The two-component system expression patterns and immune regulatory mechanism of Vibrio parahaemolyticus with different genotypes at the early stage of infection in THP-1 cells.

Vibrio parahaemolyticus must endure various challenging circumstances while being swallowed by phagocytes of the innate immune system. Moreover, bacteria should recognize and react to environmental signals quickly in host cells. Two-component system (TCS) is an important way for bacteria to perceive external environmental signals and transmit them to the interior to trigger the associated regulatory mechanism. However, the regulatory function of V. parahaemolyticus TCS in innate immune cells is unclear. Here, the expression patterns of TCS in V. parahaemolyticus-infected THP-1 cell-derived macrophages at the early stage were studied for the first time. Based on protein-protein interaction network analysis, we mined and analyzed seven critical TCS genes with excellent research value in the V. parahaemolyticus regulating macrophages, as shown below. VP1503, VP1502, VPA0021, and VPA0182 could regulate the ATP-binding-cassette (ABC) transport system. VP1735, uvrY, and peuR might interact with thermostable hemolysin proteins, DNA cleavage-related proteins, and TonB-dependent siderophore enterobactin receptor, respectively, which may assist V. parahaemolyticus in infected macrophages. Subsequently, the potential immune escape pathways of V. parahaemolyticus regulating macrophages were explored by RNA-seq. The results showed that V. parahaemolyticus might infect macrophages by controlling apoptosis, actin cytoskeleton, and cytokines. In addition, we found that the TCS (peuS/R) could enhance the toxicity of V. parahaemolyticus to macrophages and might contribute to the activation of macrophage apoptosis. IMPORTANCE This study could offer crucial new insights into the pathogenicity of V. parahaemolyticus without tdh and trh genes. In addition, we also provided a novel direction of inquiry into the pathogenic mechanism of V. parahaemolyticus and suggested several TCS key genes that may assist V. parahaemolyticus in innate immune regulation and interaction.

The effect of Chinese wild blueberry fractions on the growth and membrane integrity of various foodborne pathogens.

The objective of this study was to evaluate the antibacterial effect of Chinese wild blueberry extract and its fractions against Listeria monocytogenes, Staphylococcus aureus, Salmonella Enteritidis, and Vibrio parahaemolyticus. Chinese wild blueberry (Vaccinium uliginosum) crude extract (BBE) was obtained using methanol extraction, and sugars plus organic acids (F1), phenolics fraction (F2), and anthocyanins plus proanthocyanidins (F3) fractions were separated using C-18 Sep-Pak columns. The minimal inhibitory concentration and minimal bactericidal concentration of each fractional component were determined using a two-fold-serial dilution method. Nucleic acid leakage (OD260 nm ) and protein release (Bradford protein assay) were determined by spectrophotometry, to evaluate the permeability of the cell membrane. F3 was found to exhibit the greatest antimicrobial activity against the four tested strains, followed by F2, F1, and BBE. V. parahaemolyticus was the most sensitive to the all fractions, followed by S. Enteritidis, L. monocytogenes, and S. aureus. Survival curve analysis showed that the number of bacteria decreased from six log colony-forming units (CFU) to less than 10 CFU after bacteria were treated with fractions for 12 hr, which demonstrated the bactericidal effect of blueberry fractions. Furthermore, when the pathogens were treated with fractions for 2 hr, the OD260 nm and OD595 nm values increased significantly (P < 0.01), which indicated the significant release of nucleic acid and protein. The results from this study indicated that blueberry fractions, especially F3, inhibited the growth of foodborne pathogens by damaging their cell membrane, and may be developed as a natural preservative to prevent and control foodborne pathogens. PRACTICAL APPLICATION: A blueberry crude extract and its sugars plus organic acids, phenolics, and anthocyanins plus proanthocyanidins fractions, inhibited the growth of foodborne pathogens by destroying their cell membrane. Therefore, Chinese wild blueberries have potential as a natural preservative to prevent and control foodborne pathogens.

Investigation of Reference Genes in Vibrio parahaemolyticus for Gene Expression Analysis Using Quantitative RT-PCR.

Vibrio parahaemolyticus is a significant human pathogen capable of causing foodborne gastroenteritis associated with the consumption of contaminated raw or undercooked seafood. Quantitative RT-PCR (qRT-PCR) is a useful tool for studying gene expression in V. parahaemolyticus to characterize its virulence factors and understand the effect of environmental conditions on its pathogenicity. However, there is not a stable gene in V. parahaemolyticus that has been identified for use as a reference gene for qRT-PCR. This study evaluated the stability of 6 reference genes (16S rRNA, recA, rpoS, pvsA, pvuA, and gapdh) in 5 V. parahaemolyticus strains (O3:K6-clinical strain-tdh+, ATCC33846-tdh+, ATCC33847-tdh+, ATCC17802-trh+, and F13-environmental strain-tdh+) cultured at 4 different temperatures (15, 25, 37 and 42°C). Stability values were calculated using GeNorm, NormFinder, BestKeeper, and Delta CT algorithms. The results indicated that recA was the most stably expressed gene in the V. parahaemolyticus strains cultured at different temperatures. This study examined multiple V. parahaemolyticus strains and growth temperatures, hence the finding provided stronger evidence that recA can be used as a reference gene for gene expression studies in V. parahaemolyticus.

Odor fingerprinting of Listeria monocytogenes recognized by SPME-GC-MS and E-nose.

Microorganisms can produce species-specific microbial volatile organic compounds (MVOCs), or odor compounds, which can be characterized by odor fingerprinting. The objective of this study was to characterize the odor fingerprint of Listeria monocytogenes. Solid-phase microextraction-gas chromatography-mass spectrometry (SPME-GC-MS) and electronic nose (E-nose) were used to recognize the MVOCs of L. monocytogenes in pure culture medium. The main MVOCs of L. monocytogenes were identified by SPME-GC-MS analysis as alcohols, aldehydes, ketones, alkanes, and heterocyclics, among which the relative peak area of one compound, 3-hydroxy-2-butanone, increased along with the growth of L. monocytogenes. The odor fingerprint of L. monocytogenes at different growth stages could be clearly discriminated by E-nose. In addition, E-nose signals had a very good linear relationship with the concentration of this bacterium (R(2) = 0.9937). Our study may help to establish the analysis of the odor fingerprint of microorganisms as a potential routine method in microbiology.

Preliminary mechanism of acidic electrolyzed water ice on improving the quality and safety of shrimp.

Preliminary mechanism of acidic electrolyzed water (AEW) ice on improving the quality and safety of shrimp was investigated by examining the physicochemical and microbiological changes, sarcoplasmic proteins and enzymatic activities. The results showed that compared with tap water (TW) ice, AEW ice had an obvious (p<0.05) capability in limiting the changes of pH and shrinkage of muscle fibers in shrimp. Plate count enumeration and PCR-DGGE indicated that AEW greatly inhibited growth of bacteria on shrimp. Additionally, AEW ice had no adverse effects on sarcoplasmic proteins by SDS-PAGE method. And AEW ice displayed inhibitory activity (p<0.05) toward cathepsin B and polyphenol oxidase (PPO), although it did not present positive effects on inhibiting cathepsin D, acid phosphatase and lipase activity. Thus, this study brought new evidence to further demonstrate that AEW ice can serve as a promising technology for improving the quality of aquatic products in food industry.

Fate of Vibrio parahaemolyticus on shrimp after acidic electrolyzed water treatment.

The objective of this study was to investigate the fate of Vibrio parahaemolyticus on shrimp after acidic electrolyzed water (AEW) treatment during storage. Shrimp, inoculated with a cocktail of four strains of V. parahaemolyticus, were stored at different temperatures (4-30 °C) after AEW treatment. Experimental data were fitted to modified Gompertz and Log-linear models. The fate of V. parahaemolyticus was determined based on the growth and survival kinetics parameters (lag time, λ; the maximum growth rate, µmax; the maximum growth concentration, D; the inactivation value, K) depending on the respective storage conditions. Moreover, real-time PCR was employed to study the population dynamics of this pathogen during the refrigeration temperature storage (10, 7, 4 °C). The results showed that AEW treatment could markedly (p<0.05) decrease the growth rate (µmax) and extend the lag time (λ) during the post-treatment storage at 30, 25, 20 and 15 °C, while it did not present a capability to lower the maximum growth concentration (D). AEW treatment increased the sensitivity of V. parahaemolyticus to refrigeration temperatures, indicated by a higher (p<0.05) inactivation value (K) of V. parahaemolyticus, especially for 10 °C storage. The results also revealed that AEW treatment could completely suppress the proliferation of V. parahaemolyticus in combination with refrigeration temperature. Based on above analysis, the present study demonstrates the potential of AEW in growth inhibition or death acceleration of V. parahaemolyticus on seafood, hence to greatly reduce the risk of illness caused by this pathogen during post-treatment storage.

Use of acidic electrolyzed water ice for preserving the quality of shrimp.

Electrolyzed water ice is a relatively new concept developed in food industry in recent years. The effect of acidic electrolyzed water (AEW) ice on preserving the quality of shrimp (Litopenaeus vannamei) was investigated. Physical, chemical, and microbiological changes of the shrimp were examined during the storage. The results showed that compared with tap water (TW) ice, AEW ice displayed a potential ability in limiting the pH changes of shrimp flesh and significantly (p < 0.05) retarded the changes of color difference and the formation of total volatile basic nitrogen (TVBN). And AEW ice treatment had no adverse effects on the firmness of shrimp. Conventional plate count enumeration and PCR-DGGE demonstrated that AEW ice had a capability of inhibiting growth of bacteria on raw shrimp, and the maximum reductions of population reached >1.0 log CFU/g (>90%) on the sixth day. Moreover, AEW ice was clearly more efficient in maintaining the initial attachments between muscle fibers in shrimp according to histological section analysis. On the basis of above analysis, AEW ice can be a new alternative of traditional sanitizer to better preserve the quality of seafood in the future.

Immunomodulation of bone marrow macrophages by GLIS, a proteoglycan fraction from Lingzhi or Reishi medicinal mushroom Ganoderma lucidium (W.Curt.:Fr.) P. Karst.

The immunomodulatory effect of GLIS (Lingzhi or Reishi medicinal mushroom Ganoderma lucidum immunomodulating substance) on macrophages has been investigated as part of ongoing research into the anticancer properties of this mushroom. Proliferation of bone marrow macrophages (BMMs) was enhanced by GLIS in a dose-dependent manner. Microscopic examination revealed that numerous GLIS-treated BMMs were enlarged and formed pseudopodia. Exposure of BMMs to GLIS resulted in significant increases in NO production, induction of cellular respiratory burst activity, and increased levels of IL-1ß, IL-6, IL-12p35, IL-12p40, IL-18, and TNF-α gene expression and levels of TNF-α, IL-1ß, and IL-12 secretion. Our data indicate that GLIS activates the immune system by modulating cytokine production.

Purification, chemical modification and immunostimulating activity of polysaccharides from Tremella aurantialba fruit bodies.

Ultrafiltration and a series of chromatographic steps were used to isolate and purify polysaccharides from Tremella aurantialba fruit bodies. Three crude fractions (TAP50w, TAP10-50w, and TAP1-10w), five semi-purified fractions (TAPA-TAPE), and one purified fraction (TAPA1) were obtained. A sulfated derivative of TAPA1 (TAPA1-s) was prepared by chemical modification. The immunostimulating activity of the polysaccharide fractions in vitro was determined using the mouse spleen lymphocyte proliferation assay. Of the three crude fractions tested, cell proliferation rates were increased most by TAP50w. Furthermore, TAPA1-s was markedly more stimulatory than TAPA1, indicating that sulfonation was an effective way to enhance the immunostimulating activity of polysaccharide.

Cloning and characterization of squalene synthase (SQS) gene from Ganoderma lucidum.

This report provides the complete nucleotide sequences of the full-length cDNA encoding squalene synthase (SQS) and its genomic DNA sequence from a triterpene-producing fungus, Ganoderma lucidum. The cDNA of the squalene synthase (SQS) (GenBank Accession Number: DQ494674) was found to contain an open reading frame (ORF) of 1,404 bp encoding a 468-amino-acid polypeptide, whereas the SQS genomic DNA sequence (GenBank Accession Number: DQ494675) consisted of 1,984 bp and contained four exons and three introns. Only one gene copy was present in the G lucidum genome. The deduced amino acid sequence of Ganoderma lucidum squalene synthase (Gl-SQS) exhibited a high homology with other fungal squalene synthase genes and contained six conserved domains. A phylogenetic analysis revealed that G. lucidum SQS belonged to the fungi SQS group, and was more closely related to the SQS of U. maydis than to those of other fungi. A gene expression analysis showed that the expression level was relatively low in mycelia incubated for 12 days, increased after 14 to 20 days of incubation, and reached a relatively high level in the mushroom primordia. Functional complementation of Gl-SQS in a SQS-deficient strain of Saccharomyces cerevisiae confirmed that the cloned cDNA encoded a squalene synthase.

[The phylogenetic relationship of cultivated isolates of Ganoderma in China inferred from nuclear ribosomal DNA ITS sequences].

Nuclear ribosomal DNA ITS sequences have been used to investigate phylogenetic relationships between 34 Ganoderma isolates cultivated in China. Five distinct groups were identified: the subgenus Elfvingia, the sect. Phaeonema, and three groups within the sect. Ganoderma. Most of the Ganoderma isolates (85.7%) formed a single group within the sect. Gantoderma. The result indicated clear genetic diversity between the subgenus Elfvingia and the sects Phaeonema and Ganoderma, but a smaller degree of genetic diversity between the three groups placed within the sect. Ganoderma. Analysis of molecular data is a more effective and useful approach for studying the taxonomy of Ganoderma, and for establishing phylogenetic relationships within the genus, compared to methods based on fruiting body morphology.

A simple, rapid and effective method for total RNA extraction from Lentinula edodes.

A rapid, inexpensive and reliable method for total RNA extraction from fruiting bodies of Lentinula edodes containing large quantities of polysaccharides and secondary metabolites is described. An initial extraction step using saturated NaCl solution facilitates the separation of nucleic acids from contaminants and, after further extraction with organic solvents and precipitation with 2-propanol, total RNA of high purity and suitable for applications such as cDNA synthesis, RT-PCR and Northern blot hybridization was obtained. The procedure may also have wider applicability for total RNA extraction from the tissues of other mushrooms.

[An efficient method for DNA extraction from compost].

An efficient method for obtaining DNA from compost which contained high levels of organic matter was developed. The protocol consisted of washing with phosphate-EDTA before extraction, cell lysis with hot-SDS and enzymes (lysozyme, lywalzyme, proteinase K), removing humic acid and other inhibitors with PVPP and precipitation with PEG-8000. The compost total DNA was extracted from four different composts, the DNA yield was 63.54 +/- 12.08 to approximately 106.50 +/- 28.36 microg/g of dry compost. Molecular size of DNA obtained using this protocol was about 23kb and contained low protein and humic acid contamination with the A260/A280 ratios exceeding 1.6 and A260/A230 ratios reaching 1.8. Usually, additional purification steps such as agarose gel electrophoresis, gel permeation chromatography, or affinity chromatography were needed to get PCR-amplifiable DNA, but the DNA obtained using this protocol could directly be used to PCR-amplification and restriction enzyme digestion. Just like purity of DNA template, lower DNA yield also appears to introduce a bias towards lower community diversity. In this study compared the purified DNA the direct DNA reveals higher microbial community diversity assessed by denaturing gradient gel electrophoresis(DGGE) of amplified V3 region of 16S rDNA.

Use of intersimple sequence repeats markers to develop strain-specific SCAR markers for Lentinula edodes.

Although Lentinula edodes is the second most important cultivated mushroom worldwide, most industrially cultivated strains have been identified only through traditional phenotypic analysis. Here, we report for the first time the use of sequence characterized amplified region (SCAR) markers for strain differentiation. SCAR markers were created by first generating and sequencing single intersimple sequence repeats fragments, and then designing primers based on these sequences to amplify strain-specific fragments of a certain size. One SCAR primer pair, ISL450F/R7 (amplifying a band of c. 450 bp), was designed to identify one strain of L. edodes (strain No. 7). The SCAR primer pair was then used to correctly amplify the single unique fragment from DNA samples taken from a total of 85 strains representing three separate species. Our data provide the foundation for a precise and rapid PCR-based strain-diagnostic system for L. edodes.

Structural elucidation of a novel fucogalactan that contains 3-O-methyl rhamnose isolated from the fruiting bodies of the fungus, Hericium erinaceus.

A new heteropolysaccharide, HEPF3, was isolated from the fruiting bodies of Hericium erinaceus. HEPF3 has a molecular weight of 1.9 x 10(4) Da and is composed of fucose and galactose in a ratio of 1:4.12. Compositional analysis, methylation analysis, together with 1H and 13C NMR spectroscopy established that HEPF3 consists of a branched pentasaccharide repeating unit with the following structure: [structure: see text]. HEPF3 also contains a minor proportion of 3-O-methylrhamnose that is thought to terminate the polymer main chain.

[Activation of mouse macrophages by the alkali-extracted polysaccharide from spore of Ganoderma lucidum].

AIM: To investigate the activation of mouse macrophages by the alkali-extracted polysaccharides from the spore of Ganoderma lucidum (LZSBS). METHODS: The mouse macrophages cultured in-vitro were stimulated by LZSBS. IL-1beta and TNF-alpha in the culture supernatants were detected by ELISA. NO production was detected by Griess assay. The percentage of phagocytosis of latex beads by mouse macrophages was counted under microscope. RESULTS: The mouse macrophages stimulated by LZSBS increased in volume and darkened in appearance under phase-contrast microscope. LZSBS-activated mouse macrophages secreted IL-1beta and TNF-alpha produced a large amount of NO. The percentage of phagocytosis of latex beads by mouse macrophages was also significantly increased in the presence of LZSBS. CONCLUSION: LZSBS can activate markedly the mouse macrophages.

Assessment the genetic diversity of Auricularia strains by two PCR-based typing methods.

Two PCR-based methods, enterobacterial repetitive intergenic consensus-PCR (ERIC-PCR) and randomly amplified polymorphic DNA (RAPD), were adopted for differentiating Auricularia strains. Taken the similarity coefficient as 75%, 29 strains of three Auricularia species were grouped into 6 and 9 clusters by RAPD and ERIC, respectively. The dendrogram from ERIC exhibited two distinct parts, one representing A. auricula and the other A. polytricha, but the dendrogram from RAPD failed to clearly distinguish between these two species. However, both methods similarly revealed high homology between A. fuscosuccinea and A. auricula. The homology relationships among the three species obtained from ERIC were validated by Southern hybridization. The analyses showed that RAPD is able to differentiate mainly at the species level, while ERIC is effective at the strain level and therefore more consistent with cultivation characteristics. The results indicate that the method of ERIC-PCR is more rapid and reliable than RAPD, and may substitute for RAPD in research related to the genetic identification and genetic diversity in Auricularia.