermentation process. In the first stage, glucose was preferentially used for cell growth and no production of riboflavin was observed, while in the second stage where glucose was nearly depleted, xylose was effectively utilized for riboflavin biosynthesis. Using corn cob hydrolysate as a carbon source, the optimized riboflavin yields of strains DSM2542-DCall-MSS (full pathway dynamic control strategy) and DSM2542-DCrib (single-module dynamic control strategy) were 5.3- and 2.3-fold higher than that of the control strain DSM 2542 Rib-Gtg constitutively producing riboflavin, respectively. This GCR-DC strategy should also be applicable to the construction of cell factories that can efficiently use natural carbon sources with multiple sugar components for the production of high-value chemicals in future.

The toxicity of virgin and UV-aged PVC microplastics on the growth of freshwater algae Chlamydomonas reinhardtii.

Although more attention has been paid to plastic pollution in marine ecosystems, research on the influence of plastic in freshwater ecosystems remains limited. To help fill this information gap, this article represents an investigation of the effects of virgin polyvinyl chloride (v-PVC) microplastics (MPs) and UV-aged polyvinyl chloride (a-PVC) MPs on the growth and chlorophyll content of the freshwater algae, Chlamydomonas reinhardtii (C. reinhardtii) at different periods (0, 24, 48, 72 and 96 h). The results suggest that both virgin and aged PVC MPs have negative effects on the growth of C. reinhardtii in the range of 10 mg/L to 200 mg/L, which leads to the reduction of chlorophyll-a level in the cells. Furthermore, a-PVC MPs were more toxic than v-PVC MPs, as shown by the a-PVC MPs' lower EC50 values after 96 h (63.66 mg/L for a-PVC MPs and 104.93 mg/L for v-PVC MPs). The inhibition effect of both kinds of PVC was also testified by the enhancement of enzymatic activity of superoxide dismutase (SOD) and malondialdehyde (MDA) in algae. Meanwhile, a-PVC MPs obviously had a higher toxicity than v-PVC MPs. The aging process that affected the surface characteristics of a-PVC was identified using Fourier transform infrared (FTIR) and Zetasizer. The carbonyl groups formed on the surface and the increased zeta potential of the a-PVC MPs affected the interaction between the microplastics and the algae, which increased the toxicity of aged microplastics. The research results presented here provide more evidence of the risks microplastics bring into the freshwater ecosystem.

Reductase of Mutanobactin Synthetase Triggers Sequential C-C Macrocyclization, C-S Bond Formation, and C-C Bond Cleavage.

Mutanobactins (MUBs) and their congeners that contain a macrocycle and/or a thiazepane ring are lipopeptides from Streptococcus mutans, a major causative agent of dental caries. Here we show that the C-terminal reductase domain of MubD releases the lipohexapeptide intermediates in an aldehyde form, which enables a spontaneous C-C macrocyclization. In the presence of a thiol group, the macrocyclized MUBs can further undergo spontaneous C-S bond formation and C-C bond cleavage to generate diverse MUB congeners.

[Secondary metabolites from Streptococcus mutans and their ecological roles in dental biofilm].

Dental biofilms are composed of hundreds of bacterial species, among which Streptococcus mutans is widely recognized as the major pathogen of dental caries. The cariogenic potential of S. mutans is related to its ability to form a robust biofilm on the tooth surface and its acidogenic and acid-tolerant properties. Co-evolution of S. mutans with the host has resulted in the diversity of secondary metabolism of S. mutans in strain level. A variety of secondary metabolites, including 10 bacteriocins (mutacins) and one hybrid Polyketide/Non-Ribosomal Peptide type compound, have been characterized. Studies on these secondary metabolites indicate that they play a significant role either in interspecies or in inter-kingdom interactions in the dental biofilm. As more S. mutans strains are isolated and sequenced, additional secondary metabolites with novel functions will be discovered. The study of secondary metabolites in S. mutans is anticipated to be helpful for oral disease treatment and prevention by providing new strategies.

Genome mining unveils widespread natural product biosynthetic capacity in human oral microbe Streptococcus mutans.

Streptococcus mutans is a major pathogen causing human dental caries. As a Gram-positive bacterium with a small genome (about 2 Mb) it is considered a poor source of natural products. Due to a recent explosion in genomic data available for S. mutans strains, we were motivated to explore the natural product production potential of this organism. Bioinformatic characterization of 169 publically available genomes of S. mutans from human dental caries revealed a surprisingly rich source of natural product biosynthetic gene clusters. Anti-SMASH analysis identified one nonribosomal peptide synthetase (NRPS) gene cluster, seven polyketide synthase (PKS) gene clusters and 136 hybrid PKS/NRPS gene clusters. In addition, 211 ribosomally synthesized and post-translationally modified peptides (RiPPs) clusters and 615 bacteriocin precursors were identified by a combined analysis using BAGEL and anti-SMASH. S. mutans harbors a rich and diverse natural product genetic capacity, which underscores the importance of probing the human microbiome and revisiting species that have traditionally been overlooked as "poor" sources of natural products.