Isolation and characterization of an antimicrobial Bacillus subtilis strain O-741 against Vibrio parahaemolyticus.

Vibrio parahaemolyticus is a marine bacterium that can infect and cause the death of aquatic organisms. V. parahaemolyticus can also cause human foodborne infection via contaminated seafood, with clinical syndromes which include diarrhea, abdominal cramps, nausea and so on. Since controlling V. parahaemolyticus is important for aquaculture and human health, various strategies have been explored. This study investigates the application of antagonistic microorganisms to inhibit the growth of V. parahaemolyticus. We screened aquaculture environment samples and identified a Bacillus subtilis strain O-741 with potent antimicrobial activities. This strain showed a broad spectrum of antagonistic activities against V. parahaemolyticus and other Vibrio species. Application of the O-741 bacterium significantly increased the survival of Artemia nauplii which were infected with V. parahaemolyticus. Furthermore, the cell-free supernatant (CFS) of O-741 bacterium exhibited inhibitory ability against V. parahaemolyticus, and its activity was stable to heat, acidity, UV, enzymes, and organic solvents. Next, the O-741 CFS was extracted by ethyl acetate, and analyzed by ultra-performance liquid chromatography-mass-mass spectrometry (UPLC-MS/MS), and the functional faction was identified as an amicoumacin A compound. The organic extracts of CFS containing amicoumacin A had bactericidal effects on V. parahaemolyticus, and the treated V. parahaemolyticus cells showed disruption of the cell membrane and formation of cell cavities. These findings indicate that B. subtilis strain O-741 can inhibit the V. parahaemolyticus in vitro and in vivo, and has potential for use as a biocontrol agent for preventing V. parahaemolyticus infection.

Correlation of Beta2-Glycoprotein I With Tumor Prognosis in Breast Cancer Patients.

BACKGROUND/AIM: Beta2-glycoprotein I (ß2-GPI) is a plasma glycoprotein, which has been implicated in a variety of physiological functions. However, the connection between ß2-GPI and breast cancer is mostly unknown. Breast cancer is a malignant tumor that severely impairs women's health worldwide. The aim of the study was to investigate the role of ß2-GPI in tumor cells of breast cancer patients and its correlation with tumor prognosis. MATERIALS AND METHODS: A total of 125 female patients diagnosed with breast cancer were enrolled in the study. The expression of ß2-GPI in resected breast tissues was determined by immunohistochemistry (IHC) and correlated with clinicopathological variables by the Chi-squared test. The prognostic value of ß2-GPI for overall survival (OS) and disease-free survival (DFS) was determined by Kaplan-Meier estimates and the significance of differences was evaluated by the log-rank test. RESULTS: ß2-GPI staining was predominantly observed in tumor cells of breast cancer patients and significantly correlated with tumor stage and lymph node metastasis of breast cancer. High ß2-GPI expression was significantly correlated with better OS and DFS. Moreover, DFS was found to be significantly better in patients with higher ß2-GPI expression, especially those in the early tumor stage groups. CONCLUSION: High ß2-GPI expression levels in tumor cells of breast cancer patients were independent factors predicting a better OS and DFS. ß2-GPI activation in high-risk patients may be a potential strategy for reducing breast cancer progression.

In Vitro Assessment of the Antioxidant and Anticancer Properties of Flammulina velutipes Stipe Extracts.

BACKGROUND/AIM: Flammulina velutipes (FV), also known as the golden needle mushroom, is an edible and medicinal fungus that contains bioactive substances regulating various physiological functions. While the fruiting bodies of FV are commonly consumed, their stipes are often discarded despite containing polysaccharides. In this study, the biological functions of FV stipes (FV-S) were investigated to reduce waste and pollution while increasing their value. MATERIALS AND METHODS: The antioxidant activity of FV was evaluated using three methods: the DPPH radical-scavenging capacity assay, ferrous ion chelating assay, and reducing power analysis. The anti-cancer potential was assessed through MTT viability and immunoblotting analyses. RESULTS: Results showed that FV-S had higher polysaccharide and total phenolic contents and greater antioxidant abilities, particularly in ethanolic extracts. FV-S also exhibited significant anticancer properties, specifically in hot water extracts with high polysaccharide contents, and suppressed prostate cancer cell viability by inhibiting androgen receptor and PCa-specific antigen mRNA expression while inducing caspase-3/7 activation. CONCLUSION: FV-S is rich in bioactive components, possesses higher antioxidant and anticancer abilities, and has potential as an anticancer agent, which could enhance the value of FV.

Characterization of a lytic vibriophage VP06 of Vibrio parahaemolyticus.

Vibrio parahaemolyticus is a human enteropathogenic bacterium and is also pathogenic to shrimp and finfish. In a search for a biocontrol agent for V. parahaemolyticus and other pathogenic Vibrio species, a lytic phage VP06 was isolated from oyster using V. parahaemolyticus as the host. VP06 is a Siphoviridae phage with a polyhedral head and a long tail. The genome sequence of VP06 was 75,893 nucleotides in length and the G + C content was 49%; a total of 101 CDSs were identified in VP06, of which 39 exhibited functional domains/motifs. The genomic sequence of VP06 is similar to those of a lytic Vibrio vulnificus phage SSP002 and a temperate V. parahaemolyticus phage vB_VpaS_MAR10, although VP06 has distinct features in the CDS arrangement and 14 unique CDSs. Phylogenetic analysis revealed that VP06, SSP002 and vB_VpaS_MAR10 belong to a novel genus cluster of Siphoviridae phages. This phage lysed 28.1% of various Vibrio strains, and the efficiency of plating method revealed that VP06 was highly effective in lysing strains of Vibrio alginolyticus, Vibrio azureus, Vibrio harveyi and V. parahaemolyticus. The properties of VP06, including its broad range of hosts and resistance to environmental stresses, indicate that it may be a candidate biocontrol agent.

Molecular response of Vibrio parahaemolyticus to the sanitizer peracetic acid.

Peracetic acid (PAA) is a common oxidative sanitizer that is used in the food industry against various microorganisms. Limited information on the response of bacteria to this biocide is available. This study investigates the molecular response of the prevalent seafood-borne pathogenic Vibrio parahaemolyticus to PAA using mutants of peroxide scavenging genes. Among katE1, katE2, katG1, katG2, ahpC1 and ahpC2, and their regulator oxyR gene mutants, oxyR and katE mutants were highly susceptible to PAA. The growth and lethality of V. parahaemolyticus were harmed by 15 ppm of PAA in the △katE1E2 double mutant, and were significantly ameliorated in the presence of the katE1 gene in the wild-type strain and the gene-complementary strains that were pre-adapted in 2 ppm of PAA or 100 µM hydrogen peroxide. The application of PAA to these strains induced the accumulation of reactive oxygen species. The reduction of the level of hydrogen peroxide and gene expression during this treatment was influenced by the presence of katE genes. This investigation confirmed the major role of katE1 and a compensatory role of katE2 in the resistance of V. parahaemolyticus to PAA, and demonstrated some minor differences in the responses of this bacterium against PAA and hydrogen peroxide.

Generation of Monoclonal Antibodies against Dengue Virus Type 4 and Identification of Enhancing Epitopes on Envelope Protein.

The four serotypes of dengue virus (DENV1-4) pose a serious threat to global health. Cross-reactive and non-neutralizing antibodies enhance viral infection, thereby exacerbating the disease via antibody-dependent enhancement (ADE). Studying the epitopes targeted by these enhancing antibodies would improve the immune responses against DENV infection. In order to investigate the roles of antibodies in the pathogenesis of dengue, we generated a panel of 16 new monoclonal antibodies (mAbs) against DENV4. Using plaque reduction neutralization test (PRNT), we examined the neutralizing activity of these mAbs. Furthermore, we used the in vitro and in vivo ADE assay to evaluate the enhancement of DENV infection by mAbs. The results indicate that the cross-reactive and poorly neutralizing mAbs, DD11-4 and DD18-5, strongly enhance DENV1-4 infection of K562 cells and increase mortality in AG129 mice. The epitope residues of these enhancing mAbs were identified using virus-like particle (VLP) mutants. W212 and E26 are the epitope residues of DD11-4 and DD18-5, respectively. In conclusion, we generated and characterized 16 new mAbs against DENV4. DD11-4 and D18-5 possessed non-neutralizing activities and enhanced viral infection. Moreover, we identified the epitope residues of enhancing mAbs on envelope protein. These results may provide useful information for development of safe dengue vaccine.

An Epitope-Substituted DNA Vaccine Improves Safety and Immunogenicity against Dengue Virus Type 2.

Dengue virus (DENV), a global disease, is divided into four serotypes (DENV1-4). Cross-reactive and non-neutralizing antibodies against envelope (E) protein of DENV bind to the Fcγ receptors (FcγR) of cells, and thereby exacerbate viral infection by heterologous serotypes via antibody-dependent enhancement (ADE). Identification and modification of enhancing epitopes may mitigate enhancement of DENV infection. In this study, we characterized the cross-reactive DB21-6 and DB39-2 monoclonal antibodies (mAbs) against domain I-II of DENV; these antibodies poorly neutralized and potently enhanced DENV infection both in vitro and in vivo. In addition, two enhancing mAbs, DB21-6 and DB39-2, were observed to compete with sera antibodies from patients infected with dengue. The epitopes of these enhancing mAbs were identified using phage display, structural prediction, and mapping of virus-like particle (VLP) mutants. N8, R9, V12, and E13 are the reactive residues of DB21-6, while N8, R9, and E13 are the reactive residues of DB39-2. N8 substitution tends to maintain VLP secretion, and decreases the binding activity of DB21-6 and DB39-2. The immunized sera from N8 substitution (N8R) DNA vaccine exerted greater neutralizing and protective activity than wild-type (WT)-immunized sera, both in vitro and in vivo. Furthermore, treatment with N8R-immunized sera reduced the enhancement of mortality in AG129 mice. These results support identification and substitution of enhancing epitope as a novel strategy for developing safe dengue vaccines.

Cellulose-based diagnostic devices for diagnosing serotype-2 dengue fever in human serum.

Here, two types of cellulose-based in vitro diagnostic devices are demonstrated for the diagnosis of dengue virus infection in both buffer system and human serum: 1) paper-based ELISA for providing the semiquantitative information of the disease activity of serotype-2 dengue fever to healthcare persons (i.e., monitoring the disease activity with a specific serotype in single patients); 2) lateral flow immunoassays to screen for infection with serotype-2 dengue fever (i.e., rapid YES or NO diagnosis prepared for large populations, in terms of global public health). Paper-based ELISA (specific to serotype-2 dengue fever), which builds off of our previous studies and a revised previous ELISA procedure, owns multiple advantages: 1) high sensitivity (about 40 times higher than the current ELISA-based approaches, due to our therapeutic-based monoclonal antibody) and specificity (specific to dengue virus serotype-2 nonstructural protein-1 antigens); 2) tiny amount of sample and reagent used for single tests; 3) short operating duration (i.e., rapid diagnostic device); and, 4) inexpensiveness (appropriate for use in all developing and underdeveloped nations of the world). Due to the higher sensitivity and shorter operating duration of paper-based ELISA (compared with conventional ELISA, and lateral flow immunoassays also performed in this study), this study has not only been able to perform the diagnosis of dengue virus serotype-2 nonstructural protein-1 antigens in both buffer system and human serum but also to evaluate dengue virus serotype-2 envelope proteins in the buffer system, thus successfully achieving the first such use of these proteins as the target antigen for the development of diagnostic tools. These results provide a more comprehensive understanding for the genesis of dengue fever diagnostic tools (through antibody-antigen recognition).

Species-level identification of isolates of the Acinetobacter calcoaceticus-Acinetobacter baumannii complex by sequence analysis of the 16S-23S rRNA gene spacer region.

The species Acinetobacter calcoaceticus, A. baumannii, genomic species 3, and genomic species 13TU included in the Acinetobacter calcoaceticus-Acinetobacter baumannii complex are genetically highly related and difficult to distinguish phenotypically. Except for A. calcoaceticus, they are all important nosocomial species. In the present study, the usefulness of the 16S-23S rRNA gene intergenic spacer (ITS) sequence for the differentiation of (genomic) species in the A. calcoaceticus-A. baumannii complex was evaluated. The ITSs of 11 reference strains of the complex and 17 strains of other (genomic) species of Acinetobacter were sequenced. The ITS lengths (607 to 638 bp) and sequences were highly conserved for strains within the A. calcoaceticus-A. baumannii complex. Intraspecies ITS sequence similarities ranged from 0.99 to 1.0, whereas interspecies similarities varied from 0.86 to 0.92. By using these criteria, 79 clinical isolates identified as A. calcoaceticus (18 isolates) or A. baumannii (61 isolates) with the API 20 NE system (bioMerieux Vitek, Marcy l'Etoile, France) were identified as A. baumannii (46 isolates), genomic species 3 (19 isolates), and genomic species 13TU (11 isolates) by ITS sequencing. An identification rate of 96.2% (76 of 79 isolates) was obtained by using ITS sequence analysis for identification of isolates in the A. calcoaceticus-A. baumannii complex, and the accuracy of the method was confirmed for a subset of strains by amplified rRNA gene restriction analysis and genomic DNA analysis by AFLP analysis by using libraries of profiles of reference strains. In conclusion, ITS sequence-based identification is reliable and provides a promising tool for elucidation of the clinical significance of the different species of the A. calcoaceticus-A. baumannii complex.