Preparation and antibacterial effect of chitooligosaccharides monomers with different polymerization degrees from crab shell chitosan by enzymatic hydrolysis.

This study aimed to explore the structure and antibacterial properties of chitooligosaccharide monomers with different polymerization degrees and to provide a theoretical basis for inhibiting Salmonella infection. Chitosan was used as a raw material to prepare and separate low-molecular-weight chitooligosaccharides. Chitobiose, chitotriose, and chitotetraose were obtained by gradient elution with cation exchange resin. The molecular weights and acetyl groups of the three monomers were determined by matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) and nuclear magnetic resonance (NMR), respectively. Three chitooligosaccharide monomers were used to explore the antibacterial effect on Salmonella. The results showed that the degree of deacetylation of chitosan was 92.6%, and the enzyme activity of chitosanase was 102.53 U/g. Within 18 h, chitosan was enzymatically hydrolyzed to chitooligosaccharides containing chitobiose, chitotriose, and chitotetraose, which were analyzed by thin-layer chromatography (TLC) and MALDI-TOF. MALD-TOF and TLC showed that the separation of monomers with ion exchange resins was effective, and NMR showed that there was no acetyl group. Chitobiose had a poor inhibitory effect on Salmonella, and chitotriose and chitotetraose had equivalent antibacterial effects.

Molecular characterization, expression analysis, and bactericidal activity of the derivative peptides of TFPI-1 and TFPI-2 in half-smooth tongue sole, Cynoglossus semilaevis.

Tissue factor pathway inhibitors (TFPIs) are Kunitz-type serine protease inhibitors that reversibly regulate the blood coagulation induced by tissue factor. TFPI family contain two members, TFPI-1 and TFPI-2. Recent studies have shown TFPI-1 and TFPI-2 also play important roles in innate immunity, however, the potential function of teleost TFPI are very limited. In this study, we characterized two TFPI (CsTFPI-1 and CsTFPI-2) molecules from half-smooth tongue sole (Cynoglossus semilaevis), examined their tissue distributions and expression patterns under pathogens stimulation as well as investigated the antibacterial activity of the C-terminal peptides. Quantitative real time RT-PCR analysis showed that constitutive CsTFPI-1 expression occurred, in increasing order, in head kidney, intestine, brain, spleen, liver, skin, gills, heart, and muscle; CsTFPI-2 was expressed, in increasing order, in the gills, intestine, skin, head kidney, liver, brain, spleen, muscle, and heart. Under Vibrio anguillarum, Streptococcus agalactiae and fish megalocytivirus stimulation, both CsTFPI-1 and CsTFPI-2 expression increased significantly in a manner that depended on the pathogen, tissue type, and infection stage, which suggested CsTFPI-1 and CsTFPI-2 play important roles in anti-bacterial and anti-viral infection. Finally, C-terminal peptides of CsTFPI-1 and CsTFPI-2, were synthesized and proved to have antibacterial effect against Micrococcus luteus that were independent of host serum. Take together, these results indicate that CsTFPI-1 and CsTFPI-2 play important roles in antimicrobial immunity of this fish.

A C-type lectin that inhibits bacterial infection and facilitates viral invasion in black rockfish, Sebastes schlegelii.

C-type lectins (CTLs) are important pattern recognition receptors (PRRs) that play vital roles in innate immunity. In teleosts, a number of CTLs have been reported, but their in vivo effects on host defense are still limited. In this study, a CTL homolog (SsLec1) was identified from black rockfish, Sebastes schlegelii, and its structure, expression and biological function was analyzed. The open reading frame of SsLec1 is 633 bp, with a 5'- untranslated region (UTR) of 36 bp and a 3'- UTR of 117 bp. The deduced amino acid sequence of SsLec1 shares the highest overall identity (73.20%) with the CTL of Oplegnathus fasciatus. SsLec1 possesses conserved CTL features, including a carbohydrate-recognition domain, four disulfide bond-forming cysteine residues, the mannose-type carbohydrate-binding motif, the conserved calcium binding sites and a putative signal peptide. The expression of SsLec1 was highest in liver and could be induced by experimental infection with Listonella anguillarum. Recombinant SsLec1 (rSsLec1) purified from E. coli was able to bind and agglutinate the Gram-negative fish pathogens Vibrio ichthyoenteri and Vibrio vulnificus. The agglutinating ability of rSsLec1 was abolished in the presence of mannose or ethylenediaminetetraacetic acid. Further analysis showed that rSsLec1 could enhance phagocytosis by macrophages. In vivo experiments indicated that rSsLec1 could inhibit bacterial infection and promote viral invasion. Taken together, these results suggest that SsLec1 is a novel CTL that possesses apparent immunoregulation property and plays a critical role in host defense against pathogens invasion.

Development of a quadruplex loop-mediated isothermal amplification assay for field detection of four Vibrio species associated with fish disease.

A quadruplex loop-mediated isothermal amplification (LAMP) method was developed to detect four Vibrio species, including Vibrio ichthyoenteri, Vibrio parahaemolyticus, Vibrio scophthalmi, and Vibrio vulnificus, simultaneously. Four sets of species-specific primers were designed with different restriction sites contained in the inner primers. The quadruplex LAMP method could distinguish four Vibrio species via the subsequent restriction enzyme analysis. The sensitivity of the quadruplex LAMP method were 10(2)-10(3) times higher than the sensitivity of conventional PCR. V. scophthalmi, V. vulnificus, V. parahaemolyticus and V. ichthyoenteri could be detected in the different tissues of the infected fish by the quadruplex LAMP method simply and conveniently through using SYBR Green I to facilitate visual inspection of the LAMP products. The method we developed in this study could be a simple and convenient diagnostic tool for field detection of Vibrio infection in fish.

Study on Unit Cell Models and the Effective Thermal Conductivities of Silica Aerogel.

In this paper, two modified unit cell models, truncated octahedron and cubic array of intersecting square rods with 45-degree rotation, are developed in consideration of the tortuous path of heat conduction in solid skeleton of silica aerogel. The heat conduction is analyzed for each model and the expressions of effective thermal conductivity of the modified unit cell models are derived. Considering the random microstructure of silica aerogel, the probability model is presented. We also discuss the effect of the thermal conductivity of aerogel backbone. The effective thermal conductivities calculated by the proposed probability model are in good agreement with available experimental data when the density of the aerogel is 110 kg/m3.

The Calculation of Thermal Conductivities by Three Dimensional Direct Simulation Monte Carlo Method.

Three dimensional direct simulation Monte Carlo (DSMC) method with the variable soft sphere (VSS) collision model is implemented to solve the Boltzmann equation and to acquire the heat flux between two parallel plates (Fourier Flow). The gaseous thermal conductivity of nitrogen is derived based on the Fourier's law under local equilibrium condition at temperature from 270 to 1800 K and pressure from 0.5 to 100,000 Pa and compared with the experimental data and Eucken relation from Chapman and Enskog (CE) theory. It is concluded that the present results are consistent with the experimental data but much higher than those by Eucken relation especially at high temperature. The contribution of internal energy of molecule to the gaseous thermal conductivity becomes significant as increasing the temperature.

Isolation of differentially expressed sex genes in garden asparagus using suppression subtractive hybridization.

Garden asparagus (Asparagus officinalis L.) is a dioecious species whose male and female flowers are found in separate unisexual individuals. A region called the M-locus, located on a pair of homomorphic sex chromosomes, controls sexual dimorphism in asparagus. To date, no sex determining gene has been isolated from asparagus. To identify more genes involved in flower development in asparagus, subtractive hybridization library of male flowers in asparagus was constructed by suppression subtraction hybridization. A total of 107 expressed sequence tags (ESTs) were identified. BLASTX analysis showed that the library contained several genes that could be related to flower development. The expression patterns of seven selected genes believed to be involved in the development of asparagus male flower were further analyzed by semi-quantitative or real-time reverse-transcription polymerase chain reaction (RT-PCR). Results showed that AOEST4-5, AOEST12-40, and AOEST13-38 were strongly expressed in the male flower stage, whereas no transcript level of AOEST13-38 was detected in the female flower stage. The expression levels of AOEST13-87, AOEST13-92, AOEST13-40, and AOEST18-87 in the male flower stage were also higher than those in the female flower stage, although these transcripts were also expressed in other tissues. The identified genes can provide a strong starting point for further studies on the underlying molecular differences between the male and female flowers of asparagus.

Analysis of transposable elements in the genome of Asparagus officinalis from high coverage sequence data.

Asparagus officinalis is an economically and nutritionally important vegetable crop that is widely cultivated and is used as a model dioecious species to study plant sex determination and sex chromosome evolution. To improve our understanding of its genome composition, especially with respect to transposable elements (TEs), which make up the majority of the genome, we performed Illumina HiSeq2000 sequencing of both male and female asparagus genomes followed by bioinformatics analysis. We generated 17 Gb of sequence (12×coverage) and assembled them into 163,406 scaffolds with a total cumulated length of 400 Mbp, which represent about 30% of asparagus genome. Overall, TEs masked about 53% of the A. officinalis assembly. Majority of the identified TEs belonged to LTR retrotransposons, which constitute about 28% of genomic DNA, with Ty1/copia elements being more diverse and accumulated to higher copy numbers than Ty3/gypsy. Compared with LTR retrotransposons, non-LTR retrotransposons and DNA transposons were relatively rare. In addition, comparison of the abundance of the TE groups between male and female genomes showed that the overall TE composition was highly similar, with only slight differences in the abundance of several TE groups, which is consistent with the relatively recent origin of asparagus sex chromosomes. This study greatly improves our knowledge of the repetitive sequence construction of asparagus, which facilitates the identification of TEs responsible for the early evolution of plant sex chromosomes and is helpful for further studies on this dioecious plant.