New meroterpenoids and polyketides from the endophytic fungus Paraphaeosphaeria sp. C-XB-J-1 and their anti-inflammatory and SARS-CoV-2 Mpro inhibitory activities.

Seven new meroterpenoids, paraphaeones A-G (1-7), and two new polyketides, paraphaeones H-I (8-9), along with eight known compounds (10-17), were isolated from the endophytic fungus Paraphaeosphaeria sp. C-XB-J-1. The structures of 1-9 were identified through the analysis of 1H, 13C, and 2D NMR spectra, assisted by HR-ESI-MS data. Compounds 1 and 7 exhibited a dose-dependent decrease in lactate dehydrogenase levels, with IC50 values of 1.78 µM and 1.54 µM, respectively. Moreover, they inhibited the secretion of IL-1ß and CASP-1, resulting in a reduction in the activity levels of NLRP3 inflammasomes. Fluorescence microscopy results indicated that compound 7 concentration-dependently attenuated cell pyroptosis. Additionally, compounds 4 and 7 showed potential inhibitory effects on the severe acute respiratory syndrome coronavirus-2 main protease (SARS-CoV-2 Mpro), with IC50 values of 10.8 ± 0.9 µM and 12.9 ± 0.7 µM, respectively.

Development of na HPLC-MS/MS method for the determination of ginsenosides Rg1 and Rb1 from 'Shenmai' Injection in beagle dogs after single and multiple doses and application in pharmacokinetics.

Shenmai Injection (SMI), which tonifies Qi and prevents exhaustion, nourishes Yin and generates body fluid, is usually used in the treatment of shock with deficiency of Qi and Yin, coronary artery disease, viral myocarditis, granulocytopenia and chronic pulmonary heart disease clinically. Ginsenosides Rg1 and Rb1 are the main active ingredients of SMI. In this study, high-performance liquid chromatography tandem mass spectrometry methods for quantification of Rb1 and Rg1 in beagle dogs were developed and validated according to international regulatory guidelines. The methods were applied to measure the pharmacokinetics parameters of the two ginsenoside after intravenous administration. The linear ranges of the analytes were 3.9-1,000 ng/ml for Rg1 and Rb1. After injection of single and multiple doses of SMI (1 ml/kg), the plasma concentration-time profiles of Rg1 and Rb1 met the characteristics of one-compartment and typical two-compartment intravenous injection.

Bioactive constituents from Salacia cochinchinensis.

Two new isopimarane diterpenoids, named 1α-hydroxy-7-oxoisopimara-8, 15-diene (1), 11ß-hydroxy-7-oxoisopimara-8(14), 15-diene (2), together with six known compounds (3-8), were isolated from the medicinal plant Salacia cochinchinensis. All isolates were assayed for their cytotoxicity and α-glucosidase inhibitory activity. Results suggested compounds 1, 3 possessed significant cytotoxic activity against HepG2, HL60, and Hela cell lines with IC50 values ranging from 0.23 to 0.35 µM, and compounds 7, 8 exhibited noticeable α-glucosidase inhibitory ability with IC50 values of 0.25 and 0.31 µM, respectively.

[A new guaiane-type sesquiterpenoid from Croton yunnanensis].

Five sesquiterpenoids were isolated from 90% ethanol extract of Croton yunnanensis by silica gel,Sephadex LH-20 column chromatography,as well as prep-HPLC methods. Based on MS,1 D and 2 D NMR spectral analyses,the structures of the five compounds were identified as 11-methoxyl alismol(1),6ß,7ß-epoxy-4α-hydroxyguaian-10-ene(orientalol C,2),multisalactone D(3),arvestonol(4),and 4,5-dihydroblumenol A(5). Compound 1 was a new guaiane-type sesquiterpenoid. Compounds 2-4 were isolated from the Croton genus for the first time,and compound 5 was obtained from this plant for the first time.

Effect of high-fructose corn syrup on Streptococcus mutans virulence gene expression and on tooth demineralization.

High-fructose corn syrup-55 (HFCS-55) has been widely welcomed in recent years as a substitute for sucrose on the basis of its favourable properties and price. The objective of this study was to determine the influence of HFCS-55 on the expression of Streptococcus mutans UA159 virulence genes and on tooth demineralization. Real-time reverse-transcription PCR (real-time RT-PCR) and microhardness evaluations were performed to examine gene expression and enamel demineralization, respectively, after treatment with HFCS-55 and/or sucrose. Significant up-regulation of glucosyltransferase B (gtfB) by HFCS-55 was found. A mixture of HFCS-55 and sucrose could positively enhance expression of glucan-binding protein (gbp) genes. Regarding acidogenicity, expression of the lactate dehydrogenase (ldh) gene was unaffected by HFCS-55. A notable finding in this study was that 5% HFCS-55 significantly enhanced expression of the intracellular response gene of the two-component VicRK signal transduction system (vicR). Demineralization testing showed that the microhardness of teeth decreased by a greater extent in response to HFCS-55 than in response to sucrose. The results indicate that HFCS-55 can enhance S. mutans biofilm formation indirectly in the presence of sucrose and that HFCS-55 has a more acidogenic potential than does sucrose. Summing up the real-time PCR and demineralization results, HFCS-55 appears to be no less cariogenic than sucrose in vitro - at least, not under the conditions of our experiments.

Efficient production of butyric acid from lignocellulosic biomass by revealing the mechanisms of Clostridium tyrobutyricum tolerance to phenolic inhibitors.

Phenolic compounds (PCs) generated during pretreatment of lignocellulosic biomass severely hinder the biorefinery by Clostridia. As a hyperbutyrate-producing strain, Clostridium tyrobutyricum has excellent tolerance to PCs, but its tolerance mechanism is poorly understood. In this study, a comprehensive transcriptome analysis was applied to elucidate the response of C. tyrobutyricum to four typical PCs. The findings revealed that the expression levels of genes associated with PC reduction, HSPs, and membrane transport were significantly altered under PC stress. Due to PCs being reduced to low-toxicity alcohols/acids by C. tyrobutyricum, enhancing the reduction of PCs by overexpressing reductase genes could enhance the strain's tolerance to PCs. Under 1.0 g/L p-coumaric acid stress, compared with the wild-type strain, ATCC 25755/sdr1 exhibited a 31.2 % increase in butyrate production and a 38.5 % increase in productivity. These insights contribute to the construction of PC-tolerant Clostridia, which holds promise for improving biofuel and chemical production from lignocellulosic biomass.

luxS mutant regulation: quorum sensing impairment or methylation disorder?

AI-2-mediated quorum sensing has been identified in various bacteria, including both Gram-negative and Gram-positive species, and numerous phenotypes have been reported to be regulated by this mechanism, using the luxS-mutant strain. But the AI-2 production process confused this regulatory function; some considered this regulation as the result of a metabolic change, which refers to an important metabolic cycle named activated methyl cycle (AMC), caused by luxS-mutant simultaneously with the defect of AI-2. Herein we hypothesized that the quorum sensing system--not the metabolic aspect--is responsible for such a regulatory function. In this study, we constructed plasmids infused with sahH and induced protein expression in the luxS-mutant strain to make the quorum-sensing system and metabolic system independent. The biofilm-related genes were investigated by real-time polymerase chain reaction (PCR), and the results demonstrated that the quorum-sensing completed strain restored the gene expression of the defective strain, but the metabolically completed one did not. This evidence supported our hypothesis that the autoinducer-2-mediated, quorum-sensing system, not the AMC, was responsible for luxS mutant regulation.

Preparation and Application of a Novel Slow-Releasing with Core-Shell Deicer in Asphalt Mixtures.

The massive application of chloride salts has a direct effect on the corrosion of structures and vehicles and decreases durability as well as road pavement damage. A novel slow-release deicer with a core-shell structure was prepared to reduce the salts' impacts, subsequently characterized by scanning electron microscopy (SEM) with energy dispersive spectroscopy (EDS), differential scanning calorimetry (DSC), and thermogravimetric analysis (TG). The conductivity evaluation, moisture absorption, and the snow or ice melting performance of the deicer were also tested. The core-shell deicer with different replacement rates was used to prepare the deicing asphalt mixture based on the equivalent volume replacement method. In this study, the high- and low-temperature performance, moisture damage resistance, and snow or ice melting capacity of mixtures were evaluated in the laboratory. The results show that the low-temperature and moisture stability performances decreased, and high-temperature performance improved, as the content of the core-shell deicer was increased. It is confirmed that the replacement rate of the deicer filler should be lower than 75% to meet the specification requirements. The prepared deicing asphalt mixture has good snow and ice melting performance and can reduce the bonding strength between ice and pavement surface. Durability and cost-benefit analysis are expected in further investigations.

Four new triterpene glucosides from Salacia cochinchinensis Lour.

Four new triterpene glucosides (1-4) were isolated from the 90% ethanol extract of Salacia cochinchinensis, together with five known compounds (5-9). The structures of the new compounds were elucidated by comprehensive spectroscopic analysis including HRESIMS, IR, 1 D and 2 D NMR analysis. All isolates were assayed for their α-glucosidase inhibitory activity. Compound 9 showed remarkable α-glucosidase inhibitory activity with an IC50 value of 0.31 µM, and the triterpene glycosides (1-5) exhibited moderate α-glucosidase inhibitory activity.

Investigation of supragingival plaque microbiota in different caries status of Chinese preschool children by denaturing gradient gel electrophoresis.

This study aimed to detect differences in the richness of total supragingival plaque microbiota as well as the species composition of oral streptococci involved in the different stages of dental caries. Forty-five plaque samples were collected from caries-moderate (CM, 4 ≤ dmfs ≤ 6), caries-susceptible (CS, dmfs ≥ 10), and age-matched caries-free children separately. Total DNA was isolated directly from each sample, and polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) analyses using universal and primers specific for oral streptococci were carried out. Using 16S rDNA PCR-DGGE, 34 different species of bacteria were identified in a culture-independent manner and classified into 11 genera according to phylogenetic analysis. Among them, Mitis group streptococci and Campylobacter, which were present in health status, no longer appeared in caries-susceptible samples. In addition, Capnocytophaga, Burkholderia, and Prevotella were found significantly less frequently in the CS group samples (P < 0.05), while there were no significant differences among the prevalence of Neisseria, Leptotrichia, Haemophilus, Mutans group streptococci, Corynebacterium, and Actinomyces in the three groups. Further DGGE analysis of rnpB gene amplicons obtained with oral streptococci species-specific primers showed that a total of 23 species of oral streptococci were identified. Streptococcus sanguinis, Streptococcus mitis, and Streptococcus oralis showed a significantly higher prevalence in healthy children (P < 0.05), while that of Streptococcus mutans and Streptococcus sobrinus did not vary among the three groups. Overall, these results suggest that supragingival plaque microbiota as a whole undergoes a more complicated shift in the caries process.

Bioactive glycosides from Salacia cochinchinensis.

Four new triterpene glycosides, named salaciacochinosides A-D (1-4) were isolated from the 90% ethanol extract of Salacia cochinchinensis, together with five known compounds 2α,3ß,23-trihydroxyurs-12,18-dien-28-oic acid 28-O-ß-d-glucopyranoside (5), racemiside (6), alangiplatanoside (7), acantrifoside E (8), and syringin (9). The structures of the four new triterpenoids were characterized by chemical methods and MS, IR, 1D and 2D NMR spectral analyses. The α-glucosidase inhibitory activities of the nine compounds were assessed, compounds 6 and 7 showed remarkable α-glucosidase inhibitory activities, with IC50 values of 0.44 and 0.75 µM, respectively. Compounds 1-5 exhibited moderate α-glucosidase inhibitory activities, and compounds 8 and 9 showed none α-glucosidase inhibitory activity in our current experiments.

Porphyromonas gingivalis infection accelerates intimal thickening in iliac arteries in a balloon-injured rabbit model.

BACKGROUND: Current epidemiologic data suggest that a localized infection (periodontitis) can disseminate into the distant tissues, and subgingival bacteria can migrate in the bloodstream, thereby contributing to independent systemic disease processes. To test this hypothesis, we investigated the effect of repeated systemic inoculations with Porphyromonas gingivalis (Pg) on intimal hyperplasia in iliac arteries in a rabbit model of balloon injury. METHODS: One week after single balloon injury to the iliac artery, 30 male New Zealand rabbits were randomly assigned to intravenous inoculation with 100 microl live Pg (10(7) colony-forming units; n = 15) or vehicle (n = 15) once weekly for 4, 8, or 12 consecutive weeks. Arteries were fixed by perfusion and removed for analysis of neointimal lesion formation. We measured intimal and medial lesion areas in iliac artery cross-sections as well as the intimal/medial ratio (I/M). We also analyzed Pg 16S ribosomal DNA amplification with polymerase chain reaction, systemic proinflammatory mediators with enzyme-linked immunosorbent assay, and immunolocalization of macrophages in the balloon-injured arteries. RESULTS: At 12 weeks, iliac intimal hyperplasia was accelerated, and I/M was significantly increased in Pg-inoculated animals (I/M 3.961 +/- 0.536 in the Pg group versus 3.585 +/- 0.353 in the control animals; P <0.01). Pg-inoculated animals also had significant increases in macrophage infiltration at 12 weeks, C-reactive protein levels at all time points, and interleukin-6 levels at 12 weeks. Moreover, Pg ribosomal DNA was found in the injured arteries of Pg-inoculated animals, but only after 12 weeks. CONCLUSION: Long-term systemic challenge with Pg, an oral pathogen, may accelerate intimal hyperplasia in balloon-injured iliac arteries.

Triterpenoid saponins from the roots of Cyathula officinalis and their inhibitory effects on nitric oxide production.

The present study was designed to investigate the chemical constituents of the roots of Cyathula officinalis. Compounds were isolated by silica gel, Sephadex LH-20, ODS column chromatography, and preparative HPLC. Their structures were determined on the basis of 1D and 2D NMR techniques, mass spectrometry, and chemical methods. One new oleanane-type triterpenoid saponin, 28-O-[α-L-rhamnopyranosyl-(1→3)-ß-D-glucuronopyranosyl-(1→3)-ß-D-glucopyranosyl] hederagenin (1), was isolated from the roots of Cyathula officinalis. The anti-inflammatory activities of the isolates were evaluated for their inhibitory effects against LPS-induced nitric oxide (NO) production in RAW 264.7 macrophages cells. Compounds 2, 4, and 6 exhibited moderate anti-inflammatory activities.

Imaging of extraradicular biofilm using combined scanning electron microscopy and stereomicroscopy.

Microorganisms are able to survive and induce persistent infection in extraradicular areas. The objective of this study was to evaluate the relationship between extraradicular biofilm and persistent periapical periodontitis. Thirty-five apical samples with different stages of pulp and periapical pathology (vital pulp, pulp necrosis without radiographically visible periapical lesions, chronic periapical periodontitis that had not received root canal therapy and persistent periapical periodontitis) were initially evaluated using scanning electron microscopy. The same samples were then processed using Brown and Brenn-modified Gram staining. We detected extraradicular biofilm in all samples with persistent periapical periodontitis and in three samples with chronic periapical periodontitis. The extraradicular bacteria predominantly had rod and filament morphology and were surrounded by varying amounts of amorphous extracellular material. The surfaces outside the root of the apical samples with vital pulp and pulp necrosis were covered by fibers, and no extraradicular microorganisms were present, which suggests that extraradicular biofilm is closely associated with failed endodontic treatments, thus resulting in persistent infection.

Characterization of oral bacterial diversity of irradiated patients by high-throughput sequencing.

The objective of this study was to investigate the compositional profiles and microbial shifts of oral microbiota during head-and-neck radiotherapy. Bioinformatic analysis based on 16S rRNA gene pyrosequencing was performed to assess the diversity and variation of oral microbiota of irradiated patients. Eight patients with head and neck cancers were involved in this study. For each patient, supragingival plaque samples were collected at seven time points before and during radiotherapy. A total of 147,232 qualified sequences were obtained through pyrosequencing and bioinformatic analysis, representing 3,460 species level operational taxonomic units (OTUs) and 140 genus level taxa. Temporal variations were observed across different time points and supported by cluster analysis based on weighted UniFrac metrics. Moreover, the low evenness of oral microbial communities in relative abundance was revealed by Lorenz curves. This study contributed to a better understanding of the detailed characterization of oral bacterial diversity of irradiated patients.

Exploring the dynamic core microbiome of plaque microbiota during head-and-neck radiotherapy using pyrosequencing.

Radiotherapy is the primary treatment modality used for patients with head-and-neck cancers, but inevitably causes microorganism-related oral complications. This study aims to explore the dynamic core microbiome of oral microbiota in supragingival plaque during the course of head-and-neck radiotherapy. Eight subjects aged 26 to 70 were recruited. Dental plaque samples were collected (over seven sampling time points for each patient) before and during radiotherapy. The V1-V3 hypervariable regions of bacterial 16S rRNA genes were amplified, and the high-throughput pyrosequencing was performed. A total of 140 genera belonging to 13 phyla were found. Four phyla (Actinobacteria, Bacteroidetes, Firmicutes, and Proteobacteria) and 11 genera (Streptococcus, Actinomyces, Veillonella, Capnocytophaga, Derxia, Neisseria, Rothia, Prevotella, Granulicatella, Luteococcus, and Gemella) were found in all subjects, supporting the concept of a core microbiome. Temporal variation of these major cores in relative abundance were observed, as well as a negative correlation between the number of OTUs and radiation dose. Moreover, an optimized conceptual framework was proposed for defining a dynamic core microbiome in extreme conditions such as radiotherapy. This study presents a theoretical foundation for exploring a core microbiome of communities from time series data, and may help predict community responses to perturbation as caused by exposure to ionizing radiation.

[The influence of different alkaline pH conditions on Enterococcus faecalis in planktonic and biofilm mode].

PURPOSE: To study the influence of different pH conditions on Enterococcus faecalis(E. faecalis) in planktonic and biofilm mode. METHODS: E. faecalis were prepared in planktonic and biofilm mode and cultured in TSB mediums 2 hours at pH 7,8,9,10,11 and 12. MTT assays were applied to evaluate the survival rate of bacterial cells in different pH value. SAS 6.12 software package was used for statistical analysis. RESULTS: Mild alkaline mediums (pH7-9) had no effect on cell vitality of E. faecalis and high alkaline condition (pH>10) led to significant declines of survival rate of cells. The biofilm cells of E. faecalis were more alkaline tolerant than corresponding planktonic cells. CONCLUSION: Biofilm formation is an important step in the development of alkaline tolerance of E. faecalis.

Bacterial flora and extraradicular biofilm associated with the apical segment of teeth with post-treatment apical periodontitis.

INTRODUCTION: Microorganisms are able to survive and cause persistent infection in the extraradicular area. The aims of this study were to investigate the primary bacterial flora and the localization of extraradicular biofilm in persistent apical periodontitis lesions. METHODS: Apical root samples from root-end surgery were collected from 23 root-filled teeth with apical periodontitis. Five samples were examined for the presence of biofilm by scanning electron microscopy. Another 5 samples were examined for the presence of biofilm by Brown and Brenn-modified Gram staining. The DNA from 13 samples was processed for amplification via polymerase chain reaction and separated with denaturing gradient gel electrophoresis. Selected bands were excised from the gel and sequenced for identification. RESULTS: The extraradicular biofilm present on the external root surface of treated teeth consisted of abundant, amorphous extracellular material and multiple bacterial species. The following species were detected in the microbial community from the apical samples: Actinomyces sp. oral, Propionibacterium, Prevotella sp. oral, Streptococcus, Porphyromonas endodontalis, and Burkholderia. The prevalence of Actinomyces sp. oral and Propionibacterium were highest (84.6% and 61.5%, respectively). CONCLUSIONS: Extraradicular biofilm was present on the external root surface of treated teeth with persistent periapical lesions. Actinomyces sp. oral and Propionibacterium are likely important contributors to extraradicular biofilm formation and persistent periapical infection.

Use of the quorum sensing inhibitor furanone C-30 to interfere with biofilm formation by Streptococcus mutans and its luxS mutant strain.

Streptococcus mutans is recognised as a major aetiological agent of dental caries. One of its important virulence factors is its ability to form biofilms on tooth surfaces. The aim of this study was to evaluate the effects of the quorum sensing inhibitor furanone C-30 on biofilm formation by S. mutans and its luxS mutant strain. The effects of furanone C-30 on biofilms of both strains formed on 96-well microtitre plates at 37 °C were determined by a colorimetric technique (MTT assay). Different concentrations of furanone C-30 (0.0, 2.0 and 4.0 µg/mL) and different time points of biofilm formation (4, 14 and 24 h) were investigated. The structures and thickness of the biofilms were observed by confocal laser scanning microscopy (CLSM). Quorum sensing-related gene expression (ftf, smu630, brpA, gbpB, gtfB, vicR, comDE and relA) was investigated by real-time polymerase chain reaction (RT-PCR). The results showed that synthetic furanone C-30 can inhibit biofilm formation by S. mutans and its luxS mutant strain, although it does not affect the bacterial growth rate itself. The quantities of biofilm formed by both strains significantly decreased (P<0.05) and the biofilms became thinner and looser as revealed by CLSM with increasing concentrations of furanone C-30. Expression of the genes tested was downregulated in the biofilms by the addition of furanone C-30. These results revealed that synthetic furanone C-30 can effectively inhibit biofilm formation by S. mutans and its luxS mutant strain.

Pathogenesis could be one of the anti-cheating mechanisms for Pseudomonas aeruginosa society.

Pseudomonas aeruginosa is the major pathogen of chronic lung infections in individuals with cystic fibrosis (CF). Traditionally, it has been regarded as living in planktonic form, and as being able to perform only simple physiological activities. Recent studies in biofilm infections in CF patients, however, show that P. aeruginosa can perform many social behaviors, like cooperation and cheating. Based on the theory of "survival of the fittest", it may be presumed that every individual will take advantage of cheating instead of cooperation to increase its fitness, at the cost of group survival. In reality, however, a bacterial society can remain stable, even though cheaters arise frequently in the population. It is therefore possible that there are anti-cheating mechanisms in a bacterial society. The cheaters of P. aeruginosa will cause the loss or the decrease of the pathogenesis of the microorganism in the cystic fibrosis host. These defects in pathogenesis will be disadvantageous to bacterial colonization and compromise the resistance to host immunity. We therefore propose the hypothesis that the pathogenesis in cystic fibrosis lung infections could be one of the anti-cheating mechanisms that contribute to the hidden costs of the cheater strains. To test this hypothesis, we designed an experiment in an animal model of CF. If this hypothesis can be confirmed, it will illustrate that nontrivial analogies exist between microbial social behaviors and the social traits that are observed in the more traditional model systems for sociobiology. This will not only provide a genetic model for sociobiology research, but also cast light on the social control of chronic bacterial infections.