Discovery of 1,2,3-triazole-based pleuromutilin derivatives as potent gram-positive antibacterial agents.

A novel class of pleuromutilin derivatives possessing 1,2,3-triazole as the linker connected to phenyl analogues were designed. The antibacterial properties of the prepared compounds were assessed in vitro against five strains (E. coli, S. aureus, S. epidermidis, and E. faecalis). Most of the tested compounds displayed potent antibacterial activities against gram-positive bacteria and 14-O-[2-(4-((2,4-dinitrophenoxy)-methyl-1H-1,2,3-triazol-1-yl) acetamide)-2-methylpropan-2-yl) thioacetyl]mutilin (7c) exerted antibacterial activities against S. aureus, MRSA and S. epidermidis with MIC values 0.0625 µg/mL, representing 64-fold, 4-fold and 8-fold higher than tiamulin respectively. Compound 6e, 7c and 8c were chosen to carry out killing kinetics, which exhibited concentration-dependent effect. Subsequently, molecular modeling was conducted to further explore the binding of compound 6e, 7a, 7c, 8c and tiamulin with 50S ribosomal subunit from deinococcus radiodurans. The investigation revealed that the main interactions between compound 7c and the ribosomal residues were three hydrogen bonds, π-π, and p-π conjugate effects. Additionally, the free binding energy and docking score of 7c with the ribosome demonstrated the lowest values of -11.90 kcal/mol and -7.97 kcal/mol, respectively, consistent with its superior antibacterial activities.

The effect of thymopentin add-on in hepatitis B e antigen positive chronic hepatitis B after virus suppression by peginterferon plus entecavir therapy.

Hepatitis B virus (HBV) covalently closed circular DNA (cccDNA) was positively correlated with serological hepatitis B surface antigen (HBsAg) levels in hepatitis B e antigen (HBeAg) positive chronic hepatitis B (CHB) patients. We evaluated whether Thymopentin (TP5) and interferon (IFN-a) had a synergic effect on HBV cccDNA and the effect of TP5 addition therapy on HBsAg clearance in CHB patients. Real-time PCR experiments were performed to test cccDNA in HepG2.2.15 cells. 45 HBeAg-positive CHB patients had been distributed into two groups randomly. Treatment group: 23 patients were treated with a 24-week TP5 on the basis of the treatment entecavir (ETV) and peginterferon alfa-2a (PegIFN alpha-2a). Control group: 22 patients were treated with ETV and PegIFNa-2a. The study period was 72 weeks. In HepG2.2.15 cells, TP5 5ug/ml and 10ug/ml respectively combined with IFN-a 2ku/ml could potently inhibit cccDNA level at 72 hours (P<0.05). In clinical study, mean HBsAg levels in two groups are not significantly different at different time points (p=0.112). However, changes of mean HBsAg levels in TP5 add-on group at different time points are significantly different (p<0.05). Patients with HBsAg levels <1500IU/ml in control group had higher HBsAg levels compared with patients with HBsAg levels <1500IU/ml in TP5 add-on group (P=0.019). The latter had the most pronounced HBsAg reduction. TP5 and IFN had a synergic effect on inhibiting cccDNA levels in HepG2.2.15 cells; Patients in treatment group showed no extra side effects compared with the control group. 24 weeks TP5 add-on treatment was safe and had a tendency to accelerate the decline of HBsAg when HBV-DNA was undetectable.

Synthesis of Isorhamnetin-3-O-Rhamnoside by a Three-Enzyme (Rhamnosyltransferase, Glycine Max Sucrose Synthase, UDP-Rhamnose Synthase) Cascade Using a UDP-Rhamnose Regeneration System.

Isorhamnetin-3-O-rhamnoside was synthesized by a highly efficient three-enzyme (rhamnosyltransferase, glycine max sucrose synthase and uridine diphosphate (UDP)-rhamnose synthase) cascade using a UDP-rhamnose regeneration system. The rhamnosyltransferase gene (78D1) from Arabidopsis thaliana was cloned, expressed, and characterized in Escherichia coli. The optimal activity was at pH 7.0 and 45 °C. The enzyme was stable over the pH range of 6.5 to 8.5 and had a 1.5-h half-life at 45 °C. The Vmax and Km for isorhamnetin were 0.646 U/mg and 181 µM, respectively. The optimal pH and temperature for synergistic catalysis were 7.5 and 25 °C, and the optimal concentration of substrates were assayed, respectively. The highest titer of isorhamnetin-3-O-rhamnoside production reached 231 mg/L with a corresponding molar conversion of 100%. Isorhamnetin-3-O-rhamnoside was purified and the cytotoxicity against HepG2, MCF-7, and A549 cells were evaluated. Therefore, an efficient method for isorhamnetin-3-O-rhamnoside production described herein could be widely used for the rhamnosylation of flavonoids.

Characterization of microRNAs in orange-spotted grouper (Epinephelus coioides) fin cells upon red-spotted grouper nervous necrosis virus infection.

Nervous necrosis virus (NNV), one of the most prevalent fish pathogens, has caused fatal disease of viral nervous necrosis (VNN) in many marine and freshwater fishes, and resulted in heavy economic losses in aquaculture industry worldwide. However, the molecular mechanisms underlying the pathogenicity of NNV remain elusive. In this study, the expression profiles of microRNA (miRNA) were investigated in grouper fin (GF-1) cells infected with red-spotted grouper nervous necrosis virus (RGNNV) via deep sequencing technique. The results showed that a total of 220 miRNAs were identified by aligning the small RNA sequences with the miRNA database of zebrafish, and 18 novel miRNAs were predicted using miRDeep2 software. Compared with the non-infected groups, 51 and 16 differentially expressed miRNAs (DE-miRNAs) were identified in the samples infected with RGNNV at 3 and 24 h, respectively. Six DE-miRNAs were randomly selected to validate their expressions using quantitative reverse transcription polymerase chain reaction (qRT-PCR), the results showed that their expression profiles were consistent with those obtained by deep sequencing. The target genes of the DE-miRNAs covered a wide range of functions, such as regulation of transcription, oxidation-reduction process, proteolysis, regulation of apoptotic process, and immune response. In addition, the effects of four DE-miRNAs including miR-1, miR-30b, miR-150, and miR-184 on RGNNV replication were evaluated, and the results showed that over-expression of each of the four miRNAs promoted the replication of RGNNV. These data provide insight into the molecular mechanism of RGNNV infection, and will benefit for the development of effective strategies to control RGNNV infection.

Trichomonas vaginalis induces IL-1ß production in a human prostate epithelial cell line by activating the NLRP3 inflammasome via reactive oxygen species and potassium ion efflux.

BACKGROUND: Trichomonas vaginalis is a sexually transmitted protozoan parasite that causes vaginitis in women, and urethritis and prostatitis in men. IL-1ß is synthesized as immature pro-IL-1ß, which is cleaved by activated caspase-1. Caspase-1 is, in turn, activated by a multi-protein complex known as an inflammasome. In this study, we investigated the inflammatory response of a prostate epithelial cell line (RWPE-1) to T. vaginalis and, specifically, the capacity of T. vaginalis to activate the NLRP3 inflammasome. METHODS: RWPE-1 cells were stimulated by live T. vaginalis, and subsequent expression of pro-IL-1ß, IL-1ß, NLRP3, ASC and caspase-1 was determined by real-time PCR and Western blotting. IL-1ß and caspase-1 production was also measured by ELISA. To evaluate the effects of NLRP3 and caspase-1 on IL-1ß production, the activated RWPE-1 cells were transfected with small interfering RNAs to silence the NLRP3 and caspase-1 genes. Activation of the NLRP3 inflammasome was observed by fluorescence microscopy. Intracellular reactive oxygen species (ROS) were evaluated by spectrofluorometry. RESULTS: When RWPE-1 cells were stimulated with live T. vaginalis, the mRNA and protein expression of IL-1ß, NLRP3, ASC, and caspase-1 increased. Moreover, silencing of NLRP3 and caspase-1 attenuated T. vaginalis-induced IL-1ß secretion. The NADPH oxidase inhibitor DPI and high extracellular potassium ion suppressed the production of IL-1ß, caspase-1, and the expression of NLRP3 and ASC proteins. The specific NF-κB inhibitor, Bay 11-7082, inhibited IL-1ß production, and also inhibited the production of caspase-1, ASC and NLRP3 proteins. CONCLUSIONS: T. vaginalis induces the formation of the NLRP3 inflammasome in human prostate epithelial cells via ROS and potassium ion efflux, and this results in IL-1ß production. This is the first evidence for activation of the NLRP3 inflammasome in the inflammatory response by prostate epithelial cells infected with T. vaginalis. Prostate 76:885-896, 2016. © 2016 Wiley Periodicals, Inc.

Prevalence of Trichomoniasis by PCR in Women Attending Health Screening in Korea.

Trichomoniasis is the most common curable sexually-transmitted infection (STI) worldwide. There are few reports on the prevalence of Trichomonas vaginalis in Korea. The purpose of this study was to examine the prevalence of trichomoniasis by PCR in Guri city, Korea. All adult women who visited Hanyang University Guri Hospital for health screening within the National Health Care Service were invited to participate in the study, and 424 women were enrolled between March and June 2011. PCR was used to detect Trichomonas vaginalis using primers based on a repetitive sequence cloned from T. vaginalis (TV-E650). Fourteen women (3.3%) were found to have T. vaginalis. All were over 50, and they were significantly older on average than the 410 Trichomonas-negative women (mean ages 63.4 vs 55.3 years). It seems that T. vaginalis infection is not rare in women receiving health screening, especially among those over 50.

Spectrophotometric determination of silicon tetrahydride in the air of workplace.

A new, simple and sensitive method was developed for the determination of silicon tetrahydride in the air of workplace in this study. The alkaline resin-based spherical activated carbon was used to collect sample of silicon tetrahydride at workplace. Silicon tetrahydride was then desorbed from active carbon in 100°C hot water. After reacting with ammonium molybdate, oxalic acid and 1,2,4-trichlorobenzene alpha-naphthol amino sulfonic acid under acid condition, silicon tetrahydride was transformed into silicon molybdenum blue. The absorbance of silicon molybdenum blue was quantitatively measured at the wavelength of 680 nm. The results showed that the average sampling efficiency and desorption efficiency were 97.53% and 94.94%, respectively by this method. Detection limits were 0.054 µg/mL for the spectrophotometric method and 0.14 mg/m(3) for the determination of silicon tetrahydride in the air of workplace (sampling volume was 7.5 L). The conversion rate of silicon tetrahydride gradually decreased when storage time of samples was extended. The descent rate of sample was less than 10% when the sample was sealed for 7 days in the room temperature. It was concluded that this spectrophotometric method can be successfully used to determine silicon tetrahydride in the worksites.

Inflammatory response of prostate epithelial cells to stimulation by Trichomonas vaginalis.

BACKGROUND: Trichomonas vaginalis is known as the most common cause of sexually transmitted infection. However, its prevalence may have been underestimated. Trichomonads are detected in prostatic tissue in benign prostatic hyperplasia, prostatitis, and prostate cancer. Our objective was to investigate whether T. vaginalis could induce an inflammatory response in prostate epithelium. METHODS: The cytokine production by human prostate epithelial cell (RWPE-1) activated with T. vaginalis was determined by ELISA and real-time PCR. Intracellular ROS was evaluated by flow cytometry or spectrofluorometry. The protein levels of MAP kinase, NF-κB were analyzed by Western blot. The migration of neutrophil and monocyte were performed in 24-well microplates with filter insert. RESULTS: Incubation of cells of a human prostate epithelial cell line with a live T. vaginalis T016 isolate increased expression of the inflammatory mediators IL-1ß, CCL2, and CXCL8. In addition, ROS, MAPK, and NF-κB activities increased, while inhibitors of ROS, ERK, and NF-κB reduced IL-1ß production. Medium conditioned by incubation of RWPE-1 cells with T. vaginalis contained IL-1ß and stimulated the migration of human neutrophils and monocytes (THP-1 cell line). CONCLUSIONS: We conclude that T. vaginalis may increase IL-1ß expression in human prostate epithelium through activation of ROS, ERK, and NF-κB, and this in turn may induce the migration of neutrophils and monocytes and lead to an inflammatory response. This research is the first attempt to confirm inflammatory reaction caused by T. vaginalis in prostate epithelial cell.

Self-assembled gel microneedle formed by MS deep eutectic solvent as a transdermal delivery system for hyperpigmentation treatment.

Hyperpigmentation (HP) is an unfavorable skin disease that typically caused by injury, inflammation, or photoaging and leads to numerous physical and psychological issues in patients. Recently, development and application of natural whitening substances, particularly compound curcumin (CUR), is one of the most prevalent treatments for HP. However, it is still a formidable challenge to improve the percutaneous delivery of CUR due to its inadequate solubility in water and excellent barrier function of skin. To overcome the limitations of conventional delivery and increase the percutaneous absorption of CUR, the efficient delivery of CUR is urgently required. Herein, we developed a new malic acid-sorbitol deep eutectic solvent (MS/DES) gel microneedle loaded with CUR as a transdermal delivery system for HP treatment. The MS/DES gel produces three-dimensional (3D) network structure by self-assembly of hydrogen bond interactions, which conferred the CUR-MS/DES-GMN with sufficient mechanical properties to successfully penetrate skin tissue while also helping to enhance the drug's release rate. The CUR-MS/DES-GMN exhibit high biocompatibility and mechanical property in vivo of mice. The zebrafish experiments also show that CUR-MS/DES gel has significant effect of anti-pigmentation. Therefore, the designed CUR-MS/DES-GMN system provides a novel strategy for HP treatment based on self-assembly of naturally molecules.

[Study on synergistic effect of bactericidal effect of chlorine dioxide solution by surfactant].

OBJECTIVE: To study the effect of gemini fluorocarbon, sodium p-perfluorous nonenoxybenzene sulfonate and sodium dodecyl sulfate on the chlorine dioxide solution sterilization to object surface. METHODS: Pure chlorine dioxide solution as the reference disinfectant, carrier quantitative bactericidal test and simulated test on-site were used to carry out laboratory observation according to The disinfection technical specifications (2002). RESULTS: Carrier quantitative bactericidal test showed that the addition dosage of gemini fluoronates, sodium dodecyl sulfate surfactant and perfluorinated the nonene oxy benzene sulfonate in disinfectant solution were 60, 60 and 40 mg/L respectively, the killing log value of Staphylococcus aureus exposed to the disinfectant solution containing chlorine dioxide 50 mg/L for 10 mm were all more than 3; and the addition dosage of gemini fluorinates, sodium dodecyl sulfate and perfluorinated the nonene oxy benzene sulfonate in disinfectant solution were 60 mg/L, the killing log value of Escherichia coli exposed to the disinfectant solution containing chlorine dixoxide 20 mg/L for 10 min were all more than 3. The bactericidal effect of the mixture use of surfactant and chlorine dioxide was better than the single use of chlorine dioxide. The simulated test on-site showed that the killing log value of Escherichia coli exposed to the disinfectant solution containing perfluorinated the nonene oxy benzene sulfonate 40 mg/L and chlorine dioxide 20 mg/L for 15 min was more than 3. CONCLUSION: Surface active agent on germicidal efficacy of chlorine dioxide solution had synergistic action.

[The role of interleukin-1ß on the pulmonary fibrosis in mice exposed to crystalline silica].

OBJECTIVE: This study was designed to evaluate the role of interleukin (IL)-1ß in the development of fibrosis in mice exposed to silica. METHODS: The total of 96 Male C57BL/6 mice were divided into four groups. (1) blank control group, (2) PBS group in which mice were instilled with PBS only, (3) silica + IL-1ß mAb group in which mice were instilled with 2.5 mg silica dust and 40 µg anti-IL-1ß mAb, (4) silica group in which mice were instilled with 2.5 mg silica dust and 40 µg IgG. The final volume of suspension or PBS instilled into the mouse was 50 µl. At 7, 28 and 84 days after treatment, 8 mice were sacrificed in each group. Then BALF was collected for the count of inflammatory cells and cytokines determination. The lung tissues were collected for the detecting of mRNA levels of fibrogenic molecules. RESULTS: The collagen deposition induced by silica in the lung tissues was partly inhibited by anti-IL-1ß. A intensely pulmonary cytokines such as IL-1ß, TNF-α, MCP-1 were induced by crystalline silica exposure, and partly inhibited by anti-IL-1ß. The levels of TGF-ß and fibronectin in silica exposed mice were significantly elevated than those in control mice at days 28 and 84 after treatment (P < 0.01). And the mRNA levels of TGF-ß, collagen I and fibronectin were significantly decreased in silica+IL-1ß mAb group when compared with those in silica group at days 7, 28 and 84 (P < 0.01). There was a significant decrease of the ratios of IFN-γ/IL-4 in both silica+anti-IL-1ß mAb and silica groups when compared with those in control mice at the above three time points (P < 0.01). However, the IFN-γ/IL-4 ratios in silica+anti-IL-1ß group were significantly higher than those in silica group at 7, 28 and 84 days (P < 0.05 or P < 0.01). CONCLUSION: IL-1ß may promote the pulmonary fibrosis in mice exposed to silica.

Characterization and expression patterns of lysozymes reveal potential immune functions during male pregnancy of seahorse.

Seahorses are one of the most amazing ovoviviparous fishes in the ocean because males, and not females, have evolved a brood pouch for incubating embryos. During male pregnancy, paternal seahorses need to develop effective immune protection for embryos in the brood pouch from potential infection by pathogens. Lysozymes (Lyz) are a group of antibacterial enzymes of the innate immune system that play an important role in resisting pathogen invasion. However, the immune function of lysozymes in the brood pouch of the pregnancy-lined seahorse (Hippocampus erectus) remains unknown. In this study, we found three different lysozymes in the lined seahorse: HeLyzC, HeLyzG1, and HeLyzG2. Synteny analysis revealed that HeLyzG1 and HeLyzG2 were generated by species-specific expansion rather than tandem duplication. Tissue expression patterns showed that the highest mRNA expression levels of the three lysozymes occurred in the brood pouches. Immunostimulation-induced expression analysis showed that all three HeLyzs in the brood pouches up-regulated their mRNA expression levels after Vibrio parahaemolyticus infection, but only the HeLyzG2 was upregulated after Poly(I:C) injection. Similarly, except for HeLyzC, upregulated expressions of HeLyzG1 and HeLyzG2 were found quickly in brood pouches injected with LPS. The upregulated levels of HeLyzC and HeLyzG2 in brood pouches during pregnancy were significantly higher than those in non-pregnancy, implying that seahorse lysozymes might function in the immune defense in brood pouches during pregnancy. In addition, the expression levels of HeLyzs were low in embryos in the brood pouch but significantly increased in neonates. This implies that embryos in the brood pouch might not necessarily express more lysozymes by themselves due to paternal immune protection. In conclusion, our study demonstrated that HeLyzs play an important role in immune protection during male seahorse gestation, and the synergistic effect of multiple HeLyzs may contribute to improved neonatal survival.

Evaluation of porcine intestinal organoids as an in vitro model for mammalian orthoreovirus 3 infection.

BACKGROUND: Mammalian orthoreovirus type 3 (MRV3), which is responsible for gastroenteritis in many mammalian species including pigs, has been isolated from piglets with severe diarrhea. However, the use of pig-derived cells as an infection model for swine-MRV3 has rarely been studied. OBJECTIVES: This study aims to establish porcine intestinal organoids (PIOs) and examine their susceptibility as an in vitro model for intestinal MRV3 infection. METHODS: PIOs were isolated and established from the jejunum of a miniature pig. Established PIOs were characterized using polymerase chain reaction (PCR) and immunofluorescence assays (IFAs) to confirm the expression of small intestine-specific genes and proteins, such as Lgr5, LYZI, Mucin-2, ChgA, and Villin. The monolayered PIOs and three-dimensional (3D) PIOs, obtained through their distribution to expose the apical surface, were infected with MRV3 for 2 h, washed with Dulbecco's phosphate-buffered saline, and observed. Viral infection was confirmed using PCR and IFA. We performed quantitative real-time reverse transcription-PCR to assess changes in viral copy numbers and gene expressions linked to intestinal epithelial genes and antiviral activity. RESULTS: The established PIOs have molecular characteristics of intestinal organoids. Infected PIOs showed delayed proliferation with disruption of structures. In addition, infection with MRV3 altered the gene expression linked to intestinal epithelial cells and antiviral activity, and these effects were observed in both 2D and 3D models. Furthermore, viral copy numbers in the supernatant of both models increased in a time-dependent manner. CONCLUSIONS: We suggest that PIOs can be an in vitro model to study the infection mechanism of MRV3 in detail, facilitating pharmaceutical development.

Immunomodulatory effects of canine mesenchymal stem cells in an experimental atopic dermatitis model.

Mesenchymal stem cells (MSCs) have the potential to differentiate into multi-lineage cells, suggesting their future applicability in regenerative medicine and biotechnology. The immunomodulatory properties of MSCs make them a promising replacement therapy in various fields of animal research including in canine atopic dermatitis (AD), a skin disease with 10-15% prevalence. We investigated the immunomodulatory effects of MSCs in an experimental canine AD model induced by Dermatophagoides farinae extract ointment. Canine adipose tissue-derived MSCs (cAT-MSCs) were differentiated into mesodermal cell lineages at the third passage. Alterations in immunomodulatory factors in control, AD, and MSC-treated AD groups were evaluated using flow cytometric analysis, enzyme-linked immunosorbent assay, and quantitative reverse transcription PCR. In the MSC-treated AD group, the number of eosinophils decreased, and the number of regulatory T cells (Tregs) increased compared to those in the AD group. In addition, the immunoglobulin E (IgE) and prostaglandin E2 levels were reduced in the MSC-treated AD group compared to those in the AD group. Furthermore, the filaggrin, vascular endothelial growth factor, and interleukin-5 gene expression levels were relatively higher in the MSC-treated AD group than in the AD group, however, not significantly. cAT-MSCs exerted immunomodulatory effects in an AD canine model via a rebalancing of type-1 and -2 T helper cells that correlated with increased levels of Tregs, IgE, and various cytokines.

Biotoxicity Evaluation of a Cationic Star Polymer on a Predatory Ladybird and Cooperative Pest Control by Polymer-Delivered Pesticides and Ladybird.

Although employing nanocarriers for gene/drug delivery shows great potential in agricultural fields, the biotoxicity of nanocarriers is a major concern for large-scale applications. Herein, we synthesized a cationic star polymer (SPc) as a pesticide nanocarrier/adjuvant to evaluate its safety against a widely used predatory ladybird (Harmonia axyridis). The application of SPc at extremely high concentrations nearly did not influence the hatching of ladybird eggs but it led to the death of ladybird larvae at lethal concentration 50 (LC50) values of 43.96 and 19.85 mg/mL through the soaking and feeding methods, respectively. The oral feeding of SPc downregulated many membrane protein genes and lysosome genes significantly, and the cell membrane and nucleus in gut tissues were remarkably damaged by SPc application, revealing that the lethal mechanism might be SPc-mediated membrane damage. Furthermore, the oral feeding of SPc increased the relative abundance of Serratia bacteria in ladybird guts to result in bacterial infection. Coapplication of ladybird and SPc-loaded thiamethoxam/matrine achieved desired control efficacies of more than 80% against green peach aphids, revealing that the coapplication could overcome the slow-acting property of ladybirds. To our knowledge, this is the first attempt to investigate the polymer-mediated lethal mechanism toward natural enemies and explore the possibility of coapplying SPc-loaded pesticides and natural enemies for pest management.

A Preparation Method of Nano-Pesticide Improves the Selective Toxicity toward Natural Enemies.

Various nano-delivery systems have been designed to deliver synthetic/botanical pesticides for improved bioactivity. However, the enhanced toxicity of nanocarrier-loaded pesticides may injure the natural enemies, and their selective toxicity should be evaluated before the large-scale application. In this context, a star polymer (SPc)-based cyantraniliprole (CNAP) nano-delivery system was constructed, and its selective toxicity was evaluated using pest Frankliniella occidentalis (WFT) and predator Orius sauteri. The amide NH of CNAP could assemble with carbonyl groups or tertiary amines of SPc through hydrogen bonds to form CNAP/SPc complex spontaneously. The above self-assembly decreased the particle size of CNAP from 808 to 299 nm. With the help of SPc, the lethal concentration 50 (LC50) values of CNAP decreased from 99 to 54 mg/L and 230 to 173 mg/L toward WFTs and O. sauteri due to the enhancement of broad-spectrum bioactivity. Interestingly, the toxicity selective ratio (TSR) of CNAP increased from 2.33 to 3.23 with the help of SPc, revealing the higher selectivity of SPc-loaded CNAP. To our knowledge, it was the first successful exploration of the selective toxicity of nanocarrier-loaded pesticides, and the higher selective toxicity of SPc-loaded CNAP was beneficial for alleviating the negative impacts on predators.

Development and validity of a hazard prediction test for Chinese drivers.

BACKGROUND: Hazard perception ability, which develops with driving experience, has been proven to be associated with drivers' traffic involvement. Although classic reaction time-based hazard perception tests have been developed in many developed counties, experience-related differences may not be found in drivers from developing countries due to their increased opportunities to experience hazards on roads. Therefore, the present study aims to develop a hazard prediction test for Chinese drivers based on a predictive paradigm called "What happens next?" and assess its reliability and validity. METHOD: Thirty-six video clips filmed from drivers' perspectives of Chinese driving settings were presented to 54 novice drivers and 47 experienced drivers. Participants were asked to answer three questions after each video clip was blacked out and to then quickly press the mouse button on a reaction time-based hazard perception test. Both the differences in the test scores between novice and experienced drivers and the differences in scores between drivers with and without traffic violations were compared. RESULTS: The final hazard prediction test consisted of 20 video clips. A high internal consistency coefficient of the test, i.e., Cronbach's alpha = 0.862, was obtained. The total scores of the test were positively and significantly correlated with reaction times as measured on the video-based hazard perception test, thus providing evidence regarding the discriminant validity of the test. More importantly, drivers with traffic violations obtained significantly lower total scores on the test than did drivers without traffic violations. CONCLUSION: The newly developed hazard prediction test exhibited adequate psychometric properties and provided a practical alternative for assessing drivers' hazard perception ability in China.

Biosynthesis of 3'-O-methylisoorientin from luteolin by selecting O-methylation/C-glycosylation motif.

Glycosylation and methylation of flavonoids are the main types of structural modifications and can endow flavonoids with greater stability, bioactivity, and bioavailability. In this study, five types of O-methyltransferases were screened for producing O-methylated luteolin, and the biosynthesis strategy of 3'-O-methylisoorientin from luteolin was determined. To improve the production of 3'-O-methylluteolin, the S-adenosyl-l-methionine synthesis pathway was reconstructed in the recombinant strain by introducing S-adenosyl-l-methionine synthetase genes. After optimizing the conversion conditions, maximal 3'-O-methylluteolin production reached 641 ± 25 mg/L with a corresponding molar conversion of 76.5 %, which was the highest titer of methylated flavonoids reported to date in Escherichia coli. 3'-O-Methylluteolin (127 mg) was prepared from 250 mL of the broth by silica gel column chromatography and preparative HPLC with a yield of 79.4 %. Subsequently, we used the biocatalytic cascade of Gentiana triflora C-glycosyltransferase (Gt6CGT) and Glycine max sucrose synthase (GmSUS) to biosynthesize 3'-O-methylisoorientin from 3'-O-methylluteolin in vitro. By optimizing the coupled reaction conditions and using the fed-batch operation, maximal 3'-O-methylisoorientin production reached 226 ± 8 mg/L with a corresponding molar conversion of 98 %. Therefore, this study provides an efficient method for the production of novel 3'-O-methylisoorientin and the biosynthesis strategy for methylated C-glycosylation flavonoids by selective O-methylation/C-glycosylation motif on flavonoids.

Human papillomavirus16 E6 but not E7 upregulates GLUT1 expression in lung cancer cells by upregulating thioredoxin expression.

Background and objective: E6 and E7 proteins in human papillomavirus (HPV) 16 are major oncogenes in several types of tumors, including lung cancer. Previous studies have demonstrated that both E6 and E7 oncoproteins can upregulate GLUT1 protein and mRNA expression levels in lung cancer cells. Thus, the present study aimed to investigate the main differences in the molecular mechanisms of GLUT1 expression regulated by E6 and E7. Methods: The double directional genetic manipulation and immunofluorescence were performed to explore the molecular mechanism of E6 or E7 upregulating the expression of GLUT1 in H1299 and A549 cell lines. Results: The overexpression of E6 in well-established lung cancer cell lines upregulated thioredoxin (Trx) protein expression. Notably, plasmid transfection or small interfering RNA transfection with E7 had no regulatory effect on Trx expression. As an important disulfide reductase of the intracellular antioxidant system, Trx plays important role in maintaining oxidative stress balance and protecting cells from oxidative damage. The overexpression of Trx increased the activation of NF-κB by upregulating p65 expression and promoting p65 nuclear translocation, and further upregulated GLUT1 protein and mRNA expression levels. The results of the present study demonstrated that E6, but not E7, upregulated GLUT1 expression in lung cancer cells by activating NF-κB due to the participation of Trx. Conclusion: These results suggest that Trx plays an important role in the pathogenesis of HPV-associated lung cancer, and propose a novel therapeutic target for HPV-associated lung cancer.

Novel method to repair articular cartilage by direct reprograming of prechondrogenic mesenchymal stem cells.

Age-related cartilage loss is worsened by the limited regenerative capacity of chondrocytes. The role of cell-based therapies using mesenchymal stem cells is gaining interest. Adipose tissue-derived mesenchymal stem cells (ADSCs) are an attractive source to generate the optimal number of chondrocytes required to repair a cartilage defect and regenerate hyaline articular cartilage. Here, we report an outstanding technique to prepare chondrocytes for cartilage repair using canine ADSCs. We hypothesized that external electrical fields promote prechondrogenic condensation without requiring genetic modifications or exogenous factors. We analyzed the effect of electrical stimulation (ES) on the differentiation of ADSC micromass into chondrocytes. Highly compact structures were formed within 3 days of ES of canine ADSC micromass. The expression of type I collagen gene was abolished in these cells compared with that in control micromass cultures and monolayer cultures. We further found that ES enhanced the production of proteoglycan, a highly produced extracellular matrix component in chondrocytes. Additionally, single-cell RNA sequencing analysis showed that canine ADSC micromass undergoing ES developed a prechondrogenic cell aggregation, suggesting their metabolic conversion, biogenesis, and calcium ion change. Collectively, our findings demonstrate the capacity of ES to drive the chondrogenesis of ADSCs in the absence of exogenous factors and confirm its commercial potential as a budget-friendly therapy for the repair of cartilage defects.