Streptococcus strain D19T as a probiotic candidate to modulate oral health.

BACKGROUND: As probiotics protect host cells, they are used to treat bacterial infections. It has been indicated that probiotics may prevent or reduce the attachment of pathogens to host cells. In this study, Streptococcus strain D19T was isolated from the oropharynx of a healthy child, and its adhesion performance and Staphylococcus aureus adhesion inhibition effect were analysed using human bronchial epithelial (16-HBE) cells, as an in vitro cell model. We evaluated the probiotic properties of the D19T strain based on its acid-base, bile salt, and lysozyme tolerance; antibacterial activity; cytotoxicity; antibiotic sensitivity; in vitro adhesion to 16-HBE cells; and competitive, exclusion, and displacement effects against S. aureus. RESULTS: Streptococcus strain D19T showed tolerance to a PH range of 2-5 and 0.5-1% bile. However, it was more tolerant to 0.5% bile than to 1% bile. The strain also demonstrated an ability to adapt to maladaptive oropharyngeal conditions (i.e., tolerating 200 µg/mL lysozyme). It was resistant to 0.8 mM H2O2. The results also demonstrated that D19T exhibited inhibitory activities against various common pathogenic bacteria. Furthermore, D19T was not toxic to 16-HBE cells at different multiplicities of infection and was sensitive to most antibiotics tested. The adhesion rate of D19T cells to 16-HBE cells was 47% ± 1.2%, which was significantly higher than that of S. aureus to 16-HBE cells. The competition, exclusion, and displacement assay results showed that D19T has good inhibitory effect against S. aureus adhesion. CONCLUSIONS: The present study revealed that Streptococcus strain D19T has the potential to be developed as a respiratory microbiota preparations.

Streptococcus strain C17T as a potential probiotic candidate to modulate oral health.

In the microbiome, probiotics modulate oral diseases. In this study, Streptococcus strain C17T was isolated from the oropharynx of a 5-year-old healthy child, and its potential probiotic properties were analysed using human bronchial epithelial cells (16-HBE) used as an in vitro oropharyngeal mucosal model. The results demonstrated that the C17T strain showed tolerance to moderate pH ranges of 4-5 and 0·5-1% bile. However, it was more tolerant to 0·5% bile than 1% bile. It also demonstrated an ability to accommodate maladaptive oropharyngeal conditions (i.e. tolerating lysozyme at 200 µg ml-1 ). It was also resistant to hydrogen peroxide at 0·8 mM. In addition, we found out that the strain possesses inhibitory activities against various common pathogenic bacteria. Furthermore, C17T was not cytotoxic to 16-HBE cells at different multiplicities of infection. Scanning electron microscopy disclosed that C17T adhesion to 16-HBE cells. Competition, exclusion and displacement assays showed that it had good anti-adhesive effect against S. aureus. The present study revealed that Streptococcus strain C17T is a potentially efficacious oropharyngeal probiotic.

Systematic research of H2dedpa derivatives as potent inhibitors of New Delhi Metallo-ß-lactamase-1.

New Delhi Metallo-ß-lactamase-1 (NDM-1), a Zn (II)-dependent enzyme, can catalyze the hydrolysis of almost all ß-lactam antibiotics including carbapenems, resulting in bacterial antibiotic resistance, which threatens public health globally. Based on our finding that H2dedpa is as an efficient NDM-1 inhibitor, a series of H2dedpa derivatives was systematically prepared. These compounds exhibited significant activity against NDM-1, with IC50 values 0.06-0.94 µM. In vitro, compounds 6k and 6n could restore the activity of meropenem against Klebsiella pneumoniae, Escherichia coli and Proteus mirabilis possessing either NDM or IMP. In particular, the activity of meropenem against E. coli producing NDM-4 could be improved up to 5333 times when these two compounds were used. Time-kill cell-based assays showed that 99.9% of P. mirabilis were killed when treated with meropenem in combination with compound 6k or 6n. Furthermore, compounds 6k and 6n were nonhemolytic (HC50 > 1280 µg/mL) and showed low toxicity toward mammalian (HeLa) cells. Mechanistic studies indicated that compounds 6k and 6n inhibit NDM-1 by chelating the Zn2+ ion of the enzyme.

NOTA analogue: A first dithiocarbamate inhibitor of metallo-ß-lactamases.

The emergence of antibiotic drug (like carbapenem) resistance is being a global crisis. Among those resistance factors of the ß-lactam antibiotics, the metallo-ß-lactamases (MBLs) is one of the most important reasons. In this paper, a series of cyclic dithiocarbamate compounds were synthesized and their inhibition activities against MBLs were initially tested combined with meropenem (MEM) by in vitro antibacterial efficacy tests. Sodium 1,4,7-triazonane-1,4,7-tris(carboxylodithioate) (compound 5) was identified as the most active molecule to restore the activity of MEM. Further anti-bacterial effectiveness assessment, compound 5 restored the activity of MEM against Escherichia coli, Citrobacter freundii, Proteus mirabilis and Klebsiella pneumonia, which carried resistance genes of blaNDM-1. The compound 5 was non-hemolytic, even at a concentration of 1000 µg/mL. This compound was low toxic toward mammalian cells, which was confirmed by fluorescence microscopy image and the inhibition rate of HeLa cells. The Ki value of compounds 5 against NDM-1 MBL was 5.63 ±â€¯1.27 µM. Zinc ion sensitivity experiments showed that the inhibitory effect of compound 5 as a MBLs inhibitor was influenced by zinc ion. The results of the bactericidal kinetics displayed that compound 5 as an adjuvant assisted MEM to kill all bacteria. These data validated that this NOTA dithiocarbamate analogue is a good inhibitor of MBLs.

[Identification of tetracenomycin X from a marine-derived Saccharothrix sp. guided by genes sequence analysis].

The crude extracts of the fermentation broth from a marine sediment-derived actinomycete strain, Saccharothrix sp. 10-10, showed significant antibacterial activities against drug-resistant pathogens. A genome-mining PCR-based experiment targeting the genes encoding key enzymes involved in the biosynthesis of secondary metabolites indicated that the strain 10-10 showed the potential to produce tetracenomycin-like compounds. Further chemical investigation of the cultures of this strain led to the identification of two antibiotics, including a tetracenomycin (Tcm) analogs, Tcm X (1), and a tomaymycin derivative, oxotomaymycin (2). Their structures were identified by spectroscopic data analysis, including UV, 1D-NMR, 2D-NMR and MS spectra. Tcm X (1) showed moderate antibacterial activities against a number of drug-resistant pathogens, including methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococci (VRE) pathogens, with the MIC values in the range of 32-64 microg x mL(-1). In addition, 1 also displayed significant cytotoxic activities against human cancer cell lines, including HL60 (leukemia), HepG2 (liver), and MCF-7 (breast) with the IC 50 values of 5.1, 9.7 and 18.0 micromol x L(-1), respectively. Guided by the PCR-based gene sequence analysis, Tcm X (1) and oxotomaymycin (2) were identified from the genus of Saccharothrix and their 13C NMR data were correctly assigned on the basis of 2D NMR spectroscopic data analysis for the first time.

Correlation Between Aggressive Behavior and Impulsive and Aggressive Personality Traits in Stable Patients with Schizophrenia.

Purpose: To explore the correlation between aggressive behavior and impulsive and aggressive personality traits in inpatients with schizophrenia. Methods: In total, 367 inpatients with schizophrenia were divided into two groups: the aggressive group and the non-aggressive group. We assessed inpatients' psychotic symptoms as well as their aggressive and impulsive personality traits using the Positive and Negative Symptom Scale, the Barratt Impulsiveness Scale, and the Buss-Perry Aggression Questionnaire. Results: Compared with the scores of inpatients in the non-aggressive group, the total Buss-Perry Aggression Questionnaire, subscale, and Barratt Impulsiveness Scale behavioral factor scores in those in the aggressive group were higher (p < 0.05). The results of logistic regression analysis suggested that a high Positive and Negative Symptom Scale positive factor score (odds ratio = 1.07) and a high Buss-Perry Aggression Questionnaire physical aggression score (odds ratio = 1.02) were risk factors for aggressive behavior. Conclusion: Hospitalized patients with schizophrenia with more severe positive symptoms and aggressive traits may be more prone to aggressive behavior.

Synthesis and in-vitro antimycobacterial activity of fluoroquinolone derivatives containing a coumarin moiety.

A series of gatifloxacin, ciprofloxacin, and 8-OCH(3) ciprofloxacin coumarin derivatives with remarkable improvement in lipophilicity as compared to the parent fluoroquinolones was designed, synthesized, and characterized by (1) H-NMR, MS, and HRMS. These derivatives were evaluated for their in-vitro activity against Mycobacterium smegmatis CMCC 93202 and MTB H37Rv ATCC 27294. All of the synthesized compounds were less active than the parent compounds against M. smegmatis CMCC 93202, but the activity of compound 6 was found to be 2-8-fold more potent than ciprofloxacin, 8-OCH(3) ciprofloxacin, moxifloxacin, and rifampin, and comparable to gatifloxacin against MTB H37Rv ATCC 27294. These results indicated that the lipophilicity of the tested compounds is not the sole parameter affecting antimycobacterial activity.

Gene-specific silencing induced by parallel complementary RNA in Pseudomonas aeruginosa.

To investigate whether parallel complementary RNA (pRNA) could induce gene-specific silencing in Pseudomonas aeruginosa, pRNA of the mexA gene was expressed in it. Compared to the control strains, the strain expressing pRNA of mexA showed a 50% decrease in minimum inhibitory concentrations (MICs) of several antimicrobial agents and a twofold increase in the initial accumulation rate of ethidium bromide, all of which are substrates of the MexAB-OprM efflux pump. These results suggest that gene-specific silencing was induced by pRNA. This is the first time that such a route for gene silencing has been reported in a bacterium other than Escherichia coli. Gene-specific silencing induced by pRNA may be useful as a novel biotechnology tool for gene regulation in prokaryotes.

[Advances in the study of affinity selection-ultrafiltration/HPLC-MS].

Affinity selection-ultrafiltration/HPLC-MS is the combination of the ultrafiltration and HPLC-MS, mainly used for screening small active molecular substances from combinatorial libraries and natural product extracts, which can bind to solution-phase targets. Besides, it can be used in metabolic screening and characterization of ligand-receptor binding. It is a complement to the traditional methods of screening and identifying drugs. This review describes its principle and application in drug study.

Research on rat's pulmonary acute injury induced by lunar soil simulant.

BACKGROUND: The steps to the moon never stopped after the Apollo Project. Lessons from manned landings on the moon have shown that lunar dust has great influence on the health of astronauts. In this paper, comparative studies between the lunar soil simulant (LSS) and PM2.5 were performed to discover their harm to human biological systems and explore the methods of prevention and treatment of dust poisoning for future lunar manned landings. METHODS: Rats were randomly divided into the control group, two CAS-1 lunar soil simulant groups (tracheal perfusion with 7 mg and 0.7 mg, respectively, in a 1-mL volume) and the PM2.5 group (tracheal perfusion with 0.7 mg in a 1-mL volume). The biochemical indicators in the bronchoalveolar lavage fluid (BALF), MPO activity in the lung tissue, pathologic changes, and inflammatory cells in the BALF were measured after 4 h and 24 h. RESULTS: The LSS group showed cytotoxicity that was closely related to the concentration. The figures of the two LSS groups (4 and 24 h) show that the alveolar septa were thickened. Additionally, it was observed that neutrophils had infiltrated, and various levels of inflammation occurred around the vascular and bronchial structures. CONCLUSION: The overall results of the acute effects of the lungs caused by dust showed that the lung toxicity of LSS was greater than that of PM2.5. LSS could induce lung damage and inflammatory lesions. The biomarkers in BALF caused by acute injury were consistent with histopathologic observations.

[Preneoplastic markers of hepatitis B virus-associated hepatocellular carcinoma and their significance in clinical settings].

OBJECTIVES: To identify serologic markers that may indicate the early presence of hepatocellular carcinoma (HCC), and analyze their significance in the pathogenesis of chronic hepatitis B. METHODS: Hepatitis B x antigen (HBxAg) positive and negative HepG2 cells were subjected to PCR select cDNA subtraction to identify differentially expressed genes that may precede the development of HCC. These included the up-regulated genes URG4, URG7, URG11, and VEGFR3, and the down-regulated gene, Sui1. Specific ELISAs were constructed to measure differentially expressed antigens and their corresponding antibodies to determine whether they had prognostic and/or diagnostic value. The study population consisted of 730 people. Among them, 416 were HBsAg(-) and 298 were HBV carriers with chronic liver disease and/or HCC. In addition, 16 patients had non-viral hepatitis. Among these, serial serum samples from 53 HBsAg(+) patients with cirrhosis were collected and studied. RESULTS: Antibodies to multiple differentially regulated genes were detectable in serum samples from patients with HBV associated cirrhosis and HCC, but not in serum samples from uninfected individuals (P < 0.01). Antibodies were undetectable in serum samples from HBV patients without liver disease and in serum samples from patients with other tumor types, and among those with non viral hepatitis. Among patients at high risk of developing HCC, these antibodies were found to be independent of nationality and ethnicity. Statistical analysis of the 28 HBsAg(+) patients with HCC showed that anti-URG11 and anti-VEGFR3 were the most frequently detected antibodies. These antibodies were found to coexist in 16 (P < 0.05). In contrast, among the 25 HBsAg(+) patients without HCC, anti-Sui1 and anti-URG7 were the most prevalent antibodies. These antibodies coexisted in 11 (P < 0.05). In addition, HCC patients with four or more antibodies detected before the appearance of HCC had a poorer survival outcome. CONCLUSION: These antibodies can be detected in serum samples several months to several years before the appearance of HCC. This suggests that they may be preneoplastic markers that may help to distinguish which HBV carriers with cirrhosis are most likely to progress and develop HCC.

PM2.5-induced alterations of cell cycle associated gene expression in lung cancer cells and rat lung tissues.

The aim of the current study was to investigate the expression of cell cycle-associated genes induced by fine particulate matter (PM2.5) in lung cancer cell line and tissues. The pulmonary lymph node metastasis cells (H292) were treated with PM2.5in vitro. Wistar rats were used to perform an in vivo study. Rats were randomly assigned to experiment and control groups and those in the experiment group were exposed to PM2.5 once every 15 d, while those in the control group were exposed to normal saline. The cell cycle-associated genes expression was analyzed by real-time PCR. Trachea and lung tissues of rats were processed for scanning electron microscopic (SEM) examinations. Exposure of H292 cells to PM2.5 dramatically increased the expressions of p53 and cyclin-dependent kinase 2 (CDK2) after 24h of exposure (p<0.01) and markedly increased the expressions of the cell division cycle 2 (Cdc2) and cyclin B after 48h of exposure (p<0.01), while those genes expressions were significantly reduced after 72h of exposure, at which time the expression of p21 was predominant (p<0.01). In vivo studies further demonstrated these results. The results of SEM suggested that both of the trachea and lung tissues were damaged and the degree of damage was time-dependent. In conclusion, PM2.5 can induce significantly alterations of p53 and CDK2 in the early phase, Cdc2 and cyclin B in mid-term and p21 in long-term exposure. The degree of PM2.5-induced damage to the trachea and lung tissue was time-dependent.

Clinical research on navel application of Shehuang Paste combined with Chinese herbal colon dialysis in treatment of refractory cirrhotic ascites complicated with azotemia.

AIM: To explore the efficacy and mechanism of a novel therapeutic method of traditional Chinese medicine in patients with refractory cirrhotic ascites complicated with azotemia. METHODS: Seventy-five cases of refractory cirrhotic ascites complicated with azotemia were randomly divided into 3 groups: comprehensive treatment (n = 29), simple treatment (n = 24), and control (n = 22). The basic treatment methods were the same in all groups, including liver protecting medicines, diuretics and supportive drugs. The control group underwent only the basic treatment. Shehuang Paste (SHP) was applied to the navels of the two treatment groups once a day for 30 d. Colon dialysis with Chinese herbs was administered to the comprehensive treatment group once every two days. Before and after treatment, we measured abdominal circumference, BUN, Cr, serum Na+, urine Na+/K+, liver function, endotoxin content, NO, and ET-1. Color Doppler ultrasonography was conducted to measure the portal vein blood flow. RESULTS: The total effective rate for ascites was 72.4% in the comprehensive treatment group, 45.8% in the simple treatment, contrasting with 18.2% in the controls. Between the two treatment groups and the controls, there were significant differences in the effective rates (P < 0.01, and P < 0.05). There was also a significant difference (P < 0.05) between the two treatment groups. Measurements of Cr and BUN showed higher values for the treatment groups, with the comprehensive better than the simple group (P < 0.05). Sera Na, urine Na/K were different, P < 0.01 between pre- and post-treatment in the comprehensive group, and P < 0.05 in the simple group. The treatment groups' endotoxin content was also significantly reduced (P < 0.01, and P < 0.05), with the comprehensive group better than the simple group (P < 0.05). Portal vein blood flow and NO content significantly reduced (P < 0.05), as did ET-1 content (P < 0.01). There were no significant changes in the control group (P > 0.05). The comprehensive treatment group's pre- and post-treatment portal vein and splenic vein blood flows showed a positive correlation to NO, ET-1 and endotoxin contents. CONCLUSION: When treating refractory cirrhotic ascites complicated with azotemia, Shehuang Paste combined with Chinese herbal dialysis is better than Shehuang Paste alone for ascites resolution, azotemia, and endotoxin elimination. However, both methods on their own were also effective for reducing portal and splenic vein blood flow, and lowering the contents of NO, ET-1 in the two treatment groups.

[Bacterial resistance and study of new antibiotics].

Drug-resistant (including multidrug-resistant) bacteria increase continuously with the wide use of antibiotics, which have seriously threatened the human health. It is an important way to fight against drug-resistance by screening and developing novel drugs based on the various mechanisms of the bacterial drug tolerances. Meanwhile, the basic research related to the new drug R. & D. and studies on the new screening methods for the antimicrobial agents should be taken seriously and strengthened, so as to accelerate the process of finding new drugs and meet the challenge of new pathogens and new drug-resistant strains.

[Clone and expression of isocitrate lyase gene in Mycobacterium tuberculosis H37Rv].

OBJECTIVE: To construct recombinant plasmid with isocitrate lyase (ICL) gene of Mycobacterium tuberculosis H37Rv for stable and high level expression of ICL in prokaryotic expression system. METHODS: The recombinant plasmid with ICL gene (pET30 (a)-Rv0467) was constructed by polymerase chain reaction and cloning. The fusion protein was expressed in E. coli host strain BL21 (DE3). Activity of the fusion protein was studied after it was purified with metal chelating chromatography. RESULTS: We constructed the plasmid which could highly express Mycobacterium tuberculosis H37Rv ICL. The plasmid was highly expressed in E. coli BL21 (DE3), in which the fusion protein accounted for 30% of total protein content. After having been purified by metal chelating chromatography, the purity of the soluble fusion protein was 90%. The fusion protein had activity of ICL. CONCLUSION: Using the prokaryotic expression system, the ICL gene of Mycobacterium tuberculosis H37Rv was successfully cloned and expressed, which build the basis for screening new anti-tuberculosis drugs with ICL as the target point.

[Drug screening model acting on out-membrane protein OprM in pseudomonas aeruginosa efflux pump system].

OBJECTIVE: To establish an efflux pump inhibitor screening model with the out-membrane protein OprM in Pseudomonas aeruginosa efflux pump system as the target point. METHODS: Efflux pump out-membrane protein gene oprM was obtained from standard Pseudomonas aeruginosa PA01 strain. Expression of OprM protein was induced in E. coli strain HS151 with T-easy vector as the cloning vector, and pMMB67EH as the expression vector. In order to evaluate the function of OprM protein, we measured intracellular tetracycline concentrations with liquid scintillation counter, measured the diameters of bacteriostatic circles with paper disc, and then established a screening model accordingly. RESULTS: OprM protein was highly expressed. Using Pseudomonas aeruginosa as the main detecting bacteria, we established a drug screening model acting on OprM. A total of 1 600 microbial fermentation samples were screened with this model, among which 56 positive strains were found, with a positive rate of 3.5%. CONCLUSION: OprM plays an important role in drug efflux. The established model has good specificity and maneuverability.

Efficacy of early treatment on 52 patients with preneoplastic hepatitis B virus-associated hepatocellular carcinoma by compound Phyllanthus Urinaria L.

OBJECTIVE: To observe the change in the number of antibodies of preneoplastic hepatocellular carcinoma (HCC) using early treatment by Compound Phyllanthus Urinaria L. (CPUL) on patients with preneoplastic hepatitis B virus (HBV)-associated HCC. METHODS: A total of 102 cirrhosis patients with regenerative or dysplastic nodules whose sera were tested positive for at least one of these six proteins (five up-regulated genes URG4, URG7, URG11, URG12 and URG19, and one down-regulated gene DRG2) were assigned randomly to two groups using continual random codes by SPSS software. Fifty-two patients were in the treatment group and 50 patients were in the control group. CPUL was used in the treatment group for 3 years, while the control group did not receive any treatment. The changes in HBV-DNA level, number of antibodies, and hepatocarcinogenesis occurred were observed. Patients who did not develop HCC were followed up for another 2 years. RESULTS: HBV-DNA levels decreased ⩾2log in 22.2% (10/45) of patients in the treatment group in contrast to only 5.0% (2/40) of patients in the control group (P=0.0228). The number of antibodies that were tested positive in the treatment group (1.08±1.01) was significantly lower compared with the control group (2.11±1.12) after 24 months of drug treatment (P<0.01). Both the positive rates of anti-URG11 (33/52) and anti-URG19 (31/52) were over 60% at baseline in the two groups, and were decreased to 48.1% (25/52) and 46.2% (24/52) respectively at 36 months of drug treatment, while the rates increased to 68.0% (34/50) and 66.0% (33/50) respectively (P=0.0417, P=0.0436) in the control group. The positive rate of anti-DRG2 was increased to 55.8% (29/52) at 36 months of drug treatment, while in the control group was decreased to 36.0% (18/50, P=0.0452). Among the 102 patients who developed HCC, 2 were in the treatment group and 9 were in the control group, meaning that a significant difference between the two groups (P=0.0212). In 11 patients who developed HCC, anti-URG11 and anti-URG19 were always positive, while anti-DRG2 was negative. Patients newly developing HCC were 6 (20.0%) in the control group, and only one (2.5%) in the treatment group (P=0.0441) during 2-year follow-up after the end of the treatment. CONCLUSIONS: Anti-URG11, anti-URG19 and anti-DRG2 could be used as early markers in the prediction of the therapeutic efficacy of CPUL in treating preneoplastic HCC. CPUL is useful in preventing or delaying the development of HBV-associated cirrhosis to HCC.

Quinolone alkaloids with antibacterial and cytotoxic activities from the fruits of Evodia rutaecarpa.

Five new quinolone alkaloids, euocarpines A-E (16-20), four new natural products (1, 4, 12, and 14), and eleven known natural products were isolated from the fruits of Evodia rutaecarpa (Juss.) Benth. The structures of the new compounds were elucidated based on spectroscopic evidence. All compounds were evaluated for their antibacterial activity against three strains and for their cytotoxic activity against four human tumor cell lines. The results revealed that 5, 7-11, 13, 14, and 16-20 exhibited moderate antibacterial activities (MIC values: 4-128 µg/mL), and 9, 11, 14, and 17 exhibited moderate cytotoxic activities against HepG-2, Hela, BEL7402, and BEL7403 (IC50 values: 15.85-56.36 µM).

Synthesis, structure-activity relationship and in vitro anti-mycobacterial evaluation of 13-n-octylberberine derivatives.

Twenty-eight new 13-n-octylberberine derivatives were synthesized and evaluated for their activities against drug-susceptible Mycobacterium tuberculosis (M. tuberculosis) strain H(37)Rv. Among these compounds, compound 16e was the most effective anti-tubercular agent with a MIC value of 0.125 µg/mL. Importantly, compound 16e exhibited more potent effect against rifampicin (RIF)- and isoniazid (INH)-resistant M. tuberculosis strains than both RIF and INH, suggesting a new mechanism of action. Therefore, it has been selected as a drug candidate for further investigation, or as a chemical probe for identifying protein target and studying tuberculosis biology. We consider 13-n-octylberberine analogs to be a promising novel class of antituberculars against multi-drug-resistant (MDR) strains of M. tuberculosis.