Synergistic effects of endolysin Lysqdvp001 and ε-poly-lysine in controlling Vibrio parahaemolyticus and its biofilms.

The synergistic antibacterial effects between endolysin Lysqdvp001 and ε-poly-lysine (ε-PL) against Vibrio parahaemolyticus (V. parahaemolyticus) were investigated in this study. Lysqdvp001 combined with ε-PL exhibited a strong antibacterial synergism against V. parahaemolyticus. The combinations of Lysqdvp001 (≥60 U/mL) and ε-PL (≥0.2 mg/mL) dramatically decreased cell density of the bacterial suspensions at both 25 °C and 37 °C. Surface zeta potential increment and membrane hyperpolarization of V. parahaemolyticus were observed after treatment by ε-PL and its combination with Lysqdvp001. More ß-lactamase and ß-galactosidase were leaked from V. parahaemolyticus with combined treatment of Lysqdvp001 and ε-PL than from the bacteria treated with single Lysqdvp001 or ε-PL. Fluorescence and transmission electron microscope revealed that Lysqdvp001 and ε-PL synergistically induced the damage and morphological destruction of V. parahaemolyticus cells. When applying in Gadus macrocephalus, Penaeus orientalis and oyster, the two antimicrobials' cocktail allowed for 3.75, 4.16 and 2.50 log10CFU/g reductions of V. parahaemolyticus, respectively. Besides, Lysqdvp001 in combination with ε-PL removed approximately 44%-68% of V. parahaemolyticus biofilms on polystyrene, glass and stainless steel surfaces. These results demonstrated that Lysqdvp001 and ε-PL might be used together for controlling V. parahaemolyticus and the bacterial biofilms in food industry.

[An investigation on the need, the utilization, and the influencing factors of dental services for pre-school children in selected areas in Chongqing province].

OBJECTIVE: The aim of this study is to survey the need, the utilization, and the influencing factors of dental services for children in selected areas in Chongqing province by investigating their oral health status. The survey will provide references for preventive oral health care in targeted Chongqing areas, which may improve the level of oral health among pre-school children. METHODS: Random cluster sampling was utilized according to standards of the Fourth National Oral Health Epidemiological sampling survey, and 1 300 children between the ages of three and four years old from 24 kindergartens in 12 subdistricts of three areas in Chongqing were interviewed for free dental checkups and to participate in the survey. The questionnaires were designed according to the Anderson model and were answered by the children's parents. The results were analyzed utilizing Chi-square test logistic regression. RESULTS: The prevalence rate of caries among the pre-school children in selected areas of Chongqing was 55.4%, the decay, missing, filled surface (dmfs) was 6 696, the mean dmfs was 5.2, and the caries filling constituent ratio was 2.3%. A total of 1 173 questionnaires were analyzed. The ratio for seeing a dentist for therapeutic reasons was 6.31% (74/1 173) and for prevalence was 22.93% (269/1 173). CONCLUSIONS: The oral health service needs of pre-school children in selected areas of Chongqing are large and the oral health service utilization rate is low. Oral health care processes are arduous; thus, targeted oral prevention policies should be created.

[Prevalence and influencing factors of deciduous caries in preschool children in Chongqing city].

OBJECTIVE: This study aimed to determine the prevalence and related factors of deciduous caries in 3-5-year-old preschool children in Chongqing city. Results will be used to provide a basis for the establishment and adjustment of prevention and intervention of caries in preschool children. METHODS: We referred to the Fourth National Oral Health Epidemiological Survey. Data included caries prevalence in preschool children, and the questionnaires were distributed to children' parents in Chongqing city. Results were inputted by Epidata 3.1 and statistically analyzed using SPSS 21.0. RESULTS: A total of 1 350 preschool children were included in the study. We found that maxillary deciduous central incisor and mandibular deciduous molars were susceptible to decay. The prevalence of primary teeth caries in preschool children in Chongqing city was 51.4% (694/1 350). The mean decayed-missing-filled-teeth (dmft) index was 2.34. The caries prevalence and mean dmft between age groups were statistically significant (P<0.01) and increased with age (P<0.05). However, except the 5-year-old group (P<0.05), no significant difference in caries prevalence rate and mean dmft was found between male and female children (P>0.05). Approximately 61.7% of caries cases were concentrated in a small number (36.1%) of individuals. Multivariate Logistic regression analysis showed that age, highest educational level of parents, intake frequency of sweetened beverages and carbonated drinks, toothache or similar discomfort experience over the past year, dentist visits, and parents' assessment of teeth and oral health status of children were the factors influencing the prevalence of deciduous caries (P<0.05). CONCLUSIONS: More than half of the preschool children had dental caries. Majority of caries were concentrated in a small number of individuals. The age of children, highest educational level of parents, intake frequency of sweetened beverages and carbonated drinks, toothache or discomfort experience over the past year, dentist visits, and parents' assessment of teeth and oral health status of children were associated with the prevalence of deciduous caries.

Travel-related infections in mainland China, 2014-16: an active surveillance study.

BACKGROUND: Transmission of infection through international travel is a growing health issue, and the frequency of imported infection is increasing in China. We aimed to quantify the total number of infections imported into mainland China by arriving travellers. METHODS: We actively surveyed arriving travellers at all 272 international entry-exit ports in mainland China. Suspected cases were detected through fever screening, medical inspection, self-declaration, and reporting by on-board staff. Participants completed a standardised questionnaire with questions about demographics, their travel itinerary (including detailed information about all countries or regions visited), and clinical manifestations. Nasopharyngeal swabs, sputum samples, faecal samples, vomitus, blood, and serum were collected as appropriate for diagnoses. Diagnosis was made by specific laboratory tests according to the national technical guidelines. Infections were classified as respiratory, gastrointestinal, vector-borne, blood-transmitted and sex-transmitted, or mucocutaneous. We divided arriving travellers into two groups: travellers coming from countries other than China, and travellers coming from Hong Kong, Macau, and Taiwan. We integrated surveillance data for 2014-16, calculated incidences of travel-related infections, and compared the frequency of infections among subgroups. FINDINGS: Between Jan 1, 2014, and Dec 31, 2016, 22 797 cases were identified among 805 993 392 arriving travellers-an overall incidence of 28·3 per million. 45 pathogens were detected in participants: 18 respiratory (19 662 cases), ten gastrointestinal (189 cases), seven vector-borne (831 cases), seven blood-transmitted and sex-transmitted (1531 cases), and three mucocutaneous (584 cases). Both the overall number and incidence of infection were more than five times higher in 2016 than in 2014. Case numbers and incidences also varied substantially by province, autonomous region, and municipality. Overall, 17 643 (77%) infections were detected by fever screening, but 753 (49%) blood-transmitted and sex-transmitted infections were identified through medical inspection. 14 305 (73%) cases of respiratory infection and 96 (51%) of gastrointestinal infections were in tourists. Tuberculosis, hepatitis A virus, vector-borne, and blood-transmitted and sex-transmitted infections were common among Chinese labourers who worked abroad. Dengue and malaria were most commonly diagnosed in travellers arriving from Africa. 12 126 (93%) of the 12 985 cases arriving from Hong Kong, Macau, or Taiwan were respiratory infections. Hand, foot, and mouth disease accounted for 2·90% of infections in travellers from Hong Kong, Macau, or Taiwan and 0·31% of infections in international travellers. INTERPRETATION: This report is the first to characterise the profile of travel-related infections among arriving travellers in mainland China. Our findings should increase public awareness of the potential risk of imported infections, and help health-care providers to make evidence-based health recommendations to travellers. FUNDING: The Natural Science Foundation of China.

Dihydromyricetin inhibits microglial activation and neuroinflammation by suppressing NLRP3 inflammasome activation in APP/PS1 transgenic mice.

BACKGROUND: Activated microglia-mediated inflammation plays a key role in the pathogenesis of Alzheimer's disease (AD). In addition, chronic activation of NLRP3 inflammasomes triggered by amyloid ß peptide (Aß) in microglia contributes to persistent neuroinflammation. Here, the primary goal was to assess whether Dihydromyricetin (DHM), a plant flavonoid compound, is effective therapies for AD; it is crucial to know whether DHM will affect microglial activation and neuroinflammation in APP/PS1 transgenic mice. METHODS: After DHM was intraperitoneally injected in APP/PS1 double-transgenic mice, we assessed the effect of DHM on microglial activation, the expression of NLRP3 inflammasome components, and the production of inflammatory cytokine IL-1ß by immunofluorescence and Western blot. To determine whether DHM play roles in the Aß production and deposition, amyloid ß protein precursor (APP) and ß-site APP cleaving enzyme1 (BACE1), as well as neprilysin (NEP), were detected by Western blot. Finally, behavior was tested by Morris Water Maze to illustrate whether DHM treatment has a significantly positive effect on ameliorating the memory and cognition deficits in AD. RESULTS: Dihydromyricetin treatment significantly ameliorated memory and cognition deficits and decreased the number of activated microglia in the hippocampus and cortex of APP/PS1 mice. In addition, APP/PS1 mice show reduced activation of NLRP3 inflammasomes and reduced expression of NLRP3 inflammasome components. Furthermore, DHM could promote clearance of Aß, a trigger for NLRP3 inflammasome activation, by increasing levels of NEP and shift microglial conversion to the M2-specific agrinase-1-positive cell phenotype, which enhances microglial clearance of Aß and its aggregates but not production of Aß. CONCLUSION: Taken together, our findings suggest that DHM prevents progression of AD-like pathology through inhibition of NLRP3 inflammasome-based microglia-mediated neuroinflammation and may be a promising therapeutic drug for treating AD.

Correlations between Mitofusin 2 Expression in Fibroblasts and Pelvic Organ Prolapse: An In vitro Study.

BACKGROUND: Both Mitofusin 2 (Mfn2) and pelvic organ prolapse (POP) are related to aging. The aim of the present study was to investigate the variations of Mfn2 expression in the uterosacral ligaments of patients with and/or without POP and their correlations with the expression of procollagen. METHODS: Fibroblasts were cultured using tissue specimens that were harvested from the uterosacral ligaments of POP and non-POP (NPOP) patients (n = 10 for each group) from September 2016 to December 2016. The Cell Counting Kit-8 (CCK-8) assay was used to compare the differences in cell proliferation between the two groups. Relative quantitative reverse transcription-polymerase chain reaction and Western blotting assays were employed to assess the differences in the mRNA and protein expression levels of Mfn2 and procollagen 1A1/1A2/3A1 between the two groups. The changes in procollagen expression were assessed following the downregulation of Mfn2 in the POP group using RNAi. The data were assessed with independent sample t- test or general linear model univariate analysis using the SPSS 13.0 software. RESULTS: The results from CCK-8 assay indicated that cell viability in the POP group was significantly lower compared with that of the NPOP group (td5, 7, 9, 11= -5.925, -6.851, -9.129, and -9.661, respectively, all P < 0.001, from D5 to D11). The mRNA and protein expression levels of Mfn2 in the cultured fibroblasts of the POP group were significantly higher compared with those of the NPOP group (mRNA: t = 2.425, P = 0.032; protein: t = 2.392, P = 0.037, respectively), whereas only the expression levels of procollagen 1A1/1A2/3A1 were significantly higher in the NPOP group (mRNA: t = -2.165, P1A1 = 0.041; t = -2.741, P1A2 = 0.026; t = -2.147, P3A1 = 0.045, respectively; protein: t = -2.418, P1A1 = 0.029; t = -2.405, P1A2 = 0.033; t = -2.470, P3A1 = 0.012, respectively). The expression levels of procollagen in the POP group increased following the downregulation of Mfn2. CONCLUSIONS: The proliferation rate and cell viability of the fibroblasts in the POP group were significantly lower compared with those in the NPOP group. In the POP fibroblasts, Mfn2 expression was increased, while procollagen expression was decreased.

[Isolation and Identification of Rat Kidney Stem Cells].

OBJECTIVE: To isolate and steadily culture kidney stem cells (KSCs) from rat renal papilla, and to identify the biological characteristics of KSCs. METHODS: KSCs were isolated from the tips of renal papilla in 4 weeks-old Sprague-Dawley rats. The morphology of KSCs was observed under inversion microscope, and the phenotye characteristics of kSCs were identified through flow cytometry and immunofluorescence. The abilities of KSCs in adipogenic and osteogenic differentiation were evaluated. The differences of gene expression between KSCs and rat renal tubular epithelial cells (RTECs)were compared using quantitative real time polymerase chain reaction (qRT-PCR). RESULTS: KSCs showed a spindle-shaped and arborization-like growth pattern. Immunofluorescence indicated that KSCs staining with alpha-sooth muscle actin (α-SMA), Vimentin, N-Cadherin, Nestin, CD133 marker, and without E-cadherin, cytokeratin-18 (CK-18), zona occludens protein-1 (ZO-1). The positive staining of CD29, CD90, CD73 were 99. 0%, 95. 8%, 99. 9% respectively, the positive staining of CD45 was 3. 4%. The positive stainings of stem cell marker CD133 and Nestin were 33. 2% and 70. 2% respectively, while the double staining rate was 31. 4%., KSCs showed positive staining by oil red 0 after adipogenic differentiation, and orange calcium deposition by alizarin red staining after osteogenic differentiation. qRT-PCR showed that the expressions of embryonic stem cell marker Nanog, Oct4/pou5f1,Sox2/sry-box-2 in KSCs were higher than those in RTECs (P< 0.01), and the expressions of mesenchymal marker c-SMA, Vimentin were also higher in KSCs (P<0. 01). Compared with RTECs, the expressions of mature epithelium marker E-Cadherin, CK18 in KSCs were lower (P< 0. 01). CONCLUSION: KSCs were isolated successfully and steadily cultured from the rat renal papilla, which were identified with featured biological characteristics.

MicroRNA-125b attenuates epithelial-mesenchymal transitions and targets stem-like liver cancer cells through small mothers against decapentaplegic 2 and 4.

UNLABELLED: Emerging evidence suggests that epithelial-mesenchymal transitions (EMTs) play important roles in tumor metastasis and recurrence. Understanding molecular mechanisms that regulate the EMT process is crucial for improving treatment of hepatocellular carcinoma (HCC). MicroRNAs (miRNAs) play important roles in HCC; however, the mechanisms by which miRNAs target the EMT and their therapeutic potential remains largely unknown. To better explore the roles of miRNAs in the EMT process, we established an EMT model in HCC cells by transforming growth factor beta 1 treatment and found that several tumor-related miRNAs were significantly decreased. Among these miRNAs, miR-125b expression was most strongly suppressed. We also found down-regulation of miR-125b in most HCC cells and clinical specimens, which correlated with cellular differentiation in HCC patients. We then demonstrated that miR-125b overexpression attenuated EMT phenotype in HCC cancer cells, whereas knockdown of miR-125b promoted the EMT phenotype in vitro and in vivo. Moreover, we found that miR-125b attenuated EMT-associated traits, including chemoresistance, migration, and stemness in HCC cells, and negatively correlated with EMT and cancer stem cell (CSC) marker expressions in HCC specimens. miR-125b overexpression could inhibit CSC generation and decrease tumor incidence in the mouse xenograft model. Mechanistically, our data revealed that miR-125b suppressed EMT and EMT-associated traits of HCC cells by targeting small mothers against decapentaplegic (SMAD)2 and 4. Most important, the therapeutic delivery of synthetic miR-125b mimics decreased the target molecule of CSC and inhibited metastasis in the mice model. These findings suggest a potential therapeutic treatment of miR-125b for liver cancer. CONCLUSION: miR-125b exerts inhibitory effects on EMT and EMT-associated traits in HCC by SMAD2 and 4. Ectopic expression of miR-125b provides a promising strategy to treat HCC.

Hepatocellular carcinoma-associated mesenchymal stem cells promote hepatocarcinoma progression: role of the S100A4-miR155-SOCS1-MMP9 axis.

UNLABELLED: Cancer-associated mesenchymal stem cells (MSCs) play a pivotal role in modulating tumor progression. However, the interactions between liver cancer-associated MSCs (LC-MSCs) and hepatocellular carcinoma (HCC) remain unreported. Here, we identified the presence of MSCs in HCC tissues. We also showed that LC-MSCs significantly enhanced tumor growth in vivo and promoted tumor sphere formation in vitro. LC-MSCs also promoted HCC metastasis in an orthotopic liver transplantation model. Complementary DNA (cDNA) microarray analysis showed that S100A4 expression was significantly higher in LC-MSCs compared with liver normal MSCs (LN-MSCs) from adjacent cancer-free tissues. Importantly, the inhibition of S100A4 led to a reduction of proliferation and invasion of HCC cells, while exogenous S100A4 expression in HCC cells resulted in heavier tumors and more metastasis sites. Our results indicate that S100A4 secreted from LC-MSCs can promote HCC cell proliferation and invasion. We then found the expression of oncogenic microRNA (miR)-155 in HCC cells was significantly up-regulated by coculture with LC-MSCs and by S100A4 ectopic overexpression. The invasion-promoting effects of S100A4 were significantly attenuated by a miR-155 inhibitor. These results suggest that S100A4 exerts its effects through the regulation of miR-155 expression in HCC cells. We demonstrate that S100A4 secreted from LC-MSCs promotes the expression of miR-155, which mediates the down-regulation of suppressor of cytokine signaling 1, leading to the subsequent activation of STAT3 signaling. This promotes the expression of matrix metalloproteinases 9, which results in increased tumor invasiveness. CONCLUSION: S100A4 secreted from LC-MSCs is involved in the modulation of HCC progression, and may be a potential therapeutic target. (HEPATOLOGY 2013).

SPINDLIN1 promotes cancer cell proliferation through activation of WNT/TCF-4 signaling.

SPINDLIN1, a new member of the SPIN/SSTY gene family, was first identified as a gene highly expressed in ovarian cancer cells. We have previously shown that it is involved in the process of spindle organization and chromosomal stability and plays a role in the development of cancer. Nevertheless, the mechanisms underlying its oncogenic role are still largely unknown. Here, we first showed that expression of SPINDLIN1 is upregulated in clinical tumors. Ectopic expression of SPINDLIN1 promoted cancer cell proliferation and activated WNT/T-cell factor (TCF)-4 signaling. The Ser84 and Ser99 amino acids within SPINDLIN1 were further identified as the key functional sites in WNT/TCF-4 signaling activation. Mutation of these two sites of SPINDLIN1 abolished its effects on promoting WNT/TCF-4 signaling and cancer cell proliferation. We further found that Aurora-A could interact with and phosphorylate SPINDLIN1 at its key functional sites, Ser84 and Ser99, suggesting that phosphorylation of SPINDLIN1 is involved in its oncogenic function. Collectively, these results suggest that SPINDLIN1, which may be a novel substrate of the Aurora-A kinase, promotes cancer cell growth through WNT/TCF-4 signaling activation.

SIRT1 is required for long-term growth of human mesenchymal stem cells.

Human mesenchymal stem cells (MSCs) have therapeutic potential because of their ability to self-renew and differentiate into multiple tissues. However, senescence often occurs in MSCs when they are cultured in vitro and the molecular mechanisms underlying this effect remain unclear. In this study, we found that NAD-dependent protein deacetylase SIRT1 is differentially expressed in both human bone marrow-derived MSCs (B-MSCs) and adipose tissue-derived MSCs after increasing passages of cell culture. Using lentiviral shRNA we demonstrated that selective knockdown of SIRT1 in human MSCs at early passage slows down cell growth and accelerates cellular senescence. Conversely, overexpression of SIRT1 delays senescence in B-MSCs that have undergone prolonged in vitro culturing and the cells do not lose adipogenic and osteogenic potential. In addition, we found that the delayed accumulation of the protein p16 is involved in the effect of SIRT1. However, resveratrol, which has been used as an activator of SIRT1 deacetylase activity, only transiently promotes proliferation of B-MSCs. Our findings will help us understand the role of SIRT1 in the aging of normal diploid cells and may contribute to the prevention of human MSCs senescence thus benefiting MSCs-based tissue engineering and therapies.

Identification of a new MAGE-A10 antigenic peptide presented by HLA-A*0201 on tumor cells.

MAGE-A antigens belong to cancer/testis (CT) antigens that are expressed in tumors but not in normal tissues with the exception of testis and placenta. Among MAGE-A antigens, MAGE-A10 is extensively expressed in various histological types of tumors, representing an attractive target for tumor immunotherapy. Cytotoxic T lymphocytes (CTLs) play a key role in anti-tumor immune responses, so the identification of CTL epitopes derived from MAGE-A10 would contribute a lot to the design of epitope-based vaccines for tumor patients. In this study, we predicted HLA-A*0201-restricted CTL epitope peptides of MAGE-A10, followed by peptide/HLA-A*0201 binding affinity and complex stability assays, and induced peptide-specific CTL immune responses. Of the selected three peptides (designated P1, P2 and P3), P1 (MAGE-A10310-318, SLLKFLAKV) could elicit peptide-specific CTLs both in vitro from HLA-A*0201-positive PBMCs and in HLA-A*0201/Kb transgenic mice. And, the induced CTLs could lyse MAGE-A10-expressing tumor cells in a HLA-A*0201-restricted fashion but not MAGE-A10-negative tumor cells. Our results demonstrate that the peptide MAGE-A10310-318 is a HLA-A*0201-restricted CTL epitope of MAGE-A10 and could serve as a target for therapeutic antitumoral vaccination.

Identification of two novel HLA-A*0201-restricted CTL epitopes derived from MAGE-A4.

MAGE-A antigens belong to cancer/testis (CT) antigens that are expressed in tumors but not in normal tissues except testis and placenta. MAGE-A antigens and their epitope peptides have been used in tumor immunotherapy trials. MAGE-A4 antigen is extensively expressed in various histological types of tumors, so it represents an attractive target for tumor immunotherapy. In this study, we predicted HLA-A*0201-restricted cytotoxic T lymphocyte (CTL) epitopes of MAGE-A4, followed by peptide/HLA-A*0201 affinity and complex stability assays. Of selected four peptides (designated P1, P2, P3, and P4), P1 (MAGE-A4(286-294), KVLEHVVRV) and P3 (MAGE-A4(272-280), FLWGPRALA) could elicit peptide-specific CTLs both in vitro from HLA-A*0201-positive PBMCs and in HLA-A*0201/K(b) transgenic mice. And the induced CTLs could lyse target cells in an HLA-A*0201-restricted fashion, demonstrating that the two peptides are HLA-A*0201-restricted CTL epitopes and could serve as targets for therapeutic antitumoral vaccination.

Identification of an HLA-A*0201-restricted cytotoxic T-lymphocyte epitope in rotavirus VP6 protein.

The function of cytotoxic T lymphocytes (CTLs) in rotavirus (RV) infection in humans is poorly understood. To date, no RV-specific human leukocyte antigen (HLA) class I-restricted T-cell epitopes have been described. In this study, four peptides derived from human RV Wa strain VP6 protein were predicted by computer algorithms and verified by an HLA*0201-binding assay. Two peptides with high affinity for HLA-A*0201 molecules were further assessed. The CTLs induced in vitro by P340-348 (TLLANVTAV)-loaded autologous dendritic cells from peripheral blood lymphocytes of HLA-A*0201-matched healthy donors released gamma interferon specifically upon stimulation with P340-348-loaded T2 cells. The CTLs lysed both P340-348-loaded T2 cells and human RV Wa strain-infected HLA-A*0201(+) Caco-2 cells in an antigen-specific and HLA-A*0201-restricted manner. At the same time, P340-348 was shown to be immunogenic in vivo in HLA-A*0201/Kb transgenic mice. It is proposed that P340-348 is an HLA-A*0201-restricted CTL epitope.

[Development of transgenic oilseed plants resistant to glyphosate and insects].

In this study, the aroA-M12 gene encoding bacterial 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) and the Btslm recombinant gene encoding Bacillus thuringiensis (Bt) toxin gene were introduced into a Brassica napus variety, Xiangyou No. 15, via Agrobacterium-mediated transformation using glyphosate as a selectable agent. PCR amplification and Southern blot analyses of T0 transgenic plants showed that the alien genes were transferred and integrated stably into the genome of Xiangyou No. 15. Western blot further confirmed that the alien genes were expressed in these plants. Transgenic plants with tolerance to both glyphosate and the tested pest were demonstrated by bioassays. Our result also clearly shows that the aroA-M12 gene can be used as an efficient selectable marker gene instead of antibiotic resistant markers in plant transformation.